The Effect of Mice Adaptation Process on the Pathogenicity of Influenza A/South Africa/3626/2013 (H1N1)pdm09 Model Strain.

Influenza virus strain A/South Africa/3626/2013 (H1N1)pdm09 (SA-WT) is a non-mouse-adapted model strain that has naturally high pathogenic properties in mice. It has been suggested that the high pathogenicity of this strain for mice could be due to the three strain-specific substitutions in the polymerase complex (Q687R in PB1, N102T in PB2, and E358E/K heterogeneity in PB2). To evaluate the role of these replacements, SA-WT was passaged five times in mouse lungs, and the genome of the mouse-adapted version of the SA-WT strain (SA-M5) was sequenced. SA-M5 lost E358E/K heterogeneity and retained E358, which is the prevalent amino acid at this position among H1N1pdm09 strains. In addition, in the hemagglutinin of SA-M5, two heterogeneous substitutions (G155G/E and S190S/R) were identified. Both viruses, SA-M5 and SA-WT, were compared for their toxicity, ability to replicate, pathogenicity, and immunogenicity in mice. In mice infected with SA-M5 or SA-WT strains, toxicity, virus titer in pulmonary homogenates, and mouse survival did not differ significantly. In contrast, an increase in the immunogenicity of SA-M5 compared to SA-WT was observed. This increase could be due to the substitutions G155G/E and S190S/R in the HA of SA-M5. The prospects for using SA-M5 in studying the immunogenicity mechanisms were also discussed.

Truncation of NS1 Protein Enhances T Cell-Mediated Cross-Protection of a Live Attenuated Influenza Vaccine Virus Expressing Wild-Type Nucleoprotein.

Current seasonal influenza vaccines have suboptimal effectiveness, especially in seasons dominated by viruses that do not match the vaccine. Therefore, finding new approaches to improve the immunogenicity and efficacy of traditional influenza vaccines is of high priority for public health. Licensed live attenuated influenza vaccine (LAIV) is a promising platform for designing broadly protective vaccines due to its ability to induce cross-reactive T-cell immunity. In this study, we tested the hypothesis that truncation of the nonstructural protein 1 (NS1) and the replacement of the nucleoprotein (NP) of the A/Leningrad/17 master donor virus with a recent NP, i.e., switching to 5:3 genome composition, could improve the cross-protective potential of the LAIV virus. We generated a panel of LAIV candidates differing from the classical vaccine by the source of NP gene and/or by the length of NS1 protein. We showed that NS1-modified LAIV viruses had reduced viral replication in the respiratory tract of mice, indicating a more attenuated phenotype compared to the LAIVs with full-length NS1. Most importantly, the LAIV candidate with both NP and NS genes modified induced a robust systemic and lung-localized memory CD8 T-cell response targeting more recent viruses, and better protected immunized mice against lethal challenge with a heterosubtypic influenza virus than the control LAIV variant. Overall, these data indicate that the 5:3 LAIVs with truncated NS1 may be beneficial for protection against heterologous influenza viruses and warrant further preclinical and clinical development.

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2.

BACKGROUND: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. METHODS: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4+ and CD8+ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. RESULTS: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4+CD45RA-CCR7- phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. CONCLUSION: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8+, but not CD4+, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.