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1.  Identification of Immunodominant CD4-Restricted Epitopes Co-Located with Antibody Binding Sites in Individuals Vaccinated with ALVAC-HIV and AIDSVAX B/E 
PLoS ONE  2015;10(2):e0115582.
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.
doi:10.1371/journal.pone.0115582
PMCID: PMC4321833  PMID: 25665096
2.  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
4.  The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 
AIDS Research and Human Retroviruses  2012;28(11):1444-1457.
Abstract
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.
doi:10.1089/aid.2012.0103
PMCID: PMC3484815  PMID: 23035746
5.  Central Nervous System Viral Invasion and Inflammation During Acute HIV Infection 
The Journal of Infectious Diseases  2012;206(2):275-282.
Background. Understanding the earliest central nervous system (CNS) events during human immunodeficiency virus (HIV) infection is crucial to knowledge of neuropathogenesis, but these have not previously been described in humans.
Methods. Twenty individuals who had acute HIV infection (Fiebig stages I-IV), with average 15 days after exposure, underwent clinical neurological, cerebrospinal fluid (CSF), magnetic resonance imaging, and magnetic resonance spectroscopy (MRS) characterization.
Results. HIV RNA was detected in the CSF from 15 of 18 subjects as early as 8 days after estimated HIV transmission. Undetectable CSF levels of HIV (in 3 of 18) was noted during Fiebig stages I, II, and III, with plasma HIV RNA levels of 285 651, 2321, and 81 978 copies/mL, respectively. On average, the CSF HIV RNA level was 2.42 log10 copies/mL lower than that in plasma. There were no cases in which the CSF HIV RNA level exceeded that in plasma. Headache was common during the acute retroviral syndrome (in 11 of 20 subjects), but no other neurological signs or symptoms were seen. Intrathecal immune activation was identified in some subjects with elevated CSF neopterin, monocyte chemotactic protein/CCL2, and interferon γ–induced protein 10/CXCL-10 levels. Brain inflammation was suggested by MRS.
Conclusions. CSF HIV RNA was detectable in humans as early as 8 days after exposure. CNS inflammation was apparent by CSF analysis and MRS in some individuals during acute HIV infection.
doi:10.1093/infdis/jis326
PMCID: PMC3490695  PMID: 22551810
6.  Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial 
Journal of Virology  2013;87(13):7265-7281.
The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.
doi:10.1128/JVI.03070-12
PMCID: PMC3700312  PMID: 23576510
7.  Expression of monocyte markers in HIV-1 infected individuals with or without HIV associated dementia and normal controls in Bangkok Thailand☆ 
Journal of neuroimmunology  2008;195(0):100-107.
HIV Associated Dementia (HAD) is a complication of HIV infection in developed countries and is still poorly defined in resource-limited settings. In this study we investigated the expression of the monocyte phenotype CD14CD16HLADR and the inflammatory profiles in monocytes supernatants by surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry in a cohort of HAD and non-HAD Thai volunteers prior to the initiation of ARV. The CD14CD16HLADR phenotype was significantly increased in monocytes from HAD and non-HAD versus negative controls, but there was no difference in phenotype and in the secretion protein profiles between the two seropositive groups. In addition, monocytes supernatants from HAD and non-HAD did not induced apoptosis or cell death in brain aggregate culture. In conclusion it appears that HAD in Thai individuals has a different immunological profile then in North America cohorts.
doi:10.1016/j.jneuroim.2007.11.021
PMCID: PMC3670943  PMID: 18191233
HIV-1; Thailand; Monocyte markers; HAD
8.  A novel acute HIV infection staging system based on 4th generation immunoassay 
Retrovirology  2013;10:56.
Background
Fourth generation (4thG) immunoassay (IA) is becoming the standard HIV screening method but was not available when the Fiebig acute HIV infection (AHI) staging system was proposed. Here we evaluated AHI staging based on a 4thG IA (4thG staging).
Findings
Screening for AHI was performed in real-time by pooled nucleic acid testing (NAT, n=48,828 samples) and sequential enzyme immunoassay (EIA, n=3,939 samples) identifying 63 subjects with non-reactive 2nd generation EIA (Fiebig stages I (n=25), II (n=7), III (n=29), IV (n=2)). The majority of samples tested (n=53) were subtype CRF_01AE (77%). NAT+ subjects were re-staged into three 4thG stages: stage 1 (n=20; 4th gen EIA-, 3rd gen EIA-), stage 2 (n=12; 4th gen EIA+, 3rd gen EIA-), stage 3 (n=31; 4th gen EIA+, 3rd gen EIA+, Western blot-/indeterminate). 4thG staging distinguishes groups of AHI subjects by time since presumed HIV exposure, pattern of CD8+ T, B and natural killer cell absolute numbers, and HIV RNA and DNA levels. This staging system further stratified Fiebig I subjects: 18 subjects in 4thG stage 1 had lower HIV RNA and DNA levels than 7 subjects in 4thG stage 2.
Conclusions
Using 4th generation IA as part of AHI staging distinguishes groups of patients by time since exposure to HIV, lymphocyte numbers and HIV viral burden. It identifies two groups of Fiebig stage I subjects who display different levels of HIV RNA and DNA, which may have implication for HIV cure. 4th generation IA should be incorporated into AHI staging systems.
doi:10.1186/1742-4690-10-56
PMCID: PMC3669623  PMID: 23718762
Acute HIV infection; Primary HIV infection; Fiebig stage; 4thG stage; Enzyme immunoassay; 4th generation EIA; 2nd generation EIA; Nucleic acid testing; Reservoir; Functional cure
9.  The Thai Phase III Trial (RV144) Vaccine Regimen Induces T Cell Responses that Preferentially Target Epitopes within the V2 Region of HIV-1 Envelope 
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.
doi:10.4049/jimmunol.1102756
PMCID: PMC3383859  PMID: 22529301
Human; Vaccination; Viral; AIDS; HIV-1; T cells
10.  Analysis of V2 Antibody Responses Induced in Vaccinees in the ALVAC/AIDSVAX HIV-1 Vaccine Efficacy Trial 
PLoS ONE  2013;8(1):e53629.
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.
doi:10.1371/journal.pone.0053629
PMCID: PMC3547933  PMID: 23349725
11.  Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial 
The New England Journal of Medicine  2012;366(14):1275-1286.
BACKGROUND
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
METHODS
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
RESULTS
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
CONCLUSIONS
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
doi:10.1056/NEJMoa1113425
PMCID: PMC3371689  PMID: 22475592
12.  HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses 
PLoS ONE  2012;7(8):e42579.
Background
Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.
Methods
In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers.
Results
Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.
Conclusions
Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees.
doi:10.1371/journal.pone.0042579
PMCID: PMC3412807  PMID: 22880042
13.  Magnitude and Breadth of the Neutralizing Antibody Response in the RV144 and Vax003 HIV-1 Vaccine Efficacy Trials 
The Journal of Infectious Diseases  2012;206(3):431-441.
Background. A recombinant canarypox vector expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pro, and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E), provided modest protection against HIV-1 infection in a community-based population in Thailand (RV144 trial). No protection was observed in Thai injection drug users who received AIDSVAX B/E alone (Vax003 trial). We compared the neutralizing antibody response in these 2 trials.
Methods. Neutralization was assessed with tier 1 and tier 2 strains of virus in TZM-bl and A3R5 cells.
Results. Neutralization of several tier 1 viruses was detected in both RV144 and Vax003. Peak titers were higher in Vax003 and waned rapidly in both trials. The response in RV144 was targeted in part to V3 of gp120.vCP1521 priming plus 2 boosts with gp120 protein was superior to 2 gp120 protein inoculations alone, confirming a priming effect for vCP1521. Sporadic weak neutralization of tier 2 viruses was detected only in Vax003 and A3R5 cells.
Conclusion. The results suggest either that weak neutralizing antibody responses can be partially protective against HIV-1 in low-risk heterosexual populations or that the modest efficacy seen in RV144 was mediated by other immune responses, either alone or in combination with neutralizing antibodies.
doi:10.1093/infdis/jis367
PMCID: PMC3392187  PMID: 22634875
14.  Impact of Multi-Targeted Antiretroviral Treatment on Gut T Cell Depletion and HIV Reservoir Seeding during Acute HIV Infection 
PLoS ONE  2012;7(3):e33948.
Background
Limited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy.
Methods and Findings
We prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24.
At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm3. HIV RNA was 5.5 log10 copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/106 PBMC) vs. Fiebig I (8 copy/106 PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008).
After 24 weeks of megaHAART, HIV RNA levels of <50 copies were achieved in 14/15 in blood and 13/13 in gut. Total blood HIV DNA at week 0 predicted reservoir size at week 24 (p<0.001). Total HIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02).
Conclusions
Gut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission.
doi:10.1371/journal.pone.0033948
PMCID: PMC3316511  PMID: 22479485
15.  Human Immunodeficiency Virus Type 1 Infection Is Associated with Increased NK Cell Polyfunctionality and Higher Levels of KIR3DL1+ NK Cells in Ugandans Carrying the HLA-B Bw4 Motif ▿  
Journal of Virology  2011;85(10):4802-4811.
Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1+ CD56dim NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1+ CD56dim NK cells were decreased, and levels of KIR2DL3+ CD56dim NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1+ NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4+ patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1+ and KIR2DL3+ NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1+ CD56dim NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1+ NK cells in Ugandans carrying the HLA-B Bw4 motif.
doi:10.1128/JVI.00111-11
PMCID: PMC3126187  PMID: 21411516
16.  B Cell Depletion in HIV-1 Subtype A Infected Ugandan Adults: Relationship to CD4 T Cell Count, Viral Load and Humoral Immune Responses 
PLoS ONE  2011;6(8):e22653.
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<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.
doi:10.1371/journal.pone.0022653
PMCID: PMC3160298  PMID: 21886768
17.  Innate and Adaptive Immune Responses Both Contribute to Pathological CD4 T Cell Activation in HIV-1 Infected Ugandans 
PLoS ONE  2011;6(4):e18779.
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.
doi:10.1371/journal.pone.0018779
PMCID: PMC3079731  PMID: 21526194
18.  A Phase I/II Study of a Multiclade HIV-1 DNA Plasmid Prime and Recombinant Adenovirus-type 5 Boost Vaccine in HIV Uninfected East Africans (RV 172) 
The Journal of infectious diseases  2010;201(4):600-607.
Background
Human immunodeficiency virus (HIV) vaccine development remains a global priority. We describe the safety and immunogenicity of a multi-clade DNA vaccine prime with a replication-defective Adenovirus type 5 (rAd5) boost.
Methods
The vaccine is a 6-plasmid mixture encoding HIV envelope (env) subtypes A, B and C and subtype B gag, pol and nef, and a rAd5 expressing identical genes, with the exception of nef. Three hundred and twenty-four participants were randomized to receive placebo (n=138), a single dose of rAd5 at 1010 (n=24) or 1011 particle units (n=24), or DNA at 0, 1 and 2 months followed by rAd5 at either 1010 (n=114) or 1011 particle units (n=24) boosting at 6 months. Participants were followed for 24 weeks after the final immunization.
Results
The vaccine was safe and well tolerated. HIV-specific T cell responses were detected in 63% of vaccinees. Pre-existing Ad5 neutralizing antibody titer did not impact the frequency and magnitude of T cell responses in prime-boost recipients, but did impact the response rates in participants receiving rAd5 alone (p=0.037).
Conclusion
The DNA/rAd5 immunization regimen was safe and induced HIV-1 multi-clade T cell responses, which were not significantly affected by pre-existing rAd5 neutralizing antibody titer.
doi:10.1086/650299
PMCID: PMC2811694  PMID: 20078213
HIV-1 Vaccine; DNA plasmid vaccine; recombinant Adenovirus vaccine; Africa vaccine trial
19.  Quality Monitoring of HIV-1-Infected and Uninfected Peripheral Blood Mononuclear Cell Samples in a Resource-Limited Setting▿  
Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.
doi:10.1128/CVI.00492-09
PMCID: PMC2884426  PMID: 20200187
20.  Phase I Safety and Immunogenicity Evaluation of MVA-CMDR, a Multigenic, Recombinant Modified Vaccinia Ankara-HIV-1 Vaccine Candidate 
PLoS ONE  2010;5(11):e13983.
Background
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.
Methodology/Principal Findings
MVA-CMDR or placebo was administered intra-muscularly (IM; 107 or 108 pfu) or intradermally (ID; 106 or 107 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 51Cr-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/106 PBMC at 108 pfu IM), but high in response rate (70% 51Cr-release positive; 90% Elispot positive; 100% ICS positive, at 108 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 108 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 108 pfu IM).
Conclusions/Significance
MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses.
Trial Registration
ClinicalTrials.gov NCT00376090
doi:10.1371/journal.pone.0013983
PMCID: PMC2981570  PMID: 21085591
21.  Reference Intervals in Healthy Adult Ugandan Blood Donors and Their Impact on Conducting International Vaccine Trials 
PLoS ONE  2008;3(12):e3919.
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.
doi:10.1371/journal.pone.0003919
PMCID: PMC2593783  PMID: 19079547
22.  Reference Ranges for the Clinical Laboratory Derived from a Rural Population in Kericho, Kenya 
PLoS ONE  2008;3(10):e3327.
The conduct of Phase I/II HIV vaccine trials internationally necessitates the development of region-specific clinical reference ranges for trial enrolment 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/µl. 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/µl; 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.
doi:10.1371/journal.pone.0003327
PMCID: PMC2553265  PMID: 18833329
23.  Large-Scale Human Immunodeficiency Virus Rapid Test Evaluation in a Low-Prevalence Ugandan Blood Bank Population▿  
Journal of Clinical Microbiology  2007;45(10):3281-3285.
The use of rapid tests for human immunodeficiency virus (HIV) has become standard in HIV testing algorithms employed in resource-limited settings. We report an extensive HIV rapid test validation study conducted among Ugandan blood bank donors at low risk for HIV infection. The operational characteristics of four readily available commercial HIV rapid test kits were first determined with 940 donor samples and were used to select a serial testing algorithm. Uni-Gold Recombigen HIV was used as the screening test, followed by HIV-1/2 STAT-PAK for reactive samples. OraQuick HIV-1 testing was performed if the first two test results were discordant. This algorithm was then tested with 5,252 blood donor samples, and the results were compared to those of enzyme immunoassays (EIAs) and Western blotting. The unadjusted algorithm sensitivity and specificity were 98.6 and 99.9%, respectively. The adjusted sensitivity and specificity were 100 and 99.96%, respectively. This HIV testing algorithm is a suitable alternative to EIAs and Western blotting for Ugandan blood donors.
doi:10.1128/JCM.00894-07
PMCID: PMC2045340  PMID: 17699650
25.  Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines 
Journal of Virology  2005;79(10):6089-6101.
A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.
doi:10.1128/JVI.79.10.6089-6101.2005
PMCID: PMC1091694  PMID: 15857994

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