Vaccinia Western Reserve Research Articles

Vaccinia virus strain LC16m8 defective in the B5R gene keeps strong protection comparable to its parental strain Lister in immunodeficient mice

BackgroundAttenuated vaccinia virus strain, LC16m8, defective in the B5R envelope protein gene, is used as a stockpile smallpox vaccine strain in Japan against bioterrorism: the defect in the B5R gene mainly contributes to its highly attenuated properties. MethodsThe protective activity of LC16m8 vaccine against challenge with a lethal dose of vaccinia Western Reserve strain was assessed in wild-type and immunodeficient mice lacking CD4, MHC class I, MHC class II or MHC class I and II antigens. ResultsThe immunization with LC16m8 induced strong protective activity comparable to that of its parent strain, Lister (Elstree) strain, in wild-type mice from 2 days to 1 year after vaccination, as well as in immunodeficient mice at 2 or 3 weeks after vaccination. These results implicated that the defect in the B5R gene hardly affected the potential activity of LC16m8 to induce innate, cell-mediated and humoral immunity, and that LC16m8 could be effective in immunodeficient patients. ConclusionLC16m8 with truncated B5 protein has an activity to induce immunity, such as innate immunity and subsequent cell-mediated and humoral immunity almost completely comparable to the activity of its parental strain Lister.

Deletion of the vaccinia virus F13L gene results in a highly attenuated virus that mounts a protective immune response against subsequent vaccinia virus challenge

Vaccinia virus F13L encodes the envelope protein p37, which is the target of the anti-pox virus drug ST-246 (Yang et al., 2005) and that is required for production of extracellular vaccinia virus. The F13L (p37)-deleted (and ST-246 resistant) vaccinia virus recombinant (Vac-ΔF13L) produced smaller plaques than the wild-type vaccinia (Western Reserve vaccinia). In addition, Vac-ΔF13L proved, when inoculated either intravenously or intracutaneously in both immunocompetent and immunodeficient (athymic nude or SCID) mice, to be severely attenuated. Intravenous or intracutaneous inoculation of immunocompetent mice with the ΔF13L virus efficiently protected against a subsequent intravenous, intracutaneous or intranasal challenge with vaccinia WR (Western Reserve). This was corroborated by the observation that Vac-ΔF13L induced a humoral immune response against vaccinia following either intravenous or intracutaneous challenge. In conclusion, F13L-deleted vaccinia virus may have the potential to be developed as a smallpox vaccine.

Smallpox vaccines induce antibodies to the immunomodulatory, secreted vaccinia virus complement control protein

Vaccination with Dryvax elicits a broad humoral response against many viral proteins. Human vaccinia immune globulin was used to screen the secreted proteins from cells infected with Dryvax or the candidate smallpox vaccine LC16m8 to determine whether the protective humoral response included antibodies against secreted viral proteins. Many proteins were detected, with the primary band corresponding to a band of 28 or 30 kDa in cells infected with Dryvax or LC16m8, respectively. This was identified as the vaccinia virus complement protein (VCP), which migrated more slowly in LC16m8-infected cells due to post-translational glycosylation. Vaccinia virus deleted in VCP, vVCPko, protected mice from a lethal intranasal challenge of vaccinia Western Reserve strain. Mice vaccinated with purified VCP demonstrated a strong humoral response, but were not protected against a moderate lethal challenge of vaccinia virus, suggesting that the humoral response against VCP is not critical for protection.

Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response

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.

Comparative Analysis of Viral Gene Expression Programs during Poxvirus Infection: A Transcriptional Map of the Vaccinia and Monkeypox Genomes

BackgroundPoxviruses engage in a complex and intricate dialogue with host cells as part of their strategy for replication. However, relatively little molecular detail is available with which to understand the mechanisms behind this dialogue.Methodology/Principal FindingsWe designed a specialized microarray that contains probes specific to all predicted ORFs in the Monkeypox Zaire (MPXV) and Vaccinia Western Reserve (VACV) genomes, as well as >18,000 human genes, and used this tool to characterize MPXV and VACV gene expression responses in vitro during the course of primary infection of human monocytes, primary human fibroblasts and HeLa cells. The two viral transcriptomes show distinct features of temporal regulation and species-specific gene expression, and provide an early foundation for understanding global gene expression responses during poxvirus infection.Conclusions/SignificanceThe results provide a temporal map of the transcriptome of each virus during infection, enabling us to compare viral gene expression across species, and classify expression patterns of previously uncharacterized ORFs.

Generation and evaluation of a recombinant modified vaccinia virus Ankara vaccine for rabies

Modified vaccinia virus Ankara (MVA) has become a vaccine vector of choice for recombinant vaccine development. A MVA-based rabies vaccine would be advantageous for use as a vaccine for dogs (and wildlife), particularly if it proves innocuous and efficacious by the oral route. Here, the generation and immunological testing of a recombinant MVA expressing a rabies virus glycoprotein gene is described. In a murine model, higher dosages of recombinant MVA were needed to induce equivocal immune responses as with Vaccinia Copenhagen or Vaccinia Western Reserve recombinants, when administered by a parenteral route. The MVA recombinant was not immunogenic or efficacious when administered per os in naïve mice. The ability of the recombinant MVA to induce anamnestic responses in dogs and raccoons was also investigated. Recombinant MVA boosted humoral immune responses in these animals when administered peripherally, but not when administered orally.

Mapping and investigation of the role in pathogenesis of the major unique secreted 35-kDa protein of rabbitpox virus

Following infection, many secreted poxvirus proteins are able to modulate the host immune response through interactions with cytokines or components of the complement pathway. A comparison of the secreted protein profiles from cells infected with vaccinia Western Reserve (VV-WR), cowpox virus Brighton strain, or rabbitpox virus (RPV) showed an abundant 35-kDa protein present only in the supernatants from RPV-infected cells. The gene encoding this protein was identified and mapped by N-terminal sequencing of the protein. Examination of the predicted amino acid sequence showed it to be identical to the 35-kDa secreted protein of the Lister strain of vaccinia virus described by Patel et al. (1990, J. Gen. Virol. 71, 2013–2021). The counterpart of this gene in the commonly studied VV-WR strain is truncated and encodes a 7.5-kDa protein under control of the well-characterized p7.5 promoter. While nonessential for replication in cell culture, conservation of this gene in at least two orthopoxvirus strains suggested that this protein might play an important role in vivo. Following intranasal inoculation of Balb/c mice at several doses (10 3, 10 4, or 10 5 PFU), a mutant of RPV lacking a functional 35-kDa gene (RPVΔ35) appeared to induce an earlier onset and more severe illness at low, sublethal doses (10 3 PFU) than was observed with wild-type (wt) RPV. At higher doses (10 4 or 10 5 PFU), the behavior of wt RPV and RPVΔ35 became indistinguishable and the overall LD 50 values were similar. Intradermal infection of rabbits simultaneously, at separate sites, with RPV and RPVΔ35 showed no gross or microscopic differences between either primary skin lesions or viremic extension of each virus into the lungs. Therefore, this abundant secreted protein does not appear to play a major role in the virulence of the virus.

The Vaccinia Virus 42-kDa Envelope Protein Is Required for the Envelopment and Egress of Extracellular Virus and for Virus Virulence

Vaccinia virus gene B5R encodes a Mr 42K glycoprotein that is expressed throughout infection and forms part of the envelope of extracellular virus. In this paper deletion mutants (ΔB5R) lacking the B5R open reading frame (ORF) from the Western Reserve (WR) and IHD-J strains of vaccinia virus have been constructed and shown to form very small plaques compared with the wild-type viruses. This phenotype was directly attributable to loss of the BSR gene since re-insertion of this gene from WR or IHD-J into the WR mutant lacking B5R (W-ΔB5R) restored a normal plaque phenotype. In the latter case the failure of the revertant to form comets indicated that the nine amino acid differences in the B5R ORF between the IHD-J and WR strains of virus are not responsible for comet formation by IHD-J virus. Furthermore, the B5R deletion mutant of IHD-J (I-ΔB5R) still formed small comets. Despite the small plaque phenotype of the deletion mutants, normal yields of intracellular naked virus (INV) were produced. In contrast, deletion of B5R had a profound affect on the formation of the extracellular enveloped virus (EEV). Transmission electron microscopy indicated that INV particles were not wrapped by a double layer of Golgi-derived membrane and enveloped particles were not detected within the cell or on the cell surface without expression of the B5R protein. Biochemical measurement of EEV formation, by labeling infected cells with [ 3H]thymidine followed by cesium chloride density gradient centrifugation of particles released from the cells 24 hr postinfection, showed that only 10% of WT levels of EEV were produced by I-ΔBSR. The loss of the B5R ORF caused severe attenuation in intranasally infected mice. At doses between 10 4 and 3 × 10 7 plaque-forming units there were no signs of disease in animals infected with W-ΔB5R, whereas at comparable doses the WR parent virus caused significant mortalities. Finally, an ORF with 93.4% amino acid identity to vaccinia WR B5R is present in variola major virus strain Harvey and the B5R protein was shown by Western blotting to be expressed by all orthopoxviruses tested.