Non-neutralizing IgG to the V1V2 loop of HIV-1 gp120 correlates with a decreased risk of HIV-1 infection but the mechanism of protection remains unknown. This V1V2 IgG correlate was identified in ...RV144 Thai trial vaccine recipients, who were primed with a canarypox vector expressing membrane-bound gp120 (vCP1521) and boosted with vCP1521 plus a mixture gp120 proteins from clade B and clade CRF01_AE (B/E gp120). We sought to determine whether the mechanism of vaccine protection might involve antibody-dependent complement activation. Complement activation was measured as a function of complement component C3d deposition on V1V2-coated beads in the presence of RV144 sera. Variable levels of complement activation were detected two weeks post final boosting in RV144, which is when the V1V2 IgG correlate was identified. The magnitude of complement activation correlated with V1V2-specific serum IgG and was stronger and more common in RV144 than in HIV-1 infected individuals and two related HIV-1 vaccine trials, VAX003 and VAX004, where no protection was seen. After adjusting for gp120 IgA, V1V2 IgG, gender, and risk score, complement activation by case-control plasmas from RV144 correlated inversely with a reduced risk of HIV-1 infection, with odds ratio for positive versus negative response to TH023-V1V2 0.42 (95% CI 0.18 to 0.99, p = 0.048) and to A244-V1V2 0.49 (95% CI 0.21 to 1.10, p = 0.085). These results suggest that complement activity may have contributed in part to modest protection against the acquisition of HIV-1 infection seen in the RV144 trial.
Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) ...glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.
Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor/Ca2+-release channel RyR1. The RyR1 contains ∼100 ...Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys3636 in a manner gated by pO2. Using mice expressing a form of RyR1 with a Cys3636→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys3636 was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys3636 S-nitrosylation suppressed stimulus-evoked Ca2+ release at physiological pO2 (at least in part by altering the regulation of RyR1 by Ca2+/calmodulin), eliminated pO2 coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys3636 S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO2-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.
•Skeletal muscle RyR1 is primarily S-nitrosylated at Cys3636.•S-nitrosylation of RyR1 is gated by pO2, providing a link to activity.•S-nitrosylation of RyR1 increases force production.•Muscle size and strength are reduced in C3636A mice.•C3636A mouse muscle has altered activity-dependent gene expression.
The HIV-1 surface glycoprotein gp120 has been reported to bind and signal through α4β7 by means of a tripeptide motif in the V2 loop that mimics structures present in the natural ligands for α4β7, ...suggesting that α4β7 may facilitate HIV-1 infection of CD4(+) T cells in the gut. Furthermore, immune correlates in the RV144 vaccine efficacy trial generated the hypothesis that V1V2 antibodies to an epitope near the putative α4β7 binding motif may play a role in protection against HIV-1 infection. In the interest of developing an assay to detect antibodies that block gp120 binding to α4β7, we used retinoic acid (RA)-activated human peripheral blood mononuclear cells (PBMCs) and transfected HEK293T (293T) cells expressing the integrin complex to study the α4β7 binding properties of 16 HIV-1 envelope glycoproteins. The natural ligand for α4β7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), bound efficiently to RA-activated PBMCs and transfected 293T cells, and this binding was blocked by antibodies to α4. gp120 from multiple HIV-1 subtypes bound to RA-activated PBMCs from three donors in a CD4-dependent manner, but little or no α4β7 binding was detected. Similarly, little or no binding to α4β7 on transfected 293T cells was detected with multiple gp120s and gp140s, including gp120s from transmitted/founder strains, or when gp120 was produced in CHO, 293T, and 293S/GnT1(-/-) cells. Finally, we found no evidence that infectious HIV-1 virions produced in either PBMCs or 293T cells could bind α4β7 on transfected 293T cells. Infectious HIV-1 virions and most gp120s/gp140s appear to be poor ligands for the α4β7 integrin complex under the conditions tested here.
Certain HIV-1 gp120 envelope glycoproteins have been shown to bind the gut-homing receptor α4β7, and it has been suggested that this binding facilitates mucosal transmission and virus replication in the gut mucosa. Additional evidence has generated the hypothesis that antibodies that bind near the putative α4β7 binding motif in the V2 loop of gp120, possibly disrupting gp120-α4β7 binding, may be important for HIV-1 vaccines. Our evidence indicates that infectious HIV-1 virions and many gp120s lack detectable α4β7 binding activity, suggesting that this homing receptor may play a limited role in direct HIV-1 infection of cells.
A3R5 is a human CD4+ lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we ...describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally.
The humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source ...to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63,
= 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = -0.07 to 0.26,
= 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.
Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.
ABSTRACT
The HIV-1 surface glycoprotein gp120 has been reported to bind and signal through α
4
β
7
by means of a tripeptide motif in the V2 loop that mimics structures present in the natural ligands ...for α
4
β
7
, suggesting that α
4
β
7
may facilitate HIV-1 infection of CD4
+
T cells in the gut. Furthermore, immune correlates in the RV144 vaccine efficacy trial generated the hypothesis that V1V2 antibodies to an epitope near the putative α
4
β
7
binding motif may play a role in protection against HIV-1 infection. In the interest of developing an assay to detect antibodies that block gp120 binding to α
4
β
7
, we used retinoic acid (RA)-activated human peripheral blood mononuclear cells (PBMCs) and transfected HEK293T (293T) cells expressing the integrin complex to study the α
4
β
7
binding properties of 16 HIV-1 envelope glycoproteins. The natural ligand for α
4
β
7
, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), bound efficiently to RA-activated PBMCs and transfected 293T cells, and this binding was blocked by antibodies to α
4
. gp120 from multiple HIV-1 subtypes bound to RA-activated PBMCs from three donors in a CD4-dependent manner, but little or no α
4
β
7
binding was detected. Similarly, little or no binding to α
4
β
7
on transfected 293T cells was detected with multiple gp120s and gp140s, including gp120s from transmitted/founder strains, or when gp120 was produced in CHO, 293T, and 293S/GnT1
−/−
cells. Finally, we found no evidence that infectious HIV-1 virions produced in either PBMCs or 293T cells could bind α
4
β
7
on transfected 293T cells. Infectious HIV-1 virions and most gp120s/gp140s appear to be poor ligands for the α
4
β
7
integrin complex under the conditions tested here.
IMPORTANCE
Certain HIV-1 gp120 envelope glycoproteins have been shown to bind the gut-homing receptor α
4
β
7
, and it has been suggested that this binding facilitates mucosal transmission and virus replication in the gut mucosa. Additional evidence has generated the hypothesis that antibodies that bind near the putative α
4
β
7
binding motif in the V2 loop of gp120, possibly disrupting gp120-α
4
β
7
binding, may be important for HIV-1 vaccines. Our evidence indicates that infectious HIV-1 virions and many gp120s lack detectable α
4
β
7
binding activity, suggesting that this homing receptor may play a limited role in direct HIV-1 infection of cells.
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