Fusion Protein of Rotavirus VP6 and SARS-CoV-2 Receptor Binding Domain Induces T Cell Responses

Vaccines based on mRNA and viral vectors are currently used in the frontline to combat the ongoing pandemic caused by the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, there is still an urgent need for alternative vaccine technologies inducing/boosting long-lasting and cross-reactive immunity in different populations. As a possible vaccine candidate, we employed the rotavirus VP6-protein platform to construct a fusion protein (FP) displaying receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S) at the N-terminus of VP6. The recombinant baculovirus-insect cell produced VP6-RBD FP was proven antigenic in vitro and bound to the human angiotensin-converting enzyme 2 (hACE2) receptor. The FP was used to immunize BALB/c mice, and humoral- and T cell-mediated immune responses were investigated. SARS-CoV-2 RBD-specific T cells were induced at a high quantity; however, no RBD or S-specific antibodies were detected. The results suggest that conformational B cell epitopes might be buried inside the VP6, while RBD-specific T cell epitopes are available for T cell recognition after the processing and presentation of FP by the antigen-presenting cells. Further immunogenicity studies are needed to confirm these findings and to assess whether, under different experimental conditions, the VP6 platform may present SARS-CoV-2 antigens to B cells as well.

Expression of influenza A virus-derived peptides on a rotavirus VP6-based delivery platform

Recombinant protein technology enables the engineering of modern vaccines composed of a carrier protein displaying poorly immunogenic heterologous antigens. One promising carrier is based on the rotavirus inner-capsid VP6 protein. We explored different VP6 insertion sites for the presentation of two peptides (23 and 140 amino acids) derived from the M2 and HA genes of influenza A virus. Both termini and three surface loops of VP6 were successfully exploited as genetic fusion sites, as demonstrated by the expression of the fusion proteins. However, further studies are needed to assess the morphology and immunogenicity of these constructs.

Structural Variations Induced by Temperature Changes in Rotavirus VP6 Protein Immersed in an Electric Field and Their Effects on Epitopes of The Region 300-396

Rotavirus diarrhea is an infectious intestinal disease that causes about 215 thousand deaths annually in infants under five years old. This virus is formed by three layers of concentric proteins that envelop its genome, from which VP6 structural protein is the most conserved among rotavirus serotypes and an excellent vaccine candidate. Recent studies have shown that structural proteins are susceptible to losing their biological function when their conformation is modified by moderate temperature increments, and in the case of VP6, its antigen efficiency decreases. We performed an in silicoanalysis to identify the structural variations in the epitopes 301-315, 357-366, and 376-384 of the rotavirus VP6 protein -in a hydrated medium- when the temperature is increased from 310 K to 322 K. In the latter state, we applied an electric field equivalent to a low energy laser pulse and calculated the fluctuations per amino acid residue. We identified that the region 301-315 has greater flexibility and density of negative electrical charge; nevertheless, at 322 K it experiences a sudden change of secondary structure that could decrease its efficiency as an antigenic determinant. The applied electric field induces electrical neutrality in the region 357-366, whereas in 376-384 inverts the charge, implying that temperature changes in the range 310 K-322 K are a factor that promotes thermoelectric effects in the VP6 protein epitopes in the region 300-396.

Recombinant Porcine Rotavirus VP6 Proteins Expressed in Lactobacillus plantarum NC8 Strain Induces Specific Mucosal and Systemic Antibody Production in Experimental BALB/c mice

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Recombinant Porcine Rotavirus VP6 Proteins Expressed in Lactobacillus plantarum NC8 Strain Induces Specific Mucosal and Systemic Antibody Production in Experimental BALB/c mice

Background Rotaviruses are the main cause of animal and infant diarrhea and are widely distributed worldwide. In the pig industry, porcine rotavirus infection is a significant cause of mortality and morbidity; therefore, the optimization and well-organized distribution of vaccines for infection prevention is needed. Because immune responses related to protection are mainly mucosal in nature, the induction of mucosal immunity is significant for preventing porcine rotavirus infection. Methods The major protective VP6 antigens against porcine rotavirus (PRV) expressed by Lactobacillus plantarum NC8-pSIP409-pgsA-VP6-Dcpep were used to orally immunize mice. Western blot analysis and SDS-PAGE were used to confirm the expression of recombinant NC8-pSIP409-pgsA-VP6-Dcpep, and immunofluorescence was also used to verify its surface expression on L. plantarum NC8. Results High levels of mucosal IgA and serum immunoglobulin G (IgG) were produced in mice that were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP6-Dcpep. Mice immunized with NC8-pSIP409-pgsA-VP6-Dcpep had higher IgA titers than mice immunized with NC8-pSIP409-pgsA and PBS. The mice orally immunized with NC8-pSIP409-pgsA-VP6-Dcpep demonstrated a virus challenge-induced mean fever peak of 38.6°C in the NC8-pSIP409-pgsA-VP6-Dcpep group and 41.1°C in the NC8-pSIP409-pgsA group 4 to 6 days after the challenge, and 7 to 9 days following challenge. Conclusion Our results confirmed that with regard to stimulating mucosal immunity, the VP6 genes of the porcine rotavirus (PRV) Chinese PRV isolate DN30209 strain expressed by L. plantarum NC8 is effective and that Dcpep is superior in its ability to stimulate mucosal immunity, suggesting that this approach can be implanted in pigs.