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Biomedical subjects

David C Tscharke

Publications and source records attributed to David C Tscharke.

At least 19 recordsLinked to original sources
PubMed2006

Deletion of gene A41L enhances vaccinia virus immunogenicity and vaccine efficacy.

Vaccinia virus (VACV) is the vaccine that was used to eradicate smallpox and is being developed as a recombinant vaccine for other pathogens. Removal of genes encoding immunomodulatory proteins expressed by VACV may enhance virus immunogenicity and improve its potential as a vaccine. Protein A41 is a candidate for removal, having sequence similarity to the VACV chemokine-binding protein, vCKBP, and an association with reduced inflammation during dermal infection. Here, it is shown that, at low doses, VACV strain Western Reserve (WR) lacking A41L (vDeltaA41L) was slightly more virulent than wild-type and revertant controls after intranasal infection of BALB/c mice. The primary immune response to vDeltaA41L was marked by an increase in the percentage of VACV-specific gamma interferon-producing CD8(+) T cells and enhancement of cytotoxic T-cell responses in the spleen. However, this augmentation of cellular response was not seen in lung infiltrates. Splenic CD8(+) T-cell responses were also enhanced when VACV strain modified vaccinia virus Ankara (MVA) lacking A41L was used to immunize mice. Lastly, immunization with VACV MVA lacking A41L provided better protection than control viruses to subsequent challenge with a 300 LD(50) dose of VACV WR. This study provides insight into the immunomodulatory role of A41 and suggests that MVA lacking A41 may represent a more efficacious vaccine.

Animals↗
PubMed2005

Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara.

Many recombinant poxviral vaccines are currently in clinical trials for cancer and infectious diseases. However, these agents have failed to generate T cell responses specific for recombinant gene products at levels comparable with T cell responses associated with natural viral infections. The recent identification of vaccinia-encoded CTL epitopes, including a new epitope described in this study, allows the simultaneous comparison of CTL responses specific for poxviral and recombinant epitopes. We performed detailed kinetic analyses of CTL responses in HLA-A*0201 patients receiving repeated injections of recombinant modified vaccinia Ankara encoding a string of melanoma tumor Ag epitopes. The vaccine-driven CTL hierarchy was dominated by modified vaccinia Ankara epitope-specific responses, even in patients who had not received previous smallpox vaccination. The only recombinant epitope that was able to impact on the CTL hierarchy was the melan-A26-35 analog epitope, whereas responses specific for the weaker affinity epitope NY-ESO-1(157-165) failed to be expanded above the level detected in prevaccination samples. Our results demonstrate that immunodominant vaccinia-specific CTL responses limit the effectiveness of poxviruses in recombinant vaccination strategies and that more powerful priming strategies are required to overcome immunodominance of poxvirus-specific T cell responses.

HLA-A Antigens↗
PubMed2005

HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products.

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome approximately 20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8(+) T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.

Animals↗
PubMed2005

HLA class I-restricted responses to vaccinia recognize a broad array of proteins mainly involved in virulence and viral gene regulation.

We have analyzed by ex vivo ELISPOT the anti-vaccinia cytotoxic T lymphocyte responses of peripheral blood mononuclear cells from humans vaccinated with Dryvax vaccine. More than 6,000 peptides from 258 putative vaccinia ORFs predicted to bind the common molecules of the HLA A1, A2, A3, A24, B7, and B44 supertypes were screened with peripheral blood mononuclear cells of 31 vaccinees. A total of 48 epitopes derived from 35 different vaccinia antigens were identified, some of which (B8R, D1R, D5R, C10L, C19L, C7L, F12, and O1L) were recognized by multiple donors and contain multiple epitopes recognized in the context of different HLA types. The antigens recognized tend to be >100 residues in length and are expressed predominantly in the early phases of infection, although some late antigens were also recognized. Viral genome regulation and virulence factor were recognized most frequently, whereas few structural proteins were immunogenic. Finally, most epitopes were highly conserved among vaccinia virus Western Reserve, variola major and modified vaccinia Ankara, supporting their potential use in vaccine and diagnostic applications.

Amino Acid Sequence↗
PubMed2005

Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities.

Little is known regarding the participation of CD4+ CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.

Amino Acid Sequence↗
PubMed2005

From mice to humans - murine intelligence for human CD8+ T cell vaccine design.

There has been considerable progress in the design of vaccines capable of safely and effectively inducing CD8(+) T cells for prophylaxis and treatment of chronic infectious diseases and cancer. Much of what is known about CD8(+) T cell-mediated immunity has come from pioneering work in mice; this broad overview discusses recent work in mouse systems where lessons may be drawn for human vaccine development. The areas highlighted include antivector immunity, immunodominance, dendritic cell biology and targeting, the role of Toll-like receptors and their exploitation by novel adjuvants, the role of CD4(+) T cell help, regulatory T cells and, finally, some comments on the different requirements of prophylactic versus therapeutic vaccines.

Animals↗
PubMed2004

Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines.

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.

Amino Acid Sequence↗
PubMed2004

CD8+ T cell cross-priming via transfer of proteasome substrates.

"Cross-priming" describes the activation of naïve CD8+ T cells by professional antigen-presenting cells that have acquired viral or tumor antigens from "donor" cells. Antigen transfer is believed to be mediated by donor cell-derived molecular chaperones bearing short peptide ligands generated by proteasome degradation of protein antigens. We show here that cross-priming is based on the transfer of proteasome substrates rather than peptides. These findings are potentially important for the rational design of vaccines that elicit CD8+ T cell responses.

Acetylcysteine↗
PubMed2003

The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model.

Chordate poxviruses encode several uncharacterized POZ-kelch proteins and three of these are present in Vaccinia virus (VV) strain Western Reserve. VV gene C2L is predicted to encode a protein of 512 amino acid residues with a POZ/BTB domain in the N-terminal region and three kelch motifs in the C-terminal half of the protein. We have identified the C2L gene product as an intracellular protein of 56 kDa and constructed and characterized a VV mutant lacking the C2L gene (v Delta C2L). Compared to wild-type and revertant viruses, v Delta C2L had unaltered growth in vitro, but had a different plaque morphology due to an altered cytopathic effect (CPE) of infected cells. Deleting C2L had no effect on VV-induced formation of actin tails or enhanced cell motility, but affected the development of VV-induced cellular projections and the Ca(2+)-independent cell/extracellular matrix adhesion late during infection. In an intranasal mouse model, C2L did not contribute to virus virulence. However, in an intradermal mouse model, infection with v Delta C2L resulted in larger lesions and more cell infiltration into the infected ears during recovery from infection. Thus, in this model, C2L protein inhibits inflammation and reduces immunopathology. In summary, we found that C2L is not required for virus replication in vitro but contributes to aspects of VV-induced CPE and reduces immunopathology in vivo.

Animals↗
PubMed2002

The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model.

A bioassay that measured the interleukin (IL)-12-induced production of interferon (IFN)-gamma from mouse splenocytes was used to identify a soluble factor in the supernatants of vaccinia virus (VV)-infected cells that inhibited the production of IFN-gamma. This soluble factor was expressed by 14 out of 16 VV strains including the Western Reserve (WR) strain, but strains Copenhagen and Tashkent and a mutant of strain WR called 6/2 lacked this activity. The gene encoding this activity was identified as C12L by transferring DNA present in VV WR but missing in VV WR 6/2 into VV Copenhagen and testing for expression of the soluble factor. The C12L protein shows amino acid similarity to IL-18 binding proteins that are encoded by poxviruses, mice and humans, and C12L protein produced from VV or baculovirus inhibited the biological activity of mouse IL-18 in vitro. Thus the inhibition of IL-12-induced IFN-gamma production was due to indirect effects of C12L on IL-18, illustrating the synergistic action of these pro-inflammatory cytokines. To study the role of the C12L protein in the virus life-cycle, we constructed a deletion mutant lacking the C12L gene and a revertant virus in which the gene was reinserted into the deletion mutant. In vitro the replication and plaque size of these viruses were indistinguishable. However, infection of BALB/c mice by the intranasal route showed that the deletion mutant was attenuated and induced lower weight loss and signs of illness compared to controls.

Administration, Intranasal↗
PubMed2002

The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence.

Vaccinia virus (VV) strain Western Reserve gene B9R is shown to encode an intracellular 6 kDa protein that is expressed late during the infectious cycle. In vitro transcription and translation produced two polypeptides in the presence of microsomal membranes, but only the larger protein in the absence of membranes. The smaller protein sedimented with microsomes during centrifugation, suggesting it was inserted into the lipid membrane or into the microsomal lumen via the N-terminal hydrophobic signal sequence that was subsequently cleaved proteolytically. A VV mutant lacking B9R was constructed and found to replicate normally in cell culture and two in vivo models.

Base Sequence↗
PubMed2002

A study of the vaccinia virus interferon-gamma receptor and its contribution to virus virulence.

Vaccinia virus (VV) strain Western Reserve gene B8R encodes a 43 kDa glycoprotein that is secreted from infected cells early in infection as a homodimer. This protein has amino acid similarity with the extracellular domain of cellular IFN-gamma receptor (IFN-gammaR) and binds and inhibits IFN-gamma from a wide range of species. Here we demonstrate that the B8R protein also inhibits equine IFN-gamma. The 5' end of the B8R mRNA has been mapped by primer extension analysis and the contribution of IFN-gammaRs to VV virulence was studied by the construction of a deletion mutant lacking the B8R gene (vDeltaB8R) and a revertant virus (vB8R-R) in which the B8R gene was re-inserted into the deletion mutant. A recombinant virus that expressed a soluble form of the mouse IFN-gammaR was also constructed and studied. The virulence of these viruses was tested in rodent models of infection. In mice, the loss of the VV IFN-gammaR did not affect virulence compared with WT and revertant viruses, consistent with the low affinity of the VV IFN-gammaR for mouse IFN-gamma. However, expression of the mouse soluble IFN-gammaR increased virus virulence slightly. In rabbit skin, loss of the VV IFN-gammaR produced lesions with histological differences compared with WT and revertant viruses. Lastly, the affinity constants of the VV IFN-gammaR for human and mouse IFN-gamma were determined by surface plasmon resonance.

Amino Acid Sequence↗
PubMed2002

The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence.

The vaccinia virus (VV) N1L gene encodes a protein of 14 kDa that was identified previously in the concentrated supernatant of virus-infected cells. Here we show that the protein is present predominantly (>90%) within cells rather than in the culture supernatant and it exists as a non-glycosylated, non-covalent homodimer. The N1L protein present in the culture supernatant was uncleaved at the N terminus and was released from cells more slowly than the VV A41L gene product, a secreted glycoprotein that has a conventional signal peptide. Bioinformatic analyses predict that the N1L protein is largely alpha-helical and show that it is conserved in many VV strains, in other orthopoxviruses and in members of other chordopoxvirus genera. However, database searches found no non-poxvirus proteins with significant amino acid similarity to N1L. A deletion mutant lacking the N1L gene replicated normally in cell culture, but was attenuated in intranasal and intradermal murine models compared to wild-type and revertant controls. The conservation of the N1L protein and the attenuated phenotype of the deletion mutant indicate an important role in the virus life-cycle.

Animals↗
PubMed2002

Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes.

Previously, we developed a model for testing the virulence and immunogenicity of vaccinia virus (VV) mutants based on the intradermal injection of BALB/c mouse ear pinnae. The model is characterized by a local infection in the inoculated skin without signs of systemic illness, mimicking dermal vaccination with VV. Here a further characterization of this model is presented, including the responses of mice to infectious virus doses as low as 10 p.f.u., a quantification of the infiltrate at the site of infection and use of different virus and mouse strains. The model was then used to compare the pathogenesis of six mutants of VV strain Western Reserve (WR) lacking genes A36R, A40R, A44L, B12R, B13R or B15R with that of appropriate control viruses. All of these genes except B12R and B15R influence the outcome of dermal infection with WR and for A40R and B13R this is the first role that has been reported after infection of mammals. A comparison of new and published results from intradermal and intranasal models is presented, showing that out of 16 gene deletion or insertion mutants of VV, half have phenotypes distinct from controls in only one of these models. Thus, the intranasal and intradermal models are complementary tools for dissecting the genetic basis of VV virulence.

Age Factors↗
PubMed2001

Vaccinia virus semaphorin A39R is a 50-55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model.

Vaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytes in vitro. In this study, we show that the A39R gene of VV strain Copenhagen (COP) encodes a 50-55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including an A39R deletion mutant of VV COP and a WR mutant containing the A39R sequence from COP. Loss of the gene from COP did not affect virus growth in vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COP A39R by VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.

Amino Acid Sequence↗