Advances in gene-based vaccine platforms to address the

Advances in gene-based vaccine platforms to addressthe COVID-19 pandemic

Avanços em plataformas de vacinas baseadas em genes para abordar a pandemia COVID-19

Gustavo Motta Cabello dos Santos

Fillipe Davis Monte Mor Martins de Christo

Bruno Verri Jardine

José Carlos Botelho Monteiro

RCG0117 - Human GeneticsFMRP - USP

The article

“We review current gene-based vaccine candidates proceeding throughclinical trials, including their antigenic targets, delivery vehicles, and route ofadministration. Important features of previous gene-based vaccinedevelopments against other infectious diseases are discussed in guiding thedesign and development of effective vaccines against COVID-19 and futurederivatives.” (PUSHPARAJAH, Deborah, et al., Advances in gene-basedvaccine platforms to address the COVID-19 pandemic, 2021)

Gustavo Motta Cabello

2

Introduction

● The 2019 novel coronavirus outbreak (COVID-19),caused by SARS-CoV-2 virus, was declared a globalpandemic on March 11, 2020

● As a member of the Coronavirdae family, SARS-CoV-2 is classified as a betacoronavirus which ischaracterized by having a positive single-strandedRNA○ The virus is one of six human transmissible

coronaviruses discovered to date

● The need for protective solutions and, in particular, asafe and effective vaccine was, and still it is, ofhighest priority and paramount urgency


https://www.frontiersin.org/articles/10.3389/fimmu.2021.701501/full

3

Introduction


Overview of current COVID-19 vaccine development efforts. For each vaccine platform, the number of corresponding vaccine candidates are identified in parentheses.

Draft Landscape of COVID-19 Candidate Vaccines, https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines 2020, Accessed date: 11 December 2020.

● As of December 2020, WHO reports 215 COVID-19 vaccine candidates in development○ Diverse selection of vaccine platforms: protein-based, virus-based, and novel gene

delivery strategies such as nucleic acid, and viral vector

4

Introduction

● Conventional strategies: Tend toconfer complications withsafety,limited crossprotection andimmunogenicity

● Novel vaccine platforms:

Gene-based vaccines: greater safety andstability; potent cell-mediated protectiveimmunity; ease of manipulation, lowproduction costs; simpler and more rapiddevelopment

Gustavo Motta Cabello Next-generation vaccine platforms for COVID-19, https://www.nature.com/articles/s41563-020-0746-0, Accessed date: 15 October 2021.

5

https://www.nature.com/articles/s41563-020-0746-0

COVID-19 vaccine targets

This section will outline the general process andimportance of optimal antigen selection forvaccine development, in addition to identifyingspecific SARS-CoV-2 immunogenic targets

Gustavo Motta Cabello 6

Optimal antigen selection

Computer aided selection of candidate vaccine antigens; From: https://immunome-research.biomedcentral.com/articles/10.1186/1745-7580-6-S2-S1; Acessed date: 19 October 2021


● Gene-based vaccines (GBVs) encode specific SARS-CoV-2 antigenic epitopes and / orproteins rather than the entire viral genome○ Antigen selection is essential in the application of this strategy as it determines the

strength, type, and cross-reactivity of the immune response

7

https://immunome-research.biomedcentral.com/articles/10.1186/1745-7580-6-S2-S1

Optimal antigen selection

● Genome-based reverse vaccinology: This technique involves analyzing a pathogen genomesequence to identify relevant protein sequences that can be screened for protectiveimmunity. The sequence information can then be compared between pathogenic strains toidentify conservation levels, enabling the development of highly specialized vaccines specificto one strain, or cross-reactive vaccines conferring protection against multiple derivatives

Towards developing a vaccine for rheumatic heart disease -Scientific Figure on ResearchGate.:https://www.researchgate.net/figure/Reverse-vaccinology-The-procedure-of-reverse-vaccinology-starts-with-the-study-of-entire_fig1_316949942, Accessed date 17 October, 2021


8

COVID-19 immunogenic targets

● The genome of SARS-CoV-2 contain 14 openreading frames (ORFs) encoding 27 proteins○ ORFs represents the part of the reading frame

that has the ability to be translated● The four essential structural proteins encoded

are:○ Spike (S)○ Membrane (M)○ Envelope (E)○ Nucleocapsid proteins (N)


9

COVID-19 immunogenic targets

● The S protein facilitates viral uptake into host cells○ Binding to the host receptor, angiotensin-converting enzyme 2 (ACE2)○ The receptor binding domain (RBD) of SARS-CoV-2 has been shown to bind to the ACE2

target receptor with considerably higher affinity than that seen by SARS-CoV● The M protein regulates the shape of the viral envelope and interacts with other structural

proteins● The small E protein is involved in viral maturation, assembly, and budding● The N protein binds to the SARS-CoV-2 genome and is predominantly involved in genome

related processes


10

Amino acid sequence alignment of structural proteinsfrom Coronavirus

● In a strain-specific COVID-19 GBVs, the S1 and RBDdomains should be utilized,whereas the S2 domain ismore suitable for universalcoronavirus vaccinedevelopment


11

Novel gene-basedvaccine platforms

Fillipe Davis 12

GBVs encompass DNA, RNA, and viralvector platforms, which each conferspecific advantages and disadvantages.

Viral vector vaccines

Fillipe Davis

● A viral vector vaccine will consist of a recombinant virus, often attenuated, engineered to encode an antigen sequence for delivery into host cells for the endogenous production of high levels ofthat antigen;

● It can be either replicating (e.g. measles virus, adenovirus and vesicular stomatitis virus) or non-replicating (e.g. human adenovirus serotype 5 and serotype 26, adeno-associated virus, alphavirusand modified vaccinia virus Ankara);

● Safety concern about replicative viral vector vaccines: potential reversion to virulence;

● In the case of the non-replicative ones: higher doses are needed to confer immunity, which can result in undesirable immune reactions to the vector itself.

13

Viral vector vaccines

Fillipe Davis

● Adenovirus are the most common viral vectors used in clinical trials for vaccine, gene therapy and oncology applications;

● Benefits of viral vector vaccines in general: high levels of humoral and cellular immune responses are induced; these vaccines do not require adjuvants;

● Storage conditions for viral vector vaccines are highly dependent on the particular characteristics of each recombinant virus, which will determine the vaccine's stability, and consequently, its shelf-life. Generally, licensed viral vector vaccines are stored at approximately 2–8 °C or at low freezing temperatures (−80 to −55 °C) with a shelf-life of over a year.

14

Nucleic acid vaccines

Fillipe Davis

● Benefits of nucleic acid vaccines in general: as non-viral vectors, these vaccines are thought to confer greater safety than their viral counterparts; it can have shorter development cycles, enabling quick deployment during pandemics;

● The use of recombinant DNA vaccines requires successful transfer of the DNA vector into cell nuclei, transcription into messenger RNA (mRNA), and finally translation into the antigen of interest;

● “Naked” or purified plasmid DNA is a highly attractive vehicle for antigen presentation as it is very simple to manipulate and inexpensive to generate;

● A plasmid DNA vector typically consists of fundamental genetic components including a transcriptional promoter, RNA processing elements (polyadenylation (poly A) tail), and the geneencoding the antigen;

● The predominant challenge of utilizing DNA vaccines is that they generally impart low immunogenic responses in humans and larger animals compared to small animal systems .

15

Nucleic acid vaccines

Fillipe Davis

● An RNA vaccine consists of an mRNA molecule which encodes the selected antigen, removing the need for transcription;

● It includes the mRNA transcript encoding the gene of interest surrounded by 5′ and 3′ untranslated regions and a polyA tail;

● There are concerns regarding the instability of delivering naked RNA, in addition to the size of the delivered molecules;

● Lipo- or polyplexed DNA and RNA uptake is thought to occur primarily by endocytosis;● Methods to enhance vaccine uptake and expression have been more thoroughly investigated for

DNA vaccines compared to RNA, as DNA needs to bypass two cellular membranes to arrive at the nucleus, while RNA only needs to bypass one to enter the cytoplasm;

● As opposed to protein pharmaceuticals, tertiary structure is not generally important for DNA or RNA function, simplifying their storage requirements;

● DNA itself appears to be robustly stable;● In contrast, RNA products are very sensitive to temperature and should always be kept at

extremely cold temperatures (−70 °C) during storage and distribution;● The presence of ribonucleases can destroy the RNA vaccine product.

16

Gene-based vaccineadministration

Fillipe Davis 17

Methods and routes of administration canprofoundly impact the aforementioned GBVplatforms and are conventionally focused onparenteral and mucosal routes.

Route of administration: Mucosal Vaccination

Fillipe Davis

● Since the vast majority of pathogens, including SARS-CoV-2, invade the host through mucosal sites, using this route of administration may be an effective way to control infection and prevent disease. Both humoral and cellular immune responses are induced here, both systemically and locally;

● The immune response is characterized primarily by cytotoxic T lymphocyte and immunoglobulin A (IgA) responses. Present across many mucosal sites, IgA may aid in the prevention of pathogenic entry into the body;

● Dendritic Cells (DCs) also inhabit mucosal sites;● The expression of GBVs at mucosal sites requires penetration across the mucous and epithelial layers to

access the underlying immune cells;● Oral delivery of chitosan-encapsulated DNA vaccines appears effective;● While oral administration of GBVs seems promising, studies are sparse and thorough investigation is still

required;● Intranasal (IN) delivery is a suitable choice for respiratory-related diseases, although defense mechanisms

of the nasal cavity presents a challenge for vaccine entry;● Due to close proximity to the brain, there have been concerns that IN-delivered recombinant viral vector-

based vaccines could spread to the central nervous system (CNS) via olfactory tissue, imparting neurological side-effects.

18

Route of administration: Parenteral Vaccination

Fillipe Davis

● Certain parenteral routes of administration are preferred for GBV delivery. Similar to mucosal sites, varied levels of DCs also inhabit parenteral sites, promoting varied immune response levels;

● Administration through the ID (intradermal) route is highly effective as the skin contains enhanced levels of APCs (antigen presenting cells) that can serve as targets for the transfection of DNA and RNA vaccines;

● An enhanced immune response was found after three ID doses of a Lassa virus DNA vaccine when compared to IM (intramuscular) in guinea pigs;

● A Phase I trial for a seasonal influenza DNA vaccine demonstrated that ID administration was more favorably immunogenic, but was also associated with greater risk of adverse events;

● In a clinical trial assessing the CUTHIVAC 001 HIV-1 DNA vaccine, IM followed by ID (IM + ID) administration was found to be the least immunogenic, while IM + TC induced greater CD4+ and CD8+ T-cell responses.

19

Route of administration: Parenteral Vaccination

Fillipe Davis

● Studies employing viral vector vaccines have demonstrated similar immune responses between IM and ID routes, with heightened ID-mediated T-cell responses;

● Although the dermis may be more enriched in APCs compared to the muscle, muscle tissue is able to efficiently recruit immune cells due to its extensive network of blood vessels and the onset of temporal local inflammation upon vaccine administration;

● Local adverse reactions may be more severe with ID administration compared to IM;● This may partially explain why most viral vectors generate a more favorable response via the IM

route;● Considering the instability of many mRNA vaccines, the SC (subcutaneous) route of administration

may not be a suitable choice due to a low level of blood vessels;● Overall, both mucosal and parenteral administration of GBVs have shown promising results. So

far, many of the current COVID-19 vaccine candidates in ongoing trials are being administered parenterally, with just a few candidates using the mucosal route.

20

Novel delivery tools

Fillipe Davis

● Physical gene transfection systems are delivery platforms that transport genetic material through mechanical processes, such as biojector and electroporation (EP) devices;

● Biojector devices use CO2 pressure to deliver therapeutics via ID, IM and SC administration, which forces the vaccine into the skin, eliminating the requirement of a needle;

● Two Zika virus DNA vaccines were recently investigated in a Phase I clinical trial demonstrating enhanced T-cell responses after needle-free administration compared to needle and syringe application;

● The enhanced vaccine efficacy via jet injection could be attributed to a wider distribution of the vaccine, consequently inducing better uptake by APCs;

● EP involves the application of electrical pulses, generating pores in skin cells to enhance cell uptake of genetic material. It is able to permeabilize muscle cells to enhance the penetration and distribution of DNA vaccines;

● Several reports have shown improved antigen expression and enhanced antigen-specific immune responses by in vivo EP;

● Despite these advantages, EP has an added risk of cell death attributed to the application of high voltages● In general, if DNA or RNA COVID-19 vaccines are designed to be administered parenterally, tools such as

bioinjectors and EP may improve outcomes significantly, especially for DNA vaccines.

21

Immunity and safety ofprevious gene-basedvaccines

Bruno Jardine 22

Immunity and safety of previous gene-based vaccines

Bruno Jardine

GBVs remain novel pharmaceutical technologies. Knowledge gleaned from previousefforts evaluating GBV immunity and safety against other infectious diseases willdirect the development of a vaccine against COVID-19.

➢ Previous viral vector vaccine immunity and safety

1. Non-replicative viral vector vaccines

2. Replicative viral vector vaccines

➢ Previous DNA vaccine immunity and safety

➢ Previous RNA vaccine immunity and safety

23

Previous viral vector vaccine immunity and safety

Bruno Jardine

➢ The most frequently used Ad vector is Ad5 since it is able to induce potent CD8+ T-cell and Ab responses. Ad5-EBOV vaccine (CanSino Biologics, China) based on the Ad5 vector expressing the Ebola envelope glycoprotein (GP), was developed during the Ebola pandemic in 2016.

• Pre-existing immunity against the Ad5 vector produces NAbs, T-cells and type I IFN activated natural killer cells that inactivate the viral vector, reducing the efficacy of Ad5-based vaccines. In order to circumvent this problem, heterologous prime-boost regimens are a potential solution.

• The Ad5-vectored heterologous Ebola vaccine, GamEvac-Combi (Gamaleya Research Institute, Russia), was licensed in Russia in 2015 for emergency use. The design was supported by multiple animal models and clinical trials showing that heterologous prime-boost immunization generally elicits superior immunogenicity in comparison to repeated doses of the same vaccine.

• The authors suggest that heterologous vaccination may compensate for the negative implications of pre-existing immunity to Ad5 [188]. Indeed, a large proportion of adults worldwide have already acquired such immunity, particularly among African populations

1) Non-replicative viral vector vaccines

24


Bruno Jardine

• Although the Ad vector confers a better safety profile compared with replicative viral vectors, there are some concerns about its use in clinical trials.

• For example, an unexpected effect of pre-existing NAbs to the Ad vector was observed in a Phase II trial, where a vaccine candidate against human immunodeficiency virus (HIV) 1 showed increased HIV-1 acquisition risk in individuals with higher titers of pre-existing Ad5-Nabs. In addition, predominant sequestration of Ad vaccines within the liver has been shown to induce hepatotoxic-associated adverse effects.

• Avoiding pre-existing anti-Ad5 immunity can be addressed by the selection of rarer serotypes of Ad, such as Ad26, which has low prevalence in humans. Clinical trial results have shown that an Ad26 vector-based HIV vaccine was immunogenic and well-tolerated in healthy adults.

• The AdVac® platform (Janssen, Belgium), serving as the template vector for the Ad26CoV2-S COVID-19 vaccine, was used previously to develop the Ad26.ZEBOV vaccine against Ebola. The two-dose regimen was approved for medical use in the European Union in July 2020.

25


● ChAdOx1 is a replication-defective E1/E3 chimpanzee Ad-vector (University of Oxford, UK) developed to circumvent the issue of pre- existing anti-human Ad immunity. Ads derived from chimpanzees have a low seroprevalence rate in most human populations, hence, their use as a vector.

● ChAdOx1 is a promising vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection. To date, two Phase I trials have been performed, both demonstrating good safety and T-cell immunogenicity profiles.

● ChADOx1 has also been used to develop a MERS vaccine, which demonstrated a significant increase in T-cell and IgG responses to the MERS-CoV. A single dose was able to elicit NAbs against live MERS-CoV in 44% of participants that received the high dose, although the highest dose elicited increased severity of reactogenicity. In the majority of participants, both humoral and cellular responses against MERS-CoV were induced and maintained for at least 1 year.

➢ The data collected from the clinical studies mentioned here were relevant to support and accelerate SARS-CoV-2 vaccine development based on non-replicative viral vectors. Since most of these platforms are based on Ads, it is important to consider the pre-existing immunity problem in different populations. The use of non-human Ad vectors, heterologous prime-boost regimens and mucosal delivery of Ad vector vaccines are suitable alternatives to minimize this issue.

26

2) Replicative viral vector vaccines

Bruno Jardine

➢ To date, several replicative viral vector vaccines have been licensed for human use, including DENGVAXIA®(Dengue), IMVAMUNE® (small- pox), IMOJEV® (Japanese encephalitis) and ERVEBO® (rVSV-ZEBOV) for Ebola, supporting the use of this platform for SARS-CoV-2 vaccine development. MV is able to induce potent CD4+ T-cell responses, unlike CD8+ T-cell dominated responses toward Ad vectors.

• The first evaluation in humans of the MV vaccine platform was done with the MV-CHIK vaccine candidate (Themis Bioscience, Austria) expressing structural genes of CHIK virus. Importantly, the immunogenicity of the vaccine did not appear to be affected by pre-existing anti-MV immunity.

• Although replicative viral vector vaccines generate strong immune responses, unfortunately there is a possibility for changes of the vector to occur, inducing vaccine virulence which can then cause infection.

• Additionally, viral replication in humans may enhance the negative implications of genomic integration of viral DNA into the host genome. Although this is a predominant concern, MV replication is restricted to the cytoplasm, mitigating the risk of insertional mutagenesis.

➢ In general, studies have shown that replicative viral vector vaccines successfully induce potent immune responses, and the technology already exists for large-scale production and storage.

27

Bruno Jardine

28

Bruno Jardine

2) Replicative viral vector vaccines 29

Previous DNA vaccine immunity and safety

Bruno Jardine

➢ Plasmid-mediated Ab production was first reported in mice in 1992. A year later, plasmid-mediated cytotoxic T lymphocyte and Ab responses were also observed. Immunization with naked plasmid DNA has since demonstrated both humoral and cell-mediated immunity in preclinical studies. However, DNA vaccines have not performed as well in humans as results were not replicated in subsequent HIV vaccine trials.

● Developments against coronaviruses have been promising. A DNA vaccine encoding the SARS-CoV S protein induced CD4+ and CD8+ T-cell and anti-S Ab responses in mice. Another S protein plasmid was immunogenic in a Phase I trial: after 3 vaccinations, NAbs and CD4+ T-cell responses were detected across all participants, although CD8+ T-cell responses were present in only 20% of participants.

● More recently, a MERS DNA vaccine, INO-4700 (Inovio Phar- maceuticals, U.S.), demonstrated appreciable levels of MERS binding Abs in 94% of participants which persisted over a year in 77% of them. Unfortunately, while NAbs were detected in 43% of participants by week 14, durability proved to be an issue as this dropped drastically (3%) after a year.

➢ DNA vaccines have been examined against many pathogens without significant adverse effects including malaria, Ebola, dengue virus, West Nile virus (WNV), and Zika virus. Plasmid DNA vaccines against HIV-1 have similarly demonstrated high safety and appreciable dose-dependent CD4+ and CD8+ T-cell responses in human trials, although humoral immunity appeared to be weak in most cases.

30


Bruno Jardine

● In a WNV DNA vaccine Phase I trial for VRC-WNVD (National Institute of Allergy and In- fectious Diseases [NIAID], U.S.). No vaccine-related serious adverse events (SAEs) were reported, indicating high safety even in the older cohort. NAbs were observed in over 96% of participants, while CD4+ T-cell responses were more prevalent than CD8+.

● Seasonal influenza represents a favourable target for rapid vaccine development. VRC-FLUDNA064-00-VP (NIAID, U.S.), a trivalent DNA vaccine against seasonal influenza, was investigated in prime-boost regimens across three trials in adults and children. Participants were primed with either the plasmid vaccine or IIV3, or concurrent DNA/IIV3, followed by an IIV3 booster. Unfortunately, neither the DNA prime nor the concurrent DNA/IIV3 prime could improve upon a poor immune response in the older population.

● In a juvenile cohort (ages 6-7), priming with the DNA vaccine performed as well or better than with IIV3, demonstrating the potential for DNA-based vaccine use in an epidemic. This was further demonstrated in a Phase I trial of GTU®-MultiHIV B (Imperial College London, UK), a DNA vaccine for the 2009 pandemic influenza A H1N1 virus.

31


Bruno Jardine

➢ While generally well-tolerated, DNA vaccines may still carry other risks. Risk of insertional mutagenesis, while possible, appears low as no significant DNA vaccine integration into genomic DNA has yet been shown. FDA guidelines recommend that plasmid DNA be ≥80% supercoiled to prevent insertional mutagenesis.

➢ The biggest risks related to DNA vaccines remain relating to genotoxicity and auto-immunity. Arguably the most important instance of anti-DNA immunity is as a hallmark of systemic lupus erythematosus (SLE), an autoimmune disease. SLE is characterized by antinuclear Abs, most notably against double-stranded DNA and single-stranded DNA. Such anti-DNA Abs appear to bind specifically to antigenic DNA sequences. Therefore, COVID-19 plasmid DNA vaccination should proceed with caution in individuals with, or at risk of, autoim-mune disease.

32


Bruno Jardine

➢ Overall, plasmid DNA-based vaccines appear to be very safe, although not always immunogenic, where adequate immune response appears to require larger and more doses.

✓ Humoral immunity has not been consistent across human trials, although cell-mediated immunity appears more common.

✓ The safety profile of DNA vaccines in younger and older populations is extremely promising.

✓ Importantly, DNA vaccines can be developed faster than their inactivated equivalents, as evidenced with current COVID-19 DNA vaccine developments.

✓ Even if a DNA vaccine does not induce sufficient immunity alone, it may be useful in prime-boost regimens as development of inactivated virus vaccines catch up.

33

Bruno Jardine

34

Bruno Jardine

35

Previous RNA vaccine immunity and safety

Bruno Jardine

➢ The first mRNA vaccine to show efficacy in humans was mRNA-1944 (Moderna Therapeutics, U.S.), encapsulated with their proprietary LNP technology against the Chikungunya virus. Two mRNAs comprised mRNA- 1944, encoding the sequences for the heavy and light chains of an anti-Chikungunya IgG respectively.

● It demonstrated linear dose-dependent production Abs in the human body, where 100% of participants demonstrated NAb activity against the Chikungunya virus. While the lower doses did not induce vaccine-related sepsis-associated encephalopathy, the higher doses induced infusion-related grade 3 adverse events, indicating a sensitive balance between safety and efficacy.

● Moderna has also developed mRNA influenza vaccine candidates that have undergone Phase I trials evaluating two vaccines: one encoding the Haemagglutinin envelope glycoprotein precursor (GP) from H10N8 and the other encoding the GP from H7N9. Participants did not present significant HA- specific cell-mediated responses, but showed robust humoral immune responses and seroconversion rate.

● Previous H7N9 virus-like particle and subunit candidates required adjuvants to reach high seroconversion and seroprotective, alluding to a self-adjuvanting effect of the mRNA vaccines. Aditionally, the H7N9 mRNA vaccine demonstrated potential development of memory B cells.

36


Bruno Jardine

● Boostable levels of functional Abs were observed in a Phase I clinical trial administering three doses of the rabiesvaccine, CV7201 (CureVac, Germany), when delivered by needle-free administration, as aforementioned.

● Unfortunately, one SAE was noted after a 640 μg IM dose, but successfully resolved. T-cell immune responses were generally low, but these results were likely not representative due to blood samples that were derived two weeks from the last vaccination.

● RNA vaccines have been generally safe in humans, although unin- tended adverse reactions are the biggest fear for RNA vaccines. Clinical trials have demonstrated local and systemic moderate and severe AEs for some mRNA vaccines.

● The previously mentioned CV7201 vaccine reported one event of autoimmune thyroiditis a year after the last dose.

● Furthermore, concerns regarding mRNA vaccination have been identified, due to the fact that extracellular RNA has been shown to alter endothelial cells and damage blood vessels, consequently promoting significant health issues including oedema, coagulation and pathological thrombus formation

➢ Although RNA-based vaccines carry additional safety concerns compared to their DNA counterparts, mRNA does not need to be transported into the nucleus for transcription, dramatically improving transfection efficiency and eliminating the possibility of oncogenic events arising from random integration into host DNA.

37


Bruno Jardine

✓ While RNA vaccines overall have demonstrated strong humoral immunity and high seroconversion rates, their inconsistent cell-mediated immunity responses merit concern.

✓ Due to strong induction of potent type 1 IFN responses, COVID-19 RNA vaccine candidates require thorough safety testing to ensure protection against the onset of unexpected side effects.

✓ Similarly with DNA, auto-immunity may be a concern. Considering these caveats, close attention must be given to their formulation and more data is required to ensure their safety and efficacy in humans.

38

Bruno Jardine

39

COVID-19 Gene BasedVaccines

An overview of the COVID 19 vaccines tests,results and challenges so far.

40José Monteiro

Summary

José Monteiro

● Fundamental aspects from previous GBV studies enabled:

○ Rapid production during COVID-19 pandemics – 50% of the vaccines indevelopment are GBV.

○ Paper data shows: 26 of 52 clinical COVID-19 candidates are GBV.○ 10 non-replicative viral-vectors;○ 4 replicative viral-vectors;○ 6 DNA-based;○ 6 RNA-based.

● Also, key-roles of humoral and cellular imune responses were acquired fromvaccination during MERS-CoV and SARS-CoV pandemics.

41

Summary

José Monteiro

42

Viral Vector Vaccines

● University of Oxford/AstraZeneca

AZD1222 Vaccine

● Vector: ChAdOx1;● Antigen: S Protein;● Administration: intramuscular (IM);● Storage: between 2°C and 8 °C;

José Monteiro

43

Phase I/II

▪ Young Adults: 1077 people (ages: 18–55);

▪ A single standard dose (SD) was first delivered;

▪ T-Cell responses peaked on 14th day and neutralizing antibodies

(NAb) peaked at 28th day;

Phase II/III

▪ Adults (18+): 480 people;

▪ One or two doses regimen: standard and low doses (LD). The

second dose was delivered at the 28th day;

▪ Same phase I immune response numbers

▪ After booster dose: NAb presented in >99% of participants;

Phases I/II, II/III, III

▪ Adults (18+): 23848 people;

▪ Two doses were delivered in different ways: SD/SD and LD/SD;

▪ Efficacy results: 62.1% in SD/SD; 90% in LD/SD; 70.4% overall.

A possible neurological event that was vaccine related was being

verified (A case of Longitudinally Extensive Transverse Myelitis).


● CanSino Biologics/Beijing Instituteof Biotechnology

Adn5-CoV Vaccine

● Vector: Adenovirus Ad5(based on their Ad5-ZEBOV vac.);

● Antigen: S Protein;● Administration : IM;● Storage: between 2°C and 8 °C.

José Monteiro

44

Phase I

▪ Adults: 108 people (ages: 18–60);

▪ Three doses, in an escalation regime was delivered;

▪ T-Cell responses on 14th day and humoral immunity peaking at 28th day;

▪ 9% of the high doses participants presented fever.

Phase II

▪ Adults (18+): 508 people

▪ Two doses regimen (low and high doses);

▪ Same phase I immune response numbers;

▪ No significant difference between one or two doses immune response;

▪ The lower dose showed a better safety profile

Phase III

▪ Adults: 40000 people (18+) + 500 (18-85)

▪ Status: Ongoing

Phase II showed that participants with LOWER anti-Ad5 NAb

presented almost two times the levels of RBD-specific Ab, than the

ones presenting HIGH anti-Ad5 Nab.


● Gamalaya Research Institute

Gam-COVID-Vac (Sputnik V)

● Vector: Adenovirus Ad5 andAd26 (heterologous tests);

● Antigen: S Protein;● Administration : IM;● Storage:

Frozen formulae at -18 °C;Lyophilized formulae 2°C to 8 °C. José Monteiro

45

Phase I


▪ Single doses: rAd5-S or rAd26-S vectors;

▪ CD4+ and CD8+ T-Cell responses on 28th day. RBD-specific IgG

peaking at 42th day

Phase II


▪ Two doses regimen: rAd26-S on day 0 followed by rAd5-S on day 21;

▪ CD4+ and CD8+ T-Cell responses on 28th day and, also, RBD-specific

IgG peaking at 42th day for 85% of the participants;

▪ The heterologous vaccination was the only one which achieved NAb for

100% of the participants;

Phase III

▪ Adults: 40000 people (18+) + 500 (18-85)

▪ Same dose regimen as phase II;

▪ Preliminary data: 91.4% efficacy rate 7 days after second dose. 95%

efficacy after 21 days of second dose.

▪ Pre-existing Ad5 or Ad26 immunity didn’t affect titers of RBD-IgG


● Janssen Pharmaceutical Companies Ad26Cov2-S

● Vector: Adenovirus Ad26;● Antigen: S Protein;● Administration : IM;● Storage: between 2°C and 8 °C.

José Monteiro

46

Phase I/IIa

▪ Adults: 1045 people (ages: 18–55 and over 65);

▪ Single or two doses regimen;

▪ Status: Ongoing.

Phase II

▪ Adults: 1210 people (ages: 12-18; 18-55 and over 65);

▪ Single dose: adolescents (to assess safety and reactogenicity);

▪ Two doses schedule for adults, with 28, 56, 84 days or 1 year

after first vaccination;


Phase III

▪ Adults: 6000 people (18+);

▪ Single dose vaccination;

▪ Status: Ongoing


● Other vectors than adenosinoviruses wereused, specially for replicating viral vectors

● Also, different administration routes werebeing tested and their immunologic responses on focus;

● Vaccine candidates expressing both protein S and N.

● At the paper publication, several other vacines were still on phase I trials, with ongoingresults being collected;

José Monteiro

47

DNA Vaccines

▪ Vector: All are plasmid DNA.

PGX001 from Inovio, encodes

a consensus S sequence with

an N-terminal IgE leader

sequence, under the control of

a cytomegalovirus promoter

▪ Antigen: S protein;

▪ Intramuscular (IM) and

intradermal (ID) delivery

routes are aided with

electroporation techniques

▪ ALL Status: Ongoing.

José Monteiro

48

RNA Vaccines

● Moderna/ NIAID mRNA-1273 Vaccine

● Vector: LNP encapsulated mRNA;● Antigen: Prefusion stabilized S Protein;● Administration : IM;● Storage: six months at -20 °C;

30 days at 2°C to 8°C;12 hours at room temperature.

José Monteiro

49

Phase I

▪ 40 people with age of 18-55 and 45 with > 56 years old;

▪ First dose (25, 100 or 250µg): robust NAb and T-Cell responses

in dose dependent fashion;

▪ Safe and well tolerated: Absense of SAE;

▪ The 100µg dose had the best results, considering both

immunogenicity and reactogenicity

Phase II


▪ A second dose was indicated by the results of the phase I

▪ Two doses schedule (50 or 100µg);


Phase III


▪ Two doses schedule (100µg) on day 1 and 29;

▪ Status: Ongoing

RNA Vaccines

● BioNTech/Fosum/Pharma/Pfizer Vaccines

BNT162a1, b1, b2 and c2

● Vector: LNP encapsulated mRNA;● Antigen: two encode RBD

two encode the full S protein;● Administration : IM;● Storage: ultra-cold storage at -70 °C

After thawing: 2°C to 8°C (5 days)

José Monteiro

50

Phase I

▪ Adults: 195 people (18 – 85)

▪ Single or two doses (day 1 and 21) regimen in a dose

escalation trial (10, 20, 30 or 100 µg);

▪ Dose-dependent NAb responses;

▪ Dose-dependent local and systemic were mild to moderate

Phase I/II

▪ Adults: 501 people (18 - 85);

▪ Single or two doses (day 1 and 21) regimen in a dose

escalation trial. One trial tested doses of 10, 30 or 100 µg

and the other 1, 10, 30, 50µg;

▪ 100µg dose: increased reactogenicity and pain reports

▪ Dose-dependent RBD-IgG, T-cell and NAb titers.

Phase II/III


▪ Two dose scheduled at day 1 and 21;

▪ 30µg dose of the BNT162b2 vaccine was chosen, based

on earlier trials.

▪ 95% effectiveness in prevention of COVID-19

▪ 90-100% vaccine efficacy across different subgroups.

RNA Vaccines

José Monteiro

51

Adjuvants?

Substances that can be employed along withvaccines to ensure immune recognition andenhance immune responses.

José Monteiro 52

Adjuvants

● Are they necessary in the COVID-19 vaccination context?

● Viral Vector Vaccines

● RNA Vaccines

● DNA Vaccines

✓ No mentions within the vaccines in the paper;

✓ Viral vectors mimicks natural infection;

✓ Activates innate immune responses.

✓ Also not explicitly stated;

✓ Shows strong humoral immunity, inducing immune signals via pattern recognition receptors;

✓ LNP are suggested to have an adjuvancy effect.

✓ May have a major role in DNA vac. due to its usually insuficient immune response in humans;

✓ Aluminum salts, a common vaccine adjuvant, is not effective in DNA vaccines;

✓ Plasmid DNA may have inherent adjuvant effect -> contain bacteria CpG deoxynucleotides;

✓ However, too few DNA vaccines data are available in the paper.

José Monteiro

53

Adjuvants

● However.......Immunopathology role of adjuvants

● Some evidence shows adjuvants help to maintain balance between Th1 and Th2 cells responses;

● SARS-CoV and other respiratory deseases are known to develop Th2 immunopathology;

● As so, adjuvants may have a potential role of helping suppress this issue;

Th? –> T helper cells, derivatives from CD4+ T cell

Delta inulin (adjuvant) alleviates lung

immunopathology observed in mice with

SARS-CoV

CpG deoxynucleotides (adjuvant)

prevent induction of excessive Th2

responses, by the induction of IL-10, thus

helping Th1/Th2 balance

Pre-clinical studies showedAnd also boostered the

neutralizing antibodies

titers!

José Monteiro

54

Immune enhancement of gene-based vaccines

In some cases, vaccination could predisposeindividuals toward exacerbated disease bypriming an immune response that promotesinfection or stimulates undesirable cell-mediatedimmunopathology

Gustavo Motta Cabello 55

Antibody dependent enhancement (ADE)

● ADE has been implicated in the exacerbation of what is generally a mild viralmanifestation of flu-like symptoms in the majority of SARS-CoV-2 infections○ Has been demonstrated in previous accounts of dengue virus○ There are theoretical concerns that vaccines that generate antibodies against

the SARS-CoV-2 can bind to the virus without neutralizing it● Vaccine designs that prevent the propagation and persistence of non-neutralizing

antibodies are preferential in mitigating the possibility of ADE against SARS-CoV-2○ More substantiating data as to whether SARS-CoV recovered individuals are

susceptible to ADE is needed


56

Antibody dependent enhancement (ADE)

Gustavo Motta CabelloTwo main ADE mechanisms in viral disease, From: https://www.nature.com/articles/s41564-020-00789-5; Acessed date 18 October, 2001

Facilitate virus entry macrophage-

tropic viruses

non-macrophage-tropic viruses

57

Th2 immunopathology

Gustavo Motta CabelloLinfócitos T CD4: Th1 e Th2; From: https://www.biomedicinapadrao.com.br/2010/10/linfocitos-th1-e-th2.html; Acessed date 19 October, 2001

58

https://www.biomedicinapadrao.com.br/2010/10/linfocitos-th1-e-th2.html

Th2 immunopathology

● Many virologists believe that the more pressing matter around vaccineenhancement is the cell-mediated dysfunction phenomenon of Th2immunopathology○ Th2 immunopathology results in the recruitment of neutrophils and eosinophils to the

lung tissue, promoting local inflammation

● Th2 immunopathology was first observed in the 1960s in preparation of a vaccineagainst RSV (Respiratory syncytial virus)○ Initial vaccines produced for SARS-CoV demonstrated inflammatory responses in the

lungs, similar to those seen following immunization with the RSV vaccine


59

Th2 immunopathology

● The use of nucleic acids to encode limited and neutralizing sequences furtherstimulates protective and cross-reactive T-cell responses○ T-cell memory is critical in generating cross-reactive protection against COVID-19

● GBVs that can mimic endogenous viral protein production, minimize nonneutralizing epitopes, and adequately balance Th1 and Th2 responses andcorresponding memory may enhance efficacy, durability and cross-reactiveness ofvaccines, while further preventing vaccine enhancement


60

Conclusions

● The rapid worldwide dissemination of COVID-19 has emphasized the imperative need todevelop an efficacious vaccine to prevent infection, disease, or transmission○ Collaborative efforts around the world have contributed to unprecedented rapid vaccine

development● Clinical study results for the COVID-19 GBVs outlined in this paper demonstrate a good

immunization profile and for some, very high efficacy○ Further trials to analyze the efficacy and safety in high risk groups are needed

● There are additional challenges that the available COVID-19 vaccines need to addressincluding durability of protection, viral transmission prevention, efficacy within specificsubgroups of the population, and public acceptance


61

Bibliography

● Pushparajah D, Jimenez S, Wong S, Alattas H, Nafissi N, Slavcev RA. Advances in gene-based vaccine

platforms to address the COVID-19 pandemic. Adv Drug Deliv Rev. 170, 113-141 (2021)https://pubmed.ncbi.nlm.nih.gov/33422546/

● Corbett, K.S., Edwards, D.K., Leist, S.R. et al. SARS-CoV-2 mRNA vaccine design enabled by prototype

pathogen preparedness. Nature 586, 567–571 (2020). https://www.nature.com/articles/s41586-020-2622-0

● Dphil, M. V., Clemens, S. A. C., et al. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222)

against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and

the UK. The Lancet 397, issue 10269, 99-111 (2021). https://www.thelancet.com/journals/lancet/article/PIIS0140-

6736(20)32661-1/fulltext

● Logunov, D. Y., Dolzhikova, I. V., et al. Safety and immunogenicity of an rAd26 and rAd5 vector-based

heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2

studies from Russia. The Lancet 396, issue 10255, 887-897 (2020). https://doi.org/10.1016/S0140-6736(20)31866-3

● https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines


62

https://pubmed.ncbi.nlm.nih.gov/33422546/

https://www.nature.com/articles/s41586-020-2622-0

https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)32661-1/fulltext

https://doi.org/10.1016/S0140-6736(20)31866-3

https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines

Documents

Advances in gene-based vaccine platforms to address the