부산대학교 도서관

Summary: Antibodies are the first line of defense against invading respiratory pathogens, and their effectiveness at preventing infection and disease depends heavily on what antigens are targeted. For both influenza A virus and severe acute respiratory coronavirus 2 (SARS-CoV-2), antibodies directed at the receptor-binding surface proteins, hemagglutinin (HA) and spike, respectively, are known to neutralize virus infection and are associated with protection [1–5]. Antibodies targeting non-neutralizing epitopes on many different viral proteins also play important roles in reducing the length and severity of disease and tend to target more conserved regions than most neutralizing antibodies [1,5–11]. Here, I present my work aimed at understanding the development and interaction of neutralizing and non-neutralizing antibodies with two respiratory viruses, influenza A virus and SARS-CoV-2.First, I test the durability of antibody binding to the stem domain of HA after the accumulation of mutations naturally over 17 years in both the H1 and H3 lineages. To accomplish this, I created chimeric HA proteins combining the head domain of an avian influenza virus to which humans have not been exposed with the HA stem domain of seasonal influenza strains spanning 17 years of sequence evolution [12–16]. This allowed me to measure serum antibody binding specifically to the HA stem domain. I measured the binding of human sera collected concurrent to when the first seasonal stem strain I tested was circulating and compared that to the binding of the same sera to the stem from a seasonal strain circulating 17 years later. Based on these measurements for a number of human serum samples, we find that HA stem antibody binding wanes at a similar rate for both H1 and H3 HAs despite a faster rate of sequence evolution in the H3 lineage.In a separate set of experiments, I describe a new assay for measuring the potency of antibodies targeting the influenza A virus receptor-cleaving protein neuraminidase (NA). Our method allows for the testing of anti-NA antibodies in a format similar to traditional neutralization assays for antibodies targeting (HA). Briefly, we paired a modified HA that can perform membrane fusion but cannot bind to cellular receptors and NA containing the mutation G147R which confers the receptor-binding function to NA [17,18]. This creates a virus that is dependent on NA for attachment to cells and can thus be neutralized by anti-NA antibodies. We found that this method works best for antibodies targeting sites near the catalytic active site of NA. We were able to select for an escape mutation with one such monoclonal antibody demonstrating the utility of our methods for mapping anti-NA antibody epitopes.Finally, I address the pressing issue of antibody durability in the context of the ongoing SARS-CoV-2 pandemic by measuring neutralizing and non-neutralizing antibody responses in children for up to 52 weeks following infection. In collaboration with Seattle Children’s hospital, we performed pseudoneutralization [3,19,20] and the Abbott Laboratories SARS-CoV-2 IgG assay on samples collected from 32 children at approximately 4- and 24- weeks post-symptom onset. For both neutralizing anti-spike antibody titers and anti-nucleocapsid (N) antibodies, we observed a high degree of variability from participant to participant. Overall, neutralization titers changed very little over the observation period, while anti-N levels decrease substantially. When compared to a separate, previously characterized, adult cohort [3], we find age-related differences in the antibody responses. Specifically, children tend to have lower neutralizing antibody titers than adults early following infection that become similar to adults in the following six months. Strikingly, anti-nucleocapsid antibodies are much lower in children than adults and wane faster in children over time.