Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

Development and Immunogenicity of a Brazilian Glycoconjugate vaccine against Meningococcal W in a Pilot Scale.

Recent changes in the epidemiology of meningococcal have been reported and meningococcal group W (MenW) has become the third most prevalent group isolated in Brazil in the last 10 years. In this study we have developed a conjugate vaccine for MenW using a modified reductive amination conjugation method through a covalent linkage between periodate-oxidized MenW non-O-acetylated polysaccharide and hydrazide-activated monomeric tetanus toxoid. Process control of bulks was done by physicochemical analysis including polysaccharide and protein quantification, high performance liquid chromatography - size exclusion chromatography, capillary electrophoresis, and hydrogen nuclear magnetic resonance. Conjugate bulks were best produced with concentration of polysaccharide twice as high as protein, at room temperature, and pH approximately 6.0. A scaled-up bulk (100 mg scale) was formulated and inoculated intramuscularly in mice in a dose-response study (0.1, 0.5, 1.0 and 10.0 µg of polysaccharide/dose). The immunogenicity of conjugate bulks was determined by serum bactericidal assay and ELISA assays of serum from immunized mice. ELISA and SBA titers revealed high titers of IgG and demonstrated the functionality of the antibodies produced in all doses studied 15 days after the third dose. However, significant differences were observed among them by ELISA. In conclusion, this study established the best conditions to produce MenW conjugate bulks and showed the efficacy of the obtained conjugate bulk in induce a good immune response in mice. Further experiments will need to be done to scale up the conjugation reaction and then allow the use of this conjugate in clinical trials.

Initial development of biosimilar immune checkpoint blockers using HEK293 cells.

Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.

Brazilian meningococcal C conjugate vaccine: physicochemical, immunological, and thermal stability characteristics.

High temperature is known to cause some instability in polysaccharide-protein conjugated vaccines and studies under stress conditions may be useful in determining whether short-term accidental exposure to undesired conditions can compromise product quality. In this study, we examined the structural stability of three industrial batches of Brazilian Meningococcal C conjugate bulk (MPCT) incubated at 4, 37, and 55 °C for 5 weeks. The effect of exposure to the storage temperatures was monitored by HPLC-SEC, CZE, CD and NMR techniques. The immunological significance of any physicochemical changes observed in MPCT was determined by SBA and ELISA assays of serum from immunized mice. Fluorescence emission spectra at 4 and 37 °C were similar among all samples and compatible with the native fold of the carrier protein. Fluorescence spectra of MPCT stored at 55 °C decreased in intensity and had a significant red-shift, indicating conformational changes. Far-UV CD spectra revealed a trend toward loss of structural conformation as storage temperature was increased to 55 °C. The NMR data showed modified signal intensity of the aromatic and aliphatic residues, mainly for samples incubated at 55 °C, suggesting a partial loss of tertiary structure. About 50% free saccharide content was found in bulks stored at 55 °C, but no difference was observed in the IgG or SBA titers. The present study showed physicochemical methods alone are insufficient to predict the biological activity of a MPCT conjugate vaccine without extensive validation against immunological data. However, they provide a sensitive means of detecting changes induced in a vaccine exposed to adverse environmental condition.

Brazilian meningococcal C conjugate vaccine: Scaling up studies.

Several outbreaks caused by Neisseria meningitidis group C have been occurred in different regions of Brazil. A conjugate vaccine for Neisseria meningitidis was produced by chemical linkage between periodate-oxidized meningococcal C polysaccharide and hydrazide-activated monomeric tetanus toxoid via a modified reductive amination conjugation method. Vaccine safety and immunogenicity tested in Phase I and II trials showed satisfactory results. Before starting Phase III trials, vaccine production was scaled up to obtain industrial lots under Good Manufacture Practices (GMP). Comparative analysis between data obtained from industrial and pilot scales of the meningococcal C conjugate bulk showed similar execution times in the scaling up production process without significant losses or alterations in the quality attributes of purified compounds. In conclusion, scale up was considered satisfactory and the Brazilian meningococcal conjugate vaccine production aiming to perform Phase III trials is feasible.

Determination of hydrazine in a meningococcal C conjugate vaccine intermediary product.

In Brazil, polysaccharide-protein conjugate vaccine against Neisseria meningitidis group C (MenCPS-TT) using hydrazine-activated-tetanus toxoid (TT) as a carrier protein has been developed. Because of the toxicity of hydrazine in humans, it is necessary to monitor this substance's process control step during the vaccine production. The electroanalytical methodology was developed and validated for the determination of hydrazine during the process control of MenCPS-TT vaccine production by differential pulse polarography. The reduction potential was -0.95 V in acetone and sulphuric acid solution. The method presented linear range between 30 and 150 microgL(-1)and recovery of 93.5+/-0.8%.