Unique HIV Risk Factors and Prevention Needs for Transgender Women and Cisgender Men Who Have Sex with Men in Bangkok, Thailand.

Background: Transgender women (TGW) and cisgender men who have sex with men (cis-MSM) are often grouped together as key populations. We evaluated behavioral and other characteristics that may distinguish TGW from cis-MSM in Bangkok, Thailand. Methods: We enrolled into an 18-month cohort cis-MSM and TGW 18-35 years of age without HIV, who reported anal intercourse plus condomless anal intercourse, multiple partners, transactional sex, and/or sexually transmitted infection. Robust multivariable Poisson regression was used to estimate adjusted prevalence ratios (aPRs) and confidence intervals (95% CIs) for associations with being a TGW. Among TGW, logistic regression with generalized estimating equations was used to estimate adjusted odds ratios (aORs) and 95% CIs for associations with taking hormones and having undergone gender affirmation surgery (GAS). Results: From 2017 to 2019, 660 cis-MSM and 348 TGW were enrolled. Compared to cis-MSM, TGW were more likely to be attracted to mostly/only men (aPR: 3.79, 95% CI: 1.57-9.13), have a higher monthly income (aPR: 1.25, 95% CI: 1.04-1.50), have lived in their current residence for <1 year (aPR: 1.21, 95% CI: 1.01-1.46), have engaged in sex work (aPR: 1.48, 95% CI: 1.23-1.77), and be less likely to have ever undergone HIV testing (aPR: 0.83, 95% CI: 0.70-0.98). Among TGW, 149 (42.8%) were taking hormones and 33 (9.5%) had undergone GAS. GAS was more common among TGW who ever used methamphetamines (aOR: 1.55, 95% CI: 1.00-2.41) and those >23 years (18-20-year olds aOR: 0.17, 95% CI: 0.05-0.55; 21-23-year olds aOR: 0.36, 95% CI: 0.20-0.65). Conclusions: TGW and cis-MSM are unique populations; tailored, gender-affirming, differentiated models of HIV prevention and care are necessary to address vulnerabilities specific to each key population.

Monocyte-derived transcriptome signature indicates antibody-dependent cellular phagocytosis as a potential mechanism of vaccine-induced protection against HIV-1.

A gene signature was previously found to be correlated with mosaic adenovirus 26 vaccine protection in simian immunodeficiency virus and simian-human immunodeficiency virus challenge models in non-human primates. In this report, we investigated the presence of this signature as a correlate of reduced risk in human clinical trials and potential mechanisms of protection. The absence of this gene signature in the DNA/rAd5 human vaccine trial, which did not show efficacy, strengthens our hypothesis that this signature is only enriched in studies that demonstrated protection. This gene signature was enriched in the partially effective RV144 human trial that administered the ALVAC/protein vaccine, and we find that the signature associates with both decreased risk of HIV-1 acquisition and increased vaccine efficacy (VE). Total RNA-seq in a clinical trial that used the same vaccine regimen as the RV144 HIV vaccine implicated antibody-dependent cellular phagocytosis (ADCP) as a potential mechanism of vaccine protection. CITE-seq profiling of 53 surface markers and transcriptomes of 53,777 single cells from the same trial showed that genes in this signature were primarily expressed in cells belonging to the myeloid lineage, including monocytes, which are major effector cells for ADCP. The consistent association of this transcriptome signature with VE represents a tool both to identify potential mechanisms, as with ADCP here, and to screen novel approaches to accelerate the development of new vaccine candidates.

Longitudinal Analysis of Peripheral and Colonic CD161+ CD4+ T Cell Dysfunction in Acute HIV-1 Infection and Effects of Early Treatment Initiation.

CD161 expression on CD4+ T cells is associated with a Th17 functional phenotype, as well as with an innate capacity to respond to interleukin (IL)-12 and IL-18 without T cell receptor (TCR) stimulation. Chronic HIV-1 infection is associated with loss of the CD161+ CD4 T cell population, and non-human primate studies suggest that their depletion is associated with disease progression. However, the dynamics of the CD161+ CD4+ T cell population during acute HIV-1 infection remains unknown. In this study, we characterize peripheral blood CD161+ CD4+ T cells in detail, and examine how they are affected during the earliest stages of HIV-1 infection. Unbiased surface proteome screening and principal component analysis indicated that CD161+ CD4+ T cells are relatively phenotypically homogeneous between donors, and are intermediates between conventional CD4 T cells and innate-like T cells. In acute untreated HIV-1 infection, the circulating CD161+ CD4+ T cell population decreased in frequency, as did absolute cell counts starting from peak viral load, with elevated levels of activation and exhaustion markers expressed throughout acute HIV-1 infection. The capacity of these cells to respond to stimulation with IL-12 and IL-18 was also reduced. Early initiation of anti-retroviral treatment (ART) during acute HIV-1 infection restored the functionality of peripheral blood CD161+ CD4+ T cells, but not their frequency. In contrast, early ART initiation prevented the decline of colonic CD161+ CD4+ T cells that otherwise started during acute infection. Furthermore, loss of peripheral and colonic CD161+ CD4+ T cells in untreated infection was associated with levels of viral load. These results suggest that acute HIV-1 infection has profound effects on the CD161+ CD4+ T cell population that could not be completely prevented by the initiation of ART.

Impact of Early Antiretroviral Treatment Initiation on Performance of Cross-Sectional Incidence Assays.

Antiretroviral therapy (ART) can impact assays used for cross-sectional HIV incidence testing, causing inaccurate HIV incidence estimates. We evaluated the relationship between the timing of ART initiation and the performance of two serologic HIV incidence assays. We analyzed 302 samples from 55 individuals from the RV217 cohort (Early Capture HIV Cohort Study). Participants were grouped by ART start time: ART started <1 year after infection (N = 9); ART started 1-3 years after infection (N = 12); and never received ART (N = 34). Samples were tested using the Sedia LAg-Avidity and Johns Hopkins modified Bio-Rad-Avidity assays. Results were compared with those from the Johns Hopkins HIV Cohort in which participants initiated ART an average of 10 years after infection (N = 17). Participants on ART were virally suppressed at the time of sample collection. The increase in normalized optical density (ODn) values was an average of 2.15 U/year lower in participants who started ART <1 year after infection than in those who did not start ART. Participants who started ART 1-3 years after infection had a decline in ODn values 0.90 U/year faster compared with those who started ART an average of 10 years after infection. Timing of ART initiation did not significantly impact results obtained with the Bio-Rad-Avidity assay. ART initiation <1 year after HIV infection was associated with persistently low limiting antigen (Lag)-Avidity values; this could lead to overestimation of HIV incidence. LAg-Avidity values declined more rapidly the earlier ART was initiated. Bio-Rad-Avidity values were not impacted by the timing of ART initiation.

Late boosting of the RV144 regimen with AIDSVAX B/E and ALVAC-HIV in HIV-uninfected Thai volunteers: a double-blind, randomised controlled trial.

BACKGROUND: The RV144 phase 3 vaccine trial in Thailand demonstrated that ALVAC-HIV (vCP1521) and AIDSVAX B/E administration over 6 months resulted in a 31% efficacy in preventing HIV acquisition. In this trial, we assessed the immunological effect of an additional vaccine boost to the RV144 regimen at varying intervals between the priming vaccine series and the boost. METHODS: RV306 is a double-blind, placebo-controlled, randomised clinical trial done at three clinical sites in Thailand. Eligible volunteers were HIV-uninfected individuals aged 20-40 years who were at low risk for HIV infection and in good health. A randomisation schedule was centrally generated with fixed sized strata for Research Institute for Health Sciences Chiang Mai and combined Bangkok clinics. Participants were randomly assigned to one of five groups and then further randomly assigned to either vaccine or placebo. All participants received the primary RV144 vaccine series at months 0, 1, 3, and 6. Group 1 received no additional boost, group 2 received additional AIDSVAX B/E and ALVAC-HIV (vCP1521) or placebo at month 12, group 3 received AIDSVAX B/E alone or placebo at month 12, group 4a received AIDSVAX B/E and ALVAC-HIV or placebo at month 15, and group 4b received AIDSVAX B/E and ALVAC-HIV or placebo at month 18. Primary outcomes were safety and tolerability of these vaccination regimens and cellular and humoral immune responses compared between the RV144 series alone and regimens with late boosts at different timepoints. Safety and tolerability outcomes were assessed by evaluating local and systemic reactogenicity and adverse events in all participants. This trial is registered at ClinicalTrials.gov (NCT01931358); clinical follow-up is now complete. FINDINGS: Between Oct 28, 2013, and April 29, 2014, 367 participants were enrolled, of whom 27 were assigned active vaccination in group 1, 102 in group 2, 101 in group 3, 52 in group 4a, 51 in group 4b, and 34 combined placebo across all the groups. No vaccine-related serious adverse events were recorded. Occurrence and severity of local and systemic reactogenicity were similar across active groups. Groups with late boosts (groups 2, 3, 4a, and 4b) had increased peak plasma IgG-binding antibody levels against gp70 V1V2 relative to group 1 vaccine recipients with no late boost (gp70 V1V2 92TH023 adjusted p<0·02 for each; gp70 V1V2 CaseA2 adjusted p<0·0001 for each). Boosting at month 12 (groups 2 and 3) did not increase gp120 responses compared with the peak responses after the RV144 priming regimen at month 6; however, boosting at month 15 (group 4a) improved responses to gp120 A244gD- D11 (p=0·0003), and boosting at month 18 (group 4b) improved responses to both gp120 A244gD- D11 (p<0·0001) and gp120 MNgD- D11 (p=0·0016). Plasma IgG responses were significantly lower among vaccine recipients boosted at month 12 (pooled groups 2 + 3) than at month 15 (group 4a; adjusted p<0·0001 for each, except for gp70 V1V2 CaseA2, p=0·0142) and at month 18 (group 4b; all adjusted p<0·001). Boosting at month 18 versus month 15 resulted in a significantly higher plasma IgG response to gp120 antigens (all adjusted p<0·01) but not gp70 V1V2 antigens. CD4 functionality and polyfunctionality scores after stimulation with HIV-1 Env peptides (92TH023) increased with delayed boosting. Groups with late boosts had increased functionality and polyfunctionality scores relative to vaccine recipients with no late boost (all adjusted p<0·05, except for the polyfunctionality score in group 1 vs group 4b, p<0·01). INTERPRETATION: Taken together, these results suggest that additional boosting of the RV144 regimen with longer intervals between the primary vaccination series and late boost improved immune responses and might improve the efficacy of preventing HIV acquisition. FUNDING: US National Institute of Allergy and Infectious Diseases and US Department of the Army.

Novel Strategy To Adapt Simian-Human Immunodeficiency Virus E1 Carrying env from an RV144 Volunteer to Rhesus Macaques: Coreceptor Switch and Final Recovery of a Pathogenic Virus with Exclusive R5 Tropism.

The phase III RV144 human immunodeficiency virus (HIV) vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing the HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clade A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: the simultaneous depletion of B and CD8+ cells followed by the intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High-level viremia and CD4+ T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4+ T-cell loss; the partially adapted SHIV had become dual tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5 tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and was pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans.IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in nonhuman primate (NHP) models requires test viruses that allow the evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to nonneutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive-immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed a coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies.

Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection.

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1ß) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination.

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.

Adenovirus-based HIV-1 vaccine candidates tested in efficacy trials elicit CD8+ T cells with limited breadth of HIV-1 inhibition.

OBJECTIVES: The ability of HIV-1 vaccine candidates MRKAd5, VRC DNA/Ad5 and ALVAC/AIDSVAX to elicit CD8 T cells with direct antiviral function was assessed and compared with HIV-1-infected volunteers. DESIGN: Adenovirus serotype 5 (Ad5)-based regimens MRKAd5 and VRC DNA/Ad5, designed to elicit HIV-1-specific T cells, are immunogenic but failed to prevent infection or impact on viral loads in volunteers infected subsequently. Failure may be due in part to a lack of CD8 T cells with effective antiviral functions. METHODS: An in-vitro viral inhibition assay tested the ability of bispecific antibody expanded CD8 T cells from peripheral blood mononuclear cells to inhibit replication of a multiclade panel of HIV-1 isolates in autologous CD4 T cells. HIV-1 proteins recognized by CD8 T cells were assessed by IFNγ enzyme-linked immunospot assay. RESULTS: Ad5-based regimens elicited CD8 T cells that inhibited replication of HIV-1 IIIB isolate with more limited inhibition of other isolates. IIIB isolate Gag and Pol genes have high sequence identities (>96%) to vector HIV-1 gene inserts, and these were the predominant HIV-1 proteins recognized by CD8 T cells. Virus inhibition breadth was greater in antiretroviral naïve HIV-1-infected volunteers naturally controlling viremia (plasma viral load < 10 000/ml). HIV-1-inhibitory CD8 T cells were not elicited by the ALVAC/AIDSVAX regimen. CONCLUSION: The Ad5-based regimens, although immunogenic, elicited CD8 T cells with limited HIV-1-inhibition breadth. Effective T-cell-based vaccines should presumably elicit broader HIV-1-inhibition profiles. The viral inhibition assay can be used in vaccine design and to prioritize promising candidates with greater inhibition breadth for further clinical trials.

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Structural analysis of the unmutated ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee.

Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties.

COMPASS identifies T-cell subsets correlated with clinical outcomes.

Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells as well as the interrogation of cell population heterogeneity. However, in many instances, computational tools to analyze the wealth of data generated by these technologies are lacking. Here, we present a computational framework for unbiased combinatorial polyfunctionality analysis of antigen-specific T-cell subsets (COMPASS). COMPASS uses a Bayesian hierarchical framework to model all observed cell subsets and select those most likely to have antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, and human subject-level responses are quantified by two summary statistics that describe the quality of an individual's polyfunctional response and can be correlated directly with clinical outcome. Using three clinical data sets of cytokine production, we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals cellular 'correlates of protection/immunity' in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software.

Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines.

The human phase 2B RV144 ALVAC-HIV vCP1521/AIDSVAX B/E vaccine trial, held in Thailand, resulted in an estimated 31.2% efficacy against HIV infection. By contrast, vaccination with VAX003 (consisting of only AIDSVAX B/E) was not protective. Because protection within RV144 was observed in the absence of neutralizing antibody activity or cytotoxic T cell responses, we speculated that the specificity or qualitative differences in Fc-effector profiles of nonneutralizing antibodies may have accounted for the efficacy differences observed between the two trials. We show that the RV144 regimen elicited nonneutralizing antibodies with highly coordinated Fc-mediated effector responses through the selective induction of highly functional immunoglobulin G3 (IgG3). By contrast, VAX003 elicited monofunctional antibody responses influenced by IgG4 selection, which was promoted by repeated AIDSVAX B/E protein boosts. Moreover, only RV144 induced IgG1 and IgG3 antibodies targeting the crown of the HIV envelope V2 loop, albeit with limited coverage of breakthrough viral sequences. These data suggest that subclass selection differences associated with coordinated humoral functional responses targeting strain-specific protective V2 loop epitopes may underlie differences in vaccine efficacy observed between these two vaccine trials.

Vaccine induction of antibodies against a structurally heterogeneous site of immune pressure within HIV-1 envelope protein variable regions 1 and 2.

The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4(+) T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the ß strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.

A whole blood lymphocyte proliferation assay in healthy Thais: comparison of heparinized blood and acid citrate dextrose blood.

The lymphocyte proliferation assay (LPA) is a technique to determine T-lymphocyte functions in vitro. The standard LPA using peripheral blood mononuclear cells (PBMC) separated from heparinized blood requires a large blood sample, time consuming and expensive. It is more useful if acid citrate dextrose (ACD) blood could be used not only for LPA but also for other purposes. To determine whether whole blood composing between heparinized blood and ACD blood could be substituted for standard LPA using PBMC. Heparinized and ACD blood of 35 healthy Thai blood donors were studied herein. PBMC separated by density gradient centrifugation and diluted heparinized and ACD blood were used to test and compare for lymphoproliferative responses to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and tetanus toxoid. A stimulation index (SI) for each mitogen or antigen was calculated. All Thai blood donors demonstrated positive proliferative responses to PHA and PWM by using PBMC and whole blood culture assays from both heparinized and ACD blood. However, the difference in the frequency of positive proliferative responses to tetanus toxoid by using PBMC and whole blood culture assays was significant. Nevertheless, no significant difference in frequency of positive responses to tetanus toxoid between heparinized and ACD blood was observed. This results suggested that no significant difference between using heparinized and ACD blood in standard LPA using PBMC. However, the whole blood LPA for measuring mitogen induced lymphoproliferation could be substituted for standard LPA from heparinized andACD blood. Whole blood LPA is easy, rapid, and more cost effective than PBMC culture assay. Thus, it would be applicable in a clinical laboratory as well as in research setting.

Antibody-dependent cell-mediated cytotoxic responses in participants enrolled in a phase I/II ALVAC-HIV/AIDSVAX B/E prime-boost HIV-1 vaccine trial in Thailand.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed in volunteers participating in an ALVAC-HIV (vCP1521)/AIDSVAX B/E gp120 prime-boost vaccine trial in Thailand. ADCC activity was measured using chromium release from gp120 subtype B- and CRF01_AE-coated targets in 95 vaccinees and 28 placebo recipients. There was a significant difference in the magnitude of the ADCC response to both targets between vaccinees and placebo recipients. The frequency of responders to subtype B and to CRF01_AE was 96% and 84% in the vaccine group versus 11% and 7% in the placebo group. The results demonstrate that this HIV vaccine is a potent inducer of ADCC activity and may be an additional protection of this prime-boost vaccine in preventing HIV disease.

The development of HIV research laboratories in the Royal Thai Army Medical Department.

The development of HIV research laboratories at the Armed Forces Research Institute of Medical Sciences (AFRIMS), Royal Thai Army Medical Department in supporting of HIV-1 vaccine trials in Thailand was implemented in 1991. The collaboration between AFRIMS, Royal Thai Army Medical Department, and the US Military HIV Research Program with the ultimate goal to conduct the HIV-1 vaccine trial phase III. The HIV serology lab was set up for surveillance program in military recruits. Then, there was a need to strengthen more on the existing laboratories by training personnel to cope with the confidentiality of the lab results, specimen processing and data management which are critical. Later on, the necessary laboratory for measuring of vaccine immunogenicity was developed, such as lymphoproliferation assay. Additionally, a molecular biology lab was also developed. The HIV research laboratory management must include an ability to deal with some problems, such as late specimen receiving, fluctuating of power supply, technical staffs maintained. Good laboratory practices and safety must be strictly implemented. Communication network among facilities also played an important role in HIV laboratory strengthening at AFRIMS.

Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults.

ALVAC-HIV (vCP1521) and AIDSVAX B/E were evaluated in a phase 1/2 trial of human immunodeficiency virus (HIV)-negative Thai adults. Of 133 volunteers enrolled, 122 completed the trial. There were no serious vaccine-related adverse events, nor were there intercurrent HIV infections. Lymphoproliferative responses to glycoprotein 120 E were induced in 63% of the volunteers, and HIV-specific CD8 cytotoxic T lymphocyte responses were induced in 24%. Antibody responses increased in frequency and magnitude in association with the dose level of AIDSVAX B/E. Binding and neutralizing antibodies to the MN strain were induced in 100% and 98%, respectively, of the volunteers receiving 600 microg of AIDSVAX B/E, and such antibodies to E strains were induced in 96% and 71%, respectively, of these volunteers. This vaccine combination was well tolerated and was immunogenic, meeting milestones for advancement to phase 3 evaluation.

Safety and immunogenicity of combinations of recombinant subtype E and B human immunodeficiency virus type 1 envelope glycoprotein 120 vaccines in healthy Thai adults.

Safety and immunogenicity of 2 recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein (gp) 120 vaccines derived from SF2 (subtype B) and CM235 (CRF01_AE, Thai E) were evaluated in 370 Thai adults at low risk of HIV infection. Various doses of CM235 (25, 50, or 100 microg) and SF2 (0, 25, or 50 microg) gp120 were used. Eighty volunteers received placebo. There were no serious adverse events related to vaccination. Binding antibody developed in all vaccine recipients. There was no dose response to CM235 gp120, but a dose response to gp120 SF2 was present. Neutralizing antibodies to subtype E HIV-1 NPO3 and subtype B HIV-1 SF2 developed in 84% and 82% of vaccine recipients, respectively. Lymphoproliferative responses were detected in >95% of vaccine recipients. There was no evidence of antigenic interference in HIV-specific humoral or cellular responses. The gp120 Thai E and SF2 vaccines were safe and immunogenic in combination and could be advanced into phase 3 testing.

Longitudinal study of humoral immune responses in HIV type 1 subtype CRF01_AE (E)-infected Thai patients with different rates of disease progression.

Identification of immune correlates associated with disease progression will provide information for HIV-1 vaccine design in countries such as Thailand, where the prevalent subtypes (B and CRF01_AE [E]) are characterized. In this study, plasma viral load and humoral immune responses were measured in 20 HIV-1 subtype E-infected Thai patients with different rates of disease progression, based on CD4(+) T cell decline and clinical symptoms. Nine progressors (PRs) and 11 slower progressors (SPs) were evaluated. CD4(+) T cell counts were inversely correlated with viral load (p = 0.004) and positively correlated with p24 Ab (p = 0.022). In progressors, p24 Ab showed a significant decrease (p < 0.001) over time. V3 and gp41 Ab did not change significantly in either group. Both CD4-binding site (CD4/gp120BS) and gp120 titers correlated positively with neutralizing antibody (NAb) against both a subtype E cell line-adapted virus (NP03) and a primary isolate (TH023). However, V3 Ab correlated only with NAb against NP03 (p < 0.001). Increased NAb over time was observed more frequently in SPs as compared with PRs, against both the TH023 (p = 0.004) and NPO3 (p = 0.004) viruses. Cross-clade antibody-dependent cellular cytotoxicity was demonstrated in both groups. These data suggest that in HIV-1 subtype E infection, declining p24 Ab titer is a predictive marker of disease progression, as described for subtype B. Furthermore, in subtype E-infected patients, slower progressors retain the immune competence to develop new antibody responses to Env over time; these evolving responses may contribute to prolonged survival during HIV-1 disease progression.