Development of a Quantitative Assay for the Characterization of Human Collectin-11 (CL-11, CL-K1)

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29–18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2–459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan

Humoral and cellular immune responses eleven months after the third dose of BNT162b2 an mRNA-based COVID-19 vaccine in people with HIV – a prospective observational cohort study

BACKGROUND: We investigated long-term durability of humoral and cellular immune responses to third dose of BNT162b2 in people with HIV (PWH) and controls. METHODS: In 378 PWH with undetectable viral replication and 224 matched controls vaccinated with three doses of BNT162b2, we measured IgG-antibodies against the receptor binding domain of SARS-CoV-2 spike protein three months before third dose of BNT162b2, and four and eleven months after. In 178 PWH and 135 controls, the cellular response was assessed by interferon-γ (IFN-γ) release in whole blood four months after third dose. Differences in antibody or IFN-γ concentrations were assessed by uni- and multivariable linear regressions. FINDINGS: Before the third dose the concentration of SARS-CoV-2 antibodies was lower in PWH than in controls (unadjusted geometric mean ratio (GMR): 0.68 (95% CI: 0.54-0.86, p = 0.002). We observed no differences in antibody concentrations between PWH and controls after four (0.90 (95% CI: 0.75-1.09), p = 0.285) or eleven months (0.89 (95% CI: 0.69-1.14), p = 0.346) after the third dose. We found no difference in IFN-γ concentrations four months after the third dose between PWH and controls (1.06 (95% CI: 0.71-1.60), p = 0.767). INTERPRETATION: We found no differences in antibody concentrations or cellular response between PWH and controls up to eleven months after third dose of BNT162b2. Our findings indicate that PWH with undetectable viral replication and controls have comparable immune responses to three doses of the BNT162b2 vaccine. FUNDING: This work was funded by the Novo Nordisk Foundation (NFF205A0063505, NNF20SA0064201), the Carlsberg Foundation (CF20-476 0045), the Svend Andersen Research Foundation (SARF2021), and Bio- and Genome Bank Denmark

SARS-CoV-2 Antibodies Mediate Complement and Cellular Driven Inflammation

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to constitute a serious public health threat worldwide. Protective antibody-mediated viral neutralization in response to SARS-CoV-2 infection has been firmly characterized. Where the effects of the antibody response are generally considered to be beneficial, an important biological question regarding potential negative outcomes of a SARS-CoV-2 antibody response has yet to be answered. We determined the distribution of IgG subclasses and complement activation levels in plasma from convalescent individuals using in-house developed ELISAs. The IgG response towards SARS-CoV-2 receptor-binding domain (RBD) after natural infection appeared to be mainly driven by IgG1 and IgG3 subclasses, which are the main ligands for C1q mediated classical complement pathway activation. The deposition of the complement components C4b, C3bc, and TCC as a consequence of SARS-CoV-2 specific antibodies were depending primarily on the SARS-CoV-2 RBD and significantly correlated with both IgG levels and disease severity, indicating that individuals with high levels of IgG and/or severe disease, might have a more prominent complement activation during viral infection. Finally, freshly isolated monocytes and a monocyte cell line (THP-1) were used to address the cellular mediated inflammatory response as a consequence of Fc-gamma receptor engagement by SARS-CoV-2 specific antibodies. Monocytic Fc gamma receptor charging resulted in a significant rise in the secretion of the pro-inflammatory cytokine TNF-α. Our results indicate that SARS-CoV-2 antibodies might drive significant inflammatory responses through the classical complement pathway and via cellular immune-complex activation that could have negative consequences during COVID-19 disease. We found that increased classical complement activation was highly associated to COVID-19 disease severity. The combination of antibody-mediated complement activation and subsequent cellular priming could constitute a significant risk of exacerbating COVID-19 severity

Humoral and cellular immune responses eleven months after the third dose of BNT162b2 an mRNA-based COVID-19 vaccine in people with HIV – a prospective observational cohort studyResearch in context

Summary: Background: We investigated long-term durability of humoral and cellular immune responses to third dose of BNT162b2 in people with HIV (PWH) and controls. Methods: In 378 PWH with undetectable viral replication and 224 matched controls vaccinated with three doses of BNT162b2, we measured IgG-antibodies against the receptor binding domain of SARS-CoV-2 spike protein three months before third dose of BNT162b2, and four and eleven months after. In 178 PWH and 135 controls, the cellular response was assessed by interferon-γ (IFN-γ) release in whole blood four months after third dose. Differences in antibody or IFN-γ concentrations were assessed by uni- and multivariable linear regressions. Findings: Before the third dose the concentration of SARS-CoV-2 antibodies was lower in PWH than in controls (unadjusted geometric mean ratio (GMR): 0.68 (95% CI: 0.54–0.86, p = 0.002). We observed no differences in antibody concentrations between PWH and controls after four (0.90 (95% CI: 0.75–1.09), p = 0.285) or eleven months (0.89 (95% CI: 0.69–1.14), p = 0.346) after the third dose. We found no difference in IFN-γ concentrations four months after the third dose between PWH and controls (1.06 (95% CI: 0.71–1.60), p = 0.767). Interpretation: We found no differences in antibody concentrations or cellular response between PWH and controls up to eleven months after third dose of BNT162b2. Our findings indicate that PWH with undetectable viral replication and controls have comparable immune responses to three doses of the BNT162b2 vaccine. Funding: This work was funded by the Novo Nordisk Foundation (NFF205A0063505, NNF20SA0064201), the Carlsberg Foundation (CF20-476 0045), the Svend Andersen Research Foundation (SARF2021), and Bio- and Genome Bank Denmark

Previous immunity shapes immune responses to SARS-CoV-2 booster vaccination and Omicron breakthrough infection risk

Abstract The heterogeneity of the SARS-CoV-2 immune responses has become considerably more complex over time and diverse immune imprinting is observed in vaccinated individuals. Despite vaccination, following the emergence of the Omicron variant, some individuals appear more susceptible to primary infections and reinfections than others, underscoring the need to elucidate how immune responses are influenced by previous infections and vaccination. IgG, IgA, neutralizing antibodies and T-cell immune responses in 1,325 individuals (955 of which were infection-naive) were investigated before and after three doses of the BNT162b2 vaccine, examining their relation to breakthrough infections and immune imprinting in the context of Omicron. Our study shows that both humoral and cellular responses following vaccination were generally higher after SARS-CoV-2 infection compared to infection-naive. Notably, viral exposure before vaccination was crucial to achieving a robust IgA response. Individuals with lower IgG, IgA, and neutralizing antibody responses postvaccination had a significantly higher risk of reinfection and future Omicron infections. This was not observed for T-cell responses. A primary infection before Omicron and subsequent reinfection with Omicron dampened the humoral and cellular responses compared to a primary Omicron infection, consistent with immune imprinting. These results underscore the significant impact of hybrid immunity for immune responses in general, particularly for IgA responses even after revaccination, and the importance of robust humoral responses in preventing future infections