Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T?? values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.     

Rat liver mitochondrial F1-ATPase, an FAD containing ferroprotein.

F1-ATPase isolated from rat liver mitochondria has been found to contain approximately 1 mole of FAD and 6 g atoms of nonheme iron per mole of enzyme.     

Reversible modification of rat liver F1-ATPase by a redox reaction.

F1-ATPase from rat liver mitochondria exhibited a change in properties when reduced by dithionite: an increase in activity together with a disappearance of its sensitivity to bicarbonate stimulation was observed. A complete reversion to the original properties was achieved with the oxidizing agent 2,6-dichlorophenolindophenol.     

The contribution of induction of temperate phages to the numbers of free somatic coliphages in waters is not significant

Somatic coliphages have been proposed as indicators of water quality. But several factors have been considered a drawback for their use as indicators. We evaluated the contribution of temperate phages to the numbers of somatic coliphages detected in water by ISO (International Standards Organization) standardised methods. Prophage induction from naturally occurring bacteria was assayed with mitomycin C, ciprofloxacin and UV irradiation. Results indicate that the presence of prophages will not influence the determinations of somatic coliphages in water.     

An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must

We used experimental evolution in order to identify genes involved in the adaptation of Saccharomyces cerevisiae to the early stages of alcoholic fermentation. Evolution experiments were run for about 200 generations, in continuous culture conditions emulating the initial stages of wine fermentation. We performed whole-genome sequencing of four adapted strains from three independent evolution experiments. Mutations identified in these strains pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor specifically involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. While a loss-of-function mutation in BUL1 seems to be enough to confer a selective advantage under these assay conditions, this did not seem to be the case for RSP5 mutated strains, which required additional mutations, probably compensating for the detrimental effect of altered Rsp5p activity on essential cellular functions. The power of this experimental approach is illustrated by the identification of four independent mutants, each with a limited number of SNPs, affected within the same pathway. However, in order to obtain information relevant for a specific biotechnological process, caution must be taken in the choice of the background yeast genotype (as shown in this case for auxotrophies). In addition, the use of very stable continuous fermentation conditions might lead to the selection of a rather limited number of adaptive responses that would mask other possible targets for genetic improvement.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.