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1.  Missed opportunity to screen and diagnose PTSD and depression among deploying shipboard US military personnel 
BJPsych Open  2016;2(5):314-317.
Depression and post-traumatic stress disorder (PTSD) are significant risks for suicide and other adverse events among US military personnel, but prevalence data among ship-assigned personnel at the onset of deployment are unknown.
To determine the prevalence of shipboard personnel who screen positive for PTSD and/or major depressive disorder (MDD) at the onset of deployment, and also those who reported these diagnoses made by a physician or healthcare professional in the year prior to deployment.
Active-duty ship-assigned personnel (N = 2078) completed anonymous assessments at the beginning of deployment. Depression was measured using the Center for Epidemiologic Studies Depression Scale (CES-D; score of ≥22), and PTSD was assessed using the PTSD Checklist–Civilian Version (PCL-C; both score and symptom criteria were used).
In total, 7.3% (n = 151 of 2076) screened positive for PTSD and 22% (n = 461 of 2078) for MDD at deployment onset. Only 6% and 15% of those who screened positive for PTSD or MDD, respectively, had been diagnosed by a healthcare professional in the past year.
Missed opportunities for mental healthcare among screen-positive shipboard personnel reduce the benefits associated with early identification and linkage to care. Improved methods of mental health screening that promote early recognition and referral to care may mitigate psychiatric events in theatre.
Declaration of interest
This work was performed as part of the official duties of the authors as military service members or employees of the US Government.
Copyright and usage
This work was prepared by military service members or employees of the US Government as part of their official duties. As such, copyright protection is not available for this work (Title 17, USC, §105).
PMCID: PMC5051555  PMID: 27713833
2.  Tissue memory B cell repertoire analysis after ALVAC/AIDSVAX B/E gp120 immunization of rhesus macaques 
JCI Insight  null;1(20):e88522.
The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV‑1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env‑specific B cell clonal lineages were absent in the terminal ileum. Env‑specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.
The RV144 HIV prime-boost vaccination regimen in macaques demonstrates that vaccine-specific B cell clonal lineages are absent in terminal ileum.
PMCID: PMC5135278  PMID: 27942585
3.  Prospective Study of Acute HIV-1 Infection in Adults in East Africa and Thailand 
The New England journal of medicine  2016;374(22):2120-2130.
Acute human immunodeficiency virus type 1 (HIV-1) infection is a major contributor to transmission of HIV-1. An understanding of acute HIV-1 infection may be important in the development of treatment strategies to eradicate HIV-1 or achieve a functional cure.
We performed twice-weekly qualitative plasma HIV-1 RNA nucleic acid testing in 2276 volunteers who were at high risk for HIV-1 infection. For participants in whom acute HIV-1 infection was detected, clinical observations, quantitative measurements of plasma HIV-1 RNA levels (to assess viremia) and HIV antibodies, and results of immunophenotyping of lymphocytes were obtained twice weekly.
Fifty of 112 volunteers with acute HIV-1 infection had two or more blood samples collected before HIV-1 antibodies were detected. The median peak viremia (6.7 log10 copies per milliliter) occurred 13 days after the first sample showed reactivity on nucleic acid testing. Reactivity on an enzyme immunoassay occurred at a median of 14 days. The nadir of viremia (4.3 log10 copies per milliliter) occurred at a median of 31 days and was nearly equivalent to the viral-load set point, the steady-state viremia that persists durably after resolution of acute viremia (median plasma HIV-1 RNA level, 4.4 log10 copies per milliliter). The peak viremia and downslope were correlated with the viral-load set point. Clinical manifestations of acute HIV-1 infection were most common just before and at the time of peak viremia. A median of one symptom of acute HIV-1 infection was recorded at a median of two study visits, and a median of one sign of acute HIV-1 infection was recorded at a median of three visits.
The viral-load set point occurred at a median of 31 days after the first detection of plasma viremia and correlated with peak viremia. Few symptoms and signs were observed during acute HIV-1 infection, and they were most common before peak viremia. (Funded by the Department of Defense and the National Institute of Allergy and Infectious Diseases.)
PMCID: PMC5111628  PMID: 27192360
4.  Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection 
Journal of Virology  2016;90(8):4005-4016.
Attrition within the CD4+ T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8+ T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8+ T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38+ CD27− CD8+ T cells characterized by the low expression of the CD8 receptor (CD8dim). Interestingly, while high frequencies of HIV-specific CD8+ T cell responses occur within the CD38+ CD27− CD8dim T cell population, the minority populations of CD8bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8dim T cells, and the size of this population inversely correlates with the acute loss of CD4+ T cells. These data indicate, for the first time, that early CD4+ T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8dim T cell population less efficient in controlling HIV viremia.
IMPORTANCE A distinct population of activated CD8+ T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8+ T cell dysfunction during acute infection.
PMCID: PMC4810544  PMID: 26842474
5.  Initiation of antiretroviral therapy before detection of colonic infiltration by HIV reduces viral reservoirs, inflammation and immune activation 
Colonic infiltration by HIV occurs soon after infection, establishing a persistent viral reservoir and a barrier to cure. We investigated virologic and immunologic correlates of detectable colonic HIV RNA during acute HIV infection (AHI) and their response to antiretroviral treatment (ART).
From 49,458 samples screened for HIV, 74 participants were enrolled during AHI and 41 consented to optional sigmoidoscopy, HIV RNA was categorized as detectable (≥50 copies/mg) or undetectable in homogenized colon biopsy specimens. Biomarkers and HIV burden in blood, colon and cerebrospinal fluid were compared between groups and after 24 weeks of ART.
Colonic HIV RNA was detectable in 31 participants (76%) and was associated with longer duration since HIV exposure (median 16 vs. 11 days, p=0.02), higher median plasma levels of cytokines and inflammatory markers (CXCL10 476 vs. 148 pg/mL, p=0.02; TNF-RII 1036 vs. 649 pg/mL, p<0.01; neopterin 2405 vs. 1368 pg/mL, p=0.01) and higher levels of CD8+ T cell activation in the blood (human leukocyte antigen - antigen D related (HLA-DR)/CD38 expression 14.4% vs. 7.6%, p <0.01) and colon (8.9% vs. 4.5%, p=0.01). After 24 weeks of ART, participants with baseline detectable colonic HIV RNA demonstrated persistent elevations in total HIV DNA in colonic mucosal mononuclear cells (CMMCs) (median 61 vs. 0 copies/106 CMMCs, p=0.03) and a trend towards higher total HIV DNA in peripheral blood mononuclear cells (PBMC) (41 vs. 1.5 copies/106 PBMCs, p=0.06). There were no persistent differences in immune activation and inflammation.
The presence of detectable colonic HIV RNA at the time of ART initiation during AHI is associated with higher levels of proviral DNA after 24 weeks of treatment. Seeding of HIV in the gut may have long-lasting effects on the size of persistent viral reservoirs and may represent an important therapeutic target in eradication strategies.
PMCID: PMC5026729  PMID: 27637172
HIV; inflammation; CD4 lymphocyte count; highly active antiretroviral therapy; virus latency; infectious disease reservoirs
6.  Knowledge, attitudes, and beliefs about HIV pre-exposure prophylaxis among US Air Force Health Care Providers 
Medicine  2016;95(32):e4511.
Supplemental Digital Content is available in the text
Providers are central to effective implementation of HIV pre-exposure prophylaxis (PrEP). Primary care providers (PCP) and infectious disease physicians (ID) in the US Air Force (USAF) participated in a cross-sectional survey regarding knowledge, attitudes, and beliefs toward HIV PrEP. Characteristics associated with PrEP knowledge were assessed in univariate and multivariate analyses.
Among 403 (40% of 1015 providers) participants, 9% (PCP 383, ID 20) ever prescribed PrEP. In univariate analysis, years in practice, number of HIV-infected patients treated in the past 12 months, past prescription of antiretrovirals for HIV prevention, frequency of prescribing PrEP in the past 12 months, and ever being questioned by a patient about PrEP were associated with PrEP knowledge (P < 0.05). In multivariate analysis, providers who had ever prescribed antiretrovirals to prevent HIV (AOR: 2.37, 95% CI: 1.27–4.42) had greater odds of high PrEP knowledge. Despite concerns about medication side effects (overall 67%: PCP 68%, ID 85%) and prescribing PrEP without clear evidence (overall 60%: PCP 65%, ID 62%), 64% (PCP 65%, ID 85%) of participants indicated PrEP should be offered in the Military Health System and 68% (PCP 70%, ID 100%) disagreed with the statement that their patient population was not at risk for HIV infection.
Successful PrEP implementation in the USAF will require continued education and training of primary care providers to improve knowledge and mitigate concerns about PrEP.
PMCID: PMC4985324  PMID: 27512869
HIV pre-exposure prophylaxis; US Air Force; providers
7.  Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG 
Medicine  2016;95(32):e4346.
Supplemental Digital Content is available in the text
While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development.
The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants.
The sensitivity and specificity of MHA varied between 73–100% and 90–100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants.
CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development.
PMCID: PMC4985300  PMID: 27512845
Genetic complexity; HIV-1; Nigeria; Recombinant; Subtypes
8.  Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection 
PLoS Pathogens  2016;12(7):e1005777.
The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory.
Author Summary
The HIV-specific T cell memory response diminishes rapidly even after the initiation of anti-retroviral treatment (ART), and there is no control of viral rebound if treatment is interrupted. Restoration or preservation of memory T cells or B cells with treatment, to allow for control of virus replication after ART is stopped, is therefore very important. CD4+ T cells, in particular T follicular helper (Tfh) cells, have a major role in mediating antiviral immunity by providing help to B cells, which is key to a strong and efficient anti-HIV antibody response. The unique RV254 cohort provided the best setting to analyze immune responses during very early acute HIV, as the study was able to enroll individuals that were infected for less than 2 weeks and initiated treatment approximately 1–2 days after recruitment. We aimed to study the capacity of memory circulating Tfh (cTfh) cells to promote B cell help in acute HIV infection, and found the interaction to be dysfunctional in the later stage compared to the very early stages, accompanied by increased levels of proinflammatory cytokines and a reduction in regulatory cytokines. High levels of plasma viremia correlated with low cTfh-mediated B cell antibody production in later stage acute individuals; and memory B cells were significantly decreased but could be restored with ART, compared to chronically infected individuals, who could not normalize this compartment compared to healthy individuals. Overall, we show that the cTfh- B cell interaction and B cell memory compartment is altered in late stage acute infection, mainly attributed to an increase in inflammation and skewing of the response away from helper to proinflammatory. Identifying individuals for treatment in the earliest stages of acute infection, prior to immune damage, could preserve cTfh function and the anti-HIV B cell response, therefore reducing the chances of viral rebound upon the cessation of ART.
PMCID: PMC4963136  PMID: 27463374
9.  Human Immunodeficiency Virus Type 1 Monoclonal Antibodies Suppress Acute Simian-Human Immunodeficiency Virus Viremia and Limit Seeding of Cell-Associated Viral Reservoirs 
Journal of Virology  2016;90(3):1321-1332.
Combination antiretroviral therapy (cART) administered shortly after human immunodeficiency virus type 1 (HIV-1) infection can suppress viremia and limit seeding of the viral reservoir, but lifelong treatment is required for the majority of patients. Highly potent broadly neutralizing HIV-1 monoclonal antibodies (MAbs) can reduce plasma viremia when administered during chronic HIV-1 infection, but the therapeutic potential of these antibodies during acute infection is unknown. We tested the ability of HIV-1 envelope glycoprotein-specific broadly neutralizing MAbs to suppress acute simian-human immunodeficiency virus (SHIV) replication in rhesus macaques. Four groups of macaques were infected with SHIV-SF162P3 and received (i) the CD4-binding-site MAb VRC01; (ii) a combination of a more potent clonal relative of VRC01 (VRC07-523) and a V3 glycan-dependent MAb (PGT121); (iii) daily cART, all on day 10, just prior to expected peak plasma viremia; or (iv) no treatment. Daily cART was initiated 11 days after MAb administration and was continued for 13 weeks in all treated animals. Over a period of 11 days after a single administration, MAb treatment significantly reduced peak viremia, accelerated the decay slope, and reduced total viral replication compared to untreated controls. Proviral DNA in lymph node CD4 T cells was also diminished after treatment with the dual MAb. These data demonstrate the virological effect of potent MAbs and support future clinical trials that investigate HIV-1-neutralizing MAbs as adjunctive therapy with cART during acute HIV-1 infection.
IMPORTANCE Treatment of chronic HIV-1 infection with potent broadly neutralizing HIV-1 MAbs has been shown to significantly reduce plasma viremia. However, the antiviral effect of MAb treatment during acute HIV-1 infection is unknown. Here, we demonstrate that MAbs targeting the HIV-1 envelope glycoprotein both suppress acute SHIV plasma viremia and limit CD4 T cell-associated viral DNA. These findings provide support for clinical trials of MAbs as adjunctive therapy with antiretroviral therapy during acute HIV-1 infection.
PMCID: PMC4719604  PMID: 26581981
10.  HLA class II genes modulate vaccine-induced antibody responses to affect HIV-1 acquisition 
Science translational medicine  2015;7(296):296ra112.
In the RV144 vaccine trial, two antibody responses were found to correlate with HIV-1 acquisition. Because human leukocyte antigen (HLA) class II–restricted CD4+ T cells are involved in antibody production, we tested whether HLA class II genotypes affected HIV-1–specific antibody levels and HIV-1 acquisition in 760 individuals. Indeed, antibody responses correlated with acquisition only in the presence of single host HLA alleles. Envelope (Env)–specific immunoglobulin A (IgA) antibodies were associated with increased risk of acquisition specifically in individuals with DQB1*06. IgG antibody responses to Env amino acid positions 120 to 204 were higher and were associated with decreased risk of acquisition and increased vaccine efficacy only in the presence of DPB1*13. Screening IgG responses to overlapping peptides spanning Env 120–204 and viral sequence analysis of infected individuals defined differences in vaccine response that were associated with the presence of DPB1*13 and could be responsible for the protection observed. Overall, the underlying genetic findings indicate that HLA class II modulated the quantity, quality, and efficacy of antibody responses in the RV144 trial.
PMCID: PMC4911012  PMID: 26180102
11.  Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection 
PLoS Pathogens  2016;12(6):e1005663.
Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1β) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis.
Author Summary
HIV infection is closely associated with enhanced host susceptibility to various opportunistic infections (OIs), among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is an early and common manifestation. Even in the era of effective ART, mucosal candidiasis is still a clinically relevant presentation in HIV-infected patients. The underlying mechanisms are not well defined. CD4-mediated immunity is the major host defense mechanism against C. albicans. We here investigated a group of ART naïve, HIV-infected human subjects and examined longitudinally the impact of HIV on C. albicans-specific CD4 T-cell immunity as compared to CD4 T-cell immunity specific for CMV, another opportunistic pathogen that usually does not cause active disease in early HIV infection. We found that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo and were preferentially depleted in progressive HIV-infected individuals as compared to CMV-specific CD4 T cells. Of importance, we also found that in these HIV-infected subjects C. albicans-specific CD4 T cell response manifested a sequential dysfunction with earlier impairment of Th17, but not Th1, functions. Our study suggests an immunological basis that helps explain the earlier and more common onsets of mucosal candidiasis in progressive HIV-infected patients.
PMCID: PMC4900544  PMID: 27280548
12.  Comparable Antigenicity and Immunogenicity of Oligomeric Forms of a Novel, Acute HIV-1 Subtype C gp145 Envelope for Use in Preclinical and Clinical Vaccine Research 
Journal of Virology  2015;89(15):7478-7493.
Eliciting broadly reactive functional antibodies remains a challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development that is complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C, and novel antigenic properties have been described for subtype C Env proteins. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an infected person in the acute phase (Fiebig stage I/II) was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane-proximal external region (MPER), extended by a polylysine tail. Soluble gp145 was enriched for trimers that yielded the expected “fan blade” motifs when visualized by cryoelectron microscopy. CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16, and VRC01 HIV-1 neutralizing monoclonal antibodies (MAbs), as well as the V1/V2-specific PGT121, 697, 2158, and 2297 MAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers, and larger multimers elicited similar levels of cross-subtype binding and neutralizing antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine.
IMPORTANCE At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing, and schedule, as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing MAbs that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing good manufacturing practice production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next-generation candidate HIV immunogen.
PMCID: PMC4505676  PMID: 25972551
13.  HIV Type 1 Disease Progression to AIDS and Death in a Rural Ugandan Cohort Is Primarily Dependent on Viral Load Despite Variable Subtype and T-Cell Immune Activation Levels 
The Journal of Infectious Diseases  2014;211(10):1574-1584.
Background. Untreated human immunodeficiency virus type 1 (HIV) infection is associated with persistent immune activation, which is an independent driver of disease progression in European and United States cohorts. In Uganda, HIV-1 subtypes A and D and recombinant AD viruses predominate and exhibit differential rates of disease progression.
Methods. HIV-1 seroconverters (n = 156) from rural Uganda were evaluated to assess the effects of T-cell activation, viral load, and viral subtype on disease progression during clinical follow-up.
Results. The frequency of activated T cells was increased in HIV-1–infected Ugandans, compared with community matched uninfected individuals, but did not differ significantly between viral subtypes. Higher HIV-1 load, subtype D, older age, and high T-cell activation levels were associated with faster disease progression to AIDS or death. In a multivariate Cox regression analysis, HIV-1 load was the strongest predictor of progression, with subtype also contributing. T-cell activation did not emerge an independent predictor of disease progression from this particular cohort.
Conclusions. These findings suggest that the independent contribution of T-cell activation on morbidity and mortality observed in European and North American cohorts may not be directly translated to the HIV epidemic in East Africa. In this setting, HIV-1 load appears to be the primary determinant of disease progression.
PMCID: PMC4425824  PMID: 25404522
HIV-1; AIDS; subtype D; immune activation; PD-1; viral load
14.  HIV-1 infections with multiple founders are associated with higher viral loads than infections with single founders 
Nature medicine  2015;21(10):1139-1141.
Given the wide differences in HIV-1 viral load (VL) setpoint across subjects as opposed to fairly stable VL over time within an infected individual, it is important to identify host and viral characteristics that affect VL setpoint. While recently-infected individuals with multiple phylogenetically-linked HIV-1 founder variants represent a minority of HIV-1 infections, we found in two different cohorts that more diverse HIV-1 populations in early infection were associated with significantly higher VL one year after HIV-1 diagnosis.
PMCID: PMC4598284  PMID: 26322580
15.  Infant HIV Type 1 gp120 Vaccination Elicits Robust and Durable Anti-V1V2 Immunoglobulin G Responses and Only Rare Envelope-Specific Immunoglobulin A Responses 
The Journal of Infectious Diseases  2014;211(4):508-517.
Infant responses to vaccines can be impeded by maternal antibodies and immune system immaturity. It is therefore unclear whether human immunodeficiency virus type 1 (HIV-1) vaccination would elicit similar responses in adults and infants.
HIV-1 Env–specific antibody responses were evaluated in 2 completed pediatric vaccine trials. In the Pediatric AIDS Clinical Trials Group (PACTG) 230 protocol, infants were vaccinated with 4 doses of Chiron rgp120 with MF59 (n = 48), VaxGen rgp120 with aluminum hydroxide (alum; n = 49), or placebo (n = 19) between 0 and 20 weeks of age. In PACTG 326, infants received 4 doses of ALVAC-HIV-1/AIDSVAX B/B with alum (n = 9) or placebo (n = 13) between 0 and 12 weeks of age.
By 52 weeks of age, the majority of maternally acquired antibodies had waned and vaccine Env-specific immunoglobulin G (IgG) responses in vaccinees were higher than in placebo recipients. Chiron vaccine recipients had higher and more-durable IgG responses than VaxGen vaccine recipients or ALVAC/AIDSVAX vaccinees, with vaccine-elicited IgG responses still detectable in 56% of recipients at 2 years of age. Remarkably, at peak immunogenicity, the concentration of anti-V1V2 IgG, a response associated with a reduced risk of HIV-1 acquisition in the RV144 adult vaccine trial, was 22-fold higher in Chiron vaccine recipients, compared with RV144 vaccinees.
As exemplified by the Chiron vaccine regimen, vaccination of infants against HIV-1 can induce robust, durable Env-specific IgG responses, including anti-V1V2 IgG.
PMCID: PMC4318918  PMID: 25170104
HIV-1; vaccine; infants; antibodies
16.  Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences 
PLoS Pathogens  2016;12(2):e1005431.
Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.
Author Summary
In this study, we generated and evaluated novel simian-human immunodeficiency virus (SHIV) challenge stocks expressing env sequences from HIV-1 strains from Thailand. The lack of such SHIV challenge stocks has been a major unmet need for the field and has hindered progress to evaluate strategies aimed at preventing HIV-1 infection for this region of the world. The challenge stocks described in this manuscript should prove useful for studying the preclinical efficacy and mechanisms of vaccines, antibodies, and other interventions.
PMCID: PMC4743977  PMID: 26849216
17.  Cooperativity of HIV-Specific Cytolytic CD4 T Cells and CD8 T Cells in Control of HIV Viremia 
Journal of Virology  2015;89(15):7494-7505.
CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, nontraditional CD4+ T cell functions beyond helper effects have been described, and a role for cytolytic CD4+ T cells in the control of HIV infection has been suggested. We define here the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of CD4 T cell susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection.
IMPORTANCE The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. We show here that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypic differences compared to HIV-specific Th1 cells but with similarities to CD8+ T cells. These findings are important for our understanding of HIV immunopathology.
PMCID: PMC4505667  PMID: 25972560
18.  Markers of HIV reservoir size and immune activation after treatment in acute HIV infection with and without raltegravir and maraviroc intensification 
Journal of virus eradication  2015;1(2):116-122.
It is unclear whether intensification of standard highly active antiretroviral therapy (HAART) with entry and integrase inhibitors during acute HIV infection (AHI) could yield greater benefits in reducing markers for HIV reservoir size and immune activation.
Thai patients with Fiebig I–IV AHI were prospectively enrolled and offered treatment. They were randomised 1:1 to HAART (tenofovir/emtricitabine/efavirenz, n =31) or megaHAART, a standard regimen intensified by raltegravir/maraviroc (n =31), during the first 24 weeks of therapy. Participants were monitored at weeks 0, 2, 4, 8 and 12, then every 12 weeks. Frequencies of peripheral blood mononuclear cells (PBMCs) carrying HIV DNA (total, integrated and 2-LTR episomes), plasma C-reactive protein (CRP) concentrations, and frequencies of activated T cells were measured. Flexible sigmoidoscopy was performed in willing participants (n =25) at baseline, weeks 24 and 96, and proviral DNA and RNA were determined.
Baseline characteristics were similar in the HAART and megaHAART arms. Median age was 28 years and 95% were men. Median CD4 cell count was 388 cells/mm3. HIV RNA was 5.6 log10 copies/mL. HIV RNA declined more rapidly in the first 4 weeks with megaHAART (median −3.3 log10) than HAART (−2.6 log10). Time to achieve HIV RNA <50 copies/mL was shorter with megaHAART (median 55 days) than HAART (83 days, P =0.04). Viral suppression rates after week 12 did not differ between arms, and overall, 97% achieved suppression by week 48. The frequency of cells harbouring total HIV DNA was similarly low after 96 weeks in both treatment arms (median of 7 and 4 copies/106 PBMCs in the megaHAART and HAART arms, respectively, P =0.41). At weeks 2 and 12, frequency of cells carrying 2-LTR circles were significantly higher with megaHAART (P =0.03). In the sigmoid colon, total HIV DNA and HIV RNA declined after treatment, with no differences between arms. The frequencies of cells with 2-LTR circles were also higher in the sigmoid colon at week 24 with megaHAART. Plasma levels of CRP and frequencies of CD4+ and CD8+ T cells expressing CD38 and HLA-DR or Ki67 were similar between arms.
Intensification of standard HAART with raltegravir and maraviroc was not associated with either statistically significant reductions of markers of HIV reservoir size in blood and sigmoid colon or markers of immune activation in blood.
PMCID: PMC4729387  PMID: 26835516
Acute HIV infection; ART; raltegravir; maraviroc; reservoir; immune activation; HIV DNA
19.  Sex and Urbanicity Contribute to Variation in Lymphocyte Distribution across Ugandan Populations 
PLoS ONE  2016;11(1):e0146196.
Management of patient care and interpretation of research data require evaluation of laboratory results in the context of reference data from populations with known health status to adequately diagnose disease or make a physiological assessment. Few studies have addressed the diversity of lymphocyte subsets in rural and urban Ugandan populations. Here, 663 healthy blood bank donors from semi-urban centers of Kampala consented to participate in a study to define lymphocyte reference ranges. Whole blood immunophenotyping was performed to determine the frequency and absolute counts of T, B, and NK cells using clinical flow cytometry. Results from blood bank donors were compared to a rural cohort from the district of Kayunga and more urban clinical trial participants from the capital city, Kampala. Relationships between hematological and lymphocyte parameters were also explored. In the semi-urban blood donors, females were significantly different from males in all parameters except the frequency of CD8 T and B cells. Females had higher absolute counts of CD4 T, CD8 T and B cells, whereas males had higher NK cell counts. NK cell frequency and counts were significantly higher in semi-urban blood donors, regardless of sex, compared to more urban study participants. CD8 T cell frequency and counts were significantly higher in the blood donors compared to the rural participants, irrespective of sex. Interestingly, basophil counts were positively associated with overall T cell counts. These findings suggest that both sex and level of cohort urbanicity may influence lymphocyte subset distributions in Ugandans.
PMCID: PMC4701131  PMID: 26730706
20.  Antibody light chain-restricted recognition of the site of immune pressure in the RV144 HIV-1 vaccine trial is phylogenetically conserved 
Immunity  2014;41(6):909-918.
In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K)169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was pre-configured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.
PMCID: PMC4324565  PMID: 25526306
21.  Effect of cytokines on Siglec-1 and HIV-1 entry in monocyte–derived macrophages: the importance of HIV-1 envelope V1V2 region 
Journal of Leukocyte Biology  2015;99(6):1089-1106.
M-CSF increased Siglec-1 expression on macrophages, rendering them more permissive to HIV-1 infection due to interaction with V1V2 region of gp120 and associated sialic acids.
Monocytes and monocyte–derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte–derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor–derived monocyte–derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor–derived monocyte–derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor–derived monocyte–derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor–derived monocyte–derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1 interaction with sialic acid-binding immunoglobulin-like lectin and entry into macrophage colony-stimulating factor–derived monocyte–derived macrophages. Removal of sialic acid residues or glycans from scaffolded V1V2 protein decreased human immunodeficiency virus type 1 infectivity. These results highlight the importance of sialic acids on the V1V2 region in binding to sialic acid-binding immunoglobulin-like lectin and suggest that the unusually long surface-exposed sialic acid-binding immunoglobulin-like lectin might aid in the capture and entry of human immunodeficiency virus type 1 into monocyte–derived macrophages.
PMCID: PMC4952014  PMID: 26667473
GM-CSF; M-CSF; qRT-PCR; viral entry; flow cytometry; surface plasmon resonance
22.  COMPASS identifies T-cell subsets correlated with clinical outcomes 
Nature biotechnology  2015;33(6):610-616.
Advances in flow cytometry and other single-cell technologies have enabled high-dimensional, high-throughput measurements of individual cells and allowed interrogation of cell population heterogeneity. Computational tools to take full advantage of these technologies are lacking. Here, we present COMPASS, a computational framework for unbiased polyfunctionality analysis of antigen-specific T-cell subsets. COMPASS uses a Bayesian hierarchical framework to model all observed functional cell subsets and select those most likely to exhibit antigen-specific responses. Cell-subset responses are quantified by posterior probabilities, while subject-level responses are quantified by two novel summary statistics that can be correlated directly with clinical outcome, and describe the quality of an individual’s (poly)functional response. Using three clinical datasets of cytokine production we demonstrate how COMPASS improves characterization of antigen-specific T cells and reveals novel cellular correlates of protection in the RV144 HIV vaccine efficacy trial that are missed by other methods. COMPASS is available as open-source software.
PMCID: PMC4569006  PMID: 26006008
23.  Identification of New Regions in HIV-1 gp120 Variable 2 and 3 Loops that Bind to α4β7 Integrin Receptor 
PLoS ONE  2015;10(12):e0143895.
The gut mucosal homing integrin receptor α4β7 present on activated CD4+ T cells interacts with the HIV-1 gp120 second variable loop (V2). Case control analysis of the RV144 phase III vaccine trial demonstrated that plasma IgG binding antibodies specific to scaffolded proteins expressing the first and second variable regions (V1V2) of HIV envelope protein gp120 containing the α4β7 binding motif correlated inversely with risk of infection. Subsequently antibodies to the V3 region were also shown to correlate with protection. The integrin receptor α4β7 was shown to interact with the LDI/V motif on V2 loop but recent studies suggest that additional regions of V2 loop could interact with the α4β7. Thus, there may be several regions on the V2 and possibly V3 loops that may be involved in this binding. Using a cell line, that constitutively expressed α4β7 receptors but lacked CD4, we examined the contribution of V2 and V3 loops and the ability of V2 peptide-, V2 integrin-, V3-specific monoclonal antibodies (mAbs), and purified IgG from RV144 vaccinees to block the V2/V3-α4β7 interaction.
We demonstrate that α4β7 on RPMI8866 cells bound specifically to its natural ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) as well as to cyclic-V2 and cyclic-V3 peptides. This binding was inhibited by anti-α4β7-specific monoclonal antibody (mAb) ACT-1, mAbs specific to either V2 or V3 loops, and by purified primary virions or infectious molecular clones expressing envelopes from acute or chronic subtypes A, C, and CRF01_AE viruses. Plasma from HIV-1 infected Thai individuals as well as purified IgG from uninfected RV144 vaccinees inhibited (0–50%) the binding of V2 and V3 peptides to α4β7.
Our results indicate that in addition to the tripeptide LDI/V motif, other regions of the V2 and V3 loops of gp120 were involved in binding to α4β7 receptors and this interaction was blocked by anti-V2 peptide, anti-V2 integrin, and anti-V3 antibodies. The ability of purified IgG from some of the uninfected RV144 vaccinees to inhibit α4β7 raises the hypothesis that anti-V2 and anti-V3 antibodies may play a role in blocking the gp120-α4β7 interaction after vaccination and thus prevent HIV-1 acquisition.
PMCID: PMC4666614  PMID: 26625359
24.  Centralized HIV Program Oversight: An Investigation of a Case Series of New HIV Infections among US Army Soldiers, 2012 to 2013 
Medicine  2015;94(46):e2093.
Centralized HIV program oversight and repeal of the Department of Defense policy “Don’t Ask Don’t Tell” permitted characterization of HIV transmission among soldiers assigned to a large US Army base continental United States from 2012 to 2013. An investigation of a greater than expected number of new HIV infections among soldiers was initiated to characterize transmission and identify opportunities to disrupt transmission and deliver services.
All soldiers who were assigned to the base at the time of their first positive HIV test and who had their first positive HIV test in 2012 or in the first 6 months of 2013 and who had a clinical genotype available for analysis were eligible for inclusion in the investigation.
All patients (n = 19) were men; most were black (52%) and less than 30 years old (64%). Fifteen of the 19 patients participated in in-depth interviews. Eighty percent were men who have sex with men who reported multiple sex partners having met through social and electronic networks. All were subtype B infections. Significant knowledge gaps and barriers to accessing testing and care in the military healthcare system were identified. Most (58%) belonged to transmission networks involving other soldiers.
This investigation represents an important step forward in on-going efforts to develop a comprehensive understanding of transmission networks in the Army that can inform delivery of best practices combination prevention services. The Army is developing plans to directly engage individuals in key affected populations most at risk for HIV infection to identify and address unmet needs and expand delivery and uptake of prevention services. Further investigation is underway and will determine whether these findings are generalizable to the Army.
PMCID: PMC4652831  PMID: 26579822
25.  Accelerating HIV-1 Vaccine Efficacy Trials 
Cell  2014;159(5):969-972.
Despite major advances in HIV-1 therapeutics and prevention strategies, the development of a safe and effective prophylactic HIV-1 vaccine will likely be critical for ending the global HIV-1 epidemic. Yet only four HIV-1 vaccine concepts have been tested for clinical efficacy over the past 30 years. In this Commentary, we describe key hurdles facing the HIV-1 vaccine development field and outline strategies to accelerate efficacy evaluation of novel HIV-1 vaccine candidates.
PMCID: PMC4246198  PMID: 25416935

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