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MVDP Quarterly Report 2

1. EXECUTIVE SUMMARY

A summary of work efforts for the Malaria Vaccine Development Program (MVDP) contract for the current reporting period is detailed herein. All remaining projects have now been completed including one vaccine development project, the RCR complex vaccine development project (RCR Complex), and one project to develop human monoclonal antibodies (mAbs) to RH5 (RH5.1 Human mAb) for which both the technical work and final deliverables were completed and submitted during the reporting period. Previously completed projects for which final deliverables were submitted during the reporting period include the liver stage vaccine development project (Liver Stage Vaccine), the CSP vaccine development project (CSP Vaccine), and the RH5.1 clinical study project (RH5.1 Clinical Study). Final deliverables for two MVDP projects, the PD1 blockade inhibitor project (PD1 Blockade Inh) and the blood stage epitope-based vaccine project (Blood Stage Epitope), were previously completed and approved. As part of the MVDP, Leidos has executed collaborative agreements (e.g., information exchange under NDA, reagent exchange under MTA, and collaborative work under CRADA) to benefit the MVDP and the broader malaria research community, as well as to extend the utility of the MVDP contract. In Q3 FY2020, Leidos executed the last collaboration agreement for the MVDP prime contract (a CRADA with WRAIR involving development of a modified CSP mRNA construct) and technical work associated with this agreement was completed in Q2 FY2021 (these activities have been reported under the liver stage vaccine project). The information contained herein is intended to provide technical detail regarding activities conducted within the specified reporting period; however, at the request of USAID, Leidos retains information and data in quarterly reports for activities completed within the relevant fiscal year.

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

On October 12, 2020, Leidos submitted a second no-cost extension (NCE) request to extend the prime contract period of performance end date to April 7, 2021 due to work completion delays experienced because of impacts from COVID-19. On November 3, 2020, Leidos received the fully-executed contract modification extending the prime contract period of performance end date to April 7, 2021.

2.1. CURRENT BUDGET SUMMARY The budget summary for Q2 FY2021 (current data is only available through February 2021) is provided in Table 2.1-1. Table 2.1-2 provides FY20 to-date costs against FY21 Annual Work Plan Estimates. Detailed subcontractor spending is provided in the associated financial report.

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2.2. DELIVERABLES SUMMARY In Table 2.2-1 below, we provide the deliverables for which Leidos is responsible under the prime contract with respect to the tasks assigned by USAID. All deliverables are assigned according to the four (4) “Elements” that are outlined in the prime contract.

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Table 2-1. Deliverable Summary

ID* Deliverable/Work Product Actions/Status Notes

CONTRACTS AND ADMINISTRATION

Prime Contract

Signed: 5/28/15 Modifications: MOD 1 2/22/16, MOD 2 3/4/16 MOD 3 8/16/16 MOD 4 2/13/17 MOD 5 5/1/17 MOD 6 7/18/17 MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 MOD 10 3/1/19 MOD 11 05/04/20 MOD 12 11/05/20

The NCE modification extending the contract POP end date to 04/07/21 was received on 11/05/20.

i.

Contractor – Employee Non- Disclosure/Conflict of Interest (COI) Agreements

Delivered Signed Non-Disclosure/COI Agreements are located on SharePoint.

ii. Annual Work Plan Delivered: 09/3/19 Approved: 10/28/19 Upcoming Plans: None.

vi. Quarterly Reports Q2 FY21 Delivered: 04/07/21 Upcoming Reports: None (Q2 FY2021 report delivered as part of the Final Overall Contract Report)

vii. Quarterly Financial Reports Q2 FY21 Delivered: 04/07/21

Upcoming Reports: None (Q2 FY2021 report delivered as part of the Final Overall Contract Report)

viii. Annual Report/Q4 Report Delivered: 10/14/20 Approved: 11/20/20 Upcoming Reports: None

H.10.a Small Business Subcontracting Plan

Delivered: The Small Business Subcontracting Plan was included in Leidos’ proposal.

H.10.b.1 Individual Subcontract Reports

Due: Annually in October; submitted via eSRS by Leidos’ Small Business Liaison office.

Submitted in October FY2021

H.10.b.2 Summary Subcontract Report



ix. Final Overall Contract Report Delivered: 04/07/21 Upcoming Report: None

C.4.4 Biweekly USAID–Leidos Update Meetings

Held: See Table 2.4-1. Ad hoc meetings held: See Table 2.4-1.

Upcoming Meetings: None. Final meeting held March 17, 2021.

G.4.C

Contract Administration Meetings with Contracting Officer’s Representative (COR)

No actions in the reporting period.

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xiv. USAID Development Experience Information

Delivered: All contracts and administration deliverables will be uploaded to the DEC after 30 days from approval.

At contract completion, Leidos will upload all remaining contract deliverables to DEC. Financial information, confidential information and/or unpublished data from subcontractors, and raw data will not be uploaded to the DEC.

ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS

xii. Publications/Posters Q2 FY2021: Three manuscripts submitted See section 3.1 regarding publications.

C.3.2.2 New Project Proposals — None in the reporting period

ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS

CSP Vaccine Development Project

iii. Final Individual Project descriptions Approved

Project Plan approved by USAID on 1/29/16. Revised Project Plan approved 04/26/2017.

xii. Subcontracts: Procurement of Materials, Supplies, and Services

Subcontract/Task Order Awards: VLP Biotech, JHU, ADARC, EpiVax, Precision Antibody, ImmunoVaccine, VaxDesign Other Procurement Vehicles: CPC Scientific, Vaxine

None, project completed.

iv. Final Individual Project Report Completed: 12/10/2020 Final project report uploaded to

Google Drive.

v. Individual Project Data Sets Completed: 01/13/2021 Final raw data report uploaded to

Google Drive.

xii. Publications Completed: Submitted to Frontiers in Immunology 04/01/2021.

See section 3.1 regarding publications.

ix. Other/Ad Hoc Reports No actions in the reporting period.

RH5.1/AS01 Vaccine Clinical Study

iii. Final Individual Project descriptions Approved Project Plan approved by USAID on

2/17/16.

xii. Procurement of Materials, Supplies, and Services

Subcontract/Task Order Awards: University of Oxford (Oxford), EpiVax

None, project completed

iv. Final Individual Project Report Completed: 02/15/2021 Final version uploaded to Google

Drive.

v. Individual Project Data Set Completed: 02/15/2021 Final version delivered with Final Project Report via Google Drive.

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xii. Publications Completed: Accepted for publication by Med on 03/15/2021 See section 3.1 regarding publications.

ix. Other/Ad Hoc Reports No actions this reporting period

xii. Registration of Trials Completed Trial registered on September 5, 2016 (ClinicalTrials.gov)

Liver Stage Vaccine Development Project


Project Plan approved by USAID on 6/13/16. Attachment 1 - CD8 platform scouting plan approved by USAID on 6/6/19.


Subcontract Awards: EpiVax, VaxDesign, Precision Antibody, Multimeric BioTherapeutics, MabTech, Mimotopes, Biosynthesis, TriLink, ADARC, GeoVax, JHU

Q1 FY2021: GeoVax subcontract extension completed.

iv. Final Individual Project Report Completed: 03/30/2021 Final version delivered in Q2 FY2021

via Google Drive.





RCR Complex Vaccine Development Project-WEHI


3/21/2019.


Pending: WEHI (W3) Subcontract/Task Order Awards: ImmunoVaccine (W3 in Q4), and Precision Antibody

None, project completed


via Google Drive.


xii. Publications Not applicable


RCR Complex Vaccine Development Project-University of Oxford (Oxford)


2/13/19.


Subcontract Awards: Oxford, IMV None, project completed

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via Google Drive.




RH5.1 Human mAb Identification and Development

iii. Final Individual Project descriptions Approved Project Plan approved by USAID

(regular elements only) on 1/29/19.

xii. Procurement of Materials, Supplies, and Services Subcontract Award: Oxford Q1 FY2021: Oxford subcontract

extension completed.


via Google Drive.




Blood-Stage Epitope Vaccine Development


Project Plan approved by USAID on 3/24/2016. Addendum 1 approved on 6/23/16.


Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH, KempBio MTAs: WEHI (Alan Cowman), Swiss TPH (Gerd Pluschke), Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright)

None, project completed.

iv. Final Individual Project Report Approved

Draft delivered to USAID on 01/31/19. USAID feedback received on 3/14/19. Final Approval 5/29/19.

v. Individual Project Data Set Project Completed Raw data delivered with the final project report.




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PD1 Blockade Inhibitor


1/19/2017.


Subcontract Awards: ADARC, Oxford None, project completed.

iv. Final Individual Project Report Approved Final Project Report delivered to

USAID on 6/10/19

v. Individual Project Data Set Project Completed Raw data delivered with the final project report.

xii. Publications Project Completed Manuscripts published in FY2020. ix. Other/Ad Hoc Reports Project Completed

ELEMENT 3: SCG ANNUAL MEETING SUPPORT

x. SCG Annual Meeting Event Date: N/A x.a-e. Logistic Support N/A

ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES

xi. MVDP Reagents Repository SriSai Biopharmaceutical Solutions All materials were dispositioned in Q2

FY2021 (January 2021). *Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

2.3. MANAGEMENT TOOLS Leidos’ SharePoint document repository is a Fiscal Information Security Management Act−compliant, web-based tool that provides access to program data/documentation, deliverables, work products, and schedules. This type of interface is an especially important information exchange among study sites as vaccines progress through milestones. Leidos granted folder permissions to subcontractor technical leads and customer points of contact, commensurate with their roles, allowing direct updates to their respective folders (Table 2.3-1). Transparency is achieved via USAID access to program folders. Leidos has uploaded documents relevant to the current reporting period to SharePoint. In Q1 FY2021, USAID informed Leidos that Google Docs was the USAID official document sharing platform with their implementation partners. Leidos was able to establish Google Drive access so that large file size and final program deliverables can be submitted via a USAID-hosted Google Drive folder.

Table 2-2. Management Tools

Tool Description Location

SharePoint Cloud-based solution for exchanging and storage of documents https://vector.leidos.com/sites/ITLSO/MVDP

Conference Phone Lines

Provide USAID OCONUS line to call Scientific Consultant Group members and CONUS line to communicate with Leidos

1-855-462-5367 1778004 2013235

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2.4. TEAM MEETINGS Leidos also achieves transparency by routine copying of designated customer staff regarding email communications and teleconferences. As required, Leidos has set a standing biweekly meeting with USAID to review our MVDP activities (see Table 2.4-1). Ad hoc discussions to ensure positive study outcomes have been implemented. Leidos uses standard business tools (e.g., email, phone, teleconference, and desktop sharing) to communicate with staff and customers. Meeting agendas and summaries/minutes are available in the “Meeting Materials” folder on the MVDP SharePoint site (https://vector.leidos.com/sites/ITLSO/MVDP/Deliverables/Meeting Materials).

Table 2-3. Team Meetings

Meeting Date Topic

October 7, 2020 Leidos/USAID Bi-weekly Team Meeting


November 4, 2020 Leidos/USAID Bi-weekly Team Meeting (Canceled)

November 10, 2020 Oxford RCR Complex Project IPT Meeting

November 18, 2020 Leidos/USAID Bi-weekly Team Meeting

December 2, 2020 Leidos/USAID Bi-weekly Team Meeting (Canceled)


January 6, 2021 Leidos/USAID Bi-weekly Team Meeting

February 17, 2021 Leidos/USAID Bi-weekly Team Meeting

March 17, 2021 Leidos/USAID Bi-weekly Team Meeting

3. ELEMENT 1 ACTIVITIES

3.1. PUBLICATIONS The status of manuscripts submitted for publication is provided is provided in Table 3-1.

Table 3-1. Manuscript Listing and Status

Target Journal Title Project Status

Med Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

RH5 Clinical Study Manuscript was submitted in Q1 FY2021.

Frontiers in Immunology

Identification, Downselection and Immune Assessment of Liver-Stage CD8+ T Cell Epitopes from Plasmodium falciparum

Liver Stage Vaccine Development

Manuscript was finalized and cleared by USAID in March FY2021. This was submitted to Frontiers in Immunology on 03/22/2021 and is currently under review.


Identification and immune assessment of T cell epitopes in five Plasmodium falciparum blood stage antigens to facilitate vaccine candidate selection and optimization

Blood Stage Vaccine Development

Manuscript was finalized and cleared by USAID on 04/01/2021. This was submitted to Frontiers in Immunology on 04/05/2021.

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Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4+ T Cell Epitopes from Diverse Plasmodium falciparum Strains.

CSP Vaccine Development

Manuscript was finalized and cleared by USAID in 03/30/2021. This was submitted to Frontiers in Immunology on 04/01/2021.

3.2. ABSTRACT SUBMISSIONS/POSTER PRESENTATIONS No scientific conference abstract submissions or poster presentations are anticipated for FY2021.

3.3. NEW PROJECTS No new projects are planned.


Upon identification of viable vaccine projects to support development from proof-of-principle testing, manufacturing, and clinical trial evaluation, Leidos drafts a detailed plan and protocols, identifies subcontractors, and executes the plan. Projects completed in FY2021 are described in this section, as well as adjuvants and platforms utilized for the same.

4.1. ADJUVANT/DELIVERY PLATFORMS Adjuvants and delivery platforms used for projects completed in FY2021 are detailed in this section.

4.1.1 ADJUVANTS The DepoVaxTM platform is in use for the CSP and RCR complex projects. Matrix-MTM is in use for the RCR complex project.

4.1.1.1 DepoVax The DepoVax platform, developed by ImmunoVaccine Inc., contains lipids, cholesterol, oil, emulsifier and an immunostimulant (e.g., cGAMP, polyI:C, and/or Pam3Cys). This lipid-in-oil platform is designed to present antigen(s) and adjuvant(s) at a long-lasting depot that effectively attracts antigen-presenting cells (APCs) and from which antigen is released over an extended period of time, from weeks to months. DepoVax promotes Th2 responses and enhances Th1 immune responses without triggering regulatory T cells. DepoVax has been used in the clinic as part of a Phase I/II study for a cancer vaccine (clinicaltrials.gov identifier: NCT01095848). Of note is that there are no aqueous components in this formulation; therefore, antigen is lyophilized for use with DepoVax and components are mixed and emulsified prior to administration using materials provided as part of an administration kit. Leidos executed a purchase order with ImmunoVaccine for formulation and provision of adjuvants for preliminary efficacy studies.

4.1.1.2 Matrix-M Matrix-M is a saponin-based adjuvant comprised of purified saponin, synthetic cholesterol, and a phospholipid patented by Novavax. This adjuvant generates both cell-mediated and antibody-mediated immune responses, and has the potential to increase immune response duration as shown in numerous

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clinical trials (Shinde et al., 2018). The Draper group has an access agreement to use this adjuvant for malaria vaccine development.

4.1.2 PLATFORMS Three VLP-based vaccine delivery platforms are in use for different projects as follows: the WHcAg VLP platform is in use for the CSP project, the GeoVax MVA-VLP platform is in use for the liver stage project, and the SpyTag/SpyCatcher VLP platform is in use for the RCR complex project. In addition, development of mRNA-based vaccines using technology to enhance protein expression is also in use for the liver stage project.

4.1.2.1 WHcAg VLP The woodchuck hepatitis B core antigen (WHcAg) VLP platform, developed by VLP Biotech, is based on the core protein the of woodchuck hepatitis B virus. The core proteins self-assemble into VLPs with 240 copies of the antigen per VLP. This platform can accommodate multiple foreign sequence insertions, with long insertions possible at the N and C-termini. Inserts within the surface-exposed loop are possible, which is particularly beneficial for B cell epitopes as the VLP configuration permits cross-linking of B cells. Studies with this platform have shown it to be equal or more immunogenic than HBcAg for both B cell and T cell responses, not significantly cross-reactive with the HBcAg for B cell responses and only partially cross-reactive with HBcAg for T cell (CD4) responses, and function as a vaccine carrier platform for heterologous, B cell epitopes (Billaud et al., 2005a; Billaud et al., 2005b). In consideration of cost, WHcAg VLPs can be easily expressed at high levels in E. coli. Note that this platform has not yet been tested in the clinic.

4.1.2.2 GeoVax MVA Platform Modified Vaccinia virus Ankara (MVA)-based vaccines have been widely tested in the clinic and are known to generate high cellular responses (Gilbert, 2013). The main drawback of these platforms has been that immunogenicity is greater when these vectors are used to boost pre-existing T cell responses. However, GeoVax’s 4th generation MVA-VLP platform requires no immune response priming due to improved transgene stability during manufacture and elevated levels of expression compared to the parent platform. This is evidenced by a clinical study with GeoVax’s MVA-based HIV vaccine, where cellular (both CD8 and CD4) and humoral responses were seen in humans administered the MVA-VLP only (Goepfert et al., 2014). Such responses in animal models have also been seen (Brault et al., 2017). Also of note is that this platform does not require adjuvant.

4.1.2.3 SpyTag/SpyCatcher VLP Platform To alleviate the pitfalls of more traditional VLP development, the groups of Draper, Biswas and Howarth at Oxford (Brune et al., 2016) developed the SpyTag/SpyCatcher “plug-and-display” VLP platform, which employs use of the SpyTag peptide and SpyCatcher protein (originally generated by splitting the CnaB2 domain from the Streptococcus pyogenes fibronectin-binding protein FbaB (Zakeri et al., 2012)) to decorate the VLP surface with antigen. SpyTag-linked antigen and SpyCatcher-linked VLP carrier (resulting from the genetic fusion of SpyCatcher to VLP coat protein monomers followed by expression and self-assembly) are required for the production of VLPs using this platform. Mixing of these two components results in the spontaneous formation of an irreversible bond between the SpyTag-Antigen and SpyCatcher-VLPs, yielding VLPs decorated with the antigen of interest. The SpyTag/SpyCatcher platform has been used to generate VLPs displaying a variety of malaria-related antigens (e.g. CIDR, Pfs25, CSP) as well as self-antigens and antigens related to cancer, tick-borne encephalitis, and tuberculosis (Brune and Howarth, 2018). VLPs generated using this platform can be administered in the presence or absence of adjuvant, and studies with Pfs25-AP205 VLPs showed a higher anti-Pfs25 response than Pfs25 alone or Pfs25-SpyTag. Additionally, Pfs25-AP205 VLPs formulated in AddaVax yielded a slight increase in the anti-Pfs25 response when compared to the same VLPs without adjuvant (Brune et al., 2016). This platform has not yet been assessed in the clinic.

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4.1.2.4 RNA-based Vaccine Development Approach An RNA-based vaccine development approach involving collaboration with the Leidos Innovations Center (LInC), Promosome, and TriLink was proposed as part of the FY2020 AWP and approved by USAID. For this approach, Leidos and Promosome work together to design and test RESCUE modifications and translation enhancing elements (TEEs) to best increase expression of the proteins in the RCR complex. This will include: a) transiently transfecting mammalian cell lines with RESCUE-modified sequences and b) evaluating expression levels of each candidate protein by semi-quantitative Western blot analyses to identify the optimum modifications. The optimum sequences will be incorporated into plasmids for RNA production by TriLink. TriLink will incorporate modified nucleotides (one-methylpseudouridine-5’-triphosphate in place of UTP) and cap mRNA to increase mRNA stability. Leidos will perform quality control studies with the TriLink generated mRNAs. The resulting mRNAs will be encapsulated using a lipid nanoparticle technology.

4.2. CSP VACCINE DEVELOPMENT PROJECT: AMY NOE AND JAYNE CHRISTEN Technical work for this project was previously completed. In August 2020, the draft CSP Vaccine Development Final Project Report was circulated to USAID. Several revision cycles occurred during Q4 FY2020 and Q1 FY2021. The report was finalized on December 9, 2020; however, the consolidated report (pdf version including all attachments) could not be delivered due to size constraints for receipt of email attachments. Leidos was able to gain access to a USAID-hosted Google Drive site for provision of large file size deliverables. The finalized CSP Vaccine Development Project Final Report (including all attachments) was delivered via Google Drive on January 13, 2021. The finalized CSP Vaccine Development Project Raw Data Report (including all attachments) was delivered via Google Drive on January 13, 2021. In late March FY2021, the manuscript (see Section 3) was finalized and clearance received from USAID. This was submitted to Frontiers in Immunology in early April 2021.

4.3. RH5.1/AS01B CLINICAL STUDY: AMY NOE Technical work for this project was previously completed. In Q4 FY2020, all raw data and final report attachments were received from Oxford. Leidos circulated the draft final project report on November 5, 2020 and several revision cycles occurred during the remainder of Q1 FY2021. The finalized RH5.1 Clinical Study Project Final Report (including the all raw data and attachments) was delivered via Google Drive on February 15, 2021. In Q1 FY2021, the manuscript (see Section 3) was submitted to Med. In late March FY2021, the manuscript was accepted for publication.

4.4. LIVER STAGE VACCINE DEVELOPMENT PROJECT: KEN TUCKER/TIM PHARES A summary of the liver stage project plan is provided in Table 4.4-1. The technical work on this project was completed in Q1 FY2021. The finalized Liver Stage Project Final Report (including the all raw data and attachments) was delivered via Google Drive on April 1, 2021. In late March FY2021, the manuscript (see Section 3) was submitted to Frontiers in Immunology; review is ongoing.

Table 4.4-1. Overview of the Liver Stage Epitope-Based Vaccine Development Plan

Phase Milestone Activity

Phase 1 Epitope

Development

1.1 In silico identification of proteins (Stage 1)

In silico identification of class I and II epitopes and score of protein immunogenicity; downselection to subset of proteins

1.2 In silico identification of peptides (Stage 2)

Selection of class I and II epitopes based on in silico analysis of Treg, conservation in Plasmodium, and HLA coverage

1.3 In vitro biological response testing Testing T cell stimulation by, and immune response to peptides

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expressing Pf proteins. The vaccines were delivered and JHU performed and completed the dose titration of irradiated Py/PfCSP sporozoites study in Q4 FY2020. The active vaccination MVA-VLP Pf protein challenge study was completed in Q1 FY2021.

4.4.1.1 Construction of Individual Pf Liver Stage Protein MVA Vaccines Procurement efforts for the Pf MVA vaccines were conducted in Q1 FY2020 and GeoVax began work on constructing the recombinant MVA-VLP vaccines expressing the following Pf proteins in Q2 FY2020 (Table 4.4-2).

Table 4.4-2. Individual Pf Proteins Designation Accession Number (PlasmoDB) SPECT2 PF3D7_0408700

SPECT2.2 without MACPF domain PF3D7_0408700

Conserved protein PF3D7_1462300


Burnet Institute CSP (Pf3D7 sequence)

In Q4 FY2020, GeoVax completed scale up and characterization of the four MVA-VLPs expressing Pf proteins: MVA-PfCSP, MVA-Pf08_0081, MVA-Pf14_0593, and MVA-PfSPECT2. For each MVA-VLP, a research seed stock (RSS) and final vaccine stock (FVS) was made. The FVS was provided to JHU for the in-life challenge study. Information on characterization of the RSS and FVS for each construct was provided to USAID in Q4 FY2020.

To confirm VLP formation by the four MVA expressing Pf protein vaccines, purified virus was analyzed by transmission electron microscopy EM in Q1 FY2021. Results of the EM analysis (Figure. 4.4-1) were provided to USAID on December 23, 2020. In brief, cultured DF1 cells were infected at an MOI of 0.5 for 24 hours with each individual vaccine construct. The cells were fixed and an attempt to stain the thin layer sections with primary polyclonal serum against SPECT2, Pf08_0081, or Pf14_0593 (produced by Precision Antibody) followed by secondary immunogold anti-mouse Ab resulted in no positive staining. This result was not unexpected as previous attempts with the individual antigen-specific polyclonal serum yielded no positive staining on Western Blots when cell lysates were analyzed for Pf protein expression during vaccine construction. However, filamentous VLP formation at the cell surface was observed in all samples. Please note, oval shaped, electron dense structures are MVA and are denoted with asterisks. VLPs are less electron-dense, elongated structures budding from the cell surface and are denoted with arrows. Representative images for each vaccine construct are depicted below, scale bar is 200nm. In addition to MVA-Pf protein expressing VLPs, MVA-VP40 was used as a positive control for VLP formation. All technical work on this project is complete.

Figure 4.4-1: Electron Microscopy Analysis of Pf Protein MVA-VLPs

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4.4.1.2 Efficacy Assessment of Pf MVA-VLPs The best mouse challenge model (Py or Pb) for assessment of the Pf protein-expressing MVA-VLPs was discussed with USAID in early Q2 FY2020 and ultimately, it was agreed to move forward with the Py/PfCSP model. Use of this model required three separate experiments to (1) determine the optimal number of mosquito bites for Py/PfCSP sporozoite challenge, (2) determine the dose of irradiated Py/PfCSP sporozoites that gives ~50% protection upon Py/PfCSP sporozoite challenge, and (3) conduct the active vaccination challenge study with the Pf MVA-VLPs. In January 2020, Dr. Tsuji moved his laboratory from ADARC to Columbia University. Shortly thereafter, he informed Leidos that the NYU insectary (his source for infected mosquitoes) had closed. As Dr. Tsuji could no longer perform the studies to assess the MVA-VLPs expressing the Pf proteins, Leidos engaged Dr. Fidel Zavala (JHU) to determine if he could perform these studies. Dr. Zavala agreed to perform the studies and procurement was completed in Q2 FY2020. Updates on each of the three experiments needed for the efficacy assessment are below.

(1) Optimizing the number of mosquito bites for Py/PfCSP sporozoite challenge. In Q2 FY2020, JHU conducted a study to determine the number of mosquito bites required to ensure infection using Py/PfCSP transgenic sporozoites. This study informed the targeted infectious exposure that will be used for the subsequent studies at JHU. Results of this study were delivered to USAID on March 23 in Q2 FY2020. Based on the data Leidos and USAID agreed to move forward with using at least 6 bites/mouse to ensure that all mice are infected.

(2) Titrating the number of irradiated Py/PfCSP sporozoites. In addition to optimizing the number of mosquito bites for infection, JHU also titrated the number of irradiated Py/PfCSP sporozoites to determine the dose that gave 40-50% protection upon Py/PfCSP mosquito bite challenge. Results of this study were delivered to USAID on September 21, 2020. Based on the results Leidos and USAID agreed to move forward with using the 6.5x104 dose in the active vaccination MVA-VLP Pf protein challenge study.

(3) Performing the active vaccination MVA-VLP Pf protein challenge study

With completion of the optimizing studies and vaccines, a study to evaluate the protective efficacy or delay in patency of the individual Pf liver stage protein MVA vaccines was commenced on October 5 2020. Per the study design shown in Table 4.4-3, eight- to nine-week-old BALB/c mice (n = 7) mice were immunized IM in the hind limb with 107 of the MVA vaccine at day 0 and 28. Positive control mice were immunized IV in the tail vein with 6.5 x 104 irradiated Py/PfCSP sporozoites at day 21 and 35. At day 49 all mice were challenged via mosquitos infected with Py/PfCSP. Parasitemia in the mice was assessed via blood smears. Results of the challenge study were delivered to USAID on December 10 2020 (Table 4.4-4 and Table 4.4-5). Naïve mice that received no immunization showed no protection. Mice immunized with the 6.5 x 104 irradiated Py/PfCSP sporozoites were 100% protected. It was anticipated that none of the mice immunized with an empty MVA-VLP would be protected; however, 2 of 7 or 29% of these mice were protected after challenge. For the individual MVA-VLP Pf protein vaccines the following protection was observed: SPECT2 = 29%; PF14_0593 = 17%; PF08_0081 = 14%; and 3D7 PfCSP = 57%. Based on the 29% protection seen with the empty MVA-VLP it’s difficult to discern whether the protection seen with the individual vaccines is antigen-specific. The increased protection to 57% with the 3D7 PfCSP vaccine suggests a potential antigen-specific response; however, this cannot be assured with any certainty. Leidos proposes that if USAID decides to further explore the utility of the GeoVax platform in future work that it may be beneficial to perform a dose-titration study with the different vaccine constructs and empty MVA-VLP vaccines to establish potency with the respective constructs. All technical work on this project is complete.

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WRAIR was fully executed on February 6, 2020. Per the agreed workflow, Leidos met with WRAIR in early February 2020 to confirm logistics for shipment of the initial CSP mRNA construct; however, in early March, WRAIR informed Leidos that provision of the plasmid would need to be negotiated with TriLink®. Leidos met with TriLink in March 2020 and TriLink provided an MTA template for the material exchange. In Q3 FY2020, the MTA between Leidos and TriLink was signed. Additionally, the full-length PfCSP plasmid (starting construct reference number: 42725-2/W37-E01A, plasmid ID: TS-2019-3460) was ordered from TriLink in late June 2020. Prior to ordering the plasmid, Leidos confirmed the plasmid reference information with WRAIR. In Q4 FY2020, the starting plasmid (termed pCSP Ref) was received from TriLink. LInC modified the starting construct to introduce the designed RESCUE modifications and three different translation enhancing elements (TEEs). LInC experienced hurdles with low plasmid yield and was able to troubleshoot with the help of TriLink at the end of Q4 FY2020; these hurdles were due to the TriLink proprietary deactivated T7 promoter within pCSP Ref. In early Q1 FY2020, LInC will evaluate protein expression from the modified constructs via transfection of mouse muscle cells (C2C12) and human embryonic kidney cells (HEK293). LInC shipped the plasmid to TriLink in early Q1 FY2021 and TriLink anticipates that both mRNAs (the initial and improved) will be shipped to SriSai in January 2021, for distribution to WRAIR and LInC. Detailed development information provided by LInC in December 2020 has been included in the below.

4.4.2.1 In silico analyses and design of RESCUETM modifications A construct containing a CSP coding sequence previously developed by WRAIR, in collaboration with TriLink, was received by LInC as part of the previously noted MTA established with TriLink (Figure 4.4-2A). For simplicity LInC refers to this plasmid as pCSP Ref. LInC performed an in silico analysis of this sequence to identify cryptic translation initiation sites and identified numerous potentially inhibitory sequences. In general, RESCUETM modifications are restricted to the signal peptide of protein coding sequences and range from conservative, synonymous modifications, to aggressive non-synonymous modifications that result in altered amino acid sequences. In the latter case, the signal peptides are cleaved leaving the mature, processed protein with an unaltered protein sequence. For this evaluation, since the CSP mRNAs were to be translated in vivo in a preclinical mouse model, only conservative modifications were designed to avoid biological effects of a modified signal peptide sequence with unknown biological effects. In consideration of this and timeframe, modifications were restricted to the first ~200-nucleotides of the CSP coding sequence. This approach is known by LInC to be beneficial for increasing expression of non-secreted proteins.

Figure 4.4-2. Schematic Representation of CSP mRNA Construct Generation. (A) The pCSP Ref plasmid contains a deactivated T7 promoter, a full-length CSP coding sequence, a Kanamycin resistance gene, the pBR322 origin of replication, and TriLink® proprietary 5′ leader and 3′ untranslated regions. (B) Representative TEE/RESCUE-containing CSP plasmid. Introduction of TEEs and recoded CSP coding sequence did not alter the critical features of the reference construct including the deactivated T7 promoter, TriLink® proprietary 5′ leader, and 3′ UTRs.

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4.4.2.2 Construct generation A RESCUETM modified CSP sequence was synthesized as a gBLOCK by IDT and introduced into the CSP construct provided by TriLink along with the subsequent introduction of three different TEEs (known as E6s, 5′1s, and G11s) in the form of PCR products from TEE containing templates (Figure 4.4-2B).

4.4.2.3 Optimization of Western blotting detection conditions Western blots were used to evaluate CSP expression using anti-CSP antibodies (3B6 and 5D5), anti-CSP sera (rabbit sera provided by VLP Biotech), and VLP162 as a CSP positive control. The antibodies/sera were tested for reactivity against 50 ng of the VLP162 under denatured and non-denatured conditions. In addition, since the ultimate goal was to assess expression of secreted CSP from cultured cells, culture media from untransfected HEK293 cells that had been spiked with 50 ng of the VLP162 was also tested. The results of these optimizations are shown in Figure 4.4-3 and demonstrated that the most sensitive reagent was the rabbit antisera. For the purposes of this study, LInC performed subsequent analyses using the rabbit antisera since this appeared suitable with respect to sensitivity.

Figure 4.4-3. Optimization of Western Blot Methodology. Western blots were performed using 50 ng of reduced or unreduced VLP162 (positive control) spiked into culture media from untransfected HEK293 cells, or in PBS. For the anti-CSP rabbit antisera optimization culture media and lysate from untransfected cells was included to distinguish between true CSP reactivity and nonspecific binding with components contained within each of these samples. Fractionated proteins were transferred to PVDF membranes and probed with (A and B) a 1 µg/mL mouse monoclonal antibodies (clone 3B6 and 5D5) and (C) a 1:1,000 dilution of the anti-CSP rabbit antisera. Membranes probed with mouse anti-CSP antibodies were subsequently probed with a 1:20,000 dilution of an anti-mouse HRP-conjugated secondary antibody, while that probed with the rabbit antisera was probed with a 1:10,000 dilution of an anti-rabbit HRP conjugated secondary antibody. Blots were developed using an ECL reagent and various exposures of the chemiluminescent signal captured using an Amersham 600 imager. (D) Loading order of samples.

4.4.2.4 Evaluation of CSP expression in a C2C12 and HEK 293 cell lines These studies involved (i) generation, purification, and QC of capped and polyadenylated CSP mRNAs synthesized in vitro from the transcription templates, (ii) their transfection into C2C12 and HEK293 cells, and (iii) CSP protein expression evaluated by western blotting using optimized conditions. Expression levels from these mRNAs were normalized to account for differences in transfection efficiencies using the luciferase mRNA co-transfection control.

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4.4.2.5 In vitro transcription of CSP mRNAs Initially LInC attempted to transcribe capped and polyadenylated mRNAs from the linearized plasmids containing recoded CSP and TEEs using the HiScribe™ T7 ARCA mRNA kit. However, despite numerous attempts LInC was not able to do so. After consulting with TriLink it became apparent that TriLink had supplied LInC with a plasmid that contained a deactivated T7 promoter and that TriLink’s normal workflow involved PCR amplification of plasmids to restore the T7 promoter sequence and add a polyA tail of a defined length. Although TriLink shared the primer sequences used in their workflow, LInC was not able to specifically amplify a product of the correct length from pCSP Ref or the modified CSP plasmid (data not shown). It should be noted that TriLink also encountered this issue when the modified CSP plasmid was forwarded to them. To overcome this issue, LInC replaced the deactivated T7 promoter with the restored sequence using a cloning approach. The restored T7 plasmids were then linearized by XbaI digest, phenol:chloroform purified, and used as templates for successful in vitro transcription as described below.

4.4.2.6 Analyses of in vitro transcribed CSP RNAs. One microliter of reaction was removed after the capped in vitro transcription, and tailing reactions for analyses using the Agilent RNA 6000 Nano Assay Kit to evaluate transcription length before and after the addition of poly(A) tails, RNA integrity, and determine RNA concentrations. Figure 4.4-4D shows overlaid traces for each of the in vitro transcribed CSP RNAs before and after the tailing reaction that indicate i) the presence of non-degraded, and ii) a shift in size of RNAs indicating the successful addition of poly(A) tails. Figure 4.4-4E also shows this data as a virtual gel image alongside molecular weight standards. The inclusion of internal standards in the RNA 6000 Nano reagents provides a means for quantitation and indicated yields of capped and polyadenylated mRNAs of between ~140 to 250 ng/µL.

Figure 4.4-4. Analyses of CSP RNAs from In Vitro Transcription Reactions. In vitro transcription products for (A) the reference CSP, (B) the RESCUETM-modified CSP with the E6s TEE, (C) the RESCUETM-modified CSP with the 5′1 TEE, (D) and the RESCUETM-modified CSP with the G11s TEE were analyzed on Agilent RNA 6000 Nano bioanalyzer chips. (E) The molecular weight ladder for this experiment. For each CSP variant 1 µL of the reaction that generates the capped RNA, and 1 µL of the reaction that that adds a poly(A) tail to the capped RNA were analyzed; the peak height is used to quantitate RNA concentrations by comparison to internal standards. Images A-D show overlaid traces for the two forms of RNA for each CSP variant; the addition of a poly(A) tail is indicated by the increases in length of RNAs. (F) These data are also represented as a virtual gel image.

4.4.2.7 Transfection of C2C12 and HEK293 with in vitro transcribed CSP mRNAs Transient transfection of C2C12 and HEK293 cells with mRNAs was performed using the XfectTM RNA transfection reagent that generates biodegradable, low cytotoxicity nanoparticles containing. C2C12

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cells are a mouse muscle cell line are were selected to best model anticipated results for the malaria mouse model. HEK293 cells were included as WRAIR has previously used this line to assess transcription of pCSP Ref. In brief, 1 µg of each CSP mRNA was transfected in triplicate into cells using the protocol recommended by the manufacturer (Takara). In addition, each CSP mRNA was co-transfected alongside 100 ng of the TriLink CleanCap® Firefly Luciferase (FLuc) mRNA as a co-transfection control. This permits normalization of transfection efficiencies among replicates and different CSP mRNAs as well as due to variability in cell lysis.

4.4.2.8 Western Blot Analyses to determine CSP expression Fourteen microliters of supernatant from C2C12 and HEK293 cells transfected with the CSP mRNAs was subjected to western blot analyses using the anti-CSP rabbit antisera under the previously optimized conditions (Figure 4.4-3C). The resulting data demonstrate that, with C2C12 cells, CSP expression was higher in cells transfected with the RESCUE_E6s TEE modified mRNA relative to the cells transfected with the pCSP Ref mRNA (Figure 4.4-5A). Overall, expression in HEK293 cells was significantly less than that observed in C2C12 cells and required the use of a more sensitive ECL detection reagent (SuperSignal™ LInCst Femto ECL). For the HEK293 transfections CSP expression was higher in cells transfected the RESCUE_G11s TEE modified mRNA relative to the cells transfected with the pCSP Ref mRNA (Figure 4.4-5B). As the cell type impacts the level of expression and the C2C12 cells are thought to best model expression in mice, the RESCUE_E6s TEE modified construct was downselected.

Figure 4.4-5. Western Blot Analyses of Culture Supernatants from (A) C2C12 and (B) HEK293 Cells Transfected with In Vitro Transcribed CSP mRNAs. Western blot were performed using a 1:1000 dilution of anti-CSP rabbit antisera and a 1:10,000 dilution of goat-anti-rabbit HRP-conjugated secondary antibody. Development was via standard ECL reagents (ECL Plus) for the C2C12 cell samples (A) and the more sensitive SuperSignal™ LInCst Femto ECL reagent for HEK293 cell samples (B). Analyses was performed using 14 µL of each supernatant from transfected cells alongside 10 ng of the VLP162 positive control under reduced conditions. pCSP Ref (Ref) mRNA and modified CSP mRNAs containing RESCUE™ modifications and the different TEEs (5′1s, E6s, and G11s) were used for transfections in both cell lines.

4.4.2.9 Transfer of the optimized construct to TriLink Based on the above results and taking into consideration the downstream application, LInC provided CSP plasmid containing the RESCUE-modified coding sequence containing the E6s TEE to TriLink in

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November 2020. It should be noted that the plasmid provided contains the TriLink proprietary deactivated T7 promoter present in the pCSP Ref plasmid, which is consistent with TriLink’s workflow, as opposed to the plasmid containing a restored T7 promoter generated by LInC and used for in vitro transcription by LInC. In January 2021, TriLink shipped the mRNA to SriSai. SriSai shipped the aliquots to WRAIR and LInC, the latter for quality control (QC) assessments. In February 2021, LInC provided the QC report wherein they noted the presence aberrant expression products when the TriLink synthesized mRNAs (both the unmodified reference CSP mRNA and the RESCUE-modified CSP mRNA) were transfected into C2C12 cells. These included a smear of low molecular weight species for the former and a specific low molecular weight product at ~25 kD for the latter. Further analysis performed by LInC indicated the truncated expression products were due to frameshifted coding sequences in the CSP mRNAs introduced during the TriLink manufacturing workflow. An IPT meeting was held with WRAIR, LInC, USAID, and Leidos on February 16, 2021 to discuss the QC results. Leidos suggested that WRAIR might discuss the possibility of an alternative mRNA manufacturing workflow with TriLink and/or consider an alternate form of CSP antigen (specifically changing the repeat region) as the basis for the mRNA. With provision of the TriLink mRNA, LInC mRNA development report, and LInC mRNA QC report, all deliverables for this collaboration were received and the effort was closed.

4.4.3 SCHEDULE This project was completed in Q1 FY2021.

4.5. RCR COMPLEX VACCINE DEVELOPMENT PROJECT: VIN KOTRAIAH (WEHI) KEN TUCKER (OXFORD)

The RCR Complex vaccine development project is a multi-year effort that will continue at WEHI and Oxford into FY2021. Interdependencies between these institutions and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). The workflow diagram was updated to incorporate the decision by USAID on November 18, 2019 to proceed with the inclusion of five DPX cohorts to the Study O2 design at Oxford and removal of the penultimate study. Additionally, to incorporate the decision made by USAID on December 11, 2019 to modify Study O3 into a dose-ranging study, the workflow diagram has been updated to indicate that such a study will be performed using single antigen VLPs. The technical work and deliverables for this project were completed in Q2 FY2021. The finalized RCR Complex Vaccine Development Project Final Reports (including the all raw data and attachments) were delivered via Google Drive on April 01, 2021. All final technical/data updates and summary findings can be found in the final reports.

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Figure 4.5-1. RCR Complex Vaccine Project Workflow. Downselection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA.

4.5.1 WEHI: VIN KOTRAIAH

4.5.1.1 Study W3 The study design for W3 is shown in Table 4.5-1 and the dose assignments for W3 are shown in Table 4.5-2 are shown for reference as some of the follow-on tasks utilize the W3 sera.

Table 4.5-1 Study W3 Protein Combination Study in Rats

Cohort Formulation R # rats, strain

Study Day -2 0 28 42 56 70

1 RH5.1 + DPX4 IM 6, Wistar

Pre-

Blee

d

√ √

Tes

t bl

eed

√

Ter

min

al B

leed

2 CyRPA + DPX4 IM 6, Wistar √ √ √

3 RIPR + DPX4 IM 6, Wistar √ √ √

4 RH5.1 + RIPR + DPX4 IM 6, Wistar √ √ √

5 RH5.1 + CyRPA + DPX4 IM 6, Wistar √ √ √

6 RIPR + CyRPA + DPX4 IM 6, Wistar √ √ √

7 RH5.1 + CyRPA + RIPR + DPX4 Dose #1 By Molar Ratio IM 6, Wistar √ √ √

8 RH5.1 + CyRPA + RIPR + DPX4 Dose #2 By Mass IM 6, Wistar √ √ √

9 DPX4 alone (Negative Control) IM 6, Wistar √ √ √

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WEHI subsequently tried solubilization in Triton X-100 and DMSO without success. Due to the hydrophobic nature of the excipients, analytical method development may be needed to complete this work and an expert CRO in this area (i.e., protein analytical chemistry) would need to be utilized. Leidos also noted that such analytical method development is typically conducted as part of product release and stability test development. During further discussions, WEHI expressed their concerns that further manipulation (i.e., harsher treatment) of the material might result in complex formation/dissolution that is not representative of the current form. Because of technical challenges encountered in verifying the stability of RCR complex in DPX4 without resorting to harsher solubilization procedures, this effort was stopped.

Figure 4.5-2 Native SDS-PAGE of RCR complex stability in cohorts 7 and 8 DPX4 formulations. Bands corresponding to CyRPA and RIPR are indicated by red and green asterisks, respectively; RCR complex is marked with the purple asterisk.

4.5.1.4 Standardized ELISA In the Leidos-USAID biweekly meeting held on October 6, 2020, USAID stated their preference for a test sample exchange between the two laboratories (WEHI and Oxford) as part of the assay transfer process. Leidos then worked to coordinate the assessment test sera from WEHI by Oxford and facilitated communication between the laboratories to help finalize the sera set for evaluation at both sites. The list of samples was shared with USAID advisors who indicated their agreement on December 01, 2020.

The description of WEHI sera that were shipped to Oxford for testing in the Standardized ELISA are shown in Table 4.5-3. The inclusion of W2 sera will provide samples with a range of signal in the ELISAs and increase the sample size for the comparison analysis of data generated at the two sites. According to the current plan, these individual animal sera will be tested at both labs.

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Table 4.5-3. WEHI W3 and W2 samples shipped to Oxford for Standardized ELISA

Sera Antigen Number, volume of test samples Pre-bleed volume

W3 - cohort 1 RH5 6, 50 µL 10 µL W3 - cohort 2 CyRPA 6, 50 µL 10 µL W3 - cohort 3 RIPR 6, 50 µL 10 µL

W2 - cohorts 1-3* RH5 18, 35 µL 10 µL W2 - cohorts 4-5 CyRPA 12, 35 µL 10 µL W2 - cohorts 6-7 RIPR 12, 35 µL 10 µL

Total number of samples = 120. * Note that Dr. Julie Healer of WEHI is considering testing either the 20 µg or 2 µg W2 RH5 samples (the 0.2 µg RH5 sera will be tested). This is to keep the number of samples manageable for WEHI so they can complete all the remaining follow-on tasks by their POP end date. Dr. Healer has also noted that the nominal 20 and 2 µg dose cohorts were not very different in terms of their ELISA OD1 titers.

The WEHI sera were received at Oxford on December 14, 2020 and were partially blinded as per Oxford’s request. They were labeled with antigen (to indicate to Oxford as to which assay the sample needs to be tested on), rat ID number and as pre-bleed where applicable but the antigen dose was not specified. This will allow Oxford to test the appropriate ELISA blinded to W2/W3 and antigen dose. Following testing Oxford will request the unblinded list and WEHI’s ELISA results for concordance analysis of the data collected at both sites.

WEHI has made initial attempts at testing Oxford’s Standardized ELISA protocol. Due to COVID-related delays, delivery of the secondary antibody and detection reagent was delayed. Therefore, WEHI attempted an initial test using a different secondary antibody and detection reagent from that specified in the SOP; however, differences in signal development were significant from those described in the SOP. WEHI has since received the correct secondary antibody and is awaiting delivery of the correct detection reagent following which the Standardized ELISA will be repeated.

4.5.1.5 GIA reversal The ability of RH5.1, CyRPA and RIPR antigens to reverse GIA activity was tested by two separate methodologies.

Direct (antigen-in-well) GIA First, in accordance with advice from Kazutoyo Miura at the NIH who has published this method, the reversal assay was set up directly in the 96-well plate where antigen at 2 µM concentration was pre-incubated with IgG at a final concentration of 2 mg/mL to which parasitized red blood cells were then added as per the normal one-cycle GIA protocol. Controls for this assay were BSA as a negative control for antigen reversal and untreated IgG (cohorts 1, 2, 3 & 8) to calculate GIA without reversal, as well as normal rat IgG (NRS) at 2 mg/mL for calculation of relative growth inhibition.

Interpretation of the findings was confounded by high GIA activity in the negative control samples – BSA, BSA + NRS and the antigen-only treatments, RH5, CyRPA and RIPR (in green outline) (Figure 4.5-3). The untreated parasite controls (3D7) were assigned 100% growth. Normal rat IgG-treated wells (purified from normal rat serum (NRS), Invitrogen) at a final concentration of 2 mg/mL (NRS) showed a slight growth inhibitory effect, as seen in previous assays. Monoclonal antibody 1G12 against RIPR was used as a positive control for the assay (at 1 mg/mL) and induced a GIA of 55% as expected.

In this assay the BSA negative control antigen as well as the RH5, CyRPA and RIPR antigens caused significant growth inhibition when added at 2 µM concentration directly to the assay wells. Cohort 1 sera represented a minipool of rat sera immunized with RH5.1, cohort 2 CyRPA, cohort 3 RIPR and

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cohort 8 RCR complex (in a 1:1:1 mass ratio). While significant GIA is observed with these cohorts in line with previous GIA assays, the reversal effect of adding specific antigen was not significant. A slight increase in parasitemia relative to cohort-only wells indicated that GIA activity of cohort 2 was slightly reversed by the presence of CyRPA and cohort 3 by RIPR, but the results are difficult to interpret in the light of the ‘non-specific’ inhibition cause by antigens alone.

Since a clear interpretation of these results was not possible, the experiment was repeated (this time without the BSA control). Results were similar to the first experiment, with GIA activity in the cohort IgG only wells as seen in previous standard GIA experiments but with significant inhibitory activity in the antigen-only wells also (Figure 4.5-4). The reasons for this non-specific inhibition are not clear. The antigens were dialyzed into sterile RPMI medium and then filter sterilized after dialysis and added so that a final concentration of 2 µM was obtained in the assay, ruling out ‘contamination’ with non-sterile medium. A titration experiment of antigen only to determine the level at which no inhibition is observed and then to test this level of antigen for GIA reversal ability was suggested by Leidos. However, due to time constraints, WEHI opted to try the antigen depletion method.

Figure 4.5-3. GIA reversal by antigen co-incubation. A one cycle GIA assay was performed with IgG from minipools of 6 rats per cohort at a final concentration of 2 mg/mL (cohorts 1, 2, 3 and 8 immunized with RH5.1, CyRPA, RIPR and RCR respectively). Results are expressed in terms of percent parasite growth, with untreated 3D7 set as 100% growth. Antigen-only treatments are outlined here in green. Data were generated in a single experiment with the bars of the histogram representing the mean relative GIA ± standard deviations observed in triplicate wells.

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Figure 4.5-4. GIA reversal by antigen co-incubation - trial #2. Incubation with antigen alone caused significant growth inhibitory activity (outlined in green) relative to untreated controls. No reversal of inhibition when IgG incubated with antigen (outlined in blue and orange).

IgG Depletion Studies A second methodology was suggested by WEHI that involved depleting antigen-specific IgG in the post-immunization cohort IgG preparations by passing the IgG over a column made with antigen bound to affinity resin. This treatment would circumvent the confounding issue of having high concentrations of antigen directly in the GIA assay. Note that the data described below were received by Leidos recently and details regarding the RCR sera used in these experiments and other details have been requested from WEHI.

For RH5.1 and CyRPA, 50 μg of antigen was bound to C-tag resin and for RIPR, 50 μg of antigen was bound to Streptactin resin. The respective IgG minipool from cohort 1, 2 or 3 was passed over these columns three times to deplete specific reactivity. In a separate experiment, the IgG from cohort 1 (RH5) was passed over empty resin (C-tag) to determine non-specific depletion. The results are shown in Table 4.5-4, Table 4.5-5 and Figure 4.5-5.

While the pre-treatment GIA values are somewhat lower than seen previously for the RH5 minipool, there was a relatively small effect of the “no antigen” empty column treatment (Table 4.5-4). This indicates there is negligible depletion of the parasite neutralizing antibodies by the C-tag column resin alone.

Table 4.5-4 GIA of RH5 minipool sera post-column depletion of IgG on empty C-tag resin

Condition % GIA triplicate wells Average GIA

RH5.1 minipool pre-treatment 2 mg/mL 39.91 32.67 37.22 36.60

Post column (no antigen) 30.82 28.55 31.25 30.21

Nearly complete reversal of GIA activity was observed in both experimental replicates for the single-antigen IgG cohorts pre-treated over antigen-affinity resin (Table 4.5-5) relative to untreated IgG controls (in black), whereas a lower degree of GIA reversal was observed by single antigen affinity depletion of IgG from RCR-immunized animals. This could be due to the fact that the RCR IgG contains antibodies against individual proteins of the RCR complex as well as the complex as a whole.

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Figure 4.5-5 Antigen-specific depletion of IgG resulted in a reversal of parasite growth inhibitory activity. Mean +/- standard deviations are shown.

4.5.1.6 RCR ELISA In Q1 FY2021, assessment of W3 sera by ELISA using the Oxford RCR complex as the coating antigen (2 μg/mL) was completed. Serum from individual terminal bleeds was diluted threefold starting at 1/100 dilution to generate 12-point curve and OD1 titers were calculated using R Studio (Figure 4.5-6). Previously collected ELISA data for W3 sera with the individual antigens is shown in Figure 4.5-7. These data indicate that the antibody response generated by immunization with RH5 alone are predominantly directed against epitopes found on RH5 as single immunogen. The immune response elicited by RH5 alone reacts to a significantly lower extent towards the RCR complex than the anti-RCR complex immune response elicited by combining it with other antigens (double and triple combinations). When immunized in combination (double or triple combinations), the immune response is lower towards RH5 compared to the response achieved when immunized as single antigen. A similar trend is exhibited by CyRPA as well but to a lesser extent and with the one exception noted in the legend for Figure 4.5-6. On the other hand, RIPR appears to elicit a similar level of immune responses against the RCR complex regardless of whether it is immunized as a single antigen or in combination with other antigens. Note that complete statistical analysis of these data is pending but the Kruskal-Wallis multiple comparisons of independent samples was applied to determine significant differences. The median OD1 titer of the RH5 cohort was significantly lower than that of all the other cohorts. The OD1 titer of CyRPA was significantly different compared to all the other cohorts with the exception of the RH5+CyRPA cohort. There were no significant differences between any of the RIPR cohorts.

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penultimate study (previously Milestone 6) and to change Milestone 5 to a dose-ranging study using single antigen VLPs, respectively. Study O1 demonstrated that dosing at 2 µg RH5.1 gave a maximal response (ELISA and GIA) while dosing at 2 µg CyRPA and RIPR gave suboptimal responses. From these results and discussion during an IPT Meeting held on November 13, 2019, single antigen doses were selected for Study O2 as follows: 2 µg RH5.1, 20 µg CyRPA, 20 µg RIPR, and 20 µg RCR complex for Matrix-M formulation and 2 µg RH5.1, 2 µg CyRPA, 2 µg RIPR, and 20 µg RCR complex for DPX formulation. The results of the continuing work on Milestones 2 through 5 completed in FY2021 are reported below.

Table 4.5-6. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

Phase Milestone Activities

Study O1 1. Dose-Ranging Study • Humoral response assessments • GIA assessment • Generation of quantitative ELISA control sera

Study O2 2. Assess immunogenicity of

individual proteins and double/triple protein mixtures

• Humoral response assessments • GIA assessment

Assay Development

3. Establish quantitative method to assess antigen-specific IgG levels for the RCR proteins

• Calibration-free concentration analysis method • Affinity purification method • Technology transfer to WEHI

VLP Development

4. Generation and characterization of single antigen VLPs and RCR complex VLPs

• Generation and expression of RH5-SpyTag, CyRPA-SpyTag, and RIPR-SpyTag

• Conjugation of single antigen-SpyTag or RCR-SpyTag complexes to HBsAg-SpyCatcher VLP carrier

• Protein purification and characterization

Study O3 5. Dose-Ranging Study (VLPs) • Humoral response assessments • GIA assessment

As noted in the appropriate sections below, USAID has approved the VLP development, the Study O2 design (Table 4.5-7 and Table 4.5-8), and the Study O3 design (Table 4.5-9).

4.5.2.1 Immunogenicity Study of Individual Proteins and Double/Triple Protein Mixtures (Study O2)

An IPT Meeting was held on November 13, 2019 of Q1 FY2020 to discuss the ELISA and GIA data obtained from Study O1. In this meeting USAID decided to include five additional cohorts (RH5.1 + DPX4, RIPR + DPX4, CyRPA + DPX4, Reconstituted RCR complex + DPX4, DPX4 only) in Study O2 to allow for a comparison between DPX4 and Matrix-M and one cohort for the proposed protective domain of RIPR, RIPR (EGF 5-8). The resulting study design and antigen doses are shown in Table 4.5-7 and Table 4.5-8, respectively.

Table 4.5-7. Study O2 – Individual Proteins and Double/Triple Protein Mixtures

Cohort Formulation R # rats, strain Study Day

-2 0 28 42 56 70

1 RH5.1 + Matrix-M IM 6, Wistar

Pre-

blee

d

√ √

Tes

t bl

eed √

Ter

min

al B

leed

2 CyRPA + Matrix-M IM 6, Wistar √ √ √

3 RIPR + Matrix-M IM 6, Wistar √ √ √

4 RH5.1 + RIPR + Matrix-M IM 6, Wistar √ √ √

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-2 0 28 42 56 70 5 RH5.1 + CyRPA + Matrix-M IM 6, Wistar √ √ √

6 RIPR + CyRPA + Matrix-M IM 6, Wistar √ √ √

7 RH5.1 + CyRPA + RIPR + Matrix-M Equivalent Molar Ratio IM 6, Wistar √ √ √

8 RH5.1 + CyRPA + RIPR + Matrix-M Single Antigen Doses Combined IM 6, Wistar √ √ √

9 Reconstituted RCR + Matrix-M IM 6, Wistar √ √ √

10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

11 RH5.1 + DPX4 IM 6, Wistar √ √ √



14 Reconstituted RCR + DPX4 IM 6, Wistar √ √ √

15 DPX4 Only (Adjuvant Control) IM 2, Wistar √ √ √

16 PfRIPR (EGF 5-8) + Matrix-M IM 6, Wistar √ √ √

USAID approved the design of Study O2 on November 18, 2019 of Q1 FY2020. Note that a second approval was obtained from USAID on December 6, 2019 to account for a reduction in the RIPR (EGF 5-8) antigen dose. The dose was decreased from 6.67 µg/injection to 3.97 µg/injection to account for the difference in the molecular weight of soluble RIPR (EGF 5-8) versus the molecular weight of RIPR (EGF 5-8) plus the fusion/expression partner. The immunization schedule for Study O2 was initiated in January and completed in March of Q2 FY2020. As a result of COVID-19, all sera samples were stored at Noble Biosciences until the samples could be safely shipped and received. In February 2020, Oxford indicated concern regarding the current timeline for completion using individual samples as described in the project plan and developed timelines for the GIA assays using individual samples as well as pooled samples. These timelines were shared with USAID on February 4 and, following further discussion, USAID requested the use of individual samples and not pooled samples for all GIA assays. Due to the facility closure in response to the COVID-19 epidemic, this analysis was delayed. The laboratory reopened in early August with two benches available for this project’s research. Sera from Noble was received by Oxford on August 2, 2020 and analysis using the standard ELISA began in September 2020. GIA analysis was started in November of 2020.


Cohort Formulation Antigen Doses

RH5.1 CyRPA RIPR RCR RIPR-EGF Total

1 RH5.1 + Matrix-M 2 µg 2 µg

2 CyRPA + Matrix-M 20 µg 20 µg

3 RIPR + Matrix-M 20 µg 20 µg

4 RH5.1 + RIPR + Matrix-M 2 µg 20 µg 22 µg

5 RH5.1 + CyRPA + Matrix-M 2 µg 20 µg 22 µg

6 RIPR + CyRPA + Matrix-M 20 µg 20 µg 40 µg

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7 RH5.1 + CyRPA + RIPR + Matrix-M Equivalent Molar Ratio 5.36 µg 3.51 µg 11.11 µg 20 µg

8 RH5.1 + CyRPA + RIPR + Matrix-M Single Antigen Doses Combined 2 µg 20 µg 20 µg 42 µg

9 Reconstituted RCR + Matrix-M 20 µg 20 µg

10 Matrix-M Only (Baseline Control) NA

11 RH5.1 + DPX4 2 µg 2 µg

12 CyRPA + DPX4 2 µg 2 µg

13 RIPR + DPX4 2 µg 2 µg

14 Reconstituted RCR + DPX4 20 µg 20 µg

15 DPX4 Only (Adjuvant Control) NA

16 PfRIPR (EGF 5-8) + Matrix-M 3.97 µg 3.97 µg

Oxford presented preliminary results of analysis from studies O2 and O3 at an IPT meeting on November 10, 2020, and additional results were provided to USAID on December 15, 2020. The preliminary results are summarized below. When formulated with Matrix-M, the levels of PfRH5.1 antibody titers in Study O2 are consistent with titers to 2 µg of RH5.1 in Study O1 (Figure 4.5-10). This indicates the results between the two studies can be compared with reasonable confidence.

Figure 4.5-10. Standard ELISA titers for RH5.1 Comparing Results from Study O1 and Study O2. Bleed from Day 70 using cohort vaccinated with RH5.1 at 2 µg in Matrix-M. Each symbol represents an individual animal.

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Figure 4.5-11. Study O2 Standardized ELISA for Antibody to RH5.1. Results for bleeds taken on Day 42 (A) and Day 72 (B) are presented. Each symbol represents an individual animal. Dotted line indicates median result from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to RH5.1 when RH5.1 was administered alone and in combination with RIPR and CyRPA are presented in Figure 4.5-11. When administered with Matrix-M, RH5.1 alone resulted in the highest titers to RH5.1 on both Days 42 and 70, and combining RH5.1 with CyRPA did not decrease the response. The response to RH5.1 was decreased in the presence of RIPR. This was observed when RIPR was combined with RH5.1 or when it was combined with CyRPA in the different assortments of the three-protein combination (equimolar amount, combined total protein, or in RCR complex). When the antigen was administered with DPX4, either alone or in the RCR complex, the immune response was lower than the same antigen administered in Matrix-M.

Figure 4.5-12. Study O2 Standardized ELISA for Antibody to CyRPA. Each symbol represents an individual animal. Dotted line indicates median from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to CyRPA when CyRPA was administered alone and in combination with RH5.1 and RIPR are presented in Figure 4.5-12. When using Matrix-M, increasing the dose of CyRPA from 2 μg (Study O1) to 20 μg (Study O2) results in higher anti-CyRPA IgG titers after the second (Day

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28) and third (Day 56) administration of antigen (response on Day 42 and Day 70, respectively). CyRPA alone resulted in higher average titers relative to combination with RH5.1 and/or RIPR. When in combination with both RH5.1 and RIPR, the average immune response to CyRPA was less than the immune response to CyRPA when RH5.1 or RIPR were the only additional proteins. When administered with DPX4, the immune response to CyRPA alone at Day 70 was similar to the response when CyRPA was administered with Matrix-M. The immune response to the RCR complex in DPX4 was higher on days 42 and 70 relative to the response when administered with Matrix-M.

Figure 4.5-13. Study O2 Standardized ELISA for Antibody to RIPR. Each symbol represents an individual animal. Dotted line indicates median from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to RIPR when RIPR was administered alone and in combination with RH5.1 and CyRPA are presented in Figure 4.5-13. Increasing the dose of RIPR in Study O2 did not result in higher antibody responses than were observed in Study O1. When using Matrix-M, the immune response to RIPR reach maximal levels by Day 42 after the second administration of antigen (Day 28) when either administered alone or in combination with RH5.1 and/or CyRPA. The third administration of vaccine did not increase the immune response on Day 70. RIPR responses were unaffected by administering combinations of RH5.1 or CyRPA. When administered with DPX4, the results parallel those seen with Matrix-M, although the average immune response was slightly lower using DPX4.

Oxford established a new parasitology laboratory in mid-November to support GIA. Analysis of samples from Study O2 have been completed and the results are provided below.

Figure 4.5-14. Study O2 GIA of IgG from Rats vaccinated with Adjuvant (with no antigen). Each symbol represents an individual animal. The “Bought Negative” are results using naïve rat IgG purchased from a commercial supplier. The Matrix-M and DPX4 are the results using rat IgG isolated

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from animals immunized with Matrix-M or DPX4, respectively. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Results of analysis of IgG from rats vaccinated with either Matrix-M or DPX4 without antigen are presented in Figure 4.5-14. For reasons that are not clear three of six rats immunized with Matrix-M alone show a strong grown inhibition. However, an immune response indicated by the ELISA did not demonstrate a titer to the three proteins in these animals. This test will be repeated. Also, one rat of two immunized with DPX4 alone showed strong growth inhibition. In this case the animal demonstrated a relatively high titer to RIPR in the ELISA (Figure 4.5-13). Since both animals were immunized using DPX4 from the same vial, it is not obvious why one of two rats demonstrated immunity in both the ELISA and GIA, and possible explanations will require discussion with Noble.

For animals vaccinated using Matrix-M, the Study O2 GIA results are consistent with the Study O1 results when comparing sera from rats vaccinated with 2 µg of RH5.1 in Matrix-M (Figure 4.5-15). This indicates the GIA results between the two studies can be compared with reasonable confidence. In Study O2, administration of RH5.1 with DPX4 is of similar potency to RH5.1with Matrix-M. This is not predicted based upon the ELISA, which indicates administration of RH5.1 with Matrix-M provided higher ELISA titers relative to DPX4. Figure 4.5-16 shows that increasing the dosage of CyRPA from 2 µg to 20 µg demonstrates a trend of increasing the potency in the GIA and follows the trend of the ELISA response. However, the results of the two concentrations overlap and neither consistently results in 50% inhibition of parasite growth. Administering 2 µg of CyRPA with DPX4 shows a trend toward increased potency of the response relative to administration with Matrix-M. However, results of the two formulations overlap and the DPX4 formulation results in 50% inhibition only at the highest concentration of IgG, which was not tested in the Matrix-M formulation. For RIPR increasing the concentration or administration with DPX4 did not increase potency of the response (Figure 4.5-17 and Figure 4.5-18), which parallels the ELISA results. The GIA results from rats immunized with either RIPR or the fragment RIPR (EGF 5-8) are shown in Figure 4.5-19. Potency of the response to RIPR (EGF 5-8) trends higher than the potency of the response to RIPR. A summary of the GIA results using IgG of rats in cohorts 1-3 of Study O2 is presented in Figure 4.5-20, which presents a comparison of the potency of the response in rats immunized with either RH5.1, RIPR, or CyRPA administered with Matrix-M. This result indicates a hierarchy of response potency to be RH5.1 >RIPR =CyRPA.

Figure 4.5-15. GIA for RH5.1 Comparing Results from Study O1 and Study O2, and formulation. Bleeds are from Day 70 using cohort vaccinated with RH5.1 at 2 µg comparing Study O1 (black) and O2 (blue) results (left panel). The right panel provides the results of 2 µg in Matrix-M (black) or DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

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Figure 4.5-16. GIA for CyRPA Comparing Results from Study O1 and Study O2. Left panel is bleed from Day 70 using cohort vaccinated with CyRPA in Matrix-M using 2 µg (black) or 20 µg (blue) CyRPA. Right panel is bleed from Day 70 using cohorts immunized with 2 µg CyRPA formulated with Matrix-M (black) or with DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-17. GIA for RIPR Comparing Results from Study O1 and Study O2. Bleed from Day 70 using cohort vaccinated with RIPR at either 2 µg or 20 µg in Matrix-M. Each symbol represents an individual animal. Results from Study O2 is blue and Study O1 is black. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-18. GIA for RIPR Comparing Results from Study O1 and Study O2, and formulation with Matrix-M or DPX4. Left panel compares the bleeds from Day 70 using cohort vaccinated with RIPR at 2 µg in Matrix-M (black) in Study O1, or with 2 µg in DPX4 (blue) from Study

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O2. Right panel provides GIA results from Study O2 evaluating 20 µg RIPR with Matrix-M (black) compared to 2 µg formulated with DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-19. GIA Comparing Potency of GIA to RIPR and RIPR (EGF 5-8). Results from Study O2 bleeds on Day 70 using cohorts vaccinated with RIPR at 2 µg (black), or with 3.97 µg RIPR (EGF 5-8) in Matrix-M. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-20. Comparison of GIA for RH5.1, RIPR, or CyRPA from Study O2. Bleed from Day 70 using vaccines containing Matrix-M. CyRPA is at 20 µg (orange), RIPR is at 20 µg (blue), and RH5.1 is at 2 µg (black). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Using the GIA response to RH5.1 as a comparator, the potency of combinations of RH5.1, CyRPA, and RIPR are presented in Figure 4.5-21. In all the combinations that contain RH5.1 the potency of the mixture of proteins overlaps with the potency of RH5.1 when used as the sole antigen (Panels A, B, D, E, and F). However, when the antigen combination is only CyRPA and RIPR, the potency is less than that seen with RH5.1 alone (Panel C). The potency of the response to the vaccination with CyRPA and RIPR combination is similar to the response when the vaccine is either CyRPA or RIPR alone (data not provided). This result parallels the response seen with the individual antigens demonstrating a hierarchy of response with RH5.1>CyRPA and RIPR (Figure 4.5-20). This indicates the combination of antigens do not reduce the immune response to RH5.1. The results do not indicate a synergistic effect with the RH5.1 in combination with CyRPA and RIPR; however, the potency of the response at high concentrations of IgG does show a trend to a higher GIA response when all three antigens are used in

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the vaccine (combined either as total protein or in equimolar amounts) (Panels E and F). This trend is not seen when the RCR complex is used as the antigen (Panel D). Similarly, when DPX4 is used in combination with the RCR antigen, there is not an increase in the potency of the response (Figure 4.5-22)

Figure 4.5-21. GIA Response to combinations of RH5.1, RIPR, and CyRPA Compared to RH5.1 in Study O2. Bleed from Day 70 using vaccines containing Matrix-M. Response to RH5.1 is indicated in black, and response to combination of antigens are blue. RCR is used at 20 µg. In the analysis of the R+C+R equimolar (Panel E), RH5.1 is at 5.36 µg, CyRPA is at 3.51 µg, and RIPR is at 11.11 µg. In all other samples RH5.1 is at 2 µg, CyRPA is at 20 µg and RIPR is at 20 µg. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

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Figure 4.5-22. GIA Response to RCR complex Compared to RH5.1 in Study O2. Bleed from Day 70 using RH5.1 vaccine at 2 µg containing Matrix-M or RCR vaccine at 20 µg containing DPX4. Response to RH5.1 is indicated in black, and response to RCR is blue. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

4.5.2.2 Development of Quantitative ELISA In conversations and approval of the FY2019 annual work plan, USAID indicated support of pursuing the development of quantitative ELISAs at Oxford with subsequent assay transfer to WEHI. ELISA development was initiated during Q1 FY2020 and consists of three steps. Step 1 involved setting up the standardized ELISA for RH5.1, RIPR, and CyRPA and was completed in Q1 FY2020. Step 2 is development of calibration-free concentration analysis (CFCA) for RH5.1, CyRPA, and RIPR antigens. Step 3 is Linear Regression Analysis to develop a conversion factor to convert the standardized ELISA titer results to the concentration of IgG.

Finalized SOPs for each candidate antigen (RH5.1, CyRPA, and RIPR) were received from Oxford and circulated to USAID on March 25, 2020. Due to facility closure, all research activities remained on hold during Q3 FY2020 and resumed once the lab reopened in August. Despite this closure, a MTA between WEHI and Oxford was fully executed and, as a first step in the technology transfer of the standardized ELISA developed by Oxford, the SOPs and Gen5 protocols were provided to WEHI for review on June 15, 2020. On September 22 WEHI received from Oxford rat antisera to RH5.1, CyRPA, and RIPR (400 µL each), and 0.5 mg of each antigen. This is enough material to support analysis of Studies W2 and W3, with extra antigen and antisera to support continued use of the standardized ELISA beyond the MVDP. In addition, this shipment included 1.5 mg of C-tagged CyRPA for the GIA reversal analysis. WEHI is setting up to perform the standardized ELISA using the finalized SOPs, but did not have the alkaline phosphatase conjugate or substrate. WEHI is in the process of acquiring these reagents.

The CFCA is being developed. The approach to immobilize the antigen using antibodies to the C-tag were unsuccessful. The approach using an oligonucleotide labeled streptavidin immobilized on a chip coated with a complementary oligonucleotide was successfully used to capture biotinylated RH5.1 protein. Oxford has produced streptavidin-SpyCatcher as a first step to produce biotinylated antigens (RH5.1, RIPR, and CyRPA) for the CFCA. To develop a preliminary conversion factor for ELISA results to RH5.1, samples were run twice and averaged. The concentration of IgG verses the anti-RH5.1 ELISA Units (AU) were plotted and linear regression was performed to generate the conversion factor for antibody to RH5.1 using either sera or purified IgG (Figure 4.5-23). The results from each plot are similar, providing a preliminary conversion factor to convert ELISA results to µg/ml of IgG. Oxford will continue analysis using the purified IgG as a cleaner approach to quantitate the IgG specific ELISA. Additional data points will be evaluated in the RH5.1 plot, and work will progress to analysis using RIPR

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and CyRPA. Oxford has indicated that, due to the limited time remaining on the MVDP contract, they will not have time to develop and perform the assay for the RCR complex.

Figure 4.5-23. Plot of Antibody Concentration Verses Anti-RH5.1 ELISA Units. Left panel is the result using rat antiserum to RH5.1. The right panel is the result using rat IgG to RH5.1 purified using protein L.

4.5.2.3 VLP Development USAID approved the development of SpyCatcher-HBsAg VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019 and VLP development began in Q3 FY2019. At the conclusion of Q1 FY2020, both N- and C-term CyRPA-SpyTag as well as C-term RH5.1-SpyTag were successfully conjugated to SpyCatcher-HBsAg VLPs. Efforts to produce full length (FL) RIPR VLPs and RCR complex-HBsAg VLPs were not successful and are continuing. Outside of the MVDP contract, but in parallel to the efforts for the production of FL RIPR VLPs, C-term RIPR (EGF 5-8)-SpyTag was successfully conjugated to SpyCatcher-HBsAg VLPs and replaced FL RIPR VLPs in Study O3. For the single antigen VLPs, the conjugation efficiencies were determined to be 67%, 86%, and 44%, for RH5.1, CyRPA, and RIPR (EGF 5-8), respectively. This efficiency represents the percentage of HBsAg monomers that display the candidate antigen. For example, 67% of the HBsAg monomers in a given VLP display RH5.1. Note that formed VLPs contain between 80 and 120 monomers. As such, not all VLPs in a given population will be composed of the same number of monomers.

At the conclusion of Q2 FY2020, FL RIPR Ct SpyTag (Ct = C-terminus) was successfully conjugated to SpyCatcher-HBsAg VLPs, with a conjugation efficiency of 52%. While this was not completed in time for use in Study O3, it did allow development of the RCR complex-HBsAg VLP to continue. An initial attempt to incorporate all three proteins on a VLP was unsuccessful. The facility closure due to COVID-19 prevented further development of the RCR complex-HBsAg VLP until the laboratory reopened. At this time there is not enough protein to attempt the conjugation to the VLP and protein is being produced to repeat the attempt.

4.5.2.4 Dose-Ranging Study – VLPs (Study O3) An IPT meeting was held on November 18 and 22, 2019 of Q1 FY2020 to discuss the study design of Study O3 and included consideration of the available VLPs and what VLP dose would be most appropriate. In light of the results from Study O1, it was decided to modify the original study design to a dose-ranging study before moving forward to assess double and triple VLP combinations. Additionally, due to the tight timeline and difficulty in producing FL RIPR VLPs, it was decided to replace FL RIPR VLPs with RIPR (EGF 5-8) VLPs. Note that all RCR proteins used to generate the VLPs for this study contain a SpyTag on the C-terminus. The resulting study design is shown in Table 4.5-9 and USAID approved the design on December 11, 2019 of Q1 FY2020. In Study O3, the antibody to HBsAg-RH5.1, HBsAg-CyRPA, and HBsAg-RIPR (EGF5-8) VLPs were evaluated for immunogenicity as well as for parasite growth inhibition.

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Table 4.5-9. Study O3 – VLP Dose-Ranging Study


-2 0 14 28 42 56 70

1 HBsAg-PfRH5.1 (2 µg) + Matrix-M IM 6, Wistar

Pre-

blee

d

√

Tes

t Bl

eed

√

Tes

t bl

eed

√

Ter

min

al B

leed

2 HBsAg-PfRH5.1 (0.2 µg) + Matrix-M IM 6, Wistar √ √ √


4 HBsAg-PfCyRPA (20 µg) + Matrix-M IM 6, Wistar √ √ √


6 HBsAg-PfCyRPA (0.2 µg) + Matrix-M IM 6, Wistar √ √ √


8 HBsAg-PfRIPR(EGF 5-8) (20 µg) + Matrix-M IM 6, Wistar √ √ √


10 HBsAg-PfRIPR(EGF 5-8) (0.2 µg) + Matrix-M IM 6, Wistar √ √ √


16 HBsAg-SpyCatcher (20 µg) + Matrix-M IM 6, Wistar √ √ √

The immunization schedule for Study O3 was initiated in January and completed in March of Q2 FY2020. As a result of COVID-19, all sera samples were stored at Noble Biosciences until the samples could be safely shipped and received. Due to the COVID-19 epidemic, the Oxford facility was closed and this analysis was delayed. The laboratory reopened in early August with two benches available for this project’s research. Sera from Noble was received by Oxford on August 21. Analysis using the standard ELISA began in September, and GIA analysis began in November 2020.

During an IPT meeting on November 10, 2020, Oxford presented preliminary results of analyses from studies O2 and O3, and additional results were provided to USAID on December 15, 2020. The preliminary results of the standardized ELISA for Study O3 are summarized below. Results for bleeds taken on Day 42 and Day 72 are presented. The immune response to RH5.1 peaked by Day 42, but the immune response to 2 µg and 0.2 µg RH5.1 decreased by Day 70 (Figure 4.5-24). By Day 70 the immune response to RH5.1 in study O3 was over 10-fold less than the immune response seen in study O1 and study O2. For CyRPA and RIPR the immune response to 0.2 to 20 µg peaked by the day 42 bleed after the second immunization with little change in the average immune response by day 70, which was after the third immunization (Figure 4.5-25 and Figure 4.5-26). For CyRPA, the immune response on Day 70 to 20 µg VLP was slightly higher than the 20 µg dose of CyRPA in Study O2, and the immune response to the 2 µg VLP was slightly higher than the 2 µg dose of CyRPA in Study O1 (Figure 4.5-25). The immune response to the 20 µg dose of RIPR on Day 70 was slightly lower than the response to the 20 µg dose of RIPR and the 3.97 µg dose of RIPR (EGF 5-8) in Study O2 (Figure 4.5-26).

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Figure 4.5-24. Study O3 Standardized ELISA for Antibody to RH5.1. Each symbol represents an individual animal. Dotted line indicates median result from Study O1 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of RH5.1 is shown on the x-axis.

Figure 4.5-25. Study O3 Standardized ELISA for Antibody to CyRPA. Each symbol represents an individual animal. Dotted line indicates average result from Study O1 or Study O2 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of CyRPA is shown on the x-axis.

Figure 4.5-26. Study O3 Standardized ELISA for Antibody to RIPR (EGF 5-8). Each symbol represents an individual animal. Dotted line indicates media result from Study O2 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of RIPR (EGF 5-8) is shown on the x-axis.

GIA results for Study O3 are only available for RH5.1 (Figure 4.5-27). Interestingly, while the ELISA results indicate a lower immune response to the RH5.1-VLP relative to soluble RH5.1, the GIA for the IgG to the soluble RH5.1, and the RH5.1-VLP at dosages of 2 µg and 0.2 µg are similar, with the IgG to

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the RH5.1-VLP trending to higher GIA than the IgG to the soluble RH5.1. This suggests the immune response to the RH5.1-VLP may be a higher quality than the response to soluble RH5.1.

Figure 4.5-27. GIA for Study O3 RH5.1 and Comparing Results with those from Study O2. Bleeds are from Day 70. Cohort vaccinated with RH5.1-VLP at 0.02 µg to 2.0 µg from Study O3 are presented in the left panel. The right panel overlays these results with the GIA results from Study O2 for IgG to soluble RH5.1 (in black) at vaccinated using 2 µg. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

4.5.2.5 Schedule: RCR Complex Project Oxford The timeline for RCR Complex Project Oxford is shown in Table 4.5-10. Due to the impact of the COVID-19 epidemic, the Oxford laboratories were closed March through July 2020, and reopened in August. The timeline for the project was extended to enable completing of as much of the RCR Complex project as possible with the funds that were already committed to the project.

Table 4.5-10. Project Timeline

4.6. RH5.1 HUMAN MAB IDENTIFICATION AND DEVELOPMENT PROJECT: VIN KOTRAIAH

The RH5.1 human mAb identification and development project was initiated in FY2019 and will continue into FY2021. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project. A priority list of 20 samples was approved by USAID on April 1, 2019 (Table 4.6-2). The technical work and final deliverables for this project were completed in Q2 FY2021. The finalized RCR Complex Vaccine Development Project Final Reports (including the all raw data and attachments) were delivered via Google Drive on March 30, 2021. All final technical/data updates and summary findings can be found in the final reports.

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Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project

Phases Milestones Activities

B Cell Isolation and mAb Development

B Cell Isolation and Cloning

• Single B cell isolation (memory B cells - mBC) or plasmablasts)

• Reverse transcription and paired heavy and light chain nested PCR

• DNA purification and cloning into heavy and light chain vectors

mAb Expression and Purification

• Transfection of heavy and light chain vectors into HEK293 cells

• Harvest of supernatant and screening by ELISA • Affinity purification of antibodies from supernatant

mAb Sequencing • Sequencing of heavy and light chain variable regions • Determination of sequence maturation and germline alleles

mAb Screening

ELISA Titer and Protein Mapping

• Determination of ELISA titers • Protein region mapping using a panel of RH5 variants

GIA • Growth Inhibition Assay (using pLDH method) Epitope Similarity Screen

• Determination of similarity of B cell epitopes recognized by antibodies using competition assay and SPR/BLI

mAb Downselection • Monoclonal antibody downselection

mAb Characterization

mAb Affinity • Determination of antibody affinity by SPR mAb Inhibition of Complex Formation

• Assessment of inhibitory activity of antibodies on RCR, RH5-P113 and RH5-Basigin complexes

mAb Downselection • Selection of monoclonal antibodies for structural studies

R5.016 Immunogen Design (optional)

Computational Assessments

• Identification of sequence variants of the immunogen that are stable and have the right conformation

Production of Select Immunogen Designs • Epitope grafting and production of selected immunogens

Immunogen Downselection • SPR screening for binding to R5.016 mAb

R5.016 Immunogen Production (optional)

Production of Immunogen • Production and QC of down-selected immunogen

Coupling of Immunogen to VLP • Conjugation of down-selected immunogens to VLPs

R5.016 Immunogen Testing (optional)

Formulation of Immunogen: VLP

• Selection and procurement of adjuvant • Formulation of immunogen

Rat Immunization • Rat immunizations with RH5.1 comparator • Collection of sera

Immunogenicity and GIA assessments

• Humoral response and GIA activity with R5.016 mAb comparator

New mAb Structure Determination (optional)

Antibody Production • Recombinant antibody expression and purification • Fab fragment generation

Crystal Screening • Screening for Fab:RH5 co-crystals • Cryoprotection of crystals

Crystal Structure Determination

• X-ray diffraction studies • Model building and refinement • Epitope delineation

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Oxford proposed identification of human RH5 mAbs and conduct of a B cell analysis using VAC063 clinical samples. This project will involve surveying the B cell and Ab repertories from vaccinated subjects utilizing both B cell cloning and serum immunomics techniques in order to understand the epitopes recognized by human anti-RH5 sera, how these epitopes might contribute to functional GIA, and gather information on differences in repertoire based on dose/immunization regimen/CHMI. As part of this effort, Oxford also proposed evaluating novel immune mechanisms of protection based on the sterile protection and reduced parasite growth rate seen with unvaccinated individuals after CHMI. These activities have been incorporated into the project proposal and will be expanded on during the course of project reporting.

The initial project schedule provided by Oxford (included in the Project Plan) had a start date of December 2018 and a completion date in May 2020. Approval of the Project Plan (regular elements only) was received from USAID on January 29, 2019. Per USAID request, Leidos directed Oxford to perform a sample selection inclusive of the RH5.1 clinical trial GIA data. Oxford conducted the same in March 2019, once the RH5.1 clinical study GIA data were available for analysis. Approval of sample selection by USAID was received in early Q3 FY2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, shown in Figure 4.6-1. Note that the updated project schedule begins in April 2019 and a completion date in September 2020, extending beyond the MVDP POP end date. Leidos recommended holding an IPT meeting in Q2 FY2020 to determine the project stopping point, as needed based on progress by Oxford. However, due to COVID-related closures of lab facilities at Oxford, the project was delayed and is now expected to be completed in Q2 FY2021 well within the new MVDP POP end date.

Figure 4.6-1. Project Timeline Received from Oxford

The priority list of 20 samples approved by USAID on April 1, 2019 is shown in Table 4.6-2. Of note is that the Oxford group aims to deliver roughly 10 RH5-specific mAbs from each of these 20 samples and characterize them.

In the “Group” column of Table 4.6-2, the samples are color coded by the VAC063 Group number to which they belong. Six samples drawn from Group 7, 10 drawn from Group 5 and 4 from Group 3 constitute the total of 20 approved samples.

In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion. The color yellow indicates that the volunteer was selected on the basis of in vivo growth inhibition (IVGI) only. The green colored cells indicate that the volunteers were selected on the basis of IVGI and ELISA, Avidity or GIA. The blue colored cells indicate that the volunteers were selected on the basis of ELISA, avidity or GIA. Note that Oxford’s slide summary with the ELISA, avidity, GIA and IVGI data for all the VAC063 volunteers were provided to USAID in Q2 FY2019.

On June 17, 2019, Oxford notified Leidos that volunteer 01-809 (01-032) from Group 7 with a priority number of 7 (Table 4.6-2) had withdrawn consent and that Oxford could no longer use this volunteer’s samples. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. The consent withdrawal and proposed replacement sample were communicated to USAID in the bi-weekly meeting on June 19, 2019.

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Table 4.6-2. Priority list of samples with their VAC063 Group number and Volunteer IDs are shown. Availability of PBMC samples from the indicated day of collection are also shown. The time-points at which the ELISA, Avidity and in vitro GIA data are available are also shown. Lastly, the IVGI is shown for the samples from Groups 5 and 7.

C-1 - day before challenge; C+28 - 28 days after challenge; DOD – day of diagnosis; IVGI was calculated as % reduction in parasite multiplication rate (PMR) in individual vaccinees versus the mean PMR of the control group. Volunteer 01-809 (01-032) from Group 7 with a priority number of 7 withdrew consent in June 2019. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. In the “Group” column, the samples are color coded by the VAC063 Group number to which they belong. In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion: yellow=selection based on in vivo growth inhibition (IVGI) only; green=selection based on IVGI and ELISA, Avidity or GIA; blue=selection based on ELISA, avidity or GIA.

Due to COVID related facility closure and restrictions on non-COVID work, all research activities on this project were suspended during Q3 FY2020. Activities resumed on a limited basis in September of Q4 FY2020 and proceeded at a brisk pace in Q1 FY2021. However, due to COVID-related delays, it was agreed that no attempts will be made to isolate additional mAbs from the Priority Samples. Instead, the focus was shifted towards completing the characterization of mAbs already isolated from twelve of the Priority Samples. USAID informed Leidos that it concurred with this proposed change in focus via email on October 13, 2020. Subsequently, progress was made on completing the binding

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characterizations, single-point and dose-titration GIAs. The current status at each of the major steps in the project is shown in Table 4.6-3.

Table 4.6-3. Status of Samples that have Completed Processing for Each Project Step

Step Number of samples RH5.1 ELISA with transfection supernatants / Number of binders 290 / 126

Titration of purified mAbs in RH5.1 ELISA 126 Binding of purified mAbs with different RH5 antigens by ELISA 126 Number of mAbs tested in single-point GIA / Number of mAbs with >50% inhibition 126 / 70

Number of mAbs tested in dose titration GIA 70 With the completion of the ELISA and GIA analyses in FY2021 Q1, Oxford has begun to analyze the data from all the mAbs. Some of these data were presented by Oxford in an IPT meeting with USAID on November 04, 2020. As previously described, the mAbs were characterized in ELISA using four different RH5-based plate antigens (Figure 4.6-2).

Figure 4.6-2. RH5 plate antigens used in ELISA. Apart from RH5.1 (native and denatured) and the N-terminus alone antigen, an additional antigen called RH5.2 in which the N-terminus and intrinsic loop are deleted was used as a plate antigen. Of note is that Oxford also uses the notation “SV3” or “delta NL” when referring to the RH5.2 antigen; however, Leidos will use RH5.2 for consistency. A plate antigen called Bundle which displays the epitope of the R5.016 mAb isolated from the VAC057 trial (described in Alanine et al., Cell 2019) on a non-RH5 protein scaffold was also used as plate antigen.

Figure 4.6-3 shows the distribution of the 126 mAbs according to their RH5 binding characteristics. A majority of the mAbs recognized conformational epitopes on RH5.1 and only 8% (10) could bind to linear epitopes (Figure 4.6-3A). Nearly 82% (103 mAbs) bound the SV3 RH5 construct while 2% bound the N-terminus and 3% bound the internal loop region (Figure 4.6-3B). About 13.5% (17) mAbs require further characterization in the ELISA as they did not bind any of the RH5 constructs. Oxford is planning to repeat the ELISA for these 17 mAbs. Additionally, of the 103 mAbs that bound to SV3, 88 bound outside the Bundle region whereas the rest bound within the Bundle region.

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Figure 4.6-3. RH5 binding characteristics of the 126 mAbs. (A). Break-down of mAbs by the conformational or linear nature of their epitopes. (B). Distribution of mAbs according to their binding to different RH5 constructs.

Characterization of 74 mAbs in the single-point GIA was completed in the current quarter, bringing the total of mAbs characterized by this assay to 126 (100% of the mAbs isolated). Of the 126 mAbs tested, 70 (56%) showed GIA at or above 50% at 2 mg/mL. These were further characterized in dilution curves. The IC50s of these mAbs are reported in Figure 4.6-4. Two of the mAbs (BD5 and 8G8) were found to be the most potent human anti-RH5 mAbs isolated to date and have lower IC50 values than R5.016.

Figure 4.6-4. GIA IC50 values for 70 of the RH5 mAbs. The blue line represents the IC50 of the control R 5.016 mAb. 8G8 and the previously described BD5 mAbs have IC50s below that of R5.016.

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In addition, avidity of the mAbs towards RH5 has been characterized using the NaSCN method. Figure 4.6-5A shows the distribution of avidity IC50 values of the mAbs grouped according to their epitope. The highest avidity was displayed by 9G8, a mAb which does not have GIA activity. The BD5 mAb, which is the most potent human anti-RH5 mAb isolated to date does not have high avidity whereas 4D3, another mAb that also binds to the Bundle has slightly higher avidity than BD5. Overall, there is no correlation between avidity of binding to RH5 and GIA activity as seen in Figure 4.6-5B.

Figure 4.6-5. Avidity characteristic of RH5 mAbs. (A). Avidity according to the RH5 binding epitope location. (B). Correlative analysis of GIA at 2 mg/mL versus avidity characteristic of mAbs.

The large number of mAbs isolated in this project particularly from VAC063 groups 3, 5 and 7 allowed for identification of differences in avidity between mAbs from different groups (Figure 4.6-6A). Significantly higher avidity was observed for mAbs isolated from Groups 3 and 7 compared to those from Group 5. Interestingly, this pattern mirrored that found in the avidity of sera from the same groups (Figure 4.6-6B).

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Figure 4.6-6. Comparison of RH5 avidity of the mAbs isolated from the different VAC063 groups (A) to the serum anti-RH5 avidity of different VAC063 groups (B).

Analysis of sequences of the mAbs as well as additional characterization of mAbs that are highly potent in the GIA (currently, BD5 and possible 8G8) is planned. Characterization will include determination of their binding kinetics and their effect on RCR complex formation.

4.7. BLOOD STAGE EPITOPE-BASED VACCINE DEVELOPMENT PROJECT: VIN KOTRAIAH

All technical work on this project was completed in Q1 FY2019. Leidos submitted the final report on April 26, 2019; USAID accepted the report on May 28, 2019. Follow-on work on cloning the 3H7 RIPR mAb was completed in FY2020. Development of a manuscript on the epitope prediction analysis work performed as part of this project is ongoing. A manuscript on the structure of P113 which contained data related to P113 mAbs developed for this project was previously submitted to mBio by Dr. Gavin Wright in June 2020, accepted in August 2020, and published in September 2020. In late March the manuscript was finalized for this project with USAID clearance received on April 1, 2021. In early April 2021, the manuscript (see Section 3) was submitted to Frontiers in Immunology.

4.8. PD1 BLOCKADE INHIBITOR RESEARCH ACTIVITIES: TIM PHARES All technical work on this project was completed in Q2 FY2019 and the final report approved by USAID in Q3 FY2019. The first PD1 manuscript was submitted to Frontiers Immunology in October 2019 and accepted on January 31, 2020. A second PD1 manuscript was submitted to Frontiers Immunology in April 2020 and accepted on May 29, 2020.

5. ELEMENT 3 ACTIVITIES: AMY NOE/JESSICA SMITH

No additional SCG meetings are planned under the MVDP contract.


6.1. MVDP REAGENTS REPOSITORY SriSai Biopharmaceutical Solutions (SBS) maintained, received, and distributed the reagents/materials needed for the ongoing and future studies during Q1 FY2021. SBS furnishes all the necessary services,

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management, qualified personnel, materials, equipment, facilities, and travel required for a biologics repository and inventory management services related to cGMP and non-GMP vaccines and associated products. SBS offers Controlled Room Temperature Storage (15 to 30°C), Controlled Room Temperature with humidity control, Refrigerated Storage (2 to 8°C), Freezer Storage (-20 ±10°C), Ultra-low Freezer Storage (-80 ±10°C), Low Freezer Storage (-30±10°C) and Vapor Phase Liquid Nitrogen (≤ -140°C), as well as the option to set units at a customer-defined temperature. The current inventory consists of standard storage temperatures ranging from -80°C to 4°C.

During Q1 FY2021, Leidos initiated inventory disposition be providing USAID an inventory listing for products developed and/or procured under the prime MVDP contract and requesting information on disposition. USAID provided feedback on the same and moved forward with inventory distribution to PATH and the University of Oxford in December 2020. The shipment to Oxford in planned for January 2020. Further, for hybridomas and antibodies developed under the blood stage and RCR vaccine development projects, some materials will be transferred to BEI Resources (planned for January 2020 pending approval by NIAID) such that these can be made available to the broader research community. In December 2020, USAID was provided will all final material transfer lists via email and these will also be included as part of the final program report.

All inventory was dispositioned in January 2021. Reagents designated by USAID were provided to PATH and the University of Oxford. Final inventory information is attached to the pdf version of this document.

7. TECHNICAL CLOSEOUT ACTVITIES

In Q2 FY2020, Leidos developed an MVDP Technical Closeout and Transition Plan to clarify the end of MVDP contract (1) material and reagent disposition process and (2) document and data transfer process. This plan was circulated to USAID on February 21, 2020 for comment. USAID provided feedback on March 6, 2020 and Leidos circulated an updated plan on March 23, 2020. In Q3 FY2020, Leidos started building the MVDP Data Transfer site and populating this with documentation that will be transferred to USAID at the end of the contract. Please refer to Section 6.1 for information on disposition of physical inventory in the MVDP repository associated with technical close out.

Leidos completed technical close out in early April FY2021 with finalization of all project deliverables (Final Project Reports and Raw Data) and submission of the required manuscripts to peer-reviewed scientific journals.

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8. LEIDOS POINTS OF CONTACT

8.1. PROGRAM MANAGER Amy Noe, Ph.D., MBA Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 858-826-6105 Mobile: 858-201-9176

8.2. OPERATIONS MANAGER Shannon Robinson, MBA, PMP Leidos LInC 10260 Campus Point Drive, MS C-4 San Diego, CA 92121 Phone: (858) 826-6034

8.3. CONTRACTS MANAGER Sandra George Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-425-8546 Fax: 301-846-0794

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9. APPENDIX 1: LITERATURE CITED

Aguiar, J.C., Bolton, J., Wanga, J., Sacci, J.B., Iriko, H., Mazeika, J.K., Han, E.T., Limbach, K., Patterson, N.B., Sedegah, M., Cruz, A.M., Tsuboi, T., Hoffman, S.L., Carucci, D., Hollingdale, M.R., Villasante, E.D., Richie, T.L., 2015. Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development. PloS one 10, e0136109.

Alanine, D.G.W., Quinkert, D., Kumarasingha, R., Mehmood, S., Donnellan, F.R., Minkah, N.K., Dadonaite, B., Diouf, A., Galaway, F., Silk, S.E., Jamwal, A., Marshall, J.M., Miura, K., Foquet, L., Elias, S.C., Labbe, G.M., Douglas, A.D., Jin, J., Payne, R.O., Illingworth, J.J., Pattinson, D.J., Pulido, D., Williams, B.G., de Jongh, W.A., Wright, G.J., Kappe, S.H.I., Robinson, C.V., Long, C.A., Crabb, B.S., Gilson, P.R., Higgins, M.K., Draper, S.J., 2019. Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies. Cell 178, 216-228 e221.

Arevalo-Herrera, M., Lopez-Perez, M., Dotsey, E., Jain, A., Rubiano, K., Felgner, P.L., Davies, D.H., Herrera, S., 2016. Antibody Profiling in Naive and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites. PLoS Negl Trop Dis 10, e0004563.

Billaud, J.N., Peterson, D., Barr, M., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005a. Combinatorial approach to hepadnavirus-like particle vaccine design. J Virol 79, 13656-13666.

Billaud, J.N., Peterson, D., Schodel, F., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005b. Comparative antigenicity and immunogenicity of hepadnavirus core proteins. J Virol 79, 13641-13655.

Brault, A.C., Domi, A., McDonald, E.M., Talmi-Frank, D., McCurley, N., Basu, R., Robinson, H.L., Hellerstein, M., Duggal, N.K., Bowen, R.A., Guirakhoo, F., 2017. A Zika Vaccine Targeting NS1 Protein Protects Immunocompetent Adult Mice in a Lethal Challenge Model. Sci Rep 7, 14769.

Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432.

Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234.

Crompton, P.D., Kayala, M.A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G.E., Molina, D.M., Burk, C.R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D.L., Liang, X., Doumbo, O.K., Miller, L.H., Doolan, D.L., Baldi, P., Felgner, P.L., Pierce, S.K., 2010. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 107, 6958-6963.

Doolan, D.L., Hoffman, S.L., 2000. The complexity of protective immunity against liver-stage malaria. J Immunol 165, 1453-1462.

Doolan, D.L., Southwood, S., Freilich, D.A., Sidney, J., Graber, N.L., Shatney, L., Bebris, L., Florens, L., Dobano, C., Witney, A.A., Appella, E., Hoffman, S.L., Yates, J.R., 3rd, Carucci, D.J., Sette, A., 2003. Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data. Proceedings of the National Academy of Sciences of the United States of America 100, 9952-9957.

Gilbert, S.C., 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 31, 4241-4246. Goepfert, P.A., Elizaga, M.L., Seaton, K., Tomaras, G.D., Montefiori, D.C., Sato, A., Hural, J., DeRosa, S.C., Kalams,

S.A., McElrath, M.J., Keefer, M.C., Baden, L.R., Lama, J.R., Sanchez, J., Mulligan, M.J., Buchbinder, S.P., Hammer, S.M., Koblin, B.A., Pensiero, M., Butler, C., Moss, B., Robinson, H.L., Group, H.S., National Institutes of, A., Infectious Diseases, H.I.V.V.T.N., 2014. Specificity and 6-month durability of immune responses induced by DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis 210, 99-110.

Higbee, R.G., Byers, A.M., Dhir, V., Drake, D., Fahlenkamp, H.G., Gangur, J., Kachurin, A., Kachurina, O., Leistritz, D., Ma, Y., Mehta, R., Mishkin, E., Moser, J., Mosquera, L., Nguyen, M., Parkhill, R., Pawar, S., Poisson, L., Sanchez-Schmitz, G., Schanen, B., Singh, I., Song, H., Tapia, T., Warren, W., Wittman, V., 2009. An immunologic model for rapid vaccine assessment -- a clinical trial in a test tube. Altern Lab Anim 37 Suppl 1, 19-27.

Jin, J., Hjerrild, K.A., Silk, S.E., Brown, R.E., Labbe, G.M., Marshall, J.M., Wright, K.E., Bezemer, S., Clemmensen, S.B., Biswas, S., Li, Y., El-Turabi, A., Douglas, A.D., Hermans, P., Detmers, F.J., de Jongh, W.A., Higgins, M.K.,

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Ashfield, R., Draper, S.J., 2017. Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'. Int J Parasitol 47, 435-446.

Kariko, K., Muramatsu, H., Ludwig, J., Weissman, D., 2011. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39, e142.

Oehring, S.C., Woodcroft, B.J., Moes, S., Wetzel, J., Dietz, O., Pulfer, A., Dekiwadia, C., Maeser, P., Flueck, C., Witmer, K., Brancucci, N.M., Niederwieser, I., Jenoe, P., Ralph, S.A., Voss, T.S., 2012. Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum. Genome Biol 13, R108.

Shinde, V., Fries, L., Wu, Y., Agrawal, S., Cho, I., Thomas, D.N., Spindler, M., Lindner, E., Hahn, T., Plested, J., Flyer, D., Massare, M.J., Zhou, B., Fix, A., Smith, G., Glenn, G.M., 2018. Improved Titers against Influenza Drift Variants with a Nanoparticle Vaccine. N Engl J Med 378, 2346-2348.

Tarun, A.S., Peng, X., Dumpit, R.F., Ogata, Y., Silva-Rivera, H., Camargo, N., Daly, T.M., Bergman, L.W., Kappe, S.H., 2008. A combined transcriptome and proteome survey of malaria parasite liver stages. Proc Natl Acad Sci U S A 105, 305-310.

Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., Howarth, M., 2012. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697.

Zhang, M., Kaneko, I., Tsao, T., Mitchell, R., Nardin, E.H., Iwanaga, S., Yuda, M., Tsuji, M., 2016. A highly infectious Plasmodium yoelii parasite, bearing Plasmodium falciparum circumsporozoite protein. Malar J 15, 201.

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1. EXECUTIVE SUMMARY

A summary of efforts for the planned, ongoing, and completed projects for the Malaria Vaccine Development Program (MVDP) contract for this reporting period are detailed herein. Ongoing projects that will continue into Q2 FY2021 (contract period of performance was extended in Q1 FY2021 to April 7, 2021) include one vaccine development project, the RCR complex vaccine development project (RCR Complex), and one project to develop human monoclonal antibodies (mAbs) to RH5 (RH5.1 Human mAb). In Q1 FY2021, technical work for one project was completed, the liver stage vaccine development project (Liver Stage Vaccine). Final reporting for this project is in process and anticipated to be complete in Q2 FY2021. Final reporting for one project, the CSP vaccine development projects (CSP Vaccine) was completed in Q1 FY2021 with delivery of the compiled report (including raw data and all associated documentation) anticipated in Q2 FY2021. Final reporting for the RH5.1 clinical study project is anticipated to be completed in Q2 FY2021. Final reporting for two MVDP projects was previously completed, the PD1 blockade inhibitor project (PD1 Blockade Inh) and the blood stage epitope-based vaccine project (Blood Stage Epitope). As part of the MVDP, Leidos has executed collaborative agreements (e.g., information exchange under NDA, reagent exchange under MTA, and collaborative work under CRADA) to benefit the MVDP and the broader malaria research community, as well as to extend the utility of the MVDP contract. In FY2019, Leidos executed a CRADA with NMRC for collaboration on the Liver Stage vaccine development project. The technical work associated with this CRADA was completed in Q1 FY2021. In Q3 FY2020, Leidos executed the last collaboration agreement for the MVDP prime contract (a CRADA with WRAIR involving development of a modified CSP mRNA construct) and technical work associated with this agreement is anticipated to be completed in Q2 FY2021 (these activities have been reported under the liver stage vaccine project). The information contained herein is intended to provide technical detail regarding activities conducted within the specified reporting period; however, at the request of USAID, Leidos retains data in quarterly reports for activities completed within the relevant fiscal year.

2. CONTRACTS MANAGEMENT AND ADMINISTRATION

On October 12, 2020, Leidos submitted a second no-cost extension (NCE) request to extend the prime contract period of performance end date to April 7, 2021 due to work completion delays experienced because of impacts from COVID-19. On November 3, 2020, Leidos received the fully-executed contract modification extending the prime contract period of performance end date to April 7, 2021.

2.1. CURRENT BUDGET SUMMARY The budget summary for FY20 is provided in Table 2.1-1. Table 2.1-2 provides FY20 to-date costs against FY20 Annual Work Plan Estimates. Detailed subcontractor spending is provided in the associated Q2 financial report.

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2.2. DELIVERABLES SUMMARY In Table 2.2-1 below, we provide the deliverables for which Leidos is responsible under the prime contract with respect to the tasks assigned by USAID. All deliverables are assigned according to the four (4) “Elements” that are outlined in the prime contract.



CONTRACTS AND ADMINISTRATION

Prime Contract

Signed: 5/28/15 Modifications: MOD 1 2/22/16, MOD 2 3/4/16 MOD 3 8/16/16 MOD 4 2/13/17 MOD 5 5/1/17 MOD 6 7/18/17 MOD 7 9/28/17 MOD 8 1/09/18 MOD 9 12/20/18 MOD 10 3/1/19 MOD 11 05/04/20 MOD 12 11/05/20

The NCE modification extending the contract POP end date to 04/07/21 was received on 11/05/20.

i.

Contractor – Employee Non- Disclosure/Conflict of Interest (COI) Agreements

Delivered Signed Non-Disclosure/COI Agreements are located on SharePoint.

ii. Annual Work Plan Delivered: 09/3/19 Approved: 10/28/19 Upcoming Plans: None.

vi. Quarterly Reports Q1 FY21 Delivered: 01/14/21 Upcoming Reports: None (Q2 FY2021 information will be included with the Final Overall Contract Report)

vii. Quarterly Financial Reports Q1 FY21 Delivered: 01/14/21

Upcoming Reports: None (Q2 FY2021 information will be included with the Final Overall Contract Report)

viii. Annual Report/Q4 Report Delivered: 10/14/20 Approved: 11/20/20 Upcoming Reports: None

H.10.a Small Business Subcontracting Plan

Delivered: The Small Business Subcontracting Plan was included in Leidos’ proposal.

Subcontracts

H.10.b.1 Individual Subcontract Reports



H.10.b.2 Summary Subcontract Report



ix. Final Overall Contract Report Delivered: Upcoming Report: 04/07/21 (draft 2

weeks prior to POP end)

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C.4.4 Biweekly USAID–Leidos Update Meetings

Held: See Table 2.4-1. Ad hoc meetings held: See Table 2.4-1.

Upcoming Meetings: Regularly scheduled biweekly meetings are held the first and third Wednesdays of each month. For Q2 FY2021, this schedule will be adjusted to monthly meetings based on input from USAID.

G.4.C

Contract Administration Meetings with Contracting Officer’s Representative (COR)


xiv. USAID Development Experience Information

Due: All contracts and administration deliverables will be uploaded to the DEC after 30 days from approval.

Leidos periodically uploads final deliverables to the DEC. At contract completion, Leidos will upload all remaining contract deliverables to DEC. Financial information, confidential information and/or unpublished data from subcontractors, and raw data will not be uploaded to the DEC.

ELEMENT 1: PROTOCOL DEVELOPMENT/WHITE PAPERS

xii. Publications/Posters Q1 FY2021: One manuscript submitted, two under development

See section 3.1 regarding publications and section 3.2 regarding abstract/poster submissions.

C.3.2.2 New Project Proposals — None in the reporting period

ELEMENT 2: IMPLEMENTATION OF RESEARCH AND DEVELOPMENT PROJECTS

CSP Vaccine Development Project


Project Plan approved by USAID on 1/29/16. Revised Project Plan approved 04/26/2017.

xii. Subcontracts: Procurement of Materials, Supplies, and Services

Subcontract/Task Order Awards: VLP Biotech, JHU, ADARC, EpiVax, Precision Antibody, ImmunoVaccine, VaxDesign Other Procurement Vehicles: CPC Scientific, Vaxine

Q1 FY2021: none, project completed.

iv. Final Individual Project Report Initiated in Q2 FY2020

Finalized in Q1 FY2021. Delivery of the compiled report including all attachments anticipated in Q2 FY2021

v. Individual Project Data Sets


xii. Publications Manuscript under development See section 3.2 regarding abstract/poster submissions.

ix. Other/Ad Hoc Reports —

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RH5.1/AS01 Vaccine Clinical Study


2/17/16.


Subcontract/Task Order Awards: University of Oxford (Oxford), EpiVax

Q1 FY2021: none, project completed

iv. Final Individual Project Report Initiated in Q3 FY2020

Draft delivered November 5, 2020; several revisions occurred in Q1 FY2021. To be finalized in Q2 FY2021.

v. Individual Project Data Set Oxford quarterly reports Provided herein, circulated via email,

and/or uploaded to SharePoint.

xii. Publications Completed: manuscript submitted in Q3 FY2020; resubmitted in Q4 FY2020.

See section 3.2 regarding abstract/poster submissions.

ix. Other/Ad Hoc Reports Project Completed

xii. Registration of Trials Completed Trial registered on September 5, 2016 (ClinicalTrials.gov)

Liver Stage Vaccine Development Project


Project Plan approved by USAID on 6/13/16. Attachment 1 - CD8 platform scouting plan approved by USAID on 6/6/19.


Subcontract Awards: EpiVax, VaxDesign, Precision Antibody, Multimeric BioTherapeutics, MabTech, Mimotopes, Biosynthesis, TriLink, ADARC, GeoVax, JHU

Q1 FY2021: GeoVax subcontract extension completed.

iv. Final Individual Project Report Initiated late Q4 FY2020 Ongoing

v. Individual Project Data Set

VaxDesign, ADARC, JHU and GeoVax data sets

Provided herein, circulated via email and/or uploaded to SharePoint.

xii. Publications None during reporting period. See section 3.2 regarding abstract/poster submissions.

ix. Other/Ad Hoc Reports Data report for CRADA with NMRC Leidos/NMRC data exchange

RCR Complex Vaccine Development Project-WEHI


3/21/2019.


Pending: WEHI (W3) Subcontract/Task Order Awards: ImmunoVaccine (W3 in Q4), and Precision Antibody

Q1 FY2021: WEHI subcontract extension completed.

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iv. Final Individual Project Report Initiated in Q4 FY2020 Ongoing

v. Individual Project Data Set WEHI data sets Provided herein, circulated via email

and/or uploaded to SharePoint.



RCR Complex Vaccine Development Project-University of Oxford (Oxford)


2/13/19.


Subcontract Awards: Oxford, IMV

Q1 FY2021: Oxford subcontract extension completed.


v. Individual Project Data Set

Oxford Quarterly Reports VLP Development Data Study O1 Data

Provided herein or circulated via email.


ix. Other/Ad Hoc Reports SOPs for Standardized ELISA Circulated via email.

RH5.1 Human mAb Identification and Development

iii. Final Individual Project descriptions Approved Project Plan approved by USAID (regular

elements only) on 1/29/19.

xii. Procurement of Materials, Supplies, and Services Subcontract Award: Oxford Q1 FY2021: Oxford subcontract

extension completed.


v. Individual Project Data Set Oxford data sets mAb cloning update provided herein



Blood-Stage Epitope Vaccine Development


Project Plan approved by USAID on 3/24/2016. Addendum 1 approved on 6/23/16.


Subcontracts Awards: Agilvax, VLP Biotech, Expres2ion, NYBC, Precision Antibody, Swiss TPH, KempBio MTAs: WEHI (Alan Cowman), Swiss TPH (Gerd Pluschke), Oxford (Simon Draper), Wellcome Trust Sanger Institute (Gavin Wright)

Q1 FY2021: None, project completed.

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iv. Final Individual Project Report Approved

Draft delivered to USAID on 01/31/19. USAID feedback received on 3/14/19. Final Approval 5/29/19.

v. Individual Project Data Set Project Completed.



PD1 Blockade Inhibitor


1/19/2017.


Subcontract Awards: ADARC, Oxford Q1 FY2021: None, project completed.

iv. Final Individual Project Report Approved Final Project Report delivered to USAID

on 6/10/19

v. Individual Project Data Set Project Completed

xii. Publications Project Completed ix. Other/Ad Hoc Reports Project Completed

ELEMENT 3: SCG ANNUAL MEETING SUPPORT

x. SCG Annual Meeting Event Date: N/A x.a-e. Logistic Support N/A

ELEMENT 4: PROCURMENT OF MATERIALS, SUPPLIES AND SERVICES

xi. MVDP Reagents Repository

SriSai Biopharmaceutical Solutions

The repository is used to store and distribute materials for conduct of the MVDP program. Inventory disposition was initiated in Q1 FY2021 and is scheduled to be completed in January 2021.

*Each Roman numeral crosswalks to a deliverable, as called out by the prime contract (AID-OAA-C-15-00071).

2.3. MANAGEMENT TOOLS Leidos’ SharePoint document repository is a Fiscal Information Security Management Act−compliant, web-based tool that provides access to program data/documentation, deliverables, work products, and schedules. This type of interface is an especially important information exchange among study sites as vaccines progress through milestones. Leidos granted folder permissions to subcontractor technical leads and customer points of contact, commensurate with their roles, allowing direct updates to their respective folders (Table 2.3-1). Transparency is achieved via USAID access to program folders. Leidos has uploaded documents relevant to the current reporting period to SharePoint. In Q1 FY2021, USAID informed Leidos that Google Docs was the USAID official document sharing platform with their implementation partners. Leidos is working internally and with USAID to establish access so that large file size and final program deliverables can be submitted via a USAID-hosted Google Docs folder.

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Table 2-2. Management Tools

Tool Description Location

SharePoint Cloud-based solution for exchanging and storage of documents https://vector.leidos.com/sites/ITLSO/MVDP

Conference Phone Lines

Provide USAID OCONUS line to call Scientific Consultant Group members and CONUS line to communicate with Leidos

1-855-462-5367 1778004 2013235

2.4. TEAM MEETINGS Leidos also achieves transparency by routine copying of designated customer staff regarding email communications and teleconferences. As required, Leidos has set a standing biweekly meeting with USAID to review our MVDP activities (see Table 2.4-1). Ad hoc discussions to ensure positive study outcomes have been implemented. Leidos uses standard business tools (e.g., email, phone, teleconference, and desktop sharing) to communicate with staff and customers. Meeting agendas and summaries/minutes are available in the “Meeting Materials” folder on the MVDP SharePoint site (https://vector.leidos.com/sites/ITLSO/MVDP/Deliverables/Meeting Materials).

Table 2-3. Team Meetings

Meeting Date Topic



November 4, 2020 Leidos/USAID Bi-weekly Team Meeting (Canceled)

November 10, 2020 Oxford RCR Complex Project IPT Meeting

November 18, 2020 Leidos/USAID Bi-weekly Team Meeting




3.1. PUBLICATIONS The status of manuscripts currently under development and/or submitted for publication is provided is provided in Table 3-1.



Med Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

RH5 Clinical Study Manuscript was submitted in Q1 FY2021.

TBD TBD Liver Stage Vaccine Development

Manuscript circulation anticipated in Q2 FY2021.

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TBD TBD Blood Stage Vaccine Development



Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4+ T Cell Epitopes from Diverse Plasmodium falciparum Strains.

CSP Vaccine Development


3.2. ABSTRACT SUBMISSIONS/POSTER PRESENTATIONS No scientific conference abstract submissions or poster presentations are anticipated for FY2021.

3.3. NEW PROJECTS No new projects are planned.


Upon identification of viable vaccine projects to support development from proof-of-principle testing, manufacturing, and clinical trial evaluation, Leidos drafts a detailed plan and protocols, identifies subcontractors, and executes the plan. Projects completed and ongoing in FY2021 are described in this section, as well as adjuvants and platforms utilized for the same.

4.1. ADJUVANT/DELIVERY PLATFORMS Adjuvants and delivery platforms identified through scouting efforts and slated for use in FY2020 are detailed in this section.

4.1.1 ADJUVANTS The DepoVaxTM platform is in use for the CSP and RCR complex projects. Matrix-MTM is in use for the RCR complex project.

4.1.1.1 DepoVax The DepoVax platform, developed by ImmunoVaccine Inc., contains lipids, cholesterol, oil, emulsifier and an immunostimulant (e.g., cGAMP, polyI:C, and/or Pam3Cys). This lipid-in-oil platform is designed to present antigen(s) and adjuvant(s) at a long-lasting depot that effectively attracts antigen-presenting cells (APCs) and from which antigen is released over an extended period of time, from weeks to months. DepoVax promotes Th2 responses and enhances Th1 immune responses without triggering regulatory T cells. DepoVax has been used in the clinic as part of a Phase I/II study for a cancer vaccine (clinicaltrials.gov identifier: NCT01095848). Of note is that there are no aqueous components in this formulation; therefore, antigen is lyophilized for use with DepoVax and components are mixed and emulsified prior to administration using materials provided as part of an administration kit. Leidos executed a purchase order with ImmunoVaccine for formulation and provision of adjuvants for preliminary efficacy studies.

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4.1.1.2 Matrix-M Matrix-M is a saponin-based adjuvant comprised of purified saponin, synthetic cholesterol, and a phospholipid patented by Novavax. This adjuvant generates both cell-mediated and antibody-mediated immune responses, and has the potential to increase immune response duration as shown in numerous clinical trials (Shinde et al., 2018). The Draper group has an access agreement to use this adjuvant for malaria vaccine development.

4.1.2 PLATFORMS Three VLP-based vaccine delivery platforms are in use for different projects as follows: the WHcAg VLP platform is in use for the CSP project, the GeoVax MVA-VLP platform is in use for the liver stage project, and the SpyTag/SpyCatcher VLP platform is in use for the RCR complex project. In addition, development of mRNA-based vaccines using technology to enhance protein expression is also in use for the liver stage project.

4.1.2.1 WHcAg VLP The woodchuck hepatitis B core antigen (WHcAg) VLP platform, developed by VLP Biotech, is based on the core protein the of woodchuck hepatitis B virus. The core proteins self-assemble into VLPs with 240 copies of the antigen per VLP. This platform can accommodate multiple foreign sequence insertions, with long insertions possible at the N and C-termini. Inserts within the surface-exposed loop are possible, which is particularly beneficial for B cell epitopes as the VLP configuration permits cross-linking of B cells. Studies with this platform have shown it to be equal or more immunogenic than HBcAg for both B cell and T cell responses, not significantly cross-reactive with the HBcAg for B cell responses and only partially cross-reactive with HBcAg for T cell (CD4) responses, and function as a vaccine carrier platform for heterologous, B cell epitopes (Billaud et al., 2005a; Billaud et al., 2005b). In consideration of cost, WHcAg VLPs can be easily expressed at high levels in E. coli. Note that this platform has not yet been tested in the clinic.

4.1.2.2 GeoVax MVA Platform Modified Vaccinia virus Ankara (MVA)-based vaccines have been widely tested in the clinic and are known to generate high cellular responses (Gilbert, 2013). The main drawback of these platforms has been that immunogenicity is greater when these vectors are used to boost pre-existing T cell responses. However, GeoVax’s 4th generation MVA-VLP platform requires no immune response priming due to improved transgene stability during manufacture and elevated levels of expression compared to the parent platform. This is evidenced by a clinical study with GeoVax’s MVA-based HIV vaccine, where cellular (both CD8 and CD4) and humoral responses were seen in humans administered the MVA-VLP only (Goepfert et al., 2014). Such responses in animal models have also been seen (Brault et al., 2017). Also of note is that this platform does not require adjuvant.

4.1.2.3 SpyTag/SpyCatcher VLP Platform To alleviate the pitfalls of more traditional VLP development, the groups of Draper, Biswas and Howarth at Oxford (Brune et al., 2016) developed the SpyTag/SpyCatcher “plug-and-display” VLP platform, which employs use of the SpyTag peptide and SpyCatcher protein (originally generated by splitting the CnaB2 domain from the Streptococcus pyogenes fibronectin-binding protein FbaB (Zakeri et al., 2012)) to decorate the VLP surface with antigen. SpyTag-linked antigen and SpyCatcher-linked VLP carrier (resulting from the genetic fusion of SpyCatcher to VLP coat protein monomers followed by expression and self-assembly) are required for the production of VLPs using this platform. Mixing of these two components results in the spontaneous formation of an irreversible bond between the SpyTag-Antigen and SpyCatcher-VLPs, yielding VLPs decorated with the antigen of interest. The SpyTag/SpyCatcher platform has been used to generate VLPs displaying a variety of malaria-related antigens (e.g. CIDR, Pfs25, CSP) as well as self-antigens and antigens related to cancer, tick-borne encephalitis, and tuberculosis (Brune and Howarth, 2018). VLPs generated using this platform can be administered in the

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presence or absence of adjuvant, and studies with Pfs25-AP205 VLPs showed a higher anti-Pfs25 response than Pfs25 alone or Pfs25-SpyTag. Additionally, Pfs25-AP205 VLPs formulated in AddaVax yielded a slight increase in the anti-Pfs25 response when compared to the same VLPs without adjuvant (Brune et al., 2016). This platform has not yet been assessed in the clinic.

4.1.2.4 RNA-based Vaccine Development Approach An RNA-based vaccine development approach involving collaboration with the Leidos Innovations Center (LInC), Promosome, and TriLink was proposed as part of the FY2020 AWP and approved by USAID. For this approach, Leidos and Promosome work together to design and test RESCUE modifications and translation enhancing elements (TEEs) to best increase expression of the proteins in the RCR complex. This will include: a) transiently transfecting mammalian cell lines with RESCUE-modified sequences and b) evaluating expression levels of each candidate protein by semi-quantitative Western blot analyses to identify the optimum modifications. The optimum sequences will be incorporated into plasmids for RNA production by TriLink. TriLink will incorporate modified nucleotides (one-methylpseudouridine-5’-triphosphate in place of UTP) and cap mRNA to increase mRNA stability. Leidos will perform quality control studies with the TriLink generated mRNAs. The resulting mRNAs will be encapsulated using a lipid nanoparticle technology.

4.2. CSP VACCINE DEVELOPMENT PROJECT: AMY NOE AND JAYNE CHRISTEN Technical work for this project was previously completed. In August 2020, the draft CSP Vaccine Development Final Project Report was circulated to USAID. Several revision cycles occurred during Q4 FY2020 and Q1 FY2021. The report was finalized on December 9, 2020; however, the consolidated report (pdf version including all attachments) could not be delivered due to size constraints for receipt of email attachments. Leidos is current working with USAID to standup a site for provision of large file size deliverables.

4.3. RH5.1/AS01B CLINICAL STUDY: AMY NOE Technical work for this project was previously completed. In Q4 FY2020, all raw data and final report attachments were received from Oxford. Leidos circulated the draft final project report on November 5, 2020 and several revision cycles occurred during the remainder of Q1 FY2021. The report is expected to be finalized in early Q2 FY2021. In Q1 FY2021, the manuscript (see Section 3) submitted to Med; review is ongoing.

4.4. LIVER STAGE VACCINE DEVELOPMENT PROJECT: KEN TUCKER/TIM PHARES A summary of the liver stage project plan is provided in Table 4.4-1. The technical work on this project was completed in Q1 FY2021.

Table 4.4-1. Overview of the Liver Stage Epitope-Based Vaccine Development Plan

Phase Milestone Activity

Phase 1 Epitope

Development

1.1 In silico identification of proteins (Stage 1)

In silico identification of class I and II epitopes and score of protein immunogenicity; downselection to subset of proteins

1.2 In silico identification of peptides (Stage 2)

Selection of class I and II epitopes based on in silico analysis of Treg, conservation in Plasmodium, and HLA coverage

1.3 In vitro biological response testing Testing T cell stimulation by, and immune response to peptides

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expressing Pf proteins. The vaccines were delivered and JHU performed and completed the dose titration of irradiated Py/PfCSP sporozoites study in Q4 FY2020. The active vaccination MVA-VLP Pf protein challenge study was completed in Q1 FY2021.

4.4.1.1 Construction of Individual Pf Liver Stage Protein MVA Vaccines Procurement efforts for the Pf MVA vaccines were conducted in Q1 FY2020 and GeoVax began work on constructing the recombinant MVA-VLP vaccines expressing the following Pf proteins in Q2 FY2020 (Table 4.4-2).

Table 4.4-2. Individual Pf Proteins Designation Accession Number (PlasmoDB) SPECT2 PF3D7_0408700

SPECT2.2 without MACPF domain PF3D7_0408700



Burnet Institute CSP (Pf3D7 sequence)

In Q4 FY2020, GeoVax completed scale up and characterization of the four MVA-VLPs expressing Pf proteins: MVA-PfCSP, MVA-Pf08_0081, MVA-Pf14_0593, and MVA-PfSPECT2. For each MVA-VLP, a research seed stock (RSS) and final vaccine stock (FVS) was made. The FVS was provided to JHU for the in-life challenge study. Information on characterization of the RSS and FVS for each construct was provided to USAID in Q4 FY2020.

To confirm VLP formation by the four MVA expressing Pf protein vaccines, purified virus was analyzed by transmission electron microscopy EM in Q1 FY2021. Results of the EM analysis (Figure. 4.4-1) were provided to USAID on December 23, 2020. In brief, cultured DF1 cells were infected at an MOI of 0.5 for 24 hours with each individual vaccine construct. The cells were fixed and an attempt to stain the thin layer sections with primary polyclonal serum against SPECT2, Pf08_0081, or Pf14_0593 (produced by Precision Antibody) followed by secondary immunogold anti-mouse Ab resulted in no positive staining. This result was not unexpected as previous attempts with the individual antigen-specific polyclonal serum yielded no positive staining on Western Blots when cell lysates were analyzed for Pf protein expression during vaccine construction. However, filamentous VLP formation at the cell surface was observed in all samples. Please note, oval shaped, electron dense structures are MVA and are denoted with asterisks. VLPs are less electron-dense, elongated structures budding from the cell surface and are denoted with arrows. Representative images for each vaccine construct are depicted below, scale bar is 200nm. In addition to MVA-Pf protein expressing VLPs, MVA-VP40 was used as a positive control for VLP formation. All technical work on this project is complete.

Figure 4.4-1: Electron Microscopy Analysis of Pf Protein MVA-VLPs

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4.4.1.2 Efficacy Assessment of Pf MVA-VLPs The best mouse challenge model (Py or Pb) for assessment of the Pf protein-expressing MVA-VLPs was discussed with USAID in early Q2 FY2020 and ultimately, it was agreed to move forward with the Py/PfCSP model. Use of this model required three separate experiments to (1) determine the optimal number of mosquito bites for Py/PfCSP sporozoite challenge, (2) determine the dose of irradiated Py/PfCSP sporozoites that gives ~50% protection upon Py/PfCSP sporozoite challenge, and (3) conduct the active vaccination challenge study with the Pf MVA-VLPs. In January 2020, Dr. Tsuji moved his laboratory from ADARC to Columbia University. Shortly thereafter, he informed Leidos that the NYU insectary (his source for infected mosquitoes) had closed. As Dr. Tsuji could no longer perform the studies to assess the MVA-VLPs expressing the Pf proteins, Leidos engaged Dr. Fidel Zavala (JHU) to determine if he could perform these studies. Dr. Zavala agreed to perform the studies and procurement was completed in Q2 FY2020. Updates on each of the three experiments needed for the efficacy assessment are below.

(1) Optimizing the number of mosquito bites for Py/PfCSP sporozoite challenge. In Q2 FY2020, JHU conducted a study to determine the number of mosquito bites required to ensure infection using Py/PfCSP transgenic sporozoites. This study informed the targeted infectious exposure that will be used for the subsequent studies at JHU. Results of this study were delivered to USAID on March 23 in Q2 FY2020. Based on the data Leidos and USAID agreed to move forward with using at least 6 bites/mouse to ensure that all mice are infected.

(2) Titrating the number of irradiated Py/PfCSP sporozoites. In addition to optimizing the number of mosquito bites for infection, JHU also titrated the number of irradiated Py/PfCSP sporozoites to determine the dose that gave 40-50% protection upon Py/PfCSP mosquito bite challenge. Results of this study were delivered to USAID on September 21, 2020. Based on the results Leidos and USAID agreed to move forward with using the 6.5x104 dose in the active vaccination MVA-VLP Pf protein challenge study.

(3) Performing the active vaccination MVA-VLP Pf protein challenge study

With completion of the optimizing studies and vaccines, a study to evaluate the protective efficacy or delay in patency of the individual Pf liver stage protein MVA vaccines was commenced on October 5 2020. Per the study design shown in Table 4.4-3, eight- to nine-week-old BALB/c mice (n = 7) mice were immunized IM in the hind limb with 107 of the MVA vaccine at day 0 and 28. Positive control mice were immunized IV in the tail vein with 6.5 x 104 irradiated Py/PfCSP sporozoites at day 21 and 35. At day 49 all mice were challenged via mosquitos infected with Py/PfCSP. Parasitemia in the mice was assessed via blood smears. Results of the challenge study were delivered to USAID on December 10 2020 (Table 4.4-4 and Table 4.4-5). Naïve mice that received no immunization showed no protection. Mice immunized with the 6.5 x 104 irradiated Py/PfCSP sporozoites were 100% protected. It was anticipated that none of the mice immunized with an empty MVA-VLP would be protected; however, 2 of 7 or 29% of these mice were protected after challenge. For the individual MVA-VLP Pf protein vaccines the following protection was observed: SPECT2 = 29%; PF14_0593 = 17%; PF08_0081 = 14%; and 3D7 PfCSP = 57%. Based on the 29% protection seen with the empty MVA-VLP it’s difficult to discern whether the protection seen with the individual vaccines is antigen-specific. The increased protection to 57% with the 3D7 PfCSP vaccine suggests a potential antigen-specific response; however, this cannot be assured with any certainty. Leidos proposes that if USAID decides to further explore the utility of the GeoVax platform in future work that it may be beneficial to perform a dose-titration study with the different vaccine constructs and empty MVA-VLP vaccines to establish potency with the respective constructs. All technical work on this project is complete.

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WRAIR was fully executed on February 6, 2020. Per the agreed workflow, Leidos met with WRAIR in early February 2020 to confirm logistics for shipment of the initial CSP mRNA construct; however, in early March, WRAIR informed Leidos that provision of the plasmid would need to be negotiated with TriLink®. Leidos met with TriLink in March 2020 and TriLink provided an MTA template for the material exchange. In Q3 FY2020, the MTA between Leidos and TriLink was signed. Additionally, the full-length PfCSP plasmid (starting construct reference number: 42725-2/W37-E01A, plasmid ID: TS-2019-3460) was ordered from TriLink in late June 2020. Prior to ordering the plasmid, Leidos confirmed the plasmid reference information with WRAIR. In Q4 FY2020, the starting plasmid (termed pCSP Ref) was received from TriLink. LInC modified the starting construct to introduce the designed RESCUE modifications and three different translation enhancing elements (TEEs). LInC experienced hurdles with low plasmid yield and was able to troubleshoot with the help of TriLink at the end of Q4 FY2020; these hurdles were due to the TriLink proprietary deactivated T7 promoter within pCSP Ref. In early Q1 FY2020, LInC will evaluate protein expression from the modified constructs via transfection of mouse muscle cells (C2C12) and human embryonic kidney cells (HEK293). LInC shipped the plasmid to TriLink in early Q1 FY2021 and TriLink anticipates that both mRNAs (the initial and improved) will be shipped to SriSai in January 2021, for distribution to WRAIR and LInC. Detailed development information provided by LInC in December 2020 has been included in the below.

4.4.2.1 In silico analyses and design of RESCUETM modifications A construct containing a CSP coding sequence previously developed by WRAIR, in collaboration with TriLink, was received by LInC as part of the previously noted MTA established with TriLink (Figure 4.4-2A). For simplicity LInC refers to this plasmid as pCSP Ref. LInC performed an in silico analysis of this sequence to identify cryptic translation initiation sites and identified numerous potentially inhibitory sequences. In general, RESCUETM modifications are restricted to the signal peptide of protein coding sequences and range from conservative, synonymous modifications, to aggressive non-synonymous modifications that result in altered amino acid sequences. In the latter case, the signal peptides are cleaved leaving the mature, processed protein with an unaltered protein sequence. For this evaluation, since the CSP mRNAs were to be translated in vivo in a preclinical mouse model, only conservative modifications were designed to avoid biological effects of a modified signal peptide sequence with unknown biological effects. In consideration of this and timeframe, modifications were restricted to the first ~200-nucleotides of the CSP coding sequence. This approach is known by LInC to be beneficial for increasing expression of non-secreted proteins.

Figure 4.4-2. Schematic Representation of CSP mRNA Construct Generation. (A) The pCSP Ref plasmid contains a deactivated T7 promoter, a full-length CSP coding sequence, a Kanamycin resistance gene, the pBR322 origin of replication, and TriLink® proprietary 5′ leader and 3′ untranslated regions. (B) Representative TEE/RESCUE-containing CSP plasmid. Introduction of TEEs and recoded CSP coding sequence did not alter the critical features of the reference construct including the deactivated T7 promoter, TriLink® proprietary 5′ leader, and 3′ UTRs.

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4.4.2.2 Construct generation A RESCUETM modified CSP sequence was synthesized as a gBLOCK by IDT and introduced into the CSP construct provided by TriLink along with the subsequent introduction of three different TEEs (known as E6s, 5′1s, and G11s) in the form of PCR products from TEE containing templates (Figure 4.4-2B).

4.4.2.3 Optimization of Western blotting detection conditions Western blots were used to evaluate CSP expression using anti-CSP antibodies (3B6 and 5D5), anti-CSP sera (rabbit sera provided by VLP Biotech), and VLP162 as a CSP positive control. The antibodies/sera were tested for reactivity against 50 ng of the VLP162 under denatured and non-denatured conditions. In addition, since the ultimate goal was to assess expression of secreted CSP from cultured cells, culture media from untransfected HEK293 cells that had been spiked with 50 ng of the VLP162 was also tested. The results of these optimizations are shown in Figure 4.4-3 and demonstrated that the most sensitive reagent was the rabbit antisera. For the purposes of this study, LInC performed subsequent analyses using the rabbit antisera since this appeared suitable with respect to sensitivity.

Figure 4.4-3. Optimization of Western Blot Methodology. Western blots were performed using 50 ng of reduced or unreduced VLP162 (positive control) spiked into culture media from untransfected HEK293 cells, or in PBS. For the anti-CSP rabbit antisera optimization culture media and lysate from untransfected cells was included to distinguish between true CSP reactivity and nonspecific binding with components contained within each of these samples. Fractionated proteins were transferred to PVDF membranes and probed with (A and B) a 1 µg/mL mouse monoclonal antibodies (clone 3B6 and 5D5) and (C) a 1:1,000 dilution of the anti-CSP rabbit antisera. Membranes probed with mouse anti-CSP antibodies were subsequently probed with a 1:20,000 dilution of an anti-mouse HRP-conjugated secondary antibody, while that probed with the rabbit antisera was probed with a 1:10,000 dilution of an anti-rabbit HRP conjugated secondary antibody. Blots were developed using an ECL reagent and various exposures of the chemiluminescent signal captured using an Amersham 600 imager. (D) Loading order of samples.

4.4.2.4 Evaluation of CSP expression in a C2C12 and HEK 293 cell lines These studies involved (i) generation, purification, and QC of capped and polyadenylated CSP mRNAs synthesized in vitro from the transcription templates, (ii) their transfection into C2C12 and HEK293 cells, and (iii) CSP protein expression evaluated by western blotting using optimized conditions. Expression levels from these mRNAs were normalized to account for differences in transfection efficiencies using the luciferase mRNA co-transfection control.

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4.4.2.5 In vitro transcription of CSP mRNAs Initially LInC attempted to transcribe capped and polyadenylated mRNAs from the linearized plasmids containing recoded CSP and TEEs using the HiScribe™ T7 ARCA mRNA kit. However, despite numerous attempts LInC was not able to do so. After consulting with TriLink it became apparent that TriLink had supplied LInC with a plasmid that contained a deactivated T7 promoter and that TriLink’s normal workflow involved PCR amplification of plasmids to restore the T7 promoter sequence and add a polyA tail of a defined length. Although TriLink shared the primer sequences used in their workflow, LInC was not able to specifically amplify a product of the correct length from pCSP Ref or the modified CSP plasmid (data not shown). It should be noted that TriLink also encountered this issue when the modified CSP plasmid was forwarded to them. To overcome this issue, LInC replaced the deactivated T7 promoter with the restored sequence using a cloning approach. The restored T7 plasmids were then linearized by XbaI digest, phenol:chloroform purified, and used as templates for successful in vitro transcription as described below.

4.4.2.6 Analyses of in vitro transcribed CSP RNAs. One microliter of reaction was removed after the capped in vitro transcription, and tailing reactions for analyses using the Agilent RNA 6000 Nano Assay Kit to evaluate transcription length before and after the addition of poly(A) tails, RNA integrity, and determine RNA concentrations. Figure 4.4-4D shows overlaid traces for each of the in vitro transcribed CSP RNAs before and after the tailing reaction that indicate i) the presence of non-degraded, and ii) a shift in size of RNAs indicating the successful addition of poly(A) tails. Figure 4.4-4E also shows this data as a virtual gel image alongside molecular weight standards. The inclusion of internal standards in the RNA 6000 Nano reagents provides a means for quantitation and indicated yields of capped and polyadenylated mRNAs of between ~140 to 250 ng/µL.

Figure 4.4-4. Analyses of CSP RNAs from In Vitro Transcription Reactions. In vitro transcription products for (A) the reference CSP, (B) the RESCUETM-modified CSP with the E6s TEE, (C) the RESCUETM-modified CSP with the 5′1 TEE, (D) and the RESCUETM-modified CSP with the G11s TEE were analyzed on Agilent RNA 6000 Nano bioanalyzer chips. (E) The molecular weight ladder for this experiment. For each CSP variant 1 µL of the reaction that generates the capped RNA, and 1 µL of the reaction that that adds a poly(A) tail to the capped RNA were analyzed; the peak height is used to quantitate RNA concentrations by comparison to internal standards. Images A-D show overlaid traces for the two forms of RNA for each CSP variant; the addition of a poly(A) tail is indicated by the increases in length of RNAs. (F) These data are also represented as a virtual gel image.

4.4.2.7 Transfection of C2C12 and HEK293 with in vitro transcribed CSP mRNAs Transient transfection of C2C12 and HEK293 cells with mRNAs was performed using the XfectTM RNA transfection reagent that generates biodegradable, low cytotoxicity nanoparticles containing. C2C12

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cells are a mouse muscle cell line are were selected to best model anticipated results for the malaria mouse model. HEK293 cells were included as WRAIR has previously used this line to assess transcription of pCSP Ref. In brief, 1 µg of each CSP mRNA was transfected in triplicate into cells using the protocol recommended by the manufacturer (Takara). In addition, each CSP mRNA was co-transfected alongside 100 ng of the TriLink CleanCap® Firefly Luciferase (FLuc) mRNA as a co-transfection control. This permits normalization of transfection efficiencies among replicates and different CSP mRNAs as well as due to variability in cell lysis.

4.4.2.8 Western Blot Analyses to determine CSP expression Fourteen microliters of supernatant from C2C12 and HEK293 cells transfected with the CSP mRNAs was subjected to western blot analyses using the anti-CSP rabbit antisera under the previously optimized conditions (Figure 4.4-3C). The resulting data demonstrate that, with C2C12 cells, CSP expression was higher in cells transfected with the RESCUE_E6s TEE modified mRNA relative to the cells transfected with the pCSP Ref mRNA (Figure 4.4-5A). Overall, expression in HEK293 cells was significantly less than that observed in C2C12 cells and required the use of a more sensitive ECL detection reagent (SuperSignal™ LInCst Femto ECL). For the HEK293 transfections CSP expression was higher in cells transfected the RESCUE_G11s TEE modified mRNA relative to the cells transfected with the pCSP Ref mRNA (Figure 4.4-5B). As the cell type impacts the level of expression and the C2C12 cells are thought to best model expression in mice, the RESCUE_E6s TEE modified construct was downselected.

Figure 4.4-5. Western Blot Analyses of Culture Supernatants from (A) C2C12 and (B) HEK293 Cells Transfected with In Vitro Transcribed CSP mRNAs. Western blot were performed using a 1:1000 dilution of anti-CSP rabbit antisera and a 1:10,000 dilution of goat-anti-rabbit HRP-conjugated secondary antibody. Development was via standard ECL reagents (ECL Plus) for the C2C12 cell samples (A) and the more sensitive SuperSignal™ LInCst Femto ECL reagent for HEK293 cell samples (B). Analyses was performed using 14 µL of each supernatant from transfected cells alongside 10 ng of the VLP162 positive control under reduced conditions. pCSP Ref (Ref) mRNA and modified CSP mRNAs containing RESCUE™ modifications and the different TEEs (5′1s, E6s, and G11s) were used for transfections in both cell lines.

4.4.2.9 Transfer of the optimized construct to TriLink Based on the above results and taking into consideration the downstream application, LInC provided CSP plasmid containing the RESCUE-modified coding sequence containing the E6s TEE to TriLink in

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November 2020. It should be noted that the plasmid provided contains the TriLink proprietary deactivated T7 promoter present in the pCSP Ref plasmid, which is consistent with TriLink’s workflow, as opposed to the plasmid containing a restored T7 promoter generated by LInC and used for in vitro transcription by LInC. TriLink anticipates shipment of mRNA in January 2021.

4.4.3 SCHEDULE This project was completed in Q1 FY2021.

4.5. RCR COMPLEX VACCINE DEVELOPMENT PROJECT: VIN KOTRAIAH (WEHI) KEN TUCKER (OXFORD)

The RCR Complex vaccine development project is a multi-year effort that will continue at WEHI and Oxford into FY2021. Interdependencies between these institutions and decision points are shown in the RCR Complex Vaccine Project Workflow Figure 4.5-1, where decision points are shown as to be determined (TBD). The workflow diagram was updated to incorporate the decision by USAID on November 18, 2019 to proceed with the inclusion of five DPX cohorts to the Study O2 design at Oxford and removal of the penultimate study. Additionally, to incorporate the decision made by USAID on December 11, 2019 to modify Study O3 into a dose-ranging study, the workflow diagram has been updated to indicate that such a study will be performed using single antigen VLPs.

Figure 4.5-1. RCR Complex Vaccine Project Workflow. Downselection is based on analysis of sera samples via GIA (pLDH and flow cytometry methods) and Quantitative ELISA.

4.5.1 WEHI: VIN KOTRAIAH

4.5.1.1 Study W3 The study design for W3 is shown in Table 4.5-1 and the dose assignments for W3 are shown in Table 4.5-2 are shown for reference as some of the follow-on tasks utilize the W3 sera.

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Leidos circulated minutes for the IPT meeting on June 9, 2020. Study design development and procurement activities were completed and experiments were initiated in Q4 FY2020 and results from Q1 FY2021 are presented below.

4.5.1.3 Analytical Evaluation of Formulated RCR In order to understand if the RCR complex forms and is stable when formulated in DPX4, IMV reserved vials from study W3 to assess RCR complex stability. For this evaluation, the formulations for cohorts 7 and 8 (triple protein combinations lyophilized with DPX4 excipients) were reconstituted in water and evaluated using native gel followed by Western blot using antibodies recognizing individual proteins. However, the findings from the technical report (submitted by IMV in May 2020) were inconclusive. Per discussions with USAID, WEHI was tasked with evaluating the RCR complex using the remaining formulated vials for cohorts 7 and 8.

In Q1 FY2021, WEHI dissolved the DPX4 RCR formulations in water in preparation to load on a native SDS-PAGE gel. However, the formulations were insoluble and very little of the material could be loaded into the well of the gel and resulted in lane distortion perhaps due to the DPX4 components (Figure 4.5-1). The purified Oxford RCR and WEHI RCR complexes were run on the same gel and the Oxford RCR complex could be seen as a clear band (* in lane labeled RCR Oxford). The WEHI RCR (* in lane labeled as RCR post-IEX) was less clear and ran at a slightly higher MW due to the different affinity tags compared to the WEHI complex.

WEHI subsequently tried solubilization in Triton X-100 and DMSO without success. Due to the hydrophobic nature of the excipients, analytical method development may be needed to complete this work and an expert CRO in this area (i.e., protein analytical chemistry) would need to be utilized. Leidos also noted that such analytical method development is typically conducted as part of product release and stability test development. During further discussions, WEHI expressed their concerns that further manipulation (i.e., harsher treatment) of the material might result in complex formation/dissolution that is not representative of the current form. Because of technical challenges encountered in verifying the stability of RCR complex in DPX4 without resorting to harsher solubilization procedures, this effort was stopped.

Figure 4.5-2 Native SDS-PAGE of RCR complex stability in cohorts 7 and 8 DPX4 formulations. Bands corresponding to CyRPA and RIPR are indicated by red and green asterisks, respectively; RCR complex is marked with the purple asterisk.

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4.5.1.4 Standardized ELISA In the Leidos-USAID biweekly meeting held on October 6, 2020, USAID stated their preference for a test sample exchange between the two laboratories (WEHI and Oxford) as part of the assay transfer process. Leidos then worked to coordinate the assessment test sera from WEHI by Oxford and facilitated communication between the laboratories to help finalize the sera set for evaluation at both sites. The list of samples was shared with USAID advisors who indicated their agreement on December 01, 2020.

The description of WEHI sera that were shipped to Oxford for testing in the Standardized ELISA are shown in Table 4.5-3. The inclusion of W2 sera will provide samples with a range of signal in the ELISAs and increase the sample size for the comparison analysis of data generated at the two sites. According to the current plan, these individual animal sera will be tested at both labs.

Table 4.5-3. WEHI W3 and W2 samples shipped to Oxford for Standardized ELISA

Sera Antigen Number, volume of test samples Pre-bleed volume

W3 - cohort 1 RH5 6, 50 µL 10 µL W3 - cohort 2 CyRPA 6, 50 µL 10 µL W3 - cohort 3 RIPR 6, 50 µL 10 µL

W2 - cohorts 1-3* RH5 18, 35 µL 10 µL W2 - cohorts 4-5 CyRPA 12, 35 µL 10 µL W2 - cohorts 6-7 RIPR 12, 35 µL 10 µL

Total number of samples = 120. * Note that Dr. Julie Healer of WEHI is considering testing either the 20 µg or 2 µg W2 RH5 samples (the 0.2 µg RH5 sera will be tested). This is to keep the number of samples manageable for WEHI so they can complete all the remaining follow-on tasks by their POP end date. Dr. Healer has also noted that the nominal 20 and 2 µg dose cohorts were not very different in terms of their ELISA OD1 titers.

The WEHI sera were received at Oxford on December 14, 2020 and were partially blinded as per Oxford’s request. They were labeled with antigen (to indicate to Oxford as to which assay the sample needs to be tested on), rat ID number and as pre-bleed where applicable but the antigen dose was not specified. This will allow Oxford to test the appropriate ELISA blinded to W2/W3 and antigen dose. Following testing Oxford will request the unblinded list and WEHI’s ELISA results for concordance analysis of the data collected at both sites.

WEHI has made initial attempts at testing Oxford’s Standardized ELISA protocol. Due to COVID-related delays, delivery of the secondary antibody and detection reagent was delayed. Therefore, WEHI attempted an initial test using a different secondary antibody and detection reagent from that specified in the SOP; however, differences in signal development were significant from those described in the SOP. WEHI has since received the correct secondary antibody and is awaiting delivery of the correct detection reagent following which the Standardized ELISA will be repeated.

4.5.1.5 GIA reversal The ability of RH5.1, CyRPA and RIPR antigens to reverse GIA activity was tested by two separate methodologies.

Direct (antigen-in-well) GIA First, in accordance with advice from Kazutoyo Miura at the NIH who has published this method, the reversal assay was set up directly in the 96-well plate where antigen at 2 µM concentration was pre-incubated with IgG at a final concentration of 2 mg/mL to which parasitized red blood cells were then added as per the normal one-cycle GIA protocol. Controls for this assay were BSA as a negative control

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for antigen reversal and untreated IgG (cohorts 1, 2, 3 & 8) to calculate GIA without reversal, as well as normal rat IgG (NRS) at 2 mg/mL for calculation of relative growth inhibition.

Interpretation of the findings was confounded by high GIA activity in the negative control samples – BSA, BSA + NRS and the antigen-only treatments, RH5, CyRPA and RIPR (in green outline) (Figure 4.5-3). The untreated parasite controls (3D7) were assigned 100% growth. Normal rat IgG-treated wells (purified from normal rat serum (NRS), Invitrogen) at a final concentration of 2 mg/mL (NRS) showed a slight growth inhibitory effect, as seen in previous assays. Monoclonal antibody 1G12 against RIPR was used as a positive control for the assay (at 1 mg/mL) and induced a GIA of 55% as expected.

In this assay the BSA negative control antigen as well as the RH5, CyRPA and RIPR antigens caused significant growth inhibition when added at 2 µM concentration directly to the assay wells. Cohort 1 sera represented a minipool of rat sera immunized with RH5.1, cohort 2 CyRPA, cohort 3 RIPR and cohort 8 RCR complex (in a 1:1:1 mass ratio). While significant GIA is observed with these cohorts in line with previous GIA assays, the reversal effect of adding specific antigen was not significant. A slight increase in parasitemia relative to cohort-only wells indicated that GIA activity of cohort 2 was slightly reversed by the presence of CyRPA and cohort 3 by RIPR, but the results are difficult to interpret in the light of the ‘non-specific’ inhibition cause by antigens alone.

Since a clear interpretation of these results was not possible, the experiment was repeated (this time without the BSA control). Results were similar to the first experiment, with GIA activity in the cohort IgG only wells as seen in previous standard GIA experiments but with significant inhibitory activity in the antigen-only wells also (Figure 4.5-4). The reasons for this non-specific inhibition are not clear. The antigens were dialyzed into sterile RPMI medium and then filter sterilized after dialysis and added so that a final concentration of 2 µM was obtained in the assay, ruling out ‘contamination’ with non-sterile medium. A titration experiment of antigen only to determine the level at which no inhibition is observed and then to test this level of antigen for GIA reversal ability was suggested by Leidos. However, due to time constraints, WEHI opted to try the antigen depletion method.

Figure 4.5-3. GIA reversal by antigen co-incubation. A one cycle GIA assay was performed with IgG from minipools of 6 rats per cohort at a final concentration of 2 mg/mL (cohorts 1, 2, 3 and 8 immunized with RH5.1, CyRPA, RIPR and RCR respectively). Results are expressed in terms of percent

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parasite growth, with untreated 3D7 set as 100% growth. Antigen-only treatments are outlined here in green. Data were generated in a single experiment with the bars of the histogram representing the mean relative GIA ± standard deviations observed in triplicate wells.

Figure 4.5-4. GIA reversal by antigen co-incubation - trial #2. Incubation with antigen alone caused significant growth inhibitory activity (outlined in green) relative to untreated controls. No reversal of inhibition when IgG incubated with antigen (outlined in blue and orange).

IgG Depletion Studies A second methodology was suggested by WEHI that involved depleting antigen-specific IgG in the post-immunization cohort IgG preparations by passing the IgG over a column made with antigen bound to affinity resin. This treatment would circumvent the confounding issue of having high concentrations of antigen directly in the GIA assay. Note that the data described below were received by Leidos recently and details regarding the RCR sera used in these experiments and other details have been requested from WEHI.

For RH5.1 and CyRPA, 50 μg of antigen was bound to C-tag resin and for RIPR, 50 μg of antigen was bound to Streptactin resin. The respective IgG minipool from cohort 1, 2 or 3 was passed over these columns three times to deplete specific reactivity. In a separate experiment, the IgG from cohort 1 (RH5) was passed over empty resin (C-tag) to determine non-specific depletion. The results are shown in Table 4.5-4, Table 4.5-5 and Figure 4.5-5.

While the pre-treatment GIA values are somewhat lower than seen previously for the RH5 minipool, there was a relatively small effect of the “no antigen” empty column treatment (Table 4.5-4). This indicates there is negligible depletion of the parasite neutralizing antibodies by the C-tag column resin alone.

Table 4.5-4 GIA of RH5 minipool sera post-column depletion of IgG on empty C-tag resin

Condition % GIA triplicate wells Average GIA

RH5.1 minipool pre-treatment 2 mg/mL 39.91 32.67 37.22 36.60

Post column (no antigen) 30.82 28.55 31.25 30.21

Nearly complete reversal of GIA activity was observed in both experimental replicates for the single-antigen IgG cohorts pre-treated over antigen-affinity resin (Table 4.5-5) relative to untreated IgG controls (in black), whereas a lower degree of GIA reversal was observed by single antigen affinity depletion of IgG from RCR-immunized animals. This could be due to the fact that the RCR IgG contains antibodies against individual proteins of the RCR complex as well as the complex as a whole.

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Figure 4.5-5 Antigen-specific depletion of IgG resulted in a reversal of parasite growth inhibitory activity. Mean +/- standard deviations are shown.

4.5.1.6 RCR ELISA In Q1 FY2021, assessment of W3 sera by ELISA using the Oxford RCR complex as the coating antigen (2 μg/mL) was completed. Serum from individual terminal bleeds was diluted threefold starting at 1/100 dilution to generate 12-point curve and OD1 titers were calculated using R Studio (Figure 4.5-6). Previously collected ELISA data for W3 sera with the individual antigens is shown in Figure 4.5-7. These data indicate that the antibody response generated by immunization with RH5 alone are predominantly directed against epitopes found on RH5 as single immunogen. The immune response elicited by RH5 alone reacts to a significantly lower extent towards the RCR complex than the anti-RCR complex immune response elicited by combining it with other antigens (double and triple combinations). When immunized in combination (double or triple combinations), the immune response is lower towards RH5 compared to the response achieved when immunized as single antigen. A similar trend is exhibited by CyRPA as well but to a lesser extent and with the one exception noted in the legend for Figure 4.5-6. On the other hand, RIPR appears to elicit a similar level of immune responses against the RCR complex regardless of whether it is immunized as a single antigen or in combination with other antigens. Note that complete statistical analysis of these data is pending but the Kruskal-Wallis multiple comparisons of independent samples was applied to determine significant differences. The median OD1 titer of the RH5 cohort was significantly lower than that of all the other cohorts. The OD1 titer of CyRPA was significantly different compared to all the other cohorts with the exception of the RH5+CyRPA cohort. There were no significant differences between any of the RIPR cohorts.

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penultimate study (previously Milestone 6) and to change Milestone 5 to a dose-ranging study using single antigen VLPs, respectively. Study O1 demonstrated that dosing at 2 µg RH5.1 gave a maximal response (ELISA and GIA) while dosing at 2 µg CyRPA and RIPR gave suboptimal responses. From these results and discussion during an IPT Meeting held on November 13, 2019, single antigen doses were selected for Study O2 as follows: 2 µg RH5.1, 20 µg CyRPA, 20 µg RIPR, and 20 µg RCR complex for Matrix-M formulation and 2 µg RH5.1, 2 µg CyRPA, 2 µg RIPR, and 20 µg RCR complex for DPX formulation. The results of the continuing work on Milestones 2 through 5 completed in FY2021 are reported below.

Table 4.5-6. Overview of the RCR Complex Vaccine Development Project Plan-Oxford

Phase Milestone Activities

Study O1 1. Dose-Ranging Study • Humoral response assessments • GIA assessment • Generation of quantitative ELISA control sera

Study O2 2. Assess immunogenicity of

individual proteins and double/triple protein mixtures

• Humoral response assessments • GIA assessment

Assay Development

3. Establish quantitative method to assess antigen-specific IgG levels for the RCR proteins

• Calibration-free concentration analysis method • Affinity purification method • Technology transfer to WEHI

VLP Development

4. Generation and characterization of single antigen VLPs and RCR complex VLPs

• Generation and expression of RH5-SpyTag, CyRPA-SpyTag, and RIPR-SpyTag

• Conjugation of single antigen-SpyTag or RCR-SpyTag complexes to HBsAg-SpyCatcher VLP carrier

• Protein purification and characterization

Study O3 5. Dose-Ranging Study (VLPs) • Humoral response assessments • GIA assessment

As noted in the appropriate sections below, USAID has approved the VLP development, the Study O2 design (Table 4.5-7 and Table 4.5-8), and the Study O3 design (Table 4.5-9).

4.5.2.1 Immunogenicity Study of Individual Proteins and Double/Triple Protein Mixtures (Study O2)

An IPT Meeting was held on November 13, 2019 of Q1 FY2020 to discuss the ELISA and GIA data obtained from Study O1. In this meeting USAID decided to include five additional cohorts (RH5.1 + DPX4, RIPR + DPX4, CyRPA + DPX4, Reconstituted RCR complex + DPX4, DPX4 only) in Study O2 to allow for a comparison between DPX4 and Matrix-M and one cohort for the proposed protective domain of RIPR, RIPR (EGF 5-8). The resulting study design and antigen doses are shown in Table 4.5-7 and Table 4.5-8, respectively.



-2 0 28 42 56 70

1 RH5.1 + Matrix-M IM 6, Wistar

Pre-

blee

d

√ √

Tes

t bl

eed √

Ter

min

al B

leed

2 CyRPA + Matrix-M IM 6, Wistar √ √ √

3 RIPR + Matrix-M IM 6, Wistar √ √ √

4 RH5.1 + RIPR + Matrix-M IM 6, Wistar √ √ √

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-2 0 28 42 56 70 5 RH5.1 + CyRPA + Matrix-M IM 6, Wistar √ √ √

6 RIPR + CyRPA + Matrix-M IM 6, Wistar √ √ √

7 RH5.1 + CyRPA + RIPR + Matrix-M Equivalent Molar Ratio IM 6, Wistar √ √ √

8 RH5.1 + CyRPA + RIPR + Matrix-M Single Antigen Doses Combined IM 6, Wistar √ √ √

9 Reconstituted RCR + Matrix-M IM 6, Wistar √ √ √

10 Matrix-M Only (Baseline Control) IM 6, Wistar √ √ √

11 RH5.1 + DPX4 IM 6, Wistar √ √ √



14 Reconstituted RCR + DPX4 IM 6, Wistar √ √ √

15 DPX4 Only (Adjuvant Control) IM 2, Wistar √ √ √

16 PfRIPR (EGF 5-8) + Matrix-M IM 6, Wistar √ √ √

USAID approved the design of Study O2 on November 18, 2019 of Q1 FY2020. Note that a second approval was obtained from USAID on December 6, 2019 to account for a reduction in the RIPR (EGF 5-8) antigen dose. The dose was decreased from 6.67 µg/injection to 3.97 µg/injection to account for the difference in the molecular weight of soluble RIPR (EGF 5-8) versus the molecular weight of RIPR (EGF 5-8) plus the fusion/expression partner. The immunization schedule for Study O2 was initiated in January and completed in March of Q2 FY2020. As a result of COVID-19, all sera samples were stored at Noble Biosciences until the samples could be safely shipped and received. In February 2020, Oxford indicated concern regarding the current timeline for completion using individual samples as described in the project plan and developed timelines for the GIA assays using individual samples as well as pooled samples. These timelines were shared with USAID on February 4 and, following further discussion, USAID requested the use of individual samples and not pooled samples for all GIA assays. Due to the facility closure in response to the COVID-19 epidemic, this analysis was delayed. The laboratory reopened in early August with two benches available for this project’s research. Sera from Noble was received by Oxford on August 2, 2020 and analysis using the standard ELISA began in September 2020. GIA analysis was started in November of 2020.




1 RH5.1 + Matrix-M 2 µg 2 µg

2 CyRPA + Matrix-M 20 µg 20 µg

3 RIPR + Matrix-M 20 µg 20 µg

4 RH5.1 + RIPR + Matrix-M 2 µg 20 µg 22 µg

5 RH5.1 + CyRPA + Matrix-M 2 µg 20 µg 22 µg

6 RIPR + CyRPA + Matrix-M 20 µg 20 µg 40 µg

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7 RH5.1 + CyRPA + RIPR + Matrix-M Equivalent Molar Ratio 5.36 µg 3.51 µg 11.11 µg 20 µg

8 RH5.1 + CyRPA + RIPR + Matrix-M Single Antigen Doses Combined 2 µg 20 µg 20 µg 42 µg

9 Reconstituted RCR + Matrix-M 20 µg 20 µg

10 Matrix-M Only (Baseline Control) NA

11 RH5.1 + DPX4 2 µg 2 µg

12 CyRPA + DPX4 2 µg 2 µg

13 RIPR + DPX4 2 µg 2 µg

14 Reconstituted RCR + DPX4 20 µg 20 µg

15 DPX4 Only (Adjuvant Control) NA

16 PfRIPR (EGF 5-8) + Matrix-M 3.97 µg 3.97 µg

Oxford presented preliminary results of analysis from studies O2 and O3 at an IPT meeting on November 10, 2020, and additional results were provided to USAID on December 15, 2020. The preliminary results are summarized below. When formulated with Matrix-M, the levels of PfRH5.1 antibody titers in Study O2 are consistent with titers to 2 µg of RH5.1 in Study O1 (Figure 4.5-10). This indicates the results between the two studies can be compared with reasonable confidence.

Figure 4.5-10. Standard ELISA titers for RH5.1 Comparing Results from Study O1 and Study O2. Bleed from Day 70 using cohort vaccinated with RH5.1 at 2 µg in Matrix-M. Each symbol represents an individual animal.

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Figure 4.5-11. Study O2 Standardized ELISA for Antibody to RH5.1. Results for bleeds taken on Day 42 (A) and Day 72 (B) are presented. Each symbol represents an individual animal. Dotted line indicates median result from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to RH5.1 when RH5.1 was administered alone and in combination with RIPR and CyRPA are presented in Figure 4.5-11. When administered with Matrix-M, RH5.1 alone resulted in the highest titers to RH5.1 on both Days 42 and 70, and combining RH5.1 with CyRPA did not decrease the response. The response to RH5.1 was decreased in the presence of RIPR. This was observed when RIPR was combined with RH5.1 or when it was combined with CyRPA in the different assortments of the three-protein combination (equimolar amount, combined total protein, or in RCR complex). When the antigen was administered with DPX4, either alone or in the RCR complex, the immune response was lower than the same antigen administered in Matrix-M.

Figure 4.5-12. Study O2 Standardized ELISA for Antibody to CyRPA. Each symbol represents an individual animal. Dotted line indicates median from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to CyRPA when CyRPA was administered alone and in combination with RH5.1 and RIPR are presented in Figure 4.5-12. When using Matrix-M, increasing the dose of CyRPA from 2 μg (Study O1) to 20 μg (Study O2) results in higher anti-CyRPA IgG titers after the second (Day

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28) and third (Day 56) administration of antigen (response on Day 42 and Day 70, respectively). CyRPA alone resulted in higher average titers relative to combination with RH5.1 and/or RIPR. When in combination with both RH5.1 and RIPR, the average immune response to CyRPA was less than the immune response to CyRPA when RH5.1 or RIPR were the only additional proteins. When administered with DPX4, the immune response to CyRPA alone at Day 70 was similar to the response when CyRPA was administered with Matrix-M. The immune response to the RCR complex in DPX4 was higher on days 42 and 70 relative to the response when administered with Matrix-M.

Figure 4.5-13. Study O2 Standardized ELISA for Antibody to RIPR. Each symbol represents an individual animal. Dotted line indicates median from Study O1. Antigens were formulated with Matrix-M unless it is indicated the antigen is formulated with DPX4. CyRPA and RIPR were each at 20 µg when formulated with Matrix-M, and at 2 µg when formulated with DPX4.

The immune response of rats to RIPR when RIPR was administered alone and in combination with RH5.1 and CyRPA are presented in Figure 4.5-13. Increasing the dose of RIPR in Study O2 did not result in higher antibody responses than were observed in Study O1. When using Matrix-M, the immune response to RIPR reach maximal levels by Day 42 after the second administration of antigen (Day 28) when either administered alone or in combination with RH5.1 and/or CyRPA. The third administration of vaccine did not increase the immune response on Day 70. RIPR responses were unaffected by administering combinations of RH5.1 or CyRPA. When administered with DPX4, the results parallel those seen with Matrix-M, although the average immune response was slightly lower using DPX4.

Oxford established a new parasitology laboratory in mid-November to support GIA. Analysis of samples from Study O2 have been completed and the results are provided below.

Figure 4.5-14. Study O2 GIA of IgG from Rats vaccinated with Adjuvant (with no antigen). Each symbol represents an individual animal. The “Bought Negative” are results using naïve rat IgG purchased from a commercial supplier. The Matrix-M and DPX4 are the results using rat IgG isolated

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from animals immunized with Matrix-M or DPX4, respectively. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Results of analysis of IgG from rats vaccinated with either Matrix-M or DPX4 without antigen are presented in Figure 4.5-14. For reasons that are not clear three of six rats immunized with Matrix-M alone show a strong grown inhibition. However, an immune response indicated by the ELISA did not demonstrate a titer to the three proteins in these animals. This test will be repeated. Also, one rat of two immunized with DPX4 alone showed strong growth inhibition. In this case the animal demonstrated a relatively high titer to RIPR in the ELISA (Figure 4.5-13). Since both animals were immunized using DPX4 from the same vial, it is not obvious why one of two rats demonstrated immunity in both the ELISA and GIA, and possible explanations will require discussion with Noble.

For animals vaccinated using Matrix-M, the Study O2 GIA results are consistent with the Study O1 results when comparing sera from rats vaccinated with 2 µg of RH5.1 in Matrix-M (Figure 4.5-15). This indicates the GIA results between the two studies can be compared with reasonable confidence. In Study O2, administration of RH5.1 with DPX4 is of similar potency to RH5.1with Matrix-M. This is not predicted based upon the ELISA, which indicates administration of RH5.1 with Matrix-M provided higher ELISA titers relative to DPX4. Figure 4.5-16 shows that increasing the dosage of CyRPA from 2 µg to 20 µg demonstrates a trend of increasing the potency in the GIA and follows the trend of the ELISA response. However, the results of the two concentrations overlap and neither consistently results in 50% inhibition of parasite growth. Administering 2 µg of CyRPA with DPX4 shows a trend toward increased potency of the response relative to administration with Matrix-M. However, results of the two formulations overlap and the DPX4 formulation results in 50% inhibition only at the highest concentration of IgG, which was not tested in the Matrix-M formulation. For RIPR increasing the concentration or administration with DPX4 did not increase potency of the response (Figure 4.5-17 and Figure 4.5-18), which parallels the ELISA results. The GIA results from rats immunized with either RIPR or the fragment RIPR (EGF 5-8) are shown in Figure 4.5-19. Potency of the response to RIPR (EGF 5-8) trends higher than the potency of the response to RIPR. A summary of the GIA results using IgG of rats in cohorts 1-3 of Study O2 is presented in Figure 4.5-20, which presents a comparison of the potency of the response in rats immunized with either RH5.1, RIPR, or CyRPA administered with Matrix-M. This result indicates a hierarchy of response potency to be RH5.1 >RIPR =CyRPA.

Figure 4.5-15. GIA for RH5.1 Comparing Results from Study O1 and Study O2, and formulation. Bleeds are from Day 70 using cohort vaccinated with RH5.1 at 2 µg comparing Study O1 (black) and O2 (blue) results (left panel). The right panel provides the results of 2 µg in Matrix-M (black) or DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

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Figure 4.5-16. GIA for CyRPA Comparing Results from Study O1 and Study O2. Left panel is bleed from Day 70 using cohort vaccinated with CyRPA in Matrix-M using 2 µg (black) or 20 µg (blue) CyRPA. Right panel is bleed from Day 70 using cohorts immunized with 2 µg CyRPA formulated with Matrix-M (black) or with DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-17. GIA for RIPR Comparing Results from Study O1 and Study O2. Bleed from Day 70 using cohort vaccinated with RIPR at either 2 µg or 20 µg in Matrix-M. Each symbol represents an individual animal. Results from Study O2 is blue and Study O1 is black. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-18. GIA for RIPR Comparing Results from Study O1 and Study O2, and formulation with Matrix-M or DPX4. Left panel compares the bleeds from Day 70 using cohort vaccinated with RIPR at 2 µg in Matrix-M (black) in Study O1, or with 2 µg in DPX4 (blue) from Study

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O2. Right panel provides GIA results from Study O2 evaluating 20 µg RIPR with Matrix-M (black) compared to 2 µg formulated with DPX4 (blue). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-19. GIA Comparing Potency of GIA to RIPR and RIPR (EGF 5-8). Results from Study O2 bleeds on Day 70 using cohorts vaccinated with RIPR at 2 µg (black), or with 3.97 µg RIPR (EGF 5-8) in Matrix-M. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Figure 4.5-20. Comparison of GIA for RH5.1, RIPR, or CyRPA from Study O2. Bleed from Day 70 using vaccines containing Matrix-M. CyRPA is at 20 µg (orange), RIPR is at 20 µg (blue), and RH5.1 is at 2 µg (black). Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

Using the GIA response to RH5.1 as a comparator, the potency of combinations of RH5.1, CyRPA, and RIPR are presented in Figure 4.5-21. In all the combinations that contain RH5.1 the potency of the mixture of proteins overlaps with the potency of RH5.1 when used as the sole antigen (Panels A, B, D, E, and F). However, when the antigen combination is only CyRPA and RIPR, the potency is less than that seen with RH5.1 alone (Panel C). The potency of the response to the vaccination with CyRPA and RIPR combination is similar to the response when the vaccine is either CyRPA or RIPR alone (data not provided). This result parallels the response seen with the individual antigens demonstrating a hierarchy of response with RH5.1>CyRPA and RIPR (Figure 4.5-20). This indicates the combination of antigens do not reduce the immune response to RH5.1. The results do not indicate a synergistic effect with the RH5.1 in combination with CyRPA and RIPR; however, the potency of the response at high concentrations of IgG does show a trend to a higher GIA response when all three antigens are used in

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the vaccine (combined either as total protein or in equimolar amounts) (Panels E and F). This trend is not seen when the RCR complex is used as the antigen (Panel D). Similarly, when DPX4 is used in combination with the RCR antigen, there is not an increase in the potency of the response (Figure 4.5-22)

Figure 4.5-21. GIA Response to combinations of RH5.1, RIPR, and CyRPA Compared to RH5.1 in Study O2. Bleed from Day 70 using vaccines containing Matrix-M. Response to RH5.1 is indicated in black, and response to combination of antigens are blue. RCR is used at 20 µg. In the analysis of the R+C+R equimolar (Panel E), RH5.1 is at 5.36 µg, CyRPA is at 3.51 µg, and RIPR is at 11.11 µg. In all other samples RH5.1 is at 2 µg, CyRPA is at 20 µg and RIPR is at 20 µg. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

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Figure 4.5-22. GIA Response to RCR complex Compared to RH5.1 in Study O2. Bleed from Day 70 using RH5.1 vaccine at 2 µg containing Matrix-M or RCR vaccine at 20 µg containing DPX4. Response to RH5.1 is indicated in black, and response to RCR is blue. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

4.5.2.2 Development of Quantitative ELISA In conversations and approval of the FY2019 annual work plan, USAID indicated support of pursuing the development of quantitative ELISAs at Oxford with subsequent assay transfer to WEHI. ELISA development was initiated during Q1 FY2020 and consists of three steps. Step 1 involved setting up the standardized ELISA for RH5.1, RIPR, and CyRPA and was completed in Q1 FY2020. Step 2 is development of calibration-free concentration analysis (CFCA) for RH5.1, CyRPA, and RIPR antigens. Step 3 is Linear Regression Analysis to develop a conversion factor to convert the standardized ELISA titer results to the concentration of IgG.

Finalized SOPs for each candidate antigen (RH5.1, CyRPA, and RIPR) were received from Oxford and circulated to USAID on March 25, 2020. Due to facility closure, all research activities remained on hold during Q3 FY2020 and resumed once the lab reopened in August. Despite this closure, a MTA between WEHI and Oxford was fully executed and, as a first step in the technology transfer of the standardized ELISA developed by Oxford, the SOPs and Gen5 protocols were provided to WEHI for review on June 15, 2020. On September 22 WEHI received from Oxford rat antisera to RH5.1, CyRPA, and RIPR (400 µL each), and 0.5 mg of each antigen. This is enough material to support analysis of Studies W2 and W3, with extra antigen and antisera to support continued use of the standardized ELISA beyond the MVDP. In addition, this shipment included 1.5 mg of C-tagged CyRPA for the GIA reversal analysis. WEHI is setting up to perform the standardized ELISA using the finalized SOPs, but did not have the alkaline phosphatase conjugate or substrate. WEHI is in the process of acquiring these reagents.

The CFCA is being developed. The approach to immobilize the antigen using antibodies to the C-tag were unsuccessful. The approach using an oligonucleotide labeled streptavidin immobilized on a chip coated with a complementary oligonucleotide was successfully used to capture biotinylated RH5.1 protein. Oxford has produced streptavidin-SpyCatcher as a first step to produce biotinylated antigens (RH5.1, RIPR, and CyRPA) for the CFCA. To develop a preliminary conversion factor for ELISA results to RH5.1, samples were run twice and averaged. The concentration of IgG verses the anti-RH5.1 ELISA Units (AU) were plotted and linear regression was performed to generate the conversion factor for antibody to RH5.1 using either sera or purified IgG (Figure 4.5-23). The results from each plot are similar, providing a preliminary conversion factor to convert ELISA results to µg/ml of IgG. Oxford will continue analysis using the purified IgG as a cleaner approach to quantitate the IgG specific ELISA. Additional data points will be evaluated in the RH5.1 plot, and work will progress to analysis using RIPR

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and CyRPA. Oxford has indicated that, due to the limited time remaining on the MVDP contract, they will not have time to develop and perform the assay for the RCR complex.

Figure 4.5-23. Plot of Antibody Concentration Verses Anti-RH5.1 ELISA Units. Left panel is the result using rat antiserum to RH5.1. The right panel is the result using rat IgG to RH5.1 purified using protein L.

4.5.2.3 VLP Development USAID approved the development of SpyCatcher-HBsAg VLPs decorated with single antigens or the RCR complex on October 4, 2018 of Q1 FY2019 and VLP development began in Q3 FY2019. At the conclusion of Q1 FY2020, both N- and C-term CyRPA-SpyTag as well as C-term RH5.1-SpyTag were successfully conjugated to SpyCatcher-HBsAg VLPs. Efforts to produce full length (FL) RIPR VLPs and RCR complex-HBsAg VLPs were not successful and are continuing. Outside of the MVDP contract, but in parallel to the efforts for the production of FL RIPR VLPs, C-term RIPR (EGF 5-8)-SpyTag was successfully conjugated to SpyCatcher-HBsAg VLPs and replaced FL RIPR VLPs in Study O3. For the single antigen VLPs, the conjugation efficiencies were determined to be 67%, 86%, and 44%, for RH5.1, CyRPA, and RIPR (EGF 5-8), respectively. This efficiency represents the percentage of HBsAg monomers that display the candidate antigen. For example, 67% of the HBsAg monomers in a given VLP display RH5.1. Note that formed VLPs contain between 80 and 120 monomers. As such, not all VLPs in a given population will be composed of the same number of monomers.

At the conclusion of Q2 FY2020, FL RIPR Ct SpyTag (Ct = C-terminus) was successfully conjugated to SpyCatcher-HBsAg VLPs, with a conjugation efficiency of 52%. While this was not completed in time for use in Study O3, it did allow development of the RCR complex-HBsAg VLP to continue. An initial attempt to incorporate all three proteins on a VLP was unsuccessful. The facility closure due to COVID-19 prevented further development of the RCR complex-HBsAg VLP until the laboratory reopened. At this time there is not enough protein to attempt the conjugation to the VLP and protein is being produced to repeat the attempt.

4.5.2.4 Dose-Ranging Study – VLPs (Study O3) An IPT meeting was held on November 18 and 22, 2019 of Q1 FY2020 to discuss the study design of Study O3 and included consideration of the available VLPs and what VLP dose would be most appropriate. In light of the results from Study O1, it was decided to modify the original study design to a dose-ranging study before moving forward to assess double and triple VLP combinations. Additionally, due to the tight timeline and difficulty in producing FL RIPR VLPs, it was decided to replace FL RIPR VLPs with RIPR (EGF 5-8) VLPs. Note that all RCR proteins used to generate the VLPs for this study contain a SpyTag on the C-terminus. The resulting study design is shown in Table 4.5-9 and USAID approved the design on December 11, 2019 of Q1 FY2020. In Study O3, the antibody to HBsAg-RH5.1, HBsAg-CyRPA, and HBsAg-RIPR (EGF5-8) VLPs were evaluated for immunogenicity as well as for parasite growth inhibition.

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Table 4.5-9. Study O3 – VLP Dose-Ranging Study


-2 0 14 28 42 56 70

1 HBsAg-PfRH5.1 (2 µg) + Matrix-M IM 6, Wistar

Pre-

blee

d

√

Tes

t Bl

eed

√

Tes

t bl

eed

√

Ter

min

al B

leed











16 HBsAg-SpyCatcher (20 µg) + Matrix-M IM 6, Wistar √ √ √

The immunization schedule for Study O3 was initiated in January and completed in March of Q2 FY2020. As a result of COVID-19, all sera samples were stored at Noble Biosciences until the samples could be safely shipped and received. Due to the COVID-19 epidemic, the Oxford facility was closed and this analysis was delayed. The laboratory reopened in early August with two benches available for this project’s research. Sera from Noble was received by Oxford on August 21. Analysis using the standard ELISA began in September, and GIA analysis began in November 2020.

During an IPT meeting on November 10, 2020, Oxford presented preliminary results of analyses from studies O2 and O3, and additional results were provided to USAID on December 15, 2020. The preliminary results of the standardized ELISA for Study O3 are summarized below. Results for bleeds taken on Day 42 and Day 72 are presented. The immune response to RH5.1 peaked by Day 42, but the immune response to 2 µg and 0.2 µg RH5.1 decreased by Day 70 (Figure 4.5-24). By Day 70 the immune response to RH5.1 in study O3 was over 10-fold less than the immune response seen in study O1 and study O2. For CyRPA and RIPR the immune response to 0.2 to 20 µg peaked by the day 42 bleed after the second immunization with little change in the average immune response by day 70, which was after the third immunization (Figure 4.5-25 and Figure 4.5-26). For CyRPA, the immune response on Day 70 to 20 µg VLP was slightly higher than the 20 µg dose of CyRPA in Study O2, and the immune response to the 2 µg VLP was slightly higher than the 2 µg dose of CyRPA in Study O1 (Figure 4.5-25). The immune response to the 20 µg dose of RIPR on Day 70 was slightly lower than the response to the 20 µg dose of RIPR and the 3.97 µg dose of RIPR (EGF 5-8) in Study O2 (Figure 4.5-26).

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Figure 4.5-24. Study O3 Standardized ELISA for Antibody to RH5.1. Each symbol represents an individual animal. Dotted line indicates median result from Study O1 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of RH5.1 is shown on the x-axis.

Figure 4.5-25. Study O3 Standardized ELISA for Antibody to CyRPA. Each symbol represents an individual animal. Dotted line indicates average result from Study O1 or Study O2 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of CyRPA is shown on the x-axis.

Figure 4.5-26. Study O3 Standardized ELISA for Antibody to RIPR (EGF 5-8). Each symbol represents an individual animal. Dotted line indicates media result from Study O2 for the same day of the bleed. Antigens were formulated with Matrix-M and the concentration of RIPR (EGF 5-8) is shown on the x-axis.

GIA results for Study O3 are only available for RH5.1 (Figure 4.5-27). Interestingly, while the ELISA results indicate a lower immune response to the RH5.1-VLP relative to soluble RH5.1, the GIA for the IgG to the soluble RH5.1, and the RH5.1-VLP at dosages of 2 µg and 0.2 µg are similar, with the IgG to

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the RH5.1-VLP trending to higher GIA than the IgG to the soluble RH5.1. This suggests the immune response to the RH5.1-VLP may be a higher quality than the response to soluble RH5.1.

Figure 4.5-27. GIA for Study O3 RH5.1 and Comparing Results with those from Study O2. Bleeds are from Day 70. Cohort vaccinated with RH5.1-VLP at 0.02 µg to 2.0 µg from Study O3 are presented in the left panel. The right panel overlays these results with the GIA results from Study O2 for IgG to soluble RH5.1 (in black) at vaccinated using 2 µg. Each line represents and individual rat, each point represents the mean of three replicates, and the bars depict the standard error.

4.5.2.5 Schedule: RCR Complex Project Oxford The timeline for RCR Complex Project Oxford is shown in Table 4.5-10. Due to the impact of the COVID-19 epidemic, the Oxford laboratories were closed March through July 2020, and reopened in August. The timeline for the project was extended to enable completing of as much of the RCR Complex project as possible with the funds that were already committed to the project.

Table 4.5-10. Project Timeline

4.6. RH5.1 HUMAN MAB IDENTIFICATION AND DEVELOPMENT PROJECT: VIN KOTRAIAH

The RH5.1 human mAb identification and development project was initiated in FY2019 and will continue into FY2021. An overview of the development plan for this project is provided in Table 4.6-1 followed by a brief summary of the proposed project. A priority list of 20 samples was approved by USAID on April 1, 2019 (Table 4.6-2) and progress to date is described below.

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Table 4.6-1. Overview of the Development Plan for the RH5.1 Human mAb Isolation and Development Project

Phases Milestones Activities

B Cell Isolation and mAb Development

B Cell Isolation and Cloning

• Single B cell isolation (memory B cells - mBC) or plasmablasts)

• Reverse transcription and paired heavy and light chain nested PCR

• DNA purification and cloning into heavy and light chain vectors

mAb Expression and Purification

• Transfection of heavy and light chain vectors into HEK293 cells

• Harvest of supernatant and screening by ELISA • Affinity purification of antibodies from supernatant

mAb Sequencing • Sequencing of heavy and light chain variable regions • Determination of sequence maturation and germline alleles

mAb Screening

ELISA Titer and Protein Mapping

• Determination of ELISA titers • Protein region mapping using a panel of RH5 variants

GIA • Growth Inhibition Assay (using pLDH method) Epitope Similarity Screen

• Determination of similarity of B cell epitopes recognized by antibodies using competition assay and SPR/BLI

mAb Downselection • Monoclonal antibody downselection

mAb Characterization

mAb Affinity • Determination of antibody affinity by SPR mAb Inhibition of Complex Formation

• Assessment of inhibitory activity of antibodies on RCR, RH5-P113 and RH5-Basigin complexes

mAb Downselection • Selection of monoclonal antibodies for structural studies

R5.016 Immunogen Design (optional)

Computational Assessments

• Identification of sequence variants of the immunogen that are stable and have the right conformation

Production of Select Immunogen Designs • Epitope grafting and production of selected immunogens

Immunogen Downselection • SPR screening for binding to R5.016 mAb

R5.016 Immunogen Production (optional)

Production of Immunogen • Production and QC of down-selected immunogen

Coupling of Immunogen to VLP • Conjugation of down-selected immunogens to VLPs

R5.016 Immunogen Testing (optional)

Formulation of Immunogen: VLP

• Selection and procurement of adjuvant • Formulation of immunogen

Rat Immunization • Rat immunizations with RH5.1 comparator • Collection of sera

Immunogenicity and GIA assessments

• Humoral response and GIA activity with R5.016 mAb comparator

New mAb Structure Determination (optional)

Antibody Production • Recombinant antibody expression and purification • Fab fragment generation

Crystal Screening • Screening for Fab:RH5 co-crystals • Cryoprotection of crystals

Crystal Structure Determination

• X-ray diffraction studies • Model building and refinement • Epitope delineation

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Oxford proposed identification of human RH5 mAbs and conduct of a B cell analysis using VAC063 clinical samples. This project will involve surveying the B cell and Ab repertories from vaccinated subjects utilizing both B cell cloning and serum immunomics techniques in order to understand the epitopes recognized by human anti-RH5 sera, how these epitopes might contribute to functional GIA, and gather information on differences in repertoire based on dose/immunization regimen/CHMI. As part of this effort, Oxford also proposed evaluating novel immune mechanisms of protection based on the sterile protection and reduced parasite growth rate seen with unvaccinated individuals after CHMI. These activities have been incorporated into the project proposal and will be expanded on during the course of project reporting.

The initial project schedule provided by Oxford (included in the Project Plan) had a start date of December 2018 and a completion date in May 2020. Approval of the Project Plan (regular elements only) was received from USAID on January 29, 2019. Per USAID request, Leidos directed Oxford to perform a sample selection inclusive of the RH5.1 clinical trial GIA data. Oxford conducted the same in March 2019, once the RH5.1 clinical study GIA data were available for analysis. Approval of sample selection by USAID was received in early Q3 FY2019. Based on this updated timeframe, Leidos requested a revised schedule from Oxford, shown in Figure 4.6-1. Note that the updated project schedule begins in April 2019 and a completion date in September 2020, extending beyond the MVDP POP end date. Leidos recommended holding an IPT meeting in Q2 FY2020 to determine the project stopping point, as needed based on progress by Oxford. However, due to COVID-related closures of lab facilities at Oxford, the project was delayed and is now expected to be completed in Q2 FY2021 well within the new MVDP POP end date.

Figure 4.6-1. Project Timeline Received from Oxford

The priority list of 20 samples approved by USAID on April 1, 2019 is shown in Table 4.6-2. Of note is that the Oxford group aims to deliver roughly 10 RH5-specific mAbs from each of these 20 samples and characterize them.

In the “Group” column of Table 4.6-2, the samples are color coded by the VAC063 Group number to which they belong. Six samples drawn from Group 7, 10 drawn from Group 5 and 4 from Group 3 constitute the total of 20 approved samples.

In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion. The color yellow indicates that the volunteer was selected on the basis of in vivo growth inhibition (IVGI) only. The green colored cells indicate that the volunteers were selected on the basis of IVGI and ELISA, Avidity or GIA. The blue colored cells indicate that the volunteers were selected on the basis of ELISA, avidity or GIA. Note that Oxford’s slide summary with the ELISA, avidity, GIA and IVGI data for all the VAC063 volunteers were provided to USAID in Q2 FY2019.

On June 17, 2019, Oxford notified Leidos that volunteer 01-809 (01-032) from Group 7 with a priority number of 7 (Table 4.6-2) had withdrawn consent and that Oxford could no longer use this volunteer’s samples. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. The consent withdrawal and proposed replacement sample were communicated to USAID in the bi-weekly meeting on June 19, 2019.

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Table 4.6-2. Priority list of samples with their VAC063 Group number and Volunteer IDs are shown. Availability of PBMC samples from the indicated day of collection are also shown. The time-points at which the ELISA, Avidity and in vitro GIA data are available are also shown. Lastly, the IVGI is shown for the samples from Groups 5 and 7.

C-1 - day before challenge; C+28 - 28 days after challenge; DOD – day of diagnosis; IVGI was calculated as % reduction in parasite multiplication rate (PMR) in individual vaccinees versus the mean PMR of the control group. Volunteer 01-809 (01-032) from Group 7 with a priority number of 7 withdrew consent in June 2019. Oxford proposed use of PBMCs collected on day before challenge from volunteer 01-028 (Group 5 - priority number of 21) as a replacement. In the “Group” column, the samples are color coded by the VAC063 Group number to which they belong. In the “Volunteer” column, the volunteer IDs are color coded by the selection criterion: yellow=selection based on in vivo growth inhibition (IVGI) only; green=selection based on IVGI and ELISA, Avidity or GIA; blue=selection based on ELISA, avidity or GIA.

Due to COVID related facility closure and restrictions on non-COVID work, all research activities on this project were suspended during Q3 FY2020. Activities resumed on a limited basis in September of Q4 FY2020 and proceeded at a brisk pace in Q1 FY2021. However, due to COVID-related delays, it was agreed that no attempts will be made to isolate additional mAbs from the Priority Samples. Instead, the focus was shifted towards completing the characterization of mAbs already isolated from twelve of the Priority Samples. USAID informed Leidos that it concurred with this proposed change in focus via email on October 13, 2020. Subsequently, progress was made on completing the binding

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characterizations, single-point and dose-titration GIAs. The current status at each of the major steps in the project is shown in Table 4.6-3.

Table 4.6-3. Status of Samples that have Completed Processing for Each Project Step

Step Number of samples RH5.1 ELISA with transfection supernatants / Number of binders 290 / 126

Titration of purified mAbs in RH5.1 ELISA 126 Binding of purified mAbs with different RH5 antigens by ELISA 126 Number of mAbs tested in single-point GIA / Number of mAbs with >50% inhibition 126 / 70

Number of mAbs tested in dose titration GIA 70 With the completion of the ELISA and GIA analyses in FY2021 Q1, Oxford has begun to analyze the data from all the mAbs. Some of these data were presented by Oxford in an IPT meeting with USAID on November 04, 2020. As previously described, the mAbs were characterized in ELISA using four different RH5-based plate antigens (Figure 4.6-2).

Figure 4.6-2. RH5 plate antigens used in ELISA. Apart from RH5.1 (native and denatured) and the N-terminus alone antigen, an additional antigen called RH5.2 in which the N-terminus and intrinsic loop are deleted was used as a plate antigen. Of note is that Oxford also uses the notation “SV3” or “delta NL” when referring to the RH5.2 antigen; however, Leidos will use RH5.2 for consistency. A plate antigen called Bundle which displays the epitope of the R5.016 mAb isolated from the VAC057 trial (described in Alanine et al., Cell 2019) on a non-RH5 protein scaffold was also used as plate antigen.

Figure 4.6-3 shows the distribution of the 126 mAbs according to their RH5 binding characteristics. A majority of the mAbs recognized conformational epitopes on RH5.1 and only 8% (10) could bind to linear epitopes (Figure 4.6-3A). Nearly 82% (103 mAbs) bound the SV3 RH5 construct while 2% bound the N-terminus and 3% bound the internal loop region (Figure 4.6-3B). About 13.5% (17) mAbs require further characterization in the ELISA as they did not bind any of the RH5 constructs. Oxford is planning to repeat the ELISA for these 17 mAbs. Additionally, of the 103 mAbs that bound to SV3, 88 bound outside the Bundle region whereas the rest bound within the Bundle region.

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Figure 4.6-3. RH5 binding characteristics of the 126 mAbs. (A). Break-down of mAbs by the conformational or linear nature of their epitopes. (B). Distribution of mAbs according to their binding to different RH5 constructs.

Characterization of 74 mAbs in the single-point GIA was completed in the current quarter, bringing the total of mAbs characterized by this assay to 126 (100% of the mAbs isolated). Of the 126 mAbs tested, 70 (56%) showed GIA at or above 50% at 2 mg/mL. These were further characterized in dilution curves. The IC50s of these mAbs are reported in Figure 4.6-4. Two of the mAbs (BD5 and 8G8) were found to be the most potent human anti-RH5 mAbs isolated to date and have lower IC50 values than R5.016.

Figure 4.6-4. GIA IC50 values for 70 of the RH5 mAbs. The blue line represents the IC50 of the control R 5.016 mAb. 8G8 and the previously described BD5 mAbs have IC50s below that of R5.016.

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In addition, avidity of the mAbs towards RH5 has been characterized using the NaSCN method. Figure 4.6-5A shows the distribution of avidity IC50 values of the mAbs grouped according to their epitope. The highest avidity was displayed by 9G8, a mAb which does not have GIA activity. The BD5 mAb, which is the most potent human anti-RH5 mAb isolated to date does not have high avidity whereas 4D3, another mAb that also binds to the Bundle has slightly higher avidity than BD5. Overall, there is no correlation between avidity of binding to RH5 and GIA activity as seen in Figure 4.6-5B.

Figure 4.6-5. Avidity characteristic of RH5 mAbs. (A). Avidity according to the RH5 binding epitope location. (B). Correlative analysis of GIA at 2 mg/mL versus avidity characteristic of mAbs.

The large number of mAbs isolated in this project particularly from VAC063 groups 3, 5 and 7 allowed for identification of differences in avidity between mAbs from different groups (Figure 4.6-6A). Significantly higher avidity was observed for mAbs isolated from Groups 3 and 7 compared to those from Group 5. Interestingly, this pattern mirrored that found in the avidity of sera from the same groups (Figure 4.6-6B).

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Figure 4.6-6. Comparison of RH5 avidity of the mAbs isolated from the different VAC063 groups (A) to the serum anti-RH5 avidity of different VAC063 groups (B).

Analysis of sequences of the mAbs as well as additional characterization of mAbs that are highly potent in the GIA (currently, BD5 and possible 8G8) is planned. Characterization will include determination of their binding kinetics and their effect on RCR complex formation.

4.7. BLOOD STAGE EPITOPE-BASED VACCINE DEVELOPMENT PROJECT: VIN KOTRAIAH

All technical work on this project was completed in Q1 FY2019. Leidos submitted the final report on April 26, 2019; USAID accepted the report on May 28, 2019. Follow-on work on cloning the 3H7 RIPR mAb was completed in FY2020. Development of a manuscript on the epitope prediction analysis work performed as part of this project is ongoing. A manuscript on the structure of P113 which contained data related to P113 mAbs developed for this project was previously submitted to mBio by Dr. Gavin Wright in June 2020, accepted in August 2020, and published in September 2020.

4.8. PD1 BLOCKADE INHIBITOR RESEARCH ACTIVITIES: TIM PHARES All technical work on this project was completed in Q2 FY2019 and the final report approved by USAID in Q3 FY2019. The first PD1 manuscript was submitted to Frontiers Immunology in October 2019 and accepted on January 31, 2020. A second PD1 manuscript was submitted to Frontiers Immunology in April 2020 and accepted on May 29, 2020.

5. ELEMENT 3 ACTIVITIES: AMY NOE/JESSICA SMITH

No additional SCG meetings are planned under the MVDP contract.


6.1. MVDP REAGENTS REPOSITORY SriSai Biopharmaceutical Solutions (SBS) maintained, received, and distributed the reagents/materials needed for the ongoing and future studies during Q1 FY2021. SBS furnishes all the necessary services, management, qualified personnel, materials, equipment, facilities, and travel required for a biologics repository and inventory management services related to cGMP and non-GMP vaccines and associated

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products. SBS offers Controlled Room Temperature Storage (15 to 30°C), Controlled Room Temperature with humidity control, Refrigerated Storage (2 to 8°C), Freezer Storage (-20 ±10°C), Ultra-low Freezer Storage (-80 ±10°C), Low Freezer Storage (-30±10°C) and Vapor Phase Liquid Nitrogen (≤ -140°C), as well as the option to set units at a customer-defined temperature. The current inventory consists of standard storage temperatures ranging from -80°C to 4°C.

During Q1 FY2021, Leidos initiated inventory disposition be providing USAID an inventory listing for products developed and/or procured under the prime MVDP contract and requesting information on disposition. USAID provided feedback on the same and moved forward with inventory distribution to PATH and the University of Oxford in December 2020. The shipment to Oxford in planned for January 2020. Further, for hybridomas and antibodies developed under the blood stage and RCR vaccine development projects, some materials will be transferred to BEI Resources (planned for January 2020 pending approval by NIAID) such that these can be made available to the broader research community. In December 2020, USAID was provided will all final material transfer lists via email and these will also be included as part of the final program report.

7. TECHNICAL CLOSEOUT ACTVITIES

In Q2 FY2020, Leidos developed an MVDP Technical Closeout and Transition Plan to clarify the end of MVDP contract (1) material and reagent disposition process and (2) document and data transfer process. This plan was circulated to USAID on February 21, 2020 for comment. USAID provided feedback on March 6, 2020 and Leidos circulated an updated plan on March 23, 2020. In Q3 FY2020, Leidos started building the MVDP Data Transfer site and populating this with documentation that will be transferred to USAID at the end of the contract. Please refer to Section 6.1 for information on disposition of physical inventory in the MVDP repository associated with technical close out.

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8. LEIDOS POINTS OF CONTACT

8.1. PROGRAM MANAGER Amy Noe, Ph.D., MBA Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 858-826-6105 Mobile: 858-201-9176

8.2. OPERATIONS MANAGER Shannon Robinson, MBA, PMP Leidos LInC 10260 Campus Point Drive, MS C-4 San Diego, CA 92121 Phone: (858) 826-6034

8.3. CONTRACTS MANAGER Sandra George Leidos Life Sciences 5202 Presidents Court, Suite 110 Frederick, MD 21703-8398 Phone: 240-425-8546 Fax: 301-846-0794

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9. APPENDIX 1: LITERATURE CITED

Aguiar, J.C., Bolton, J., Wanga, J., Sacci, J.B., Iriko, H., Mazeika, J.K., Han, E.T., Limbach, K., Patterson, N.B., Sedegah, M., Cruz, A.M., Tsuboi, T., Hoffman, S.L., Carucci, D., Hollingdale, M.R., Villasante, E.D., Richie, T.L., 2015. Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development. PloS one 10, e0136109.

Alanine, D.G.W., Quinkert, D., Kumarasingha, R., Mehmood, S., Donnellan, F.R., Minkah, N.K., Dadonaite, B., Diouf, A., Galaway, F., Silk, S.E., Jamwal, A., Marshall, J.M., Miura, K., Foquet, L., Elias, S.C., Labbe, G.M., Douglas, A.D., Jin, J., Payne, R.O., Illingworth, J.J., Pattinson, D.J., Pulido, D., Williams, B.G., de Jongh, W.A., Wright, G.J., Kappe, S.H.I., Robinson, C.V., Long, C.A., Crabb, B.S., Gilson, P.R., Higgins, M.K., Draper, S.J., 2019. Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies. Cell 178, 216-228 e221.

Arevalo-Herrera, M., Lopez-Perez, M., Dotsey, E., Jain, A., Rubiano, K., Felgner, P.L., Davies, D.H., Herrera, S., 2016. Antibody Profiling in Naive and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites. PLoS Negl Trop Dis 10, e0004563.

Billaud, J.N., Peterson, D., Barr, M., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005a. Combinatorial approach to hepadnavirus-like particle vaccine design. J Virol 79, 13656-13666.

Billaud, J.N., Peterson, D., Schodel, F., Chen, A., Sallberg, M., Garduno, F., Goldstein, P., McDowell, W., Hughes, J., Jones, J., Milich, D., 2005b. Comparative antigenicity and immunogenicity of hepadnavirus core proteins. J Virol 79, 13641-13655.

Brault, A.C., Domi, A., McDonald, E.M., Talmi-Frank, D., McCurley, N., Basu, R., Robinson, H.L., Hellerstein, M., Duggal, N.K., Bowen, R.A., Guirakhoo, F., 2017. A Zika Vaccine Targeting NS1 Protein Protects Immunocompetent Adult Mice in a Lethal Challenge Model. Sci Rep 7, 14769.

Brune, K.D., Howarth, M., 2018. New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue. Front Immunol 9, 1432.

Brune, K.D., Leneghan, D.B., Brian, I.J., Ishizuka, A.S., Bachmann, M.F., Draper, S.J., Biswas, S., Howarth, M., 2016. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234.

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