Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb

Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb View ORCID Profile Swarandeep Singh , View ORCID Profile Sanjeev Kumar , View ORCID Profile Arnab Chatterjee , View ORCID Profile Lakshay Malhotra , View ORCID Profile Muneeb Pervez , View ORCID Profile Abdul Rahman , Ifrah Andrabi , View ORCID Profile Bapan Mondal , Sanket Katpara , View ORCID Profile Shaifali Sharma , Harsh Bhakri , Anjli Gaur , View ORCID Profile Tanu Bansal , Vikram Iyer , Abdul Wahid Hussain , View ORCID Profile Shubbir Ahmed , Rajesh Kumar , Ethayathulla Abdul Samath , Rakesh Lodha , View ORCID Profile Somnath Dutta , View ORCID Profile Kalpana Luthra doi: https://doi.org/10.1101/2025.04.26.649701 Swarandeep Singh 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Swarandeep Singh Sanjeev Kumar 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India 2 ICGEB-Emory Vaccine Center, International Center for Genetic Engineering and Biotechnology , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sanjeev Kumar Arnab Chatterjee 3 Molecular Biophysics Unit, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnab Chatterjee Lakshay Malhotra 4 Department of Biophysics, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lakshay Malhotra Muneeb Pervez 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Muneeb Pervez Abdul Rahman 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Abdul Rahman Ifrah Andrabi 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bapan Mondal 3 Molecular Biophysics Unit, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bapan Mondal Sanket Katpara 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shaifali Sharma 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shaifali Sharma Harsh Bhakri 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anjli Gaur 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tanu Bansal 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tanu Bansal Vikram Iyer 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abdul Wahid Hussain 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shubbir Ahmed 6 Centralized Core Research Facility, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shubbir Ahmed Rajesh Kumar 5 Indian Institute of Technology , Roorkee, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ethayathulla Abdul Samath 4 Department of Biophysics, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rakesh Lodha 7 Department of Pediatrics, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Somnath Dutta 3 Molecular Biophysics Unit, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Somnath Dutta For correspondence: kalpanaluthra{at}gmail.com somnath{at}iisc.ac.in Kalpana Luthra 1 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kalpana Luthra For correspondence: kalpanaluthra{at}gmail.com somnath{at}iisc.ac.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT The envelope (Env) glycoprotein derived from circulating viruses in elite-neutralizers (who naturally develop broadly neutralizing antibodies (bNAbs)), serves as potential template for HIV-1 vaccine design. Herein, we report the structure and immunogenicity of soluble clade-C HIV-1 Env trimer (330) derived from a pediatric elite-neutralizer (AIIMS_330). Using SOSIP, NFL and ferritin-nanoparticle (NP) platforms, we engineered immunogens that preserved native-like Env conformation, exhibited high thermostability and nanomolar affinity for diverse bNAbs, with minimal reactivity to non-neutralizing antibodies. Cryo-EM structure solved at 5 Å resolution of 330-SOSIP trimer-autologous bNAb 44m complex revealed interactions at GDIR motif, N332 supersite along with additional contacts at E293, K337, K446 and glycans N326, N442, N448, as compared to our previously reported BG505–44m structure. Rabbit immunizations with soluble and NP-displayed formats elicited autologous, and heterologous neutralization of tier-1 clade-C viruses. Herein we define a structurally resolved HIV-1 clade C Env that supports multivalent vaccine strategies and provides mechanistic insights toward rational HIV-1 immunogen design. INTRODUCTION A critical goal in HIV vaccine development is to elicit broadly neutralizing antibodies (bNAbs) in uninfected and high-risk individuals 1 . Elite neutralizers (ENs), a rare group of HIV-1-infected individuals who develop exceptionally potent bNAb responses, provide valuable blueprints for rational HIV-1 immunogen design, aimed at inducing similar bNAb responses upon vaccination 1 – 3 . Studies involving simian-human immunodeficiency virus (SHIV) challenges in nonhuman primates (NHPs) have indicated that passive immunization with bNAbs can effectively prevent infection, supporting the development of an HIV-1 vaccine that induces such protective immune responses in humans upon vaccination 4 – 8 . Stabilized HIV-1 envelope (Env) glycoproteins have emerged as promising candidates for immunogen design, with the potential to elicit protective immune responses through vaccination, based on insights from the RV144 Thai trial 9 , 10 . Native like HIV-1 Env trimers, particularly the clade-A BG505 Env have shown the ability to induce neutralizing antibody (nAb) responses in animal models and are currently under evaluation in clinical trials 11 – 18 . HIV-1 clade C (HIV-1C), predominates global HIV-1 infection with more than 50% infections 19 , 20 . A limited number of clade-C Envs has been stabilized; with low expression levels and instability, posing hurdles to develop clade C Env-based vaccines 21 – 24 . Recent investigations highlight distinct differences in surface antigen presentation between HIV-1 and HPV, contributing to HIV-1’s limited ability to elicit a strong immune response compared to HPV 25 . In contrast, currently approved multivalent vaccines for HPV and HBV generate robust immune responses with >95% efficacy 26 – 28 . Recent studies suggest that single Env-based vaccine regimens may not be the most effective approach for HIV-1 prevention. Notably, presenting HIV-1 Env trimers on self-assembling ferritin nanoparticles has shown promise in eliciting robust immune response in rabbits, suggesting improved immunogenicity 29 , 30 . Studies also suggest that HIV-infected children who often acquire the infection through vertical transmission, are inherently better at generating bNAbs than adults 31 – 36 . The relative immaturity of the immune system in children may allow for more effective protective immune responses, as founder virus encounters minimal selection pressure, providing the evolving B-cells an advantage in generating NAbs that effectively neutralize contemporary viruses 31 – 33 , 37 . Env trimers from contemporaneous viruses can serve as effective tools for immunogen design, and structural characterization of Env in complex with autologous bNAbs from the same donor can provide valuable insights for vaccine development 36 . We previously reported on a pair of HIV-1-infected monozygotic twins (AIIMS_330 and AIIMS_329) demonstrating elite neutralizing activity, with longitudinal sampling of AIIMS_330 revealing sustained elite neutralizing activity. The Env from this EN exhibited multiple broad bNAb epitope specificities, making it an ideal template for designing and characterizing HIV-1 Env mimics 33 , 35 . In this study, we designed and characterized an HIV-1 Clade-C 330 Env trimer derived from a pediatric EN AIIMS_330, as soluble SOSIP & NFL Env trimers, alongside tailoring the Env to display a repetitive array of HIV-1 trimers on self-assembling ferritin nanoparticles (NPs) 11 , 29 , 38 . Designed Env constructs, stabilized by trimer-stabilizing mutations, exhibited enhanced expression and a greater propensity to assemble into native-like HIV-1 Env mimics 12 , 15 , 39 , 40 . The 330 Env constructs exposed multiple epitopes and demonstrated strong affinity to bNAbs while occluding the immunodominant epitopes of non-neutralizing antibodies (non-NAbs). Ferritin NPs exhibited strong immunogenicity and tier-1 autologous neutralizing activity, upon immunization in rabbits compared to soluble counterparts. Negative stain-electron microscopy (NS-EM) showed that both soluble Envs and multivalent NPs adopted native-like morphology. A cryo-EM structure at 5 Å resolution, revealed a stable, well-organized Envelope structure in the prefusion closed conformation of the 330 SOSIP Env trimer. Structural insights from the cryo-EM model of the 330 SOSIP Env trimer in complex with the autologous bNAb 44m, a V3-glycan-specific potent bNAb identified from the AIIMS_330 EN 36 , define the key determinants of autologous bNAb recognition. Collectively, this study provides mechanistic insights into Env–autologous bnAb interactions and expands our understanding of the immunogenicity of HIV-1C Env derived from pediatric elite neutralizers. The 330 Env represents a valuable antigenic probe for HIV-1 bNAb discovery and a potential HIV-1C based Env for a multivalent vaccine design. RESULTS Design & biophysical characterization of 330 Env trimers and ferritin nanoparticles Previously we reported a pair of HIV-1 chronically infected monozygotic twins AIIMS_330 & AIIMS_329 exhibiting elite plasma neutralizing activity 35 . Over five years of longitudinal sampling, the AIIMS_330 EN showed sustained evolution of plasma bNAbs targeting major neutralizing determinants on the HIV-1 Env glycoprotein 33 , 35 , 41 . The pseudoviral Env clone 330.16.E6, derived from the AIIMS_330 EN, exhibited high susceptibility to neutralization by a broad range of HIV-1 bNAbs with multiple epitope specificity, along with autologous contemporaneous plasma antibodies, and resistance to the panel of non-nAbs tested 33 , 35 . Therefore, we engineered this Env in membrane soluble forms, as SOSIP.664 gp120-gp41 cleaved Env trimers, NFL.664 gp140 single-chain uncleaved Env trimers and SOSIP Env trimers on self-assembling ferritin NPs to achieve multivalency ( Figure 1 A-C ). The 330 Env trimers were stabilized by incorporating mutations for increased expression and trimerization, as illustrated in Figure 1A-D , Table S1. The SOSIP Env was designed by introducing a SOS disulfide bond (A501C-T605C) 42 , an I559P mutation 43 , and an optimized furin cleavage site (RRRRRR) 44 . We designed the NFL Env construct, that carried the I559P mutation, by linking the gp120 and gp41 using a flexible glycine-serine linker (GGGGSGGGGS) 38 . Both Env constructs were human codon-optimized to enhance expression in mammalian systems. Download figure Open in new tab Figure 1: Design & biochemical characterization of 330 gp140 Env trimers and ferritin nanoparticles. Linear representation of three 330 Envelope variants (A) Design of SOSIP with the SOS-bond (A605-T605C), IP (I559P) stabilizing mutation along with TD8 mutations, a intraprotomer disulfide bond (I201C-A433C), an improved furin cleavage (R6) with CD5 Signal Peptide (SP) and Twin Strep-Tag (tst) tag (B) Design of NFL covalently linking gp120 to gp41 with flexible glycine serine (GGGGSGGGGS) linker (C) Design of H. pylori ferritin nanoparticle linked glycine serine linker to SOSIP construct (D) A surface structure highlighting the TD8 stabilizing mutations (right) in the 330 Envelope construct. Structure was illustrated using PyMOL and based on the BG505 SOSIP.664 crystal structure (PDB ID: 4ZMJ) (E) Size-exclusion chromatography (SEC) profiles of AIIMS 330 Env trimers (SOSIP and NFL) and Ferritin nanoparticles (NPs), eluted between 8–12 mL on a Superdex 200 Increase 10/300 GL column (F) Negative Stain-EM 2-D class averages of NFL & SOSIP gp140 Env trimers and ferritin nanoparticles (G) Guided by NS-TEM 2D class averages, four SOSIP gp140 Env trimers were manually placed onto the multivalent ferritin nanoparticle using ChimeraX to produce a 3D representation of the complex (H) Melting curve̴ around 74°C of AIIMS 330 Envelope The stabilized and optimized 330 Envs were expressed in HEK293F cells, followed by a two-step purification viz. HIV-1 Env trimer specific bNAb PGT145-Immunoaffinity chromatography followed by Size Exclusion Chromatography (SEC). The SEC purification of 330 Env trimers (SOSIP and NFL) yielded a peak at an elution volume of 10 mL, which shifted further upward for ferritin nanoparticles, eluting at 8.5 mL on a Superdex 200 10/300 column ( Figure 1E ). The purification yield of 330 Env trimers following SEC was around ∼1 mg/L for SOSIP and ∼1.45 mg/L for NFL (Figure S2), which was higher or comparable to the Indian and South African Clade-C strains (CZA97: ∼0.4-0.6 mg/mL, 329 SOSIP: ∼0.6 mg/mL, 16055 NFL: ∼2 mg/mL) 23 , 45 – 47 . The NFL trimer of Env 330 had better expression yields than the SOSIP version, likely due to the increased stability and functionality provided by the covalent linkage between the gp120 and gp41 subunits, resulting in more functional gp140 molecules 38 , 47 . However, low yield of Ferritin NPs was observed, around 0.05 mg/L, likely due to the complex assembly of Env trimers into synthesized ferritin nanocages as reported previously 48 , 49 . The 330 Env trimers displayed single band on Blue Native-PAGE (BN-PAGE) stained with Coomassie, whereas the 330 ferritin NPs were too large to efficiently enter the gels and visible at the top of the lanes, indicating the high purity of Env trimers and NPs preparations (Figure S2-Left). The SDS-PAGE analysis revealed intact trimeric nature of the 330 Env proteins under non-reducing conditions that separated as monomers in the presence of DTT under reducing conditions (Figure S2-Right). Negative-Stain Electron Microscopy (NS-EM) reference-free two-dimensional (2D) class averages demonstrated that over 99% of the gp140 proteins adopted a native-like trimeric morphology, with multiple copies of the Env spikes efficiently displayed on the self-assembling ferritin NPs ( Figure 1F , Supplementary S3). The 3D reconstruction of soluble gp140 trimers reveals a trilobular structure and while predicted model of ferritin NPs display multiple copies of Env spikes ( Figure 1G ). Thermal stability & secondary structures of 330 Envs were evaluated using sypro-orange Differential Scanning Fluorimetry (DSF) and by Circular Dichroism (CD) spectroscopy. The 330 SOSIP Env exhibited a Tm of ∼74°C indicating its ability to retain its structural integrity even at elevated temperatures ( Figure 1H , S4 A-B). The CD spectra of 330 SOSIP Env reveal ∼25% alpha-helical and ∼25% antiparallel beta-sheet content, contributing to the stability and rigidity of the Env, while random coils impart the flexibility required for molecular recognition, collectively suggesting a thermodynamically stable secondary structure for the Envelope protein (Figure S4 C-D). Overall, biophysical characterization demonstrated that the 330 Envs are well-stabilized, and effectively displayed on ferritin NP cages. Antigenicity of 330 Env trimers and ferritin nanoparticles indicated strong binding affinity to HIV-1 bNAbs Next, we assessed the antigenicity of the 330 Envs using binding ELISA with an extensive panel of HIV-1 bNAbs and non-nAbs. The 330 Env trimers exhibited well-exposed epitopes recognized by potent bNAbs, including the V1V2 apex (PGT145, PGDM1400, CAP256.25), the V3-glycan supersite (2G12, PGT121, 10-1074, AIIMS-P01), the CD4 binding site (VRC01, N6), and the gp120-gp41 interface (PGT151), while showing minimal reactivity to non-NAbs (F105, A32, 48d, 17b+sCD4), suggesting that the 330 Env trimers adopt native-like conformations ( Figure 2 ). The antigenic profile of ferritin NPs was found to resemble that of SOSIP, though with less saturation at the tested monoclonal antibodies(mAbs) concentration and allowing all the conformational epitopes to be accessible to HIV-1 neutralizing mAbs targeting to the Envs presented on the nanoparticle. A minor degree of binding of the non-NAbs was observed with NFL and ferritin particles, with negligible binding to SOSIP. Notably, the 330 Env trimers and particles exhibited preferential affinity for neutralizing mAbs over non-neutralizing mAbs. Download figure Open in new tab Figure 2: Antigenicity of 330 gp140 Env trimers and ferritin nanoparticles to HIV-1 bNAbs and non-NAbs. Representative AIIMS 330 Env binding curves with conformational specific bNAbs (PGT145, CAP256.25, PGDM1400, PGT151), CD4 specific bNAbs (VRC01, N6), V3 Supersite (10.1074, AIIMS P01) along with non-nAbs (17b+sCD4, F105, A32, 48d) assessed by ELISA. Subsequently, we assessed the binding affinity of 330 Envs to neutralizing & non-neutralizing mAbs with distinct specificities by BLI-Octet analysis. To assess binding affinity and kinetics, we selected conformation-specific bNAbs (PGT145, PGDM1400, and PGT151) as well as non-neutralizing antibodies (F105, 17b, and sCD4). The gp140 Envs (affinity) & ferritin NPs (avidity) exhibited strong binding affinity to the tested bNAbs ( Figure 3 ). The SOSIP variants (Ferritin NPs) (KD 1.07 nm) & NFL (KD 1.39 nm) showed a high affinity of ∼1nM to the V1V2 directed bNAb PGT145. A similar trend in binding of PGDM1400 with SOSIP (KD 1.34 nm) & NFL Env (KD 3.32 nm) was observed respectively. The interface-directed bNAb PGT151 demonstrated an exceptionally high affinity to SOSIP variants (Ferritin NPs), with an affinity of ∼5 pM, indicating that the PGT151 epitopes on the Env proteins displayed on the nanoparticle were highly accessible (Table S2). We observed that PGT145-purified SOSIP trimers were efficiently cleaved at the gp120-gp41 junction, as indicated by strong binding to the cleavage-specific bNAb PGT151, while no binding was detected with NFL trimer ( Figure 2 & 3). The introduction of intraprotomer locking 201C –433C disulfide bond as described previously 47 , in these constructs prevented the CD4-induced Env rearrangements as assessed by low reactivity with sCD4 & 17b non-nAbs, indicating the prefusion conformation of the Envelopes. As expected, and consistent with the ELISA binding results, the 330 Envs exhibited minimal reactivity to the non-NAbs F105 and 17b+sCD4 ( Figure 2 & 3, Table S2). Collectively, these findings suggest that both the soluble Envs and multivalent NPs adopt a native-like conformation, exposing multiple key bNAb epitopes while shielding the immunodominant epitopes targeted by non-NAbs. Based on the negligible antigenic reactivity of the SOSIP Env with non-nAbs, we next performed the structural analysis of the SOSIP Env. Download figure Open in new tab Figure 3: Octet sensograms kinetics of 330 Env to HIV-1 bNAbs and non-NAbs. (B) Octet Sensograms showing response shift (nm) over time (sec) of AIIMS 330 Env trimers and particles with varied concentrations (500 nM, 166.6 nM, 55.5 nM, 18.5 nM, 6.1 nM) against the quaternary specific bNAbs PGT145, PGD1400, cleavage dependent bNAb PGT151 and the non-nAbs F105 & 17b+sCD4. Cryo-EM structure of 330 SOSIP Env in complex with autologous bNAb 44m The structure of the natively glycosylated 330 SOSIP Env was solved using single-particle cryo-electron microscopy (cryo-EM). A total of 109004 particles were analyzed to determine the 2D class averages and reconstruct the trilobular 3D model of 330 SOSIP Env in its prefusion closed conformations (Figure S5 A-C). In a recent study, we reported heavy chain matured HIV-1 lineage bNAb 44m, which effectively neutralizes its autologous pseudovirus, 330.16.E6, from which the 330 Env trimer is derived 36 . The cryo EM structure was solved at 5 Å, to map the key interacting epitopes on the clade C Env 330 with its autologous bnAb 44m. The architecture of 330 Env displayed the trilobular shapes of trimers with three extra densities of corresponding bNAb 44m Fab bound moieties ( Figure 5 A-B , Supply S6). The model showed that three 44m Fab’s bind to each gp120 monomeric subunit, enabling effective neutralization ( Figure 5C-D ). The interacting surface area covered by the mainly by heavy chain of 44m and 330 Env are 690 Ų, and 613 Ų respectively ( Figure 5C-D ). The model further reveals that the 44m CDRH1 is positioned near the 330 Env V1V2 loop, while CDRH2 and CDRH3 are located near the V3 region ( Figure 5E ). The 330-SOSIP trimer-44m bNAb complex revealed interactions at the GDIR motif, N332 supersite along with additional contacts at E293, K337, K446 and glycans N326, N442, N448, as compared to our previously reported BG505–44m structure 36 . Within the GDIR motif of the 330 env, we found interactions of 44m with the R327 in addition to the D325 residue, while earlier solved cryo EM structure of the BG505-44m complex showed D325 dependence alone. The distinct key interacting residues identified in case of autologous Env 330 as compared to the heterologous clade-A Env BG505 include S30 in the CDRH1 loop, which forms a contact site with R327 in the V3 loop; Y52 and Y53 in the CDRH2 loop, which interact with R327, and H330 in the V3 region; R109 in the CDRH3 loop, which engages with E293 of C2 region, and K337 in the C3 region; and K446 in the C4 of gp120; R78 residues from FR3 loop interacts with D325 in the V3 region ( Figure 5E-F ). Therefore, both CDRH2 and FR3 loops interact with the GDIR motif in the V3 region of envelope. These interactions are primarily stabilized through electrostatic interactions, hydrogen bonding, and van der Waals forces. Epitope mapping of the 44m bNAb on its autologous Env revealed that the antibody engaged a peptide surface area of 1305.3 Ų and a glycan surface area of 788.65 Ų on gp120 (Figure S7). The electrostatic surface potential map of 44m showed the facing of E293, D325, R327, H330, N332, K337, and K446 residues towards the charged and hydrophilic regions of the 44m bnAb ( Figure 5G ). The glycan interaction at the 330 Env-44m Fab junction involves N301 and N332 from the V3 loop, and N397 in V4 loop, N442, and N448 from the C4 region ( Figure 5H ). Next, we performed structural superimposition of the 44m-BG505 atomic model into our atomic model (Figure S8). This analysis allowed us to investigate the binding modes of 44m 36 and other V3-glycan specific bNAbs 10-1074 50 , PGT121 51 , 438-B11 52 , BG18 53 , mature BG18 54 , DH270.6 55 , and BF520.1 56 to a 330 Env superimposed BG505 model. Our findings suggest that 44m interacts with the V3 loop via a slightly declining shift, similar to BF520 and BG18, but exhibits a distinct binding mode compared to PGT121 and 35O22 (Figure S8). Structural and glycan homology of 330 Env to BG505 To assess structural similarity, The HIV-1 Clade C 330 SOSIP trimer was superimposed onto a clade C Env 16055 NFL 57 (PDB: 5UM8) and a Clade-A Env BG505 SOSIP 58 (PDB: 4TVP). We observed that the 330 Env trimer exhibited greater structural similarity to BG505 Env, with lower root-mean-square deviations (RMSDs) of 0.685 Å for gp120 and 0.478 Å for gp41 compared to modelled 16055 Env, which had RMSDs of 0.798 Å for gp120 and 0.739 Å for gp41 ( Figure 4A-D ). Upon glycan prediction analysis by Los Alamos HIV-1 sequence database (LANGT), we observed that 330 Env lacked conserved PNGS at positions 295 and 339 in the V3/C4 region and at position 130 in the V1V2 apex, in contrast to BG505 Env, which lacks PNGS at positions 241 and 289 ( Figure 4E ). Next, we calculated the total glycan hole area in the 330 Env using Los Alamos glycan shield mapping tool (LAGSM), suggesting that the optimal protein accessible area was 967 Ų, compared to BG505, B41, C9Z7A & Du422 counterparts ( Figure 4F ). The potential N-glycosylation sites (PNGS) conserved in the 330 Env were determined by Los Alamos HIV-1 sequence database (LANGT) using N-Glycosite tool. Conserved PNGS analysis revealed that 330 Env comprises of 28 PNGS comparable to that reported in the BG505 59 & 16055 60 Envs ( Figure 4F , Table S3). Targeting exposed protein surfaces within glycan holes can be an effective strategy to boost the immune response against these vulnerable sites. Overall, our structural homology modelling and glycan analysis demonstrate that the stabilized clade-C Env 330 shares numerous features with the well-studied BG505 Env and suggesting its capacity to adopt well-ordered structures. Download figure Open in new tab Figure 4: Structural and glycan homology of 330 SOSIP with BG505 & 16055 trimers (A-B) Atomic model comparison of 330 SOSIP Env trimer with BG505 SOSIP (PDB: 4TVP) (RMSD gp120: 0.685 A⁰, gp41: 0.478 A⁰) (C-D) 16055 NFL (PDB: 5UM8) (RMSD gp120: 0.798 A⁰, gp41: 0.739 A⁰) SOSIP trimer. Colour coding of SOSIP trimers corresponding to segmented EM densities are: dodger blue, AIIMS 330 SOSIP, deep pink, BG505 SOSIP and 16055. Within each protomer, gp120 regions are highlighted in blue and pink, while gp41 regions are shown in yellow and green (E) Presence of glycan hole in 330 Env at position 130, 295, 339 in HXB2 reference coordinates showed in top, front & side view (F) Figure depicting the total protein accessible area across cross-clade Envelopes (G) Presence of total 28 PNGS in AIIMS 330 & BG505 Envelope. An N indicates the presence of a PNGS, if NXT/S motif in a.a sequence whereas X should not be proline. The glycan structure and alignment created by N-Glycosite tool from Los Alamos HIV-1 sequence database. Download figure Open in new tab Figure 5: Cryo-EM structure of 330 SOSIP Env trimer in complex with autologous bNAb AIIMS 44m (A) Top and side views of Cryo-EM reconstructed map of AIIMS 330 SOSIP Env trimer in complex with 44m bNAb. Colour coding corresponding to segmented EM densities are: deep pink, 44m bNAb; dodger blue, gp120; golden rod, gp41 (B) The corresponding atomic model fitted in the EM map (in gray) of AIIMS 330 SOSIP trimer in complex with 44m bNAb. The model illustrates that three 44m bNAbs bind to each gp120 monomeric subunit, facilitating effective neutralization (C) Atomic model of AIIMS 330 SOSIP trimer in complex with three 44m bNAbs. (D) Atomic model of one protomer of gp120 binding to one 44m bnAb (E) Location of CDRH and FR3 regions are shown here in different colours (CDRH1-cyan, CDRH2-deep green, CDRH3-light green, FR3-light brown) (F) Key interacting residues AIIMS 44m bNAb – S30, Y52, Y53, R78 and R109 highlighted in heteroatom colour, and corresponding interacting residues of gp120 loops – E293, D325, R327, H330, N332, K337 and K446 highlighted in heteroatom colour (G) Electrostatic surface potential map of 44m bnAb showing interaction of residues from gp120 loops with charged portion of 44m bnAb (H) The N-glycans N301, N332, N397, N442, and N448 located at the junction of AIIMS 44m bNAb and AIIMS 330 gp120 protomers. 330 Ferritin nanoparticles and SOSIP trimer exhibit enhanced immunogenicity compared to NFL in rabbits Following the establishment of native-like prefusion-closed Env structures, we further evaluated and compared the immunogenicity of soluble Env trimers (SOSIP & NFL) and multivalent ferritin NPs in New Zealand White (NZW) rabbits. Four groups of female rabbits were immunized, primed at week 0, and boosted at weeks 4 and 20 and administered a dose of 20 µg of 330 gp140 Env trimers or an equimolar amount (25 µg) of ferritin nanoparticles, formulated with AddaVax™ adjuvant ( Figure 6A ). The AddaVax™ adjuvant is a squalene-based oil-in-water nano-emulsion and has been shown not to impact the structural integrity or antigenicity of the Env trimers, as demonstrated in previous studies 61 , 62 . Rabbits were bled at weeks 0, 4, 6, 16, 20, and 22 and sera samples were subsequently evaluated for binding reactivity with the Env trimer using capture ELISA and for neutralizing activity against Env pseudotyped viruses. First, we evaluated the binding titers of the collected immune sera from different timepoints against the immunogens tested and measured as median titers across each group. The binding titers of immune sera exhibited a significant increase between 4 to 8 weeks and then again between 20 to 22 weeks, immediately after the second and third boosters ( Figure 6B ). The polyclonal immune sera demonstrated sustained binding reactivity to the autologous Envelope trimer and with the cross-clade Envelopes, though with a lower extent to heterologous Envs. The half-lives ( t 1/2) of the immune sera titers against the autologous Env were significantly longer (p value: 0.007) as compared to that of the heterologous Envs (SF162, 6240, 1086) tested. The overall immune sera binding titers were comparable across the test groups; however, rabbits immunized with 330 SOSIP and ferritin NPs exhibited statistically significantly antibody titers (p value: 0.01) compared to those immunized with NFL counterparts. Peak serum antibody binding to 330 Env was observed at week 22, prompting further analysis of week 22 serum samples for their neutralization activity. Download figure Open in new tab Figure 6: Immunogenicity & Neutralizing potential of 330 SOSIP, NFL Env trimers & Ferritin NP’s in NZW rabbits. (A) Rabbit immunization Schedule. Four groups of rabbits were immunized at week 0 (prime), 4 & 20 (boost), gp140 SOSIP and NFL Env immunogens (G1 and G2) were administered at a dose of 20 µg of trimers, or an equivalent molar amount SOSIP-ferritin nanoparticles (25 µg) (G3) formulated with the AddaVax adjuvant (1:1 v/v). Sera was obtained at indicated intervals of week 4, 6, 8, 12, 16, 20 & 22 (red arrows). (B) Binding endpoint titers (OD at A450) over time against autologous virus (330.16.E6) Dots and error bars represent the median binding titers and standard deviations. The assay cut-off is marked with a dotted line (C) Midpoint neutralization titers (ID 50 ) for week 22 sera of the immunized rabbits against autologous (330.16.E6) and closely related (329.14.B1), and heterologous clade C (MW965.26 and 25925) and Tier 1 clade B (SF162) pseudoviruses. Median ID50 titers NFL, SOSIP & Ferritin NP group was 375, 830 & 1160. Statistical analysis was done using an unpaired two-tailed Student’s t-test and the assay cut-off is marked with a dotted line. Neutralization breadth was further evaluated against autologous (330.16.E6), closely related (329.14.B1), and heterologous clade C (MW965.26 and 25925), Tier 1 clade B (SF162), and Tier 2 clade A (BG505.T332N) pseudoviruses. No immune sera response was detected against murine leukemia virus (MLV) used as a negative control and all animals exhibited neutralizing antibody responses following immunization, except for one in the NFL group (NZW5273) (Table S4). Multivalent 330 ferritin NPs and SOSIP elicited a strong neutralizing response against autologous (330.16.E6) and closely related Envs (329.14.B1) as compared to NFL counterpart. The Ferritin NP group elicited the most robust autologous neutralizing antibody response, with a median ID 50 titer of 1160 compared to the soluble gp140 trimer groups (SOSIP and NFL) with median ID 50 titers of 830 and 375, respectively ( Figure 6C ). The Ferritin NP group demonstrated a statistically significant increase in neutralizing antibody titers compared to both the SOSIP (p = 0.014691) and NFL (p = 0.010323) groups, with fold differences of 2.5-fold and 3.5-fold, respectively. These findings were consistent with the results from the capture ELISA, where the NP and SOSIP groups exhibited sustained binding reactivity, whereas the NFL group showed a decline in response after boosters. The NP and SOSIP groups demonstrated moderate heterologous neutralizing responses against the clade C Tier 1 viruses (MW965.26 and 25925) and the Tier 1 clade B virus (SF162), with responses comparable to each other and higher than the NFL group although the differences were not statistically significant. None of the 330 immunogens induced neutralizing responses against the Tier 2 BG505 virus, which aligns with the low BG505 nAb activity observed in the plasma of the AIIMS_330 HIV-1-infected elite neutralizer 35 . Overall, we observed that both 330 SOSIP Env trimer and ferritin NPs elicited strong autologous NAb responses, as well as cross-clade neutralization, though with a limited magnitude. Notably, the presentation of these antigens on ferritin NPs were associated with a higher trend in NAb response induction. DISCUSSION HIV-1 clade-C accounts for more than half of global HIV-1 infections, with particularly high prevalence in South Africa and India 19 , 20 . Comparative analyses of clade-C viruses from these regions reveal the significant differences in their sequences, highlighting distinct evolutionary trajectories 63 . These regional variations in intra-clade-C Env sequences likely contribute to the differential sensitivity of circulating strains to HIV-1 nAbs. HIV-1 Clade-C characterized by unique features such as enhanced transmission efficiency, extensive genetic diversity within the V1-V3 region of the Env gene, and a distinct profile of drug resistance mutations compared to other clades 63 – 66 . Understanding these differences is crucial for tailoring the prevention and treatment strategies in clade C prevalent regions 19 , 20 . Structural information on the HIV-1 Env trimer from clade-C is crucial for structure-guided immunogen design. However, only a limited number of high-resolution structures are available for native-like clade-C Env trimers, such as 16055 47 , 67 , DU172.17 68 , 426c 69 , and CZA97.12 70 . Although many HIV-1 Env immunogens have been stabilized, most studies have extensively characterized BG505, a Clade-A transmitted founder Env derived from a 6-week-old infant with elite neutralizing activity 11 . Such elite individuals from the pediatric group could provide valuable Env immunogen templates, as HIV-1 infection and disease progression in children differs from that in adults 31 , 34 , 35 . Notably, neutralizing antibodies tend to arise early in the course of infection in pediatric individuals, with the ability to neutralize contemporaneous viral Envelopes 33 , 37 . We previously reported a pair of monozygotic twins (AIIMS_329 and AIIMS_330) with elite neutralizing activity, whose plasma demonstrated exceptionally potent bNAb responses with multi-epitope specificities against a diverse panel of multi-clade heterologous Env pseudoviruses 33 , 35 . These Envs hold significant potential as templates for vaccine design and as antigenic baits to isolate HIV-1 bNAbs for immunotherapeutic applications 46 . Recent studies demonstrated that, multimeric presentation of viral class I fusion viral glycoproteins, such as, Ebola virus Glycoprotein (GP) 71 , 72 , influenza hemagglutinin (HA) 73 , 74 , Respiratory Syncytial Virus (RSV) F protein 75 , 76 , SARS-CoV-2 S protein 77 – 80 and HIV-1 Env 29 , 30 on ferritin NPs have enabled efficient production of well-ordered NP-based vaccine candidates with a capacity to induce robust immune response. Here we report a soluble HIV-1C 330 native-like Env trimer on SOSIP and NFL platform, derived from a circulating virus in an Indian pediatric EN AIIMS_330 35 , by stabilizing the Env using version 7 mutations and displaying the SOSIP Env on self-assembling ferritin NPs 29 , 30 . The purification yield of Env trimers post SEC was around ∼1 mg/L for SOSIP and ∼1.45 mg/L for NFL, which was higher or comparable to the Indian and South African Clade-C strains (CZA97: ∼0.4-0.6 mg/mL, 329 SOSIP: ∼0.6 mg/mL, 16055 NFL: ∼2 mg/mL, 1PGE-THIVC SOSIP) 23 , 45 – 47 . Using Negative stain Electron Microscopy, we demonstrated that SOSIP and NFL Envs displaying key features of native-like trimeric conformation and Env trimers were presented efficiently on ferritin NPs as observed in stabilized BG505 Env-particles 29 . The structure of the 330 SOSIP, resolved by single-particle cryo-EM, revealed trilobular Env structures in their prefusion, closed conformation. The generated Env trimer and particles displays a desirable antigenic profile, with nanomolar affinity to conformation specific bNAbs (PGT145 & PGT151), with negligible reactivity to non-nAbs (F105 & 17b+sCD4), shielding the Env immunodominant regions. Biophysical characterization showed that the 330 Env exhibits a balanced thermodynamic profile and high degree of thermal stability with a Tm of ∼74°C which is notably higher as compared to other reported clade C Envs ZM197 (69°C) 12 , IPGE-THIVC (62°C) 23 and 16055 (68°C) 81 . We observed greater structural similarity of 330 Env to clade-A BG505 Env (RMSD gp120-0.685 Å & gp41-0.478 Å) compared to clade-B AMC009 Env reported (RMSD gp120-2.623 Å & gp41-1.624 Å), suggesting well-ordered structures 14 . Cryo-EM structure of 330 Env in complex with autologous bNAb 44m at 5 Å resolution revealed the well-exposed epitopes; the three trilobular structures occupied by the three Fab arms. We identified herein, additional key antigenic determinants E293, K337, K446 in the autologous 330 Env, that interacted with 44m; in addition to D325, R327 and N332 dependency, as observed with heterologous BG505 Env 36 . Further, the additional interaction within the GDIR motif, of the R327 along with the D325 residue as observed in the solved structure of 330 with 44m, plausibly contributed to the potent neutralization of the autologous virus 330.16.E6. Our findings offer crucial insights into the antigenic determinants in the clade C virus Env, that interact with the autologous antibody 44m, that can inform a clade-based vaccine design. Furthermore, using a reverse vaccinology approach, these insights can guide the structure-based engineering of neutralizing antibodies 82 . Rabbit immunizations demonstrated that the 330 Env immunogens elicited robust immune responses against autologous and other tier-1 viruses tested, with limited cross-clade neutralization. This may be attributed to distinct antigenic determinants on heterologous Envs, adjuvant used or by varied glycan compositions 83 , 84 . Further insight can be achieved by investigating the specific epitopes targeted by NAbs in the immune sera, however it has not been addressed herein and is a limitation of this study. The multivalent presentation of HIV-1 antigens on ferritin NPs correlated with enhanced neutralization potency, likely attributed to augmented B-cell activation signals 85 . This observation aligns with findings from germline-targeting eOD-GT8 NPs, suggesting a potential mechanism for eliciting more robust and coordinated immune responses 85 . These findings emphasize the need for further exploration of the role of glycans and structure guided mutations in shaping immunogenicity. In summary, we designed and characterized soluble HIV-1C Env trimers derived from a pediatric elite-neutralizer from India. Structural analysis of the 330 Env trimer in complex with autologous bNAb 44m, a heavy chain matured version of the first bNAb AIIMS_PO1, identified from the AIIMS_330 donor, revealed molecular features of 330 Env-44m bNAb interaction which could inform structure-based vaccine design. Our findings indicate that the HIV-1C 330 SOSIP Env trimer can serve as a valuable antigenic probe for bNAb discovery and is a promising candidate for multivalent HIV-1 vaccine design and development. MATERIALS AND METHODS Institutional Review Board & Ethics approval This study was conducted after obtaining approval from the institutional ethics committee, All India Institute of Medical Sciences (AIIMS), New Delhi, India (250/IAEC-1/2020). Rabbit immunization was outsourced to a contract research organization (CRO) Liveon Biolabs Private Limited, Bengaluru, Karnataka, India with IAEC (IAEC-approved protocol No.: LBPL-IAEC-008-01/2021; study number: LBPL/NG-1736 (EF)). The immunization studies described here were carried out on female naive New Zealand White rabbits of 2.0–2.5 kg and aged 4 months. All animal experiments adhered to the guidelines for the care and use of laboratory animals as outlined by the Committee for Control and Supervision of Experiments on Animals (CCSEA), Government of India 86 . Construct design The 330 Env gene was derived from a previously identified Indian clade C HIV-1 Env (330.16.E6, GenBank: MK076720 ) obtained from a pediatric elite-neutralizer AIIMS_330, as previously described 35 . The 330 Env construct were design in soluble forms as SOSIP.664 11 gp120-gp41 cleaved Env trimers and NFL.664 38 gp140 single-chain Env trimers along with tailoring of SOSIP Env trimers on self-assembling ferritin nanoparticles 29 to achieve multivalency. SOSIP & NFL constructs were stabilize by incorporating I559P & ΔMPER (Env prefusion stabilizing mutation) 43 , 87 , R315Q (reduced V3 non-nAb immunodominant exposure) 88 , I201C-A433C (intraprotomer locking disulfide bond, reduced V3 exposure) 12 , and clade C stabilizing B505-Trimer Derived (TD8) mutations (E47D, T49E, V65K, I165L, K429R, K432Q, K500R) 39 . The SOSIP construct was designed by introducing an SOS disulfide bond (A501C-T605C) 42 , an I559P mutation 43 , and an optimized furin cleavage site (RRRRRR) 44 . In contrast, the NFL (Native Flexible Linker) construct retained the I559P mutation and linked gp120 and gp41 using a flexible glycine-serine linker (GGGGSGGGGS) 38 . Native signal sequence is replaced by the CD5 signal peptide (MGSLQPLATLYLLGMLVASVLA) for enhanced secretion of Envelope proteins, constructs were codon optimized for mammalian expression and synthesized commercially by GeneArt (Thermo Fischer Scientific). For binding assays, 330 Env constructs were tagged with either a His-tag (GSGSGGSGHHHHHHHH) or a twin strep tag (tst) (WSHPQFEKGGGSGGGSWSHPQFEK) at the C-terminus of gp41ECTO, preceding the stop codon. These constructs were subsequently cloned into pPI4 expression vector using Pst1 and Not1 restriction sites. The 330 ferritin NP’s construct was generated by fusing the N-terminus from Helicobacter pylori ferritin (GenBank: WP_000949190.1), starting from Asp5 to Ser167, to the SOSIP.664 C-terminus (truncated at position 664), separated by a Gly-Ser ( GS G) linker, as described previously 29 . Envelope Protein Expression The 330 Env encoding plasmids were transiently expressed in 293F suspension cells (Gibco, R790-07). 293F cells were thawed and maintained in Freestyle Expression Medium (Gibco, K9000-01) in a Shaker incubator at 37°C, with 120 r.p.m. and 8% CO2. For transfection, HIV-1 Env and furin protease encoding plasmids mixed in a 3:1 ratio (w/w) with NFL (500 µg), SOSIP Env (333.3 µg), to furin (166.6 µg) incubated with transfection reagent PEImax (Polysciences, 24765) in a 3:1 ratio (w/ w) PEI max to DNA for 20 min at room temperature (RT) and then added to 1L culture at density of 1 million cells/mL. Seven days post-transfection, the supernatants were harvested, centrifuged, and filtered using 0.45-mm filters (Nalgene, 2954545). Envelope Protein Purification 330 SOSIP Env and SOSIP Env-NP fusion proteins were purified by PGT145 bNAb immunoaffinity chromatography, as described previously 45 . Briefly, HIV-1 pre-fusion closed trimer-specific bNAb PGT145 89 was covalently coupled to the CNBr-activated Sepharose 4B beads (GE Healthcare, 17043001) according to the manufacturer’s protocol for antibody coupling. The PGT145-coupled CNBr Sepharose 4B bead column was used to purify HIV-1 Env trimers or Env trimer NPs. Env proteins contained in Expi293F filtered supernatants were captured on PGT145-coupled CNBr-activated sepharose beads by overnight rolling incubation at 4 °C. Subsequently, the mixture of supernatants and beads was passed over Econo-columns (Biorad, 7371512). The columns were then washed with three column volumes of 0.5 M NaCl and 20 mM Tris HCl pH 8.0 solution. After elution with 3 M MgCl 2 pH 7.5, the proteins were buffer-exchanged into TN75 (75 mM NaCl, 20 mM Tris HCl pH 8.0) or PBS buffer by ultrafiltration with Vivaspin concentrators MWCO 100 kDa (Cytivia, 28932363). Protein concentrations were determined from the A280 values measured using a NanoDrop One (Thermo Fisher Scientific, 29018226) and the molecular weight and extinction coefficient values were calculated by the ProtParam Expasy webtool. Env proteins used for immunization were first purified using PGT145 affinity chromatography, followed by size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (28990944, GE Healthcare Life Sciences) using an Äkta Pure 25M system (Cytiva, 29018226). Envs containing fractions were pooled, concentrated, filter-sterilized, snap-frozen in liquid nitrogen, and stored at –80°C until further use. SDS-PAGE and BN-PAGE Analyses For SDS-PAGE and blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses, 2 µg of 330 gp140 Env trimers, or equimolar amounts of ferritin NP’s (2.5 µg) was ran over precast native gel 4–20 % Bis-Tris gradient gel (Biorad, 4561094). The degree gp120-gp41 cleavage was examined by incubating the SOSIP & NFL protein with 0.1M dithiothreitol (DTT) and analysed by SDS-PAGE under reducing conditions. Subsequently, gels were stained with the Colloidal Blue Staining Kit (Invitrogen, LC6025), respectively. Enzyme linked Immunosorbent assay (ELISA) ELISAs were performed as previously described 90 using tst-tagged Env constructs on StrepTactinXT-coated microplates (IBA Lifesciences, 2-4101-001). Purified proteins (1 μg/mL) were diluted in NaHCO3 buffer and immobilized on 96-well Streptactin plates by O/N incubation at 4°C. Following a three wash step with PBST (0.05% Tween20) to remove unbound trimers, plates were blocked by 1% BSA in PBST for 2 hours at RT. Further the serial dilutions of test antibodies ranging from 10ug – 0.004 ug/ml in PBST/1% BSA solution were added and incubated for 2 h. After 3 washes with PBST, HRP-labelled goat anti-human IgG (Jackson ImmunoResearch, AB_2337578) diluted 1:3000 in PBST/1% BSA was added and incubated for 30 mins, followed by 3 washes with PBST and 3 washes of PBS. A developing solution (1% TMB (Mabtech, 3652-F10), allowed the colorimetric reaction, which was stopped by the addition of 2 N H2SO4. Finally, colour development (absorption at 450 nm, OD450) was measured at NanoDrop One (Thermo Fisher Scientific, 29018226) to obtain the different binding curves. Biolayer Interferometry (BLI) Binding kinetics were measured using an Octet K2 (ForteBio, RED96e) device at RT with 1000 rpm agitation. Anti-human Fc sensors (Sartorius AHC, 18-5060) were used to capture mAbs on the Octet K2, were equilibrated in kinetics buffer for 60 s to obtain a baseline prior to protein association. 330 Envelope protein was used as analyte in concentrations ranging from 210 to 2.6 nM in the HEPES buffer (pH 7.2) background supplemented with 0.02% Tween 20 and 0.1% BSA. Binding was assessed by incubating mAb-captured sensors in wells containing 10 μg/ml Env for 120 seconds with 1000 rpm agitation. Association was recorded for 150 s followed by dissociation for 450 s. Data were analysed using the ForteBio Data analysis software (Forte-Bio Inc, Version 9.0) and a global fit was performed using a 1:1 binding model to fit the association and dissociation curves. Sypro orange differential scanning fluorimetry (DSF) 330 SOSIP protein were prepared at a concentration of 0.2 mg/mL in 1XPBS and transferred into 0.1 mL polypropylene PCR tube strips (Tarsons, 611020) with a final volume of 25 μL. Sypro Orange protein stain (Invitrogen, 4461146) was added at a 1:200 dilution. The samples were analyzed using a Rotor-Gene Q system (Qiagen, 9001862) with a high-resolution melt (HRM) protocol, starting at 25°C and increasing to 90°C in 1°C increments, with each step lasting 4 seconds. Melt curve data were processed using Rotor-Gene Q Series Software. Circular dichroism (CD) spectroscopy The Far-UV spectrum was recorded in the wavelength range of 200–250 nm using a CD Spectrophotometer (JASCO, J-1500) with a 0.1 cm path length Quartz cuvette (Sigma Aldrich, I0285). Measurements were conducted for the 330 protein in a 20 mM phosphate buffer (pH 7.5) with100 mM NaCl concentration at 4°C for 30 minutes. Each spectral scan was averaged over 2–3 measurements. The CD spectra were visualized using SigmaPlot 10.0, and the secondary structure was analyzed through the BESTSEL online server. For thermal studies, measurements were performed between 20°C and 80°C using the same Quartz cuvette, and the melting curve was generated using SigmaPlot 10.0. Negative Stain Microscopy (NS-EM) Negative-stain electron microscopy (NS-EM) was performed essentially as described elsewhere 91 . Briefly, 3 μl of purified 330 gp140 Env trimers or ferritin NPs (∼0.03 mg/mL) were applied to glow discharged (20 mA for 30 s) carbon-coated 400 Cu mesh grid. After 5 s, grids were negatively stained with 2% (w/v) uranyl formate for 60 s. Imaging was performed using a 120 kV Talos L120C electron microscope (TALOSL120CMS) equipped with a 4k x 4k Ceta camera at a magnification of 73kx (3.84 Å/pixel). The collected images were processed in EMAN 2.1 92 . Particle extraction and reference-free 2D class averages were generated using simple_prime2D in SIMPLE 2.1 software 93 with a mask diameter of 30 pixels at 3.84 Å/pix. Preparation of 44m Fab fragment Fab fragments were generated from 4 mg of AIIMS-44m IgG using a Fab fragmentation kit (G Biosciences, 786-273) following the manufacturer’s protocol. Briefly, 0.5 ml of 44m IgG was desalted using a SpinOUT™ GT-600 column. Cysteine.HCl was added to the Fab Digestion Buffer (pH) to a final concentration of 20 mM before papain digestion. The 0.5 ml IgG sample was then incubated with Immobilized Papain in the spin column at 37°C for 6 hours with end-over-end mixing. After incubation, the digested antibody solution was collected by centrifugation at 5,000 x g for 1 minute. The flow-through was further incubated with Protein A resin for 10-15 minutes at room temperature (RT) with end-over-end mixing. Subsequently, the Fab fragments were collected in the flow-through while Fc fragments and undigested IgG remained bound to the resin. The purity and size of the Fab fragments were assessed by SDS-PAGE and the purified Fab fragments were stored at –80°C. Cryo-EM sample preparation, data acquisition, data analysis and model fitting 330 Env trimer (1.5 mg/ml) incubated with 44m Fab at 1:3 molar ratio. 3 μl of sample were applied to glow discharged R1.2/R1.3 300 mesh copper grids and immediately blotted for 6 secs with 5 seconds of blotting force using FEI Vitrobot Mark IV plunger at 22°C and 100% humidity. The grid was immediately plunge-frozen into liquid ethane at liquid nitrogen temperature. Cryo-EM data were collected using 200 kV Talos Arctica transmission electron microscope (Thermo Scientific, TALOSARCTICA) equipped with Gatan K2 Summit Direct Electron Detector. Movies were recorded automatically using Latitude-S (DigitalMicrograph – GMS 3.5) at a nominal magnification of 45,000x at the effective pixel size of 1.17 Å 93 . Micrographs were acquired in counting mode with a total dose of 40 e - /Å 2 , with an exposure time of 8 sec distributed for 20 frames. A total of 6046 movies were acquired for the 330 SOSIP Env trimer and 44m Fab complexes, respectively. Single-Particle Analysis (SPA) was performed for the acquired Cryo-EM movies using the cryoSPARC 4.5 94 . At first, drift and gain corrections of the individual movies were performed with Patch Motion Correction and estimated Contrast transfer function (CTF) parameters using Patch Ctf estimation. Subsequently, CTF-estimated micrographs were subjected to analysis to eliminate poor-resolution micrographs with a fit resolution threshold of 8 Å using curate exposure. The particles from the best micrographs were chosen for automated picking using 2D reference in Relion and extracted with the box sizes of 320 pixels for the 44m Fab bound 330 SOSIP Env trimer complexes. The extracted particles were imported to CryoSPARC v4.5, and the rest of the processing was performed there. After four rounds of rigorous 2D classification, 363,761 particles were selected from the best 2D classes with high-resolution features of the Ab-bound complex. This dataset was divided into 4 classes for Ab initio reconstruction with C3 symmetry. The class 04, with 118572 particles, had the best Ab-bound features among all the classes (Figure S6). Therefore, we considered class 04 for non-uniform refinement by imposing C3 symmetry. The resolution of the 3D map was estimated at FSC 0.143. The local resolution estimation tool in cryoSPARC was used to determine local resolution. The Alphafold model of AIIMS 330 env and 44m bnAb were used for atomic model building using Phenix1.19.2 95 . For model fitting, the Cryo-EM maps of 330 Env trimers were docked on ferritin structure (PDB: 3BVE) symmetrically on four sites. Overviews of Cryo-EM data processing are shown in S. Table S4. The atomic model fitting was done and visualized using ChimeraX 96 . Immunizations The rabbit immunization studies were conducted at a contract research organization (CRO), Liveon Biolabs Private Limited, Karnataka, India, under GLP-compliant conditions. Liveon Biolabs is an AAALAC International-accredited facility and is registered with the CCSEA, Government of India. Female, naive New Zealand White rabbits, aged 4 months and weighing between 2.0–2.5 kg, were used for the study. The protocol for animal use was approved by the Institutional Animal Ethics Committee (IAEC) of Liveon Biolabs (Approval No.: LBPL-IAEC-008-01/2021; Study No.: LBPL/NG-1736 (EF)). Prior to housing, the experimental room underwent fumigation for decontamination, and microbial load was assessed using the settle plate method. The rabbits were kept in a temperature-controlled Environment (20 ± 3 °C) with relative humidity ranging from 30–70%, a 12-hour light/dark cycle, 12–15 air changes per hour, and a noise level below 80 dBs. Mortality and morbidity were monitored twice daily, while clinical observations were conducted at least once per day throughout the study. Body weight measurements were recorded for all animals prior to the administration of the test item on Day 1 and subsequently on a weekly basis (±1 day) during the treatment period. The gp140 SOSIP and NFL Env immunogens (G1 and G2) were administered at a dose of 20 µg of trimers, or an equivalent molar amount SOSIP-ferritin nanoparticles (25 µg) (G3) formulated with the AddaVax adjuvant (1:1 v/v) (vac-adx-10, Invivogen). The placebo group (G4) received only adjuvant with sterile 1XPBS. Doses were calculated based on the molecular weight of the protein peptides, excluding glycans, as described elsewhere 60 , 60 . Blood samples were collected from rabbits at weeks 0, 4, 6, 16, 20, and 22. Before blood collection from the marginal ear vein, a local anaesthetic cream containing 4% lidocaine was applied for 5 minutes. The immunization process, including bleeding protocols, caused minimal and brief pain or distress, with no significant impact on animal health. At the end of the experimental period, the rabbits were euthanized using a lethal dose of sodium thiopental. Complement-deactivated serum samples were subsequently analysed for their binding reactivity and neutralization potential. HIV-1 pseudovirus generation The HIV-1 pseudoviruses were produced in HEK293T cells as described previously 97 by co-transfecting the corresponding full HIV-1 gp160 Envelope plasmid and a pSG3ΔEnv backbone plasmid. Briefly, 1 × 10 5 cells in 2 mL complete DMEM (10% fetal bovine serum (FBS) (Gibco, 26140079 and 1% penicillin and streptomycin (Gibco, 15140-122) were seeded per well of a 6-well cell culture plate (Costar, 3506) the day before transfection. For transfection, Envelope (1.25 μg) and delta Envelope (pSG3ΔEnv backbone) plasmids (2.50 μg) were mixed in a 1:2 ratio in Opti-MEM (Gibco, 31985062), with a final volume of 200 μL per well, and incubated for 5 min at room temperature. Next, 3 μL of PEI-Max transfection reagent (Polysciences, 24765) (1 mg/mL) was added to this mixture before further incubation for 15 min at room temperature. This mixture was then added dropwise to the HEK 293T cells supplemented with fresh complete DMEM growth media and incubated at 37 °C for 48 h. Pseudoviruses were then harvested by filtering cell supernatants with 0.45 μm sterile filters (mdi), aliquoted, and stored at −80 °C until usage. HIV-1 Neutralization assay Neutralization assays were carried out using TZM-bl reporter cells which is described elsewhere 98 . The autologous and heterologous NAb response of end point sera was measured in TZM-bl cell neutralization assays. Neutralization was measured as a reduction in luciferase gene expression after a single round of infection of TZM-bl cells (NIH AIDS Reagent Program) with HIV-1 Env pseudoviruses. The TCID 50 of the HIV-1 pseudoviruses was calculated and 200 TCID 50 of the virus was used in the neutralization assays through incubation with three-fold serially diluted rabbit sera starting at a 1:20 dilution. Next, freshly trypsinized TZM-bl cells in a complete DMEM (10% fetal bovine serum (FBS) (Gibco, 26140079 and 1% penicillin and streptomycin (Gibco, 15140-122) containing 50 μg/mL DEAE-dextran (Sigma, D9885) at 10 5 cells/well were added and the plates were incubated at 37 °C for 48 h. Virus controls (cells with HIV-1 virus only) and cell controls (cells without virus and antibody) were included. MuLV was used as a negative control. After the incubation of the plates for 48 h, luciferase activity was measured using the Bright-Glow Luciferase Assay System (Promega, E2610). ID 50 for antibodies were calculated from a dose–response curve fit with a non-linear function using GraphPad Prism 8 software (San Diego, CA, USA). All neutralization assays were repeated at least 2 times, and the data shown are from representative experiments. Statistical analysis Statistical significance between groups and subgroups was assessed using the Mann–Whitney U test, Student’s t-test, and one-way analysis of variance for continuous variables. For representative results, experiments was done in replicates or repeated twice. All statistical analyses and plotting were performed using GraphPad Prism (version 8.0; La Jolla, CA, USA) FUNDING This study was funded by the Department of Biotechnology (DBT), India (BT/PR39156/DRUG/134/91/2021, BT/PR30120/MED/29/1339/2018) grants awarded to K.L. This work was also supported by Department of Science & Technology-FIST (SR/FST/LSII-039/2015) and DBT-BUILDER Program (BT/INF/22/SP22844/2017) grants awarded to S.D. S.K. is supported by the DBT-Wellcome Trust India Alliance Early Career Fellowship Grant IA/E/18/1/504307. The funders played no role in the study design, data collection, analysis, or interpretation, nor in the writing of the manuscript or the decision to publish the results. AUTHOR CONTRIBUTIONS Conceptualization and implementation; S.D.S., S.Ku, and K.L., EM work & data acquisition; S.D.S., A.C., Formal analysis; S.D.S., S.Ku, K.L., S.D., Biophysical analysis; A.C., S.D.S., L.M., B.M., E.A.S., Expression and purifications: S.D.S., M.P., A.R., I.A., S.S., V.I., A.G., T.B., & R.Ku., ELISA & neutralization assays; S.D.S., S.S, A.R., S.Ka, A.W.H., Animal studies; S.D.S., S.Ka, H.B., A.G., & A.W.H. Octet experimentation; S.D.S., R.Ku., S.A., and M.P., Original manuscript writing; S.D.S., S.Ku, A.C., & K.L., Supervision; S.D., & K.L., Project administration; S.D.S. & S.Ku, funding acquisition; S.D., & K.L. All authors have read and agreed to the published version of the manuscript. CONFLICT OF INTERESTS An Indian patent application (202211004612) has been filed on the 330 SOSIP Env trimer described in this study. The following authors: K.L., S.D.S., S.Ku. and S.S. are listed as inventors on the patent. The other authors declare no conflicts of interest. DATA AVAILABILITY The modified 330 Envelope protomer sequences have been deposited in GenBank under accession numbers: PV575182 (330_SOSIP), PV575183 (330_NFL), and PV575184 (330_SOSIP Ferritin Nanoparticle). The cryo-EM maps and atomic coordinates of the reported structures have been deposited in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) under accession codes EMD-66717 and 9XC1, respectively, for the 330 SOSIP Env trimer in complex with bNAb 44m. Additional data are available from the corresponding authors upon reasonable request. ACKNOWLEDGMENTS The authors express gratitude to Prof. R.W. Sanders for generously providing the PPI4 expression vector and the training provided to S.D.S as a part of his HIV-1 Trust Fellowship. We are grateful to the NIH AIDS reagent program for providing the HIV-1 research reagents and the Neutralizing Antibody Consortium(NAC), IAVI, USA for providing the HIV-1 neutralizing antibodies donated by Michel Nussenzweig, Hermann Katinger, Mark Connors, James Robinson, Dennis Burton, John Mascola, Peter Kwong, and William Olson. We appreciate the support of Liveon Biolabs for conducting the immunization studies. We also extend our appreciation for the IAVI-Leader Development Program awarded to S.D.S. Funder Information Declared Department of Biotechnology , BT/PR39156/DRUG/134/91/2021 , BT/PR30120/MED/29/1339/2018 , BT/INF/22/SP22844/2017 Department of Science and Technology, https://ror.org/0101xrq71 , SR/FST/LSII-039/2015 Footnotes This revised version of the preprint includes updated structural data of the 330 SOSIP Env trimer in complex with the autologous bNAb 44m, with improved resolution and additional insights into epitope interactions. Figures, results, and data files have been updated accordingly. The title has been modified to reflect the inclusion of the new structural findings of the HIV-1C env-autologous bnAb complex. All other content remains consistent with the original version (May 2025) REFERENCES 1. ↵ Burton , D. R. et al. A Blueprint for HIV Vaccine Discovery . Cell Host Microbe 12 , 396 – 407 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Simek , M. D. et al. Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm . J. Virol . 83 , 7337 – 7348 ( 2009 ). OpenUrl Abstract / FREE Full Text 3. ↵ van den Kerkhof , T. L. G. M. , et al. Early development of broadly reactive HIV-1 neutralizing activity in elite neutralizers . AIDS 28 , 1237 ( 2014 ). OpenUrl CrossRef PubMed 4. ↵ Mascola , J. R. et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies . Nat. Med . 6 , 207 – 210 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 5. ↵ Mascola , J. R. et al. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies . J. Virol . 73 , 4009 – 4018 ( 1999 ). OpenUrl Abstract / FREE Full Text 6. Parren , P. W. et al. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro . J. Virol . 75 , 8340 – 8347 ( 2001 ). OpenUrl Abstract / FREE Full Text 7. Moldt , B. et al. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo . Proc. Natl. Acad. Sci. U. S. A . 109 , 18921 – 18925 ( 2012 ). OpenUrl Abstract / FREE Full Text 8. ↵ Hessell , A. J. et al. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers . PLoS Pathog . 5 , e1000433 ( 2009 ). OpenUrl CrossRef PubMed 9. ↵ Karasavvas , N. et al. The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 . AIDS Res. Hum. Retroviruses 28 , 1444 – 1457 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 10. ↵ Kim , J. , Excler , J.-L. & Michael , N . Lessons from the RV144 Thai Phase III HIV-1 Vaccine Trial and the search for correlates of protection . Annu. Rev. Med . 66 , ( 2014 ). 11. ↵ Sanders , R. W. et al. A Next-Generation Cleaved, Soluble HIV-1 Env Trimer, BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies . PLOS Pathog . 9 , e1003618 ( 2013 ). OpenUrl CrossRef PubMed 12. ↵ Torrents de la Peña , A. , et al. Improving the Immunogenicity of Native-like HIV-1 Envelope Trimers by Hyperstabilization . Cell Rep . 20 , 1805 – 1817 ( 2017 ). OpenUrl CrossRef PubMed 13. Torrents de la Peña , A. , et al. Immunogenicity in Rabbits of HIV-1 SOSIP Trimers from Clades A, B, and C, Given Individually, Sequentially, or in Combination . J. Virol . 92 , ( 2018 ). 14. ↵ Schorcht , A. et al. Neutralizing Antibody Responses Induced by HIV-1 Envelope Glycoprotein SOSIP Trimers Derived from Elite Neutralizers . J. Virol . 94 , doi: 10.1128/jvi.01214-20 ( 2020 ). OpenUrl CrossRef 15. ↵ de Taeye , S. W. et al. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes . Cell 163 , 1702 – 1715 ( 2015 ). OpenUrl CrossRef PubMed 16. Sok , D. et al. Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows . Nature 548 , 108 – 111 ( 2017 ). OpenUrl CrossRef PubMed 17. Sanders , R. W. et al. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers . Science 349 , aac4223 ( 2015 ). OpenUrl Abstract / FREE Full Text 18. ↵ Houser , K. V. , et al. Safety and immunogenicity of an HIV-1 prefusion-stabilized envelope trimer (Trimer 4571) vaccine in healthy adults: A first-in-human open-label, randomized, dose-escalation, phase 1 clinical trial . eClinicalMedicine 48 , ( 2022 ). 19. ↵ Gartner , M. J. , Roche , M. , Churchill , M. J. , Gorry , P. R. & Flynn , J. K . Understanding the mechanisms driving the spread of subtype C HIV-1 . EBioMedicine 53 , 102682 ( 2020 ). OpenUrl PubMed 20. ↵ Hemelaar , J. , Gouws , E. , Ghys , P. D. & Osmanov , S . Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004 . AIDS Lond. Engl . 20 , W13 – 23 ( 2006 ). OpenUrl CrossRef 21. ↵ Ahmed , S. et al. Stabilization of a soluble, native-like trimeric form of an efficiently cleaved Indian HIV-1 clade C envelope glycoprotein . J. Biol. Chem . 292 , 8236 – 8243 ( 2017 ). OpenUrl Abstract / FREE Full Text 22. Rutten , L. et al. A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers . Cell Rep . 23 , 584 – 595 ( 2018 ). OpenUrl CrossRef PubMed 23. ↵ Kumar , R. et al. 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 Pathog . 17 , e1008977 ( 2021 ). OpenUrl CrossRef PubMed 24. ↵ Kumar , S. et al. Design and immunogenicity of an HIV-1 clade C pediatric envelope glycoprotein stabilized by multiple platforms . BioRxiv Prepr. Serv. Biol . 2024.09.14.613016 ( 2024 ) doi: 10.1101/2024.09.14.613016 . OpenUrl Abstract / FREE Full Text 25. ↵ Schiller , J. & Chackerian , B . Why HIV Virions Have Low Numbers of Envelope Spikes: Implications for Vaccine Development . PLOS Pathog . 10 , e1004254 ( 2014 ). OpenUrl CrossRef PubMed 26. ↵ Zhao , Q. , Li , S. , Yu , H. , Xia , N. & Modis , Y . Virus-like particle-based human vaccines: quality assessment based on structural and functional properties . Trends Biotechnol . 31 , 654 – 663 ( 2013 ). OpenUrl CrossRef PubMed 27. Ludwig , C. & Wagner , R . Virus-like particles-universal molecular toolboxes . Curr. Opin. Biotechnol . 18 , 537 – 545 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 28. ↵ Jennings , G. T. & Bachmann , M. F . The coming of age of virus-like particle vaccines . Biol. Chem . 389 , 521 – 536 ( 2008 ). OpenUrl CrossRef PubMed 29. ↵ Sliepen , K. et al. Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity . Retrovirology 12 , 82 ( 2015 ). OpenUrl CrossRef PubMed 30. ↵ He , L. et al. Presenting native-like trimeric HIV-1 antigens with self-assembling nanoparticles . Nat. Commun . 7 , 12041 ( 2016 ). 31. ↵ Goo , L. , Chohan , V. , Nduati , R. & Overbaugh , J . Early development of broadly neutralizing antibodies in HIV-1–infected infants . Nat. Med . 20 , 655 – 658 ( 2014 ). OpenUrl CrossRef PubMed 32. Ditse , Z. et al. HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes . J. Virol . 92 , ( 2018 ). 33. ↵ Kumar , S. et al. An HIV-1 Broadly Neutralizing Antibody from a Clade C-Infected Pediatric Elite Neutralizer Potently Neutralizes the Contemporaneous and Autologous Evolving Viruses . J. Virol . 93 , e01495 – 18 ( 2019 ). OpenUrl PubMed 34. ↵ Makhdoomi , M. A. et al. Neutralization resistant HIV-1 primary isolates from antiretroviral naïve chronically infected children in India . Virology 499 , 105 – 113 ( 2016 ). OpenUrl CrossRef PubMed 35. ↵ Mishra , N. et al. Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins . J. Virol . 93 , e00654 – 19 , /jvi/93/17/JVI.00654-19.atom ( 2019 ). OpenUrl PubMed 36. ↵ Kumar , S. et al. Recognition determinants of improved HIV-1 neutralization by a heavy chain matured pediatric antibody . iScience 26 , 107579 ( 2023 ). OpenUrl PubMed 37. ↵ Simonich , C. A. et al. HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant . Cell 166 , 77 – 87 ( 2016 ). OpenUrl CrossRef PubMed 38. ↵ Sharma , S. K. et al. Cleavage-Independent HIV-1 Env Trimers Engineered as Soluble Native Spike Mimetics for Vaccine Design . Cell Rep . 11 , 539 – 550 ( 2015 ). OpenUrl CrossRef PubMed 39. ↵ Guenaga , J. et al. Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers . J. Virol . 90 , 2806 – 2817 ( 2015 ). OpenUrl CrossRef PubMed 40. ↵ Joyce , M. G. et al. Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse HIV-1 Strains . Cell Rep . 21 , 2992 – 3002 ( 2017 ). OpenUrl CrossRef PubMed 41. ↵ Kumar , S. et al. B cell repertoire sequencing of HIV-1 pediatric elite-neutralizers identifies multiple broadly neutralizing antibody clonotypes . Front. Immunol . 15 , 1272493 ( 2024 ). OpenUrl CrossRef PubMed 42. ↵ A Recombinant Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Complex Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits Is an Antigenic Mimic of the Trimeric Virion-Associated Structure | Journal of Virology . doi: 10.1128/JVI.74.2.627-643.2000 . OpenUrl Abstract / FREE Full Text 43. ↵ Sanders , R. W. et al. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1 . J. Virol . 76 , 8875 – 8889 ( 2002 ). OpenUrl Abstract / FREE Full Text 44. ↵ Binley , J. M. et al. Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins . J. Virol . 76 , 2606 – 2616 ( 2002 ). OpenUrl Abstract / FREE Full Text 45. ↵ Ringe , R. P. et al. Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes . J. Virol . 91 , ( 2017 ). 46. ↵ The Design and Immunogenicity of an HIV-1 Clade C Pediatric Envelope Glycoprotein Stabilized by Multiple Platforms . https://www.mdpi.com/2076-393X/13/2/110 . 47. ↵ Guenaga , J. et al. Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein . Immunity 46 , 792 – 803 .e3 ( 2017 ). OpenUrl CrossRef PubMed 48. ↵ Rodrigues , M. Q. , Alves , P. M. & Roldão , A . Functionalizing Ferritin Nanoparticles for Vaccine Development . Pharmaceutics 13 , 1621 ( 2021 ). OpenUrl CrossRef PubMed 49. ↵ López-Sagaseta , J. , Malito , E. , Rappuoli , R. & Bottomley , M. J . Self-assembling protein nanoparticles in the design of vaccines . Comput. Struct. Biotechnol. J . 14 , 58 – 68 ( 2016 ). OpenUrl CrossRef PubMed 50. ↵ Schommers , P. et al. Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody . Cell 180 , 471 – 489 .e22 ( 2020 ). OpenUrl CrossRef PubMed 51. ↵ Julien , J.-P. et al. Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans . PLoS Pathog . 9 , e1003342 ( 2013 ). OpenUrl CrossRef PubMed 52. ↵ Kumar , S. et al. A V H 1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite . Sci. Adv . 6 , eabb1328 ( 2020 ). OpenUrl FREE Full Text 53. ↵ Barnes , C. O. et al. Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope . Nat. Commun . 9 , 1251 ( 2018 ). OpenUrl CrossRef PubMed 54. ↵ Steichen , J. M. et al. A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses . Science 366 , eaax4380 ( 2019 ). OpenUrl Abstract / FREE Full Text 55. ↵ Saunders , K. O. et al. Targeted selection of HIV-specific antibody mutations by engineering B cell maturation . Science 366 , eaay7199 ( 2019 ). OpenUrl FREE Full Text 56. ↵ Simonich , C. A. et al. Kappa chain maturation helps drive rapid development of an infant HIV-1 broadly neutralizing antibody lineage . Nat. Commun . 10 , 2190 ( 2019 ). OpenUrl CrossRef PubMed 57. ↵ Guenaga , J. et al. Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein . Immunity 46 , 792 – 803 .e3 ( 2017 ). OpenUrl CrossRef PubMed 58. ↵ Pancera , M. et al. Structure and immune recognition of trimeric pre-fusion HIV-1 Env . Nature 514 , 455 – 461 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 59. ↵ Behrens , A.-J. et al. Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein . Cell Rep . 14 , 2695 – 2706 ( 2016 ). OpenUrl CrossRef PubMed 60. ↵ Brouwer , P. J. M. et al. Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles . Npj Vaccines 6 , 1 – 14 ( 2021 ). OpenUrl CrossRef PubMed 61. ↵ Ozorowski , G. et al. Effects of Adjuvants on HIV-1 Envelope Glycoprotein SOSIP Trimers In Vitro . J. Virol . 92 , ( 2018 ). 62. ↵ Ahmed , S. et al. Enhancing anti-viral neutralization response to immunization with HIV-1 envelope glycoprotein immunogens . Npj Vaccines 8 , 1 – 10 ( 2023 ). OpenUrl PubMed 63. ↵ Sutar , J. et al. Geospatial HIV-1 subtype C gp120 sequence diversity and its predicted impact on broadly neutralizing antibody sensitivity . PLOS ONE 16 , e0251969 ( 2021 ). OpenUrl CrossRef PubMed 64. Ariën , K. K. , Vanham , G. & Arts , E. J . Is HIV-1 evolving to a less virulent form in humans? Nat. Rev. Microbiol . 5 , 141 – 151 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 65. Taylor , B. S. , Sobieszczyk , M. E. , McCutchan , F. E. & Hammer , S. M . The challenge of HIV-1 subtype diversity . N. Engl. J. Med . 358 , 1590 – 1602 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 66. ↵ Huang , A. et al. Global Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes . Open Forum Infect. Dis . 3 , ofv158 ( 2016 ). OpenUrl CrossRef PubMed 67. ↵ Julien , J.-P. et al. Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens . Proc. Natl. Acad. Sci. U. S. A . 112 , 11947 – 11952 ( 2015 ). OpenUrl Abstract / FREE Full Text 68. ↵ Kumar , S. et al. Neutralizing Antibodies Induced by First-Generation gp41-Stabilized HIV-1 Envelope Trimers and Nanoparticles . mBio 12 , doi: 10.1128/mbio.00429-21 ( 2021 ). OpenUrl CrossRef 69. ↵ Borst , A. J. et al. Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core . eLife 7 , e37688 ( 2018 ). OpenUrl CrossRef PubMed 70. ↵ Ringe , R. P. et al. Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization . PLOS Pathog . 19 , e1011601 ( 2023 ). OpenUrl CrossRef PubMed 71. ↵ Xu , D. et al. Design of universal Ebola virus vaccine candidates via immunofocusing . Proc. Natl. Acad. Sci. U. S. A . 121 , e2316960121 . 72. ↵ Powell , A. E. et al. Multimerization of Ebola GPΔmucin on protein nanoparticle vaccines has minimal effect on elicitation of neutralizing antibodies . Front. Immunol . 13 , 942897 ( 2022 ). OpenUrl CrossRef PubMed 73. ↵ Casazza , J. P. et al. Phase 1 dose-escalation trial evaluating a group 2 influenza hemagglutinin stabilized stem nanoparticle vaccine . Npj Vaccines 9 , 1 – 10 ( 2024 ). OpenUrl CrossRef PubMed 74. ↵ Tang , P. et al. A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs . Front. Immunol . 15 , ( 2024 ). 75. ↵ Stephens , L. M. & Varga , S. M . Nanoparticle vaccines against respiratory syncytial virus . Future Virol . 15 , 763 – 778 . 76. ↵ Swanson , K. A. , et al. A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain . Sci. Immunol . 5 , eaba6466 ( 2020 ). OpenUrl Abstract / FREE Full Text 77. ↵ Joyce , M. G. et al. SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity . bioRxiv 2021.05.09.443331 ( 2021 ) doi: 10.1101/2021.05.09.443331 . OpenUrl Abstract / FREE Full Text 78. Weidenbacher , P. A.-B. et al. A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates . Nat. Commun . 14 , 2149 ( 2023 ). OpenUrl CrossRef PubMed 79. Yu , J. et al. Ad26.COV2.S and SARS-CoV-2 spike protein ferritin nanoparticle vaccine protect against SARS-CoV-2 Omicron BA.5 challenge in macaques . Cell Rep. Med . 4 , 101018 ( 2023 ). OpenUrl PubMed 80. ↵ Johnston , S. C. et al. A SARS-CoV-2 Spike Ferritin Nanoparticle Vaccine Is Protective and Promotes a Strong Immunological Response in the Cynomolgus Macaque Coronavirus Disease 2019 (COVID-19) Model . Vaccines 10 , 717 ( 2022 ). OpenUrl PubMed 81. ↵ Guenaga , J. et al. Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers . J. Virol . 90 , 2806 – 2817 ( 2016 ). OpenUrl Abstract / FREE Full Text 82. ↵ Rappuoli , R. , Bottomley , M. , D’Oro , U. , Finco , O. & Gregorio , E . Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design . J. Exp. Med . 213 , jem.20151960 ( 2016 ). OpenUrl 83. ↵ Klasse , P. J. , Ozorowski , G. , Sanders , R. W. & Moore , J. P . Env Exceptionalism: Why Are HIV-1 Env Glycoproteins Atypical Immunogens? Cell Host Microbe 27 , 507 – 518 ( 2020 ). OpenUrl CrossRef PubMed 84. ↵ Seabright , G. E. , Doores , K. J. , Burton , D. R. & Crispin , M . Protein and Glycan Mimicry in HIV Vaccine Design . J. Mol. Biol . 431 , 2223 – 2247 ( 2019 ). OpenUrl CrossRef PubMed 85. ↵ Veneziano , R. et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami . Nat. Nanotechnol . 15 , 716 – 723 ( 2020 ). OpenUrl CrossRef PubMed 86. ↵ ACTS, RULES AND GUIDELINES: Committee for the Purpose of Control And Supervision of Experiments on Animals . https://ccsea.gov.in/Content/54_1_ACTS,RULESANDGUIDELINES.aspx . 87. ↵ Klasse , P. J. et al. Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein . J. Virol . 87 , 9873 – 9885 ( 2013 ). OpenUrl Abstract / FREE Full Text 88. ↵ de Taeye , S. W. et al. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes . Cell 163 , 1702 – 1715 ( 2015 ). OpenUrl CrossRef PubMed 89. ↵ Sok , D. et al. Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex . Proc. Natl. Acad. Sci. U. S. A . 111 , 17624 – 17629 ( 2014 ). OpenUrl Abstract / FREE Full Text 90. ↵ del Moral-Sánchez , I. et al. Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines . Npj Vaccines 9 , 1 – 18 ( 2024 ). OpenUrl CrossRef PubMed 91. ↵ Kumar , S. et al. Recognition determinants of improved HIV-1 neutralization by a heavy chain matured pediatric antibody . iScience 26 , 107579 ( 2023 ). OpenUrl PubMed 92. ↵ Tang , G. et al. EMAN2: An extensible image processing suite for electron microscopy . J. Struct. Biol . 157 , 38 – 46 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 93. ↵ Kumar , A. , P., S., Gulati , S. & Dutta , S. User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy . J. Vis. Exp . 62832 ( 2021 ) doi: 10.3791/62832 . OpenUrl CrossRef 94. ↵ Punjani , A. , Rubinstein , J. L. , Fleet , D. J. & Brubaker , M. A . cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination . Nat. Methods 14 , 290 – 296 ( 2017 ). OpenUrl CrossRef PubMed 95. ↵ Liebschner , D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix . Acta Crystallogr. Sect. Struct. Biol . 75 , 861 – 877 ( 2019 ). OpenUrl CrossRef 96. ↵ Goddard , T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis . Protein Sci. Publ. Protein Soc . 27 , 14 – 25 ( 2018 ). OpenUrl CrossRef 97. ↵ Mishra , N. et al. Broadly neutralizing plasma antibodies effective against autologous circulating viruses in infants with multivariant HIV-1 infection . Nat. Commun . 11 , 4409 ( 2020 ). OpenUrl CrossRef PubMed 98. ↵ Prasad , V. R. Montefiori , D. C . Measuring HIV Neutralization in a Luciferase Reporter Gene Assay . in HIV Protocols (eds. Prasad , V. R. & Kalpana , G. V. ) 395 – 405 ( Humana Press , Totowa, NJ , 2009 ). doi: 10.1007/978-1-59745-170-3_26 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted December 02, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb Swarandeep Singh , Sanjeev Kumar , Arnab Chatterjee , Lakshay Malhotra , Muneeb Pervez , Abdul Rahman , Ifrah Andrabi , Bapan Mondal , Sanket Katpara , Shaifali Sharma , Harsh Bhakri , Anjli Gaur , Tanu Bansal , Vikram Iyer , Abdul Wahid Hussain , Shubbir Ahmed , Rajesh Kumar , Ethayathulla Abdul Samath , Rakesh Lodha , Somnath Dutta , Kalpana Luthra bioRxiv 2025.04.26.649701; doi: https://doi.org/10.1101/2025.04.26.649701 Share This Article: Copy Citation Tools Immunogenicity and Structure of stabilized HIV-1 Clade-C Env from pediatric Elite-neutralizer complexed with autologous bNAb Swarandeep Singh , Sanjeev Kumar , Arnab Chatterjee , Lakshay Malhotra , Muneeb Pervez , Abdul Rahman , Ifrah Andrabi , Bapan Mondal , Sanket Katpara , Shaifali Sharma , Harsh Bhakri , Anjli Gaur , Tanu Bansal , Vikram Iyer , Abdul Wahid Hussain , Shubbir Ahmed , Rajesh Kumar , Ethayathulla Abdul Samath , Rakesh Lodha , Somnath Dutta , Kalpana Luthra bioRxiv 2025.04.26.649701; doi: https://doi.org/10.1101/2025.04.26.649701 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42005) Biophysics (21472) Cancer Biology (18624) Cell Biology (25534) Clinical Trials (138) Developmental Biology (13391) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40437) Molecular Biology (17200) Neuroscience (88697) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4829) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9827) Zoology (2272)