Dendritic cells focus CTL responses toward highly conserved and topologically important HIV-1 epitopes.

BACKGROUND: During early HIV-1 infection, immunodominant T cell responses to highly variable epitopes lead to the establishment of immune escape virus variants. Here we assessed a type 1-polarized monocyte-derived dendritic cell (MDC1)-based approach to selectively elicit cytotoxic T lymphocyte (CTL) responses against highly conserved and topologically important HIV-1 epitopes in HIV-1-infected individuals from the Thailand RV254/SEARCH 010 cohort who initiated antiretroviral therapy (ART) during early infection (Fiebig stages I-IV). METHODS: Autologous MDC1 were used as antigen presenting cells to induce in vitro CTL responses against HIV-1 Gag, Pol, Env, and Nef as determined by flow cytometry and ELISpot assay. Ultra-conserved or topologically important antigens were respectively identified using the Epigraph tool and a structure-based network analysis approach and compared to overlapping peptides spanning the Gag proteome. FINDINGS: MDC1 presenting either the overlapping Gag, Epigraph, or Network 14-21mer peptide pools consistently activated and expanded HIV-1-specific T cells to epitopes identified at the 9-13mer peptide level. Interestingly, some CTL responses occurred outside known or expected HLA associations, providing evidence of new HLA-associated CTL epitopes. Comparative analyses demonstrated more sequence conservation among Epigraph antigens but a higher magnitude of CTL responses to Network and Gag peptide groups. Importantly, CTL responses against topologically constrained Gag epitopes contained in both the Network and Gag peptide pools were selectively enhanced in the Network pool-initiated cultures. INTERPRETATION: Our study supports the use of MDC1 as a therapeutic strategy to induce and focus CTL responses toward putative fitness-constrained regions of HIV-1 to prevent immune escape and control HIV-1 infection. FUNDING: A full list of the funding sources is detailed in the Acknowledgment section of the manuscript.

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.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P < 0.001 compared to CD8+ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gagp24 regions, more conserved between prime and boost, compared to those of regions within Gagp15 (not primed) and Gagp17 (less conserved; P < 0.0001 for both). Four regions within Gagp24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 protein.IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

Antibody to HSV gD peptide induced by vaccination does not protect against HSV-2 infection in HSV-2 seronegative women.

BACKGROUND: In the HIV-1 vaccine trial RV144, ALVAC-HIV prime with an AIDSVAX® B/E boost reduced HIV-1 acquisition by 31% at 42 months post first vaccination. The bivalent AIDSVAX® B/E vaccine contains two gp120 envelope glycoproteins, one from the subtype B HIV-1 MN isolate and one from the subtype CRF01_AE A244 isolate. Each envelope glycoprotein harbors a highly conserved 27-amino acid HSV-1 glycoprotein D (gD) tag sequence that shares 93% sequence identity with the HSV-2 gD sequence. We assessed whether vaccine-induced anti-gD antibodies protected females against HSV-2 acquisition in RV144. METHODS: Of the women enrolled in RV144, 777 vaccine and 807 placebo recipients were eligible and randomly selected according to their pre-vaccination HSV-1 and HSV-2 serostatus for analysis. Immunoglobulin G (IgG) and IgA responses to gD were determined by a binding antibody multiplex assay and HSV-2 serostatus was determined by Western blot analysis. Ninety-three percent and 75% of the vaccine recipients had anti-gD IgG and IgA responses two weeks post last vaccination, respectively. There was no evidence of reduction in HSV-2 infection by vaccination compared to placebo recipients over 78 weeks of follow-up. The annual incidence of HSV-2 infection in individuals who were HSV-2 negative at baseline or HSV-1 positive and HSV-2 indeterminate at baseline were 4.38/100 person-years (py) and 3.28/100 py in the vaccine and placebo groups, respectively. Baseline HSV-1 status did not affect subsequent HSV-2 acquisition. Specifically, the estimated odds ratio of HSV-2 infection by Week 78 for female placebo recipients who were baseline HSV-1 positive (n = 422) vs. negative (n = 1120) was 1.14 [95% confidence interval 0.66 to 1.94, p = 0.64)]. No evidence of reduction in the incidence of HSV-2 infection by vaccination was detected. CONCLUSIONS: AIDSVAX® B/E containing gD did not confer protection from HSV-2 acquisition in HSV-2 seronegative women, despite eliciting anti-gD serum antibodies.

Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte-derived macrophages: the importance of HIV-1 envelope V1V2 region.

Monocytes and monocyte-derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte-derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor-derived monocyte-derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor-derived monocyte-derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor-derived monocyte-derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1 interaction with sialic acid-binding immunoglobulin-like lectin and entry into macrophage colony-stimulating factor-derived monocyte-derived macrophages. Removal of sialic acid residues or glycans from scaffolded V1V2 protein decreased human immunodeficiency virus type 1 infectivity. These results highlight the importance of sialic acids on the V1V2 region in binding to sialic acid-binding immunoglobulin-like lectin and suggest that the unusually long surface-exposed sialic acid-binding immunoglobulin-like lectin might aid in the capture and entry of human immunodeficiency virus type 1 into monocyte-derived macrophages.

Designing the epitope flanking regions for optimal generation of CTL epitopes.

The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.

Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys.

The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of such global HIV-1 vaccine antigens has not previously been evaluated. Here, we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of infection following heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection correlated with vaccine-elicited binding, neutralizing, and functional nonneutralizing antibodies, suggesting that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy for the development of a global HIV-1 vaccine. PAPERCLIP:

Transfusion-transmitted human T-lymphotropic virus Type I infection in a United States military emergency whole blood transfusion recipient in Afghanistan, 2010.

BACKGROUND: The United States introduced human T-lymphotropic virus Type I (HTLV-I) screening of blood donors in 1988. The US military uses freshly collected blood products for life-threatening injuries when available stored blood components in theater have been exhausted or when these components are unsuccessful for resuscitation. These donors are screened after donation by the Department of Defense (DoD) retrospective testing program. All recipients of blood collected in combat are tested according to policy soon after and at 3, 6, and 12 months after transfusion. CASE REPORT: A 31-year-old US Army soldier tested positive for HTLV-I 44 days after receipt of emergency blood transfusions for severe improvised explosive device blast injuries. One donor's unit tested HTLV-I positive on the DoD-mandated retrospective testing. Both the donor and the recipient tested reactive with enzyme immunoassay and supplemental confirmation by HTLV-I Western blot. The donor and recipient reported no major risk factors for HTLV-I. Phylogenetic analysis of HTLV-I sequences indicated Cosmopolitan subtype, Subgroup B infections. Comparison of long terminal repeat and env sequences revealed molecular genetic linkage of the viruses from the donor and recipient. CONCLUSION: This case is the first report of transfusion transmission of HTLV-I in the US military during combat operations. The emergency fresh whole blood policy enabled both the donor and the recipient to be notified of their HTLV-I infection. While difficult in combat, predonation screening of potential emergency blood donors with Food and Drug Administration-mandated infectious disease testing as stated by the DoD Health Affairs policy should be the goal of every facility engaged with emergency blood collection in theater.

HyperPack: a software package for the study of levels, contexts, and patterns of APOBEC-mediated hypermutation in HIV.

G-to-A hypermutation, a massive substitution of G for A, was first observed in HIV genomes 15 years ago. Initially ascribed to fluctuating nucleotide pools or PCR errors, it has now been recognized as the result of an important host-defense mechanism mediated by APOBECs, a family of sequence-specific cytidine deaminases. Levels of hypermutation vary, and APOBECs have different preferences for the sequence context of cytidines targeted for deamination; analysis of hypermutation provides important information about the host-virus interaction during viral replication. Here we present HyperPack, a software package to support systematic characterization of hypermutated sequences. Users can define the context of substitution for independent study of the effects of different APOBECs. The SubstrateScan and HyperScan modules are overlapping sliding-window strategies to analyze the distribution of targeted motifs and their substitution levels, respectively, across the viral genome. HyperPack is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/hyperpack/.

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response.

BACKGROUND: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. RESULTS: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets. CONCLUSION: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.

HIV infection among female drug users in Northern Thailand.

Reports on HIV infection and risk behaviors among female drug users in developing countries, particularly in Asia, are limited. In this study, we investigated HIV prevalence and risk factors for HIV infection among 200 women admitted for 21-day inpatient drug detoxification in Chiang Mai, Thailand. Volunteers completed a face-to-face interview using a structured interview, HIV pre-test counseling, specimen collection for HIV and STD tests, and were provided test results and HIV post-test counseling 1 week later. Two-third of participants (68%) were ethnic minorities with no formal education. Overall, 14 (7%) were HIV positive: 25% among 28 heroin injectors and 4.1% among 172 opium or methamphetamine smokers (p<0.001). History of drug injection and sexual abuse were associated with HIV infection. HIV prevention strategies for drug-using women in Thailand should consider both harm reduction strategies for drug use and promoting safer sex measures in a culturally appropriate context.