Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

Prior vaccination increases the epitopic breadth of the cytotoxic T-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection

Although recent evidence has confirmed the importance of cytotoxic T-lymphocyte (CTL) responses in controlling human immunodeficiency virus type 1 and simian immunodeficiency virus replication, the relevance of the epitopic breadth of those CTL responses remains unexplored. In the present study, we sought to determine whether vaccination can expand CTL populations which recognize a repertoire of viral epitopes that is greater than is typically generated in the course of a viral infection. We demonstrate that potent secondary CTL responses to subdominant epitopes are rapidly generated following a pathogenic simian-human immunodeficiency virus challenge of rhesus monkeys vaccinated with plasmid DNA or recombinant modified vaccinia virus Ankara vaccines. These data indicate that prior vaccination can increase the breadth of the CTL response that evolves after an AIDS virus infection.     

Simian immunodeficiency virus (SIV) gag DNA-vaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection

The potential contribution of a plasmid DNA construct to vaccine-elicited protective immunity was explored in the simian immunodeficiency virus (SIV)/macaque model of AIDS. Making use of soluble major histocompatibility class I/peptide tetramers and peptide-specific killing assays to monitor CD8(+) T-lymphocyte responses to a dominant SIV Gag epitope in genetically selected rhesus monkeys, a codon-optimized SIV gag DNA vaccine construct was shown to elicit a high-frequency SIV-specific cytotoxic T-lymphocyte (CTL) response. This CTL response was demonstrable in both peripheral blood and lymph node lymphocytes. Following an intravenous challenge with the highly pathogenic viral isolate SIVsm E660, these vaccinated monkeys developed a secondary CTL response that arose with more rapid kinetics and reached a higher frequency than did the postchallenge CTL response in control plasmid-vaccinated monkeys. While peak plasma SIV RNA levels were comparable in the experimentally and control-vaccinated monkeys during the period of primary infection, the gag plasmid DNA-vaccinated monkeys demonstrated better containment of viral replication by 50 days following SIV challenge. These findings indicate that a plasmid DNA vaccine can elicit SIV-specific CTL responses in rhesus monkeys, and this vaccine-elicited immunity can facilitate the generation of secondary CTL responses and control of viral replication following a pathogenic SIV challenge. These observations suggest that plasmid DNA may prove a useful component of a human immunodeficiency virus type 1 vaccine.     

Magnitude and diversity of cytotoxic-T-lymphocyte responses elicited by multiepitope DNA vaccination in rhesus monkeys

In an effort to develop an AIDS vaccine that elicits high-frequency cytotoxic-T-lymphocyte (CTL) responses with specificity for a diversity of viral epitopes, we explored two prototype multiepitope plasmid DNA vaccines in the simian-human immunodeficiency virus/rhesus monkey model to determine their efficiency in priming for such immune responses. While a simple multiepitope vaccine construct demonstrated limited immunogenicity in monkeys, this same multiepitope genetic sequence inserted into an immunogenic simian immunodeficiency virus gag DNA vaccine elicited high-frequency CTL responses specific for all of the epitopes included in the vaccine. Both multiepitope vaccine prototypes primed for robust epitope-specific CTL responses that developed following boosting with recombinant modified vaccinia virus Ankara vaccines expressing complete viral proteins. The natural hierarchy of immunodominance for these epitopes was clearly evident in the boosted monkeys. These studies suggest that multiepitope plasmid DNA vaccine-based prime-boost regimens can efficiently prime for CTL responses of increased breadth and magnitude, although they do not overcome predicted hierarchies of immunodominance.     

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4(+) and CD8(+) HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.     

Enhanced presentation of major histocompatibility complex class I-restricted human immunodeficiency virus type 1 (HIV-1) Gag-specific epitopes after DNA immunization with vectors coding for vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 Gag particles

In vivo priming of cytotoxic T lymphocytes (CTL) by DNA injection predominantly occurs by antigen transfer from DNA-transfected cells to antigen-presenting cells. A rational strategy for increasing DNA vaccine potency would be to use a delivery system that facilitates antigen uptake by antigen-presenting cells. Exogenous antigen presentation through the major histocompatibility complex (MHC) class I-restricted pathway of some viral antigens is increased after adequate virus-receptor interaction and the fusion of viral and cellular membranes. We used DNA-based immunization with plasmids coding for human immunodeficiency virus type 1 (HIV-1) Gag particles pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) to generate Gag-specific CTL responses. The presence of the VSV-G-encoding plasmid not only increased the number of mice displaying anti-Gag-specific cytotoxic response but also increased the efficiency of specific lysis. In vitro analysis of processing confirmed that exogenous presentation of Gag epitopes occurred much more efficiently when Gag particles were pseudotyped with the VSV-G envelope. We show that the VSV-G-pseudotyped Gag particles not only entered the MHC class II processing pathway but also entered the MHC class I processing pathway. In contrast, naked Gag particles entered the MHC class II processing pathway only. Thus, the combined use of DNA-based immunization and nonreplicating pseudotyped virus to deliver HIV-1 antigen to the immune system in vivo could be considered in HIV-1 vaccine design.     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.     

Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines.     

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.     

Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.     

Lack of Viral Escape and Defective In Vivo Activation of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes in Rapidly Progressive Infection

Human immunodeficiency virus type 1 (HIV-1)-specific immune responses over the course of rapidly progressive infection are not well defined. Detailed longitudinal analyses of neutralizing antibodies, lymphocyte proliferation, in vivo-activated and memory cytotoxic T-lymphocyte (CTL) responses, and viral sequence variation were performed on a patient who presented with acute HIV-1 infection, developed an AIDS-defining illness 13 months later, and died 45 months after presentation. Neutralizing-antibody responses remained weak throughout, and no HIV-1-specific lymphocyte proliferative responses were seen even early in the disease course. Strong in vivo-activated CTL directed against Env and Pol epitopes were present at the time of the initial drop in viremia but were quickly lost. Memory CTL against Env and Pol epitopes were detected throughout the course of infection; however, these CTL were not activated in vivo. Despite an initially narrow CTL response, new epitopes were not targeted as the disease progressed. Viral sequencing showed the emergence of variants within the two targeted CTL epitopes; however, viral variants within the immunodominant Env epitope were well recognized by CTL, and there was no evidence of viral escape from immune system detection within this epitope. These data demonstrate a narrowly directed, static CTL response in a patient with rapidly progressive disease. We also show that disease progression can occur in the presence of persistent memory CTL recognition of autologous epitopes and in the absence of detectable escape from CTL responses, consistent with an in vivo defect in activation of CTL.     

Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.     

Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8(+) T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8(+) T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8(+) T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D(b)-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D(b)-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.     

Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8⁺ T-lymphocyte responses in Mamu-A*01⁺ rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01⁺ rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8⁺ T-lymphocyte response in the absence of secondary Gag p11C-specific CD8⁺ T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8⁺ T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8⁺ T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.CD8⁺ T lymphocyte responses play a central role in controlling human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) infections in nonhuman primates (18, 20, 29, 41). Naturally occurring virus-specific CD8⁺ T-lymphocyte responses typically focus on a limited number of dominant epitopes (52). However, accumulating data indicate that a broad cellular immune response, in which multiple viral epitopes are recognized by CD8⁺ T lymphocytes, confers better protection against viral replication than a restricted cellular immune response (26, 33). Therefore, it has been suggested that increasing the magnitude of subdominant epitope-specific responses may increase the breadth of a cellular immune response and provide enhanced protection against HIV/SIV replication.An understanding of the factors that influence the immunodominance hierarchy of viral epitopes will be needed to develop vaccination strategies that can generate the greatest breadth of virus-specific CD8⁺ T-lymphocyte responses. Differences in antigen processing, competition between epitope peptides for major histocompatibility complex (MHC) class I molecules, T-cell receptor (TCR) repertoire, TCR affinity for peptide class I complexes, and immunodomination have been shown to contribute to the dominance of an epitope-specific response (6, 10, 24, 32, 45, 52). In addition, studies have shown that immunodominance patterns for T-lymphocyte epitopes may differ following a primary and secondary exposure to the same viral antigen (4, 5, 43).In the present study, we observed that Mamu-A*01⁺ rhesus monkeys primed with plasmid DNA and boosted with recombinant adenovirus (rAd) vaccines encoding SIVmac239 Gag-Pol-Nef and HIV-1 Env proteins generated Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses of comparable magnitude. However, while there was a significant expansion of Gag p11C-specific CD8⁺ T-lymphocyte populations following challenge with pathogenic simian-human immunodeficiency virus 89.6P (SHIV-89.6P), there was no significant expansion of the Env p41A-specific CD8⁺ T-lymphocyte populations. We hypothesized that factors influencing the relative immunodominance of the primed Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses after viral challenge may have contributed to the observed differences in their secondary expansion. In the present study, we sought to identify the potential factors contributing to this immunodominance.     

Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Vaccination reduces simian-human immunodeficiency virus sequence reversion through enhanced viral control

It has been suggested that vaccination prior to infection may direct the mutational evolution of human immunodeficiency virus type 1 (HIV-1) to a less fit virus, resulting in an attenuated course of disease. The present study was initiated to explore whether prior immunization might prevent the reversion of the virus to the wild-type form. Mamu-A*01 monkeys were vaccinated to generate a cytotoxic T-lymphocyte response to the immunodominant Gag p11C epitope and were then challenged with a cloned pathogenic CXCR4-tropic simian-human immunodeficiency virus (SHIV) expressing a mutant Gag p11C sequence (Δp11C SHIV). The epitopic and extraepitopic compensatory mutations introduced into gag of Δp11C SHIV resulted in attenuated replicative capacity and eventual reversions to the wild-type Gag p11C sequence in naïve rhesus monkeys. However, in vaccinated rhesus monkeys, no reversions of the challenge virus were observed, an effect that may have been a consequence of significantly decreased viral replication rather than a redirection of the mutational evolution of the virus. These findings highlight the multifactorial pressures that affect the evolution of primate immunodeficiency viruses.CD8⁺ cytotoxic T-lymphocyte (CTL) responses are important for controlling replication of human immunodeficiency virus type 1 (HIV-1) in humans and simian immunodeficiency virus (SIV) in rhesus monkeys (6, 15, 19, 25, 32, 37, 39-41). However, the accumulation of mutations in dominant epitopes of these viruses can undermine this immune control (1, 8, 13, 18, 28). It has been proposed that a preexisting memory-specific CTL response elicited by vaccination prior to HIV-1/SIV infection might change the epitope specificity or the mutational pattern of the infecting virus (9). It is also possible that vaccine-induced cellular immunity might diminish the level of virus replication in individuals following infection and in doing so decrease the rate of accumulation of viral mutations and the likelihood of emergence of viruses that can escape CTL recognition.Our laboratory has previously described a rare SHIV-89.6P escape virus that contains a mutation in the dominant Mamu-A*01-restricted Gag p11C C-M (CTPYDINQM) epitope (3, 4). The emergence of this viral variant was associated with an increase in viral load and the eventual death of the previously vaccinated rhesus monkey 798. Analysis of the escape virus demonstrated a threonine-to-isoleucine mutation at amino acid position 47 (T47I) of the SIV capsid protein, which corresponds to position 2 of the Gag p11C epitope. This T47I mutation abrogated binding to the Mamu-A*01 class I molecule, allowing the virus to escape from recognition by the dominant epitope-specific CTL population (4). In addition to the T47I mutation, a downstream isoleucine-to-valine (I71V) substitution was found to be required for the viability of the escape virus in vitro (12, 29, 30, 42).The present studies were initiated to study the effects of prior vaccination on Gag p11C sequence reversion by infecting monkeys with a simian-human immunodeficiency virus (SHIV) clone containing the gag mutations found in the escape virus that evolved in monkey 798. We first explored the effects of these mutations in vivo by infecting naïve Mamu-A*01⁺ rhesus monkeys with a cloned SHIV (Δp11C SHIV) containing both the Gag p11C T47I mutation and the downstream I71V compensatory substitutions. We then determined whether vaccination prior to infection could generate a cellular immune response that might alter the expected pattern of virus mutation in the immunodominant Mamu-A*01-restricted Gag p11C epitope of Δp11C SHIV.     

Relative dominance of epitope-specific cytotoxic T-lymphocyte responses in human immunodeficiency virus type 1-infected persons with shared HLA alleles.

Cytotoxic T lymphocytes (CTL) target multiple epitopes in human immunodeficiency virus (HIV)-infected persons, and are thought to influence the viral set point. The extent to which HLA class I allele expression predicts the epitopes targeted has not been determined, nor have the relative contributions of responses restricted by different class I alleles within a given individual. In this study, we performed a detailed analysis of the CTL response to optimally defined CTL epitopes restricted by HLA class I A and B alleles in individuals who coexpressed HLA A2, A3, and B7. The eight HIV-1-infected subjects studied included two subjects with acute HIV infection, five subjects with chronic HIV infection, and one long-term nonprogressor. Responses were heterogeneous with respect to breadth and magnitude of CTL responses in individuals of the same HLA type. Of the 27 tested epitopes that are presented by A2, A3, and B7, 25 were targeted by at least one person. However, there was wide variation in the number of epitopes targeted, ranging from 2 to 17. The A2-restricted CTL response, which has been most extensively studied in infected persons, was found to be narrowly directed in most individuals, and in no cases was it the dominant contributor to the total HIV-1-specific CTL response. These results indicate that HLA type alone does not predict CTL responses and that numerous potential epitopes may not be targeted by CTL in a given individual. These data also provide a rationale for boosting both the breadth and the magnitude of HIV-1-specific CTL responses by immunotherapy in persons with chronic HIV-1 infection.