Gene Deletions in Mycobacterium bovis BCG Stimulate IncreasedCD8� T Cell Responses

Michael W. Panas,a* Jaimie D. Sixsmith,a KeriAnn White,a Birgit Korioth-Schmitz,a Shana T. Shields,a Brian T. Moy,a Sunhee Lee,b

Joern E. Schmitz,a William R. Jacobs, Jr.,c Steven A. Porcelli,c Barton F. Haynes,b Norman L. Letvin,a† Geoffrey O. Gillarda*

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USAa; Human Vaccine Institute andDepartment of Medicine, Duke University Medical Center, Durham, North Carolina, USAb; Howard Hughes Medical Institute, Department of Microbiology andImmunology, Albert Einstein College of Medicine, Bronx, New York, USAc

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and havenumerous ways of suppressing their host’s immune response. It has been suggested that mycobacteria may contain genes thatreduce the host’s ability to elicit CD8� T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus CalmetteGuerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobac-terium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgeneproduct-specific CD8� T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Recon-struction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotypeof the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novelBCG strains, we observed enhanced SIV Gag-specific CD8� T cell responses in vivo. This study demonstrates that mycobacteriacarry numerous genes that act to dampen CD8� T cell responses and suggests that genetic modification of these genes may gen-erate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Slow-growing mycobacteria, including Mycobacterium tubercu-losis and Mycobacterium bovis, have evolved mechanisms to

evade host immune responses. These mycobacteria have beenshown to alter vesicle trafficking and acidification, allowing themto survive for extended periods of time in the phagocytic compart-ments of macrophages (1–3). They evade host cellular immuneresponses by modulating type I interferon production (4, 5) andinhibit apoptosis of infected macrophages (6). A number of stud-ies have shown that these mycobacteria can inhibit major histo-compatibility complex (MHC) class II-restricted peptide presen-tation (7–10). It has also been suggested that mycobacteria may becapable of evading MHC class I-restricted T cell responses, but themechanisms mycobacteria utilize to do so have not yet been de-fined.

M. bovis bacillus Calmette-Guerin (BCG), an attenuated strainof M. bovis, has been used as a vaccine for M. tuberculosis foralmost a century, and technologies have been developed thatshould allow for its use as a vaccine vector. However, BCG hasdemonstrated only limited efficacy as a tuberculosis vaccine, and ithas proven disappointing in preclinical studies as a vaccine vector.We have shown that CD8� T cell responses are critical for BCG-mediated protection against M. tuberculosis in rhesus monkeys(11). This observation raises the possibility that the efficacy ofBCG, used both as an M. tuberculosis vaccine and as a vaccinevector, could be improved by increasing its ability to induce morerobust CD8� T cell responses.

Questions about the efficacy of BCG as a vaccine and a vaccinevector may in part be explained by the possibility that BCG hasevolved mechanisms to minimize its induction of MHC class I-re-stricted cellular immune responses. In the present study, we usedtransposon mutagenesis to identify a number of genes in BCGwhose expression diminishes productive MHC class I presenta-tion of BCG-vectored proteins, and we show that the selective

elimination of these genes enhances the induction of CD8� T cellresponses by recombinant BCG (rBCG) vaccine constructs.

MATERIALS AND METHODSMice. Age-matched adult C57BL/6 mice were obtained from The JacksonLaboratory (Bar Harbor, ME). All mice were maintained in the Beth IsraelDeaconess Medical Center (BIDMC) Animal Research Facilities and usedin accordance with protocols approved by the Institutional Animal Careand Use Committee.

BCG transformation. A 10-ml culture of BCG Danish 1331 wasgrown to an optical density at 600 nm (OD600) of 1, pelleted, and washedthree times with a solution of 10% glycerol in distilled water. The pelletwas resuspended in 200 �l of 10% glycerol, mixed with 200 ng of plasmidDNA of the plasmid pMV261-19kDaSIINFEKL (12) or pSL10, and incu-bated at room temperature for 20 min. The pSL10 plasmid is a multicopyepisomal Escherichia coli-mycobacterial shuttle plasmid that carries a fu-sion protein gene containing the signal sequence from the Ag85b secretedmycobacterial protein, the full-length simian immunodeficiency virus(SIV) Gag protein, and a hemagglutinin (HA) tag (S. Lee, unpublisheddata). Cells were then transformed by electroporation (2.5 kV, 25 mF,

Received 27 May 2014 Returned for modification 5 July 2014Accepted 30 September 2014

Published ahead of print 6 October 2014

Editor: J. L. Flynn

Address correspondence to Geoffrey O. Gillard, geoff.gillard1@gmail.com.

* Present address: Michael W. Panas, Department of Microbiology andImmunology, Stanford Medical School, Palo Alto, California, USA; Geoffrey O.Gillard, Biogen Idec Inc., Cambridge, Massachusetts, USA.

† Deceased.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02100-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02100-14

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1,000 �), incubated in Middlebrook 7H9 medium (Sigma-Aldrich) over-night at 37°C and plated on 7H10-ADS (albumin-dextrose-saline) plateswith 20 �g/ml kanamycin (pMV261-19kDaSIINFEKL) or 30 �g/ml apra-mycin (pSL10). Three weeks later, colonies were selected, cultures weregrown to an OD600 of 1, and transgene expression was assessed by Westernblotting.

Transposon library. Cultures (100 ml) of BCG Danish-pMV261-19kDaSIINFEKL were grown to an OD600 of 1, pelleted in 10 tubes at3,000 rpm, and washed twice with buffer MP (50 mM Tris, 150 mM NaCl,10 mM MgCl2, 2 mM CaCl2). Pelleted cells were resuspended in 3 ml ofbuffer MP containing 1010 PFU of the transposon-containing phagepCS9Hygro1Mar, incubated for 24 h, and plated on 10 plates of 7H10 –oleic acid-albumin-dextrose-catalase (Sigma-Aldrich) containing 100�g/ml hygromycin and 20 �g/ml kanamycin. A total of 5,000 colonieswere selected, and each was grown in 10 ml 7H9 with 20 �g/ml kanamycinin a 15-ml conical tube maintained on a roller at 37°C.

MHC class I presentation assay. A3.1A7 cells (an H-2Kb macrophagecell line kindly provided by Kenneth Rock, University of MassachusettsMedical School, Worcester, MA) were washed with RPMI–10% fetal calfserum (FCS) and resuspended at 5 � 105 cells/ml, and 5 � 104 cells (100�l) were aliquoted into each well of a 96-well plate. Cells were activatedwith 250 U/ml beta interferon (IFN-�) for 2 h at 37°C. Mycobacterialstrains expressing SIINFEKL were washed with phosphate-buffered saline(PBS)– 0.02% Tween 20 and resuspended at 4 � 107 CFU/ml in RPMIwithout antibiotics, and 50 �l (2 � 106 CFU) was added to each well(multiplicity of infection [MOI] of 20). RF33.70 T cells (Kenneth Rock)were resuspended in RPMI–10% FCS at a concentration of 2 � 106/ml,and 50 �l was added to each well (1 � 105 cells/well). Ninety-six-wellplates were incubated at 37°C for 24 h and then frozen at �20°C untilinterleukin-2 (IL-2) production was assayed via an enzyme-linked immu-nosorbent assay (ELISA). Aliquots of 100 �l of supernatant were assayedfor IL-2 production with an IL-2 ELISA kit (Invitrogen). IL-2 levels weredetermined by comparison to the standard curve calculated using an IL-2protein control. Strain-specific IL-2 production was compared to that ofMycobacterium smegmatis pMV261-19kDaSIINFEKL (positive control)and rBCG transformed with pMV361 (no SIINFEKL) as a negative con-trol.

Identification of transposon insertion site. The culture (10 ml) ofeach transposon mutant was grown to an OD600 of 0.8 before 1% (wt/vol)glycine was added for 24 h. Cultures were then pelleted and incubatedwith lysis buffer for 1 h at 65°C, and genomic DNA was isolated using theDNeasy Qiagen kit. Genomic DNA was digested for 2 h with BssHII,circularized by ligation with T4 DNA ligase for 1 h, transformed intopir-116 electrocompetent cells (Epicenter, Madison, WI), and plated onLB agar plates with 100 �g/ml hygromycin. pir-116 cells containing viableplasmids grew into colonies, and plasmid DNA was isolated using theQiagen Miniprep spin kit. Plasmids were sequenced using primers thatbound to the left inverted repeat and right inverted repeat of the trans-poson. Sequences were aligned with the BCG Pasteur sequenceAM408590 by using the NCBI BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the genes surrounding the site of transposondisruption, allowing the identification of the disrupted pair of nucleo-tides.

Complementation of ICO M (AZ11). PCR was used to amplify theAZ11 locus containing echA18 and amiD. The primers created a PCRproduct with a HindIII site at both ends. This product was digested andligated into the HindIII site in pYUB1141 and used to transform DH5� E.coli. The plasmid was isolated from E. coli colonies selected on LB agarplates containing 100 �g/ml apramycin. Sequencing was used to confirmsuccessful cloning, and plasmids were transformed into the ICO M trans-poson strain and selected on 7H10-ADS plates containing 20 �g/mlkanamycin and 30 �g/ml apramycin. Successful complementation wasassessed by PCR using primers specific for echA18.

AERAS 401-SIINFEKL construction. AERAS 401, a recombinantstrain of BCG lacking the urease C gene and containing the perfringolysin

O gene (PfoAG137Q), was obtained from the AERAS Foundation, Rock-ville, MD. Plasmid pMV261-19kDa-SIINFEKL was transformed intoAERAS 401 via electroporation and selected on kanamycin-containingplates. Single colonies were picked and assessed by Western blotting be-fore injection in vivo.

Plasmid DNA vaccine. A plasmid DNA vaccine expressing the 19-kDaSIINFEKL sequence was created by PCR using the template pMV261-19kDa-SIINFEKL. The PCR product was digested, ligated into the multi-ple -cloning site in the pVRC2000 backbone, and transformed into DH5�cells (New England BioLabs, Ipswich, MA). The plasmid transgene regionwas sequenced prior to large-scale preparation, and sufficient quantitieswere obtained by using a Qiagen Maxiprep kit. The plasmid pVRC4307(pVR1012x/s SIVmac239 Gag [delFS] Pol Nef/h), containing codon-op-timized SIV gag under the control of the cytomegalovirus promoter, was akind gift of Gary Nabel, Vaccine Research Center, NIAID, Bethesda, MD.

AES experiment. The allelic exchange substrate (AES) pAES2589 wascreated to reconstruct ICO J. Flanking regions of the target operonBCG_2587-2590 were amplified by PCR and cloned into p0004S on eitherside of the hygromycin resistance gene. The AES was confirmed by se-quencing and then cloned into the PacI restriction enzyme site in thephasmid phAES159. High-titer phage was prepared in M. smegmatis andthen used to transduce BCG Danish. Confirmation was done by PCR andby Southern blotting.

Colony PCR. Forty-nine microliters of a stock PCR mixture contain-ing the BD Advantage Taq polymerase (BD Bioscience, San Jose, CA),deoxynucleoside triphosphates (New England BioLabs, Ipswich, MA),buffer, and primers to amplify the gene of interest were aliquoted intoPCR tubes. A small but visible amount of bacterial colony was taken offthe plate (estimated volume, 1 �l) and added to the appropriate tube. Forculture PCR, 1 �l of BCG culture at an OD600 of 0.5 was added to thePCR mixture. Samples were denatured at 95°C for 10 min prior to PCR.Fifteen microliters of a 50-�l PCR mixture was run in each lane.

Western blotting. For Western blot assays assessing SIINFEKL ex-pression, 2.5 � 106 CFU (10 �l of a culture at an OD600 of 0.5) wereisolated from actively growing log-phase BCG cultures. For Western blotassays assessing SIV Gag expression, 2.5 � 107 CFU (100 �l of a culture atan OD600 of 0.5) were isolated. Bacilli were pelleted and washed with 100�l of extraction buffer containing a protease inhibitor. The pellet was thenresuspended in extraction buffer containing a 1� final concentration ofComplete mini protease inhibitor cocktail (Roche), lithium dodecyl sul-fate, and reducing agent (Nupage; Invitrogen), and heated to 95°C for 10min. Samples were run on a 10% bis-Tris 15-lane gel (Invitrogen) for 90min at 100 V. Protein was transferred to a polyvinylidene difluoride mem-brane at 30 V for 1 h and stained with antibody for 1 h. For blot assaysassessing SIINFEKL expression, a rabbit polyclonal serum raised againstthe SIINFEKL peptide conjugated to keyhole limpet hemocyanin wasused as a primary antibody, and a secondary rat anti-rabbit antibody-conjugated antibody (Invitrogen) was used for detection. For blot assaysassessing SIV Gag expression, a high-affinity rat monoclonal antibodydirected against the HA tag (clone 3F10) conjugated to horseradish per-oxidase was used for detection. Antibody binding was detected by chemi-luminescence using the BM chemiluminescence Western blotting sub-strate (Roche) according to the manufacturer’s instructions.

Immunization protocol. A total of 1 � 108 CFU from an activelygrowing log-phase BCG culture (OD600 between 0.6 and 0.9) were iso-lated, pelleted, and resuspended in 1 ml PBS containing 0.2% Tween 20. A100-�l aliquot (107 CFU) was injected intravenously (i.v.) into 4 to 8C57BL/6 mice per group. For immunization with naked plasmid DNA, 25�g of plasmid DNA was injected into each hind leg quadriceps muscle fora total of 50 �g DNA/mouse. Replication-incompetent adenovirus sero-type 5 (Ad5) containing full-length ovalbumin (rAd5-OVA) was obtainedfrom the Gene Transfer Vector Core (University of Iowa, Iowa City, IA).Heterologous boost was accomplished by injecting 106 virus particles (vp)of rAd5-SIINFEKL intramuscularly (i.m.) or 107 vp rAd5-SIVGag into thehind leg quadriceps of mice.

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Tetramer staining. At various time points after vaccination, 100 �l ofperipheral blood was collected from each mouse into RPMI plus 40 U/mlheparin, treated with lysis buffer to remove red blood cells, and stainedusing an H-2Kb–SIINFEKL–phycoerythrin (PE) conjugate or H-2Db–AL11–PE tetramer, prepared as previously described (13) and anti-CD8 –peridinin chlorophyll protein-Cy5.5 antibodies (catalog number 53-6.7;Ebioscience). Results are displayed as the percentage of tetramer-positivecells gated on peripheral CD8� T lymphocytes. The gating strategy em-ployed is described in Fig. S1 in the supplemental material.

Statistical analysis. Statistical analyses were performed by one-wayanalysis of variance (ANOVA) testing using Prism software (GraphPadSoftware, La Jolla, CA).

RESULTSAssay for macrophage MHC class I presentation of epitope pep-tide by mycobacterial vector. To identify BCG genes that influ-ence the infected host cell MHC class I presentation pathway, weestablished an in vitro assay for quantifying MHC class I presen-tation of a mycobacterium-vectored transgenic epitope (Fig. 1A).This assay consisted of incubating a macrophage cell line withtransposon-mutagenized BCG strains expressing a transgenic an-tigen and then coincubating the infected cells with a T cell hybrid-oma expressing a T cell receptor specific for that transgenic anti-gen. The efficiency of antigen presentation can be assessed byquantifying the amount of IL-2 produced by the hybridoma inresponse to the infected macrophage cell line. Variations of thisassay have been previously used to examine TAP1 processing, my-cobacterial viability, and the MOI with regard to MHC class I andclass II antigen presentation during infection with M. tuberculosisand BCG (14).

More specifically, the A3.1A7 macrophage cell line has beenshown to present the immunodominant epitope of chickenovalbumin, SIINFEKL, in association with the H-2Kb MHC class Imolecule (15). As previously demonstrated, when this antigen-presenting cell (APC) line is pulsed with SIINFEKL and then in-cubated with the T cell hybridoma RF33.70, IL-2 is produced bythe RF33.70 T cell hybridoma at levels proportional to the numberof H-2Kb:SIINFEKL complexes expressed on the surface of thepulsed APCs (16). The IL-2 production of the RF33.70 cells, asdetected by ELISA, provides a measure of the efficiency of SIINFEKL presentation by the APC line.

When tested over a range of SIINFEKL concentrations, adetectable IL-2 response was generated by the RF33.70 T cellhybridoma to A3.1A7 cells pulsed with less than 0.1 pg/ml ofSIINFEKL peptide, and there was a linear increase in IL-2 pro-duction at concentrations between 0.1 pg/ml and 1 pg/ml SIINFEKL (Fig. 1B).

We sought to determine if this assay was capable of detectingSIINFEKL presentation when the SIINFEKL epitope was ex-pressed by recombinant mycobacteria. Our initial studies usingparental rBCG strains expressing SIINFEKL resulted in a level ofIL-2 production that was at or below the level of detection. Tooptimize several of the parameters in this assay, we utilized thenonpathological strain M. smegmatis mc2155 as a surrogate forBCG, because M. smegmatis expressing SIINFEKL elicited a de-tectable IL-2 response. We reasoned that M. smegmatis, as a non-pathological saprophytic strain of mycobacteria, would not con-tain the theoretical mechanisms to suppress antigen presentation

FIG 1 In vitro screen for identifying mutant rBCG strains with enhanced MHC class I presentation of transgenic protein. (A) Schematic representation of the invitro screening of a transposon-mutagenized rBCG library for enhanced MHC class I presentation. Macrophages infected with rBCG-SIINFEKL transposonmutant strains (yellow) are compared to macrophages infected with the parental rBCG-SIINFEKL strain (blue) for their ability to stimulate IL-2 production bythe RF33.70 T cell hybridoma. Presentation of increased amounts of SIINFEKL on H-2Kb elicits more IL-2 production, one of several mechanisms through whichthe mutated bacteria may increase RF33.70 hybridoma activity. (B) IL-2 production by RF33.70 cells in response to exogenous SIINFEKL peptide over a rangeof concentrations. (C) IL-2 production by RF33.70 cells. A3.1A7 cells were activated with various concentrations of exogenous rIFN-� and infected withrecombinant M. smegmatis expressing the 19-kDa SIINFEKL fusion protein (rSmeg-SIINFEKL) over a range of MOIs.

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for which we were searching in BCG. In testing M. smegmatismc2155 strains expressing SIINFEKL from various shuttle plas-mids, we determined that the highest levels of IL-2 were producedin response to M. smegmatis expressing the SIINFEKL epitopefused to the full-length 19-kDa lipoprotein (Rv3763) under thecontrol of the Hsp60 promoter, a plasmid first published byHinchey et al. (12).

The sensitivity of this assay for detecting SIINFEKL presenta-tion in association with MHC class I following ingestion of therecombinant M. smegmatis by the A3.1A7 line was tested over arange of MOIs and after activation by increasing concentrations ofIFN-� (Fig. 1C). The addition of IFN-� was critical for the pro-duction of IL-2 by infected APCs. In APCs activated with IFN-�,limited production of IL-2 was detected at an MOI as low as 2.5.IL-2 production increased as the MOI increased and was highestwhen the A3.1A7 cells were preactivated with the highest concen-tration of IFN-�, 250 U/ml. The highest IL-2 production, approx-imately 15 pg/ml, was detected in response to A3.1A7 cells infectedat an MOI of 40 in the presence of 250 U/ml of IFN-�; however, atMOI levels above 20, the host cell viability was compromised. Forscreening of BCG mutant strains, infection of A3.A17 cells wasdone at an MOI of 20, because this MOI elicited the highestamount of IL-2 without compromising host cell viability.

The level of IL-2 production in response to mycobacteriallyvectored SIINFEKL (0 to 15 pg IL-2/ml) was lower than the level ofIL-2 production generated in response to free SIINFEKL peptide(0 to 275 pg IL-2/ml) (data not shown). We adapted this in vitropresentation assay into a high-throughput 96-well format forscreening a large library of transposon-mutagenized rBCG strainsto determine which mutant rBCG strains generated increased pre-sentation of the transgenic protein.

Creation of the rBCG-SIINFEKL transposon mutant library.We created a parental strain of rBCG by transforming BCG Dan-ish with the pMV261-19kDaSIINFEKL plasmid and then gener-ated an rBCG library that could be tested in the in vitro presenta-tion assay by transposon mutagenesis of this parental strain withthe phage pCS9Hygro1-MAR (phAE180) (17). This created a li-brary of several thousand mariner transposon mutant strains, eachhaving a disruption at a random TA dinucleotide (18). The trans-posase enzyme is lost following insertion of the transposon intothe BCG genome, fixing the insertion site. Transposon mutagen-esis did not appear to be a source of significant variation in SIINFEKL expression from rBCG, as tested by Western blotting (seeFig. S2 in the supplemental material).

In vitro screening of antigen presentation by APCs infectedwith individual clones of the rBCG mutant library. A total of3,290 individual transposon-mutagenized rBCG strains were usedto infect A3.1A7 cells, and the infected cells were then tested twicein duplicate in the peptide/MHC class I presentation assay. Datashown in Fig. 2A are representative of the data obtained from atypical assay. The parental rBCG-SIINFEKL-infected A3.1A7 cellsreproducibly elicited 4 to 6 pg/ml of IL-2. Most mutant strainselicited a level of IL-2 comparable to that induced by the parentalstrain, and some mutant strains generated little or no IL-2 pro-duction. However, mutant strains such as C60, J13, and K14 (Fig.2A) elicited a markedly higher level of IL-2 production and weretherefore selected for in vivo testing. These mutant strains wereselected for further in vivo analysis based on their outperformingthe parental strain and their peers within the individual ELISAplates. Figure S3 in the supplemental material summarizes theIL-2 responses elicited by the selected pool of mutants that in-

FIG 2 Identification of novel rBCG transposon mutant strains that generate enhanced MHC class I-mediated presentation in vitro and increased CD8� T cellresponses in vivo. (A) Representative results of IL-2 production by the T cell hybridoma RF33.70 stimulated by the presentation of SIINFEKL from A3.1A7 cellsexposed to exogenous SIINFEKL at 312 pg/ml or 62.5 pg/ml and infected with various rBCG transposon mutant strains. The dotted line indicates the level of IL-2production in response to the parental rBCG-SIINFEKL strain. (B) Representative results for the primary SIINFEKL-specific CD8� T cell response generated inperipheral blood 7 days after vaccination with 107 CFU of selected rBCG mutant clones (n 4). (C) Secondary SIINFEKL-specific CD8� T cell responses in miceprimed with selected rBCG transposon mutant clones. SIINFEKL-specific CD8� T cell responses were assessed 10 days after boosting with a suboptimal dose ofrAd-SIINFEKL (106 vp) in mice primed with the indicated rBCG strain (n 4). Data are presented as means � standard errors of the means.

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creased primary and secondary CD8� T cell responses in the invivo portion of this work.

In vivo screening of selected rBCG mutant strains. Based onthe results of the in vitro screen, 76 strains with increased IL-2production in vitro were tested for their ability to induce increasedantigen presentation to CD8� T cells in vivo. This increased anti-gen presentation by APCs and the resulting increased T cell ex-pansion in vivo can be measured by quantifying SIINFEKL-spe-cific T cells as a percentage of the total T cell population inperipheral blood by using H-2Kb:SIINFEKL tetramers.

Groups of four C57BL/6 (H-2b) mice were inoculated intrave-nously with 107 CFU of each selected strain, and the primarySIINFEKL-specific CD8� T cell responses were assessed byH-2Kb:SIINFEKL tetramer staining 7, 14, and 21 days later. Theparental strain consistently generated mean peak tetramer re-sponses ranging from 0.4% to 0.8% of total peripheral bloodCD8� T cells at day 7. Nearly half of the strains selected for in vivoimmunogenicity studies (36/76) elicited greater SIINFEKL-spe-cific CD8� T cell responses than those induced by the parentalstrain. Representative data from an assay in which mutant strainswere tested for their ability to generate primary in vivo tetramerresponses are shown in Fig. 2B. In contrast, only 17% (2 of 12) ofunselected strains generated enhanced tetramer responses whentested in vivo. Thus, the in vitro screen enriched the pool of mu-tants with increased immunogenicity for CD8� T cell responses.

Several months after the primary vaccination, all immunizedmice were boosted with a suboptimal dose of 106 vp of rAd5-SIINFEKL i.m. to provide the greatest discrimination between thesevarious mutant rBCG strains for their ability to prime in a rBCG/rAd5 prime-boost vaccine regimen. Upon boosting with rAd5-SIINFEKL, 16 of the 36 novel strains inducing elevated primaryresponses (44%) also primed for increased secondary responsesrelative to the parental strain of rBCG. Figure 2C shows represen-tative data following boosting of one cohort of mice. Ten daysafter boosting with rAd5-SIINFEKL, mice primed with mutant

strains had tetramer responses between 4 and 7%, significantlygreater than those of mice primed with the parental rBCG strain.

Identification of BCG genes that are associated with in-creased MHC class I-restricted CD8� T cell responses. To deter-mine the genes disrupted by transposon insertions, we isolatedgenomic DNA from the 16 mutant strains of rBCG that generatedaugmented primary and secondary SIINFEKL-specific CD8� Tcell responses in vivo and sequenced the insertion site. The 16strains identified through the screening process had disruptions in14 unique genes/operons. We named the 14 unique strains Inhib-itor of Class One (ICO) strains A to N. These strains are listed inFig. 3A, along with the specific library clone number, the BCGopen reading frames (ORFs) disrupted, and the correspondinghomologous genes in M. tuberculosis H37Rv. Genes encodingORFs that were identified and characterized encode proteins thatperform cellular functions ranging from pathogenicity to DNArepair (ICOs A, B, D, F, G, and M). ORFs that have not beenpreviously characterized encode proteins with putative functionsranging from transcriptional regulation, DNA modification, pro-tein modification, or metabolic regulation to protein transport.Two of the immunomodulatory operons identified in the screen-ing process were independently disrupted by two transposon in-sertions (ICO J, C57 and AF25; ICO C, A79 and AK27). The loca-tions of the mutated genes in the ICO strains within the BCGgenome are shown in Fig. 3B. Consistent with the wide variety offunctions associated with the affected genes, the gene locations aredistributed throughout the BCG genome. No “islands of pathoge-nicity” could be identified in the suppression of CD8� T cell re-sponses.

Enhanced CD8� T cell responses primed by mutant strainAZ11 (ICO M) were abrogated by complementation. To confirmthat the phenotype of increased immunogenicity was a conse-quence of the transposon disruption of a single locus in theseselected mutants, we complemented the ICO M strain (cloneAZ11). In this strain, a transposon insertion disrupted a two-gene

FIG 3 Summary of gene disruptions associated with both enhanced primary and secondary MHC class I-restricted transgene product-specific CD8� T cellresponses. (A) Disrupted genes in mutant rBCG strains that were associated with increased MHC class I presentation in vitro and increased transgene product-specific primary and secondary CD8� T cell responses in vivo. (B) Map of transposon insertion sites within the mycobacterial genome that gave rise to mutantclones which elicited increased transgene product-specific CD8� T cell responses.

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operon containing echA18 (BCG_3445) and amiD (BCG_3446),which we complemented by using a cloned fragment that includedboth genes as well as their endogenous promoter. The echA18 geneis predicted to encode an enzyme involved in fatty acid metabo-lism, while amiD is one of the better-characterized genes identi-fied in our screen and has been demonstrated to play a role inpeptidoglycan (PG) synthesis and turnover (19, 20). We hypoth-esized that this operon might be important for regulating the gen-eration and composition of the mycobacterial cell wall andthereby influence the host response through differential engage-ment of host PRRs. Besides the existence of a potential mechanismfor this mutant, this mutant was attractive for complementationbecause of the short length of the operon that facilitated cloning.The cloned complementing fragment was inserted into the inte-grating vector pYUB114 and transformed into ICO M (Fig. 4A).PCR analysis showed that complementation by pYUB1141-ICOM resulted in the introduction of the BCG_3445 gene in the ICOM transposon strain of rBCG (Fig. 4B). Seven days after vaccina-tion with these strains, the transposon-mutated ICO M strain gen-erated a mean tetramer response of 0.9%, approximately twice themagnitude of the responses generated by the parental BCG-SIINFEKL strain (0.5%) and the ICO M strain with pYUB1141 (0.5%)(Fig. 4C). Therefore, the enhanced CD8� T cell immunogenicityof the ICO M strain reverted to wild-type levels when a functionalcopy of the ICO M gene was introduced into this mutant strain ofrBCG. This finding demonstrates that the disruption of the two-gene operon containing echA18 and amiD was responsible for theenhanced immunogenicity of the ICO M rBCG mutant strain.

Comparison of the immunogenicities of one novel mutantrBCG strain and that of an existing modified BCG with plasmidDNA. One rationale for generating and selecting these novelstrains of BCG was to employ them as priming vectors in a heter-ologous prime-boost vaccination regimen. We compared the abil-ity of one of these novel strains to prime for adenoviral boostagainst the priming agents plasmid DNA and AERAS 401 (21).AERAS 401 is an rBCG strain being considered for evaluation inhuman trials and has been modified to express the perfringolysingene from Clostridium perfringens, allowing the bacterium to formpores in the endosomal compartments and enhancing antigen accessto the MHC class I pathway. In order to assess CD8� T cell responsesto a common transgene across all three vector platforms, we trans-formed AERAS 401 with pMV261-19kDaSIINFEKL and created apVRC2000 plasmid expressing SIINFEKL.

At the time of priming, mutant strain ICO J demonstrated theability to induce primary SIINFEKL-specific CD8� T cell responsesin vivo that were significantly greater than the parental BCG strain(1.0% versus 0.4%) as well as significantly greater than that withAERAS 401 (1.0% versus 0.2%) (Fig. 5B). The peak of the primaryCD8� T cell response to DNA vaccine occurred after 14 days, 7 dayslater than the peak primary CD8� T cell response to rBCG. We ob-served that the peak SIINFEKL-specific response to ICO J at day 7 wasof comparable magnitude to the peak response to plasmid DNA vac-cine at day 14 (1.6% versus 1.4%) (Fig. 5B).

When boosted with a suboptimal dose of rAd5-SIINFEKL (1 �106 vp) 10 weeks after the priming vaccination, mice primed withICO J generated mean secondary SIINFEKL-specific CD8� T cell

FIG 4 Complementation of a novel rBCG transposon-mutated strain reduces CD8� T cell immunogenicity. (A) Schematic diagram of the genomic DNAsegment cloned from wild-type BCG containing wild-type copies of both genes in the two-gene operon disrupted in ICO M. The cloned fragment was insertedinto the integrating plasmid pYUB1411 to construct the complemented rBCG strain ICO M with pYUB1141-ICO M. (B) Culture PCRs for amplification of afragment of the wild-type BCG genome containing the BCG_3445 gene (echA18) from wild-type BCG, BCG-SIINFEKL, ICO M, and the complemented strainICO M with pYUB1141-ICO M indicated successful complementation of the operon containing the BCG_3445 and BCG_3446 genes. (C) Primary SIINFEKL-specific CD8� T cell responses at day 7 induced by the ICO M strain of rBCG were greater than those induced by the parental strain. Complementation of the ICOM strain of rBCG reduced the SIINFEKL-specific CD8� T cell responses to the level of the parental rBCG. n 7 (for BCG-SIINFEKL); n 8 (for all othergroups). Data are presented as means � standard errors of the means and represent the results of two independent experiments. P values were determined byANOVA: *, P � 0.05; **, P � 0.01.

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responses that were significantly greater than in mice primed witheither the parental or the AERAS 401 SIINFEKL-expressing strain(9.7% versus 3.9% and 3.4%, respectively) (Fig. 5C). The magni-tude of the secondary response in mice primed with ICO J wascomparable to that of mice primed by plasmid DNA vaccination(9.7% versus 9.7%). Moreover, mice immunized with the subop-timal dose of rAd5-SIINFEKL without a SIINFEKL prime did notgenerate significant SIINFEKL-specific CD8� T cell responses(Fig. 5C), indicating that responses in SIINFEKL-primed micerepresent secondary CD8� T cell responses rather than de novoprimary responses to the rAd5 vector.

Reconstruction of transposon mutant strains by site-di-rected mutagenesis. Upon repeated testing, rBCG strains withtransposon insertions in the ICO J operon (mutant strains C57and AF25) consistently primed for primary and secondary SIINFEKL-specific CD8� T cell responses that were greater than thoseelicited by the parental strain of rBCG. C57 was one of the firstmutants to be identified in our screen, and besides the fact thatthese two independent transposon mutations located in a singlelocus yielded one consistent phenotype, we were intrigued be-cause the genes in this locus have yet to be characterized. To con-firm that the increased immunogenicity of these rBCG strains wasa consequence of disrupting this specific locus rather than unre-lated mutations at other gene loci, we reconstructed the mutantrBCG strain ICO J from wild-type BCG Danish by site-directedmutagenesis using the well-established technical strategy of spe-cialized transduction (22). An AES targeting the entire ICO Joperon spanning BCG_2587-2590 was synthesized, allowing re-placement of the targeted genes with a hygromycin antibiotic re-sistance gene (Fig. 6A). This AES was subsequently cloned into theprophage phAE159 and used to transduce wild-type BCG. Theresulting reconstructed rBCG strain lacking BCG_2587-2590 wastermed ICO J Rec. Successful reconstruction was confirmed by

PCR and by Southern blotting (see Fig. S4 in the supplementalmaterial).

Vaccination with the reconstructed strain ICO J Rec elicitsincreased CD8� T cell responses comparable to those from thetransposon mutant ICO J. We assessed the ability of the ICO J Recstrain to stimulate CD8� T cell responses to the model transgenicantigen SIINFEKL. Clones of the reconstructed strain were trans-formed with the plasmid pMV261–19kDaSIINFEKL. The result-ing strain, ICO J Rec-SIINFEKL, was assessed for SIINFEKL ex-pression (see Fig. S5 in the supplemental material) and thenevaluated as an immunogen in mice. The original transposon mu-tant strain ICO J elicited a significantly enhanced primary SIINFEKL-specific CD8� T cell response compared to the parentalBCG-SIINFEKL strain (1.1% versus 0.4%) (Fig. 6B). Importantly,the reconstructed rBCG strain ICO J Rec was comparable in itsimmunogenicity to the transposon mutant strain (1.0% versus1.1%) and significantly above that of the parental BCG-SIINFEKL. These results demonstrated that the targeted deletion of theoperon spanning BCG_2587-2590 resulted in a novel rBCG strain(ICO J Rec) that recapitulated the enhanced immunogenicity ob-served in the original transposon mutant strains C57 and AF25.

Vaccination with novel rBCG vectors efficiently primesCD8� T cell responses to an SIV vaccine antigen. A major goal ofthis work was to develop novel vectors for use as vaccine primingimmunogens. To determine if the novel strains of rBCG identifiedand reconstructed are effective vectors for priming CD8� T cellresponses to SIV, we transformed the reconstructed BCG strainwith the plasmid pSL10 containing the SIV gag transgene andassessed transgene expression by Western blotting (see Fig. S6 inthe supplemental material). The SIV Gag protein includes theH-2Db-restricted immunodominant AL11 epitope, and thereforethe immunogenicity of this vaccine construct could be evaluatedin C57BL/6 mice. The pSL10 plasmid is a multicopy episomal E.

FIG 5 The transposon-mutated strain ICO J generates primary and secondary SIINFEKL-specific CD8� T cell responses that are greater in magnitude than thoseelicited by another mutant rBCG vector strain and comparable to those generated by plasmid DNA. (A) Transposon mutant ICO J generated greater SIINFEKL-specific CD8� T cell responses than did recombinant AERAS 401 expressing SIINFEKL in mice. BCG wild type, n 4; BCG-SIINFEKL, n 9; AERAS, n 10; ICO J, n 5. (B) Primary SIINFEKL-specific CD8� T cell responses to ICO J were comparable to the peak primary response elicited by vaccination withplasmid DNA. Peak tetramer responses occurred on day 7 following inoculation with the rBCG constructs and on day 14 following inoculation with the plasmidDNA construct. BCG wild type, n 8; BCG-SIINFEKL, n 7; ICO J, n 7; DNA-SIINFEKL, n 8. (C) Secondary responses in mice, after receiving aheterologous prime-boost regimen. ICO J-primed, rAd-boosted SIINFEKL-specific CD8� T cell responses were comparable to secondary responses generatedby DNA prime and rAd boost and were greater than responses generated by rAERAS 401 prime with rAd boost. All secondary SIINFEKL-specific CD8� T cellresponses were tested on day 10 following boosting with a suboptimal dose of rAd5-SIINFEKL. BCG wild type, n 7; BCG-SIINFEKL, n 8; AERAS, n 6; ICOJ, n 5; DNA-SIINFEKL, n 7. Data are presented as means � standard errors of the means and represent the results of three independent experiments. P valueswere determined by ANOVA: ***, P � 0.001.

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coli-mycobacterium shuttle plasmid that encodes a fusion proteinconsisting of the signal sequence from the Ag85b secreted myco-bacterial protein and the full-length SIV Gag protein, as well as anHA tag.

Wild-type BCG, BCG-pSL10, and ICO J Rec-pSL10 (Fig. 6C)were administered to age-matched C57BL/6 mice to assess theability of these strains to prime for SIV Gag-specific CD8� T cellresponses in a prime-boost regimen with rAd5-SIV gag. Re-sponses to the SIV Gag AL11 epitope presented on H-2Db weregenerally lower in magnitude than the strongly immunodominantresponses elicited by SIINFEKL presented on H-2Kb; conse-quently, we did not observe significant SIV Gag AL11-specificprimary CD8� T cell responses following a priming inoculation.However, since rBCG strains can efficiently prime for robust sec-ondary responses following a heterologous boost even in the ab-sence of detectable primary transgene-specific responses (23),these mice were boosted with a suboptimal dose of rAd5-SIV Gag8 weeks after vaccination with BCG.

Ten days after boosting with rAd5-SIV Gag, we observed sig-nificantly enhanced AL11-specific CD8� T cell responses in micethat had been primed with the strain ICO J Rec-pSL10 comparedto mice primed with the parental BCG-pSL10. Vaccination withICO J Rec-pSL10 primed for an AL11-specific CD8� T cell re-sponse (9%) that was approximately 4-fold higher than after vac-cination with the parental rBCG-pSL10 strain (2.2%). These re-sults demonstrated that the novel strain ICO J Rec was a moreeffective priming vector for SIV Gag-specific responses than theunmutated recombinant BCG strain.

DISCUSSION

In this study, we examined the immunogenicity of rBCG strains inorder to identify genes that impact the ability of BCG-infectedcells to present peptides through the MHC class I pathway, first byperforming a high-throughput assessment of MHC class I presen-tation in vitro and then by evaluating transgene product-specificCD8� T cell responses in vivo. We were able to screen over 3,200individual mutant strains of BCG, and we isolated 16 strains thatdemonstrated enhanced prime and boost CD8� T cell responsesin vivo, which mapped to 14 different genetic loci. Given the largenumber of strains that appeared to elicit increased IL-2 by thehybridoma, this study is by no means an exhaustive assessment ofthe genes that impact MHC class I antigen presentation. Due tolimitations in time and resources, only a select subset of strainswere chosen to be tested in vivo. Therefore, we believe it is likelythat there will be additional mutants that demonstrate enhancedimmunogenicity through the MHC class I pathway in vivo. Thestrains identified so far are those that were characterized as capa-ble of mediating enhanced primary CD8� T cell responses as wellas enhanced priming for secondary CD8� responses to vectoredantigens, and they represent promising novel candidate vaccinevectors for further development.

Among the elements necessary for further development areexploration of the route of immunization, removal of antibioticresistance cassettes, and assessment of these mutations in othermycobacterial vaccine strains. The enhanced immunogenicity ofthese strains must be assessed using traditional vaccination routes

FIG 6 The site-directed mutant rBCG strain ICO J Rec can generate greater primary SIINFEKL-specific CD8� T cell responses and prime for greater secondarySIV Gag-specific CD8� T cell responses than wild-type BCG. (A) Schematic diagram of the region of the BCG genome targeted for site-directed mutagenesis toreconstruct the ICO J transposon mutant strain in wild-type BCG. Specialized transduction was used to delete genes BCG_2587 to BCG_2590 from wild-typeBCG. (B) The site-directed mutant ICO J Rec expressing SIINFEKL elicited primary SIINFEKL-specific CD8� T cell responses that were comparable inmagnitude to the respective transposon mutant strain ICO J, both of which generated significantly higher responses than wild-type BCG-SIINFEKL. n 8 (allgroups). (C) Secondary SIV Gag-specific responses in a heterologous prime-boost regimen, priming with rBCG strains, followed by a suboptimal dose ofrAd5-SIV Gag. ICO J Rec expressing SIV Gag primed for SIV Gag epitope-specific CD8� T cell responses more efficiently than wild-type BCG-expressing SIVGag. n 8 (all groups). Data are presented as means � standard errors of the means, and represent the results of two independent experiments. P values weredetermined by ANOVA: *, P � 0.05; **, P � 0.01.

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(e.g., subcutaneous or intradermal) from verified lyophilized orfrozen vaccine lots. In addition, modern safety standards requirethe elimination of any antibiotic drug resistance from the bacte-rium, and the use of auxotrophic mutations may be warranted tolimit replication, particularly if these will be administered to pa-tients who are at risk of contracting HIV. Furthermore, recentadvances in live attenuated M. tuberculosis strains combine astrong safety profile with a more immunogenic phenotype andmay be improved by the mutations identified in our study.

In the multitiered testing strategy used to identify these 14 loci,the library of transposon-mutated BCG strains was created usingan MOI of the transposon that would induce on average a singletransposon disruption per cell. It is important to exclude the pos-sibility that secondary spontaneous mutations are responsible forthe observed phenotype, or secondary unidentified transposondisruptions are present. We performed several studies to demon-strate that the genes identified as disrupted by transposon mu-tagenesis had the ability to alter immunogenicity. First, in map-ping the location of transposon disruptions in these strains thatgenerated increased immunogenicity, we identified independenttransposon strains whose disruptions mapped to the same operon(ICO C, A79 and AK27; ICO J, C57 and AF25). Second, we per-formed complementation of a mutant strain with a wild-type copyof the disrupted gene and observed reversion to the wild-typephenotype. Third, we employed site-directed mutagenesis to de-lete an operon in wild-type BCG that resulted in a recombinantstrain that recapitulated the phenotype of the transposon-dis-rupted mutant strains. Thus, we confirmed the contributions ofthe genes identified in the screen of the transposon mutant libraryof rBCG strains.

The transposon insertion sites in the 16 selected rBCG strainswere sequenced and mapped to 14 unique genes/operons. We sawno evidence of clustering of these 14 genes or operons in the ge-nome, and the 14 unique loci encoded proteins with a wide rangeof predicted functions, including several genes whose putativefunctions have no obvious immunomodulatory activity. Thisleaves open the question as to how each of these genes influencesimmunogenicity in the host. Potential mechanisms include inter-ference with the host cell antigen processing and presentationpathway, active suppression of the host immune responses, alter-ation in the growth rate of the bacterium, alterations in the fitnessof the bacterium in the intracellular environment, and alterationsin the shedding of antigen into the MHC presentation pathway.We did identify the gene pks12, which contributes to phthioceroldimycocerosate (PDIM) synthesis, and the gene cmaA2, whichgives rise to cyclopropanation within mycolic acid; both of theseenzymes have been implicated in masking highly immunogenicepitopes (24, 25). In the mutant strain ICO M, which we comple-mented in these studies, the genes affected were found in a two-gene operon containing BCG_3445 (echA18) and BCG_3446(amiD). EchA18 is predicted to be a hydratase that metabolizesfatty acids, while AmiD plays a role in PG synthesis and turnover(19, 20). Both of these processes may affect the generation andmaintenance of the mycobacterial cell wall, and as such they mayincrease immunogenicity by altering which host PRRs are engagedupon encountering mycobacteria. Based on the identification ofthis diverse array of genes, we hypothesized that the effect of themajority of BCG genes identified in our screen on CD8� T cellresponses is not through direct interference with the antigen pro-cessing and presentation machinery itself, but rather via alterna-

tive mechanisms. However, these proposed mechanisms need fur-ther evaluation; additional studies will be required to determinehow each of the genes we identified influences the ability of theinfected host cells to present antigen through the MHC class Ipathway and generate CD8� T cell responses. Our studies werealso limited to analysis of CD8� T cell responses, and the influenceof these strains on the generation of transgene-specific CD4� Tcell responses has not been tested in mice. Thus, it is possible thatsome mutations affect MHC class I presentation exclusively, whileother mutations may increase the availability of antigen for allantigen presentation pathways, attenuate the fitness of the bacte-rium, or modulate the host immune response through the cyto-kine pathways.

The observation that such a wide array of genes involved insuch diverse cellular functions influences the immunogenicity ofrBCG constructs underscores the complexity of the interactionsbetween BCG and the host. It also suggests that it may be possibleto construct viable compound BCG strains by disrupting combi-nations of the genes identified in this study. Targeted mutation ofseveral genes involved in diverse functional pathways may facili-tate a synergistic enhancement of the immunogenicity of rBCGconstructs.

In conclusion, in this study we have identified a number ofgenes that modulate the MHC class I presentation of mycobacte-rium-vectored proteins, and we showed that vaccine vectors cre-ated through the deletion of these genes mediate enhancedepitope-specific CD8� T cell responses. The ability of these se-lected strains to prime for robust secondary responses in a heter-ologous prime-boost strategy indicates that these rBCG strainsmay prove useful as enhanced M. tuberculosis vaccines or, givenfurther development, as a novel class of vaccine vectors.

ACKNOWLEDGMENTS

Funding for this study was provided by the Collaboration for AIDS Vac-cine Discovery program grants OPP38614 and OPP1033104 of the Bill &Melinda Gates Foundation and the NIH-funded program (P30AI060354) to the Harvard University Center for AIDS Research (CFAR).Portions of this work were also supported by grants from NIH/NIAIDgrants RO1 AI093649, RO1 AI070258, and PO1 AI063537. In addition,W.R.J. acknowledges generous support from the NIH Centers for AIDSResearch Grant (CFAR) AI-051519 at the Albert Einstein College of Med-icine and NIH grant AI26170.

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