Distinct anti-NP, anti-RBD and anti-Spike antibody profiles discriminate death from survival in COVID-19.

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

Distinct anti-NP, anti-RBD and anti-spike antibody profiles discriminate death from survival in COVID-19

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Anti-SARS-CoV-2 equine F (Ab′)2 immunoglobulin as a possible therapy for COVID-19

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab′)2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab′)2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56°C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100°C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20°C). At frozen temperatures, large aggregates (>200nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56°C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100°C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20°C). At frozen temperatures, large aggregates (>200nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Utilização da sílica nanoestruturada SBA-15 como adjuvante em imunizações com a vacina para hepatite B / Nanostructured SBA-15 silica as an adjuvant in immunizations with hepatitis B vaccine

Objective: To evaluate the applicability of SBA-15 silica as an adjuvant in immunizations with purified particles of the viral protein HBsAg, the main component of hepatitis B vaccine, Butang®, produced by Instituto Butantan. Methods: BALB/c mice orally or subcutaneously received 0.5 mug of HBsAg adsorbed/encapsulated to SBA-15 or adsorbed to Al(OH)3. To assess the secondary immune response, a subcutaneous booster was administered 30 days after the first immunization. Individual serum and fecal samples of each group were periodically collected for specific antibody titration by ELISA. Results: Analysis of secretory IgA showed that mice orally primed with HBsAg on SBA-15 had increased levels of specific antibodies in primary and secondary immune responses. Specific serum IgA and IgG titers in HBsAg:SBA-15-orally immunized mice reached higher levels after the booster, demonstrating the effectiveness of oral vaccination with the use of silica. All immunized groups showed higher IgG1 levels. Conclusion: Our results clearly indicate the promising use of SBA-15 as an adjuvant, especially in oral immunizations.

Objetivo: Demonstrar a aplicabilidade da sílica do tipo SBA-15 como adjuvante nas imunizações com a proteína recombinante HBsAg do vírus da hepatite B, principal componente da vacina Butang® produzida pelo Instituto Butantan. Métodos: Camundongos BALB/c receberam, pela via oral ou subcutânea, 0,5 mig do HbsAg adsorvido/encapsulado à SBA-15 ou adsorvido ao Al(OH)3. Para avaliar a resposta imune secundária, uma dose de reforço foi administrada subcutaneamente 30 dias após a primeira imunização. Amostras individuais de soro e fezes foram coletadas periodicamente para titulação de anticorpos específicos por ELISA. Resultados: A análise de IgA secretada mostrou que camundongos imunizados pela via oral com HbsAg em SBA-15 apresentaram aumento nos níveis de anticorpos específicos nas respostas primária e secundária. Ainda, após o reforço, observaram-se maiores níveis de IgA e IgG séricas anti-HBsAg no grupo preparado com HBsAg:SBA-15 pela via oral. Todos os grupos imunizados apresentaram maior produção de IgG1. Conclusão: Os resultados indicam o uso promissor da sílica SBA-15 como adjuvante, especialmente nas imunizações pela via oral.

Nanostructured SBA-15 silica as an adjuvant in immunizations with hepatitis B vaccine.

OBJECTIVE: To evaluate the applicability of SBA-15 silica as an adjuvant in immunizations with purified particles of the viral protein HBsAg, the main component of hepatitis B vaccine, Butang®, produced by Instituto Butantan. METHODS: BALB/c mice orally or subcutaneously received 0.5 µg of HBsAg adsorbed/encapsulated to SBA-15 or adsorbed to Al(OH)3. To assess the secondary immune response, a subcutaneous booster was administered 30 days after the first immunization. Individual serum and fecal samples of each group were periodically collected for specific antibody titration by ELISA. RESULTS: Analysis of secretory IgA showed that mice orally primed with HBsAg on SBA-15 had increased levels of specific antibodies in primary and secondary immune responses. Specific serum IgA and IgG titers in HBsAg:SBA-15-orally immunized mice reached higher levels after the booster, demonstrating the effectiveness of oral vaccination with the use of silica. All immunized groups showed higher IgG1 levels. CONCLUSION: Our results clearly indicate the promising use of SBA-15 as an adjuvant, especially in oral immunizations.