Serum IgG anti-SARS-CoV-2 Binding Antibody Level Is Strongly Associated With IgA and Functional Antibody Levels in Adults Infected With SARS-CoV-2.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide (p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients.

Background: Cleavage of the inactive precursor fusion protein (F0) of respiratory syncytial virus (RSV) at two furin-recognition sites is required for membrane fusion activity, and the cleavage releases the twenty-seven amino acid peptide (p27). However, a recent study shows that p27 was an immunodominant epitope in RSV infected children, indicating that p27 was recognized as an immunogen. In the present study, we investigated the immunogenicity of p27 in an immunocompromised population of adults by measuring serum and mucosal antibody responses to p27 in samples from adult hematopoietic cell transplant (HCT) recipients. Methods: We prospectively enrolled a cohort of RSV infected HCT recipients. Serum and nasal-wash samples were obtained within the first week of RSV infection (acute) and 3 to 5 weeks post-infection (convalescent). We quantified the serum and mucosal IgG and IgA anti-p27 antibodies by a RSV/A p27 peptide enzyme-linked immunosorbent assay (ELISA) and serum and mucosal p27 like antibodies (P27LA) by a p27 competitive antibody (P27CA) assay. Results: The lower limit of detection for the ELISA and P27CA assays was 0.2 and 50 ng/mL, respectively with no cross-reaction detected with a panel of monoclonal antibodies targeting pre-fusion and post-fusion antigenic sites. P27 antibodies were detected at nanogram concentration in sera and nasal washes in the majority of RSV infected HCT recipients. However, there was no significant difference in the geometric mean antibody concentrations between the acute and convalescent sera (except for serum P27LA), between HCT recipients who shed RSV <14 days and ≥14 days, as well as between RSV/A and RSV/B infected HCT recipients. In addition, approximately 30% of HCT recipients had a 4-fold or greater decrease in mucosal IgG and IgA anti-p27 antibodies during viral clearance. Conclusion: In conclusion, in RSV naturally infected adult HCT recipients, the antibodies against p27 were detectable in both serum and nasal wash samples with higher concentration in serum than that in nasal washes. However, nearly 30% of RSV infected HCT recipients had a significant decrease in their mucosal anti-p27 antibody, suggesting that IgG and IgA anti-p27 antibodies were binding to either free viruses or RSV infected cells containing p27, and that anti-p27 antibodies in the respiratory tract were part of the mucosal antibody response in controlling RSV infection.