Emergence of two distinct variants of SARS-CoV-2 and an explosive second wave of COVID-19: the experience of a tertiary care hospital in Pune, India.

The emergence of novel variants of SARS-CoV-2 in several countries has been associated with increased transmissibility or reduced neutralization potential of antibodies against the Wuhan virus (wild type). From August 2021 onwards, India experienced a progressive decline in the number of active SARS-CoV-2 infections, indicative of a downward trend in the explosive second wave. This prospective study was conducted quarterly for one year (May 2020 to June 2021) at a tertiary care hospital in the city of Pune in western India. Receptor-binding domain (RBD, n = 319) and full genome (n = 20) sequences from viral-RNA-positive nasopharyngeal swabs of COVID-19 patients representing the first and second waves were used for analysis. No Brazilian, South African, or California variants were detected in this study. Until December 2020, only the wild-type strain was prevalent. Concurrent with the upsurge of the second wave in March 2021, 73% (33/45) of RBD sequences harboured L452R/E484Q mutations characteristic of the Kappa variant. In April 2021, co-circulation of Kappa (37%) and Delta (L452R/T478K, 59%) variants was recorded. During May and June 2021, the Delta variant became the predominant circulating variant, and this coincided with a significant decline in the number of COVID-19 cases. Of the 20 full genome sequences, six isolates each exhibited signature mutations of the Kappa and Delta variant. With several states witnessing a reduction in the number of COVID-19 cases, continuous monitoring of newer mutations and assessment of their effect on virus transmissibility and their impact on vaccinated or previously exposed individuals is necessary.

Evaluation of Liposome, Heat-Killed Mycobacterium w, and Alum Adjuvants in the Protection Offered by Different Combinations of Recombinant HA, NP proteins, and M2e Against Homologous H5N1 Virus.

Continued evolution of highly pathogenic H5N1 viruses causing high mortality in humans obviates need for broadly cross-reactive vaccines. For this, hemagglutinin (HA) inducing specific protective antibodies, highly conserved nucleoprotein (NP), and ectodomain of matrix (M2e) protein, either singly or in combination, were evaluated in BALB/c mice. Recombinant HA and NP (baculovirus system) and M2e (synthetic peptide) and 3 adjuvants, that is, liposomes, Mw (heat killed Mycobacterium w), and alum were utilized for the homologous virus challenge. Additional immunogens included liposome-encapsulated HA/NP proteins and corresponding DNAs. Mice groups received two doses of respective formulations given at 3-week intervals and challenged intranasally with 100LD50 of H5N1 virus strain. Dynamics of weight loss, lung viral load, titres of IgG-anti-HA, NP, and M2e antibodies (ELISA), and IgG-subtype analysis was done. Two doses of all the formulations led to 100% seroconversion against the immunogens evaluated (100% seroconversion after the first dose in majority). Antibody titres against the components were dependent on the adjuvant and combination. HA-driven Th2 response with all the adjuvants, balanced Th1/Th2 response to NP protein, and Th2-bias with alum were noted. Low anti-M2e antibody titres did not allow subtype analysis. On challenge, complete protection was observed with Mw-HA, alum-HA+NP, Lipo-HA+NP+M2e, alum-HA+NP+M2e, and HA-DP formulations with 12-fold, 8-fold, 720-fold, 17-fold, and no reduction, respectively, in lung viral load. In conclusion, the results identify several adjuvant-immunogen combinations conferring 100% protection in mice that need further evaluation in higher animals.

Inter-Clade Protection Offered by Mw-Adjuvanted Recombinant HA, NP Proteins, and M2e Peptide Combination Vaccine in Mice Correlates with Cellular Immune Response.

We documented earlier that Mw (heat-killed suspension of Mycobacterium indicus pranii) adjuvant when used with conserved antigens, nucleoprotein (NP), and ectodomain of matrix (M2) protein (M2e) provided complete protection against homologous (clade 2.2) virus challenge in mice. The present study extends these observations to inter-clade challenge (clade 2.3.2.1) H5N1 virus and attempts to understand preliminary immunologic basis for the observed protection. Female BALB/c mice immunized with a single or two doses of vaccine formulations (clade 2.2 antigens) were challenged with 100LD50 homologous or heterologous (clade 2.3.2.1) virus. To understand the preliminary immunologic mechanism, we studied proportions of selected immune cell types, immune response gene expression, and Th1/Th2 cytokines induced by antigen-stimulated splenocytes from immunized mice, at different time points. Complete protection was conferred by Mw-HA, Mw-HA + NP, and Mw-HA + NP + M2e against homologous challenge. The protection correlated with IgG2a antibody titers indicating important role of Th1 response. Despite high inter-cladal antigenic differences, complete protection against the heterologous strain was achieved with Mw-HA + NP + M2e. Of note, a single dose with higher antigen concentrations (50 µg HA + 50 µg NP + 50 µg M2e) led to 80% protection against clade 2.3.2.1 strain. The protection conferred by Mw-HNM correlated with induction of IFN-γ, CD8+ T cytotoxic cells, and CD4+ T helper cells. Mw-adjuvanted HA + NP + M2e combination represents a promising vaccine candidate deserving further evaluation.