ARTEMIS: A Novel Mass-Spec Platform for HLA-Restricted Self and Disease-Associated Peptide Discovery.

Conventional immunoprecipitation/mass spectroscopy identification of HLA-restricted peptides remains the purview of specializing laboratories, due to the complexity of the methodology, and requires computational post-analysis to assign peptides to individual alleles when using pan-HLA antibodies. We have addressed these limitations with ARTEMIS: a simple, robust, and flexible platform for peptide discovery across ligandomes, optionally including specific proteins-of-interest, that combines novel, secreted HLA-I discovery reagents spanning multiple alleles, optimized lentiviral transduction, and streamlined affinity-tag purification to improve upon conventional methods. This platform fills a middle ground between existing techniques: sensitive and adaptable, but easy and affordable enough to be widely employed by general laboratories. We used ARTEMIS to catalog allele-specific ligandomes from HEK293 cells for seven classical HLA alleles and compared results across replicates, against computational predictions, and against high-quality conventional datasets. We also applied ARTEMIS to identify potentially useful, novel HLA-restricted peptide targets from oncovirus oncoproteins and tumor-associated antigens.

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 ß-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083.

BACKGROUND: Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. METHODS: A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. RESULTS: T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6-1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2-5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). CONCLUSIONS: These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. CLINICAL TRIALS REGISTRATION: NCT01095224.