[Show abstract][Hide abstract] ABSTRACT: Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104--120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.
[Show abstract][Hide abstract] ABSTRACT: Smallpox vaccination might be associated with adverse reactions, ranging in severity from benign to lethal. One of the most
serious complications is postvaccinal encephalitis. The aim of this study was to identify early markers for vaccinia virus
(VACV) induced encephalitis. For this purpose we infected mice intracranially with VACV-Lister or VACV-WR. Histopathological
analysis showed that following infection with VACV-WR tissue damage in the brain spatially and temporally correlated with
virus replication, infiltration of white blood cells and apoptosis. None of the above markers was observed upon infection
with the vaccine strain Lister.
MMP-9 is a serum factor known for its correlation to BBB integrity and encephalitis in humans. We found that in sera of VACV-Lister
infected animals, MMP-9 levels did not change throughout the infection. However, during VACV-WR infection, in the first 2
days levels of MMP-9 were significantly low than the controls and subsequently rose to levels which were significantly higher
than the controls. Elevated MMP-9 was associated with damage to the brain and to BBB integrity.
In conclusion, efficient virus replication in the brain causes significant brain damage followed by BBB brake-down and release
of MMP-9 to the serum, markers which were not observed when the attenuated strain was examined. Thus, we suggest MMP-9 as
a possible non-invasive serum indicator for encephalitis caused by VACV virus.
[Show abstract][Hide abstract] ABSTRACT: Immunization of BALB/c mice with vaccinia virus protein A33 (A33VACV) protects mice from intranasal challenge with the WR strain of vaccinia virus or with ectromelia virus making A33 an important
candidate to be included in experimental smallpox subunit vaccines. Single vaccination with a recombinant Sindbis virus expressing
A33VACV protect mice against lethal VACV-WR and ectromelia virus (ECTV) but not against the closely related cowpox virus (CPXV).
Furthermore, even recombinant Sindbis virus expressing the cowpox virus A33 ortholog (A33CPXV) failed to protect either against cowpox or against VACV-WR challenge. Our attempts to map the regions which may account
for this differential behavior were directed against a region of difference between the two orthologs. A stretch of 7 amino
acids in A33 was mapped as important for protection which contain the following changes in A33CPXV: L112F, Q117K and L118S. This region maps to a single putative prevalent 9-mer CTL epitope with L112 as an essential anchoring
residue, and a major target epitope for neutralizing antibodies encompassing L118. Vaccination with A33 harboring these individual
substitutions highlighted the crucial role of L118 in induction of protective immunity.
[Show abstract][Hide abstract] ABSTRACT: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain.
We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV).
In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses.
ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.
The Journal of Infectious Diseases 12/2008; 199(1):39-48. DOI:10.1086/595565 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since smallpox eradication by the WHO during the 1980s, potency of new vaccines is compared to vaccines that were used during the eradication campaign. In this work we characterize the tail scarification technique in mice as a model for scarification in humans. Similar to humans, mice develop "clinical take" which is dependent on the vaccination dose. Appearance of anti-Vaccinia IgM is followed by IgG antibodies 10 days post scarification and lasting more then 1(1/2) years. Mice with "clinical take" are 100% protected against lethal respiratory challenge (100LD(50)) of Vaccinia WR indicating that the "clinical take" can serve as a correlate of protective immunity. Reducing the vaccination dose and using Cowpox virus as a more virulent strain, enabled us to draw the limit of the vaccine potency in mice. Similar to humans, in revaccinated mice the development of "clinical take" was inversely correlated to the level of pre-existing antibodies. These results indicate that tail scarification of mice can be used as a model for evaluation of smallpox vaccines. High correlation between "clinical take" and protective immunity allows the use of visual inspection to evaluate vaccine potency.