Pinghuang Liu

Initial B-Cell Responses to Transmitted Human Immunodeficiency Virus Type 1: Virion-Binding Immunoglobulin M (IgM) and IgG Antibodies Followed by Plasma Anti-gp41 Antibodies with Ineffective Control of Initial Viremia

ABSTRACT A window of opportunity for immune responses to extinguish human immunodeficiency virus type 1 (HIV-1) exists from the moment of transmission through establishment of the latent pool of HIV-1-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus), but, to date, this period has been logistically difficult to analyze. To probe B-cell responses immediately following HIV-1 transmission, we have determined envelope-specific antibody responses to autologous and consensus Envs in plasma donors from the United States for whom frequent plasma samples were available at time points immediately before, during, and after HIV-1 plasma viral load (VL) ramp-up in acute infection, and we have modeled the antibody effect on the kinetics of plasma viremia. The first detectable B-cell response was in the form of immune complexes 8 days after plasma virus detection, whereas the first free plasma anti-HIV-1 antibody was to gp41 and appeared 13 days after the appearance of plasma virus. In contrast, envelope gp120-specific antibodies were delayed an additional 14 days. Mathematical modeling of the earliest viral dynamics was performed to determine the impact of antibody on HIV replication in vivo as assessed by plasma VL. Including the initial anti-gp41 immunoglobulin G (IgG), IgM, or both responses in the model did not significantly impact the early dynamics of plasma VL. These results demonstrate that the first IgM and IgG antibodies induced by transmitted HIV-1 are capable of binding virions but have little impact on acute-phase viremia at the timing and magnitude that they occur in natural infection.

HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities.

ABSTRACT The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission. IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.

Diversion of HIV-1 vaccine–induced immunity by gp41-microbiota cross-reactive antibodies

Microbiota can mislead antibodies Unlike the response to many viral infections, most people do not produce antibodies capable of clearing HIV-1. Non-neutralizing antibodies that target HIV-1s envelope glycoprotein (Env) typically dominate the response, which is generated by B cells that cross-react with Env and the intestinal microbiota. Williams et al. analyzed samples from individuals who had received a vaccine containing the Env protein, including the gp41 subunit. Most of the antibodies were non-neutralizing and targeted gp41. The antibodies also reacted to intestinal microbiota, suggesting that preexisting immunity to microbial communities skews vaccineinduced immune responses toward an unproductive target. Science, this issue 10.1126/science.aab1253. The antibody response to an HIV-1 vaccine is dominated by preexisting immunity to microbiota. INTRODUCTION Inducing protective antibodies is a key goal in HIV-1 vaccine development. In acute HIV-1 infection, the dominant initial plasma antibody response is to the gp41 subunit of the envelope (Env) glycoprotein of the virus. These antibodies derive from polyreactive B cells that cross-react with Env and intestinal microbiota (IM) and are unable to neutralize HIV-1. However, whether a similar gp41-IM cross-reactive antibody response would occur in the setting of HIV-1 Env vaccination is unknown. RATIONALE We studied antibody responses in individuals who received a DNA prime vaccine, with a recombinant adenovirus serotype 5 (rAd5) boost (DNA prime–rAd5 boost), a vaccine that included HIV-1 gag, pol, and nef genes, as well as a trivalent mixture of clade A, B, and C env gp140 genes containing both gp120 and gp41 components. This vaccine showed no efficacy. Thus, study of these vaccinees provided an opportunity to determine whether the Env-reactive antibody response in the setting of Env vaccination was dominated by gp41-reactive antibodies derived from Env-IM cross-reactive B cells. RESULTS We found that vaccine-induced antibodies to HIV-1 Env dominantly focused on gp41 compared with gp120 by both serologic analysis and by vaccine-Env memory B cells sorted by flow cytometry (see the figure). Remarkably, the majority of HIV-1 Env-reactive memory B cells induced by the vaccine produced gp41-reactive antibodies, and the majority of gp41-targeted antibodies used restricted immunoglobulin heavy chain variable genes. Functionally, none of the gp41-reactive antibodies could neutralize HIV, and the majority could not mediate antibody-dependent cellular cytotoxicity. Most of the vaccine-induced gp41-reactive antibodies cross-reacted with host and IM antigens. Two of the candidate gp41-intestinal cross-reactive antigens were bacterial RNA polymerase and pyruvate-flavodoxin oxidoreductase, which shared sequence similarities with the heptad repeat 1 region of HIV gp41. Next-generation sequencing of vaccinee B cells demonstrated a prevaccination antibody that was reactive to both IM and the vaccine–Env gp140, which demonstrated the presence of a preexisting pool of gp41-IM cross-reactive B cells from which the vaccine gp41-reactive antibody response was derived. CONCLUSION In this study, we found that the DNA prime–rAd5 boost HIV-1 vaccine induced a gp41-reactive antibody response that was mainly non-neutralizing and derived from an IM-gp41 cross-reactive B cell pool. These findings have important implications for HIV-1 vaccine design. Because IM antigens shape the B cell repertoire from birth, our data raise the hypothesis that neonatal immunization with HIV-1 envelope may be able to imprint the B cell repertoire to respond to envelope antigenic sites that may otherwise be subdominant or disfavored, such as Env broadly neutralizing antibody epitopes. Our data also suggest that deleting or modifying amino acids in the gp41 heptad repeat 1 region of Env-containing vaccine immunogens may avoid IM-gp41 cross-reactivity. Thus, an obstacle that may need to be overcome for development of a successful HIV vaccine is diversion of potentially protective HIV-1 antibody responses by preexisting envelope-IM cross-reactive pools of B cells. Diversion of HIV-1 vaccine–induced immunity by Env gp41–microbiota cross-reactive antibodies. Immunization of humans with a vaccine containing HIV-1 Env gp120 and gp41 components, including the membrane-proximal external region (MPER) of Env, induced a dominant B cell response primarily from a preexisting pool of gp41-IM cross-reactive B cells. This response diverted the vaccine-stimulated antibody response away from smaller subdominant B cell pools capable of reacting with potentially protective epitopes on HIV-1 Env. An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1–reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1–reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Prolonged exposure of the HIV-1 gp41 membrane proximal region with L669S substitution

The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13, and 4E10 monoclonal antibodies (mAbs). One strategy to elicit such antibodies is to design an immunogen with increased exposure of the 2F5 and 4E10 mAb epitopes. In this study we characterize a single leucine to serine substitution at position 669 (L669S) in the gp41 Env MPER that confers >250-fold more neutralization sensitivity to 2F5 and 4E10 mAbs than does the wild-type gp41 sequence. On synthetic liposomes, increased solvent exposure of MPER tryptophan residues and stable docking of 2F5 and 4E10 mAbs to mutant MPER peptide liposomes indicate more favorable membrane orientation of MPER neutralizing epitopes with L669S substitution. The time during which virus is sensitive to 2F5 mAb-mediated neutralization is approximately 3-fold longer when the mutation is present. These data suggest that a major contribution to the L669S mutant virus phenotype of enhanced susceptibility to MPER mAbs is prolonged exposure of the MPER neutralizing epitope during viral entry.

Infectious Virion Capture by HIV-1 gp120-Specific IgG from RV144 Vaccinees

ABSTRACT The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.

Dynamic Antibody Specificities and Virion Concentrations in Circulating Immune Complexes in Acute to Chronic HIV-1 Infection

ABSTRACT Understanding the interactions between human immunodeficiency virus type 1 (HIV-1) virions and antibodies (Ab) produced during acute HIV-1 infection (AHI) is critical for defining antibody antiviral capabilities. Antibodies that bind virions may prevent transmission by neutralization of virus or mechanically prevent HIV-1 migration through mucosal layers. In this study, we quantified circulating HIV-1 virion-immune complexes (ICs), present in approximately 90% of AHI subjects, and compared the levels and antibody specificity to those in chronic infection. Circulating HIV-1 virions coated with IgG (immune complexes) were in significantly lower levels relative to the viral load in acute infection than in chronic HIV-1 infection. The specificities of the antibodies in the immune complexes differed between acute and chronic infection (anti-gp41 Ab in acute infection and anti-gp120 in chronic infection), potentially suggesting different roles in immunopathogenesis for complexes arising at different stages of infection. We also determined the ability of circulating IgG from AHI to bind infectious versus noninfectious virions. Similar to a nonneutralizing anti-gp41 monoclonal antibody (MAb), purified plasma IgG from acute HIV-1 subjects bound both infectious and noninfectious virions. This was in contrast to the neutralizing antibody 2G12 MAb that bound predominantly infectious virions. Moreover, the initial antibody response captured acute HIV-1 virions without selection for different HIV-1 envelope sequences. In total, this study demonstrates that the composition of immune complexes are dynamic over the course of HIV-1 infection and are comprised initially of antibodies that nonselectively opsonize both infectious and noninfectious virions, likely contributing to the lack of efficacy of the antibody response during acute infection.

Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection in vivo.

Capacity for Infectious HIV-1 Virion Capture Differs by Envelope Antibody Specificity

ABSTRACT Antibody capacity to recognize infectious virus is a prerequisite of many antiviral functions. We determined the infectious virion capture index (IVCI) of different antibody specificities. Whereas broadly neutralizing antibodies (bNAbs), except for an MPER bNAb, selectively captured infectious virions, non-bNAbs and mucosal human immunodeficiency virus type 1 (HIV-1)-positive IgG captured subsets of both infectious and noninfectious virions. Infectious virion capture was additive with a mixture of antibodies, providing proof of concept for vaccine-induced antibodies that together have improved capacity to recognize infectious virions.

Aggregate complexes of HIV-1 induced by multimeric antibodies

BackgroundAntibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry.ResultsThe potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA.ConclusionsThese results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.