Panels of HIV-1 Subtype C Env Reference Strains for Standardized Neutralization Assessments

ABSTRACT In the search for effective immunologic interventions to prevent and treat HIV-1 infection, standardized reference reagents are a cost-effective way to maintain robustness and reproducibility among immunological assays. To support planned and ongoing studies where clade C predominates, here we describe three virus panels, chosen from 200 well-characterized clade C envelope (Env)-pseudotyped viruses from early infection. All 200 Envs were expressed as a single round of replication pseudoviruses and were tested to quantify neutralization titers by 16 broadly neutralizing antibodies (bnAbs) and sera from 30 subjects with chronic clade C infections. We selected large panels of 50 and 100 Envs either to characterize cross-reactive breadth for sera identified as having potent neutralization activity based on initial screening or to evaluate neutralization magnitude-breadth distributions of newly isolated antibodies. We identified these panels by downselection after hierarchical clustering of bnAb neutralization titers. The resulting panels represent the diversity of neutralization profiles throughout the range of virus sensitivities identified in the original panel of 200 viruses. A small 12-Env panel was chosen to screen sera from vaccine trials or natural-infection studies for neutralization responses. We considered panels selected by previously described methods but favored a computationally informed method that enabled selection of viruses representing diverse neutralization sensitivity patterns, given that we do not a priori know what the neutralization-response profile of vaccine sera will be relative to that of sera from infected individuals. The resulting 12-Env panel complements existing panels. Use of standardized panels enables direct comparisons of data from different trials and study sites testing HIV-1 clade C-specific products. IMPORTANCE HIV-1 group M includes nine clades and many recombinants. Clade C is the most common lineage, responsible for roughly half of current HIV-1 infections, and is a focus for vaccine design and testing. Standard reference reagents, particularly virus panels to study neutralization by antibodies, are crucial for developing cost-effective and yet rigorous and reproducible assays against diverse examples of this variable virus. We developed clade C-specific panels for use as standardized reagents to monitor complex polyclonal sera for neutralization activity and to characterize the potency and breadth of cross-reactive neutralization by monoclonal antibodies, whether engineered or isolated from infected individuals. We chose from 200 southern African, clade C envelope-pseudotyped viruses with neutralization titers against 16 broadly neutralizing antibodies and 30 sera from chronic clade C infections. We selected panels to represent the diversity of bnAb neutralization profiles and Env neutralization sensitivities. Use of standard virus panels can facilitate comparison of results across studies and sites.

Download Full-text

Griffithsin Retains Anti-HIV-1 Potency with Changes in gp120 Glycosylation and Complements Broadly Neutralizing Antibodies PGT121 and PGT126

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01084-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 1

Author(s):

Kathryn Fischer ◽

Kimberly Nguyen ◽

Patricia J. LiWang

Keyword(s):

Neutralizing Antibodies ◽

Glycosylation Site ◽

Significant Loss ◽

Broadly Neutralizing Antibodies ◽

Clade B ◽

Key Factor ◽

Clade C ◽

Cd4 Binding Site ◽

Gp120 Glycosylation ◽

Hiv 1

ABSTRACT Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft’s role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft’s ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic.

Download Full-text

Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection

PLoS Pathogens ◽

10.1371/journal.ppat.1005520 ◽

2016 ◽

Vol 12 (3) ◽

pp. e1005520 ◽

Cited By ~ 84

Author(s):

Kshitij Wagh ◽

Tanmoy Bhattacharya ◽

Carolyn Williamson ◽

Alex Robles ◽

Madeleine Bayne ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

Prevention And Treatment ◽

Clade C ◽

Hiv 1

Download Full-text

New Member of the V1V2-Directed CAP256-VRC26 Lineage That Shows Increased Breadth and Exceptional Potency

Journal of Virology ◽

10.1128/jvi.01791-15 ◽

2015 ◽

Vol 90 (1) ◽

pp. 76-91 ◽

Cited By ~ 115

Author(s):

Nicole A. Doria-Rose ◽

Jinal N. Bhiman ◽

Ryan S. Roark ◽

Chaim A. Schramm ◽

Jason Gorman ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

New Members ◽

Isolation And Characterization ◽

The Family ◽

Clade C ◽

Potential Clinical Utility ◽

Hiv 1 ◽

Viral Isolates

ABSTRACT The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55–62, 2014, http://dx.doi.org/10.1038/nature13036 ). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

Download Full-text

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

PLoS Pathogens ◽

10.1371/journal.ppat.1008977 ◽

2021 ◽

Vol 17 (4) ◽

pp. e1008977

Author(s):

Rajesh Kumar ◽

Suprit Deshpande ◽

Leigh M. Sewall ◽

Gabriel Ozorowski ◽

Christopher A. Cottrell ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralizing Antibody ◽

Viral Envelope ◽

Negative Stain ◽

Clade C ◽

Negative Stain Electron Microscopy ◽

Env Protein ◽

Hiv 1

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

Download Full-text

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

10.1101/2020.09.15.297697 ◽

2020 ◽

Author(s):

Rajesh Kumar ◽

Suprit Deshpande ◽

Leigh M. Sewall ◽

Gabriel Ozorowski ◽

Christopher A. Cottrell ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Clade C ◽

Immunogen Design ◽

Env Protein ◽

Immunogenic Properties ◽

Hiv 1

AbstractEvaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The thermostable 1PGE-THIVC Env displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. While the specificity of antibodies elicited in rabbits associated with neutralizing autologous viruses were distinct to those developed in the elite neutralizer, EMPEM analysis demonstrated significant changes to Env conformations when incubated with polyclonal antibody sera from the elite neutralizer, suggesting these antibodies lead to the destabilization of Env trimers. Our study not only shows distinct mechanisms associated with potent neutralization of sequence matched and unmatched autologous viruses by antibodies induced in rabbits and in the elite neutralizer, but also highlights how neutralizing antibodies developed during the course of natural infection can impact viral Env conformations.Author SummaryThe interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a thermostable soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. The novel clade C SOSIP induced polyclonal neutralizing antibody response developed in rabbits not only differed in its epitope specificity compared to that elicited in natural infection in presence of pool of viral quasispecies but also showed how they differ in their ability to influence Env structure and conformation. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies.

Download Full-text

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

PLoS Pathogens ◽

10.1371/journal.ppat.1009736 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009736

Author(s):

Jelle van Schooten ◽

Marlies M. van Haaren ◽

Hui Li ◽

Laura E. McCoy ◽

Colin Havenar-Daughton ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Broadly Neutralizing Antibodies ◽

Antibody Diversity ◽

Infected Infant ◽

Immunodeficiency Virus ◽

Shiv Infection ◽

Hiv 1

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.

Download Full-text

Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

Journal of Virology ◽

10.1128/jvi.00369-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00369-18 ◽

Cited By ~ 7

Author(s):

Christine A. Bricault ◽

James M. Kovacs ◽

Alexander Badamchi-Zadeh ◽

Krisha McKee ◽

Jennifer L. Shields ◽

...

Keyword(s):

Guinea Pigs ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Tier 2 ◽

Broadly Neutralizing Antibodies ◽

Vaccination Regimen ◽

Tier 1 ◽

Clade C ◽

Hiv 1

ABSTRACTA vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCEThe elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

Download Full-text

Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from an HIV-1 Clade C-Infected Elite Neutralizer Mediate Autologous Virus Escape through Mutations in the V1 Loop

Journal of Virology ◽

10.1128/jvi.03090-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3446-3457 ◽

Cited By ~ 21

Author(s):

Shilpa Patil ◽

Rajesh Kumar ◽

Suprit Deshpande ◽

Sweety Samal ◽

Tripti Shrivastava ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Core Protein ◽

Natural Infection ◽

Viral Envelope ◽

External Region ◽

Membrane Proximal External Region ◽

Virus Escape ◽

Clade C ◽

Immunogen Design ◽

Hiv 1

ABSTRACTBroadly neutralizing antibodies isolated from infected patients who are elite neutralizers have identified targets on HIV-1 envelope (Env) glycoprotein that are vulnerable to antibody neutralization; however, it is not known whether infection established by the majority of the circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of the known targets. In the present study, we examined the specificity of a broad and potent cross-neutralizing plasma obtained from an Indian elite neutralizer infected with HIV-1 clade C. This plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Env from distinct HIV clades of different geographical origins. Mapping studies using gp120 core protein, single-residue knockout mutants, and chimeric viruses revealed that G37080 broadly cross-neutralizing (BCN) plasma lacks specificities to the CD4 binding site, gp41 membrane-proximal external region, N160 and N332 glycans, and R166 and K169 in the V1-V3 region and are known predominant targets for BCN antibodies. Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (but with neither monomeric gp120 nor clade C membrane-proximal external region peptides) resulted in significant reduction of virus neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved trimeric Env. Further examination of autologous circulating Envs revealed the association of mutation of residues in the V1 loop that contributed to neutralization resistance. In summary, we report the identification of plasma antibodies from a clade C-infected elite neutralizer that mediate neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous neutralization escape via mutation of residues in the V1 loop.IMPORTANCEA preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope glycoproteins are targets of neutralizing antibodies and represent a key component for immunogen design. The mapping of epitopes on viral envelopes vulnerable to immune evasion will aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on the viral envelope targeted by broadly cross-neutralizing antibodies elicited in natural infection in an elite neutralizer infected with HIV-1 clade C. Our data extend our knowledge on neutralizing epitopes associated with virus escape and potentially contribute to immunogen design and antibody-based prophylactic therapy.

Download Full-text

Broadly neutralizing plasma antibodies effective against diverse autologous circulating viruses in infants with multivariant HIV-1 infection

10.1101/837039 ◽

2019 ◽

Cited By ~ 3

Author(s):

Nitesh Mishra ◽

Shaifali Sharma ◽

Ayushman Dobhal ◽

Sanjeev Kumar ◽

Himanshi Chawla ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

B Cell Maturation ◽

Indian Origin ◽

Immunodeficiency Virus ◽

Perinatally Infected ◽

Clade C ◽

One Year ◽

Polyvalent Vaccination ◽

Hiv 1

AbstractDue to the extensive antigenic diversity of human immunodeficiency virus-1 (HIV-1), broadly neutralizing antibodies (bnAbs) develop in a subset of infected individuals over 2-3 years of infection. Interestingly, infected infants have been shown to develop plasma bnAbs frequently and as early as one-year post-infection, with features atypical than adult bnAbs, suggesting that the factors governing bnAb induction in infants are distinct from that in adults. Understanding the viral characteristics in infected infants with early bnAb responses will provide key information on the antigenic triggers driving B cell maturation pathways towards the induction of bnAbs. Herein, we evaluated the presence of plasma bnAbs in a cohort of 51 HIV-1 clade C perinatally infected infants of Indian origin and identified viral factors associated with early bnAb responses. Plasma bnAbs targeting V2-apex on the env were predominant in infant elite and broad neutralizers. Circulating viral variants in infant elite neutralizers were susceptible to known bnAbs against V2-apex while varied resistance profile to other bnAb classes was observed. In infant elite neutralizers, multivariant infection was associated with plasma bnAbs targeting diverse autologous viruses. Our data provides information supportive of polyvalent vaccination approaches capable of inducing V2-apex bnAbs against HIV-1.

Download Full-text