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1.  Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family 
Journal of Virology  2012;86(21):11521-11532.
The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.
doi:10.1128/JVI.01023-12
PMCID: PMC3486290  PMID: 22896626
2.  A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays 
Journal of Virology  2012;86(22):12039-12052.
The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.
doi:10.1128/JVI.01650-12
PMCID: PMC3486484  PMID: 22933282
3.  A Nonfucosylated Variant of the anti-HIV-1 Monoclonal Antibody b12 Has Enhanced FcγRIIIa-Mediated Antiviral Activity In Vitro but Does Not Improve Protection against Mucosal SHIV Challenge in Macaques 
Journal of Virology  2012;86(11):6189-6196.
Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.
doi:10.1128/JVI.00491-12
PMCID: PMC3372207  PMID: 22457527
4.  ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge 
PLoS Pathogens  2012;8(8):e1002890.
Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIVmac251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.
Author Summary
Live-attenuated vaccines can prevent simian immunodeficiency virus (SIV) infection upon experimental challenge of rhesus macaques. Although safety considerations preclude vaccinating humans with live-attenuated HIV-1, it may be possible to replicate the types of immunity induced by live-attenuated SIV through an alternative approach. Thus, identifying the immune responses underlying protection by live-attenuated SIV and understanding their induction would provide guidance for HIV-1 vaccine design. An important role for the maturation of virus-specific antibody responses could explain the time-dependent development of protection by live-attenuated SIV. However, antibodies that block the entry of the challenge virus into cells are usually undetectable. Antibodies can also direct the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Here we show that live-attenuated SIV induces progressive increases in ADCC over time, and that the development of these antibodies is dependent upon the persistent replication of the vaccine strain. In two different experiments, the animals immunized with live-attenuated SIV that remained uninfected after pathogenic SIV challenge had higher measures of ADCC than those that became infected. Our results suggest that antibodies contribute to protection by live-attenuated SIV, and that persistent stimulation of antibody responses may be essential for HIV-1 vaccines to induce high ADCC activity.
doi:10.1371/journal.ppat.1002890
PMCID: PMC3426556  PMID: 22927823
5.  A Panel of IgG1 b12 Variants with Selectively Diminished or Enhanced Affinity for Fcγ Receptors To Define the Role of Effector Functions in Protection against HIV ▿  
Journal of Virology  2011;85(20):10572-10581.
Passive transfer of neutralizing antibodies is effective in protecting rhesus macaques against simian/human immunodeficiency virus (SHIV) challenge. In addition to neutralization, effector functions of the crystallizable fragment (Fc) of antibodies are involved in antibody-mediated protection against a number of viruses. We recently showed that interaction between the Fc fragment of the broadly neutralizing antibody IgG1 b12 and cellular Fcγ receptors (FcγRs) plays an important role in protection against SHIV infection in rhesus macaques. The specific nature of this Fc-dependent protection is largely unknown. To investigate, we generated a panel of 11 IgG1 b12 antibody variants with selectively diminished or enhanced affinity for the two main activating FcγRs, FcγRIIa and FcγRIIIa. All 11 antibody variants bind gp120 and neutralize virus as effectively as does wild-type b12. Binding studies using monomeric (enzyme-linked immunosorbent assay [ELISA] and surface plasmon resonance [SPR]) and cellularly expressed Fcγ receptors show decreased (up to 5-fold) and increased (up to 90-fold) binding to FcγRIIa and FcγRIIIa with this newly generated panel of antibodies. In addition, there was generally a good correlation between b12 variant affinity for Fcγ receptor and variant function in antibody-dependent cell-mediated virus inhibition (ADCVI), phagocytosis, NK cell activation assays, and antibody-dependent cellular cytotoxicity (ADCC) assays. In future studies, these b12 variants will enable the investigation of the protective role of individual FcγRs in HIV infection.
doi:10.1128/JVI.05541-11
PMCID: PMC3187489  PMID: 21849450
6.  A National Survey of Veterans Affairs Rheumatologists for Relevance of Quality of Care Indicators for Gout Management 
Arthritis care & research  2010;62(9):1306-1311.
Objective
To determine the relevance of current gout Quality indicators (QIs).
Methods
Members of the Veterans Affairs Rheumatology Consortium were invited to participate in an online survey and provide opinions (rank 0–10) regarding existing gout QIs. Opinions sought on each QI were 1) relevance to United States Veterans, 2) likelihood to improve gout care, and 3) ease of electronic capture. Participants were also asked to rank their top 3 gout QIs.
Results
Participating VA rheumatologists were mainly male, of mean age 51.3 years and experienced in the management of gout. All 10 gout QIs were considered relevant, with a score of 8.2 of higher. The initiation of urate lowering therapy, monitoring of urate levels after initiation of urate lowering therapy, and treatment of acute gout with anti-inflammatory agents scored the highest with regards to likely to improving gout care, with the first 2 QIs also felt to be most relevant. Adjustment of initial allopurinol dosing in patients with renal impairment and in those receiving concurrent azathioprine/6-mercaptopurine were perceived as the QIs most amenable to electronic capture. The top ranked QIs were initiation of urate-lowering therapy with frequent gout attacks, serum urate monitoring after initiation of urate lowering therapy and adjustment of initial allopurinol dose to renal function.
Conclusions
In a national survey of VA rheumatologists, most gout QIs were thought to be highly relevant. QIs related to initiation of urate lowering therapy, serum urate monitoring, and initial dosing of allopurinol were ranked the most important for veterans with gout.
doi:10.1002/acr.20192
PMCID: PMC2943024  PMID: 20235197
Quality Indicators; Gout; Veterans Affairs
7.  Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated, Low-Dose Vaginal Challenge ▿  
Journal of Virology  2010;84(20):10748-10764.
Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIVmac239 or SIVmac251UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIVmac251UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.
doi:10.1128/JVI.00945-10
PMCID: PMC2950576  PMID: 20702641
8.  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

Results 1-8 (8)