A novel therapeutic vaccine composed of a rearranged human papillomavirus type 16 E6/E7 fusion protein and Fms-like tyrosine kinase-3 ligand induces CD8+ T cell responses and antitumor effect

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8⁺ T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer.     

Vaccination with combination of Fit3L and RANTES in a DNA prime-protein boost regimen elicits strong cell-mediated immunity and antitumor effect

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in the antitumor response, strategies are being pursued to elicit augmented CD8⁺ T-cell responses against tumors with tumor vaccines. Here, we report the protective efficacy of vaccine-elicited antitumor immune responses with an aggressive HBc-expressing B16-HBc melanoma, which expressed HBc as a self and model antigen, tumor model. We demonstrated that the significantly better memory responses or marked inhibition on tumor growth could be achieved after coadministration of cytokine adjuvants RANTES and Flt3L in a DNA prime-protein boost regimen. Furthermore, the augmentation of DNA prime-protein boost regimens by cytokines gene was due to the improvement the immunopotency of DNA vaccine and subsequently the augmented Ag-specific and IFN-γ mediating CD8⁺ T-cell responses after protein boosting. Hence, this study demonstrates for the first time that combinatorial use of chemotactic and potent DC-specific growth factor molecules provides a useful strategy for enhancing antitumor responses.     

Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model

Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, and subsets of anogenital and oropharyngeal cancers. HPV18 is the second most prevalent high-risk HPV type after HPV16. Furthermore, HPV18 is responsible for approximately 12% of cervical squamous cell carcinoma and 37% of cervical adenocarcinoma cases worldwide. In this study, we aimed to characterize the HPV18-E6-specific epitope and establish an HPV18 animal tumor model to evaluate the E6-specific immune response induced by our DNA vaccine. We vaccinated naïve C57BL/6 mice with a prototype DNA vaccine, pcDNA3-HPV18-E6, via intramuscular injection followed by electroporation, and analyzed the E6-specific CD8+ T cell responses by flow cytometry using a reported T cell epitope. We then characterized the MHC restriction element for the characterized HPV18-E6 epitope. Additionally, we generated an HPV18-E6-expressing tumor cell line to study the antitumor effect mediated by E6-specific immunity. We observed a robust HPV18-E6aa67-75 peptide-specific CD8+ T cell response after vaccination with pcDNA3-HPV18-E6. Further characterization demonstrated that this epitope was mainly restricted by H-2K^b, but was also weakly presented by HLA-A¹0201, as previously reported. We observed that vaccination with pcDNA3-HPV18-E6 significantly inhibited the growth of HPV18-E6-expressing tumor cells, TC-1/HPV18-E6, in mice. An antibody depletion study demonstrated that both CD4+ and CD8+ T cells are necessary for the observed antitumor immunity. The characterization of HPV18-E6-specific T cell responses and the establishment of HPV18-E6-expressing tumor cell line provide infrastructures for further development of HPV18-E6 targeted immunotherapy.     

Enhancing therapeutic HPV DNA vaccine potency through depletion of CD4CD25 T regulatory cells

Therapeutic human papillomavirus (HPV) vaccines targeting E6 and/or E7 antigens represent an opportunity to control HPV-associated lesions. We have previously generated several therapeutic DNA vaccines targeting HPV-16 E7 antigen and generated significant antitumor effects. Since regulatory T cells (Tregs) play an important role in suppressing immune responses against tumors by immunotherapy, such as DNA vaccines, we tested if the therapeutic effects of a DNA vaccine encoding E7 linked to heat shock protein 70 (Hsp70) can be improved by a strategy to deplete Tregs using a anti-CD25 monoclonal antibody (PC61) in vaccinated mice. We found that administration of PC61 prior to vaccination with E7/Hsp70 DNA was capable of generating higher levels of E7-specific CD8⁺ T cells compared to the control antibody, leading to significantly improved therapeutic and long-term protective antitumor effects against an E7-expressing tumor, TC-1. Thus, a strategy to deplete CD4⁺CD25⁺ Tregs in conjunction with therapeutic tumor antigen-specific DNA vaccine may represent a potentially promising approach to control tumor. The clinical implications of our study are discussed.     

A Mage3/Heat Shock Protein70 DNA vaccine induces both innate and adaptive immune responses for the antitumor activity

Heat shock proteins (HSPs) are highly effective and versatile molecules in promoting antitumor immune responses. We tested whether a HSP-based DNA vaccine can induce effective immune response against Mage3, a cancer testis (CT) antigen frequently expressed in many human tumors, thereby controlling the Mage3-expressing tumor. The vaccine was constructed by linking human inducible HSP70 to the C-terminus of a modified Mage3 gene (sMage3) that was attached at its N-terminus with the signal leader sequence of the human RANTES for releasing the expressed fusion protein from the transduced cells. Intramuscular injection of sMage3Hsp DNA induced CD4⁺/CD8⁺ T cell and antibody responses. Vaccination with sMage3Hsp DNA was more effective in inhibiting Mage3-expressing TC-1 tumors. When we dissected the antitumor activity of CD4⁺ and CD8⁺ T cells by immunizing CD4⁺ and CD8⁺ knockout mice with sMage3Hsp DNA, we found that both CD8⁺ T and CD4⁺ T cells played a role in control of inoculated tumor, but did not constitute the whole of immune protection in the prophylactic immunization. Instead, depletion of natural killer (NK) cells led to a major loss of antitumor activity in the immunized mice. These results indicate that the HSP-based Mage3 DNA vaccine can more effectively inhibit tumor growth by inducing both the innate immune responses and Mage3-specific adaptive immune responses via the Hsp-associated adjuvant function.     

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 μg) with 50 μg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon γ producing CD8⁺ T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5 × 10⁴ HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 μg of TA-CIN and 1000 μg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.     

The combination of Pleurotus ferulae water extract and CpG-ODN enhances the immune responses and antitumor efficacy of HPV peptides pulsed dendritic cell-based vaccine

Our previous study reported that the combination of Pleurotus ferulae water extract (PFWE) and CpG (PFWE + CpG) enhanced the maturation and function of dendritic cells (DCs). Here, we investigated the effects of PFWE + CpG on the immune responses and antitumor efficacy of DC-based vaccine. We observed that all of HPV E6 and E7 peptides pulsed DCs (HPV-immature DCs, HPV + PFWE-, +CpG- or +PFWE + CpG-DCs) induced antigen-specific CD8⁺ T cell responses and HPV + PFWE + CpG-DCs induced highest level of CD8⁺ T cell responses. The antitumor efficacy of HPV-DCs vaccines was evaluated in TC-1 tumor mouse model. The early therapeutic study showed that HPV + PFWE-, +CpG- and +PFWE + CpG-DCs greatly inhibited tumor growth. Moreover, HPV + PFWE + CpG-DCs controlled tumor growth at a faster rate compared to other groups. These three groups induced HPV-specific CD8⁺ T cell responses and significantly decreased the frequencies of induced regulatory T cells (iTregs: CD4⁺CD25⁻Fopx3⁺). However, only HPV + PFWE + CpG-DCs significantly decreased the frequency of natural Tregs (nTregs: CD4⁺CD25⁺Fopx3⁺). Furthermore, HPV + PFWE + CpG-DCs also significantly inhibited tumor growth in the late therapeutic study. The results showed that PFWE + CpG enhanced the immune responses and antitumor efficacy of DC-based vaccine, suggesting that PFWE + CpG might be the potential candidate for the generation of clinical-grade mature DCs.     

SA-4-1BBL as the immunomodulatory component of a HPV-16 E7 protein based vaccine shows robust therapeutic efficacy in a mouse cervical cancer model

Cervical cancer is the leading cause of cancer-related deaths among women worldwide. Current prophylactic vaccines based on HPV (Human papillomavirus) late gene protein L1 are ineffective in therapeutic settings. Therefore, there is an acute need for the development of therapeutic vaccines for HPV associated cancers. The HPV E7 oncoprotein is expressed in cervical cancer and has been associated with the cellular transformation and maintenance of the transformed phenotype. As such, E7 protein represents an ideal target for the development of therapeutic subunit vaccines against cervical cancer. However, the low antigenicity of this protein may require potent adjuvants for therapeutic efficacy. We recently generated a novel chimeric form of the 4-1BBL costimulatory molecule engineered with core streptavidin (SA-4-1BBL) and demonstrated its safe and pleiotropic effects on various cells of the immune system. We herein tested the utility of SA-4-1BBL as the immunomodulatory component of HPV-16 E7 recombinant protein based therapeutic vaccine in the E7 expressing TC-1 tumor as a model of cervical cancer in mice. A single subcutaneous vaccination was effective in eradicating established tumors in approximately 70% of mice. The therapeutic efficacy of the vaccine was associated with robust primary and memory CD4⁺ and CD8⁺ T cell responses, Th1 cytokine response, infiltration of CD4⁺ and CD8⁺ T cells into the tumor, and enhanced NK cell killing. Importantly, NK cells played an important role in vaccine mediated therapy since their physical depletion compromised vaccine efficacy. Collectively, these data demonstrate the utility of SA-4-1BBL as a new class of multifunctional immunomodulator for the development of therapeutic vaccines against cancer and chronic infections.     

Nonlytic Fc-fused IL-7 synergizes with Mtb32 DNA vaccine to enhance antigen-specific T cell responses in a therapeutic model of tuberculosis

Improvement to the immunogenicity of DNA vaccines was evaluated in a Mycobacterium tuberculosis (MTB) infection mouse model examining the combined effects of nonlytic Fc-fused IL-7 DNA (IL-7-nFc) and Flt3-ligand fused Mtb32 (F-Mtb32) DNA. Mice were treated with conventional chemotherapy for 6 weeks from 4 weeks after aerosol infection of MTB. Following the start of chemotherapy, DNA immunizations were administered five times with 2-week intervals. Coadministration of IL-7-nFc and F-Mtb32 DNA given during chemotherapy synergistically enhanced the magnitude of Mtb32-specific T cell responses and sustained for one-year after the last immunization assessed by IFN-γ ELISPOT assay. After dexamethasone treatment, a significantly reduced MTB reactivation was observed in mice received both IL-7-nFc and F-Mtb32 DNA, compared with F-MTb32 DNA alone or with control mice. In addition, mice treated with IL-7-nFc and F-Mtb32 DNA together showed improved lung pathology and reduced pulmonary inflammation values relative to F-Mtb32 DNA or saline injected mice. Intracellular cytokine staining revealed that the protection levels induced by combination therapy with IL-7-nFc and F-Mtb32 DNA was associated with enhanced Mtb32-specific IFN-γ secreting CD4⁺ T cell responses and CD8⁺ T cell responses stimulated with CTL epitope peptide in the lungs and spleens. These data suggest that IL-7-nFc as a novel TB adjuvant may facilitate therapeutic TB DNA vaccine to the clinics through significant enhancement of codelivered DNA vaccine-induced T cell immunity.     

Comparison of the immune responses in BALB/c mice following immunization with DNA-based and live attenuated vaccines delivered via different routes

The objective of this study was to compare immune responses induced in BALB/c mice following immunization with pcDNA-GPV-VP2 DNA by gene gun bombardment (6 μg) or by intramuscular (im) injection (100 μg) with the responses to live attenuated vaccine by im injection (100 μl). pcDNA3.1 (+) and physiological saline were used as controls. Peripheral blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63, 77 and 105 d after immunization. T lymphocyte proliferation was analyzed by MTT assay and enumeration of CD4⁺, and CD8⁺ T cell populations in peripheral blood was performed by flow cytometric analysis. Indirect ELISA was used to detect IgG levels. Cellular and humoral responses were induced by pcDNA-GPV-VP2 DNA and live virus vaccines. No differences were observed in T cell proliferation and CD8⁺ T cell responses induced by the genetic vaccine regardless of the route of delivery. However, CD4⁺ T cell responses and humoral immunity were enhanced in following gene gun immunization compared with im injection of the genetic vaccine. Cellular and humoral immunity was enhanced in following gene gun delivery of the genetic vaccine compared with the live attenuated vaccine. In conclusion, the pcDNA-GPV-VP2 DNA vaccine induced enhanced cellular and humoral immunity compared with that induced by the live attenuated vaccine.     

GTL001 and bivalent CyaA-based therapeutic vaccine strategies against human papillomavirus and other tumor-associated antigens induce effector and memory T-cell responses that inhibit tumor growth

GTL001 is a bivalent therapeutic vaccine containing human papillomavirus (HPV) 16 and HPV18 E7 proteins inserted in the Bordetella pertussis adenylate cyclase (CyaA) vector intended to prevent cervical cancer in HPV-infected women with normal cervical cytology or mild abnormalities. To be effective, therapeutic cervical cancer vaccines should induce both a T cell-mediated effector response against HPV-infected cells and a robust CD8⁺ T-cell memory response to prevent potential later infection. We examined the ability of GTL001 and related bivalent CyaA-based vaccines to induce, in parallel, effector and memory CD8⁺ T-cell responses to both vaccine antigens. Intradermal vaccination of C57BL/6 mice with GTL001 adjuvanted with a TLR3 agonist (polyinosinic-polycytidylic acid) or a TLR7 agonist (topical 5% imiquimod cream) induced strong HPV16 E7-specific T-cell responses capable of eradicating HPV16 E7-expressing tumors. Tumor-free mice also had antigen-specific memory T-cell responses that protected them against a subsequent challenge with HPV18 E7-expressing tumor cells. In addition, vaccination with bivalent vaccines containing CyaA-HPV16 E7 and CyaA fused to a tumor-associated antigen (melanoma-specific antigen A3, MAGEA3) or to a non-viral, non-tumor antigen (ovalbumin) eradicated HPV16 E7-expressing tumors and protected against a later challenge with MAGEA3- and ovalbumin-expressing tumor cells, respectively. These results show that CyaA-based bivalent vaccines such as GTL001 can induce both therapeutic and prophylactic anti-tumor T-cell responses. The CyaA platform can be adapted to different antigens and adjuvants, and therefore may be useful for developing other therapeutic vaccines.     

DNA/Ad5 vaccination with SIV epitopes induced epitope-specific CD4⁺ T cells, but few subdominant epitope-specific CD8⁺ T cells

The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8⁺ T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8⁺ T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4⁺ T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8⁺ T cells against subdominant epitopes and CD4⁺ T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01⁺ animals with five Mamu-A*01-restricted subdominant SIV-specific CD8⁺ T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8⁺ T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4⁺ T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4⁺ T cell responses, vaccination with subdominant MHC class I epitopes elicited few detectable CD8⁺ T cell responses. Broadening the CD8⁺ T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.     

The efficacy of a novel vaccine approach using tumor cells that ectopically express a codon-optimized murine GM-CSF in a murine tumor model

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that is known to facilitate vaccine efficacy by promoting the development and prolongation of both humoral and cellular immunity. Here, we investigated a novel vaccine approach using a human papillomavirus (HPV)-16 E6/E7-transformed cell line, TC-1, that ectopically expresses a codon-optimized 26-11-2015 murine GM-CSF (cGM-CSF). Ectopically expressing cGM-CSF in TC-1 (TC-1/cGM) cells significantly increased expression of a GM-CSF that was functionally identical to wt GM-CSF by 9-fold compared with ectopically expressed wild type GM-CSF in TC-1 cells (TC-1/wt). Mice vaccinated with irradiated TC-1/cGM cells exhibited enhanced survival compared with mice vaccinated with TC-1/wt cells when both groups were subsequently injected with live TC-1. Consistently, mice vaccinated with irradiated TC-1/cGM cells exhibited stronger IFN-γ production in HPV E7-specific CD8⁺ T cells. More dendritic cells were recruited to the draining lymph nodes (dLNs) of mice vaccinated with TC-1/cGM cells than C-1/wt cells. Regarding dLN cell recall responses, both proliferation and IFN-γ production in the HPV E7-specific CD8⁺ T cells were enhanced in mice that were vaccinated with TC-1/cGM cells. Our results demonstrate that a novel practical molecular strategy utilizing a codon-optimized GM-CSF gene overcomes the limitation and improves the efficacy of tumor cell-based vaccines.     

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes

DNA vaccination is highly efficient at inducing CD8⁺ T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8⁺ T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D^b-restricted CD8⁺ CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.     

Profound CD8+ T cell immunity elicited by sequential daily immunization with exogenous antigen plus the TLR3 agonist poly(I:C)

The development of vaccines that elicit robust CD8⁺ T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as useful for eliciting CD8⁺ T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8⁺ T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100 μg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10 μg of poly(I:C) mounted remarkable antigen-specific CD8⁺ T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8⁺ T cells being antigen specific within 7-8 days of vaccination. CD8⁺ T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8⁺ T cells were also able to regress large, established tumors in vivo. Together these data suggest that ‘cluster’ vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design.     

DNA vaccine with α-galactosylceramide at prime phase enhances anti-tumor immunity after boosting with antigen-expressing dendritic cells

DNA vaccines contribute to a promising new approach for the generation of cytotoxic T lymphocytes (CTL). DNA vaccines do have several disadvantages, including poor immunogenicity and oncogene expression. We used the natural killer T-cell (NKT) ligand α-galactosylceramide (α-GalCer) as an adjuvant to prime initial DNA vaccination; and used the potent immune-stimulatory tumor antigen-expressing dendritic cells (DCs) as a booster vaccination. A DNA vaccine expressing human papillomavirus (HPV) type 16 E7 (pcDNA3-CRT/E7) was combined with α-GalCer at the prime phase, and generated a higher number of E7-specific CD8⁺ T-cells in vaccinated mice than vaccine used at boost phase. Therefore, priming with a DNA vaccine in the presence of α-GalCer and boosting with E7-pulsed DC-1 led to a significant enhancement of E7-specific CD8⁺ effector and memory T-cells as well as significantly improved therapeutic and preventive effects against an E7-expressing tumor model (TC-1) in vaccinated mice. Our findings suggested that the potency of a DNA vaccine combined with α-GalCer could be further enhanced by boosting with an antigen-expressing DC-based vaccine to generate anti-tumor immunity.     

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX™ adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with “high risk” human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX™ adjuvant (HPV16 Immunotherapeutic) for patients with CIN.Experimental design: Patients with CIN (n = 31) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy.Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients.Conclusions: The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX™ adjuvant is safe and induces vaccine antigen specific cell mediated immunity.     

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8–22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8–22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8–22) hybrids or the epitope-only peptide HPV16 E7(8–22). As measured by IFN-γ ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8–22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8–22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D^b-restricted HPV16 E7(49–57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8–22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.     

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8 T cells and antibody when expressed from modified vaccinia Ankara

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8⁺ T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8⁺ T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8⁺ T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8⁺ T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8⁺ T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.