Search

ABSTRACTWe propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates.IMPORTANCELive vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Determining energy requirements are an important component of nutritional support for patients with malnutrition; however, the validity of prediction equations for resting energy expenditure (REE) is disputed in older hospitalized patients. We aimed to assess the validity of these equations in older hospitalized patients in Japan. This was a single-center, cross-sectional study of 100 patients aged ≥70 years, hospitalized between January 2020 and December 2021. REE was measured using an indirect calorimeter and was compared to the predicted values calculated from five REE prediction equations. The mean (95% confidence interval) measured REE was 968.1 (931.0, 1005.3) kcal/day, and the mean predicted REE was higher for the FAO/WHO/UNU (1014.3 [987.1, 1041.6] kcal/day, p = 0.164) and Schofield (1066.0 [1045.8, 1086.2] kcal/day, p < 0.001) equations and lower for the Harris-Benedict (898.6 [873.1, 924.1] kcal/day, p = 0.011), Ganpule (830.1 [790.3, 869.9] kcal/day, p < 0.001), and body weight (kg) × 20 (857.7 [821.9, 893.5] kcal/day, p < 0.001) equations. In the age group analysis, none of the predicted values were within a 10% error for more than 80% of patients aged 70–89 years and ≥90 years. The five REE prediction equations did not provide accurate estimates. Validated REE prediction equations need to be developed for older hospitalized patients.

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3. [ABSTRACT FROM AUTHOR]