---
title: "Immunogenicity and structure of a stabilized HIV-1 clade C Env bound to an autologous bnAb — Front"
id: "frontiers-in-immunology-13-immunogenicity-and-structure-of-a-stabilized-hiv-1-clade-c-env-from-a-pediatric"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-13-immunogenicity-and-structure-of-a-stabilized-hiv-1-clade-c-env-from-a-pediatric"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1919627"
published_at: "2026-10-21T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Immunogenicity and structure of a stabilized HIV-1 clade C Env bound to an autologous bnAb — Front
## Provenance & Clinical Metadata
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- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1919627)
- **Published At:** 2026-10-21T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The source article title and citation were available, identifying a study of a stabilized **HIV-1** clade C **Env** from a pediatric elite neutralizer analyzed in complex with an autologous **bnAb**. - The online record appeared in Frontiers in Immunology; the publication metadata (journal, title, URL) were present in the source. - The accessible text did not include the study abstract, methods, sample characteristics, or numeric results; those elements were not present in the provided source content. - Investigators' experimental details—protein stabilization strategy, structural determination method, neutralization assays, and immunogenicity endpoints—were not reported in the provided excerpt. - The source excerpt contained navigational and journal pages but omitted figures, tables, and author-reported conclusions; therefore no factual performance metrics, sample sizes, or statistical outcomes can be summarized. - Critical unanswered items from the source: how the Env was stabilized, which structural technique was used (e.g., cryo-EM or X-ray), the bnAb binding footprint, immunogenicity readouts, and whether the construct elicited cross-clade neutralization. - Because the source text lacked substantive results, this summary lists documented metadata and specifies absent data elements that would be required for clinical or translational interpretation.
## Clinical Analysis & Structured Key Points
Frontiers | Immunogenicity and structure of a stabilized HIV-1 clade C Env from a pediatric elite neutralizer in complex with an autologous bnAb ORIGINAL RESEARCH article Front. Immunol. , 21 October 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1919627 Published in Frontiers in Immunology Vaccines and Molecular Therapeutics 7 impact factor 11.3 citescore Part of a Research Topic Broad Spectrum Viral Vaccines Submission open 53k views 13 articles Editor & Reviewers Edited by D C Diego Cantoni Reviewed by S P Swayam Prakash S K Soohyun Kim Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Figure 7 View in article ORIGINAL RESEARCH article Front. Immunol. , 21 October 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1919627 Immunogenicity and structure of a stabilized HIV-1 clade C Env from a pediatric elite neutralizer in complex with an autologous bnAb S S Swarandeep Singh 1 † S K Sanjeev Kumar 1 † A C Arnab Chatterjee 2 † L M Lakshay Malhotra 3 M P Muneeb Pervez 1 A R Abdul Rahman 1 I A Ifrah Andrabi 1 B M Bapan Mondal 2 S K Sanket Katpara 1 S S Shaifali Sharma 1 H B Harsh Bhakhri 1 A G Anjli Gaur 1 T B Tanu Bansal 1 V I Vikram Iyer 1 A W Abdul Wahid Hussain 1 S A Shubbir Ahmed 4 R K Rajesh Kumar 5 A S Abdul Samath Ethayathulla 3 R L Rakesh Lodha 6 S D Somnath Dutta 2 * +12 more K L Kalpana Luthra 1 * 1. Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India 2. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India 3. Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India 4. Centralized Core Research Facility, All India Institute of Medical Sciences, New Delhi, India 5. Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, India 6. Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India See more Article metrics View details Abstract Introduction: The envelope (Env) glycoproteins derived from circulating viruses in elite-neutralizers (ENs), who naturally develop potent broadly neutralizing antibody (bnAb) responses, serve as a potential template for HIV-1 vaccine design. Here, we investigated the immunogenicity and structure of a clade C HIV-1 Env trimer (330) derived from the pediatric elite-neutralizer AIIMS_330. Methods: The 330 Env was engineered as SOSIP.664, NFL.664, and multivalent ferritin-nanoparticle (NP) immunogens. Biophysical and antigenic properties were assessed using HIV-1 trimer-specific bnAbs and non-neutralizing antibodies. Immunogenicity was evaluated in rabbits immunized with Addavax-formulated SOSIP.664, NFL.664, or ferritin NP immunogens (n = 4), with an adjuvant only placebo group (n = 2). Structural characterization was performed by cryo-electron-microscopy (cryo-EM) using the autologous Env 330 SOSIP.664 trimer in complex with the bnAb 44m. Results: The 330 Env trimers exhibited native-like antigenic properties, high thermostability, strong binding to trimer-specific bnAbs, and limited reactivity with non-neutralizing antibodies. Immunization of rabbits elicited strong autologous neutralizing antibody responses together with modest heterologous Tier-1 neutralization. Autologous neutralizing responses were significantly higher for ferritin-NP (P = 0.019) and SOSIP.664 (P = 0.048) than for NFL.664, with no significant difference between ferritin-NP and SOSIP.664 (P = 0.681). A 5.1 Å cryo-EM reconstruction of the autologous Env 330 SOSIP.664–44m complex revealed native-like presentation of the N332-V3 supersite. The structural analysis further suggested the interacting Env contacts E293, R327, K337, and K446, along with glycans N332, N326, N442, and N448 with the bnAb-44m. We identified R327 of the conserved GDIR-motif and the N332 glycan, within the V3-loop, as neutralizing determinants on the 330 Env, with a fold reduction of 116, on testing the mutant 330 viruses with autologous bnAb 44m. Discussion: Pediatric elite-neutralizer AIIMS_330 derived Env trimers represent structurally and antigenically well-characterized clade C immunogens with ability to elicit strong autologous neutralizing antibodies, with modest heterologous Tier-1 neutralization. Together, these findings advance our understanding of clade C Env antigenic variability. Further, the structural and immunological insights support the potential utility of AIIMS_330 derived Env as an antigenic probe and may inform future rational design of Clade C HIV-1 vaccines and structure-guided strategies for eliciting neutralizing antibodies. Introduction A critical goal in HIV vaccine development is to elicit broadly neutralizing antibodies (bnAbs) in uninfected and high-risk individuals ( 1 ). Despite the challenges associated with this goal, two key observations in humans provide a strong rationale for developing vaccines that can elicit bnAbs. First, the identification of elite neutralizers (ENs), a rare group of HIV-1-infected individuals who develop exceptionally potent bnAb responses, has provided valuable insights for rational HIV-1 immunogen design aimed at eliciting similar protective bnAb responses through vaccination ( 1 – 3 ). Second, simian-human immunodeficiency virus (SHIV) challenge studies in nonhuman primates (NHPs) have demonstrated that passive immunization with bnAbs can confer protection against infection, thereby providing strong support for HIV-1 vaccine strategies designed to elicit similar protective bnAb responses in humans following vaccination ( 4 – 8 ). Stabilized HIV-1 envelope (Env) glycoproteins are considered promising candidates for immunogen design to elicit such bnAb responses. The extensively studied clade A pediatric-origin Env BG505 has demonstrated the ability to elicit autologous and, occasionally, Tier-2 heterologous neutralizing responses across multiple animal models and is currently under evaluation in clinical trials ( 9 – 17 ). It is believed that single Env-based immunization regimens are unlikely to induce such bnAb responses; therefore, multi-stage, multi-component immunization approaches appear to be a promising strategy to guide rare precursor B cells toward bnAb development ( 18 , 19 ). HIV-1 clade C predominates global HIV-1 infection and accounts for approximately 50% of the infections worldwide ( 20 , 21 ). However, a limited number of clade C Envs have been successfully stabilized and characterized as native-like trimers, often with relatively low expression and limited intrinsic stability, thereby posing challenges for the development of clade C Env-based immunogens ( 22 – 25 ). Multivalent display of HIV-1 Env trimers on self-assembling ferritin nanoparticles (NPs) in a repetitive array has proven to be a promising approach, with several studies demonstrating enhanced humoral immune responses and improved immunogenicity in animal models ( 26 , 27 ). Studies have suggested that HIV-1-infected children who often acquire the infection through vertical transmission develop bnAbs more frequently and earlier than adults ( 28 – 33 ). Such early development of bnAb responses may be attributed to the relative immaturity of the pediatric immune system, in which the founder virus encounters minimal selection pressure compared with adults, thereby giving evolving B cells an advantage in generating neutralizing antibodies against contemporary viruses ( 28 , 30 ). Recent studies suggest that bnAb development is driven by reciprocal virus–host co-evolution, whereby evolving Env variants and the host B-cell repertoire jointly shape the maturation of bnAb responses ( 34 ). Characterization of antigenic variability in viral Envs is of particular interest for investigating antigenic determinants associated with bnAb development. Previously, we reported a pair of chronically HIV-1-infected monozygotic twins, AIIMS_330 & AIIMS_329, exhibiting elite plasma neutralizing activity ( 32 ). Longitudinal analysis over 5 years revealed sustained evolution of plasma bnAbs in the AIIMS_330 EN, targeting multiple major neutralizing epitopes, including V1V2, V3-glycan, and CD4bs on the HIV-1 Env glycoprotein ( 30 , 32 , 35 ). Pseudoviral HIV-1 Env clones derived from circulating viruses in the AIIMS_329 and AIIMS_330 pediatric donors exhibited high susceptibility to neutralization by a select panel of HIV-1 bnAbs with diverse epitope specificities, as well as to autologous contemporaneous plasma antibodies ( 30 , 32 ). Env derived from such contemporaneous viruses can serve as valuable templates for HIV-1 immunogen design, while structural characterization of Env trimers in complex with autologous bnAbs from the same donor can provide valuable insights for vaccine development. We recently characterized the antigenicity and immunogenicity of a native- like Env trimer, generated from a virus from AIIMS_329 ( 25 ). In earlier studies, we had evaluated the longitudinal antibody response in AIIMS_330 (a pediatric elite neutralizer identical twin of AIIMS_329) ( 32 ), and the evolving 44m bnAb lineage ( 33 , 35 ) and defined the interaction of 44m with BG505 Env. The structural, antigenic, and immunogenic properties of the native-like clade C Env, derived from the viruses isolated from AIIMS_330, along with its interaction with the autologous bnAb 44m, remain to be determined, in order to evaluate the potential of the 330 Env to elicit similar responses on immunization. Here, we designed and characterized an HIV-1 clade C 330 Env trimer derived from a pediatric elite donor, AIIMS_330. Among the longitudinally derived autologous viruses from AIIMS_330, the 330.16.E6 envelope (described as 330 Env in this study) was selected, as it was susceptible to neutralization by multiple bnAbs and contemporaneous autologous plasma antibodies, distinguishing it from several other autologous viral variants that were comparatively resistant, as previously reported ( 30 , 32 ). The 330 Env was engineered as soluble SOSIP.664 and NFL.664 gp140 trimers, and a ferritin NP displaying SOSIP.664 Env trimers in a repetitive array. The designed 330 Envs adopted native-like trimeric conformation and exhibited strong binding to key HIV-1 bnAbs, while showing limited accessibility to the immunodominant epitopes targeted by non-neutralizing antibodies (non-NAbs). The 330 Env constructs elicited strong autologous neutralizing responses, with modest heterologous Tier-1 responses, but did not induce bnAb responses. The multimerization of Env on ferritin NPs resulted in an improved autologous response compared with soluble gp140 trimers. A 5.1 Å cryo-EM reconstruction of the autologous Env 330 SOSIP.664–44m complex revealed a native-like presentation.Collectively, these findings provide mechanistic insights into clade C Env-autologous bnAb interactions and define the structural, antigenic, and immunogenic properties of an Env derived from a pediatric elite neutralizer, while providing a potential antigenic template for future sequential or multicomponent HIV-1 immunogen strategies. Results 330 Env construct designs and biophysical characterization We previously reported a pseudoviral Env clone 330.16.E6 derived from an HIV-1 Indian clade C-infected pediatric EN AIIMS_330 that exhibited potent neutralization susceptibility to a select panel of HIV-1 bnAbs targeting V1V2, V3-glycan N332 supersite, CD4bs, gp120-gp41 interface, and MPER epitopes, as well as with autologous contemporaneous plasma antibodies, while showing limited exposure to the panel of non-nAbs tested ( 30 , 32 ). In this study, using 330.16.E6 as a template, we designed and engineered 330 Env constructs in three formats: as soluble cleaved 330 SOSIP.664 gp140 Env trimers, uncleaved 330 NFL.664 gp140 Env trimers, and SOSIP.664 Env trimers displayed on self-assembling 330 ferritin nanoparticles (NPs) to enable multivalent antigen presentation ( Figures 1A–C ; Supplementary Figure S1 ). The 330 Env constructs were stabilized by introducing strategic mutations to increase expression and trimerization, as illustrated in Figures 1A–D ; Supplementary Table S1 . The codon-optimized Env constructs were expressed in Expi293F cells and initially purified by Galanthus nivalis lectin (GNL) affinity chromatography. This initial purification yielded ~15 mg/L of SOSIP.664 Env and ~16.5 mg/L of NFL.664 Env, comprising a heterogeneous population of Env oligomers, including monomers, dimers, trimers, and aggregates. To select properly folded Env trimers in the prefusion-closed conformation, Expi293F supernatant was subjected to PGT145-based immunoaffinity chromatography, followed by size-exclusion chromatography (SEC). PGT145 recognizes a quaternary apex epitope preferentially presented by native-like, prefusion-closed Env trimers, thereby enabling enrichment of trimers displaying an intact apex architecture. The resulting PGT145-enriched material was further resolved by SEC to obtain purified Env trimers for subsequent biochemical, biophysical, and antigenic characterization. The SEC profiles of 330 SOSIP.664 and NFL.664 Env trimers showed elution peaks at 10.2 mL volume, whereas ferritin NPs eluted earlier, around 8.7 mL, on a Superdex 200 10/300 column ( Figure 1E ). The purification yields of 330 Env constructs following SEC were ~1 mg/L for SOSIP.664, ~1.45 mg/L for NFL.664, and ~0.05 mg/L for ferritin NPs. Purity analyses confirmed the intact trimeric nature of the gp140 Env trimers, which appeared as crisp bands on Coomassie-stained blue native polyacrylamide gel electrophoresis (BN-PAGE) ( Supplementary Figure S2A ), whereas the 330 ferritin NPs were too large to efficiently enter the gel and were visible at the top of the lanes. ( Supplementary Figure S2A , right). SDS-PAGE analysis showed that the 330 gp140 Env proteins migrated as monomers under reducing conditions, suggesting the high purity of the Env trimer preparations ( Supplementary Figure S2B ). Negative-stain electron microscopy (ns-EM) reference-free two-dimensional (2D) class averages demonstrated that most of the gp140 proteins adopted a native-like trimeric conformation, with multiple copies of the Env spikes efficiently displayed on the self-assembling ferritin NPs ( Figure 1F ; Supplementary Figure S3 ). The 3D reconstruction of soluble gp140 trimers further confirmed a trimeric structure, while the predicted model of ferritin NPs displays multiple copies of SOSIP.664. Env spikes ( Figure 1G ). The thermal stability of 330 SOSIP.664 was evaluated by differential scanning fluorimetry (DSF), which revealed a melting temperature (Tm) of ~74 °C ( Figure 1H ). Overall, biophysical characterization demonstrated that the 330 Envs are well-stabilized and adopt native-like trimeric conformations. Figure 1 Design and biophysical characterization of 330 gp140 Env trimers and ferritin nanoparticles. (A-C) Linear representation of the three 330 Envelope construct design. (A) SOSIP.664 design with the SOS-bond (A501C-T605C), IP (I559P) stabilizing mutation along with TD8 stabilizing mutations, an intraprotomer disulfide bond (I201C-A433C), an optimized furin cleavage site (R6), the CD5 Signal Peptide (SP), and Twin Strep-tag (tst). (B) NFL.664 design covalently linking gp120 to gp41 with a flexible glycine-serine (GGGGSGGGGS) linker. (C) Multivalent design with SOSIP.664 Env displayed on (H) pylori ferritin nanoparticle covalently linked with glycine-serine linker at the C-terminal. (D) Structural representation of 330 Env highlighting the TD8 stabilizing mutations and I201C-A433C disulfide bond. The structure was illustrated using PyMOL and was based on the BG505 SOSIP.664.664 crystal structure (PDB ID: 4ZMJ). (E) SEC profiles of 330 Env trimers (SOSIP.664 and NFL.664) eluted around 10.2 mL, and Ferritin NP showed a major peak at 8.7 mL, using a Superdex 200 Increase 10/300 GL column. (F) Negative stain-electron microscopy (ns-EM) 2-D class averages of NFL.664 and SOSIP.664 gp140 Env trimers and ferritin NP. (G) Predicted 3D model of Env trimers and ferritin NP displaying SOSIP.664 Env trimers using ns-EM 2D class averages using ChimeraX. (H) Thermal stability profile of 330 SOSIP.664 Env trimer determined by DSF. 330 gp140 Env trimers and ferritin nanoparticles exhibit strong binding to HIV-1 bnAbs Next, we evaluated the antigenic profiles of the three 330 Envs (SOSIP.664, NFL.664, and ferritin NPs) by ELISA assays using a select panel of HIV-1 bnAbs including those targeting the V1V2 apex (PGT145, PGDM1400, CAP256.25), the V3-glycan (2G12, PGT121, 10-1074, AIIMS P01), the CD4 binding site (VRC01, N6), and the gp120-gp41 interface (PGT151). We also evaluated the binding of Envs with a panel of non-nAbs, including F105, which targets CD4bs on open Env conformations, A32, which recognizes the gp120 inner domain for open states, and the CD4-induced antibodies 48d and 17b, assessed in the presence of soluble CD4 (sCD4) ( Figure 2 ). All the three 330 Env constructs were recognized by the tested HIV-1 bnAbs, with the exception of PGT151, which did not bind the uncleaved NFL.664 trimer, consistent with the absence of the native gp120–gp41 cleavage-dependent interface required for PGT151 recognition. In contrast, the constructs showed minimal binding reactivity with the CD4-induced non-neutralizing antibody 17b in the presence of sCD4, supporting their predominantly closed, native-like prefusion conformation. Overall, reactivity to the non-neutralizing antibodies was lower than that observed for broadly neutralizing antibodies, although some differences in epitope exposure were evident among the platforms. For F105, NFL, and ferritin NP, AUCs were modestly higher than SOSIP, at 2.43, 3.00, and 1.78, corresponding to 1.37- and 1.69-fold increases, respectively. Similarly, A32 binding increased 1.44-fold for NFL (AUC = 2.39) and 1.27-fold for ferritin NP (AUC = 2.11) compared with SOSIP (AUC = 1.66). For 48d, reactivity was modestly higher with NFL (1.23-fold; AUC = 2.31) and ferritin NP (1.45-fold; AUC = 2.71) than with SOSIP (AUC = 1.88). In contrast, 17b binding in the presence of sCD4 showed a marked increase for NFL, with a 3.22-fold higher AUC (3.41) than SOSIP (1.06), whereas ferritin NP showed only a modest 1.35-fold increase (AUC = 1.43). Overall, the 330 gp140 SOSIP, NFL, and ferritin NP platforms showed high binding reactivity with the bnAbs tested, with negligible binding to non-neutralizing antibodies. Figure 2 Antigenicity of 330 gp140 Env trimers and ferritin nanoparticles to HIV-1 broadly neutralizing and non-neutralizing antibodies. ELISA binding curves showing the antigenicity of 330 Env trimers and ferritin nanoparticles with conformation-specific bnAbs, including V1V2-directed (PGT145, CAP256.25, PGDM1400), gp120-gp41 interface (PGT151), CD4bs (VRC01, N6), V3 Supersite directed (10-1074, AIIMS P01), along with non-nAbs, including CD4-induced co-receptor binding site (CD4i) non-nAbs (17b+sCD4, A32, 48d) and CD4bs non-nAb (F105). Data are presented as mean values ± SD from two independent experiments, each performed in duplicate. Subsequently, we evaluated the binding kinetics of all three 330 Env constructs using octet biolayer interferometry (BLI) analysis. We used selected HIV-1 trimer conformation-specific bnAbs PGT145, PGDM1400, and PGT151, along with non-nAbs F105 and 17b, in the presence of sCD4 ( Figure 3 ). Sensograms were fitted using a 1:1 binding model, and the resulting kinetic parameters are reported as KD for SOSIP.664 & NFL.664 Env trimers and apparent dissociation constants ( K D app ) for Ferritin NPs, as multivalent interactions may introduce avidity and rebinding effects that can influence the observed binding kinetics. The 330 Envs SOSIP.664 and NFL.664 exhibited comparable binding to the V2-apex-directed bnAb PGT145, with KD values of
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