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A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Unlabelled: Cellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination., Importance: HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Mucosal immunity is central to sexual transmission and overall pathogenesis of HIV-1 infection, but the ability of vaccines to induce immune responses in mucosal tissue compartments is poorly defined. Because macaque vaccine studies suggest that inguinal (versus limb) vaccination may better target sexually-exposed mucosa, we performed a randomized, double-blinded, placebo-controlled Phase I trial in HIV-1-uninfected volunteers, using the recombinant Canarypox (CP) vaccine vCP205 delivered by different routes. 12 persons received vaccine and 6 received placebo, divided evenly between deltoid-intramuscular (deltoid-IM) or inguinal-subcutaneous (inguinal-SC) injection routes. The most significant safety events were injection site reactions (Grade 3) in one inguinal vaccinee. CP-specific antibodies were detected in the blood of all 12 vaccinees by Day 24, while HIV-1-specific antibodies were observed in the blood and gut mucosa of 1/9 and 4/9 evaluated vaccinees respectively, with gut antibodies appearing earlier in inguinal vaccinees (24-180 versus 180-365 days). HIV-1-specific CD8(+) T lymphocytes (CTLs) were observed in 7/12 vaccinees, and blood and gut targeting were distinct. Within blood, both deltoid and inguinal responders had detectable CTL responses by 17-24 days; inguinal responders had early responses (within 10 days) while deltoid responders had later responses (24-180 days) in gut mucosa. Our results demonstrate relative safety of inguinal vaccination and qualitative or quantitative compartmentalization of immune responses between blood and gut mucosa, and highlight the importance of not only evaluating early blood responses to HIV-1 vaccines but also mucosal responses over time., Trial Registration: ClinicalTrials.gov NCT00076817.

HLA-A 0201-restricted virus-specific CD8(+) CTL do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag(19-27)), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9-specific CTL primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA-A 0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro-primed CTL showed that TV9p6 consistently activated cross-reactive and equally functional CTL as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTL primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a "public" TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional cross-reactive clonotypes that are less readily recruited from the naïve T-cell pool by the corresponding WT epitope. Mimotope-induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine-induced responses to conserved but poorly immunogenic determinants within the HIV proteome.

A likely key factor in the failure of a HIV-1 vaccine based on cytotoxic T lymphocytes (CTL) is the natural immunodominance of epitopes that fall in variable regions of the proteome, which both increases the chance of epitope sequence mismatch with the incoming challenge strain and replicates the pathogenesis of early CTL failure due to epitope escape mutation during natural infection. To identify potential vaccine sequences to focus the CTL response on highly conserved epitopes, the whole proteomes of HIV-1 clades A1, B, C, and D were assessed for Shannon entropy at each amino acid position. Highly conserved regions in Gag (cGag-1, Gag 148-214, and cGag-2, Gag 253-331), Env (cEnv, Env 521-606), and Nef (cNef, Nef 106-148) were identified across clades. Inter- and intra-clade variability of amino acids within the regions tended to overlap, suggesting that polyvalent representation of consensus sequences for the four clades would allow broad HIV-1 strain representation. These four conserved regions were rich in both known and predicted CTL epitopes presented by a breadth of HLA types, and screening of 54 persons with chronic HIV-1 infection revealed that these regions are commonly immunogenic in the context of natural infection. These data suggest that vaccine delivery of a 16-valent mixture of these regions could focus the CTL response against conserved epitopes that are broadly representative of circulating HIV-1 strains.

Mucosal immune responses induced by HIV-1 vaccines are likely critical for prevention. We report a Phase 1 safety and immunogenicity trial in eight participants using the vaccinia-based TBC-3B vaccine given subcutaneously to determine the relationship between HIV-1 specific systemic and gastrointestinal mucosal responses. Across all subjects, detectable levels of blood vaccinia- and HIV-1-specific antibodies were elicited but none were seen mucosally. While the vaccinia component was immunogenic for CD8(+) T lymphocyte (CTL) responses in both blood and mucosa, it was greater in blood. The HIV-1 component of the vaccine was poorly immunogenic in both blood and mucosa. Although only eight volunteers were studied intensively, the discordance between mucosal and blood responses may highlight mechanisms contributing to recent vaccine failures.

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.

Many vaccine approaches emphasize producing HIV-1-specific CD8+ T-lymphocyte (CTL) responses. Towards this goal, many studies simply classify vaccinees as "responders" or "nonresponders," based on arbitrary cutoff criteria. HIV-1-uninfected participants receiving the TBC-3B vaccine were assessed for HIV-1-specific CTL by interferon-gamma ELISpot, and compared to HIV-1-infected control subjects not on antiretroviral therapy. Vaccinees also were tested for HIV-1-specific antibody responses and generalized CD8+ T-lymphocyte activation. Different criteria for vaccine "responder" status were applied to the measured CTL values. The vaccinees showed evidence of vaccine exposure by CD8+ T-lymphocyte activation and HIV-1-specific antibodies. Considering any single positive HIV-1-specific CTL measurement a vaccine "response," all vaccinees could be classified as "responders," but even slight increases in the stringency of response criteria resulted in a steep decline of the "response" rate. In contrast, HIV-1-infected persons were clearly "responders" against the same proteins by the same criteria. Quantitative assessment of CTL demonstrated low and transient HIV-1-specific CTL compared to natural infection. These analyses emphasize the pitfalls of summarizing vaccine study results using simple cutoff criteria to define response rates, and suggest the utility of more comprehensive descriptions to describe vaccine immunogenicity and persistence of responses.

Many HIV-1 vaccine efforts have heavily emphasized class I-restricted CD8(+) cytotoxic T lymphocytes (CTLs) as a potentially important arm of immunity. Commonly used CTL assays describe only specificity and frequency, and not antiviral effects. However, increasing evidence indicates that CTL antiviral function is determined by the complex interplay of multiple virologic and cellular factors, and thus these measurements are an incomplete reflection of CTL efficacy. Our current understanding of the factors determining HIV-1-specific CTL antiviral efficacy is inadequate to interpret fully the significance of detected CTLs in vaccine and pathogenesis studies. To assess CTLs as a protective immune response, it will be crucial to elucidate these mechanisms and/or devise new assays that directly reflect the interaction of CTLs and HIV-1.