Breadth and magnitude of antigen-specific antibody responses in the control of plasma viremia in simian immunodeficiency virus infected macaques

HIV-1 infection is associated with polyclonal B-cell activation, hypergammaglobulinemia, the presence of immature/transitional CD10+ or exhausted CD27 negative B-cells in blood [1, 2], exhaustion of tissue-like memory (CD20(hi)/CD27(−)/CD21(lo)) B-cells [3], loss of total B-cell populations [4, 5], and nonspecific switching from IgM to IgG, IgA and IgE responses. Our recent data demonstrated defective memory (CD21 + CD27+) B-cell proliferation in selective tissues in simian immunodeficiency virus (SIV)-infected macaques [6, 7]. Therefore, the maintenance of normal and effective humoral immune responses may be the key to the prevention and control of HIV/SIV infection. Recent reports emphasize that HIV-specific antibodies (Abs), instead of T-cell responses, may correlate better with protection in seronegative partners of HIV-1 infected individuals [8]. Moreover, other emerging studies demonstrate a correlation between anti-HIV antibodies and protection from infection, although these protective Abs are not strictly neutralizing in vitro [9]. Furthermore, lymphocytic choriomeningitis virus infection in the mouse model has also shown that non-neutralizing Abs elicited early in infection are capable of binding to the virus and limiting it's spread [10].

B-cell epitopes are described either as conformational (discontinuous, assembled) epitopes where multiple discontinuous amino-acids (aa) segments are folded to produce a unique conformational epitope complementary to the antibody, termed a contact epitope [11‐14], or linear epitopes (continuous, sequential) where epitopes do not incorporate protein folding and can be represented by linear peptide sequence [15]. The continuous maturation of the immune response following SIV infection emphasizes the need to study the generation of SIV-specific Ab responses, antigen-antibody binding efficacy, and their potential importance in regulating disease progression.

The present study was designed to determine the importance of total immunoglobulin, antigen-specific immunoglobulin responses against whole viral lysate (WVL), peptides corresponding to Env, Gag, Nef, and Tat and neutralizing antibodies (NAbs) in controlling plasma viral load (pVL) in SIV_MAC251 infected rhesus macaques (RMs). Regions containing binding epitopes for antigen-specific IgG, IgM and IgA responses during different stages of SIV infection were determined, and the minimum size of linear Env epitope responsible for binding Abs was identified. Our findings suggest that conformational IgG and IgM responses as well as breadth of different peptide-specific functional IgG responses are indicative of disease outcome. The presence of NAbs against neutralization-sensitive and resistant pseudovirus did not predict the outcome of the disease.

Identification of broadly neutralizing antibodies (bNAbs) directed at the Env protein provides a possible solution to generate antibody based vaccine strategies. However, all HIV vaccines assessed to date were unable to generate bNAbs. The contribution of bNAbs to the control of early HIV-1 infection remains uncertain, as bNAbs develop relatively late in infection. Approximately 10–25% of HIV-1 infected people develop bNAbs within 3-years post infection [33]. Moreover, SIV_MAC251 and SIV_MAC239 were shown to be relatively resistant to antibody-mediated neutralization by both autologous and MAbs treatment [34, 35]. In our earlier study with either SHIV immunized or naïve SIV_MAC251 challenged macaques, none of the macaques were able to generate significant levels of neutralizing Abs against either pathogenic SIV_MAC251 or laboratory-adapted SIV_MAC251 [23]. In this study, the presence of NAbs did not predict the outcome of the disease and moreover the occurrence of those antibodies might be too late to control the infection at that point.

GN91 was able to control pVL to approximately 1 x 10³ RNA copies/ml of plasma and had significantly upregulated WVL-specific IgG and IgM responses compared to CL87 suggesting that binding Ab responses might be playing an important role in clearing pVL. Our overall data implies that combination of different peptide-specific IgG responses are predictive of the control of plasma viremia compared to the peptide-specific IgM and IgA responses. Our findings corroborate prior studies showing that while gp41 and gp120 show high levels of binding to IgG, there appears to be no correlation with control of early viral load, and subsequent Env-specific IgG responses had little impact on disease progression [36‐38] when present without additional responses. Gag has been shown to have little antiviral function [39], however, it appears to be a strong target for antigen-specific immunoglobulin responses. The majority of immunoglobulins bind around the conserved NC, p8, and p6 regions of the Gag protein, which have several important functions in viral pathogenesis, such as viral replication, encapsulating the viral genome, aiding in reverse transcription, viral genome packaging, and viral budding [40]. While Gag is not a target for bNAbs, B-cell recognition of these highly important and conserved regions of the Gag protein may be indicative of a potential target for vaccine design. Limited data are available on the role of Nef-specific Abs in HIV disease progression. Absence of anti-Nef Abs was found to be associated with symptomatic HIV infection [41]. In our study, we identified increased and early Nef-specific IgG responses in GN91, which controlled pVL much earlier than other disease progressing RMs. Our findings, along with the recent single domain antibody study for the inhibition of Nef protein in a mouse model [42], indicate that Nef needs to be considered as an important target for a novel therapeutic approach in the prevention and control of HIV. Several studies demonstrated that Tat-specific antibodies are more common in individuals who are successfully controlling the disease, suggesting that Tat-specific Abs have a beneficial role in preventing disease progression [43‐48]. In this study, we detected the presence of Tat-specific IgG in several animals, but as we have shown, it appears to be part of a large overall antigen-specific response that plays role in controlling pVL. Early induction of IgG responses against important targets and maintenance of those responses at higher magnitude might be crucial in reducing plasma viral loads and a possible predictor of disease outcome.

We also asked whether linear epitopes for antigen-specific immunoglobulins identified in this study corresponded to bNAb targets. The b12 bNAb recognizes almost exactly the same region as CD4, where these conserved regions of the CD4bs [49] fall within Env-PP10 and PP11 and induced very limited peptide-specific IgM responses (Fig. 4). However, the CD4bs is highly conformational, whereas only linear epitopes were examined in this study. Similarly, three series of bNAbs, specifically PG9, PG16 [50], and CH01-CH04 [51] target conserved conformational regions within the variable loops (V1-V3) of gp120. While these interactions have been shown to be highly conformational, the analogous V1-V3 regions in SIV_MAC239 were shown to be strong targets for peptide-specific IgG, IgA and IgM responses in SIV infected macaques (Fig. 4). Two bNAbs, 2F5 and 4E10, target the highly conserved MPER region of gp41 [52]. Linear epitopes for 4E10 and 2F5 on HIV-1 strain HXB2 represent NWFNIT and ELDKWA peptide sequences respectively [52], and these regions correspond to PP17 region in SIV_MAC239 (665–678aa). Seventy percent of SIV-infected macaques generated antigen-specific IgG responses to this region, and several macaques also generated antigen-specific IgA responses to this region. Therefore, Env-specific immunoglobulins bind to several linear epitopes of recently identified bNAbs, with the exception of the CD4bs. In future studies, it will be important to define the plasma viral sequence data in SIV infected animals and determine if the presence of escape variant(s) have any correlation with the antigen-specific immunoglobulin responses.

In summary, quantitative and qualitative immunoglobulin responses were detected in SIV-infected macaques, where increased IgG responses were measured specifically against the gp41 region followed by variable regions V1-V3 of gp120, and NC and p6 region of Gag protein. Antigen-specific IgM and IgA responses were more limited but targeted towards similar regions of the Env and Gag proteins. Several regions in Env protein strongly bind to immunoglobulins and are important epitopes for bNAbs. Early induction and increased breadth of antigen-specific IgG responses might be crucial to the control of plasma viral load and predictive of disease progression in SIV/HIV infection.

Our data strongly suggest that an early induction and increased breadth of peptide-specific IgG responses are indicative of disease progression. While Gag-specific responses may be argued to be an indirect indicator of HIV disease progression, recent identification of Abs against Nef and Tat proteins suggests that it may be possible to prevent and control HIV/AIDS by targeting the function of Tat and Nef proteins. Moreover, the presence of NAbs did not predict the outcome of the disease in our study where the generation of those antibodies might be too late to control the SIV infection. The experimental design does not address the role of T-cell responses and other innate immune responses in regulating plasma viral load in these SIV-infected animals.