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.
The WHO ‘Global Strategy for Dengue Prevention and Control, 2012–2020’ addresses the growing need for the treatment of dengue, and targets a 25% reduction in morbidity and 50% in mortality (using 2010 estimates as baseline). Achieving these goals requires future dengue prevention strategies that will employ both potential vaccines and sustainable vector-control measures. Maternally transferred dengue antibody is an important factor in determining the optimal age for dengue vaccination.
To estimate the seroprevalence of dengue antibodies among mothers living in an area of high endemicity – Ban Pong, Ratchaburi Province – and to assess maternal dengue antibodies transferred to cord blood.
Materials & Methods
A cross-sectional study was conducted with 141 pregnant women who delivered at Ban Pong Hospital, Ratchaburi, Thailand. Maternal-cord paired sera were tested for dengue neutralizing (NT) antibody by PRNT50 assay. A ratio of ≥ 1:10 NT titer to dengue serotype was considered seropositive.
Most mothers (137/141, 97.2%) had NT antibodies to at least one dengue serotype in their sera. At birth, the proportion of cord sera with NT antibodies to DEN-1, DEN-2, DEN-3, and DEN-4, were high and similar to the sera of their mothers, at 93.6%, 97.2%, 97.9%, and 92.2%, respectively. The dengue geometric mean titers (GMT) in cord blood were significantly higher than the maternal antibodies (p<0.001): highest in DEN-2, followed by DEN-3, and then DEN-1. The GMT of DEN-4 was the lowest among all four serotypes.
Dengue infection is highly prevalent among pregnant women in this dengue-endemic area. Most of the cord blood had transferred dengue antibodies, which may have an impact on the disease burden in this population.
Dengue is the fastest spreading mosquito-borne viral infection. Infections cause mild to severe diseases, including dengue hemorrhagic fever (DHF), a severe form that may kill infants and young children. Dengue virus antibody transfer from mother to fetus in pregnancy confers protection at birth, thereafter subsiding to a lower level that may cause DHF in infants. Infant dengue antibodies levels also influence the optimal age for dengue vaccination because of neutralization of the proposed live virus vaccine by the protective antibody levels in the newborn. To establish the optimal age, we identified mother-child pairs in which maternal dengue antibodies were transferred from mother to fetus in this study. Then a follow-up study would measure the infant antibody levels. Our study found that 97.2% of pregnant women giving birth in a dengue-endemic area had evidence of previous dengue infection. All umbilical cord blood from fetuses had the same proportion of positive tests for the presence of dengue antibodies, but had a higher dengue antibody levels compared to their mothers. The period of protection provided by maternally transferred dengue antibodies might affect the disease burden among infants and offer a better understanding of the optimal age for dengue vaccination.
The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or “signatures” and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.
We present an analysis of the genomes of the HIV viruses that infected some participants of the RV144 Thai trial, which was the first study to show efficacy of a vaccine to prevent HIV infection. We analyzed the HIV genomes of infected vaccine recipients and infected placebo recipients, and found differences between them. These differences coincide with previously-studied genetic features that are relevant to the biology of HIV infection, including features involved in immune recognition of the virus. The findings presented here generate testable hypotheses about the mechanism of the partial protection seen in the Thai trial, and may ultimately lead to improved vaccines. The article also presents a toolkit of methods for computational analyses that can be applied to other vaccine efficacy trials.
Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site.
IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.
Studies of undervaccinated children of minority/stateless populations have highlighted significant barriers at individual, community, and state levels. These include geography-related difficulties, poverty, and social norms/beliefs.
The objective of this study was to assess project outcomes regarding immunization coverage, as well as maternal attitudes and practices toward immunization.
The “StatelessVac” project was conducted in Thailand-Myanmar-Laos border areas using cell phone-based mechanisms to increase immunization coverage by incorporating phone-to-phone information sharing for both identification and prevention. With limitation of the study among vulnerable populations in low-resource settings, the pre/post assessments without comparison group were conducted. Immunization coverage was collected from routine monthly reports while behavior-change outcomes were from repeat surveys.
This study revealed potential benefits of the initiative for case identification; immunization coverage showed an improved trend. Prevention strategies were successfully integrated into the routine health care workflows of immunization activities at point-of-care. A behavior-change-communication package contributes significantly in raising both concern and awareness in relation to child care.
The mobile technology has proven to be an effective mechanism in improving a children’s immunization program among these hard-to-reach populations. Part of the intervention has now been revised for use at health centers across the country.
expanded program on immunization; EPI; hill tribes; stateless; behavioral change communication; mobile technology
Tensions between researchers and ethics committees have been reported in several institutions. Some reports suggest researchers lack confidence in the quality of institutional review board (IRB) reviews, and that emphasis on strict procedural compliance and ethical issues raised by the IRB might unintentionally lead to delays in correspondence between researchers and ethics committees, and/or even encourage prevarication/equivocation, if researchers perceive committee concerns and criticisms unjust. This study systematically analyzed the efficiency of different IRB functions, and the relationship between efficiency and perceived quality of the decision-making process. The major purposes of this study were thus (1) to use the IRB Metrics developed by the Faculty of Tropical Medicine, Mahidol University, Thailand (FTM-EC) to assess the operational efficiency and perceived effectiveness of its ethics committees, and (2) to determine ethical issues that may cause the duration of approval process to be above the target limit of 60 days. Based on a literature review of definitions and methods used and proposed for use, in assessing aspects of IRB quality, an “IRB Metrics” was developed to assess IRB processes using a structure-process-outcome measurement model. To observe trends in the indicators evaluated, data related to all protocols submitted to the two panels of the FTM-EC (clinical and non-clinical), between January 2010–September 2013, were extracted and analyzed. Quantitative information based on IRB Metrics structure-process-outcome illuminates different areas for internal-process improvement. Ethical issues raised with researchers by the IRB, which were associated with the duration of the approval process in protocol review, could be considered root causes of tensions between the parties. The assessment of IRB structure-process-outcome thus provides a valuable opportunity to strengthen relationships and reduce conflicts between IRBs and researchers, with positive outcomes for all parties involved in the conduct of human-subject research.
To assess qualities and outcomes of women participating in a large, community-based HIV vaccine trial, the present study was conducted among female participants of the RV 144 prime-boost trial in Thailand from 2003 to 2009. Qualities of participation refer to complete vaccination, retention, and status change. Outcomes of participation refer to incident rate, adverse event, and participation impact event. A total of 6,334 (38.6%) women participated in the trial, of whom about 50% were classified as low risk and 11% as high risk. About 85% of participants completed four vaccinations and 76% were included in the per-protocol analysis of the on-time vaccination schedule. More women (88%) completed 42 months follow-up compared with men (85%). Women aged 21 and above had more adverse events compared to younger age groups. More women (5%) compared with men (3%) reported participation impact events (PIEs). High-risk women had more PIEs and a higher infection rate compared to the low-risk group. Complete vaccination and retention on last follow-up were more common in married women aged above 21, and being a housewife. Female volunteers showed the same qualities and outcomes of participation as males in the HIV vaccine trial. There was no statistically significant difference in vaccine efficacy between men and women, especially among the high-risk and married women. The study highlighted the important behavioral, social, and cultural issues that could be considered for future HIV vaccine trial designs.
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.
Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry.
The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA.
These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-014-0078-8) contains supplementary material, which is available to authorized users.
HIV-1; Mucosal immunity; Immunoglobulin A; Aggregation
HIV-1–specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1–specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1–specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.
The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.
Melioidosis is among the most common causes of septicemia in Thailand, but data on economic burden are limited. We describe the economic impact of bacteremic melioidosis hospitalizations in two Thailand provinces during 2006–2008. Costs are presented in US dollars ($1 = 30.49 Thai Baht). The average annual incidence of bacteremic melioidosis cases per 100,000 persons in Sa Kaeo and Nakhon Phanom was 4.6 and 14.4, respectively. The annual cost of bacteremic melioidosis hospitalizations from the societal perspective, including direct and indirect costs, was $152,159 in Sa Kaeo and $465,303 in Nakhon Phanom. The average cost per fatal case was $14,182 and $14,858 in Sa Kaeo and Nakhon Phanom, respectively. In addition to the high morbidity and mortality, the substantial economic burden of melioidosis further supports the need for investments to identify improved prevention and control strategies for melioidosis.
The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission.
IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.
School absenteeism is a common source of data used in syndromic surveillance, which can eventually be used for early outbreak detection. However, the absenteeism reporting system in most schools, especially in developing countries, relies on a paper-based method that limits its use for disease surveillance or outbreak detection.
The objective of this study was to develop an electronic real-time reporting system on school absenteeism for syndromic surveillance.
An electronic (Web-based) school absenteeism reporting system was developed to embed it within the normal routine process of absenteeism reporting. This electronic system allowed teachers to update students' attendance status via mobile tablets. The data from all classes and schools were then automatically sent to a centralized database for further analysis and presentation, and for monitoring temporal and spatial patterns of absent students. In addition, the system also had a disease investigation module, which provided a link between absenteeism data from schools and local health centers, to investigate causes of fever among sick students.
The electronic school absenteeism reporting system was implemented in 7 primary schools in Bangkok, Thailand, with total participation of approximately 5000 students. During May-October 2012 (first semester), the percentage of absentees varied between 1% and 10%. The peak of school absenteeism (sick leave) was observed between July and September 2012, which coincided with the peak of dengue cases in children aged 6-12 years being reported to the disease surveillance system.
The timeliness of a reporting system is a critical function in any surveillance system. Web-based application and mobile technology can potentially enhance the use of school absenteeism data for syndromic surveillance and outbreak detection. This study presents the factors that determine the implementation success of this reporting system.
syndromic surveillance; schools; absenteeism; tablets; reporting system
Emerging antimalarial drug resistance in mobile populations remains a significant public health concern. We compared two regimens of dihydroartemisinin-piperaquine in military and civilians on the Thai-Cambodian border to evaluate national treatment policy.
Efficacy and safety of two and three-day regimens of dihydroartemisinin-piperaquine were compared as a nested open-label evaluation within a malaria cohort study in 222 otherwise healthy volunteers (18% malaria-infected at baseline). The first 80 volunteers with slide-confirmed Plasmodium falciparum or vivax malaria were randomized 1:1 to receive either regimen (total dose 360mg dihydroartemisinin and 2880mg piperaquine) and followed weekly for up to 6 months. The primary endpoint was malaria recurrence by day 42. Volunteers with vivax infection received primaquine at study discharge with six months follow-up.
Eighty patients (60 vivax, 15 falciparum, and 5 mixed) were randomized to dihydroartemisinin-piperaquine. Intention-to-treat all-species efficacy at Day 42 was 85% for the two-day regimen (95% CI 69–94) and 90% for the three-day regimen (95% CI 75–97). PCR-adjusted falciparum efficacy was 75% in both groups with nearly half (45%) still parasitemic at Day 3. Plasma piperaquine levels were comparable to prior published reports, but on the day of recrudescence were below measurable in vitro piperaquine IC50 levels in all falciparum treatment failures.
In the brief period since introduction of dihydroartemisinin-piperaquine, there is early evidence suggesting declining efficacy relative to previous reports. Parasite IC50 levels in excess of plasma piperaquine levels seen only in treatment failures raises concern for clinically significant piperaquine resistance in Cambodia. These findings warrant improved monitoring of clinical outcomes and follow-up, given few available alternative drugs.
Public health surveys are often conducted using paper-based questionnaires. However, many problems are associated with this method, especially when collecting data among ethnic groups who speak a different language from the survey interviewer. The process can be time-consuming and there is the risk of missing important data due to incomplete surveys.
This study was conducted as a proof-of-concept to develop a new electronic tool for data collection, and compare it with standard paper-based questionnaire surveys using the research setting of assessing Knowledge Attitude and Practice (KAP) toward the Expanded Program on Immunization (EPI) among 6 ethnic groups in Chiang Rai Province, Thailand. The two data collection methods were compared on data quality in terms of data completeness and time consumed in collecting the information. In addition, the initiative assessed the participants’ satisfaction toward the use of a smartphone customized-language voice-based questionnaire in terms of perceived ease of use and perceived usefulness.
Following a cross-over design, all study participants were interviewed using two data collection methods after a one-week washout period. Questions in the paper-based questionnaires in Thai language were translated to each ethnic language by the interviewer/translator when interviewing the study participant. The customized-language voice-based questionnaires were programmed to a smartphone tablet in six, selectable dialect languages and used by the trained interviewer when approaching participants.
The study revealed positive data quality outcomes when using the smartphone, voice-based questionnaire survey compared with the paper-based questionnaire survey, both in terms of data completeness and time consumed in data collection process. Since the smartphone questionnaire survey was programmed to ask questions in sequence, no data was missing and there were no entry errors. Participants had positive attitudes toward answering the smartphone questionnaire; 69% (48/70) reported they understood the questions easily, 71% (50/70) found it convenient, and 66% (46/70) reported a reduced time in data collection. The smartphone data collection method was acceptable by both the interviewers and by the study participants of different ethnicities.
To our knowledge, this is the first study showing that the application of specific features of mobile devices like smartphone tablets (including dropdown choices, capturing pictures, and voiced questions) can be successfully used for data collection. The mobile device can be effectively used for capturing photos of secondary data and collecting primary data with customized-language and voiced questionnaire survey. Using smartphone questionnaires can minimize or eliminate missing data and reduce the time consumed during the data collection process. Smartphone customized-language, voice-based questionnaires for data collection can be an alternative and better approach than standard translated paper-based questionnaires for public health surveys, especially when collecting data among ethnic and hard-to-reach groups residing in multilanguage-speaking settings.
expanded program on immunization; EPI; ethnicity; mobile technology; smartphone questionnaire survey; voiced question
In the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008–0.05; estimated odds ratios of 0.53–0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.
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%, that 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 residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the beta 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.
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
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.
Recruiting minorities into research studies requires special attention, particularly when studies involve “extra-vulnerable” participants with multiple vulnerabilities, e.g., pregnant women, the fetuses/neonates of ethnic minorities, children in refugee camps, or cross-border migrants. This study retrospectively analyzed submissions to the Ethics Committee of the Faculty of Tropical Medicine (FTM-EC) in Thailand. Issues related to the process and outcomes of proposal review, and the main issues for which clarification/revision were requested on studies, are discussed extensively.
The study data were extracted from proposals and amendments submitted to the FTM-EC during the period October 2009 – September 2012, and then analyzed qualitatively and quantitatively. The main issues for clarification/revision were analyzed by thematic content analysis.
373 proposals were submitted; 44 studies involved minority groups with 21 extra-vulnerable minorities. All clinical and 2/3 of non-clinical studies submitted for initial review underwent full-board review. For combined clinical and non-clinical study submissions, 92.1% were referred back to the investigators and approved after clarification/revision, while 2.7% were deferred due to major/critical changes, and 2.1% not approved due to substantial violations of ethical principles. The main issues needing clarification/revision differed between all studies and those involving minorities: participant information sheet (62.2% vs. 86.4%), informed consent/assent form (51.2% vs. 86.4%), and research methodology (80.7% vs. 84.1%), respectively. The main ethical issues arising during the meetings, regarding studies involving minorities, included ensuring no exploitation, coercion, or pressure on the minority to participate; methodology not affecting their legal status; considering ethnicity and cultural structure; and providing appropriate compensation.
Delays in the approval or non-approval of studies involving minorities were mainly due to major or minor deviations from acceptable ethical standards and/or unclear research methodology. The FTM-EC has employed several mechanisms in its operations, including transparency in the review process, building good relationships via open communication with investigators, requesting investigators to consider closely the necessity to enroll minority groups and the risk-benefits for individuals and their communities, and the inclusion of minority-community engagement when developing the proposal. Other effective activities include annual study-site inspections, and offering refresher courses to raise awareness of minority and vulnerability issues among researchers.
Ethical considerations; Minority groups; Vulnerable populations; Research proposal
An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.
The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.
The Thai phase III HIV vaccine trial's modest efficacy (VE 31.2% 95% CI 1.1, 51.2) represents the first demonstration that a vaccine can protect against HIV acquisition. Baseline variables of age, gender, marital status, and risk did not modify vaccine efficacy (VE). Here we explore behavioral risk and efficacy at 6 monthly intervals following vaccination.
Behavioral risk was assessed with a self-administered questionnaire every 6 months during trial participation. Both the acquisition endpoint and the early viral load endpoint are examined for interactions with risk status over time and temporal effects following vaccination.
Risk for HIV acquisition is low in each risk group, but the majority of participants reported higher-risk behavior at least once during the study (N= 9187, 58%). In post-hoc analyses, comparing those participants categorized as high or rising risk at least once during study follow-up versus those who maintained low or medium risk behavior as a time-varying covariate, the interaction of risk status and acquisition efficacy is significant (P = 0.010) with greater benefit in the lower risk individuals. VE appears to peak early with an estimate of cumulative VE = 60% through 12 months after initial vaccination (95% CI 22 –80%), and declines quickly. Vaccination did not appear to affect viral load in either early or late infections.
Future HIV vaccine trials must recognize potential interactions between challenge intensity and risk heterogeneity in the population and treatment effects. The regimen tested in the Thai phase III trial may benefit from extended immunization schedules.
The Thai HIV phase III prime-boost trial (RV144) using ALVAC-HIV® (vCP1521) and AIDSVAX B/E® was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT were performed on PBMC from HIV-1 uninfected vaccine (N=61) and placebo (N=10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4+ T cell mediated. Responses were targeted within the HIV Env region, with 15/25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α4β7 integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19/30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4+ T cells, with the majority of responders producing both IL-2 and IFN-γ (12/19; 63%). HIV-Env Ab titers were higher in subjects with IL-2 compared to those without IL-2 secreting HIV-Env specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4+ with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality and functional cytolytic capacity. While the RV144 T cell responses were modest in frequency compared to humoral immune responses, the CD4+ T cell response was directed to HIV-1 Env and more particularly the V2 region.
Human; Vaccination; Viral; AIDS; HIV-1; T cells