To all, 4 Stressgen abstracts from 5th Annual Conference on Vaccine Research - Baltimore, MD May 6-8, 2002
1. Mycobacterial Heat Shock Protein (Hsp) Fusions: A Novel Class of Immunotherapeutics for the Treatment of Human Papillomavirus (HPV) Infection
Chu, N.R., Wu B., Liu H., Wisniewski J., Recktenwald A., Rowse G., Boux H.A., *Boux L., Mizzen L., *Siegel M.I.
Stressgen Biotechnologies, Victoria British Columbia, CANADA. And *Collegeville, PA.
Cell-mediated immunity (CMI) to HPV16 E7 (E7) is associated with the resolution of HPV-related lesions. An in-frame fusion protein comprised of mycobacterial Hsp65 and E7 (SGN-00101) induces CMI and tumor regression preclinically and provides clinical benefit in phase II immunotherapy trials of anogenital HPV diseases. To test other Hsp fusions, a series of constructs using different Hsp fused to E7 were tested for activity. DNA constructs or recombinant proteins comprised of full-length Hsp10, Hsp40, Hsp65 or Hsp70 from M. tuberculosis fused with full-length HPV16 E7 were used (without adjuvant) to treat C57Bl/6 mice with pre-established, E7-expressing TC-1 tumor. Primed splenocytes from mice immunized with Hsp fusion proteins were tested for cytolytic and cytokine-producing activity. Tumor regression was observed in mice treated with pCMV/SGN-00101, but not pCMV/Hsp65, pCMV/E7, or pCMV/Hsp65 + pCMV/E7, confirming that the activity is a property intrinsic to the Hsp fusion. Treatment with SGN-00101, but not its constituent proteins, Hsp65, and E7, regresses tumor. Similarly, Hsp10-E7, Hsp40-E7 and E7-Hsp70 fusion protein treatment leads to tumor regression. Primed splenocytes from E7-Hsp70- or SGN-00101-immunized mice were cytolytic for E7-expressing targets and released IFN-gamma upon E7 restimulation. Immunization of mice with plasmids encoding Hsp-E7 fusion proteins, or Hsp-E7 fusion proteins, induces E7-specific CMI and regresses E7-expressing tumor. These results suggest that Hsp-E7 fusions are a potentially new class of immunotherapeutics for HPV-associated disease.
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2. Induction of HBcAg-Specific CTL Responses by a Heat Shock Protein Fused to the Core Antigen of the Hepatitis B Virus
Anthony L.S.D., Liu H., Rowse G., Wu B., Recktenwald A., Mizzen L.A., Boux H. and *Siegel M.I.
Stressgen Biotechnologies, Victoria British Columbia, CANADA. And *Collegeville, PA.
Individuals acutely infected with hepatitis B virus (HBV) typically recover from infection and exhibit strong, broadly specific CD8+ CTL responses, while chronically infected patients display relatively weak, narrowly focused responses. Resolution of chronic hepatitis may be induced by immunotherapy that primes effective HBV-specific CTL responses. Heat shock protein (Hsp) fusion proteins have been shown to prime potent CTL activity in experimental animals. We generated recombinant fusion proteins containing sequences from the HBV core antigen (HBc) and the 65 kDa Hsp from Mycobacterium bovis BCG. Spleen cells from mice immunized without exogenous adjuvant were restimulated with HBc-derived CTL epitope peptides. Cells from fusion protein-primed mice exhibited a high degree of lytic activity, whereas cells from mice immunized with HBc antigen alone displayed poor target cell lysis. The lytic activity was specific for multiple CTL epitopes, including an HLA-A2-restricted epitope. Effector cells lysed both peptide-pulsed target cells and also target cells transfected to express endogenous HBc antigen. The CTL released IFN-g and TNF-a, cytokines thought to be involved in inhibition of hepatic viral gene expression. In comparison with control mice, Tlr4 deficient C57BL/10ScNCr mice responded relatively poorly to fusion protein immunization, indicating that CTL priming is dependent, at least in part, upon this toll-like receptor. The results of these experiments clearly demonstrate the potential value of Hsp fusion proteins in the immunotherapy of chronic HBV hepatitis.
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3. HLA-A2 and H-2Db-restricted CTL activity induced by SGN-00101 - a fusion protein comprised of heat shock protein and human papillomavirus E7
Liu H., Rowse G., N. Chu N.R., Wu B., Boux H., and *Siegel M.I.
Stressgen Biotechnologies, Victoria British Columbia, CANADA. And *Collegeville, PA.
Infection with human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. It is believed that induction of T cell-mediated immunity, particularly cytolytic T lymphocytes (CTL), is important in eradication of HPV-induced lesions. Previous studies have shown that heat shock protein (Hsp) fusion proteins are capable of inducing potent antigen-specific CTL activity in experimental animals. In addition, the outgrowth of E7-expressing murine tumor cell line (TC-1) can be prevented by treatment with SGN-00101, an Hsp fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7. In the present study, we investigated the ability of SGN-00101 to prime E7-specific CTL activity. Splenocytes from mice immunized with SGN-00101 were restimulated in vitro with HPV16 E7-derived CTL epitope peptides. CTL activities were measured using 51Cr-labeled peptide-pulsed or E7-expressing target cells. Results indicated that SGN-00101 was effective in priming HLA-A2-restricted CTL in A2.1/Kb transgenic mice and H-2Db-restricted CTL in C57BL/6 mice. In addition to lysing target cells pulsed with peptides, the E7 specific CTL were capable of lysing target cells expressing E7 endogenously. Significant CTL activity was observed (60% at 100:1 E:T ratio) in both mouse strains. The present study supports the previous findings that Hsp fusion proteins are capable of inducing peptide-specific CTL activity. This study provides additional support for the use of SGN-00101 to treat HPV-related disease.
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4. Immunostimulatory actions of SGN-00101 and the separation of these effects from Lipopolysaccharide.
Rowse G., Chu N.R., Wiebe E., Recktenwald A., Mizzen L., Boux H. and *Siegel M.I.
Stressgen Biotechnologies, Victoria British Columbia, CANADA. And *Collegeville, PA.
Immunization with SGN-00101, an Hsp fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7, results in eradication of HPV E7 expressing cells in both preclinical models and human phase II clinical trials. Effective treatment with SGN-00101 requires activation of CD8+ve T cells. Since SGN-00101 is active without adjuvant, we wished to determine if HspE7 could stimulate the release of proinflammatory cytokines from cells of the innate immune system. THP-1 cells were stimulated with LPS, SGN-00101 or it's component parts. For desensitization studies, cells were stimulated with LPS or SGN-00101, washed and then restimulated. For serum dependence studies, THP-1 cells were stimulated with SGN-00101 or LPS in 10% or 1% FCS. TNFa production was assessed by ELISA.
HspE7, but not Hsp65, E7 (or the admixture) induced the release of TNFa from THP-1 cells. While SGN-00101 was able to elicit TNFa release from THP-1 cells cultured in low serum containing media, LPS did not elicit TNFa under these conditions. Pretreatment with SGN-00101 desensitized the THP-1 cells to restimulation with either LPS or SGN-00101, while LPS pretreatment resulted in complete desensitization to restimulation with LPS, but only partial desensitization to SGN-00101. These results demonstrate that SGN-00101 is able to stimulate cells of the monocyte/macrophage lineage to secrete proinflamatory cytokines such as TNFa. Further, actions of SGN-00101 on THP-1 cells cannot be solely attributed to LPS as the stimulatory properties of SGN-00101 can be differentiated from those of LPS.
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