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1.  Adenovirus Type 4 and 7 Vaccination or Adenovirus Type 4 Respiratory Infection Elicits Minimal Cross-Reactive Antibody Responses to Nonhuman Adenovirus Vaccine Vectors 
Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd). Small statistically significant increases in titers to ChAd63, ChAd3, human Ad35, and human Ad5 were observed. Neutralizing antibodies elicited by Ad4 infection or immunization results in a small amount of adenovirus cross-reactivity.
doi:10.1128/CVI.00011-14
PMCID: PMC4018874  PMID: 24623627
2.  Effect of Antiretroviral Therapy on HIV Reservoirs in Elite Controllers 
The Journal of Infectious Diseases  2013;208(9):1443-1447.
Elite controllers suppress human immunodeficiency virus (HIV) viremia to below the limit of detection in the absence of antiretroviral therapy (ART). However, precise frequencies of CD4+ T cells carrying replication-competent HIV and/or the dynamics of the infectious viral reservoirs in response to initiation and discontinuation of ART in elite controllers are unknown. We show that the size of the pool of CD4+ T cells harboring infectious HIV diminished significantly after initiation of ART and rebounded to baseline upon cessation of therapy. Our data provide compelling evidence that persistent viral replication occurs in untreated elite controllers even in the absence of detectable plasma viremia.
doi:10.1093/infdis/jit306
PMCID: PMC3789563  PMID: 23847057
human immunodeficiency virus; viral reservoirs; antiretroviral therapy; elite controllers
3.  Pathogen-specific T cell susceptibility to HIV influences the natural history of opportunistic infections 
Cell  2013;155(3):10.1016/j.cell.2013.09.045.
During HIV infection, the timing of opportunistic infections is not always associated with severity of CD4 T cell depletion and different opportunistic pathogens reactivate at different CD4 T cell thresholds. Here we review how differences in the phenotype and function of pathogen-specific CD4 T cells influence susceptibility to HIV infection. By focusing on three common opportunistic infections (Mycobacterium tuberculosis, human papillomavirus, and cytomegalovirus) we examine how differential depletion of pathogen-specific CD4 T cells impacts the natural history of these pathogens in HIV infection. A broader understanding of this relationship can better inform treatment strategies against co-pathogens.
doi:10.1016/j.cell.2013.09.045
PMCID: PMC3858849  PMID: 24243010
4.  Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages 
Journal of Virology  2014;88(8):4047-4057.
ABSTRACT
An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles.
IMPORTANCE Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.
doi:10.1128/JVI.03422-13
PMCID: PMC3993745  PMID: 24501410
5.  A computational framework for the analysis of peptide microarray antibody binding data with application to HIV vaccine profiling 
We present an integrated analytical method for analyzing peptide microarray antibody binding data, from normalization through subject-specific positivity calls and data integration and visualization. Current techniques for the normalization of such data sets do not account for non-specific binding activity. A novel normalization technique based on peptide sequence information quickly and effectively reduced systematic biases. We also employed a sliding mean window technique that borrows strength from peptides sharing similar sequences, resulting in reduced signal variability. A smoothed signal aided in the detection of weak antibody binding hotspots. A new principled FDR method of setting positivity thresholds struck a balance between sensitivity and specificity. In addition, we demonstrate the utility and importance of using baseline control measurements when making subject-specific positivity calls. Data sets from two human clinical trials of candidate HIV-1 vaccines were used to validate the effectiveness of our overall computational framework.
doi:10.1016/j.jim.2013.06.001
PMCID: PMC3999921  PMID: 23770318
Peptide microarrays; Antibodies; Normalization; Positivity calls; Software; Visualization
6.  Homologous Boosting with Adenoviral Serotype 5 HIV Vaccine (rAd5) Vector Can Boost Antibody Responses despite Preexisting Vector-Specific Immunity in a Randomized Phase I Clinical Trial 
PLoS ONE  2014;9(9):e106240.
Background
Needle-free delivery improves the immunogenicity of DNA vaccines but is also associated with more local reactogenicity. Here we report the first comparison of Biojector and needle administration of a candidate rAd5 HIV vaccine.
Methods
Thirty-one adults, 18–55 years, 20 naive and 11 prior rAd5 vaccine recipients were randomized to receive single rAd5 vaccine via needle or Biojector IM injection at 1010 PU in a Phase I open label clinical trial. Solicited reactogenicity was collected for 5 days; clinical safety and immunogenicity follow-up was continued for 24 weeks.
Results
Overall, injections by either method were well tolerated. There were no serious adverse events. Frequency of any local reactogenicity was 16/16 (100%) for Biojector compared to 11/15 (73%) for needle injections. There was no difference in HIV Env-specific antibody response between Biojector and needle delivery. Env-specific antibody responses were more than 10-fold higher in subjects receiving a booster dose of rAd5 vaccine than after a single dose delivered by either method regardless of interval between prime and boost.
Conclusions
Biojector delivery did not improve antibody responses to the rAd5 vaccine compared to needle administration. Homologous boosting with rAd5 gene-based vectors can boost insert-specific antibody responses despite pre-existing vector-specific immunity.
Trial Registration
Clinicaltrials.gov NCT00709605 NCT00709605
doi:10.1371/journal.pone.0106240
PMCID: PMC4179264  PMID: 25264782
7.  Dendritic cell recruitment in response to skin antigen tests in HIV-1 infected individuals correlates with the level of T cell infiltration 
AIDS (London, England)  2013;27(7):1071-1080.
Objectives
To study whether in vivo recruitment of dendritic cells (DCs) in response to antigen administration in the skin is altered during HIV-1 infection.
Design
Skin punch biopsies were collected from HIV-1+ as well as seronegative individuals at 48 hours post intradermal injection of inactivated antigens of mumps virus, Candida albicans or purified protein derivate (PPD) from Mycobacterium tuberculosis.
Methods
Cryosections were analyzed by in situ staining and computerized imaging.
Results
Control skin biopsies showed that there was no difference in the number of skin-resident DCs between seronegative and HIV-1+ individuals. Antigen injection resulted in substantial infiltration of DCs compared to the frequencies found in donor-matched control skin. In HIV-1+ individuals, CD123+/CD303+ plasmacytoid DCs and CD11c+ myeloid DCs, including the CD141+ cross-presenting subset, were recruited at lower levels compared to healthy controls in response to PPD and mumps but not C. albicans. The level of DC recruitment correlated with the frequencies of T cells infiltrating the respective antigen sites. Ki67+ cycling T cells at the injection sites were much more frequent in response to each of the antigens in the HIV-1+ individuals, including those with AIDS, compared to healthy controls.
Conclusions
Multiple DC subsets infiltrate the dermis in response to antigen exposure. There was no obvious depletion or deficiency in mobilization of DCs in response to antigen skin tests during chronic HIV-1 infection. Instead, the levels of antigen-specific memory T cells that accumulate at the antigen site may determine the level of DC infiltration.
doi:10.1097/QAD.0b013e32835ecaca
PMCID: PMC4176731  PMID: 23324660
HIV-1; dendritic cells; plasmacytoid; skin; skin test; delayed-type hypersensitivity reaction; Ki67
8.  Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials 
A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein– Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
doi:10.1016/j.jim.2014.04.005
PMCID: PMC4152919  PMID: 24787274
Peripheral blood mononuclear cells; Cryopreservation; Repository
9.  Differential Impact of Magnitude, Polyfunctional Capacity, and Specificity of HIV-Specific CD8+ T Cell Responses on HIV Set Point 
Journal of Virology  2014;88(3):1819-1824.
Defining the characteristics of HIV-specific CD8+ T cell responses that lead to viral control is crucial for vaccine development. We evaluated the differential impact of magnitude, polyfunctional capacity, and specificity of the CD8+ response at approximately 6 months postinfection on the viral set point at 12 months in a cohort of HIV-infected individuals. High frequencies of Gag and Nef responses endowed with four functions were the best predictors of a low viral set point.
doi:10.1128/JVI.02968-13
PMCID: PMC3911608  PMID: 24227857
10.  Prime-Boost Interval Matters: A Randomized Phase 1 Study to Identify the Minimum Interval Necessary to Observe the H5 DNA Influenza Vaccine Priming Effect 
The Journal of Infectious Diseases  2013;208(3):418-422.
Background. H5 DNA priming was previously shown to improve the antibody response to influenza A(H5N1) monovalent inactivated vaccine (MIV) among individuals for whom there was a 24-week interval between prime and boost receipt. This study defines the shortest prime-boost interval associated with an improved response to MIV.
Methods. We administered H5 DNA followed by MIV at intervals of 4, 8, 12, 16, or 24 weeks and compared responses to that of 2 doses of MIV (prime-boost interval, 24 weeks).
Results. H5 DNA priming with an MIV boost ≥12 weeks later showed an improved response, with a positive hemagglutination inhibition (HAI) titer in 91% of recipients (geometric mean titer [GMT], 141–206), compared with 55%–70% of recipients with an H5 DNA and MIV prime-boost interval of ≤8 weeks (GMT, 51–70) and 44% with an MIV-MIV prime-boost interval of 24 weeks (GMT, 27).
Conclusion. H5 DNA priming enhances antibody responses after an MIV boost when the prime-boost interval is 12–24 weeks.
Clinical Trials Registration. NCT01086657.
doi:10.1093/infdis/jit180
PMCID: PMC3699006  PMID: 23633407
Avian influenza; DNA vaccine; H5N1; boost interval; hemagglutination inhibition
11.  Immunological and Virological Mechanisms of Vaccine-Mediated Protection Against SIV and HIV 
Nature  2013;505(7484):502-508.
Summary
A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a non-human primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV Envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two amino acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of HIV-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine- or naturally-elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials.
doi:10.1038/nature12893
PMCID: PMC3946913  PMID: 24352234
12.  T-bet and Eomes Are Differentially Linked to the Exhausted Phenotype of CD8+ T Cells in HIV Infection 
PLoS Pathogens  2014;10(7):e1004251.
CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation.
Author Summary
CD8+ T cells display numerous traits of severe dysfunction in both treated and untreated HIV infection. Previous studies have demonstrated that HIV-specific CD8+ T cells in most individuals possess poor polyfunctionality, and an immature/skewed maturation phenotype. However, it remains unclear which transcriptional programming governs the regulation of CD8+ T cell differentiation and exhaustion in HIV infection. T-bet and Eomes represent two key transcription factors for CD8+ T cell differentiation and function, but surprisingly little is known about their influence of effector immunity following chronic viral infections in humans. In this study, we demonstrate that HIV-specific CD8+ T cells possess highly elevated levels of Eomes, but low T-bet expression. This differential relationship is linked to the up-regulation of several inhibitory receptors, impaired functional characteristics and a transitional memory differentiation phenotype for virus-specific CD8+ T cells. Importantly, these characteristics of HIV-specific CD8+ T cells remained stable despite suppressive ART for many years. These results implicate that reinvigoration of these cells might fail to elicit efficient responses to eradicate the viral reservoir.
doi:10.1371/journal.ppat.1004251
PMCID: PMC4102564  PMID: 25032686
13.  Therapeutic Vaccination Expands and Improves the Function of the HIV-Specific Memory T-Cell Repertoire 
The Journal of Infectious Diseases  2013;207(12):1829-1840.
Background. The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy.
Methods. In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4+ T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (1010 particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8+ cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4+ T cells were determined.
Results. Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed.
Conclusions. Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control.
Clinical Trials Registration. NCT00270465
doi:10.1093/infdis/jit098
PMCID: PMC3654747  PMID: 23482645
HIV; vaccination; therapy; cytotoxic T lymphocytes; humoral immunity; viral latency
14.  Highly multiplexed quantitation of gene expression on single cells 
Journal of immunological methods  2013;391(0):10.1016/j.jim.2013.03.002.
Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a “bulk” approach that measures expression patterns from 100 cells at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells.
doi:10.1016/j.jim.2013.03.002
PMCID: PMC3814038  PMID: 23500781
Single cell; Gene expression; T-cell activation; qPCR
15.  Efficacy Trial of a DNA/rAd5 HIV-1 Preventive Vaccine 
The New England journal of medicine  2013;369(22):2083-2092.
Background
A safe and effective vaccine for the prevention of human immunodeficiency virus type 1 (HIV-1) infection is a global priority. We tested the efficacy of a DNA prime–recombinant adenovirus type 5 boost (DNA/rAd5) vaccine regimen in persons at increased risk for HIV-1 infection in the United States.
Methods
At 21 sites, we randomly assigned 2504 men or transgender women who have sex with men to receive the DNA/rAd5 vaccine (1253 participants) or placebo (1251 participants). We assessed HIV-1 acquisition from week 28 through month 24 (termed week 28+ infection), viral-load set point (mean plasma HIV-1 RNA level 10 to 20 weeks after diagnosis), and safety. The 6-plasmid DNA vaccine (expressing clade B Gag, Pol, and Nef and Env proteins from clades A, B, and C) was administered at weeks 0, 4, and 8. The rAd5 vector boost (expressing clade B Gag-Pol fusion protein and Env glycoproteins from clades A, B, and C) was administered at week 24.
Results
In April 2013, the data and safety monitoring board recommended halting vaccinations for lack of efficacy. The primary analysis showed that week 28+ infection had been diagnosed in 27 participants in the vaccine group and 21 in the placebo group (vaccine efficacy, −25.0%; 95% confidence interval, −121.2 to 29.3; P = 0.44), with mean viral-load set points of 4.46 and 4.47 HIV-1 RNA log10 copies per milliliter, respectively. Analysis of all infections during the study period (41 in the vaccine group and 31 in the placebo group) also showed lack of vaccine efficacy (P = 0.28). The vaccine regimen had an acceptable side-effect profile.
Conclusions
The DNA/rAd5 vaccine regimen did not reduce either the rate of HIV-1 acquisition or the viral-load set point in the population studied. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00865566.)
doi:10.1056/NEJMoa1310566
PMCID: PMC4030634  PMID: 24099601
17.  Intrinsic TGF-β signaling promotes age-dependent CD8+ T cell polyfunctionality attrition 
The Journal of Clinical Investigation  2014;124(6):2441-2455.
Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell–extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-β1. Furthermore, TGF-β depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-β signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-β1 promote apoptosis of CD8+ effector T cells and high TGF-β1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-β levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-β represents an evolutionarily conserved negative regulator of the immune response in aging organisms.
doi:10.1172/JCI70522
PMCID: PMC4038564  PMID: 24762437
18.  Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation 
Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively.
doi:10.4049/jimmunol.1202861
PMCID: PMC3594325  PMID: 23390298
19.  Phase I Randomized Clinical Trial of VRC DNA and rAd5 HIV-1 Vaccine Delivery by Intramuscular (IM), Subcutaneous (SC) and Intradermal (ID) Administration (VRC 011) 
PLoS ONE  2014;9(3):e91366.
Background
Phase 1 evaluation of the VRC HIV DNA and rAd5 vaccines delivered intramuscularly (IM) supported proceeding to a Phase 2 b efficacy study. Here we report comparison of the IM, subcutaneous (SC) and intradermal (ID) routes of administration.
Methods
Sixty subjects were randomized to 6 schedules to evaluate the IM, SC or ID route for prime injections. Three schedules included DNA primes (Wks 0,4,8) and 3 schedules included rAd5 prime (Wk0); all included rAd5 IM boost (Wk24). DNA vaccine dosage was 4 mg IM or SC, but 0.4 mg ID, while all rAd5 vaccinations were 1010 PU. All injections were administered by needle and syringe.
Results
Overall, 27/30 subjects completed 3 DNA primes; 30/30 subjects completed rAd5 primes. Mild local pruritus (itchiness), superficial skin lesions and injection site nodules were associated with ID and SC, but not IM injections. All routes induced T-cell and antibody immune responses after rAd5 boosting. Overall, >95% had Env antibody and >80% had Env T-cell responses.
Conclusions
The pattern of local reactogenicity following ID and SC injections differed from IM injections but all routes were well-tolerated. There was no evidence of an immunogenicity advantage following SC or ID delivery, supporting IM delivery as the preferred route of administration.
Trial Registration
Clinicaltrials.gov NCT00321061
doi:10.1371/journal.pone.0091366
PMCID: PMC3951381  PMID: 24621858
20.  Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection 
PLoS Pathogens  2014;10(1):e1003853.
The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.
Author Summary
Follicular T helper cells (TFH) interact with B cells within germinal centers of lymphoid tissue to promote the survival, isotype switching and generation of high affinity memory B cells and plasma cells. Recently, a population of circulating CD4 T cells that shares phenotypic and functional characteristics with TFH cells, named peripheral TFH cells, has been identified. The relationship between peripheral TFH cells in the blood and TFH cells within the lymphoid tissue remains unclear, and whether or not peripheral TFH cells can provide insight into T cell and B cell dynamics within lymphoid tissue during infection or vaccination is not understood. Here we characterize peripheral TFH cells and show that unlike TFH cells, peripheral TFH cells secrete a diverse array of cytokines and decrease, rather than increase, during chronic HIV infection. Furthermore, we did not observe a relationship between peripheral TFH cells and memory B cells, or with the production of neutralizing antibodies to HIV. Overall, our data indicate that while peripheral TFH cells share some characteristics with TFH cells, they may not represent a good surrogate to study T cell and B cell dynamics within lymphoid tissue.
doi:10.1371/journal.ppat.1003853
PMCID: PMC3911819  PMID: 24497824
21.  The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains 
Journal of Virology  2013;87(13):7471-7485.
Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the “immune paralysis” observed during EBOV infections results from a cooperative effect of two or more individual IRADs.
doi:10.1128/JVI.03316-12
PMCID: PMC3700277  PMID: 23616668
22.  HIV integration and T cell death: additional commentary 
Retrovirology  2013;10:150.
Estaquier et al. provide commentary on our paper that elucidated the mechanism by which HIV-1 causes cell death in activated CD4 T lymphocytes. We showed that proviral DNA integration triggers DNA-PK dependent death signaling, leading to p53 phosphorylation and cell demise (Cooper A et al. Nature498:376-379, 2013). They have raised several hypothetical points that we further clarify here.
doi:10.1186/1742-4690-10-150
PMCID: PMC3878915  PMID: 24321564
23.  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
24.  High Production Rates Sustain In Vivo Levels of PD-1high Simian Immunodeficiency Virus-Specific CD8 T Cells in the Face of Rapid Clearance 
Journal of Virology  2013;87(17):9836-9844.
Programmed Death 1 (PD-1) expression by human/simian immunodeficiency virus (HIV/SIV)-specific CD8 T cells has been associated with defective cytokine production and reduced in vitro proliferation capacity. However, the cellular mechanisms that sustain PD-1high virus-specific CD8 T cell responses during chronic infection are unknown. Here, we show that the PD-1high phenotype is associated with accelerated in vivo CD8 T cell turnover in SIV-infected rhesus macaques, especially within the SIV-specific CD8 T cell pool. Mathematical modeling of 5-bromo-2′ deoxyuridine (BrdU) labeling dynamics demonstrated a significantly increased generation rate of PD-1high compared to PD-1low CD8 T cells in all memory compartments. Simultaneous analysis of Ki67 and BrdU kinetics revealed a complex in vivo turnover profile whereby only a small fraction of PD-1high cells, but virtually all PD-1low cells, returned to rest after activation. Similar kinetics operated in both chronic and acute SIV infection. Our data suggest that the persistence of PD-1high SIV-specific CD8 T cells in chronic infection is maintained in vivo by a mechanism involving high production coupled with a high disappearance rate.
doi:10.1128/JVI.01001-13
PMCID: PMC3754085  PMID: 23824823
25.  Type I Interferon Induced by Adenovirus Serotypes 28 and 35 Has Multiple Effects on T Cell Immunogenicity 
Recombinant adenovirus vectors (rAds) are being investigated as vaccine delivery vehicles in pre-clinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. Here, we compared the infectivity and cell stimulatory capacity of rAd serotype 5 (rAd5), rAd28 and rAd35 in association with their respective immunogenicity profiles. We found that rAd28 and rAd35 infected, and led to the in vitro maturation and activation, of both human and mouse dendritic cells (DCs) more efficiently compared to rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of interferon alpha (IFNα) and stimulated interferon-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFNα signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T-cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFNα signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFNα signaling. Taken together, our results demonstrate that rAd-induced IFNα production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors.
doi:10.4049/jimmunol.1103717
PMCID: PMC3370104  PMID: 22586038

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