Induction of HIV-1 gag specific immune responses by cationic micelles mediated delivery of gag mRNA.

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA.

A common path to innate immunity to HIV-1 induced by Toll-like receptor ligands in primary human macrophages.

Toll-like receptors (TLR) represent the best characterized receptor family transducing innate immune responses, the first line of defense against microbial invaders. This study was designed to investigate whether responses through TLR inhibit HIV-1 replication in its primary target cells. Primary human macrophages and lymphocytes from several different donors and HIV-1 infection in tissue culture were used exclusively in this work. We report that ligands of three different TLR: LPS, R848, and double stranded RNA, induce a common antiviral response in macrophages as assayed by measurement of HIV-1 p24 protein, gag DNA, and entry into cells. HIV-1 infection is arrested after efficient entry but prior to reverse transcription. TLR-ligand activated cells secrete antiviral factors that induce a similar restriction. HIV-1 infection of lymphocytes is not affected by exposure to TLR ligands or to antiviral factors secreted by activated macrophages. TBK1, but neither NF-κB nor JAK-STAT activity, is required in macrophages to mount this antiviral response; the combination of p38 MAPK and JNK are partially required for induction of antiviral activity. Based on transcriptional induction and inhibition, the TLR-linked antiviral activity is different from APOBEC3 A or G, interferon-ß, NAMPT, or p21(Cip1). The cell-type specificity, site of action, and requirement for signaling intermediates suggest that the TLR-linked antiviral activity is novel.

Gag-specific CD4+ T-cell responses are associated with virological control of paediatric HIV-1 infection.

HIV-specific Elispot responses were investigated in 57 antiretroviral therapy-naive children, of median age 9.9 years. CD8(+) T-cell responses were detected in 96% children; Nef was the immunodominant protein. Responses broadened over time, but there was no association between magnitude, breadth or specificity of response and viraemia. Gag-specific CD4(+) T-cell responses, detectable in 26% children, correlated inversely with viraemia (R = -0.43, P < 0.001), suggesting that preservation of this cell population may be an important goal of therapeutic/vaccine strategies.

Comparative cell-mediated immunogenicity of DNA/DNA, DNA/adenovirus type 5 (Ad5), or Ad5/Ad5 HIV-1 clade B gag vaccine prime-boost regimens.

BACKGROUND: We report composite results from the Merck phase I program of near-consensus clade B human immunodeficiency virus (HIV) type 1 gag vaccines. METHODS: Healthy HIV-uninfected adults were enrolled in 6 blinded placebo-controlled studies evaluating the immunogenicity of (1) a 4-dose regimen of a DNA vaccine, (2) a 3-dose priming regimen of the DNA vaccine with a booster dose of an adenovirus type 5 (Ad5)-vectored vaccine, or (3) a 3-dose regimen of the Ad5 vaccine. The DNA plasmid was provided with or without an aluminum phosphate or CRL1005 adjuvant. The primary end point was the unfractionated HIV-1 gag-specific interferon gamma enzyme-linked immunospot (ELISpot) response 4 weeks after the final dose. RESULTS: Overall, 254 (83%) of 307 subjects randomized to the vaccine groups were evaluable. Adjuvants did not enhance immunogenicity of the DNA vaccine. Postboost ELISpot responder frequencies were higher for Ad5-containing regimens than for the DNA/DNA regimen (33%) but were similar for DNA/Ad5 (55%) and Ad5/Ad5 (50%). DNA/DNA elicited mainly a CD4 response, whereas Ad5/Ad5 elicited mainly a CD8 response; DNA/Ad5 generated CD4 and CD8 responses comparable to those of DNA/DNA and Ad5/Ad5, respectively. CONCLUSIONS: The DNA vaccine alone or as a priming regimen for the Ad5 vaccine did not increase unfractionated ELISpot responses compared with the Ad5 vaccine alone. Qualitative T cell responses to different vaccine regimens deserve further study.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes.

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Potent specific immune responses induced by prime-boost-boost strategies based on DNA, adenovirus, and Sendai virus vectors expressing gag gene of Chinese HIV-1 subtype B.

To study the immune responses elicited by multiple vectors and develop vaccines strategies against prevalent HIV-1 strains in China, we have examined the potency of vaccine regimens of plasmid DNA, adenovirus, and Sendai virus vectors expressing HIV-1 gag consensus sequence of HIV-1 isolates from China for inducing specific immune responses. In BALB/c mice, combination of these vectors induced higher Gag-specific cellular immune response than any regimen using single vector alone. The prime-boost-boost regimen consisting of the triple heterologous vectors induced Gag-specific T-cell responses the most efficiently. In rhesus macaques, the prime-boost-boost regimen induced potent Gag-specific cellular immune responses as well as long lasting humoral immune response, and each booster resulted in rapid and efficient expansion of Gag-specific T cells. These results indicate that this prime-boost-boost regimen using triple heterologous vectors is a promising AIDS vaccine candidate for efficiently inducing HIV-1-specific cellular and humoral immune responses. Its further studies as a promising scheme for therapeutic and/or prophylactic HIV-1 vaccines should be grounded.

HIV Gag-specific immune responses predict the rate of CD4 decline.

In the present study, we assessed whether Gag-specific interferon (IFN)-gamma secreting responses correlate with the rate of disease progression as defined by the annual rate of CD4 decline. Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-gamma response correlated with viral load or rate of CD4 decline, the preferential targeting of Gag is associated with slower annual CD4 T cell decline.

Reduction of anti-HIV-1 Gag immune responses during co-immunization: immune interference by the HIV-1 envelope.

Immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens. However, immune interference has been reported using multi-plasmid DNA immunizations. HIV-1 envelope (Env) and Gag gene products are the predominant immunogens used in current AIDS vaccines, although, few studies have evaluated possible immune interference when these two antigens are co-administered. Therefore, in this study, immune interference during co-inoculation was examined using DNA vaccines expressing lentiviral Envs and Gag from gene sequences optimized for efficient expression in mammalian cells (codon-optimized). BALB/c mice vaccinated in separate hind legs with each plasmid individually elicited high titer immune responses, however, when HIV-1 Env(gp120) and HIV-1 Gag(p55) DNA plasmids were co-inoculated, there was a reduction in the immune responses elicited to HIV-1 Gag(p55). To determine if the anti-HIV-1 Gag(p55) immune interference was specific to HIV-1 Env(gp120), mice were co-immunized with plasmids expressing the surface envelope protein from two additional lentiviruses, Env(gp130)-SIV or Env(gp90)-EIAV, or a soluble form of hemagglutinin (sHA) from influenza virus and HIV-1 Gag(p55)- or SIV Gag(p55)-DNA. Interestingly, there was no reduction in anti-HIV-1 Gag(p55) immune responses using other lentiviral envelopes or the influenza sHA. Also, none of the lentiviral envelopes reduced anti-SIV Gag(p55) immune responses during co-immunization. Therefore, anti-HIV-1 Gag immune interference appears specific to co-immunizations with HIV-1 Env(gp120) and may involve a yet undefined immunological mechanism(s).

In a mixed subtype epidemic, the HIV-1 Gag-specific T-cell response is biased towards the infecting subtype.

OBJECTIVES: Southwest Tanzania is affected by an HIV-1 epidemic consisting of subtypes A, C, and D, and their recombinant forms. This study was designed to assess whether the Gag- and Nef-specific T-cell response is biased towards recognizing the infecting subtype. METHODS: The infecting subtypes were characterized with a Multi-hybridization assay that discriminates between subtypes A, C and D. The interferon-gamma ELISPOT assay was used to detect the Gag- and Nef-specific T-cell responses in freshly isolated peripheral blood mononuclear cells in 56 seropositive patients. To study the HIV-specific T-cell responses, isolate-based Gag and Nef peptide sets representative of the locally occurring subtypes were used. The results were analysed at the total protein and single peptide level. RESULTS: In the study population, 35% were infected with a pure C subtype, 24% and 23% with ACD or AC recombinant forms, respectively. The total magnitude (P < 0.01) and breadth (P < 0.01) of the Gag-specific T-cell response detected with the subtype C-Gag peptide set was significantly greater than that detected with either the subtype A-Gag or D-Gag peptide sets. No significant difference was observed in the Nef-specific response. In 85% of responses targeting the most immunodominant Gag epitopes with subtype-specific sequence differences, the best recognized epitope variant corresponded to the infecting subtype. CONCLUSIONS: The Gag-specific T-cell response had a preference for recognizing peptides related to the infecting subtype.

Recombinant vaccinia DIs expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate.

A highly attenuated vaccinia virus substrain of Dairen-I (DIs) shows promise as a candidate vector for eliciting positive immunity against immune deficiency virus. DIs was randomly obtained by serial 1-day egg passages of a chorioarantoic membrane-adapted Dairen strain (DIE), resulting in substantial genomic deletion, including various genes regulating the virus-host-range. To investigate the impact of that deletion and of the subsequent insertion of a foreign gene into that region of DIs on the ability of the DIs recombinant to induce antigen-specific immunity, we generated a recombinant vaccinia DIs expressing fulllength gag and pol genes of simian immunodeficiency virus (SIV) (rDIsSIV gag/pol) and studied the biological and immunological characteristics of the recombinant natural mutant. The rDIsSIV gag/pol developed a tiny plaque on the chick embryo fibroblast (CEF). Viral particles of rDIsSIV gag/pol as well as SIV Gag-like particles were electromicroscopically detected in the cytoplasm. Interestingly, the recombinant DIs strain grows well in CEF cells but not in mammalian cells. While rDIsSIV gag/pol produces SIV proteins in mammalian HeLa and CV-1 cells, recombinant modified vaccinia Ankara strain (MVA) expressing SIV gag and pol genes (MVA/SIV239 gag/pol) clearly replicates in HeLa and CV-1 cell lines under synchronized growth conditions and produces the SIV protein in all cell lines. Moreover, intradermal administration of rDIsSIV gag/pol or of MVA/SIV239 gag/pol elicited similar levels of IFN-gamma spot-forming cells specific for SIV Gag. If the non-productive infection characteristically induced by recombinant DIs is sufficient to trigger immune induction, as we believe it is, then a human immunodeficiency virus vaccine employing the DIs recombinant would have the twin advantages of being both effective and safe.

Group M-based HIV-1 Gag peptides are frequently targeted by T cells in chronically infected US and Zambian patients.

BACKGROUND: The enormous sequence diversity of HIV-1 has been a major obstacle in the development of a globally useful vaccine for AIDS. The consensus and ancestral sequence-based immunogens minimize the genetic distance between contemporary isolates and vaccine strains. Hence these sequences may be promising candidates for HIV vaccines or serve as a universal reagent set for evaluating Gag-specific responses. METHODS: In this study, we measured the T-cell reactivity to consensus (subtype A, B, C and group M), ancestral (group M and subtype B) and HXB2 Gag peptides (15-mers overlapping by 11) in HIV-1-infected subjects from two reference populations. We evaluated the Gag-specific T-cell responses in 43 chronically infected US (subtype B) and 13 Zambian (subtype C) subjects using an interferon-gamma enzyme-linked immunosorbent spot assay. RESULTS: Our findings demonstrate a broad cross-reactivity of nearly 70% among all the seven Gag immunogens evaluated. Consensus M sequences elicited similar levels of responses as did the consensus B, ancestral subtype B and HXB2 peptides in subtype B-infected US patients. In subtype C-infected Zambian subjects, responses of similar breadth and magnitude were elicited by consensus C, consensus M and ancestral M peptides. CONCLUSION: Our data demonstrate that peptide pools based on consensus or ancestral M-based sequences can be used to evaluate Gag-specific responses elicited by subtype B or subtype C-based immunogens.

Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial.

In this study, the design and preclinical development of a multigene human immunodeficiency virus type 1 (HIV-1) subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of two plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8+ gamma interferon responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime-boost regimen that is scheduled for clinical trials in humans in the USA and South Africa.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate.

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naïve animals. Further, one of six immunized animals was completely protected whereas all six naïve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).

Therapeutic immunization of highly active antiretroviral therapy-treated HIV-1-infected patients: safety and immunogenicity of an HIV-1 gag/poly-epitope DNA vaccine.

In view of the global emergency posed by lack of access to highly active antiretroviral therapy (HAART) and the limitations of current drug regimens, alternative therapeutic strategies are urgently needed. Cellular immune responses elicited by HIV-1 exert some control over virus replication, therefore the enhancement of HIV-1-specific responses by therapeutic vaccination might lead to viral containment without HAART. We evaluated the safety and immunogenicity, in HIV-1-infected individuals under HAART suppression, of a DNA vaccine, pTHr.HIVA.

Pseudovirion particle production by live poxvirus human immunodeficiency virus vaccine vector enhances humoral and cellular immune responses.

Live-vector-based human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation. Live vectors that carry an intact gag gene are capable of eliciting HIV pseudovirion particle formation from infected host cells. The impact of pseudovirion particle formation on the immune response generated by live HIV vaccine vectors has not been established. In this study, a canarypox HIV vaccine candidate vector expressing HIV gag and env genes, vCP205, was modified by the introduction of a glycine-to-alanine coding change in the N-terminal myristylation site of gag to create Myr- vCP205. This substitution effectively eliminated particle formation without altering the level of protein production. vCP205 and Myr- vCP205 were then directly compared for the ability to induce HIV-specific immune responses in mice. The particle-competent vector vCP205 elicited higher levels of CD8+ T-cell responses, as indicated by gamma interferon enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining. Humoral responses to Gag and Env were also markedly higher from animals immunized with the particle-competent vector. Furthermore, HIV-specific CD4+ T-cell responses were greater among animals immunized with the particle-competent vector. Using a human dendritic cell model of antigen presentation in vitro, vCP205 generated greater ELISPOT responses than Myr- vCP205. These results demonstrate that pseudovirion particle production by live-vector HIV vaccines enhances HIV-specific cellular and humoral immune responses.

Soluble HIV-specific T cell receptor: expression, purification and analysis of the specificity.

We have produced soluble T cell receptor (TCR) derived from a human CD8(+) cytotoxic T lymphocyte (CTL) clone D3 that recognizes the immunodominant HIV Gag peptide SLYNTVATL (SL9) in association with major histocompatibility complex (MHC) class I protein HLA-A2. Drosophila Schneider cells (S2) were used to express genes coding the TCR alpha and beta chains under an inducible promoter. Both chains were labeled with two different tags: a (His)(6) was introduced at the C-terminal end of alpha chain, while beta chain was terminated by c-myc. Since an isolated alpha chain is unstable unless it is associated with a beta chain, this design permits rapid separation of alpha,beta-heterodimer from unpaired beta chain in a single step of Ni-NTA Agarose chromatography yielding 90% pure alpha,beta-TCR. Introduction of the c-myc epitope to the beta chain allows capture of soluble D3 from the culture supernatant by immobilized anti-c-myc antibody, without the need for receptor purification. The TCR specificity was then examined by analyzing the binding of peptide-HLA-A2/tetramer in an ELISA assay. Using this assay, we have also evaluated the binding of monomeric SL9-HLA-A2 complex to the immobilized D3 TCR and determined that the affinity measurement of the D3-SL9-HLA-A2 reaction is similar to that obtained by a biosensor instrument. We propose that the approach described here is generally useful for purification of other soluble TCRs and will allow rapid analysis of their specificity.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Quantitative assessment of antigen-specific CD8+ T cells in the mouse: application to vaccine research.

An effective HIV vaccine will likely need to induce potent and broad-based immunity, including CD8+ T cell responses. Hence, a quantitative assay to measure such responses in animal models will be important in the evaluation of candidate HIV vaccines. We show here that a single immunization with HIV DNA vaccines, followed by challenge with recombinant vaccinia virus expressing the relevant HIV antigen, allows quantitative assessment of CD8+ T cell responses. These responses can be profound (>30% of total CD8+ T cells) and directly reflect the level of memory CD8+ T cells at the time of challenge. Therefore, this assay will facilitate the selection of promising HIV vaccine candidates for further evaluation.

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.