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The prediction of the durability of immunity against COVID-19 is relevant, and longitudinal studies are essential for unraveling the details regarding protective SARS‐CoV‐2 antibody responses. It has become challenging to discriminate between COVID-19 vaccine- and infection-induced immune responses since all approved vaccines in Europe and the USA are based on the viral spike (S) protein, which is also the most commonly used antigen in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a nucleocapsid (N) protein-based sandwich ELISA for detecting pan anti-SARS-CoV-2 Ig with a sensitivity and specificity of 97%. Generalized mixed models were used to determine the degree of long‐term humoral immunity against the N protein and the receptor-binding domain (RBD) of the S protein in a cohort of infected individuals to distinguish between COVID-19 vaccine- and infection-induced immunity. N-specific waning could be observed in individuals who did not experience reinfection, while individuals who experienced reinfection had a new significant increase in N-specific Ig levels. In individuals that seroconverted without a reinfection, 70.1% remained anti-N seropositive after 550 days. The anti-RBD Ig dynamics were unaffected by reinfection but exhibited a clear increase in RBD-specific Ig when vaccination was initiated. In conclusion, a clear difference in the dynamics of the antibody response against N protein and RBD was observed over time. Anti-N protein-specific Igs can be detected up to 18 months after SARS-CoV-2 infection allowing long-term discrimination of infectious and vaccine antibody responses., The prediction of the durability of immunity against COVID-19 is relevant, and longitudinal studies are essential for unraveling the details regarding protective SARS‐CoV‐2 antibody responses. It has become challenging to discriminate between COVID-19 vaccine- and infection-induced immune responses since all approved vaccines in Europe and the USA are based on the viral spike (S) protein, which is also the most commonly used antigen in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a nucleocapsid (N) protein-based sandwich ELISA for detecting pan anti-SARS-CoV-2 Ig with a sensitivity and specificity of 97%. Generalized mixed models were used to determine the degree of long‐term humoral immunity against the N protein and the receptor-binding domain (RBD) of the S protein in a cohort of infected individuals to distinguish between COVID-19 vaccine- and infection-induced immunity. N-specific waning could be observed in individuals who did not experience reinfection, while individuals who experienced reinfection had a new significant increase in N-specific Ig levels. In individuals that seroconverted without a reinfection, 70.1% remained anti-N seropositive after 550 days. The anti-RBD Ig dynamics were unaffected by reinfection but exhibited a clear increase in RBD-specific Ig when vaccination was initiated. In conclusion, a clear difference in the dynamics of the antibody response against N protein and RBD was observed over time. Anti-N protein-specific Igs can be detected up to 18 months after SARS-CoV-2 infection allowing long-term discrimination of infectious and vaccine antibody responses. IMPORTANCE Longitudinal studies are essential to unravel details regarding the protective antibody responses after COVID-19 infection and vaccination. It has become challenging to distinguish long-term immune responses to SARS-CoV-2 infection and vaccination since most approved vaccines are based on the viral spike (S) protein, which is also

Background Factors influencing SARS-CoV-2 antibody dynamics, transmission, waning and long COVID-19 symptomatology are still not fully understood. Methods In the Danish section of the Novo Nordisk Group, we performed a prospective seroepidemiological study during the first and second waves of the COVID-19 pandemic. All employees and their household members (>18 years) were invited to participate in a baseline (June–August 2020), 6-month follow-up (December 2020–January 2021), and 12-month follow-up (August 2021) sampling. In total, 18,614 accepted and provided at least one blood sample and completed a questionnaire regarding socioeconomic background, health status, previous SARS-CoV-2 infection, and persistent symptoms. Total antibody and specific IgM, IgG and IgA levels against recombinant receptor binding domain were tested. Results At baseline, the SARS-CoV-2-antibody seroprevalence was 3.9%. At 6-month follow-up, the seroprevalence was 9.1%, while at 12-month follow-up, the seroprevalence was 94.4% (after the vaccine roll-out). Male sex and younger age (18–40 years) were significant risk factors for seropositivity. From baseline to the 6-month sampling, we observed a substantial waning of IgM, IgG and IgA levels (p < 0.001), regardless of age, sex and initial antibody level. An increased antibody level was found in individuals infected prior to vaccination compared to vaccinated infection naïves (p < 0.0001). Approximately a third of the seropositive individuals reported one or more persistent COVID-19 symptoms, with anosmia and/or ageusia (17.5%) and fatigue (15.3%) being the most prevalent. Conclusion The study provides a comprehensive insight into SARS-CoV-2 antibody seroprevalence following infection and vaccination, waning, persistent COVID-19 symptomatology and risk factors for seropositivity in large working environments., Background: Factors influencing SARS-CoV-2 antibody dynamics, transmission, waning and long COVID-19 symptomatology are still not fully understood. Methods: In the Danish section of the Novo Nordisk Group, we performed a prospective seroepidemiological study during the first and second waves of the COVID-19 pandemic. All employees and their household members (>18 years) were invited to participate in a baseline (June–August 2020), 6-month follow-up (December 2020–January 2021), and 12-month follow-up (August 2021) sampling. In total, 18,614 accepted and provided at least one blood sample and completed a questionnaire regarding socioeconomic background, health status, previous SARS-CoV-2 infection, and persistent symptoms. Total antibody and specific IgM, IgG and IgA levels against recombinant receptor binding domain were tested. Results: At baseline, the SARS-CoV-2-antibody seroprevalence was 3.9%. At 6-month follow-up, the seroprevalence was 9.1%, while at 12-month follow-up, the seroprevalence was 94.4% (after the vaccine roll-out). Male sex and younger age (18–40 years) were significant risk factors for seropositivity. From baseline to the 6-month sampling, we observed a substantial waning of IgM, IgG and IgA levels (p < 0.001), regardless of age, sex and initial antibody level. An increased antibody level was found in individuals infected prior to vaccination compared to vaccinated infection naïves (p < 0.0001). Approximately a third of the seropositive individuals reported one or more persistent COVID-19 symptoms, with anosmia and/or ageusia (17.5%) and fatigue (15.3%) being the most prevalent. Conclusion: The study provides a comprehensive insight into SARS-CoV-2 antibody seroprevalence following infection and vaccination, waning, persistent COVID-19 symptomatology and risk factors for seropositivity in large working environments.

Background: The durability of SARS-CoV-2 antibody response and the resulting immunity to COVID-19 is unclear. Objectives: To investigate long-term humoral immunity to SARS-CoV-2. Methods: In this nationwide, longitudinal study, we determined antibody response in 411 patients aged 0–93 years from two waves of infections (March to December 2020) contributing 1063 blood samples. Each individual had blood drawn on 4–5 occasions 1–15 months after disease onset. We measured total anti-SARS-CoV-2 receptor-binding domain (RBD) antibody using a qualitative RBD sandwich ELISA, IgM, IgG and IgA levels using an quantitative in-house ELISA-based assay and neutralizing antibodies (NAbs) using an in-house ELISA-based pseudoneutralizing assay. IgG subclasses were analyzed in a subset of samples by ELISA-based assay. We used nonlinear models to study the durability of SARS-CoV-2 antibody responses and its influence over time. Results: After 15 months, 94% still had detectable circulating antibodies, mainly the IgG isotype, and 92% had detectable NAbs. The distribution of IgG antibodies varied significantly over time, characterized by a biphasic pattern with an initial decline followed by a plateau after approximately 7 months. However, the NAbs remained relatively stable throughout the period. The strength of the antibody response was influenced by smoking and hospitalization, with lower IgG levels in smokers and higher levels in hospitalized individuals. Antibody stability over time was mainly associated with male sex and older age with higher initial levels but more marked decrease. Conclusions: The humoral immune response to SARS-CoV-2 infection varies depending on behavioral factors and disease severity, and antibody stability over 15 months was associated with sex and age.

Background: People with HIV (PWH) are at increased risk of severe COVID-19. We aimed to determine humoral responses in PWH and controls who received two doses of BNT162b2. Methods: In 269 PWH and 538 age-matched controls, we measured IgG and neutralizing antibodies specific for the receptor-binding domain of SARS-CoV-2 at baseline, 3 weeks and 2 months after the first dose of BNT162b2. Results: IgG antibodies increased from baseline to 3 weeks and from 3 weeks to 2 months in both groups, but the concentrations of IgG antibodies were lower in PWH than that in controls at 3 weeks and 2 months (p = 0.025 and <0.001), respectively. The IgG titres in PWH with a humoral response at 2 months were 77.9% (95% confidence interval [62.5%–97.0%], age- and sex-adjusted p = 0.027) of controls. Conclusions: Reduced IgG antibody response to vaccination with BNT162b2 was found in PWH, and thus increased awareness of breakthrough infections in PWH is needed.

The recent identification and rise to dominance of the P.1 and B.1.351 SARS-CoV-2 variants have brought international concern because they may confer fitness advantages. The same three positions in the receptor-binding domain (RBD) are affected in both variants, but where the 417 substitution differs, the E484K/N501Y have co-evolved by convergent evolution. Here we characterize the functional and immune evasive consequences of the P.1 and B.1.351 RBD mutations. E484K and N501Y result in gain-of-function with two different outcomes: The N501Y confers a ten-fold affinity increase towards ACE-2, but a modest antibody evasion potential of plasma from convalescent or vaccinated individuals, whereas the E484K displays a significant antibody evasion capacity without a major impact on affinity. On the other hand, the two different 417 substitutions severely impair the RBD/ACE-2 affinity, but in the combined P.1 and B.1.351 RBD variants, this effect is partly counterbalanced by the effect of the E484K and N501Y. Our results suggest that the combination of these three mutations is a two-step forward and one step back in terms of viral fitness.

Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.

The ficolins are an important family of pattern recognition molecules which contributes to complement activation via the lectin pathway. How the ficolins respond to inflammatory stimuli remains only partly understood. In the present study, we investigated the ficolin A and ficolin B expression and protein distribution patterns in mice during LPS-induced mouse model of systemic inflammation. The tissue-specific expression of ficolin A and B was determined by real time PCR. Furthermore, ficolin protein levels in serum and bone marrow extracts was determined in LPS challenged mice by novel in-house developed sandwich ELISAs. The newly developed assays exhibited great specificity and can be used for reliable quantification of murine ficolin protein levels in the future. In agreement with previous observations, ficolin A was mainly expressed in liver and spleen. However, our data also suggested that ficolin A is expressed in bone marrow, which was the main site of ficolin B expression. Ficolin A and B expression in the investigated tissues increased after stimulation with LPS. This was followed by a downregulation of expression, causing mRNA levels to reach baseline 24 hours post LPS challenge. Protein levels appeared to follow the same pattern as the mRNA profiles, with an exception of ficolin B protein levels in serum, which kept increasing for 24 hours. The results collectively demonstrate that systemic inflammation due to LPS challenge can induce a significant ficolin response. The data suggest that the murine ficolins are acute phase reactants with an increase in both mRNA and protein levels upon induced systemic inflammation.