Biology subjects

Capozzola, T.

Publications and source records attributed to Capozzola, T..

11 recordsLinked to original sources

bioRxiv2026

Protective pan-betacoronavirus neutralizing antibodies by vaccination

The continued emergence of betacoronaviruses underscores the urgent need for vaccines that provide broadly protective immunity. Here, we present an epitope-focused vaccine strategy targeting the conserved S2 stem-helix region of the spike fusion machinery, a broadly neutralizing antibody-(bnAb) epitope shared across betacoronaviruses yet partially occluded on the native spike. Immunization of non-human primates with engineered S2 stem-helix nanoparticle immunogens, alone or followed by a SARS-CoV-2 BA.1 spike mRNA boost, elicited broadly cross-reactive antibody responses against sarbecoviruses, merbecoviruses, and embecoviruses and neutralized SARS-CoV-2, multiple variants, other sarbecoviruses, and MERS-CoV. Vaccine-elicited monoclonal antibodies displayed broad in-vitro neutralizing activity and protected against both SARS-CoV-2 and MERS-CoV in-vivo. Structural analyses revealed conserved features between rhesus and human stem-helix bnAbs, supporting the translational potential. Overall, our findings provide proof-of-concept that epitope-focused nanoparticle immunogens can target partially occluded, immunoquiescent bnAb epitopes, laying the groundwork for pan-betacoronavirus vaccines that provide broad protection and strengthen pandemic preparedness. ONE SENTENCE SUMMARYEpitope-focused S2 stem-helix nanoparticle immunogens elicit protective broadly neutralizing antibodies (bnAbs) against diverse betacoronaviruses in non-human primates, establishing a framework for development of pan-betacoronavirus vaccines.

immunology↗

bioRxiv2026

Heterologous betacoronavirus spike immunization in nonhuman primates elicits cross-reactive antibodies that neutralize both sarbeco- and merbecoviruses

In anticipation of future coronavirus (CoV) pandemics, developing vaccines that elicit broadly neutralizing antibodies (bnAbs) against diverse CoVs is critical. Here, we vaccinated rhesus macaques with SARS-CoV-2 spike (S)-protein, then boosted with heterologous {beta}-CoV S-proteins to focus responses to common conserved S2 bnAb epitopes. Initial SARS-CoV-2 priming elicited receptor-binding domain (RBD)-focused responses, while MERS-CoV boosting redirected responses toward the S2 region, including the stem-helix bnAb site. Although S2-directed serum cross-neutralization was undetectable and most isolated cross-reactive monoclonal antibodies (mAbs) targeted non-neutralizing epitopes, two S2 stem-helix mAbs were identified from memory B cells. These bnAbs neutralized diverse sarbeco- and merbecoviruses, including MERS-CoV, and conferred robust in vivo protection against SARS-CoV-2 challenge. Structural studies revealed that these macaque bnAbs closely mimic human S2-stem bnAbs induced by infection. These findings provide proof-of-principle for vaccination strategies that elicit broadly protective {beta}-coronavirus responses and highlight non-human primates as a translational model for evaluating S2-targeted immunogens.

immunology↗

bioRxiv2025

Structure-guided design of native-like HIV Env Single Chain Trimers for enhanced stability, immunogenicity, and versatile vaccine delivery

Stabilizing the HIV envelope (Env) trimer in its native prefusion conformation is key to eliciting broadly neutralizing antibodies (bnAbs). We present a generalizable single-chain trimer-(SCT) design platform that enables the production of stable, native-like Env trimers across diverse HIV strains. Using a structure-guided approach, we developed CAP256.SU-SCT9, based on a CAP256.SU Env shown to induce V2-apex bnAbs in humans and rhesus macaques. CAP256.SU-SCT9 trimer exhibited native-like structure, bnAb-specific antigenicity, authentic glycosylation, and compatibility with nucleic acid and nanoparticle delivery. We extended this design strategy to a panel of HIV Envs representing global diversity, and immunogens known to initiate bnAb responses in humans. Structural analyses confirmed that SCTs consistently maintained prefusion-closed conformations, favorable glycan profiles, strong binding to trimer-specific bnAbs, and low reactivity with non-neutralizing antibodies. These findings establish SCT as a versatile platform for HIV trimer immunogen design, with potential to support next-generation vaccines aimed at eliciting protective bnAb responses.

immunology↗

bioRxiv2025

Germline-targeting HIV immunogen induces cross-neutralizing antibodies in outbred macaques

Germline-targeting-(GT) is a promising strategy to activate rare broadly neutralizing antibody (bnAb)-producing B cells against HIV, but induction of such responses in outbred animals has not been achieved. Using antibody-guided structure-based design, we engineered a germline-targeting trimer immunogen Q23-APEX-GT2 that primes diverse V2-apex bnAb precursors. Q23-APEX-GT2 efficiently activated V2-apex-specific B cells in humanized knock-in mice and consistently elicited immunofocused antibody responses in rhesus macaques, priming multiple long CDRH3-loop bnAb-B cell lineages. Monoclonal antibodies from immunized macaques exhibited broad heterologous HIV trimer binding and cross-neutralization. Atomic-level structural studies confirmed precise epitope targeting and revealed CDRH3-paratope configurations that mirrored those of human V2-apex bnAbs. This study provides proof-of-principle for successful priming and maturation of authentic V2-apex bnAb precursors in outbred macaques, underscoring the potential of V2-apex-targeted vaccines. HIGHLIGHTSO_LIEngineered Q23-APEX-GT2 trimer to stimulate diverse V2-apex bnAb B cell precursors C_LIO_LIQ23-APEX-GT2 primed rare V2-apex bnAb B cells in mice and outbred rhesus macaques C_LIO_LIQ23-APEX-GT2 elicited immunofocused antibody responses and diverse V2-apex B cell lineages with desirable long-CDRH3 paratope properties C_LIO_LIQ23-APEX-GT2 alone induced V2-apex antibodies with broad HIV trimer binding and modest neutralization breadth C_LIO_LIStructural analysis confirmed bnAb site targeting, mirroring human and rhesus V2-apex bnAbs C_LI

immunology↗

bioRxiv2025

Directed evolution of a stem-helix targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats. Author SummaryThe persistent emergence of new SARS-CoV-2 variants of concern that evade neutralizing antibody responses and other zoonotic betacoronaviruses with pandemic potential have provided strong motivation to develop broadly neutralizing antibodies (bnAbs) that target more conserved regions of the spike protein in sarbecoviruses and other betacoronaviruses. Here, we employed a directed evolution strategy to engineer the sarbecovirus-neutralizing antibody CC65.1, which targets the conserved S2 stem helix, to enhance its binding affinity with the MERS-CoV stem helix region, thereby conferring MERS-CoV neutralization. High-resolution structural studies of engineered CC65.1 revealed that key mutations reshape the paratope to better accommodate and stabilize the MERS-CoV S2 stem helix, resulting in increased binding affinity and neutralization potency. This study emphasizes the critical role of affinity maturation in expanding neutralization breadth and provides valuable insights for design of bnAbs to prevent and treat pandemic threats by betacoronaviruses.

immunology↗

bioRxiv2023

Germline-targeting chimpanzee SIV Envelopes induce V2-apex broadly neutralizing-like B cell precursors in a rhesus macaque infection model

Eliciting broadly neutralizing antibodies-(bnAbs) remains a major goal of HIV-1 vaccine research. Previously, we showed that a soluble chimpanzee SIV Envelope-(Env) trimer, MT145K, bound several human V2-apex bnAb-precursors and stimulated an appropriate response in V2-apex bnAb precursor-expressing knock-in mice. Here, we tested the immunogenicity of three MT145 variants (MT145, MT145K, MT145K.dV5) expressed as chimeric simian-chimpanzee-immunodeficiency-viruses-(SCIVs) in rhesus macaques-(RMs). All three viruses established productive infections with high setpoint vRNA titers. RMs infected with the germline-targeting SCIV_MT145K and SCIV_MT145K.dV5 exhibited larger and more clonally expanded B cell lineages featuring long anionic heavy chain complementary-determining-regions-(HCDR3s) compared with wildtype SCIV_MT145. Moreover, antigen-specific B cell analysis revealed enrichment for long-CDHR3-bearing antibodies in SCIV_MT145K.dV5 infected animals with paratope features resembling prototypic V2-apex bnAbs and their precursors. Although none of the animals developed bnAbs, these results show that germline-targeting SCIVs can activate and preferentially expand B cells expressing V2-apex bnAb-like precursors, the first step in bnAb elicitation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/558743v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@17673c8org.highwire.dtl.DTLVardef@1ab61a8org.highwire.dtl.DTLVardef@98781forg.highwire.dtl.DTLVardef@1f63e9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Broad-Spectrum Heavily Mutated Monoclonal Antibody Isolated from COVID-19 Convalescent Vaccinee with Capacity to Neutralize SARS-CoV2 Variants Ranging from B.1 to BQ.1.1

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/539267v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@f7dab8org.highwire.dtl.DTLVardef@388861org.highwire.dtl.DTLVardef@181a7c7org.highwire.dtl.DTLVardef@12e02cb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG In briefChoudhary et al. have isolated and characterized Acovimab, a broadly neutralizing RBM-specific human monoclonal antibody with a relatively high level of somatic hypermutation, which potently neutralizes SARS-CoV2 variants ranging from WuhanB.1 to OmicronBQ.1.1, but not the XBB.1.5 variant. Acovimab also possesses strong synergistic neutralizing activity against some Omicron variants when combined with Sotrovimab. Polyclonal plasma antibodies from COVID-19 vaccinees who had recovered from SARS-CoV2 infection were shown to possess low neutralizing titers of antibodies against conserved RBD targets of CoV2 variants including XBB.1.5, which also synergistically neutralize with Sotrovimab against this variant. SummaryThe increasing prevalence of the highly antibody-evasive Omicron sublineages increases the risk of breakthrough infections and leaves high-risk and vulnerable immunocompromised individuals with no effective options for prophylactic or therapeutic antibody treatments. Here, we report a heavily mutated anti-RBD monoclonal antibody, Acovimab, directed against a site in the receptor-binding motif (RBM) region of the CoV2 receptor-binding domain (RBD), that possesses very broad and highly potent neutralizing activity against CoV2 variants, including many Omicron variants. This antibody is derived from the IGHV1-58*01 germline sequence and possesses a relatively high level of mutation (15.5% of the VH aa sequence), which is unusual for anti-RBD antibodies. Neutralizing activity was very potent (IC50s range of 1-9 ng/ml) for early Omicron subvariants that possess an unmutated F486 residue and is retained but less potent (IC50s of 200-650 ng/ml) for more resistant Omicron subvariants which contain the F486V mutation (BA4/5, BA4.6, and BQ1.1), but is lost for the later ultra-resistant variants that contain F486S (XBB) or F486P (XBB.1.5) mutations. Based on these specificities, it is predicted that Acovimab by itself should protect against CoV2 infections other than those caused by the XBB/XBB.1.5 family. Acovimab also shows strong synergy in neutralization when combined with Sotrovimab, which neutralizes all Omicron variants, including XBB.1.5. Plasma from subjects with hybrid immunity (induced by vaccination + infection) possessed low levels of XBB.1.5 RBM-targeting plasma-neutralizing antibodies, and these also neutralized synergistically when combined with Sotrovimab. These results suggest potentially novel immunotherapeutic options for treating most of the CoV2 variants responsible for current infections.

immunology↗

bioRxiv2023

Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift

Developing broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized "site V" on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV- 2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.

immunology↗

bioRxiv2023

Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple epitopes

Development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection against in vivo challenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

immunology↗

bioRxiv2022

A broad and potent neutralization epitope in SARS-related coronaviruses

Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.

microbiology↗

bioRxiv2022

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

immunology↗