Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.     

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.     

Characterization of HPV-16 E6 DNA vaccines employing intracellular targeting and intercellular spreading strategies

Summary Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8⁺ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8⁺ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.     

Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects

The use of DNA vaccines for generating antigen-specific CD8+ T cell responses has been well established. However, little is known about the quantitative and qualitative aspects of CD8+ T cell responses and protective immunity generated after repeated DNA vaccinations. We used human papillomavirus (HPV) type-16 E7 as a model tumor antigen in an E7-expressing tumor model, TC-1, to assess the influence of the frequency of DNA vaccinations on E7-specific immunological and antitumor responses. Mice were vaccinated with 1–4 inoculations of pcDNA3-E7 DNA. Immunological assays and tumor protection experiments were performed to assess the effect of repeated E7 DNA vaccination on E7-specific T cells and E7-expressing tumors. Our results demonstrated that mice receiving an increased number of E7 DNA vaccinations exhibited higher E7-specific CTL activity, a rapid expansion of E7-specific IFN--secreting CD8+ T cells upon stimulation with E7 antigen, and a stronger antitumor effect against an E7-expressing tumor. Furthermore, we found that increasing the number of E7 DNA vaccinations followed by vaccinia booster enhanced the functional avidity of E7-specific CD8+ T cells. Our data suggest that quantitative and qualitative characteristics of antigen-specific CD8+ T cell responses and the ensuing protective antitumor effect can be influenced by the frequency of DNA vaccinations. These results have important clinical implications for the use of naked DNA vaccines in cancer immunotherapy.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen

We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Marek's disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8(+)-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency.     

Cancer Immunotherapy Employing an Innovative Strategy to Enhance CD4+ T Cell Help in the Tumor Microenvironment

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.     

Successful tumor eradication was achieved by collaboration of augmented cytotoxic activity and anti-angiogenic effects following therapeutic vaccines containing helper-activating analog-loaded dendritic cells and tumor antigen DNA

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4⁺ and CD8⁺ T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.     

Treatment with Imiquimod enhances antitumor immunity induced by therapeutic HPV DNA vaccination

Background

There is an urgent need to develop new innovative therapies for the control of advanced cancer. The combination of antigen-specific immunotherapy with the employment of immunomodulatory agents has emerged as a potentially plausible approach for the control of advanced cancer.

Methods

In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the TLR7 agonist imiquimod for their ability to generate E7-specific immune responses and antitumor effects in tumor-bearing mice.

Results

We observed that treatment with CRT/E7 DNA in combination with imiquimod leads to an enhancement in the E7-specific CD8+ T cell immune responses and a decrease in the number of myeloid-derived suppressor cells in the tumor microenvironment of tumor-bearing mice. Furthermore, treatment with CRT/E7 DNA in combination with imiquimod leads to significantly improved antitumor effects and prolonged survival in treated mice. In addition, treatment with imiquimod led to increased number of NK1.1+ cells and F4/80+ cells in the tumor microenvironment. Macrophages and NK1.1+ cells were found to play an important role in the antitumor effects mediated by treatment with CRT/E7 DNA in combination with imiquimod.

Conclusions

Thus, our data suggests that the combination of therapeutic HPV DNA vaccination with topical treatment with the TLR7 agonist imiquimod enhances the antitumor immunity induced by DNA vaccination. The current study has significant implications for future clinical translation.

     

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.     

Development of a DNA vaccine targeting human papillomavirus type 16 oncoprotein E6

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8(+) T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8(+) T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2K(b), is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8(+) T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.     

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.     

Enhancement of gp120-specific immune responses by genetic vaccination with the human immunodeficiency virus type 1 envelope gene fused to the gene coding for soluble CTLA4

DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8(+)) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8(+) T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8(+) T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120-specific CD8(+) T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice.     

Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8⁺ T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Low-dose cyclophosphamide administered as daily or single dose enhances the antitumor effects of a therapeutic HPV vaccine

Although therapeutic HPV vaccines are able to elicit systemic HPV-specific immunity, clinical responses have not always correlated with levels of vaccine-induced CD8⁺ T cells in human clinical trials. This observed discrepancy may be attributable to an immunosuppressive tumor microenvironment in which the CD8⁺ T cells are recruited. Regulatory T cells (Tregs) are cells that can dampen cytotoxic CD8⁺ T-cell function. Cyclophosphamide (CTX) is a systemic chemotherapeutic agent, which can eradicate immune cells, including inhibitory Tregs. The optimal dose and schedule of CTX administration in combination with immunotherapy to eliminate the Treg population without adversely affecting vaccine-induced T-cell responses is unknown. Therefore, we investigated various dosing and administration schedules of CTX in combination with a therapeutic HPV vaccine in a preclinical tumor model. HPV tumor-bearing mice received either a single preconditioning dose or a daily dose of CTX in combination with the pNGVL4a-CRT/E7(detox) DNA vaccine. Both single and daily dosing of CTX in combination with vaccine had a synergistic antitumor effect as compared to monotherapy alone. The potent antitumor responses were attributed to the reduction in Treg frequency and increased infiltration of HPV16 E7-specific CD8⁺ T cells, which led to higher ratios of CD8⁺/Treg and CD8⁺/CD11b⁺Gr-1⁺ myeloid-derived suppressor cells (MDSCs). There was an observed trend toward decreased vaccine-induced CD8⁺ T-cell frequency with daily dosing of CTX. We recommend a single, preconditioning dose of CTX prior to vaccination due to its efficacy, ease of administration, and reduced cumulative adverse effect on vaccine-induced T cells.     

Generation and characterization of DNA vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a serious threat to public health and the economy on a global scale. The SARS coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Thus, vaccination against SARS-CoV may represent an effective approach to controlling SARS. DNA vaccines are an attractive approach for SARS vaccine development, as they offer many advantages over conventional vaccines, including stability, simplicity, and safety. Our investigators have previously shown that DNA vaccination with antigen linked to calreticulin (CRT) dramatically enhances major histocompatibility complex class I presentation of linked antigen to CD8(+) T cells. In this study, we have employed this CRT-based enhancement strategy to create effective DNA vaccines using SARS-CoV nucleocapsid (N) protein as a target antigen. Vaccination with naked CRT/N DNA generated the most potent N-specific humoral and T-cell-mediated immune responses in vaccinated C57BL/6 mice among all of the DNA constructs tested. Furthermore, mice vaccinated with CRT/N DNA were capable of significantly reducing the titer of challenging vaccinia virus expressing the N protein of the SARS virus. These results show that a DNA vaccine encoding CRT linked to a SARS-CoV antigen is capable of generating strong N-specific humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV.     

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.T.W.K. and C.-F.H. contributed equally to this work.     

Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70–80% of mice. The anti-tumor activity was shown to be dependent on CD8⁺ cells and NK1.1⁺. Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer⁺ cell numbers, cytokine release and CTL lysis of CEA⁺ targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8⁺ T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8⁺ T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.