Obtaining a high titer of polyclonal antibodies from rats to the SARS-CoV-2 nucleocapsid protein and its N- and C-terminal domains for diagnostic test development

Author(s)

de Almeida, Michelle Teixeira ; Barbosa, Ana Paula ; Bomfim, Camila Gasque ; Visnardi, Aline Biazola ; Vinces, Tania Churasacari ; Ceroni, Alexandre ; Durigon, Edison Luiz ; Guzzo, Cristiane Rodrigues

• Document type: Journal article
• Source: JOURNAL OF IMMUNOLOGICAL METHODS; v. 522, p. 9-pg., 2023-09-27.

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped, plus-stranded RNA virus responsible for the Coronavirus Disease 2019 (COVID-19). Patients infected with COVID-19 may be asymptomatic or have symptoms ranging from mild manifestations to severe cases of the disease that could lead to death. The SARS-CoV-2 genome encodes 4 structural proteins, including the Spike protein (S), the Nucleocapsid protein (N), Membrane protein (M) and, the Envelope protein (E). The N protein forms a major component of the ribonucleoprotein complex within the virus particle and play a vital role in its transcription and replication. Nevertheless, the S protein was the most important protein in the development of vaccines against COVID-19. However, the decrease in number of registered immunizations against the disease and the rapid drop in neutralizing antibody titers together with looser preventive measures for virus transmission, favored the rapid appearance of new variants of concerns (VOCs) that primarily show mutations in the S protein. This fact makes the N protein a good candidate for the development of diagnostic tests, due to its stability, amino acid conservation, high immunogenicity, and the smaller likelihood of mutation. With the aim of developing a new diagnostic kit based on the N protein, we evaluated the humoral response in female Wistar rats against this target. Three constructions of the N protein were used to inoculate the animals: the full-length protein (Cfull), the N(NTD), and the C-terminal (CTD) portion of the protein. The immunizations induced the animal's immune response, with specific polyclonal IgG antibodies against the Cfull protein and its fragments. There were not nonspecific bind to the protein used as negative control. Anti-Cfull antibodies demonstrated high efficiency in binding to the NTD protein and the antibodies present in the anti-CTD and anti-NTD sera have recognized the Cfull protein, but they were not able to recognize the NTD and CTD proteins, respectively. Our results indicate an efficient protocol for obtaining high antibody titers against the N recombinant protein of SARS-CoV-2 and its fragments highlighting the Cfull protein, which can be used in the development of new diagnostic kits. (AU)

• FAPESP's process: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center
• Grantee: Shaker Chuck Farah
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 19/21739-0 - Antimicrobial activity of proteins secreted by Xanthomonas citri against microorganisms of medical interest
• Grantee: Camila Gasque Bomfim
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 22/08730-7 - Protein structural biology applied in the study of c-di-GMP signaling and bacterial secretion systems
• Grantee: Cristiane Rodrigues Guzzo Carvalho
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/14914-8 - Characterization of LIC_11920 and its role in c-di-GMP signaling
• Grantee: Tania Geraldine Churasacari Vinces
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training
• FAPESP's process: 19/00195-2 - Understanding the c-di-GMP signaling networks and the Type II secretion system present into the human pathogen Leptospira interrogans Copenhageni
• Grantee: Cristiane Rodrigues Guzzo Carvalho
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/09047-8 - Comparative genomics of bacterial toxins associated with type IV secretion systems
• Grantee: Robson Francisco de Souza
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/04680-0 - Design of different fragments of SARS-CoV-2 surface protein spike for the development of fast diagnostic test and vaccine
• Grantee: Cristiane Rodrigues Guzzo Carvalho
• Support Opportunities: Regular Research Grants