Biology subjects

Rappazzo, C. G.

Publications and source records attributed to Rappazzo, C. G..

6 recordsLinked to original sources

bioRxiv2023

Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2

Characterization of functional antibody responses to the N-terminal domain (NTD) of the SARS-CoV-2 spike (S) protein has included identification of both potent neutralizing activity and putative enhancement of infection. Fc{gamma}-receptor (Fc{gamma}R)-independent enhancement of SARS-CoV-2 infection mediated by NTD-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is not clear. Here we studied 1,213 S-binding mAbs derived from longitudinal sampling of B-cells collected from eight COVID-19 convalescent patients and identified 72 (5.9%) mAbs that enhanced infection in a VSV-SARS-CoV-2-S-Wuhan pseudovirus (PV) assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least five months post-infection. Enhancement of PV infection by NTD-binding mAbs was not observed using intestinal (Caco-2) and respiratory (Calu-3) epithelial cells as infection targets and was diminished or lost against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire from two of the convalescent subjects identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum (i.e., functionally secreted antibody). Functional analysis of these mAbs demonstrated robust activation of Fc{gamma}RIIIa associated with antibody binding to recombinant S proteins. Taken together, these findings suggest functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display diverse attributes that include Fc{gamma}R activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

immunology↗

bioRxiv2022

Identification of highly cross-reactive mimotopes for a public T cell response in murine melanoma

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

immunology↗

bioRxiv2021

A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

microbiology↗

bioRxiv2020

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.

immunology↗

bioRxiv2020

Vaccination reshapes the virus-specific T cell repertoire in unexposed adults

How baseline T cell characteristics impact human T cell responses to novel pathogens remains unknown. Here, we address this question by studying the CD4+ T cell response in unexposed individuals to live attenuated yellow fever virus (YFV) vaccine. We quantified virus-specific population dynamics over time using class II peptide-MHC tetramers. Our data revealed that, even in the absence of known viral exposure, memory phenotype T cells were found in the majority of virus-specific precursors in healthy adults. Pre-existing memory T cells can be divided into two groups; abundant pre-vaccine populations that underwent limited overall expansion and rare cells that generated naive-like responses and preferentially contributed to the memory repertoire after vaccination. Single cell T cell receptor (TCR) sequencing was used to track the evolution of immune responses to different epitopes and showed an association between preservation of unexpanded TCRs before exposure and the robustness of post-vaccine responses. Instead of a further increase in pre-established TCR clones, vaccination boosted the representation of rare TCRs. Thus, vaccine restructures the abundance and clonal hierarchy of virus-specific T cells. Our results link T cell precursor states to post-exposure responses, identifying peripheral education of virus-specific repertoire as a key component of effective vaccination. {circ}YFV-specific precursors contain abundant memory phenotype cells in healthy adults. {circ}Precursor frequency and phenotype are linked to post-immune response. {circ}Vaccination recruits rare virus-specific populations. {circ}Vaccination overrides pre-established clonal hierarchy

immunology↗

bioRxiv2019

Longitudinal single cell profiling of regulatory T cells identifies IL-33 as a driver of tumor immunosuppression

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we used single cell RNA-Seq to longitudinally profile conventional CD4+ T cells (Tconv) and Tregs in a genetic mouse model of lung adenocarcinoma. Tissue-infiltrating and peripheral CD4+ T cells differed, highlighting divergent pathways of activation during tumorigenesis. Longitudinal shifts in Treg heterogeneity suggested increased terminal differentiation and stabilization of an effector phenotype over time. In particular, effector Tregs had enhanced expression of the interleukin 33 receptor ST2. Treg-specific deletion of ST2 reduced effector Tregs, increased infiltration of CD8+ T cells into tumors, and decreased tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting new potential paths for therapeutic intervention.

cancer biology↗