Biology subjects

Purushotorman, K.

Publications and source records attributed to Purushotorman, K..

5 recordsLinked to original sources

bioRxiv2025

Intranasal sarbecovirus vaccine booster elicits cross-clade, durable and protective systemic and mucosal immunity

Short-lived, clade-specific immune responses with limited mucosal priming are limitations faced by current COVID-19 mRNA vaccines against sarbecoviruses. We have developed a nasal booster vaccine candidate that induced robust and sustained, cross-clade, systemic and mucosal protective immunity. Two recombinant Clec9A-specific monoclonal antibodies fused to the Receptor Binding Domain (RBD) from Omicron XBB.1.5 and SARS-CoV-1, respectively were generated. In Comirnaty mRNA-vaccinated mice, boosting with each individual Clec9A-RBD construct induced immune responses that either were limited in breadth or waned over time; while boosting with both constructs combined (Clec9AOMNI) elicited robust cross-clade neutralizing antibodies (nAb) and T cell responses that were significantly more sustained compared to Bivalent Comirnaty (BC) mRNA vaccine booster. The persistence of RBD-specific follicular helper CD4+ T cells, germinal centre B cells, and long-lived plasma cells that facilitated affinity maturation in Clec9AOMNI-boosted mice, correlated with the detection of triple cross-reactive B cells that bind to ancestral SARS-CoV-2 ancestral, SARS-CoV-2 XBB.1.5 and SARS-CoV-1 RBD. Remarkably, intranasal boosting with Clec9AOMNI generated robust and sustained mucosal immune responses in the upper and lower respiratory compartments, including RBD-specific IgA, cross-clade nAb and cellular immunity together with functional tissue-resident memory T cells, without compromising the systemic immune responses. Correspondingly, Clec9AOMNI booster conferred superior protection against Omicron BA.1 compared to BC booster when challenge was performed at six months post-boost. Hence, Clec9AOMNI is a promising nasal booster vaccine candidate that has the potential to mitigate pandemic threats from emerging sarbecoviruses. One Sentence SummaryNasal booster immunization with dendritic cell-targeting vaccine candidate in mRNA-vaccinated mice induced cross-clade, sustained, systemic and mucosal protective immunity.

immunology↗

bioRxiv2023

Single shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad and durable systemic and mucosal immune responses

Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+ cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+ T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.

immunology↗

bioRxiv2021

Mapping the allosteric effects that define functional activity of SARS-CoV-2 specific antibodies

Previous studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies. TeaserDifferent neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein

biochemistry↗

bioRxiv2020

The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for the optimal therapeutic efficacy of the antibody

SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFN{gamma}-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite Fc{gamma}R binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development. One Sentence SummaryAnti-SARS-CoV-2 IgG1 antibody SC31 controls infection in vivo by blocking SP:ACE2 binding and triggering a Fc-mediated anti-viral response.

microbiology↗

bioRxiv2020

SARS-CoV-2 S protein ACE2 interaction reveals novel allosteric targets

The Spike (S) protein is the main handle for SARS-CoV-2 to enter host cells through surface ACE2 receptors. How ACE2 binding activates proteolysis of S protein is unknown. Here, we have mapped the S:ACE2 interface and uncovered long-range allosteric propagation of ACE2 binding to sites critical for viral host entry. Unexpectedly, ACE2 binding enhances dynamics at a distal S1/S2 cleavage site and flanking protease docking site ~27 [A] away while dampening dynamics of the stalk hinge (central helix and heptad repeat) regions ~ 130 [A] away. This highlights that the stalk and proteolysis sites of the S protein are dynamic hotspots in the pre-fusion state. Our findings provide a mechanistic basis for S:ACE2 complex formation, critical for proteolytic processing and viral-host membrane fusion and highlight protease docking sites flanking the S1/S2 cleavage site, fusion peptide and heptad repeat 1 (HR1) as allosterically exposed cryptic hotspots for potential therapeutic development. One Sentence SummarySARS-CoV-2 spike protein binding to receptor ACE2 allosterically enhances furin proteolysis at distal S1/S2 cleavage sites

biochemistry↗