The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Lasting immunity will be critical for overcoming COVID-19. However, the factors associated with the development of high titers of anti-SARS-CoV-2 Abs and how long those Abs persist remain incompletely defined. In particular, an understanding of the relationship between COVID-19 symptoms and anti-SARS-CoV-2 Abs is limited. To address these unknowns, we quantified serum anti-SARS-CoV-2 Abs in clinically diverse COVID-19 convalescent human subjects 5 wk (n 5 113) and 3 mo (n 5 79) after symptom resolution with three methods: a novel multiplex assay to quantify IgG against four SARS-CoV-2 Ags, a new SARS-CoV-2 receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralizing assay. We then identified clinical and demographic factors, including never-before-assessed COVID-19 symptoms, that consistently correlate with high anti-SARS-CoV-2 Ab levels. We detected anti-SARS-CoV-2 Abs in 98% of COVID-19 convalescent subjects 5 wk after symptom resolution, and Ab levels did not decline at 3 mo. Greater disease severity, older age, male sex, higher body mass index, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 Ab levels. Moreover, we report for the first time (to our knowledge) that COVID-19 symptoms, most consistently fever, body aches, and low appetite, correlate with higher anti-SARS-CoV-2 Ab levels. Our results provide robust and new insights into the development and persistence of anti-SARS-CoV-2 Abs. ImmunoHorizons, 2021, 5: 466-476.

The consequences of past COVID-19 infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. We evaluated anti-membrane antibodies in COVID-19 naïve, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. We found that anti-membrane antibodies persist for at least a year and are a sensitive and specific marker of past COVID-19 infection. Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naïve states to advance public health and research.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), orf3a, and orf8 proteins are also humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here we show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which one epitope achieved near-perfect diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that anti-SARS-CoV-2 antibodies appear to bind homologous peptide sequences in the 6 known human CoVs. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins, especially M, which should be considered in diagnostic, vaccine, and therapeutic development.

Neutrophils (PMN) regulate inflammation in many ways, including communication with other immune cells via extracellular vesicles (EVs). EVs released by human neutrophils activated with N‐formylmethionyl‐leucyl‐phenylalanine (fMLF) (PMN‐fMLF EVs) had an outside‐out orientation and contained functionally important neutrophil plasma membrane proteins, including flavocytochrome b558, and enzymatically active granule proteins, elastase, and myeloperoxidase. Treatment of naïve PMN with PMN‐fMLF EVs primed fMLF‐stimulated NADPH oxidase activity, increased surface expression of the complement receptors CD11b/CD18 and CD35, the specific granule membrane protein CD66, and flavocytochrome b558, and promoted phagocytosis of serum‐opsonized Staphylococcus aureus. The primed oxidase activity reflected increased surface expression of flavocytochrome b558 and phosphorylation of SER345 in p47phox, two recognized mechanisms for oxidase priming. Taken together, these data demonstrate that stimulated PMN released EVs that altered the phenotype of naïve phagocytes by priming of the NADPH oxidase activity and augmenting phagocytosis, two responses that are integral to optimal PMN host defense.