The role of Antibody-dependent cellular cytotoxicity (ADCC) responses in HIV-1 controllers is still unclear due to the heterogeneity of these patients. We analyzed 67 HIV-1 controllers and found significantly higher levels of ADCC antibodies in controllers versus viremic subjects (p = 0.017). Moreover, multivariate analysis revealed significantly higher ADCC titers in HLA B57- controllers compared to HLA-B57+ ones (p = 0.0086). These data suggest a role for ADCC in immune control of HIV, especially in HLA B57 negative controllers.
In 2009, the United Nations Estimated that 33.2 Million People worldwide were living with human immunodeficiency virus type 1 (HIV-1) infection and that 2.6 million people had been newly infected.1 The need for effective HIV-1 prevention has never been greater. In this review, we address recent critical advances in our understanding of HIV-1 transmission and acute HIV-1 infection. Fourth-generation HIV-1 testing, now available worldwide,2,3 will allow the diagnosis of infection in many patients and may lead to new treatments and opportunities for prevention.
Tuberculosis (TB) remains a global health burden for which safe vaccines are needed. BCG has limitations as a TB vaccine so we have focused on live attenuated Mycobacterium tuberculosis mutants as vaccine candidates. Prior to human studies, however, it is necessary to demonstrate safety in non-human primates (NHP). In this study, we evaluate the safety and efficacy of two live attenuated M. tuberculosis double deletion vaccine strains mc26020 (ΔlysA ΔpanCD) and mc26030 (ΔRD1 ΔpanCD) in cynomolgus macaques. In murine models, mc26020 is rapidly cleared while mc26030 persists. Both mc26020 and mc26030 were safe and well tolerated in cynomolgus macaques. Following a high-dose intrabronchial challenge with virulent M. tuberculosis, mc26020-vaccinates were afforded a level of protection intermediate between that elicited by BCG vaccination and no vaccination. BCG vaccinates had reduced tuberculosis-associated pathology and improved clinical scores as compared to saline and mc26030 vaccinates, but survival did not differ among the groups.
Vaccine; Mycobacteria; Mycobacterium; Tuberculosis; Non-human primate; BCG; Safety
Development of a safe and effective vaccine for HIV-1 infection is a critical global priority. However, the nature of host-virus interactions that lead to early immunosuppression and CD4 depletion, HIV-1 diversity, and the inability of the immune system to eliminate the latently infected CD4 pool of cells has to date thwarted successful vaccine development. Moreover, both the initial antibody-inducing vaccine (protein envelope gp120) and cell-mediated vaccine (recombinant adenovirus containing HIV-1 genes) strategies have failed in efficacy trials, and the latter cell-mediated vaccine appeared to have caused enhanced HIV-1 acquisition. Thus basic and translational research to understand why current vaccines have failed and elucidation of new mechanisms of virus control at mucosal surfaces is essential for eventual successful development of a preventive HIV-1 vaccine.
HIV-1; vaccine; mucosal; gastrointestinal tract; T cells; antibody; innate; adaptive; immunity
Immunological tolerance to self-antigen impairs humoral responses to HIV-1.
Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance because mice expressing the VH and VL regions of 2F5 have a block in B cell development that is characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. We identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes an epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but they retain the SF3B3 4E10 epitope. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motifs shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1.
Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140ΔCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.
HIV-1 has evolved many ways to evade protective host immune responses, thus creating a number of problems for HIV vaccine developers. In particular, durable, broadly specific neutralizing antibodies to HIV-1 have proved difficult to induce with current HIV-1 vaccine candidates. The recent observation that some broadly neutralizing anti-HIV-1 envelope monoclonal antibodies have polyspecific reactivities to host antigens have raised the hypothesis that one reason antibodies against some of the conserved HIV-1 envelope trimer neutralizing epitopes are not routinely made may be down-regulation of some specificities of anti-HIV-1 antibody producing B cells by host B cell tolerance mechanisms.
We have developed a high throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4+ T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6±2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.
ADCC; HIV; SIV; NK; Fc gamma receptors; Granzyme B; high throughput
Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with Kd values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.
Different HIV-1 antigen specificities appear in sequence after HIV-1 transmission and the immunoglobulin G (IgG) subclass responses to HIV antigens are distinct from each other. The initial predominant IgG subclass response to HIV-1 infection consists of IgG1 and IgG3 antibodies with a noted decline in some IgG3 antibodies during acute HIV-1 infection. Thus, we postulate that multiple antigen-specific IgG3 responses may serve as surrogates for the relative time since HIV-1 acquisition.
We determined the magnitude, peak, and half-life of HIV-1 antigen-specific IgG1 and IgG3 antibodies in 41 HIV-1-infected individuals followed longitudinally from acute infection during the first appearance of HIV-1-specific antibodies through approximately 6 months after infection.
We used quantitative HIV-1-binding antibody multiplex assays and exponential decay models to estimate concentrations of IgG1 and IgG3 antibodies to eight different HIV-1 proteins including gp140 Env, gp120 Env, gp41 Env, p66 reverse transcriptase, p31 Integrase, Tat, Nef, and p55 Gag proteins during acute/recent HIV-1 infection.
Among HIV-1-specific IgG3 responses, anti-gp41 IgG3 antibodies were the first to appear. We found that anti-gp41 Env IgG3 and anti-p66 reverse transcriptase IgG3 antibodies, in addition to anti-Gag IgG3 antibodies, each consistently and measurably declined after acute infection, in contrast to the persistent antigen-specific IgG1 responses.
The detailed measurements of the decline in multiple HIV-specific IgG3 responses simultaneous with persistent IgG1 responses during acute and recent HIV-1 infection could serve as markers for detection of incident HIV infection.
HIV-1 acute infection; HIV-1 incidence; immunoglobulin G subclass
The HIV-1 envelope (Env) is a key determinant in mediating viral entry and fusion to host cells and is a major target for HIV vaccine development. While Env is typically about 50% glycan by mass, glycosylation sites are known to evolve, with some glycosylation profiles presumably being more effective at facilitating neutralization escape than others.1 Thus, characterizing glycosylation patterns of Env and native virions and correlating glycosylation profiles with infectivity and Env immunogenicity are necessary first steps in designing effective immunogens. Herein, we describe a mass spectrometrybased strategy to determine HIV-1 Env glycosylation patterns and have compared two mammalian cell expressed recombinant Env immunogens, one a limited immunogen and one that induces crossclade neutralizing antibodies. We have used a glycopeptide-based mass mapping approach to identify and characterize Env’s glycosylation patterns by elucidating which sites are utilized and what type of glycan motif is present at each glycosylation site. Our results show that the immunogens displayed different degrees of glycosylation as well as a different characteristic set of glycan motifs. Thus, these techniques can be used to (1) define glycosylation profiles of recombinant Env proteins and Env on mature virions, (2) define specific carbohydrate moieties at each glycosylation site, and (3) determine the role of certain carbohydrates in HIV-1 infectivity and in modulation of Env immunogenicity.
HIV; envelope glycoprotein; glycosylation; vaccine; mass spectrometry
A vaccine against human immunodeficiency virus (HIV) seems to be on the horizon. Correlates of risk of infection for the RV144 trial have been found. There is understanding of what makes HIV envelope–specific antibodies broadly neutralizing and new T cell vaccine approaches can overcome virus variability.
A single-nucleotide polymorphism (rs2395029) in the HCP5 gene associated with HLA-B*5701 is correlated with lower HIV-1 viral set point. The two allelic forms of coding region were ectopically expressed in TZM-bl cells for an effect on HIV-1 replication. No significant HIV-1 restriction was observed in the cells with infectivity assays throughout HIV-1 life cycle, suggesting that the association of HCP5 variant with viral control is likely due to HLA-B*5701-related effect or other functional variants in the haplotype or both.
To determine the spectrum of antiviral antibodies in HIV-1-infected individuals in whom viral replication is spontaneously undetectable, termed HIV controllers (HICs).
Multicenter French trial ANRS EP36 studying the viral control in HICs.
Neutralizing Antibody (nAb) activities (neutralization assay, competition with broadly reactive monoclonal antibodies, and reactivity against the viral MPER gp41 region), FcγR-mediated antiviral activities, antibody-dependent cell cytotoxicity (ADCC), as well as autoantibody levels, were quantified in plasma from 22 controllers and from viremic individuals. The levels of these different antibody responses and HIV-specific CD8 T cell responses quantified by enzyme-linked immunosorbent spot (ELISPOT) IFNγ assay were compared in each controller.
The levels of antibody against the gp120 CD4 binding site, gp41, as well as Env epitopes near to the sites bound by broadly nAbs 2F5 and 1b12 were not different between HICs and viremic individuals. We did not find significant autoantibody levels in HICs. The magnitude and breadth of nAbs were heterogeneous in HICs but lower than in viremic individuals. The levels of nAbs using FcγR-mediated assay inhibition were similar in both groups. Regardless of the type of antibody tested, there was no correlation with HIV-specific CD8 T cell responses. ADCC was detectable in all controllers tested and was significantly higher than in viremic individuals (P <0.0002).
There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers. Rather, for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.
antibody-dependent cell cytotoxicity; FcγR; HIV controller; humoral immunity; neutralizing antibodies
Human (h) CD7 is a 40 kDa single chain Ig superfamily molecule that is expressed on thymocytes, a major subunit of peripheral T cells, and most natural killer cells. Ligands for hCD7 include the epithelial cell-produced molecule, K-12, and galectin. Mice deficient in CD7 have been shown to be resistant to LPS-induced endotoxic shock syndromes. However, monoclonal antibodies (MAb) to mouse (m) CD7 have yet to be produced, nor is the distribution of mCD7 protein in mice known. We have raised a panel of three rat MAbs to mCD7 by immunizing rats with recombinant mCD7 protein. However, using Western blot and immunoprecipitation of tissue extracts from mouse thymus, spleen, liver, brain, lymph node and skin, these anti-mouse CD7 MAbs bound only to murine heat shock protein 60 (HSP-60) present both in wild-type (CD7+/+) and CD7-deficient (CD7−/−) mice. Epitope mapping of the sites on HSP-60 and recombinant mCD7 recognized by mCD7 MAbs demonstrated non-homologous amino acid sequence epitopes recognized by anti-CD7 MAbs on both proteins. These data demonstrated molecular mimicry of mCD7 with HSP-60, and leave open the question of surface expression of mCD7.
Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although non-human primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, thereby improving the characterization of promising vaccine candidates and the identification of correlates of immune protection.
CD7 is an immunoglobulin superfamily molecule involved in T and natural killer (NK) cell activation and cytokine production. CD7-deficient animals develop normally but have antigen-specific defects in interferon (IFN)-γ production and CD8+ CTL generation. To determine the in vivo role of CD7 in systems dependent on IFN-γ, the response of CD7-deficient mice to lipopolysaccharide (LPS)-induced shock syndromes was studied. In the high-dose LPS-induced shock model, 67% of CD7-deficient mice survived LPS injection, whereas 19% of control C57BL/6 mice survived LPS challenge (P < 0.001). CD7-deficient or C57BL/6 control mice were next injected with low-dose LPS (1 μg plus 8 mg D-galactosamine [D-gal] per mouse) and monitored for survival. All CD7-deficient mice were alive 72 h after injection of LPS compared with 20% of C57BL/6 control mice (P < 0.001). After injection of LPS and D-gal, CD7-deficient mice had decreased serum IFN-γ and tumor necrosis factor (TNF)-α levels compared with control C57BL/6 mice (P < 0.001). Steady-state mRNA levels for IFN-γ and TNF-α in liver tissue were also significantly decreased in CD7-deficient mice compared with controls (P < 0.05). In contrast, CD7-deficient animals had normal liver interleukin (IL)-12, IL-18, and interleukin 1 converting enzyme (ICE) mRNA levels, and CD7-deficient splenocytes had normal IFN-γ responses when stimulated with IL-12 and IL-18 in vitro. NK1.1+/ CD3+ T cells are known to be key effector cells in the pathogenesis of toxic shock. Phenotypic analysis of liver mononuclear cells revealed that CD7-deficient mice had fewer numbers of liver NK1.1+/CD3+ T cells (1.5 ± 0.3 × 105) versus C57BL/6 control mice (3.7 ± 0.8 × 105; P < 0.05), whereas numbers of liver NK1.1+/CD3− NK cells were not different from controls. Thus, targeted disruption of CD7 leads to a selective deficiency of liver NK1.1+/ CD3+ T cells, and is associated with resistance to LPS shock. These data suggest that CD7 is a key molecule in the inflammatory response leading to LPS-induced shock.
CD7; lipopolysaccharide; septic shock; NK1.1; T cells
HIV infection is characterized by a gradual deterioration of immune function mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4+ and CD8+ T cell responses in 12 subtype C-infected individuals with different disease progression profiles from acute to chronic HIV infection. The frequencies of Gag-responsive CD4+ and CD8+ T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFNγ+CD4+ T cell was observed at a median of 28 days (IQR: 21-81) post Fiebig I/II staging, whilst Gag-specific IFNγ+CD8+ T cell responses peaked at a median of 253 days (IQR: 136-401 and showed a significant biphasic expansion. The proportion of TNFα-expressing cells within the IFNγ+CD4+ T cell population increased (p=0.001) over time, whilst TNFα-expressing cells within IFNγ+CD8+ T cells declined (p=0.005). Both Gag-responsive CD4+ and CD8+ T cells showed decreased Ki67 expression within the first 120 days post Feibig I/II staging. Prior to the disappearance of Gag-responsive Ki67+CD4+ T cells, these cells positively correlated (p=0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8+ T cell compartment. Overall, these observations indicate that circulating Gag-responsive CD4+ and CD8+ T cell frequencies and functions are not synchronous and properties change rapidly at different tempos during early HIV infection.
Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion (“high secretors”) and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8+ T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8+ T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8+ T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses.
Antibody PG9 is a prototypical member of a class of V1/V2-directed antibodies that effectively neutralizes diverse strains of HIV-1. We analyzed strain-specific resistance to PG9 using sequence and structural information. For multiply resistant strains, mutations in a short segment of V1/V2 resulted in gain of sensitivity to PG9 and related V1/V2 neutralizing antibodies, suggesting both a common mechanism of HIV-1 resistance to and a common mode of recognition by this class of antibodies.
Purpose of review
Major roadblocks persist in the development of vaccines that elicit potent neutralizing antibodies targeting diverse HIV-1 strains, similar to known broadly neutralizing HIV-1 human monoclonal antibodies. Alternatively, other types of anti-HIV-1 envelope antibodies that may not neutralize HIV-1 in traditional neutralization assays but have other anti-HIV-1 activities (hereafter termed HIV-1 inhibitory antibodies) can be elicited by current vaccine strategies, and numerous studies are exploring their roles in preventing HIV-1 acquisition. We review examples of strategies for eliciting potentially protective HIV-1 inhibitory antibodies.
Heterologous prime-boost strategies can yield anti-HIV immune responses; although only one (canarypox prime, Env protein boost) has been tested and shown positive results in an efficacy trial (RV144). Although the immune correlates of protection are as yet undefined, the reduced rate of acquisition without a significant effect on initial viral loads or CD4+ T cell counts, have raised the hypothesis of an RV144 vaccine-elicited transient protective B cell response.
In light of the RV144 trial, there is a critical need to define the entire functional spectrum of anti-HIV-1 antibodies, how easily each can be elicited, and how effective different types of antibody effector mechanisms can be in prevention of HIV-1 transmission.
Vaccines; B-cells; Neutralizing Antibodies; Inhibitory Antibodies; Mucosal
Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity.
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.
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.
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.
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.
The HIV-1 broad neutralizing antibody (bnAb) 2F5 has been shown to be poly/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 VH knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. Here, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 VHxVL KI mice, and find an even higher degree of tolerance control than observed in the 2F5 VH KI strain. Although B-cell development was severely impaired in 2F5 VHxVL KI animals, we demonstrate rescue of their B-cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B-cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B-cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 VH/VL expression by immature/transitional B-cells. Importantly, 7% of rescued B-cells retained 2F5 VH/VL-expression and secreted Env-specific mAbs with HIV-1 neutralizing activity. This “partial” rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B-cells in 2F5 VHxVL KI × Eμ-bcl2 tg mice, and significant (yet modest) enrichment of Env-specific B-cells and serum Igs. The rescued 2F5 mAb-producing B-cell clones in this study are the first examples of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs, and provide a starting point for design of strategies aimed at rescuing such B-cells.