Zhen-Kun Pan

Two Listeria monocytogenes Vaccine Vectors That Express Different Molecular Forms of Human Papilloma Virus-16 (HPV-16) E7 Induce Qualitatively Different T Cell Immunity That Correlates with Their Ability to Induce Regression of Established Tumors Immortalized by HPV-16

Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2Db E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-β and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-β and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.

Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site

ABSTRACT Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+ T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+ T cells but may also increase the number of antigen-specific CD8+ T cells in the tumor, the principle site of antigen expression.

Recombinant Listeria Vaccines Containing PEST Sequences Are Potent Immune Adjuvants for the Tumor-Associated Antigen Human Papillomavirus-16 E7

Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH2 terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8+ T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8+ T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8+ T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.

Cancer Immunotherapy Targeting the High Molecular Weight Melanoma-Associated Antigen Protein Results in a Broad Antitumor Response and Reduction of Pericytes in the Tumor Vasculature

The high molecular weight melanoma-associated antigen (HMW-MAA), also known as melanoma chondroitin sulfate proteoglycan, has been used as a target for the immunotherapy of melanoma. This antigen is expressed on the cell surface and has a restricted distribution in normal tissues. Besides its expression in a broad range of transformed cells, this antigen is also found in pericytes, which are important for tumor angiogenesis. We generated a recombinant Listeria monocytogenes (Lm-LLO-HMW-MAA-C) that expresses and secretes a fragment of HMW-MAA (residues 2,160-2,258) fused to the first 441 residues of the listeriolysin O (LLO) protein. Immunization with Lm-LLO-HMW-MAA-C was able to impede the tumor growth of early established B16F10-HMW-MAA tumors in mice and both CD4(+) and CD8(+) T cells were required for therapeutic efficacy. Immune responses to a known HLA-A2 epitope present in the HMW-MAA(2160-2258) fragment was detected in the HLA-A2/K(b) transgenic mice immunized with Lm-LLO-HMW-MAA-C. Surprisingly, this vaccine also significantly impaired the in vivo growth of other tumorigenic cell lines, such as melanoma, renal carcinoma, and breast tumors, which were not engineered to express HMW-MAA. One hypothesis is that the vaccine could be targeting pericytes, which are important for tumor angiogenesis. In a breast tumor model, immunization with Lm-LLO-HMW-MAA-C caused CD8(+) T-cell infiltration in the tumor stroma and a significant decrease in the number of pericytes in the tumor blood vessels. In conclusion, a Lm-based vaccine against HMW-MAA can trigger cell-mediated immune responses to this antigen that can target not only tumor cells but also pericytes in the tumor vasculature.

Listeria monocytogenes-Based Antibiotic Resistance Gene-Free Antigen Delivery System Applicable to Other Bacterial Vectors and DNA Vaccines

ABSTRACT Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of d-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the d-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.

A Novel Human Her-2/neu Chimeric Molecule Expressed by Listeria monocytogenes Can Elicit Potent HLA-A2 Restricted CD8-positive T cell Responses and Impact the Growth and Spread of Her-2/neu-positive Breast Tumors

Purpose: The aim of this study was to efficiently design a novel vaccine for human Her-2/neu-positive (hHer-2/neu) breast cancer using the live, attenuated bacterial vector Listeria monocytogenes. Experimental Design: Three recombinant L. monocytogenes–based vaccines were generated that could express and secrete extracellular and intracellular fragments of the hHer-2/neu protein. In addition, we generated a fourth construct fusing selected portions of each individual fragment that contained most of the human leukocyte antigen (HLA) epitopes as a combination vaccine (L. monocytogenes–hHer-2/neu chimera). Results: Each individual vaccine was able to either fully regress or slow tumor growth in a mouse model for Her-2/neu-positive tumors. All three vaccines could elicit immune responses directed toward human leukocyte antigen-A2 epitopes of hHer-2/neu. The L. monocytogenes–hHer-2/neu chimera was able to mimic responses generated by the three separate vaccines and prevent spontaneous outgrowth of tumors in an autochthonous model for Her-2/neu-positive breast cancer, induce tumor regression in transplantable models, and prevent seeding of experimental lung metastases in a murine model for metastatic breast cancer. Conclusion: This novel L. monocytogenes–hHer-2/neu chimera vaccine proves to be just as effective as the individual vaccines but combines the strength of all three in a single vaccination. These encouraging results support future clinical trials using this chimera vaccine and may be applicable to other cancer types expressing the Her-2/neu molecule such as colorectal and pancreatic cancer.

The Induction of HIV Gag-Specific CD8+ T Cells in the Spleen and Gut-Associated Lymphoid Tissue by Parenteral or Mucosal Immunization with Recombinant Listeria monocytogenes HIV Gag

The induction of mucosal immunity is crucial in controlling viral replication during HIV infection. In this study we compare the ability of a recombinant Listeria monocytogenes that expresses and secretes the HIV Ag Gag to induce CD8+ T cells against this Ag in the spleen, mesenteric lymph nodes, and Peyer’s patches and the ability to provide effector Gag-specific CD8+ T cells to the lamina propria after i.v., oral, or rectal administration of the vaccine. The levels of Ag-specific CD8+-activated T cells were measured ex vivo using intracellular cytokine staining for IFN-γ and H-2Kd Gag peptide tetramer staining. We found that all routes of immunization induced Gag-specific CD8+ T cells in the spleen. After secondary infection, we observed substantial increases in splenic levels of CD8+ T cells, and levels of Gag-specific cells were similar to those against listeriolysin O, the immunodominant Ag of L. monocytogenes. Both primary and secondary oral immunization resulted in abundant Gag-specific CD8+-activated T cells in the lamina propria that constituted ∼35% of the CD8 compartment. However, significant levels of Gag and listeriolysin O-specific CD8+ T cells were observed in mucosal lymphoid tissue only after two immunizations, perhaps because they had already entered the lamina propria compartment after a single immunization. In the context of HIV, a mucosally administered vaccine seems best calculated to prompt an immune response that is capable of preventing infection. The data presented in this report demonstrate that mucosally administered Listeria can prompt such a response and that booster doses can maintain this response.

Tumor sensitivity to IFN-γ is required for successful antigen-specific immunotherapy of a transplantable mouse tumor model for HPV-transformed tumors

Purpose: Many human tumors lose responsiveness to IFN-γ, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN-γ in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. Methods: To investigate the role of IFN-γ in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN-γ due to overexpression of a dominant negative IFN-γ receptor. Results: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN-γ is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN-γ is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4+ and CD8+ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. Conclusions: Our findings identify a role for IFN-γ in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN-γ poses a challenge to antigen-based immunotherapy.

Targeting tumor vasculature with novel Listeria-based vaccines directed against CD105

The FDA approval of bevacizumab (Avastin®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-β receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

Listeria monocytogenes Delivery of HPV-16 Major Capsid Protein L1 Induces Systemic and Mucosal Cell-Mediated CD4+ and CD8+ T-Cell Responses After Oral Immunization

Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyers patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.