Broadly protective vaccines against known and pre-emergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy. We longitudinally profiled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)–specific serological and memory B cell (MBC) responses in individuals who received either homologous (ChAdOx1:ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous messenger RNA (mRNA) booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared with that of homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273–boosted participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relative to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced after heterologous mRNA booster vaccination.

HIV replication is suppressed in vitro by a CD8 + cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8 + cell subsets having strong CNAR activity. CD8 + cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8 + cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA − CD27 + and PD-1 + (CD279 + ) cells. Functional assessments of CD8 + cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA − CCR7 − CD27 + and PD-1 + CD8 + cells. T cell receptor (TCR) repertoire profiles of CD8 + cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8 + cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8 + cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pansarbecovirus vaccines. In December 2019, a novel pathogen emerged in the city of Wuhan in China's Hubei province, causing an outbreak of atypical pneumonia (a disease known as COVID-19). The infectious agent was rapidly characterized as a lineage B betacoronavirus, named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and shown to be closely related to SARS-CoV and several SARS-like bat CoVs(1). Despite the urgent need, there are currently no approved vaccines or therapeutics available for the prevention or treatment of COVID-19. Furthermore, the recurrent zoonotic spillover of CoVs into humans, along with the broad diversity of SARS-like CoV strains circulating in animal reservoirs, suggests that novel pathogenic CoVs are likely to emerge in the future and underscores the need for broadly active countermeasures.CoV entry into host cells is mediated by the viral S glycoprotein, which forms trimeric spikes on the viral surface(2). Each monomer in the trimeric S assembly is a heterodimer of S1 and S2 subunits. The S1 subunit is composed of four domains: an N-terminal domain (NTD), a C-terminal domain (CTD), and subdomains I and II(3-5). The CTD of both SARS-CoV and

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.