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Aberrant expression of regulatory receptors programmed death-1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) is linked with dysregulation and exhaustion of T lymphocytes during chronic human immunodeficiency virus type 1 (HIV-1) infection; however, less is known about whether a similar process impacts B-lymphocyte function during HIV-1 infection. We reasoned that disruption of the peripheral B cell compartment might be associated with decreased neutralizing antibody activity. Expression of markers that indicate dysregulation (BTLA and PD-1), immune activation (CD95), and proliferation (Ki-67) was evaluated in B cells from HIV-1-infected viremic and aviremic subjects and healthy subjects, in conjunction with immunoglobulin production and CD4 T cell count. Viral load and cross-clade neutralizing activity in plasma from viremic subjects were also assessed. Dysregulation of B lymphocytes was indicated by a marked disruption of peripheral B cell subsets, increased levels of PD-1 expression, and decreased levels of BTLA expression in viremic subjects compared to aviremic subjects and healthy controls. PD-1 and BTLA were correlated in a divergent fashion with immune activation, CD4 T cell count, and the total plasma IgG level, a functional correlate of B cell dysfunction. Within viremic subjects, the total IgG level correlated directly with cross-clade neutralizing activity in plasma. The findings demonstrate that even in chronically infected subjects in which B lymphocytes display multiple indications of dysfunction, antibodies that mediate cross-clade neutralization breadth continue to circulate in plasma.

Antibodies that can neutralize diverse viral strains are likely to be an important component of a protective human immunodeficiency virus type 1 (HIV-1) vaccine. To this end, preclinical simian immunodeficiency virus (SIV)-based nonhuman primate immunization regimens have been designed to evaluate and enhance antibody-mediated protection. However, these trials often rely on a limited selection of SIV strains with extreme neutralization phenotypes to assess vaccine-elicited antibody activity. To mirror the viral panels used to assess HIV-1 antibody breadth, we created and characterized a novel panel of 14 genetically and phenotypically diverse SIVsm envelope (Env) glycoproteins. To assess the utility of this panel, we characterized the neutralizing activity elicited by four SIVmac239 envelope-expressing DNA/modified vaccinia virus Ankara vector- and protein-based vaccination regimens that included the immunomodulatory adjuvants granulocyte-macrophage colony-stimulating factor, Toll-like receptor (TLR) ligands, and CD40 ligand. The SIVsm Env panel exhibited a spectrum of neutralization sensitivity to SIV-infected plasma pools and monoclonal antibodies, allowing categorization into three tiers. Pooled sera from 91 rhesus macaques immunized in the four trials consistently neutralized only the highly sensitive tier 1a SIVsm Envs, regardless of the immunization regimen. The inability of vaccine-mediated antibodies to neutralize the moderately resistant tier 1b and tier 2 SIVsm Envs defined here suggests that those antibodies were directed toward epitopes that are not accessible on most SIVsm Envs. To achieve a broader and more effective neutralization profile in preclinical vaccine studies that is relevant to known features of HIV-1 neutralization, more emphasis should be placed on optimizing the Env immunogen, as the neutralization profile achieved by the addition of adjuvants does not appear to supersede the neutralizing antibody profile determined by the immunogen. IMPORTANCE Many in the HIV/AIDS vaccine field believe that the ability to elicit broadly neutralizing antibodies capable of blocking genetically diverse HIV-1 variants is a critical component of a protective vaccine. Various SIV-based nonhuman primate vaccine studies have investigated ways to improve antibody-mediated protection against a heterologous SIV challenge, including administering adjuvants that might stimulate a greater neutralization breadth. Using a novel SIV neutralization panel and samples from four rhesus macaque vaccine trials designed for cross comparison, we show that different regimens expressing the same SIV envelope immunogen consistently elicit antibodies that neutralize only the very sensitive tier 1a SIV variants. The results argue that the neutralizing antibody profile elicited by a vaccine is primarily determined by the envelope immunogen and is not substantially broadened by including adjuvants, resulting in the conclusion that the envelope immunogen itself should be the primary consideration in efforts to elicit antibodies with greater neutralization breadth.

The HIV-1 envelope (Env) glycoprotein gp120 is heavily glycosylated, displaying an array of high mannose and complex carbohydrate moieties that are tightly packed and arranged into spatially distinct clusters. It is well established that together these glycans form an “evolving shield” that protects vulnerable receptor-binding sites from recognition by neutralizing antibodies. Paradoxically, the absence of glycans has been consistently associated with HIV-1 transmission in certain settings, but the underlying benefit for transmission remains poorly understood. A less glycosylated form of the viral Env may be more sensitive to neutralizing antibodies (nAbs) in an established infection; yet in the absence of adaptive immunity, this could be advantageous by providing evasion from mucosal immune defenses directed against pathogen-associated glycans or an enhanced ability to attach to and infect its primary target, the CCR5+ CD4+ T cell. Thus, HIV-1 must continually maintain a delicate balance between the number, position, and type of glycans in Env in response to host-selective pressures. Here we will explore the complex roles that glycans play in establishing new infections, maintaining Env function, and evading sequential waves of neutralizing antibodies.

Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection., Author Summary Since cases were first recognized in the United States in 1981, human immunodeficiency virus (HIV-1) has infected over one million Americans. Globally, this scale reaches into the tens of millions, but no effective vaccine exists. Of those infected, approximately 20–30% of patients will develop broadly neutralizing antibodies. The reasons for maturation of these potentially protective responses are presently unknown, but being able to elicit such antibodies via vaccination could curb the pandemic. Here, we defined the earliest neutralizing antibody targets and the consequent routes of viral escape in one subtype A HIV-1 infected subject who developed modest breadth. We also determined the genetic and structural characteristics of early neutralizing monoclonal antibodies circulating in this subject and found that subtle light chain alteration enhanced target contact and neutralization. Overall, our data support the idea that exposure to a specific sequence of viral variants, which have escaped from immune pressure, could program long-term potential for antibody breadth.

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.

Methods In 16 viremic seroconverters, the cross-clade neutralizing activity of plasma was investigated using a panel of thirteen clade A, B, and C HIV-1 envelope (Env) pseudotyped virions, which represented three tiers of sensitivity. The neutralization IC50 was calculated for each plasma-Env combination, and these data were used to determine a breadth (how many Envs were neutralized) and potency (the strength of neutralization) score for each seroconverter. Additionally, the level of plasma antibodies that bound to the monomeric form of a subtype B Env gp120 (HIV-1 BaL) was quantitated.

Background Understanding of the autologous neutralizing antibody (Nab) targets during HIV-1 infection remains fragmentary and warrants further investigation in order to be incorporated during development of a successfully protective vaccine. In subtype C infection, the eighteen amino acid amphipathic alpha2 helix, encoded immediately downstream of gp120's V3 domain, undergoes strong positive selection where mutations track with the neutralization resistant phenotype; such characteristics would seem to make the alpha2 helix an attractive potential site for Nab targeting and escape.

Methods Here we characterized the initial nAb response in a subtype A HIV-1-infected Rwandan seroconverter and investigated how consequent immune events influenced the downstream development of cross-clade breadth. Autologous envelope (Env) glycoproteins from the transmitted/ founder virus and twenty longitudinal nAb escape variants were utilized to define the neutralization targets of autologous plasma and monoclonal antibodies (mAbs), the latter of which were also examined genetically and structurally through crystallization. Heterologous Env glycoproteins from nine cross-clade variants were used to determine neutralization breadth.

Address: 1Centers for Disease Control and Prevention, Atlanta, GA, USA, 2Zambia Blood Transfusion Service, Lusaka, Zambia, 3Public Health Research Institute, UMDNJ-New Jersey Medical School, Newark, NJ, USA, 4University of Alabama at Birmingham, Birmingham, USA, 5Los Alamos National Laboratory, Los Alamos, USA, 6Tulane University, New Orleans, LA, USA and 7Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA * Corresponding author

Background The potential role of neutralizing antibody in protecting against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody responses in three individuals, who were superinfected within one year of primary infection, to ten case-matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length env showed minimal diversification in the individuals who became superinfected with the same subtype of HIV-1 within year one postseroconversion. We hypothesized that these superinfected individuals had a muted neutralizing antibody response that elicited little pressure on the founder virus to escape. Methods We molecularly cloned envs from virus at the time of seroconversion and from virus at the time point that superinfection was detected. Using a TZM-BL pseudovirus reporter assay, we tested plasma neutralization of these autologous variants over the first year of infection. Results Neutralization assays showed that autologous plasma NAb titers to founder virus were low to undetectable in all three superinfected individuals prior to superinfection. In contrast, neutralizing antibodies with a median IC50 of 1:1896 were detected as early as three months postseroconversion in non-superinfected matched controls. There was no evidence, prior to superinfection, of crossneutralization of superinfecting variants in any of the three cases, although cross-neutralization breadth and potency to the subtype C pseudovirus reference panel was also limited in the plasma from non-superinfected individuals. Although there was a trend towards superinfected individuals having reduced levels of gp120 binding antibodies prior to superinfection compared to nonsuperinfected controls, this difference was not statistically significant between the groups. Conclusion These data suggest that development of antibodies, as reflected in autologous neutralizing antibodies to the primary infection variants, may provide protection and decrease susceptibility to superinfection.