Patient-derived monoclonal antibodies to SARS-CoV-2 nucleocapsid protein N-terminal and C-terminal domains cross-react with their counterparts of SARS-CoV, but not other human betacoronaviruses.

Introduction: SARS-CoV-2 nucleocapsid (N) protein plays a key role in multiple stages of the viral life cycle such as viral replication and assembly. This protein is more conserved than the Spike protein of the virus and can induce both humoral and cell-mediated immune responses, thereby becoming a target for clinical diagnosis and vaccine development. However, the immunogenic characteristics of this protein during natural infection are still not completely understood. Methods: Patient-derived monoclonal antibodies (mAbs) against SARS-CoV-2 N protein were generated from memory B cells in the PBMCs using the antigen-specific B cell approach. For epitope mapping of the isolated hmAbs, a panel of series-truncated N proteins were used , which covered the N-terminal domain (NTD, aa 46-174 ) and C-terminal domain (CTD, aa 245-364 ), as well as the flanking regions of NTD and CTD. NTD- or CTD-specific Abs in the plasma from COVID-19 patients were also tested by ELISA method. Cross-binding of hmAbs or plasma Abs in COVID-19 patients to other human ß-CoV N proteins was determined using the capture ELISA. Results: We isolated five N-specific monoclonal antibodies (mAbs) from memory B cells in the peripheral blood of two convalescent COVID-19 patients. Epitope mapping revealed that three of the patient-derived mAbs (N3, N5 and N31) targeted the C-terminal domain (CTD), whereas two of the mAbs (N83 and 3B7) targeted the N-terminal domain (NTD) of SARS-CoV-2 N protein. All five patient-derived mAbs were cross-reactive to the N protein of SARS-CoV but showed little to no cross-reactivity to the N proteins of other human beta coronaviruses (ß-CoVs). We also tested 52 plasma samples collected from convalescent COVID-19 patients for Abs against the N proteins of human ß-CoVs and found that 78.8% of plasma samples showed detectable Abs against the N proteins of SARS-CoV-2 and SARS-CoV. No plasma sample had cross-reactive Abs to the N protein of MERS-CoV. Cross-reactive Abs to the N proteins of OC43 and HKU1 were detected in 36.5% (19/52) and 19.2% (10/52) of plasma samples, respectively. Discussion: These results suggest that natural SARS-CoV-2 infection elicits cross-reactive Abs to the N protein of SARS-CoV and that the five patient-derived mAbs to SARS-CoV-2 N protein NTD and CTD cross-react with their counterparts of SARS-CoV, but not other human ß-CoVs. Thus, these five patient-derived mAbs can potentially be used for developing the next generation of COVID-19 At-Home Test kits for rapid and specific screening of SARS-CoV-2 infection.

Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA.

BACKGROUND: Zika virus (ZIKV) had spread rapidly in the past few years in southern hemisphere where dengue virus (DENV) had caused epidemic problems for over half a century. The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it challenging to perform differential diagnosis between the two infections using standard ELISA method for E protein. METHODS: Using an IgG capture ELISA, we investigated the kinetics of nonstructural protein 1 (NS1) antibody response during natural ZIKV infection and the cross-reactivity to NS1 proteins using convalescent sera obtained from patients infected by either DENV or ZIKV. RESULTS: The analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared 2 weeks later than E specific Abs. Notably, human sera from ZIKV infected individuals did not contain cross-reactivity to NS1 proteins of any of the four DENV serotypes. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited amount of cross-reactivity to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution. CONCLUSIONS: The high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV infections.

Phylogenetic and genetic characterization of a 2017 clinical isolate of H7N9 virus in Guangzhou, China during the fifth epidemic wave.

Pathogenic H7N9 influenza viruses continue to pose a public health concern. The H7N9 virus has caused five outbreak waves of human infections in China since 2013. In the present study, a novel H7N9 strain (A/Guangdong/8H324/2017) was isolated from a female patient with severe respiratory illness during the fifth wave of the 2017 H7N9 epidemic. Phylogenetic analysis showed that the H7N9 viruses collected during the fifth wave belong to two different lineages: the Pearl River Delta lineage and the Yangtze River Delta lineage. The novel isolate is closely related to the Pearl River Delta H7N9 viruses, which were isolated from patients in Guangdong Province. The novel H7N9 isolate has an insertion of three basic amino acids in the cleavage site of hemagglutinin (HA), which may enhance virulence in poultry. The 2017 isolate also possesses an R292K substitution in the neuraminidase (NA) protein, which confers oseltamivir resistance. This study highlights the pandemic potential of the novel H7N9 virus in mammals; thus, future characterization and surveillance is warranted.

Delineating antibody recognition against Zika virus during natural infection.

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that shares a considerable degree of homology with dengue virus (DENV). Here, we examined longitudinal antibody response against ZIKV during natural infection in 2 convalescent individuals. By decomposing the antibody recognition into DI/DII and DIII of the E glycoprotein, we showed their development in humans followed a spatiotemporal hierarchy. Plasma binding to DI/DII appeared to peak and wane during early infection with extensive cross-reactivity with DI/DII of DENV. Binding to DIII, however, peaked early but persisted months into the infection without detectable cross-reactivity with DIII of DENV. A clear trend of increase in DIII-specific neutralizing activity was observed over the course of infection. mAbs isolated during early infection are largely DI/DII specific, weakly neutralizing, and highly cross-reactive with DENV, while those from later infection are more diverse in recognition, potently neutralizing, and ZIKV specific. The most potent neutralizing mAb targeting the DIII provided 100% protection in mice from lethal ZIKV infection and could therefore serve as a promising candidate for antibody-based therapy and prevention. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.