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1.  Cautious Optimism for HIV Vaccine Science 
Current opinion in HIV and AIDS  2013;8(5):367-368.
doi:10.1097/01.COH.0000432529.38768.1f
PMCID: PMC4089146  PMID: 23924996
2.  Rare serotype adenoviral vectors for HIV vaccine development 
Human adenoviral vectors are being developed for use in candidate vaccines for HIV-1 and other pathogens. However, this approach suffered a setback when an HIV-1 vaccine using an adenovirus type 5 (Ad5) vector failed to reduce, and might even have increased, the rate of HIV infection in men who were uncircumcised and who had preexisting antibodies specific for Ad5. This increased interest in the evaluation of serologically distinct adenoviral vectors. In this issue of the JCI, Frahm and coworkers report evidence that preexisting cellular immune responses directed toward Ad5 reduce the immunogenicity of antigens expressed in Ad5-vectored vaccines and have cross-reacting potential with non-Ad5 adenoviral vectors. The implications of this observation need to be carefully evaluated in future clinical trials of all serotypes of adenovirus-vectored vaccines.
doi:10.1172/JCI60988
PMCID: PMC3248308  PMID: 22201675
3.  TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1 
Scientific Reports  2015;5:10443.
CD4+ T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5+PD-1++) and precursor-TFH (CXCR5+PD-1+) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer’s patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines.
doi:10.1038/srep10443
PMCID: PMC4451806  PMID: 26034905
4.  Accuracy of Clinical Diagnosis of Dengue Episodes in the RV144 HIV Vaccine Efficacy Trial in Thailand 
PLoS ONE  2015;10(5):e0127998.
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.
doi:10.1371/journal.pone.0127998
PMCID: PMC4444125  PMID: 26011728
5.  Epidemiology of HIV among US Air Force Military Personnel, 1996–2011 
PLoS ONE  2015;10(5):e0126700.
Objective
The objectives of this study were to describe the epidemiology of HIV in the United States Air Force (USAF) from 1996 through 2011 and to assess whether socio-demographic characteristics and service-related mobility, including military deployments, were associated with HIV infection.
Methods
We conducted a retrospective cohort analysis of USAF personnel who were HIV-infected during the study period January 1, 1996 through December 31, 2011 and a matched case-control study. Cases were USAF personnel newly-diagnosed with HIV during the study period. Five randomly-selected HIV-uninfected controls were matched to each case by age, length of service, sex, race, service, component, and HIV test collection date. Socio-demographic and service-related mobility factors and HIV diagnosis were assessed using conditional logistic regression.
Results
During the study period, the USAF had 541 newly diagnosed HIV-infected cases. HIV incidence rate (per 100,000 person-years) among 473 active duty members was highest in 2007 (16.78), among black/ African-American USAF members (26.60) and those aged 25 to 29 years (10.84). In unadjusted analysis restricted to personnel on active duty, 10 characteristics were identified and considered for final multivariate analysis. Of these single (adjusted odds ratio [aOR], 8.15, 95% confidence interval [CI] 5.71–11.6) or other marital status (aOR 4.60, 95% CI 2.72–7.75), communications/ intelligence (aOR 2.57, 95% CI 1.84–3.60) or healthcare (aOR 2.07, 95% CI 1.28–3.35) occupations, and having no deployment in the past 2 years before diagnosis (aOR 2.02, 95% CI 1.47–2.78) conferred higher odds of HIV infection in adjusted analysis.
Conclusion
The highest risk of HIV infection in the USAF was among young unmarried deployment-naïve males, especially those in higher risk occupation groups. In an era when worldwide military operations have increased, these analyses identified potential areas where targeted HIV prevention efforts may be beneficial in reducing HIV incidence in the USAF military population.
doi:10.1371/journal.pone.0126700
PMCID: PMC4427109  PMID: 25961564
6.  Machine Learning Methods Enable Predictive Modeling of Antibody Feature:Function Relationships in RV144 Vaccinees 
PLoS Computational Biology  2015;11(4):e1004185.
The adaptive immune response to vaccination or infection can lead to the production of specific antibodies to neutralize the pathogen or recruit innate immune effector cells for help. The non-neutralizing role of antibodies in stimulating effector cell responses may have been a key mechanism of the protection observed in the RV144 HIV vaccine trial. In an extensive investigation of a rich set of data collected from RV144 vaccine recipients, we here employ machine learning methods to identify and model associations between antibody features (IgG subclass and antigen specificity) and effector function activities (antibody dependent cellular phagocytosis, cellular cytotoxicity, and cytokine release). We demonstrate via cross-validation that classification and regression approaches can effectively use the antibody features to robustly predict qualitative and quantitative functional outcomes. This integration of antibody feature and function data within a machine learning framework provides a new, objective approach to discovering and assessing multivariate immune correlates.
Author Summary
Antibodies are one of the central mechanisms that the human immune system uses to eliminate infection: an antibody can recognize a pathogen or infected cell using its Fab region while recruiting additional immune cells through its Fc that help destroy the offender. This mechanism may have been key to the reduced risk of infection observed among some of the vaccine recipients in the RV144 HIV vaccine trial. In order to gain insights into the properties of antibodies that support recruitment of effective functional responses, we developed and applied a machine learning-based framework to find and model associations among properties of antibodies and corresponding functional responses in a large set of data collected from RV144 vaccine recipients. We characterized specific important relationships between antibody properties and functional responses, and demonstrated that models trained to encapsulate relationships in some subjects were able to robustly predict the quality of the functional responses of other subjects. The ability to understand and build predictive models of these relationships is of general interest to studies of the antibody response to vaccination and infection, and may ultimately lead to the development of vaccines that will better steer the immune system to produce antibodies with beneficial activities.
doi:10.1371/journal.pcbi.1004185
PMCID: PMC4395155  PMID: 25874406
7.  Rapid Seeding of the Viral Reservoir Prior to SIV Viremia in Rhesus Monkeys 
Nature  2014;512(7512):74-77.
The viral reservoir represents a critical challenge facing HIV-1 eradication strategies1–5. However, it remains unclear when and where the viral reservoir is seeded during acute infection and the extent to which it is susceptible to early antiretroviral therapy (ART). Here we show that the viral reservoir is seeded very early following mucosal SIV infection of rhesus monkeys and prior to systemic viremia. We initiated suppressive ART in groups of monkeys on days 3, 7, 10, and 14 following intrarectal SIVmac251 infection. Treatment on day 3 blocked the emergence of viral RNA and proviral DNA in peripheral blood and also substantially reduced levels of proviral DNA in lymph nodes and gastrointestinal mucosa as compared with treatment at later timepoints. In addition, treatment on day 3 abrogated the induction of SIV-specific humoral and cellular immune responses. Nevertheless, following discontinuation of ART after 24 weeks of fully suppressive therapy, virus rebounded in all animals, although animals treated on day 3 exhibited a delayed viral rebound as compared with animals treated on days 7, 10 and 14. The time to viral rebound correlated with total viremia during acute infection and with proviral DNA at the time of ART discontinuation. These data demonstrate that the viral reservoir is seeded very early following intrarectal SIV infection of rhesus monkeys, during the “eclipse” phase, and prior to viremia. This strikingly early seeding of the refractory viral reservoir raises important new challenges for HIV-1 eradication strategies.
doi:10.1038/nature13594
PMCID: PMC4126858  PMID: 25042999
8.  Comprehensive Sieve Analysis of Breakthrough HIV-1 Sequences in the RV144 Vaccine Efficacy Trial 
PLoS Computational Biology  2015;11(2):e1003973.
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.
Author Summary
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.
doi:10.1371/journal.pcbi.1003973
PMCID: PMC4315437  PMID: 25646817
9.  Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide 
Journal of Virology  2014;88(16):9406-9417.
ABSTRACT
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.
doi:10.1128/JVI.01031-14
PMCID: PMC4136246  PMID: 24920809
10.  Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation 
PLoS Pathogens  2014;10(12):e1004543.
Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation.
Author Summary
Persistent systemic immune activation is a hallmark of chronic HIV infection and an independent predictor of disease progression. The underlying mechanism is not yet completely understood but thought to be associated with the loss of Th17 cells leading to the disruption of the mucosal barrier and subsequent microbial translocation. However, it remains unclear when these events take place in HIV infection, as the only data available to date are from SIV models. We evaluated the kinetics of Th17 depletion, microbial translocation and subsequent immune activation in early acute HIV infection and the effect of early initiated ART on these events. We discovered that a collapse of Th17 cell number and function, accompanied by local and systemic immune activation, occurs already during acute HIV infection. However, early initiation of ART preserved Th17 number and function and fully reversed any initial HIV-related immune activation. These findings argue for the importance of early events during HIV infection setting the stage for chronic immune activation and for early and aggressive treatment during acute HIV infection.
doi:10.1371/journal.ppat.1004543
PMCID: PMC4263756  PMID: 25503054
11.  Human Immunodeficiency Virus Vaccine Trials 
More than 2 million AIDS-related deaths occurred globally in 2008, and more than 33 million people are living with HIV/AIDS. Despite promising advances in prevention, an estimated 2.7 million new HIV infections occurred in that year, so that for every two patients placed on combination antiretroviral treatment, five people became infected. The pandemic poses a formidable challenge to the development, progress, and stability of global society 30 years after it was recognized. Experimental preventive HIV-1 vaccines have been administered to more than 44,000 human volunteers in more than 187 separate trials since 1987. Only five candidate vaccine strategies have been advanced to efficacy testing. The recombinant glycoprotein (rgp)120 subunit vaccines, AIDSVAX B/B and AIDSVAX B/E, and the Merck Adenovirus serotype (Ad)5 viral-vector expressing HIV-1 Gag, Pol, and Nef failed to show a reduction in infection rate or lowering of postinfection viral set point. Most recently, a phase III trial that tested a heterologous prime-boost vaccine combination of ALVAC-HIV vCP1521 and bivalent rgp120 (AIDSVAX B/E) showed 31% efficacy in protection from infection among community-risk Thai participants. A fifth efficacy trial testing a DNA/recombinant(r) Ad5 prime-boost combination is currently under way. We review the clinical trials of HIV vaccines that have provided insight into human immunogenicity or efficacy in preventing HIV-1 infection.
Clinical trials of HIV vaccines have provided insight into human immunogenicity and efficacy in preventing HIV-1 infection.
doi:10.1101/cshperspect.a007351
PMCID: PMC3543076  PMID: 23209178
12.  Vaccine-induced Human Antibodies Specific for the Third Variable Region of HIV-1 gp120 Impose Immune Pressure on Infecting Viruses 
EBioMedicine  2014;1(1):37-45.
To evaluate the role of V3-specific IgG antibodies (Abs) in the RV144 clinical HIV vaccine trial, which reduced HIV-1 infection by 31.2%, the anti-V3 Ab response was assessed. Vaccinees' V3 Abs were highly cross-reactive with cyclic V3 peptides (cV3s) from diverse virus subtypes. Sieve analysis of CRF01_AE breakthrough viruses from 43 vaccine- and 66 placebo-recipients demonstrated an estimated vaccine efficacy of 85% against viruses with amino acids mismatching the vaccine at V3 site 317 (p = 0.004) and 52% against viruses matching the vaccine at V3 site 307 (p = 0.004). This analysis was supported by data showing that vaccinees' plasma Abs were less reactive with I307 when replaced with residues found more often in vaccinees' breakthrough viruses. Simultaneously, viruses with mutations at F317 were less infectious, possibly due to the contribution of F317 to optimal formation of the V3 hydrophobic core. These data suggest that RV144-induced V3-specific Abs imposed immune pressure on infecting viruses and inform efforts to design an HIV vaccine.
Highlights
•The RV144 vaccine reduced infection by viruses with isoleucine in V3 position 307.•Many vaccine-induced antibodies are cross-reactive and target an epitope including I307.•There was selection for breakthrough viruses carrying F317 in V3 (p = 0.004).•F317 is needed to maintain optimal infectivity.•F317 is a poor or non-contact residue for vaccine induced V3 antibodies.
doi:10.1016/j.ebiom.2014.10.022
PMCID: PMC4293639  PMID: 25599085
HIV; Antibody; Vaccine; Clinical trial
13.  CD8 and CD4 Epitope Predictions in RV144: No Strong Evidence of a T-Cell Driven Sieve Effect in HIV-1 Breakthrough Sequences from Trial Participants 
PLoS ONE  2014;9(10):e111334.
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.
doi:10.1371/journal.pone.0111334
PMCID: PMC4211711  PMID: 25350851
14.  Protective Efficacy of a Global HIV-1 Mosaic Vaccine Against Heterologous SHIV Challenges in Rhesus Monkeys 
Cell  2013;155(3):10.1016/j.cell.2013.09.061.
SUMMARY
The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of global HIV-1 vaccine antigens has not previously been evaluated. Here we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection was correlated with vaccine-elicited binding, neutralizing, and functional non-neutralizing antibodies. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy towards the development of a global HIV-1 vaccine. Moreover, our findings suggest that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses.
doi:10.1016/j.cell.2013.09.061
PMCID: PMC3846288  PMID: 24243013
15.  Natural Killer Cell–Mediated Innate Sieve Effect on HIV-1: The Impact of KIR/HLA Polymorphism on HIV-1 Subtype-Specific Acquisition in East Africa 
The Journal of Infectious Diseases  2013;208(8):1250-1254.
Here we explore the association between killer cell immunoglobulin-like receptor (KIR)/HLA and human immunodeficiency virus type 1 (HIV-1) acquisition with different viral subtypes circulating in East Africa. In the prospective Cohort Development (CODE) cohort (Mbeya, Tanzania), carriers of KIR3DS1 and its putative ligand (HLA-A or HLA-B Bw4-80Ile alleles) showed increased HIV-1 acquisition risk (odds ratio [OR] = 3.46; 95% confidence interval [CI], 1.12–10.63; P = .04) and a trend for enrichment for subtype A and A-containing recombinants (78% vs 46%; OR = 4.05; 95% CI, .91–28.30; P = .09) at the expense of subtype C (11% vs 43%; OR = 0.17; 95% CI, .01–.97; P = .08). In vitro, only natural killer cells from KIR3DS1(+)/HLA-Bw4-80Ile(+) healthy donors showed a 2-fold increased capacity to inhibit replication of subtype C vs subtype A viruses (P = .01). These findings suggest the presence of an innate sieve effect and may inform HIV-1 vaccine development.
doi:10.1093/infdis/jit349
PMCID: PMC3778971  PMID: 23922366
HIV-1; innate immunity; KIR; HLA; sieve effect; subtypes; East Africa
16.  High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging 
BMC Genomics  2014;15(1):864.
Background
Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.
Results
Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.
Conclusions
The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-864) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-864
PMCID: PMC4196003  PMID: 25283548
HLA; NGS; HLA typing; Illumina MiSeq
17.  Aggregate complexes of HIV-1 induced by multimeric antibodies 
Retrovirology  2014;11:78.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/s12977-014-0078-8
PMCID: PMC4193994  PMID: 25274446
HIV-1; Mucosal immunity; Immunoglobulin A; Aggregation
18.  Nautilus: A Bioinformatics Package for the Analysis of HIV Type 1 Targeted Deep Sequencing Data 
AIDS Research and Human Retroviruses  2013;29(10):1361-1364.
Abstract
The advent of next generation sequencing technologies is providing new insight into HIV-1 diversity and evolution, which has created the need for bioinformatics tools that could be applied to the characterization of viral quasispecies. Here we present Nautilus, a bioinformatics package for the analysis of HIV-1 targeted deep sequencing data. The DeepHaplo module determines the nucleotide base frequency and read depth at each position and computes the haplotype frequencies based on the linkage among polymorphisms in the same next generation sequence read. The Motifs module computes the frequency of the variants in the setting of their sequence context and mapping orientation, which allows for the validation of polymorphisms and haplotypes when strand bias is suspected. Both modules are accessed through a user-friendly GUI, which runs on Mac OS X (version 10.7.4 or later), and are based on Python, JAVA, and R scripts. Nautilus is available from www.hivresearch.org/research.php?ServiceID=5&SubServiceID=6.
doi:10.1089/aid.2013.0175
PMCID: PMC3785804  PMID: 23809062
19.  Cryptic Determinant of α4β7 Binding in the V2 Loop of HIV-1 gp120 
PLoS ONE  2014;9(9):e108446.
The peptide segment of the second variable loop of HIV-1 spanning positions 166–181 harbors two functionally important sites. The first, spanning positions 179–181, engages the human α4β7 integrin receptor which is involved in T-cell gut-homing and may play a role in human immunodeficiency virus (HIV)-host cell interactions. The second, at positions 166–178, is a major target of anti-V2 antibodies elicited by the ALVAC/AIDSVAX vaccine used in the RV144 clinical trial. Notably, these two sites are directly adjacent, but do not overlap. Here, we report the identity of a second determinant of α4β7 binding located at positions 170–172 of the V2 loop. This segment – tripeptide QRV170–172– is located within the second site, yet functionally affects the first site. The absence of this segment abrogates α4β7 binding in peptides bearing the same sequence from position 173–185 as the V2 loops of the RV144 vaccines. However, peptides exhibiting V2 loop sequences from heterologous HIV-1 strains that include this QRV170–172 motif bind the α4β7 receptor on cells. Therefore, the peptide segment at positions 166–178 of the V2 loop of HIV-1 viruses appears to harbor a cryptic determinant of α4β7 binding. Prior studies show that the anti-V2 antibody response elicited by the RV144 vaccine, along with immune pressure inferred from a sieve analysis, is directed to this same region of the V2 loop. Accordingly, the anti-V2 antibodies that apparently reduced the risk of infection in the RV144 trial may have functioned by blocking α4β7-mediated HIV-host cell interactions via this cryptic determinant.
doi:10.1371/journal.pone.0108446
PMCID: PMC4180765  PMID: 25265384
20.  Vaccine-Induced Env V1–V2 IgG3 Correlates with Lower HIV-1 Infection Risk and Declines Soon After Vaccination 
Science translational medicine  2014;6(228):228ra39.
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.
doi:10.1126/scitranslmed.3007730
PMCID: PMC4116665  PMID: 24648342
21.  FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial 
The Journal of Clinical Investigation  2014;124(9):3879-3890.
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.
doi:10.1172/JCI75539
PMCID: PMC4151214  PMID: 25105367
22.  HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities 
Journal of Virology  2014;88(14):7715-7726.
ABSTRACT
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.
doi:10.1128/JVI.00156-14
PMCID: PMC4097802  PMID: 24807721
23.  HIV-1 Variable Loop 2 and its Importance in HIV-1 Infection and Vaccine Development 
Current HIV research  2013;11(5):427-438.
A vaccine that can prevent the transmission of HIV-1 at the site of exposure to the host is one of the best hopes to control the HIV-1 pandemic. The trimeric envelope spike consisting of heterodimers, gp120 and gp41, is essential for virus entry and thus has been a key target for HIV-1 vaccine development. However, it has been extremely difficult to identify the types of antibodies required to block the transmission of various HIV-1 strains and the immunogens that can elicit such antibodies due to the high genetic diversity of the HIV-1 envelope. The modest efficacy of the gp120 HIV-1 vaccine used in the RV144 Thai trial, including the studies on the immune correlates of protection, and the discovery of vaccine-induced immune responses to certain signature regions of the envelope have shown that the gp120 variable loop 2 (V2) is an important region. Since there is evidence that the V2 region interacts with the integrin α4β7 receptor of the host cell, and that this interaction might be important for virus capture, induction of antibodies against V2 loop could be postulated as one of the mechanisms to prevent the acquisition of HIV-1. Immunogens that can induce these antibodies should therefore be taken into consideration when designing HIV-1 vaccine formulations.
PMCID: PMC4086350  PMID: 24191938
24.  Differential loss of invariant NKT cells and FoxP3+ regulatory T cells in HIV-1 subtype A and subtype D infections 
HIV-1 subtype D is associated with faster disease progression as compared to subtype A. Immunological correlates of this difference remain undefined. We investigated invariant natural killer T cells and FoxP3+ regulatory T cells in Ugandans infected with either subtype. Loss of iNKT cells was pronounced in subtype D, whereas Tregs displayed more profound loss in subtype A infection. iNKT cell levels were associated with CD4 T cell IL-2 production in subtype A, but not D, infection. Thus, these viral subtypes are associated with differential loss of iNKT cells and Tregs that may influence the quality of the adaptive immune response.
doi:10.1097/QAI.0b013e31828b2073
PMCID: PMC3683089  PMID: 23403863
HIV-1; viral subtype; AIDS; iNKT cell; CD1d; T regulatory cell

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