Repertoire and immunofocusing of CD8 T cell responses generated by HIV-1 gag-pol and expression library immunization vaccines

Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.     

Broad cross-clade T-cell responses to gag in individuals infected with human immunodeficiency virus type 1 non-B clades (A to G): importance of HLA anchor residue conservation

We aimed to identify cross-clade human immunodeficiency virus type 1 (HIV-1) specific T-cell responses among 10 HLA-typed individuals who were infected with non-B HIV-1 strains (A, AG, C, D, G, or F) and to correlate these responses with genetic variation in documented T-cell epitopes. T-cell reactivity was tested against peptide pools spanning clade B Gag, Pol, Nef, Rev, and Tat consensus, with Gag and Nef providing the highest responses. Nine individuals who responded to clade B Gag demonstrated cross-reactive T-cell responses against clade A and C Gag pools, while six of seven responders to Nef-B reacted to clade A and C Nef pools. An inverse correlation between the height of the T-cell responses and the sequence divergence of the HLA class I-restricted epitopes was identified when we compared autologous Gag and Nef sequences with the reactive consensus pools. This could be explained for the Gag sequences through observed variations in the HLA anchor residues. Through mapping of 30 amino acid cross-clade-reactive regions using Gag-B pools, we were able to link 58% (14/24) of the T-cell responses to regions containing previously described HLA class I-restricted epitopes. Forty-two percent (10/24) of the responses were directed to regions containing new epitopes, for which predicted HLA class I motifs could be recognized in 70% (7/10) of individuals. We demonstrate here that cross-clade T-cell responses are frequently induced in individuals infected with distinct HIV-1 clades, suggesting that interclade variation outside of HLA anchor residues may have less impact on vaccine-induced T-cell reactivity than previously thought.     

Improved vaccine protection from simian AIDS by the addition of nonstructural simian immunodeficiency virus genes

An HIV-1 vaccine able to induce broad CD4+ and CD8+ T cell responses may provide long-term control of viral replication. In this study we directly assess the relative benefit of immunization with vaccines expressing three structural Ags (Gag, Pol, and Env), three early regulatory proteins (Rev, Tat, and Nef), or a complex vaccine expressing all six Ags. The simultaneous administration of all six Ags during vaccination resulted in Ag competition manifested by a relative reduction of CD8+ T cell and lymphoproliferative responses to individual Ags. Despite the Ag competition, vaccination with all six Ags resulted in a delay in the onset and a decrease in the extent of acute viremia after mucosal challenge exposure to highly pathogenic SIV(mac251). Reduced levels of acute viremia correlated with lower post-set point viremia and long-term control of infection. In immunized animals, virus-specific CD4+ T cell and lymphoproliferative responses were preserved during acute viremia, and the maintenance of these responses predicted the long-term virological outcome. Taken together, these results suggest that the breadth of the immune response is probably more important than high frequency responses to a limited number of epitopes. These data provide the first clear evidence of the importance of nonstructural HIV Ags as components of an HIV-1 vaccine.     

Identification and antigenicity of broadly cross-reactive and conserved human immunodeficiency virus type 1-derived helper T-lymphocyte epitopes

Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4(+) T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the Step study

T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial registration: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.     

Th1 T cell responses to HIV-1 Gag protein delivered by a Listeria monocytogenes vaccine are similar to those induced by endogenous listerial antigens.

Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-gamma at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.     

The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens

Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.     

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8⁺ T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8⁺ T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8⁺ T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8⁺ T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.     

Vaccines expressing the innate immune modulator EAT-2 elicit potent effector memory T lymphocyte responses despite pre-existing vaccine immunity

The mixed results from recent vaccine clinical trials targeting HIV-1 justify the need to enhance the potency of HIV-1 vaccine platforms in general. Use of first-generation recombinant adenovirus serotype 5 (rAd5) platforms failed to protect vaccinees from HIV-1 infection. One hypothesis is that the rAd5-based vaccine failed due to the presence of pre-existing Ad5 immunity in many vaccines. We recently confirmed that EAT-2-expressing rAd5 vectors uniquely activate the innate immune system and improve cellular immune responses against rAd5-expressed Ags, inclusive of HIV/Gag. In this study, we report that use of the rAd5-EAT-2 vaccine can also induce potent cellular immune responses to HIV-1 Ags despite the presence of Ad5-specific immunity. Compared to controls expressing a mutant SH2 domain form of EAT-2, Ad5 immune mice vaccinated with an rAd5-wild-type EAT-2 HIV/Gag-specific vaccine formulation significantly facilitated the induction of several arms of the innate immune system. These responses positively correlated with an improved ability of the vaccine to induce stronger effector memory T cell-biased, cellular immune responses to a coexpressed Ag despite pre-existing anti-Ad5 immunity. Moreover, inclusion of EAT-2 in the vaccine mixture improves the generation of polyfunctional cytolytic CD8(+) T cell responses as characterized by enhanced production of IFN-γ, TNF-α, cytotoxic degranulation, and increased in vivo cytolytic activity. These data suggest a new approach whereby inclusion of EAT-2 expression in stringent human vaccination applications can provide a more effective vaccine against HIV-1 specifically in Ad5 immune subjects.     

The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.     

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.     

融合表达小鼠IgG2b-Fc可增强人类免疫缺陷病毒1型Gag DNA疫苗的免疫原性

为阐明小鼠IgG2b-Fc对DNA疫苗免疫原性的增强作用,首先构建表达人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)CN54Gag基因的DNA疫苗及表达Gag与小鼠IgG2b-Fc融合基因的DNA疫苗,限制性酶切和DNA测序结果表明这两个疫苗均构建成功,蛋白免疫印迹结果也显示其正确表达。然后,利用上述DNA疫苗接种C57BL/6小鼠,比较两个DNA疫苗所诱导的特异性体液免疫反应和特异性细胞免疫反应。结果显示,融合表达小鼠IgG2b-Fc对特异性细胞免疫和体液免疫反应均有增强作用,但只有对特异性体液免疫反应的增强作用有统计学意义。     

Comparison of human immunodeficiency virus (HIV)-specific T-cell responses in HIV-1- and HIV-2-infected individuals in Senegal

Human immunodeficiency virus type 2 (HIV-2) infection is typically less virulent than HIV-1 infection, which may permit the host to mount more effective, sustained T-cell immunity. We investigated antiviral gamma interferon-secreting T-cell responses by an ex vivo Elispot assay in 68 HIV-1- and 55 HIV-2-infected Senegalese patients to determine if differences relate to more efficient HIV-2 control. Homologous HIV-specific T cells were detected in similar frequencies (79% versus 76%, P = 0.7) and magnitude (3.12 versus 3.08 log(10) spot-forming cells/10(6) peripheral blood mononuclear cells) in HIV-1 and HIV-2 infection, respectively. Gag-specific responses predominated in both groups (>/=64%), and significantly higher Nef-specific responses occurred in HIV-1-infected (54%) than HIV-2-infected patients (22%) (P < 0.001). Heterologous responses were more frequent in HIV-1 than in HIV-2 infection (46% versus 27%, P = 0.04), but the mean magnitude was similar. Total frequencies of HIV-specific responses in both groups did not correlate with plasma viral load and CD4(+) T-cell count in multivariate regression analyses. However, the magnitude of HIV-2 Gag-specific responses was significantly associated with lower plasma viremia in HIV-1-infected patients (P = 0.04). CD4(+) T-helper responses, primarily recognizing HIV-2 Gag, were detected in 48% of HIV-2-infected compared to only 8% of HIV-1-infected patients. These findings indicate that improved control of HIV-2 infection may relate to the contribution of T-helper cell responses. By contrast, the superior control of HIV-1 replication associated with HIV-2 Gag responses suggests that these may represent cross-reactive, higher-avidity T cells targeting epitopes within Gag regions of functional importance in HIV replication.     

HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines

Vaccine strategies designed to elicit strong cell-mediated immune responses to HIV Ags are likely to lead to protective immunity against HIV infection. Dendritic cells (DC) are the most potent APCs capable of priming both MHC class I- and II-restricted, Ag-specific T cell responses. Utilizing a system in which cultured DC from HIV-seronegative donors were used as APC to present HIV-1 Ags to autologous T cells in vitro, the strength and specificity of primary HIV-specific CTL responses generated to exogenous HIV-1 Nef protein as well as intracellularly expressed nef transgene product were investigated. DC expressing the nef gene were able to stimulate Nef-specific CTL, with T cells from several donors recognizing more than one epitope restricted by a single HLA molecule. Primary Nef-specific CTL responses were also generated in vitro using DC pulsed with Nef protein. T cells primed with Nef-expressing DC (via protein or transgene) were able to lyse MHC class I-matched target cells pulsed with defined Nef epitope peptides as well as newly identified peptide epitopes. The addition of Th1-biasing cytokines IL-12 or IFN-alpha, during priming with Nef-expressing DC, enhanced the Nef-specific CTL responses generated using either Ag-loading approach. These results suggest that this in vitro vaccine model may be useful in identifying immunogenic epitopes as vaccine targets and in evaluating the effects of cytokines and other adjuvants on Ag-specific T cell induction. Successful approaches may provide information important to the development of prophylactic HIV vaccines and are envisioned to be readily translated into clinical DC-based therapeutic vaccines for HIV-1.     

Control of human immunodeficiency virus type 1 is associated with HLA-B*13 and targeting of multiple gag-specific CD8+ T-cell epitopes

To better understand relationships between CD8+ T-cell specificity and the immune control of human immunodeficiency virus type 1 (HIV-1), we analyzed the role of HLA-B*13, an allele associated with low viremia, in a cohort of 578 C clade-infected individuals in Durban, South Africa. Six novel B*13-restricted cytotoxic T lymphocyte epitopes were defined from analyses of 37 B*13-positive subjects, including three Gag epitopes. These B*13-restricted epitopes contribute to a broad Gag-specific CD8+ response that is associated with the control of viremia. These data are consistent with data from studies of other HLA-class I alleles associated with HIV control that have shown that the targeting of multiple Gag epitopes is associated with relative suppression of viremia.     

A Phase I Randomized Therapeutic MVA-B Vaccination Improves the Magnitude and Quality of the T Cell Immune Responses in HIV-1-Infected Subjects on HAART

Trial Design

Previous studies suggested that poxvirus-based vaccines might be instrumental in the therapeutic HIV field. A phase I clinical trial was conducted in HIV-1-infected patients on highly active antiretroviral therapy (HAART), with CD4 T cell counts above 450 cells/mm³ and undetectable viremia. Thirty participants were randomized (2:1) to receive either 3 intramuscular injections of MVA-B vaccine (coding for clade B HIV-1 Env, Gag, Pol and Nef antigens) or placebo, followed by interruption of HAART.

Methods

The magnitude, breadth, quality and phenotype of the HIV-1-specific T cell response were assayed by intracellular cytokine staining (ICS) in 22 volunteers pre- and post-vaccination.

Results

MVA-B vaccine induced newly detected HIV-1-specific CD4 T cell responses and expanded pre-existing responses (mostly against Gag, Pol and Nef antigens) that were high in magnitude, broadly directed and showed an enhanced polyfunctionality with a T effector memory (TEM) phenotype, while maintaining the magnitude and quality of the pre-existing HIV-1-specific CD8 T cell responses. In addition, vaccination also triggered preferential CD8⁺ T cell polyfunctional responses to the MVA vector antigens that increase in magnitude after two and three booster doses.

Conclusion

MVA-B vaccination represents a feasible strategy to improve T cell responses in individuals with pre-existing HIV-1-specific immunity.

Trial Registration

ClinicalTrials.gov NCT01571466     

Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.     

A Novel Rabbit Monoclonal Antibody Platform To Dissect the Diverse Repertoire of Antibody Epitopes for HIV-1 Env Immunogen Design

The majority of available monoclonal antibodies (MAbs) in the current HIV vaccine field are generated from HIV-1-infected people. In contrast, preclinical immunogenicity studies have mainly focused on polyclonal antibody responses in experimental animals. Although rabbits have been widely used for antibody studies, there has been no report of using rabbit MAbs to dissect the specificity of antibody responses for AIDS vaccine development. Here we report on the production of a panel of 12 MAbs from a New Zealand White (NZW) rabbit that was immunized with an HIV-1 JR-FL gp120 DNA prime and protein boost vaccination regimen. These rabbit MAbs recognized a diverse repertoire of envelope (Env) epitopes ranging from the highly immunogenic V3 region to several previously underappreciated epitopes in the C1, C4, and C5 regions. Nine MAbs showed cross-reactivity to gp120s of clades other than clade B. Increased somatic mutation and extended CDR3 were observed with Ig genes of several molecularly cloned rabbit MAbs. Phylogenic tree analysis showed that the heavy chains of MAbs recognizing the same region on gp120 tend to segregate into an independent subtree. At least three rabbit MAbs showed neutralizing activities with various degrees of breadth and potency. The establishment of this rabbit MAb platform will significantly enhance our ability to test optimal designs of Env immunogens to gain a better understanding of the structural specificity and evolution process of Env-specific antibody responses elicited by candidate AIDS vaccines.     

For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.