Association of mutations in V3/C3 domain with enhanced sensitivity of HIV-1 clade C primary envelopes to autologous broadly neutralizing plasma antibodies
- Suprit Deshpande†1,
- Shilpa Patil†1,
- Rajesh Kumar†1,
- Tripti Shrivastava1,
- Aylur K. Srikrishnan2,
- Kailapuri G. Murugavel2,
- Wayne C. Koff3,
- Bimal K. Chakrabarti1, 3 and
- Jayanta Bhattacharya1, 3Email author
© The Author(s) 2016
Received: 3 May 2016
Accepted: 2 June 2016
Published: 15 June 2016
Broadly neutralizing antibodies to HIV-1 elicited in infected individuals evolves through shifts in their molecular specificities to viral envelope (Env) in the disease course. Recently, we showed that resistance of circulating HIV-1 clade C to the autologous plasma obtained from one Indian elite neutralizer is associated with mutations in V1 loop. In the present study, we examined the genetic attributes associated with exceptional sensitivity of pseudoviruses expressing an env gene obtained from the follow up visit contemporaneous plasma of the same donor.
Examination of chimeric autologous Envs, we found that enhanced neutralization sensitivity is associated with mutations in the V3/C3 region. A positive association between V3/C3 mutation mediated enhanced autologous neutralization of autologous viruses with their sensitivity to both neutralizing and non-neutralizing monoclonal antibodies was found. Interestingly, we found that depletion of autologous plasma with trimeric and monomeric Envs conferred the sensitive Env with resistance indicating that mutations in V3/C3 region altered Env conformation towards optimal exposure of epitopes targeted by the neutralizing and non-neutralizing antibodies.
In summary, we found distinct vulnerabilities associated with evasion of circulating viruses to broadly neutralizing antibodies mounted in an Indian elite neutralizer.
Vulnerabilities associated with shift in molecular specificities of the broadly neutralizing antibodies elicited in a small proportion of the Human Immunodeficiency Virus Type 1 (HIV-1) infected individuals (also known as elite neutralizers) in natural infection provide vital information of host-virus interaction towards developmental pathway of protective antibodies [1–4]. The HIV-1 uses its surface trimeric envelope (Env) protein composed of three heterodimers of gp120 and gp41 subunits attached non-covalently for viral entry via interaction with CD4 receptor present on T cells. These Env glycoproteins are heavily glycosylated and are targets of the neutralizing antibodies. Although HIV-1 infected individuals typically develop type-specific autologous neutralizing antibodies [5–7], however rarely few of them move on to develop cross-neutralizing antibodies [8–12]. It is believed that a better understanding of immune evasion mechanisms and events leading to development of broadly neutralizing antibodies in natural infection would rationally inform effective Env based immunogen design that can elicit protective antibodies upon vaccination .
Recently in an IAVI Protocol G study, by examining Envs obtained from broadly cross neutralizing (BCN) plasma of an elite neutralizer (G37080) collected at two different time points and spaced between 8 months, we showed that mutations in the V1 loop was associated with autologous neutralization escape . In the present study, we examined an HIV-1 clade C Env (HVTR-PG80v2.eJ7) obtained from the follow up plasma of the same donor (G37080) that significantly differed in its sensitivity to the contemporaneous autologous plasma antibodies compared to the two contemporaneous resistant Envs (HVTR-PG80v2.eJ38 and HVTR-PG80v2.eJ41). The enhanced sensitivity of pseudoviruses expressing HVTR-PG80v2.eJ7 Env to the contemporaneous autologous broadly neutralizing plasma antibodies was found to be associated with mutations in the V3/C3 region and exposure of discontinuous epitopes targeted by neutralizing antibodies with multiple specificities.
The blood samples were collected under the IAVI Protocol G study as described before  following approval and clearance from the Y R Gaitonde Care, Chennai Institutional Review Board (IRB) and the Ethics Committee. The informed consents were obtained from the participating donors to use the samples for assessing neutralizing antibody responses and publishing data keeping anonymity of every donor. The serum and plasma samples collected were shipped to the HIV Vaccine Translational Research Laboratory, Translational Health Science and Technology Institute for further assessment and research on the neutralizing antibody response.
Plasmids, viruses, antibodies, proteins and cells
Plasmids encoding patient (G37080) derived HIV-1 envelopes representing distinct clades were described previously . TZM-bl cells and monoclonal antibodies were obtained from the NIH AIDS Research Reagents Reference program and from the IAVI Neutralizing Antibody Consortium (NAC). 293T cells were obtained from the American Type Culture Collection (ATCC). Purified BG505-SOSIP.664-D7324 protein was kindly provided by Prof John Moore, Weill Cornell Medical College, New York.
Depletion of plasma antibodies by monomeric, trimeric Env proteins and MPER peptide
The depletion of the G37080 BCN plasma with soluble Env proteins and MPER peptide was done as described previously . Briefly, 30 mg of beads were used to couple with 1 mg of both monomeric (4-2.J41) and trimeric Env (BG505-SOSIP.664) proteins and 0.5 mg of MPER peptide (C1C) in coupling buffer (0.1M NaBO4, 1M (NH4)2SO4; pH 9.4) overnight at 37 °C for 16–24 h. Beads bound to Env proteins were next incubated with blocking buffer [PBS (pH 7.4), 0.1 % bovine serum albumin (BSA; Sigma) and 0.05 % Tween 20] at 37 °C to block the unbound sites. For depletion studies, G37080 visit 2 plasma was diluted to 1:50 in DMEM containing 10 % Fetal Bovine Sera (FBS) and 500 µl of diluted plasma was incubated with 20 µl of beads at room temperature. Unbound plasma antibodies were separated from ones those are bound to protein coated beads using a DynaMag™ 15 magnet as described previously . This step was repeated 4–5 times for depletion of plasma antibodies by monomeric gp120 and 10–12 times in case of BG505-SOSIP.664 coated beads respectively.
Neutralization assays were carried out using TZM-bl cells as described before . Env-pseudotyped viruses were incubated with varying dilutions of depleted plasma antibodies and incubated for an hour at 37 °C CO2 incubator under humidified condition and subsequently 1 × 104 TZM-bl cells were added into the mixture in presence of 25 μg/ml DEAE-dextran (Sigma, Inc.).The plates were further incubated for 48 h and the degree of virus neutralization was assessed by measuring relative luminescence units (RLU) in a Luminometer (Victor X2, PerkinElmer Inc.). All the neutralization assays were done in duplicate and repeated at least two times. The effect of point substitutions on neutralization of Env-pseudotyped viruses (as shown in Table 2) were done in triplicates and was repeated three times. A fold change in neutralization titer (ID50) (due to amino acid substitution/s) that is equal to or greater than twofold (≥twofold) in our study was considered positive.
PCR amplification, cloning and mutagenesis of autologous env
Autologous env clones were obtained from G37080 plasma by limited dilution PCR with slight modification to the methodology described previously . Briefly, viral RNA were extracted using High Pure viral RNA kit (Roche Inc.) following manufacturer’s protocol and cDNA prepared by RT-PCR using Superscript-III first strand synthesis kit (Invitrogen Inc.). rev-gp160 env genes were amplified from the maximally diluted plasma sample using a Phusion hi fidelity DNA polymerase (New England Biolabs Inc.). The complete env gene was purified and ligated into pcDNA 3.1/V5-His-TOPO (Invitrogen Inc.) vector. Chimeric Envs were prepared (Fig. 2a) by overlapping PCR and point substitutions were made by Quikchange II kit (Agilent technologies Inc.) following manufacturer’s protocol and as described previously .
Preparation of Env-pseudotyped viruses
Pseudotyped viruses were prepared by co-transfection of envelope expressing plasmid with env-deleted HIV-1 backbone plasmid (pSG3ΔEnv) into 293T cells in 6-well tissue culture plates using FuGENE6 Transfection Reagent (Promega Inc). Cell supernatants containing pseudotyped viruses were used subsequently in neutralization assays in TZM-bl cells. The reduction of infection of TZM-bl cells by the Env-pseudotyped viruses were determined by measuring luciferase activity using Britelite luciferase substrate (Perkin Elmer) with a Victor X2 Luminometer (Perkin Elmer).
Results and discussion
An HIV-1 clade C Env obtained from broadly neutralizing plasma showed exceptional degree of sensitivity to contemporaneous and retrospective autologous plasma antibodies
Similarity of amino acid sequence of PG80 v2.eJ7 env with that of other autologous envs
Donor visit #
Visit # 2
Visit # 1
V3/C3 region conferred resistant autologous Env with enhanced sensitivity to contemporaneous plasma antibodies
Mapping amino acid residues in the V3/C3 region associated with neutralization sensitivity
Env-pseudotyped viruses (Env chimeras & point mutants)
Fold changes in ID50
HVTR-PG80v2.eJ7 wild type
HVTR-PG80v2.eJ38 wild type
Effect of depletion of G37080 BCN plasma antibodies on autologous virus neutralization
G37080 depleted plasma (fold reduction in ID50)
gp140 Trimer (BG505-SOSIP.664)
gp120 Monomer (4-2.J41)
MPER (C1C peptide)
Changes in V3/C3 sequence conferred autologous Envs vulnerable to neutralizing and non-neutralizing mAbs
Association of presence of D167 in V2 apex with resistance to the PGT145 mAb
In summary, we report an HIV-1 clade C Env (HVTR-PG80v2.eJ7) from the G37080 BCN plasma of an elite neutralizer of Indian origin which displayed exceptional sensitivity to its contemporaneous autologous plasma antibodies. The HVTR-PG80v2.eJ38 resistant Env expressing the V3/C3 sequence of the HVTR-PG80v2.eJ7 Env was found to accommodate conformational epitopes targeted by the neutralizing antibodies present in the G37080 plasma as evident by the BCN plasma depletion study. Overall, we found that initially while the viruses circulating early escaped from the broadly neutralizing autologous antibodies via mutations in V1 loop that we recently reported , they subsequently evaded the antibody pressure in the disease course by accommodating mutations in V3/C3 region. Understanding pathways of co-evolution of circulating virus and neutralizing antibodies specific to them will complement strategies of rational immunogen design towards achieving elicitation of antibodies with similar breadth and potency.
JB, SD, SP designed study; SD,SP, RK,TS performed experiments; AKS, KGM analyzed clinical parameters including CD4 counts; BKC and WCK analyzed data along with JB,SD, SP, RK; JB wrote the manuscript with help of all the authors. All authors read and approved the final manuscript.
We thank all the Protocol G study participants registered with YRG Care, Chennai, and all the research staffs at the Protocol G clinical center at the YRG Care, Chennai and every member of the IAVI Protocol G team. We thank Dr. Albert Cupo, Prof. John P. Moore and the members the SOSIP trimer HIVRAD team, Weill Cornell Medical College, New York for providing us with BG505.SOSIP.664 plasmid DNA and purified protein. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs. John C. Kappes and Xiaoyun Wu: pSG3Δenv. Many of the reagents used in the present study were obtained from the IAVI Neutralizing Antibody Consortium (NAC) at The Scripps Research Institute, La Jolla, CA, USA. The International AIDS Vaccine Initiative has filed a patent relating to the autologous HIV-1 clade C envelope clones: U.S. Provisional Application no. 62/189,418, titled “HIV-1 clade C envelope glycoproteins,” with inventors J. Bhattacharya, S. Deshpande, S. Patil, R. Kumar, B.K. Chakrabarti. We sincerely thank all the HVTR laboratory members for support. IAVI’s work was made possible by generous support from many donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United Kingdom Department for International Development (DFID); and the United States Agency for International Development (USAID). The full list of IAVI donors is available at www.iavi.org. The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government. The contents of this manuscript are the responsibility of IAVI and do not necessarily reflect the views of USAID or the US Government.
The authors declare that they have no competing interests.
This work was funded by IAVI and made possible by the support of the United States Agency for International Development (USAID) and other donors including the support from the THSTI-IAVI HIV vaccine design program grant through the Department of Biotechnology, Govt. of India. The full list of IAVI donors is available at http://www.iavi.org. This work was partly supported by the DBT National Bioscience Research Award Grant (BT/HRD/NBA34/01/2012-13(iv) to Jayanta Bhattacharya). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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