Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Autophagy is involved in the early step of Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry-LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge.

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.

Characterization of a highly attenuated Salmonella enterica serovar Typhimurium mutant strain.

Salmonella enterica serovar Typhimurium ATCC 13311 is virulent at a dose as low as 10(2) colony-forming units when administered intraperitoneally to BALB/c mice. In order to develop highly attenuated mutant strain through the combination of 2 phenotypically attenuated markers, we constructed a number of amino acid requiring auxotrophic strains of S. enterica serovar Typhimurium by means of UV-induced mutations. One of them, strain NDMC-B1, was highly attenuated for mice, with an LD50-value of 6 and 3 log units lower for mice than the wild-type strain and S. enterica serovar Typhimurium aroA strain, respectively. This strain still contained the Salmonella O- and H-antigens but had a requirement for cysteine and was unable to utilize citrate as its sole carbon source. NDMC-B1 colonized the gut-associated lymphoid tissue more efficiently than the wild-type strain, but its capacities to colonize spleen and liver were significantly reduced. Mice intraperitoneally or orally vaccinated with NDMC-B1 were highly protected against either an intraperitoneal challenge with 10(6) colony-forming units or an oral challenge with 10(9) colony-forming units of the wild-type strain. Taken together, the results illustrate that through the combination of 2 independently phenotypical attenuating markers, the requirement for cysteine and the inability to use citrate, we have successfully constructed a highly attenuated, stable, and immunogenic S. enterica serovar Typhimurium vaccine strain which can induce protective immunity in a mouse model against lethal challenge of wild-type strain.