The human CD1-restricted T cell repertoire is limited to cross-reactive antigens: implications for host responses against immunologically related pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.     

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.     

Conservation of CD1 intracellular trafficking patterns between mammalian species

Dendritic cells (DC) are potent APCs that sample Ags from the surrounding environment and present them to naive T cells using cell surface Ag-presenting molecules. The DC in both lymphoid and nonlymphoid tissues express high levels of CD1, a cell surface glycoprotein capable of presenting lipids and glycolipids to T cells. Distinct group 1 CD1 isoforms (CD1a, -b, -c) in man are known to traffic to different parts of the endocytic system where microbial Ags may be sampled. Guinea pigs are the only known rodent species that express the group 1 CD1 proteins. Therefore, we examined the expression and trafficking of guinea pig CD1 (gpCD1) isoforms on isolated DC. Confocal microscopy using mAbs specific for individual gpCD1 isoforms revealed differential trafficking of two distinct CD1b isoforms within DC. Colocalization of MHC class II was observed with the gpCD1b1 isoform, consistent with localization in the late endosomes of DC. In contrast, the gpCD1b3 isoform lacks an endosomal sorting motif and remains on the cell surface. Following incubation with Mycobacterium tuberculosis lipoarabinomannan, colocalization of endocytosed lipoarabinomannan with the gpCD1b1 isoform was observed but not with the gpCD1b3 isoform, which remained primarily on the cell surface. These data demonstrate that guinea pig DC express CD1 isoforms with unique trafficking patterns that recapitulate the patterns seen for human CD1 isoforms. This suggests evolutionary pressure for a conserved mechanism in mammals that allows CD1 to sample lipid Ags from various subcompartments of the endocytic system.     

Cutting edge: major CD8 T cell response to live bacillus Calmette-Guérin is mediated by CD1 molecules

MHC class I-restricted CD8(+) T cells are a crucial component of the host defense against mycobacterial infection in mice, but it has often proved very difficult to identify the CD8 T cell response in humans. Human group 1 CD1 molecules (CD1a, -b, -c) mediate MHC-independent presentation of mycobacteria-derived lipid and glycolipid Ags to CD8(+) T cells, and their intracellular localization to the endocytic system may favor efficient monitoring of phagosome-resident mycobacteria. Here, we show that bacillus Calmette-Guérin (BCG)-immunized subjects contain a significant circulating pool of CD8(+) T cells that recognize BCG-infected DCs in a CD1-dependent, but MHC-independent, manner. These CD1-restricted T cells efficiently detected live, rather than dead, BCG and produced IFN-gamma, an important cytokine for protection against mycobacterial infection. These results emphasize that lipid-reactive CD8(+) T cells may contribute to host defense against mycobacterial infection.     

The role of group 1 and group 2 CD1-restricted T cells in microbial immunity

Group 1 and group 2 CD1 present both self and microbial lipid antigens to T cells. While group 1 CD1-restricted T cells are known for their ability to recognize mycobacterial glycolipid antigens, group 2 CD1-restricted T cells are recognized as regulatory T cells that can influence the outcome of innate and adaptive immune responses. The evidence that these T cells contribute to host defense against infectious diseases is reviewed.     

CD1a-, b-, and c-restricted TCRs recognize both self and foreign antigens

Individual CD1-restricted T cells can recognize either endogenous or foreign lipid Ags, but the extent to which the same CD1-restricted TCR can react to both self and microbial lipids is unknown. In this study, we have identified CD1a-, CD1b-, and CD1c-restricted T cells from normal human donors that induce cytolysis and secrete copious IFN-gamma in response to self-CD1 expressed on monocyte-derived dendritic cells. Remarkably, microbial Ags presented by CD1 are even more potent agonists for these same T cells. The alphabeta T cell receptors from such clones are diverse and confer specificity for both self-CD1 and foreign lipid Ags. The dual reactivity of these CD1-restricted cells suggests that the capacity for rapid responses to inflammatory stimuli without memory coexists with the capacity for strong Ag-specific responses and the generation of memory in vivo.     

The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-gamma and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, alpha-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.     

Mycobacterial lipopeptides elicit CD4+ CTLs in Mycobacterium tuberculosis-infected humans

In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-gamma+ T cells/10(5) PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.     

Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.     

IL-4 and T cells are required for the generation of IgG1 isotype antibodies against cardiolipin

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either alphabeta TCR(+), gammadelta TCR(+), CD4(+), CD8(+), or NK1.1(+) T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.     

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.     

Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.     

Airway delivery of soluble mycobacterial antigens restores protective mucosal immunity by single intramuscular plasmid DNA tuberculosis vaccination: role of proinflammatory signals in the lung

Protection by parenteral immunization with plasmid DNA vaccines against pulmonary tuberculosis (TB) is very modest. In this study, we have investigated the underlying mechanisms for the poor mucosal protective efficacy and the avenues and mechanisms to improve the efficacy of a single i.m. immunization with a monogenic plasmid DNA TB vaccine in a murine model. We show that i.m. DNA immunization fails to elicit accumulation of Ag-specific T cells in the airway lumen despite robust T cell responses in the spleen. Such systemically activated T cells cannot be rapidly mobilized into the airway lumen upon Mycobacterium tuberculosis exposure. However, airway deposition of low doses of soluble mycobacterial Ags in previously immunized mice effectively mobilizes the systemically activated T cells into the airway lumen. A fraction of such airway luminal T cells can persist in the airway lumen, undergo quick, robust expansion and activation and provide marked immune protection upon airway M. tuberculosis exposure. Airway mucosal deposition of soluble mycobacterial Ags was found to create a tissue microenvironment rich in proinflammatory molecules including chemokines and hence conducive to T cell recruitment. Thus, in vivo neutralization of MIP-1alpha or IFN-inducible protein-10 markedly inhibited the accumulation of Ag-specific T cells in the airway lumen. Our data suggest that immunoprotective efficacy on the mucosal surface by i.m. plasmid DNA immunization could be substantially improved by simple mucosal soluble Ag inoculation and restoration of mucosal luminal T cells. Our study holds implication for the future design of DNA vaccination strategies against intracellular infections.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

The secreted lipoprotein, MPT83, of Mycobacterium tuberculosis is recognized during human tuberculosis and stimulates protective immunity in mice

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83(127-135) (PTNAAFDKL) as the dominant H-2(b)-restricted CD8(+) T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8(+) T cell responses to MPT83(127-135). Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines.     

Mucosal luminal manipulation of T cell geography switches on protective efficacy by otherwise ineffective parenteral genetic immunization

Genetic immunization holds great promise for future vaccination against mucosal infectious diseases. However, parenteral genetic immunization is ineffective in control of mucosal intracellular infections, and the underlying mechanisms have remained unclear. By using a model of parenteral i.m. genetic immunization and pulmonary tuberculosis (TB), we have investigated the mechanisms that determine the failure and success of parenteral genetic immunization. We found that lack of protection from pulmonary Mycobacterium tuberculosis (M.tb) challenge by i.m. immunization with a recombinant adenovirus-vectored tuberculosis vaccine was linked to the absence of M.tb Ag-specific T cells within the airway lumen before M.tb challenge despite potent T cell activation in the systemic compartments. Furthermore, pulmonary mycobacterial challenge failed to recruit CD8 T cells into the airway lumen of i.m. immunized mice. Such defect in T cell recruitment, intra-airway CTL, and immune protection was restored by creating acute inflammation in the airway with inflammatory agonists such as virus. However, the Ag-specific T cells recruited as such were not retained in the airway lumen, resulting in a loss of protection. In comparison, airway exposure to low doses of soluble M.tb Ags not only recruited but retained Ag-specific CD8 T cells in the airway lumen over time that provided robust protection against M.tb challenge. Thus, our study reveals that mucosal protection by parenteral immunization is critically determined by T cell geography, i.e., whether Ag-specific T cells are within or outside of the mucosal lumen and presents a feasible solution to empower parenteral immunization strategies against mucosal infectious diseases.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Generation of genome-wide CD8 T cell responses in HLA-A*0201 transgenic mice by an HIV-1 ubiquitin expression library immunization vaccine.

HIV-1 is a fundamentally difficult target for vaccines due to its high mutation rate and its repertoire of immunoevasive strategies. To address these difficulties, a multivalent, proteasome-targeted, live genetic vaccine was recently developed against HIV-1 using the expression library immunization approach. In this HIV-1 vaccine all open reading frames of HIV-1 are expressed from 32 plasmids as Ag fragments fused to the ubiquitin protein to increase Ag targeting to the proteasome to enhance CTL responses. In this work we demonstrate the ability of the HIV-1 library vaccine to simultaneously provoke robust HLA-A*0201-restricted T cell responses against all 32 HIV-1 library vaccine Ags after single immunization by gene gun. These CD8 T cell responses included HLA-A*0201-restricted CTL activity, CD8/IFN-gamma T cell responses, and HLA tetramer binding against defined immunodominant epitopes in gag, pol, env, and nef as well as potent CD8/IFN-gamma responses against undefined HLA-A*0201-restricted epitopes in all remaining Ags of the library. CD8 responses mediated by single gag, pol, env, and nef plasmids from the vaccine demonstrated little reduction in specific T cell responses when these plasmids were diluted into the context of the full 32-plasmid library, suggesting that Ag dominance or immune interference is not an overt problem to limit the efficacy of this complex vaccine. Therefore, this work demonstrates the ability of the HIV-1 library vaccine to generate robust multivalent genome-wide T cell responses as one approach to control the highly mutable and immunoevasive HIV-1 virus.