Treatment with the smallpox antiviral tecovirimat (ST-246) alone or in combination with ACAM2000 vaccination is effective as a postsymptomatic therapy for monkeypox virus infection.

The therapeutic efficacies of smallpox vaccine ACAM2000 and antiviral tecovirimat given alone or in combination starting on day 3 postinfection were compared in a cynomolgus macaque model of lethal monkeypox virus infection. Postexposure administration of ACAM2000 alone did not provide any protection against severe monkeypox disease or mortality. In contrast, postexposure treatment with tecovirimat alone or in combination with ACAM2000 provided full protection. Additionally, tecovirimat treatment delayed until day 4, 5, or 6 postinfection was 83% (days 4 and 5) or 50% (day 6) effective.

Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge.

The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections, and would likely have relevance for vaccine design against complex human and animal pathogens.

Examination of the effect of a naturally occurring mutation in glycoprotein L on Marek's disease virus pathogenesis.

We recently reported a comparison of glycoprotein-encoding genes of different Marek's disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four-amino acid) deletion in the glycoprotein L (gL)-encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK1HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test groups, these data suggest that replication of the TK strain and HVT, when coadministered, might incrementally affect the virulence of MDV-1 strains. The strict correlation of this enhancement of virulence with the mutation in gL, however, requires additional experiments with genetically identical MDV background strains.

Ptch1 is required locally for mammary gland morphogenesis and systemically for ductal elongation.

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1(mes)), we found that Ptch1(mes) homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1(mes) homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.

Characterization of an alternative oxidase activity of Histoplasma capsulatum.

Histoplasma capsulatum possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consuming activities. The latter, carried out by a single subunit enzyme termed 'alternative oxidase', is the focus of this report. AOX1 cDNA clones were isolated and direct evidence that the cDNA ORF encodes functional alternative oxidase is reported. Also reported are the generation of an antiserum to the AOX1 protein product, and specific detection in vivo of the mRNA and protein products of the AOX1 gene. Finally, initial studies of regulation of H. capsulatum AOX1 gene expression demonstrated that RNA abundance was increased after hydrogen peroxide-mediated oxidative stress and after inhibition of mitochondrial electron transport enzymes with antimycin A or sodium cyanide. This pattern of regulation is consistent with the hypothesis that alternative oxidase contributes to survival of H. capsulatum after oxidative or metabolic stress and may be important for virulence of this pathogenic organism. The GenBank Accession Nos for the cDNA sequences reported in this paper are AF133236, AF133237 (AOX1).