A novel beta coronavirus that emerged in late December 2019 triggered a global pandemic. Diagnostic methods for rapid identification of infected individuals were established in new biotechnological approaches. Vaccine production and application to individuals and measurement of SARS-CoV-2 antibodies also began. Serum samples from 240 health care workers were collected at three-month intervals over nine months. Indirect SARS-CoV-2 nucleocapsid IgG ELISA tests were used to identify humoral immune responses. All seropositive individuals and those with borderline ELISA values were tested with a specifically generated multipanel nucleocapsid fragment immunoblot. Of the 240 individuals, 24 showed seroconversion in ELISA after experiencing COVID-19. All of them showed a positive reaction against the full-length nucleocapsid protein in the immunoblot. The highest reactivity was seen either against fragment N(100–300) or in a minority against the posterior part N(200–419). In general, the staining pattern of COVID-19 patients showed four phenotypes. In contrast, three individuals classified as borderline by ELISA reacted exclusively with fragments N(1–220) and N(100–300) containing the octamer amino acid sequence FYYLGTGP, which is identical in human coronaviruses sharing this sequence with SARS-CoV-2. These represent a unique and thus fifth phenotype. This work suggests the existence of distinct phenotypic patterns of IgG production towards N-protein subdomains.

First-generation vaccines against SARS-CoV-2 do not provide adequate immune protection. Therefore, we engineered a divalent gene construct combining the receptor-binding domain (RBD) of the spike protein and the immunodominant region of the viral nucleocapsid. This fusion protein was produced in either E. coli or a recombinant baculovirus system. Subsequently, the fusion protein was mixed with adjuvant and administered to mice in a prime-booster mode. Mice (72%) produced an IgG response against both proteins (titer: 10⁻⁴–10⁻⁵) 14 days after the first booster injection, which was increased to 100% by a second booster. Comparable IgG responses were detected against the delta, gamma and omicron variants of the RBD region. Durability testing revealed IgGs beyond 90 days. In addition, cytolytic effector cell molecules were increased in lymphocytes isolated from peripheral blood. Ex vivo stimulation of T cells by nucleocapsid and RBD peptides showed antigen-specific upregulation of CD44 among the CD4⁺ and CD8⁺ T cells of vaccinated mice. No side effect was documented in the central nervous system. Cumulatively, these data represent a proof-of-principle approach alternative to existing mRNA vaccination strategies.

SARS-CoV-2 variants of concern (VOCs) have caused a significant increase in infections worldwide. Despite high vaccination rates in industrialized countries, the fourth VOC, Omicron, has outpaced the Delta variant and is causing breakthrough infections in individuals with two booster vaccinations. While the magnitude of morbidity and lethality is lower in Omicron, the infection rate and global spread are rapid. Using a specific IgG multipanel-ELISA with the spike protein’s receptor-binding domain (RBD) from recombinant Alpha, Gamma, Delta, and Omicron variants, sera from health-care workers from the Medical University of Vienna were tested pre-pandemic and post-vaccination (BNT162b2; ChAdOx1 nCoV-19). The cohort was continuously monitored by SARS-CoV-2 testing and commercial nucleocapsid IgG ELISA. RBD IgG ELISA showed significantly lower reactivity against the Omicron-RBD compared to the Alpha variant in all individuals (p < 0.001). IgG levels were independent of sex, but were significantly higher in BNT162b2 recipients <45 years of age for Alpha, Gamma, and Delta (p < 0.001; p = 0.040; p = 0.004, respectively). Pre-pandemic cross-reactive anti-Omicron IgG was detected in 31 individuals and was increased 8.78-fold after vaccination, regardless of vaccine type. The low anti-RBD Omicron IgG level could explain the breakthrough infections and their presence could also contribute to a milder COVID-19 course by cross-reactivity and broadening the adaptive immunity.