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BackgroundClinical laboratory reference intervals (RIs) are essential for diagnosing and managing patients in routine clinical care as well as establishing eligibility criteria and defining adverse events in clinical trials, but may vary by age, gender, genetics, nutrition and geographic location. It is, therefore, critical to establish region-specific reference values in order to inform clinical decision-making.MethodsWe analyzed data from a prospective observational HIV incidence cohort study in Kombewa, Kenya. Study participants were healthy males and females, aged 18-35 years, without HIV. Median and 95% reference values (2.5th percentile to 97.5th percentile) were calculated for laboratory parameters including hematology, chemistry studies, and CD4 T cell count. Standard Deviation Ratios (SDR) and Bias Ratios (BR) are presented as measures of effect magnitude. Findings were compared with those from the United States and other Kenyan studies.ResultsA total of 299 participants were analyzed with a median age of 24 years (interquartile range: 21-28). Ratio of males to females was 0.9:1. Hemoglobin range (2.5th-97.5th percentiles) was 12.0-17.9 g/dL and 9.5-15.3 g/dL in men and women respectively. In the cohort, MCV range was 59-95fL, WBC 3.7-9.2×103/μL, and platelet 154-401×103/μL. Chemistry values were higher in males; the creatinine RI was 59-103 μmol/L in males vs. 46-76 μmol/L in females (BRUL>.3); and the alanine transferase range was 8.8-45.3 U/L in males vs. 7.5-36.8 U/L in females (SDR>.3). The overall CD4 T cell count RI was 491-1381 cells/μL. Some parameters including hemoglobin, neutrophil, creatinine and ALT varied with that from prior studies in Kenya and the US.ConclusionThis study not only provides clinical reference intervals for a population in Kisumu County but also highlights the variations in comparable settings, accentuating the requirement for region-specific reference values to improve patient care, scientific validity, and quality of clinical trials in Africa.

IntroductionVaccine efficacy testing requires engagement of willing volunteers with high disease incidence. We evaluated factors associated with willingness to participate in potential future HIV vaccine trials in Maputo, Mozambique.MethodsAdults aged 18-35 years without HIV and who reported at least two sexual partners in the 3 months prior to screening were enrolled into a 24-month observational study. They were asked at screening and exit if they would be willing to participate in a theoretical HIV vaccine study. Bivariate and multivariate logistic regression analyses were done between willingness to participate, demographic, sexual behavior, and motivational factors for screening visit data. Logistic regression with generalized estimating equations (GEE) was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for factors potentially associated with willingness to participate for data from both visits.ResultsA total of 577 participants without HIV were eligible, including 275 (48%) women. The mean age was 22.2 (SD ± 3.9) years. At screening 529 (92%) expressed willingness to participate and the proportion remained stable at 378 (88%) of the 430 participants retained through the exit visit (p = 0.209). Helping the country (n = 556) and fear of needles (n = 26) were the top motive and barrier for willingness to participate, respectively. Results from the GEE binary logistic regression (screening visit and exit visit) showed that wanting to learn how to avoid risk behaviors (aOR 3.33, 95% CI: 1.61-6.86) and feeling protected against HIV infection (aOR 2.24, 95% CI: 1.07-4.7) were associated with willingness to participate in HIV vaccine studies.ConclusionThe majority of our study population in Mozambique expressed willingness to participate in a theoretical HIV vaccine trial. Participation in a HIV vaccine trial was seen as a way to contribute to the fight against HIV but was associated with some unrealistic expectations such as protection against HIV. This reinforces the need for continuous mobilization and awareness of potential participants to HIV vaccine trial.

BACKGROUND:Knowledge of HIV risk factors and reduction strategies is essential for prevention in key populations such as men who have sex with men (MSM) and transgender women (TGW). We evaluated factors associated with HIV-related knowledge among MSM and TGW and the impact of engagement in care at trusted community health centers in Nigeria. METHODS:The TRUST/RV368 cohort recruited MSM and TGW in Lagos and Abuja, Nigeria via respondent driven sampling. During study visits every three months, participants underwent structured interviews to collect behavioral data, received HIV education, and were provided free condoms and condom compatible lubricants. Five HIV-related knowledge questions were asked at enrollment and repeated after 9 and 15 months. The mean number of correct responses was calculated for each visit with 95% confidence intervals (CIs). Multivariable Poisson regression was used to calculate adjusted risk ratios and CIs for factors associated with answering more knowledge questions correctly. RESULTS:From March 2013 to April 2018, 2122 persons assigned male sex at birth were enrolled, including 234 TGW (11.2%). The mean number of correct responses at enrollment was 2.36 (95% CI: 2.31-2.41) and increased to 2.95 (95% CI: 2.86-3.04) and 3.06 (95% CI: 2.97-3.16) after 9 and 15 months in the study, respectively. Among 534 participants who completed all three HIV-related knowledge assessments, mean number of correct responses rose from 2.70 (95% CI: 2.60-2.80) to 3.02 (95% CI: 2.93-3.13) and then 3.06 (95% CI: 2.96-3.16). Factors associated with increased overall HIV-related knowledge included longer duration of study participation, HIV seropositivity, higher education level, and more frequent internet use. CONCLUSIONS:There was suboptimal HIV-related knowledge among Nigerian MSM and TGW at that improved modestly with engagement in care. These data demonstrate unmet HIV education needs among Nigerian MSM and TGW and provide insights into modalities that could be used to address these needs.

BackgroundMen who have sex with men (MSM) and transgender women (TGW) are at risk for sexually transmitted infections (STIs), including those of the oropharynx. We estimated the prevalence and factors associated with oral sex practices and characterized oropharyngeal STIs among a cohort of MSM and TGW in Nigeria.MethodsFrom 2013 to 2018, TRUST/RV368 recruited MSM and TGW into HIV/STI diagnosis and treatment at community-based clinics in Nigeria. Participants who completed HIV testing and oral sex questions at enrollment were selected. Cross-sectional analyses with bivariate and multivariable logistic regression models estimated odds ratios (ORs) and 95% confidence intervals (CIs). Oropharyngeal swab testing for Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) began in 2014 and for those with diagnostic results at enrollment, the unadjusted association of oral sex practices with oropharyngeal STIs was conducted.ResultsA total of 1342 participants had a median age of 25 years (interquartile range: 22-29), 58% were living with HIV, and 69% reported oral sex practices. Factors associated with increased odds of engaging in oral sex included living with HIV (adjusted [a]OR: 1.4, 95% CI: 1.1-1.8), self-identifying as a woman (aOR:1.8, 95% CI: 1.1-2.8), mobile phone ownership (aOR:2.3, 95% CI: 1.3-3.9), receptive anal sex (aOR:1.7, 95% CI:1.3-2.3) and multiple male sexual partners (2 to 4 vs. ≤1, aOR:1.5, 95% CI: 1.0-2.2; 5+ vs ≤1, aOR:2.9, 95% CI:1.9-4.3). Oropharyngeal STI prevalence was 7% (52/752) and higher among those who engaged in oral sex compared to those who did not (unadjusted OR: 2.5, 95% CI:1.2-5.3).ConclusionsOral sex was common and associated with an increased odds of oropharyngeal STIs among MSM and TGW from Nigeria. In the absence of screening and treatment guidelines, condoms continue to be the mainstay for oral STI prevention. A pre-exposure prophylaxis for bacterial STIs would complement current prevention strategies to curb transmission.

BackgroundHIV vaccine trials routinely measure multiple vaccine-elicited immune responses to compare regimens and study their potential associations with protection. Here we employ unsupervised learning tools facilitated by a bidirectional power transformation to explore the multivariate binding antibody and T-cell response patterns of immune responses elicited by two pox-protein HIV vaccine regimens. Both regimens utilized a recombinant canarypox vector (ALVAC-HIV) prime and a bivalent recombinant HIV-1 Envelope glycoprotein 120 subunit boost. We hypothesized that within each trial, there were participant subgroups sharing similar immune responses and that their frequencies differed across trials.Methods and findingsWe analyzed data from three trials-RV144 (NCT00223080), HVTN 097 (NCT02109354), and HVTN 100 (NCT02404311), the latter of which was pivotal in advancing the tested pox-protein HIV vaccine regimen to the HVTN 702 Phase 2b/3 efficacy trial. We found that bivariate CD4+ T-cell and anti-V1V2 IgG/IgG3 antibody response patterns were similar by age, sex-at-birth, and body mass index, but differed for the pox-protein clade AE/B alum-adjuvanted regimen studied in RV144 and HVTN 097 (PAE/B/alum) compared to the pox-protein clade C/C MF59-adjuvanted regimen studied in HVTN 100 (PC/MF59). Specifically, more PAE/B/alum recipients had low CD4+ T-cell and high anti-V1V2 IgG/IgG3 responses, and more PC/MF59 recipients had broad responses of both types. Analyses limited to "vaccine-matched" antigens suggested that some of the differences in responses between the regimens could have been due to antigens in the assays that did not match the vaccine immunogens. Our approach was also useful in identifying subgroups with unusually absent or high co-responses across assay types, flagging individuals for further characterization by functional assays. We also found that co-responses of anti-V1V2 IgG/IgG3 and CD4+ T cells had broad variability. As additional immune response assays are standardized and validated, we anticipate our framework will be increasingly valuable for multivariate analysis.ConclusionsOur approach can be used to advance vaccine development objectives, including the characterization and comparison of candidate vaccine multivariate immune responses and improved design of studies to identify correlates of protection. For instance, results suggested that HVTN 702 will have adequate power to interrogate immune correlates involving anti-V1V2 IgG/IgG3 and CD4+ T-cell co-readouts, but will have lower power to study anti-gp120/gp140 IgG/IgG3 due to their lower dynamic ranges. The findings also generate hypotheses for future testing in experimental and computational analyses aimed at achieving a mechanistic understanding of vaccine-elicited immune response heterogeneity.

HIV-1 strain diversity in Bulgaria is extensive and includes contributions from nearly all major subtypes and the Circulating Recombinant Forms (CRF): 01_AE, 02_AG, and 05_DF. Prior to this study, HIV-1 sequence information from Bulgaria has been based solely on the pro-RT gene, which represent less than 15% of the viral genome. To further characterize HIV-1 in Bulgaria, assess participant risk behaviors, and strengthen knowledge of circulating strains in the region, the study "Genetic Subtypes of HIV-1 in Bulgaria (RV240)" was conducted. This study employed the real time-PCR based Multi-region Hybridization Assay (MHA) B/non-B and HIV-1 sequencing to survey 215 of the approximately 1100 known HIV-1 infected Bulgarian adults (2008-2009) and determine if they were infected with subtype B HIV-1. The results indicated a subtype B prevalence of 40% and demonstrate the application of the MHA B/non-B in an area containing broad HIV-1 strain diversity. Within the assessed risk behaviors, the proportion of subtype B infection was greatest in men who have sex with men and lowest among those with drug use risk factors. During this study, 15 near full-length genomes and 22 envelope sequences were isolated from study participants. Phylogenetic analysis shows the presence of subtypes A1, B, C, F1, and G, CRF01_AE, CRF02_AG, CRF05_DF, and one unique recombinant form (URF). These sequences also show the presence of two strain groups containing participants with similar risk factors. Previous studies in African and Asian cohorts have shown that co-circulation of multiple subtypes can lead to viral recombination within super-infected individuals and the emergence of new URFs. The low prevalence of URFs in the presence of high subtype diversity in this study, may be the result of successful infection prevention and control programs. Continued epidemiological monitoring and support of infection prevention programs will help maintain control of the HIV-1 epidemic in Bulgaria.

IntroductionThe 2016 WHO consolidated guidelines on the use of antiretroviral drugs defines HIV virologic failure for low and middle income countries (LMIC) as plasma HIV-RNA ≥ 1000 copies/mL. We evaluated virologic failure and predictors in four African countries.Materials and methodsWe included HIV-infected participants on a WHO recommended antiretroviral therapy (ART) regimen and enrolled in the African Cohort Study between January 2013 and October 2017. Studied outcomes were virologic failure (plasma HIV-RNA ≥ 1000 copies/mL at the most recent visit), viraemia (plasma HIV-RNA ≥ 50 copies/mL at the most recent visit); and persistent viraemia (plasma HIV-RNA ≥ 50 copies/mL at two consecutive visits). Generalized linear models were used to estimate relative risks with their 95% confidence intervals.Results2054 participants were included in this analysis. Viraemia, persistent viraemia and virologic failure were observed in 396 (19.3%), 160 (7.8%) and 184 (9%) participants respectively. Of the participants with persistent viraemia, only 57.5% (92/160) had confirmed virologic failure. In the multivariate analysis, attending clinical care site other than the Uganda sitebeing on 2nd line ART (aRR 1.8, 95% CI 1·28-2·66); other ART combinations not first line and not second line (aRR 3.8, 95% CI 1.18-11.9), a history of fever in the past week (aRR 3.7, 95% CI 1.69-8.05), low CD4 count (aRR 6.9, 95% CI 4.7-10.2) and missing any day of ART (aRR 1·8, 95% CI 1·27-2.57) increased the risk of virologic failure. Being on 2nd line therapy, the site where one receives care and CD4 count < 500 predicted viraemia, persistent viraemia and virologic failure.ConclusionIn conclusion, these findings demonstrate that HIV-infected patients established on ART for more than six months in the African setting frequently experienced viraemia while continuing to be on ART. The findings also show that being on second line, low CD4 count, missing any day of ART and history of fever in the past week remain important predictors of virologic failure that should trigger intensified adherence counselling especially in the absence of reliable or readily available viral load monitoring. Finally, clinical care sites are different calling for further analyses to elucidate on the unique features of these sites.

IntroductionMozambique continues to have a significant burden of HIV. Developing strategies to control the HIV epidemic remains a key priority for the Mozambican public health community. The primary aim of this study was to determine HIV prevalence and risk behavior among males and females screened for a HIV vaccine preparedness study in Maputo, Mozambique.MethodsMale and female participants between 18-35 years old were recruited from the general community and from female sex worker (FSW) and lesbian, gay, bisexual, and transgender (LGBT) associations in Maputo. All participants were screened for HIV and a questionnaire was administered to each participant to assess HIV risk behavior.ResultsA total of 1125 adults were screened for HIV infection, among whom 506 (45%) were male. Among men, 5.7% reported having had sex with men (MSM) and 12% of female participants reported having exchanged sex for money, goods or favors in the past 3 months. The overall HIV prevalence was 10.4%; 10.7% of women, and 10.1% of men were HIV infected; 41.4% of MSM were seropositive. HIV infection was associated with older age (25-35 years old) (OR: 6.13, 95% CI: 3.01, 12.5), MSM (OR: 9.07, 95% CI: 3.85, 21.4), self-perception of being at high-risk for HIV (OR: 3.99, 95% CI: 1.27, 12.5) and self-report of a history of a diagnosis of sexually transmitted infection (OR: 3.75, 95% CI: 1.57, 8.98).ConclusionIn our cohort, HIV prevalence was much higher among MSM compared to the overall prevalence. Behavioral factors were found to be more associated with HIV prevalence than demographic factors. The study findings demonstrate the critical importance of directing services to minority communities, such as MSM, when prevention strategies are being devised for the general population.

BackgroundWe evaluated the safety and immunogenicity of (i) an intradermal HIV-DNA regimen given with/without intradermal electroporation (EP) as prime and (ii) the impact of boosting with modified vaccinia virus Ankara (HIV-MVA) administered with or without subtype C CN54rgp140 envelope protein adjuvanted with Glucopyranosyl Lipid A (GLA-AF) in volunteers from Tanzania and Mozambique.MethodsHealthy HIV-uninfected adults (N = 191) were randomized twice; first to one of three HIV-DNA intradermal priming regimens by needle-free ZetaJet device at weeks 0, 4 and 12 (Group I: 2x0.1mL [3mg/mL], Group II: 2x0.1mL [3mg/mL] plus EP, Group III: 1x0.1mL [6mg/mL] plus EP). Second the same volunteers received 108 pfu HIV-MVA twice, alone or combined with CN54rgp140/GLA-AF, intramuscularly by syringe, 16 weeks apart. Additionally, 20 volunteers received saline placebo.ResultsVaccinations and electroporation did not raise safety concerns. After the last vaccination, the overall IFN-γ ELISpot response rate to either Gag or Env was 97%. Intradermal electroporation significantly increased ELISpot response rates to HIV-DNA-specific Gag (66% group I vs. 86% group II, p = 0.026), but not to the HIV-MVA vaccine-specific Gag or Env peptide pools nor the magnitude of responses. Co-administration of rgp140/GLA-AF with HIV-MVA did not impact the frequency of binding antibody responses against subtype B gp160, C gp140 or E gp120 antigens (95%, 99%, 79%, respectively), but significantly enhanced the magnitude against subtype B gp160 (2700 versus 300, pConclusionIntradermal electroporation increased DNA-induced Gag response rates but did not show an impact on Env-specific responses nor on the magnitude of responses. Co-administration of HIV-MVA with rgp140/GLA-AF significantly enhanced antibody responses.

Sexual transmission is the principal driver of the human immunodeficiency virus (HIV) pandemic. Understanding HIV vaccine-induced immune responses at mucosal surfaces can generate hypotheses regarding mechanisms of protection, and may influence vaccine development. The RV144 (ClinicalTrials.gov NCT00223080) efficacy trial showed protection against HIV infections but mucosal samples were not collected, therefore, the contribution of mucosal antibodies to preventing HIV-1 acquisition is unknown. Here, we report the generation, magnitude and persistence of antibody responses to recombinant gp120 envelope and antigens including variable one and two loop scaffold antigens (gp70V1V2) previously shown to correlate with risk in RV144. We evaluated antibody responses to gp120 A244gD and gp70V1V2 92TH023 (both CRF01_AE) and Case A2 (subtype B) in cervico-vaginal mucus (CVM), seminal plasma (SP) and rectal secretions (RS) from HIV-uninfected RV144 vaccine recipients, who were randomized to receive two late boosts of ALVAC-HIV/AIDSVAX®B/E, AIDSVAX®B/E, or ALVAC-HIV alone at 0 and 6 months. Late vaccine boosting increased IgG geometric mean titers (GMT) to gp120 A244gD in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (28 and 17 fold, respectively), followed by SP and RS. IgG to gp70V1V2 92TH023 increased in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (11-17 fold) and SP (2 fold) two weeks post first boost. IgG to Case A2 was only detected in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM. Mucosal IgG to gp120 A244gD (CVM, SP, RS), gp70V1V2 92TH023 (CVM, SP), and Case A2 (CVM) correlated with plasma IgG levels (p

Non-neutralizing IgG to the V1V2 loop of HIV-1 gp120 correlates with a decreased risk of HIV-1 infection but the mechanism of protection remains unknown. This V1V2 IgG correlate was identified in RV144 Thai trial vaccine recipients, who were primed with a canarypox vector expressing membrane-bound gp120 (vCP1521) and boosted with vCP1521 plus a mixture gp120 proteins from clade B and clade CRF01_AE (B/E gp120). We sought to determine whether the mechanism of vaccine protection might involve antibody-dependent complement activation. Complement activation was measured as a function of complement component C3d deposition on V1V2-coated beads in the presence of RV144 sera. Variable levels of complement activation were detected two weeks post final boosting in RV144, which is when the V1V2 IgG correlate was identified. The magnitude of complement activation correlated with V1V2-specific serum IgG and was stronger and more common in RV144 than in HIV-1 infected individuals and two related HIV-1 vaccine trials, VAX003 and VAX004, where no protection was seen. After adjusting for gp120 IgA, V1V2 IgG, gender, and risk score, complement activation by case-control plasmas from RV144 correlated inversely with a reduced risk of HIV-1 infection, with odds ratio for positive versus negative response to TH023-V1V2 0.42 (95% CI 0.18 to 0.99, p = 0.048) and to A244-V1V2 0.49 (95% CI 0.21 to 1.10, p = 0.085). These results suggest that complement activity may have contributed in part to modest protection against the acquisition of HIV-1 infection seen in the RV144 trial.

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.

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.

BackgroundA vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF) in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA).MethodsForty volunteers (35 vaccinees and five placebo recipients) were given two CN54rgp140/GLA-AF immunizations 30-71 weeks after the last HIV-MVA vaccination. These immunizations were delivered intramuscularly four weeks apart.ResultsThe vaccine was safe and well tolerated except for one episode of asymptomatic hypoglycaemia that was classified as severe adverse event. Two weeks after the second HIV-MVA vaccination 34 (97%) of the 35 previously vaccinated developed Env-specific binding antibodies, and 79% and 84% displayed IFN-γ ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C Env (included in HIV-DNA and protein boost), subtype B Env (included only in HIV-DNA) and CRF01_AE Env (included only in HIV-MVA) were significantly boosted by the CN54rgp140/GLA-AF immunizations. Functional antibodies detected using an infectious molecular clone virus/peripheral blood mononuclear cell neutralization assay, a pseudovirus/TZM-bl neutralization assay or by assays for antibody-dependent cellular cytotoxicity (ADCC) were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-γ ELISpot responses to Env peptides were significantly enhanced. Four volunteers not primed with HIV-DNA and HIV-MVA before the CN54rgp140/GLA-AF immunizations mounted an antibody response, while cell-mediated responses were rare. After the two Env subtype C protein immunizations, a trend towards higher median subtype C Env binding antibody titers was found in vaccinees who had received HIV-DNA and HIV-MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07).ConclusionWe report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA.Trial registrationInternational Clinical Trials Registry PACTR2010050002122368.

Management of patient care and interpretation of research data require evaluation of laboratory results in the context of reference data from populations with known health status to adequately diagnose disease or make a physiological assessment. Few studies have addressed the diversity of lymphocyte subsets in rural and urban Ugandan populations. Here, 663 healthy blood bank donors from semi-urban centers of Kampala consented to participate in a study to define lymphocyte reference ranges. Whole blood immunophenotyping was performed to determine the frequency and absolute counts of T, B, and NK cells using clinical flow cytometry. Results from blood bank donors were compared to a rural cohort from the district of Kayunga and more urban clinical trial participants from the capital city, Kampala. Relationships between hematological and lymphocyte parameters were also explored. In the semi-urban blood donors, females were significantly different from males in all parameters except the frequency of CD8 T and B cells. Females had higher absolute counts of CD4 T, CD8 T and B cells, whereas males had higher NK cell counts. NK cell frequency and counts were significantly higher in semi-urban blood donors, regardless of sex, compared to more urban study participants. CD8 T cell frequency and counts were significantly higher in the blood donors compared to the rural participants, irrespective of sex. Interestingly, basophil counts were positively associated with overall T cell counts. These findings suggest that both sex and level of cohort urbanicity may influence lymphocyte subset distributions in Ugandans.

Development of a globally effective HIV-1 vaccine will need to encompass Nigeria, one of the hardest hit areas, with an estimated 3.2 million people living with HIV. This cross-sectional Institutional Review Board (IRB) approved study was conducted in 2009-12 at four market sites and two highway settlements sites in Nigeria to identify and characterize populations at high risk for HIV; engage support of local stakeholders; and assess the level of interest in future vaccine studies. Demographic, HIV risk data were collected by structured interviewer-administered questionnaires. Blood samples were tested on site by HIV rapid diagnostic tests, followed by rigorous confirmatory testing, subtype evaluation and testing for HBV and HCV markers in a clinical reference laboratory. Of 3229 study participants, 326 were HIV infected as confirmed by Western Blot or RNA, with a HIV prevalence of 15.4%-23.9% at highway settlements and 3.1%-9.1% at market sites. There was no observable correlation of prevalence of HIV-1 (10.1%) with HBV (10.9%) or HCV (2.9%). Major HIV-1 subtypes included CRF02_AG (37.5%); G (27.5%); G/CRF02_AG (25.9%); and non-typeable (8.9%), with 0.3% HIV-2. Univariate analysis found age, gender, marital status, level of education, and sex under substance influence as significant risk factors for HIV (p

BackgroundIntradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools.MethodsIn this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46.Results129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups.ConclusionsA simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA.Trial registrationWorld Health Organization International Clinical Trials Registry Platform PACTR2010050002122368.

Characterization of HIV-1 subtype diversity in regions where vaccine trials are conducted is critical for vaccine development and testing. This study describes the molecular epidemiology of HIV-1 within a tea-plantation community cohort in Kericho, Kenya. Sixty-three incident infections were ascertained in the HIV and Malaria Cohort Study conducted in Kericho from 2003 to 2006. HIV-1 strains from 58 of those individuals were full genome characterized and compared to two previous Kenyan studies describing 41 prevalent infections from a blood bank survey (1999-2000) and 21 infections from a higher-risk cohort containing a mix of incident and prevalent infections (2006). Among the 58 strains from the community cohort, 43.1% were pure subtypes (36.2% A1, 5.2% C, and 1.7% G) and 56.9% were inter-subtype recombinants (29.3% A1D, 8.6% A1CD, 6.9% A1A2D, 5.2% A1C, 3.4% A1A2CD, and 3.4% A2D). This diversity and the resulting genetic distance between the observed strains will need to be addressed when vaccine immunogens are chosen. In consideration of current vaccine development efforts, the strains from these three studies were compared to five candidate vaccines (each of which are viral vectored, carrying inserts corresponding to parts of gag, pol, and envelope), which have been developed for possible use in sub-Saharan Africa. The sequence comparison between the observed strains and the candidate vaccines indicates that in the presence of diverse recombinants, a bivalent vaccine is more likely to provide T-cell epitope coverage than monovalent vaccines even when the inserts of the bivalent vaccine are not subtype-matched to the local epidemic.

UNLABELLED:Vaccine-induced HIV antibodies were evaluated in serum samples collected from healthy Tanzanian volunteers participating in a phase I/II placebo-controlled double blind trial using multi-clade, multigene HIV-DNA priming and recombinant modified vaccinia Ankara (HIV-MVA) virus boosting (HIVIS03). The HIV-DNA vaccine contained plasmids expressing HIV-1 gp160 subtypes A, B, C, Rev B, Gag A, B and RTmut B, and the recombinant HIV-MVA boost expressed CRF01_AE HIV-1 Env subtype E and Gag-Pol subtype A. While no neutralizing antibodies were detected using pseudoviruses in the TZM-bl cell assay, this prime-boost vaccination induced neutralizing antibodies in 83% of HIVIS03 vaccinees when a peripheral blood mononuclear cell (PBMC) assay using luciferase reporter-infectious molecular clones (LucR-IMC) was employed. The serum neutralizing activity was significantly (but not completely) reduced upon depletion of natural killer (NK) cells from PBMC (p=0.006), indicating a role for antibody-mediated Fcγ-receptor function. High levels of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies against CRF01_AE and/or subtype B were subsequently demonstrated in 97% of the sera of vaccinees. The magnitude of ADCC-mediating antibodies against CM235 CRF01_AE IMC-infected cells correlated with neutralizing antibodies against CM235 in the IMC/PBMC assay. In conclusion, HIV-DNA priming, followed by two HIV-MVA boosts elicited potent ADCC responses in a high proportion of Tanzanian vaccinees. Our findings highlight the potential of HIV-DNA prime HIV-MVA boost vaccines for induction of functional antibody responses and suggest this vaccine regimen and ADCC studies as potentially important new avenues in HIV vaccine development. TRIAL REGISTRATION:Controlled-Trials ISRCTN90053831 The Pan African Clinical Trials Registry ATMR2009040001075080 (currently PACTR2009040001075080).

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

We performed fine epitope mapping of the CD4+ responses in the ALVAC-HIV-AIDSVAX B/E prime-boost regimen in the Thai Phase III trial (RV144). Non-transformed Env-specific T cell lines established from RV144 vaccinees were used to determine the fine epitope mapping of the V2 and C1 responses and the HLA class II restriction. Data showed that there are two CD4+ epitopes contained within the V2 loop: one encompassing the α4β7 integrin binding site (AA179-181) and the other nested between two previously described genetic sieve signatures (AA169, AA181). There was no correlation between the frequencies of CD4+ fine epitope responses and binding antibody.

RV144 was a community-based HIV vaccine efficacy trial conducted in HIV-uninfected adults in Thailand, where dengue virus continues to cause a large number of infections every year. We attempted to document the accuracy of clinically diagnosed dengue episodes reported as serious adverse events (SAEs) and adverse events (AEs) and examine whether dengue serology would support the clinical diagnosis. Subjects without a clinical dengue diagnosis but with an infection or idiopathic fever were selected as a control population. Dengue serology was performed by hemagglutination inhibition on plasma samples. A total of 124 clinical dengue episodes were reported (103 SAEs and 21 AEs). Overall 82.6% of the clinically diagnosed dengue episodes were supported by a positive dengue serology: 71.4% of the AEs and 85.0% of the SAEs. Of the 100 subjects with both clinical dengue and positive serology, all presented with fever, 83% with leucopenia, 54% with thrombocytopenia, and 27% with hemorrhagic symptoms. All episodes resolved spontaneously without sequellae. Only two of 15 subjects with a negative serology presented with fever. The sensitivity and specificity of clinical dengue diagnosis were 90.9% and 74.4%, respectively, when compared to the control population, and with a positive predictive value of 82.6% and negative predictive value of 84.7% when compared to dengue serology. Clinical diagnosis of dengue is an accurate method of dengue diagnosis in adults in Thailand. Large-scale clinical trials offer the opportunity to systematically study infectious diseases such as dengue and other infections that may occur during the trial.

Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow-up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines.We included participant-level data from all three efficacy trials, and three Phase 1-2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests.Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99-1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11-1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61-1.26, P = 0.48). Results were similar when including the Phase 1-2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5 vaccine-increased risk was concentrated in Ad5-positive or uncircumcised men early in follow-up, and in Ad5-negative or circumcised men later. Overall, MRKAd5 vaccine-increased risk was evident across subgroups except in circumcised Ad5-negative men (HR 0.97, 95% CI 0.58-1.63, P = 0.91); there was little evidence that the DNA/rAd5 vaccine, that was tested in this subgroup, increased risk (HR 0.88, 95% CI 0.61-1.26, P = 0.48). When restricting the analysis of Step and Phambili to follow-up time before unblinding, 114 (n = 65 vaccine; n = 49 placebo) of 3770 MITT participants acquired HIV-1, with a non-significantly higher incidence in MRKAd5 vaccine recipients (HR 1.30, 95% CI 0.89-1.14, P = 0.18).The data support increased risk of HIV-1 infection by MRKAd5 over all follow-up time, but do not support increased risk of HIV-1 infection by DNA/rAd5. This study provides a rationale for including monitoring plans enabling detection of increased susceptibility to infection in HIV-1 at-risk populations.

HIV is prevalent in Sofala Province, Mozambique. To inform future prevention research, we undertook a study in the provincial capital (Beira) to measure HIV incidence in women at higher risk of HIV and assess the feasibility of recruiting and retaining them as research participants.Women age 18-35 were recruited from schools and places where women typically meet potential sexual partners. Eligibility criteria included HIV-seronegative status and self-report of at least 2 sexual partners in the last month. History of injection drug use was an exclusion criterion, but pregnancy was not. Participants were scheduled for monthly follow-up for 12 months, when they underwent face-to-face interviews, HIV counseling and testing, and pregnancy testing.387 women were eligible and contributed follow-up data. Most were from 18-24 years old (median 21). Around one-third of participants (33.8%) reported at least one new sexual partner in the last month. Most women (65.5%) reported not using a modern method of contraception at baseline. Twenty-two women seroconverted for a prospective HIV incidence of 6.5 per 100 woman-years (WY; 95% confidence interval (CI): 4.1-9.9). Factors associated with HIV seroconversion in the multivariable analysis were: number of vaginal sex acts without using condoms with partners besides primary partner in the last 7 days (hazard ratio (HR) 1.7; 95% CI: 1.2-2.5) and using a form of contraception at baseline other than hormonal or condoms (vs. no method; HR 25.3; 95% CI: 2.5-253.5). The overall retention rate was 80.0% for the entire follow-up period.We found a high HIV incidence in a cohort of young women reporting risky sexual behavior in Beira, Mozambique. HIV prevention programs should be strengthened. Regular HIV testing and condom use should be encouraged, particularly among younger women with multiple sexual partners.

BACKGROUND: Reliable HIV incidence estimates for Mozambique are limited. We conducted a prospective HIV incidence study as part of a clinical research site development initiative in Chókwè district, Gaza Province, southern Mozambique. METHODS: Between June 2010 and October 2012, we recruited women at sites where women at higher risk of HIV infection would likely be found. We enrolled and tested 1,429 sexually active women in the screening phase and 479 uninfected women in the prospective phase. Participants were scheduled for 12+ months follow-up, when they underwent face-to-face interviews, HIV counseling and testing, and pregnancy testing. We observed a total of 373.1 woman-years (WY) of follow-up, with mean (median) of 9.4 (9.7) women-months per participant. RESULTS: The prevalence of HIV was 29.4% (95% confidence interval [CI]: 27.0-31.8%). In multivariable logistic regression analysis, factors that remained significantly associated with prevalent HIV were: older age (OR: 0.6; 95% CI: 0.4-0.7), lower educational level (OR: 0.4; 95% CI: 0.3-0.7), and using hormonal contraception (OR: 0.6; 95% CI: 0.4-0.7) or condoms (OR: 0.5; 95% CI: 0.3-0.7). We observed an HIV incidence rate of 4.6 per 100 WY (95% CI: 2.7, 7.3). The HIV incidence was 4.8 per 100 WY (95% CI: 2.5, 8.3) in women aged 18-24 years, 4.5 per 100 WY (95% CI: 1.2, 11.4) in women aged 25-29 years and 3.2 per 100 WY (95% CI: 0.1, 18.0) in the 30-35 years stratum. None of the demographic factors or time-varying behavioral factors examined was significantly associated with incident HIV infection in bivariable analysis at p ≤ 0.10. CONCLUSIONS: We found a high HIV incidence among sexually active young women in Chókwè, Mozambique. HIV prevention programs should be strengthened in the area, with more comprehensive reproductive health services, regular HIV testing, condom promotion, and messaging about multiple sexual partners.

The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84-91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.

Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.

The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165-178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.

We characterized prime-boost vaccine regimens using heterologous and homologous vector and gene inserts. Heterologous regimens offer a promising approach that focuses the cell-mediated immune response on the insert and away from vector-dominated responses. Ad35-GRIN/ENV (Ad35-GE) vaccine is comprised of two vectors containing sequences from HIV-1 subtype A gag, rt, int, nef (Ad35-GRIN) and env (Ad35-ENV). MVA-CMDR (MVA-C), MVA-KEA (MVA-K) and MVA-TZC (MVA-T) vaccines contain gag, env and pol genes from HIV-1 subtypes CRF01_AE, A and C, respectively. Balb/c mice were immunized with different heterologous and homologous vector and insert prime-boost combinations. HIV and vector-specific immune responses were quantified post-boost vaccination. Gag-specific IFN-γ ELISPOT, intracellular cytokine staining (ICS) (CD107a, IFN-γ, TNF-α and IL-2), pentamer staining and T-cell phenotyping were used to differentiate responses to inserts and vectors. Ad35-GE prime followed by boost with any of the recombinant MVA constructs (rMVA) induced CD8+ Gag-specific responses superior to Ad35-GE-Ad35-GE or rMVA-rMVA prime-boost combinations. Notably, there was a shift toward insert-focus responses using heterologous vector prime-boost regimens. Gag-specific central and effector memory T cells were generated more rapidly and in greater numbers in the heterologous compared to the homologous prime-boost regimens. These results suggest that heterologous prime-boost vaccination regimens enhance immunity by increasing the magnitude, onset and multifunctionality of the insert-specific cell-mediated immune response compared to homologous vaccination regimens. This study supports the rationale for testing heterologous prime-boost regimens in humans.

HIV-1 disease progression is associated with persistent immune activation. However, the nature of this association is incompletely understood. Here, we investigated immune activation in the CD4 T cell compartment of chronically HIV-1 infected individuals from Rakai, Uganda. Levels of CD4 T cell activation, assessed as co-expression of PD-1, CD38 and HLA-DR, correlated directly to viral load and inversely to CD4 count. Deeper characterization of these cells indicated an effector memory phenotype with relatively frequent expression of Ki67 despite their PD-1 expression, and levels of these cells were inversely associated with FoxP3+ regulatory T cells. We therefore use the term deregulated effector memory (DEM) cells to describe them. CD4 T cells with a DEM phenotype could be generated by antigen stimulation of recall responses in vitro. Responses against HIV-1 and CMV antigens were enriched among the DEM CD4 T cells in patients, and the diverse Vβ repertoire of DEM CD4 T cells suggested they include diverse antigen-specificities. Furthermore, the levels of DEM CD4 T cells correlated directly to soluble CD14 (sCD14) and IL-6, markers of innate immune activation, in plasma. The size of the activated DEM CD4 T cell subset was predictive of the rate of disease progression, whereas IL-6 was only weakly predictive and sCD14 was not predictive. Taken together, these results are consistent with a model where systemic innate immune activation and chronic antigen stimulation of adaptive T cell responses both play important roles in driving pathological CD4 T cell immune activation in HIV-1 disease.

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p

We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone.Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production.ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes.ClinicalTrials.gov NCT00013572.

A prime-boost vaccination regimen with ALVAC-HIV (vCP1521) administered intramuscularly at 0, 4, 12, and 24 weeks and gp120 AIDSVAX B/E at 12 and 24 weeks demonstrated modest efficacy of 31.2% for prevention of HIV acquisition in HIV-uninfected adults participating in a community-based efficacy trial in Thailand.Reactogenicity was recorded for 3 days following vaccination. Adverse events were monitored every 6 months for 3.5 years, during which pregnancy outcomes were recorded. Of the 16,402 volunteers, 69% of the participants reported an adverse event any time after the first dose. Only 32.9% experienced an AE within 30 days following any vaccination. Overall adverse event rates and attribution of relatedness did not differ between groups. The frequency of serious adverse events was similar in vaccine (14.3%) and placebo (14.9%) recipients (p = 0.33). None of the 160 deaths (85 in vaccine and 75 in placebo recipients, p = 0.43) was assessed as related to vaccine. The most common cause of death was trauma or traffic accident. Approximately 30% of female participants reported a pregnancy during the study. Abnormal pregnancy outcomes were experienced in 17.1% of vaccine and 14.6% (p = 0.13) of placebo recipients. When the conception occurred within 3 months (estimated) of a vaccination, the majority of these abnormal outcomes were spontaneous or elective abortions among 22.2% and 15.3% of vaccine and placebo pregnant recipients, respectively (p = 0.08). Local reactions occurred in 88.0% of vaccine and 61.0% of placebo recipients (p

We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand.MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7) or 10(8) pfu) or intradermally (ID; 10(6) or 10(7) pfu) at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2). Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51)Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i) moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6) PBMC at 10(8) pfu IM), but high in response rate (70% (51)Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8) pfu IM); (ii) predominantly HIV Env-specific CD4(+) T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route); (iv) dose- and route-dependent with 10(8) pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8) pfu IM).MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses.ClinicalTrials.gov NCT00376090.

The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA).A total of 73 healthy adults ages 18-40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA.The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses.ClinicalTrials.gov NCT00057525.

The W-curve was originally developed as a graphical visualization technique for viewing DNA and RNA sequences. Its ability to render features of DNA also makes it suitable for computational studies. Its main advantage in this area is utilizing a single-pass algorithm for comparing the sequences. Avoiding recursion during sequence alignments offers advantages for speed and in-process resources. The graphical technique also allows for multiple models of comparison to be used depending on the nucleotide patterns embedded in similar whole genomic sequences. The W-curve approach allows us to compare large numbers of samples quickly.We are currently tuning the algorithm to accommodate quirks specific to HIV-1 genomic sequences so that it can be used to aid in diagnostic and vaccine efforts. Tracking the molecular evolution of the virus has been greatly hampered by gap associated problems predominantly embedded within the envelope gene of the virus. Gaps and hypermutation of the virus slow conventional string based alignments of the whole genome. This paper describes the W-curve algorithm itself, and how we have adapted it for comparison of similar HIV-1 genomes. A treebuilding method is developed with the W-curve that utilizes a novel Cylindrical Coordinate distance method and gap analysis method. HIV-1 C2-V5 env sequence regions from a Mother/Infant cohort study are used in the comparison.The output distance matrix and neighbor results produced by the W-curve are functionally equivalent to those from Clustal for C2-V5 sequences in the mother/infant pairs infected with CRF01_AE.Significant potential exists for utilizing this method in place of conventional string based alignment of HIV-1 genomes, such as Clustal X. With W-curve heuristic alignment, it may be possible to obtain clinically useful results in a short time-short enough to affect clinical choices for acute treatment. A description of the W-curve generation process, including a comparison technique of aligning extremes of the curves to effectively phase-shift them past the HIV-1 gap problem, is presented. Besides yielding similar neighbor-joining phenogram topologies, most Mother and Infant C2-V5 sequences in the cohort pairs geometrically map closest to each other, indicating that W-curve heuristics overcame any gap problem.

HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.

The conduct of Phase I/II HIV vaccine trials internationally necessitates the development of region-specific clinical reference ranges for trial enrollment and participant monitoring. A population based cohort of adults in Kericho, Kenya, a potential vaccine trial site, allowed development of clinical laboratory reference ranges. Lymphocyte immunophenotyping was performed on 1293 HIV seronegative study participants. Hematology and clinical chemistry were performed on up to 1541 cohort enrollees. The ratio of males to females was 1.9:1. Means, medians and 95% reference ranges were calculated and compared with those from other nations. The median CD4+ T cell count for the group was 810 cells/microl. There were significant gender differences for both red and white blood cell parameters. Kenyan subjects had lower median hemoglobin concentrations (9.5 g/dL; range 6.7-11.1) and neutrophil counts (1850 cells/microl; range 914-4715) compared to North Americans. Kenyan clinical chemistry reference ranges were comparable to those from the USA, with the exception of the upper limits for bilirubin and blood urea nitrogen, which were 2.3-fold higher and 1.5-fold lower, respectively. This study is the first to assess clinical reference ranges for a highland community in Kenya and highlights the need to define clinical laboratory ranges from the national community not only for clinical research but also care and treatment.

BACKGROUND: Clinical trials are increasingly being conducted internationally. In order to ensure enrollment of healthy participants and proper safety evaluation of vaccine candidates, established reference intervals for clinical tests are required in the target population. METHODOLOGY/PRINCIPAL FINDINGS: We report a reference range study conducted in Ugandan adult blood bank donors establishing reference intervals for hematology and clinical chemistry parameters. Several differences were observed when compared to previously established values from the United States, most notably in neutrophils and eosinophils. CONCLUSIONS/SIGNIFICANCE: In a recently conducted vaccine trial in Uganda, 31 percent (n = 69) of volunteers screened (n = 223) were excluded due to hematologic abnormalities. If local reference ranges had been employed, 83% of those screened out due to these abnormalities could have been included in the study, drastically reducing workload and cost associated with the screening process. In addition, toxicity tables used in vaccine and drug trial safety evaluations may need adjustment as some clinical reference ranges determined in this study overlap with grade 1 and grade 2 adverse events.