ABSTRACT: In an event of a smallpox outbreak in humans, the window for efficacious treatment by vaccination with vaccinia viruses (VACV) is believed to be limited to the first few days post-exposure (p.e.). We recently demonstrated in a mouse model for human smallpox, that active immunization 2-3 days p.e. with either VACV-Lister or modified VACV Ankara (MVA) vaccines, can rescue animals from lethal challenge of ectromelia virus (ECTV), the causative agent of mousepox. The present study was carried out in order to determine whether a single dose of the anti-viral cidofovir (CDV), administered at different times and doses p.e. either alone or in conjunction with active vaccination, can rescue ECTV infected mice.
Animals were infected intranasally with ECTV, treated on different days with various single CDV doses and monitored for morbidity, mortality and humoral response. In addition, in order to determine the influence of CDV on the immune response following vaccination, both the "clinical take", IFN-gamma and IgG Ab levels in the serum were evaluated as well as the ability of the mice to withstand a lethal challenge of ECTV. Finally the efficacy of a combined treatment regime of CDV and vaccination p.e. was determined.
A single p.e. CDV treatment is sufficient for protection depending on the initiation time and dose (2.5 - 100 mg/kg) of treatment. Solid protection was achieved by a low dose (5 mg/kg) CDV treatment even if given at day 6 p.e., approximately 4 days before death of the control infected untreated mice (mean time to death (MTTD) 10.2). At the same time point complete protection was achieved by single treatment with higher doses of CDV (25 or 100 mg/kg). Irrespective of treatment dose, all surviving animals developed a protective immune response even when the CDV treatment was initiated one day p.e.. After seven days post treatment with the highest dose (100 mg/kg), virus was still detected in some organs (e.g. lung and liver) yet all animals survived, suggesting that efficacious single CDV treatment requires a potent immune system. The combination of CDV and vaccination provided no additional protection over CDV alone. Yet, combining CDV and vaccination maintained vaccination efficacy.
Altogether, our data substantiate the feasibility of single post-exposure antiviral treatment to face orthopoxvirus infection.
Virology Journal 06/2012; 9:119. · 2.34 Impact Factor
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.
06/2010: pages 147-155;
ABSTRACT: Vaccinia Immune Globulin (VIG) is currently used to treat severe complications of smallpox vaccines. In this study we compare
the therapeutic potential of vaccinia virus rabbit hyper immune sera (RHIS) with that of human VIG. The clearance rate of
RHIS from mouse circulation is only slightly slower than that of VIG (t
1/2=10 and 7.5 days respectively). Like VIG, passively administered RHIS can protect mice against lethal respiratory and dermal
Ectromelia virus (ECTV) challenge. Administration of both homologous (anti ECTV) and heterologous (anti VACV-WR or VACV-Lister)
anti-sera conferred efficient protection against a subsequent lethal respiratory ECTV challenge. These observations formed
the basis for passive cross protection studies against ECTV, conducted in mice. RHIS conferred better protection as compared
to VIG as a result of its better specific activity which is about 100 folds higher than that of VIG, allowing for significant
protection even if administered 5 days post infection. This study emphasizes the advantage of a hyper immune product and validates
the potential use of VIG and other antibody based therapeutics, not only as prophylactic measures against post-vaccination
complications but also for post-exposure treatment of smallpox disease.
KeywordsECTV-Hairless mice-Hyper immune sera-Smallpox-Vaccinia Immune Globulin (VIG)
06/2010: pages 253-260;
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.
06/2010: pages 115-123;
ABSTRACT: Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium.
We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection.
Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.
Virology Journal 09/2009; 6:151. · 2.34 Impact Factor
ABSTRACT: The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.
Vaccine 03/2009; 27(11):1691-9. · 3.77 Impact Factor
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. · 6.41 Impact Factor
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.
Vaccine 12/2007; 25(45):7743-53. · 3.77 Impact Factor
ABSTRACT: Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.
Journal of Bacteriology 12/2003; 185(22):6513-21. · 3.83 Impact Factor