Anti-cancer activity of plant-produced HPV16 E7 vaccine

The E7 oncoprotein from Human Papilloma Virus (HPV) is an attractive candidate for anti-cancer vaccine development. In this study, we engineered HPV16 E7 coding sequence (wild type or mutagenized sequence, E7GGG) as fusions to beta-1,3-1,4-glucanase (LicKM) of Clostridium thermocellum and produced in Nicotiana benthamiana plants using a transient expression system. Target antigens were purified and evaluated in mice for their potential as prophylactic and therapeutic vaccine candidates. Both fusion proteins induced E7-specific IgG and cytotoxic T-cell responses and protected mice against challenge with E7-expressing tumor cells. Furthermore, when administered after challenge, these plant-produced antigens prevented tumor development.     

An improved rearranged Human Papillomavirus Type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response

A new and very promising approach in vaccine development is the application of naked DNA. In comparison to conventional vaccines it offers several advantages, especially if there is a need for the development of low cost vaccines. Infection with high-risk human papillomaviruses (hr-HPVs) is the major risk factor for the development of cervical cancer (cc), the third most common cancer in women worldwide. The HPV E7 oncogene is constitutively expressed in HPV-infected cells and represents an excellent target for immune therapy of HPV-related disease. Therefore, we chose the HPV-16 E7 as model antigen in the development of a therapeutic DNA vaccine candidate. For safety reasons the use of a transforming gene like the HPV-16 E7 for DNA vaccination is not feasible in humans. In consequence we have generated an artificial ("shuffled") HPV-16 E7-gene (HPV-16 E7SH), containing all putative cytotoxic T-lymphocyte (CTLs) epitopes and exhibiting high safety features. Here, we show the induction of a strong E7-wildtype (E7WT) directed cellular and humoral immune response including tumor protection and regression after in vivo immunization in the murine system. Moreover, the vaccine candidate demonstrated immunogenicity in humans, demonstrated by priming of antigen-specific T cells in vitro. Importantly, the artificial HPV-gene has completely lost its transforming properties as measured in soft agar transformation assays. These results may be of importance for the development of vaccines based on oncogenes or oncoproteins.     

Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7

Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.     

Enhanced tumour growth after DNA vaccination against human papilloma virus E7 oncoprotein: evidence for tumour-induced immune deviation

We have examined the induction of anti-tumour immunity in a murine model using a gene vaccine approach to deliver a well defined tumour antigen. The vaccines expressed the human papilloma virus type 16 (HPV 16) E7 oncoprotein, and protection was measured against HPV 16-expressing C3R tumour cell line in vivo. In control mice injected with saline, C3R cells initially formed tumours but then regressed completely. As expected, animals injected with a peptide that represents the D(b)-presented CTL epitope from E7 (RAHYNIVTF) were completely protected from tumour growth. Contrary to expectation, however, we consistently saw enhanced tumour growth, delayed regression, or tumour outgrowth in mice vaccinated with two different E7-expressing DNA vaccines. We found no evidence for loss of D(b) or K(b) class I MHC molecules from C3R cells recovered from outgrown tumours, and fluorescent MHC/peptide tetramer staining revealed E7 gene vaccination did not delete RAHYNIVTF-specific CD8(+) T cells. However, we did observe an effect on cytokine production. Splenocytes from E7 gene vaccinated animals responded to re-stimulation in vitro with C3R cells by producing IL-4 but background levels of IFN-gamma. We also observed that cytokine production and E7 peptide-specific CTL were only detectable in vaccinated animals after C3R challenge, but not after DNA priming alone. We conclude that 'prime-boosting' is necessary to observe tumour-specific T cell responses with the gene vaccine approach, but that boosting with tumour cells causes skewing of the primed cells in a T2 direction that is incompatible with protective anti-tumour immunity.     

A single amino acid substitution improves the in vivo immunogenicity of the HPV16 oncoprotein E7(11-20) cytotoxic T lymphocyte epitope

One of the few and most extensively studied human papilloma virus (HPV) type 16 oncoprotein E7-derived cytotoxic T lymphocyte (CTL) epitopes is YMLDLQPETT, presented to CTL by HLA-A2.1. We previously identified an altered peptide ligand (APL) of this epitope with increased binding affinity for HLA-A2.1, YMLDLQPETV. Herein, the in vivo immunogenicity of this APL was investigated in HLA-A2.1 transgenic HHD mice. Both in vitro and direct ex vivo analysis, performed using newly generated HHD tetramers, showed a significant increase in the number of specific CD8+ T cells upon vaccination with the APL as compared to its unmodified counterpart. Improved immunogenicity of the APL was also observed in functional analyses, including antigen-specific lytic activity and cytokine production of primed CD8+ T cells. Consequently, the YMLDLQPETV peptide may prove useful when included in vaccination strategies against HPV16-induced cervical carcinoma.     

Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7

Although many clinical trials on human papillomavirus (HPV) therapeutic vaccines have been performed, clinical responses have not been consistent. We have addressed mucosal cytotoxic cellular immune responses to HPV16 E7 after oral immunization of mice with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7). C57BL/6 mice were orally exposed to 0.1–100 mg/head of attenuated LacE7 or vehicle (Lac) vaccines at weeks 1, 2, 4, and 8. Responses to subcutaneous or intramuscular injection of an HPV16 E7 fusion protein using the same timing protocol were used for comparison. Oral immunization with LacE7 elicited E7-specific IFNγ-producing cells (T cells with E7-type1 immune responses) among integrin α4β7⁺ mucosal lymphocytes collected from gut mucosa. An induction of E7-specific granzyme B-producing cells (E7-CTL) exhibiting killer responses toward HPV16 E7-positive cells was also observed. The induction of T cells with specific mucosal E7-type1 immune responses was greater after oral immunization with LacE7 when compared to subcutaneous or intramuscular antigen delivery. Oral immunization with Lactobacillus-based vaccines was also able to induce mucosal cytotoxic cellular immune responses. This novel approach at a therapeutic HPV vaccine may achieve more effective clinical responses through its induction of mucosal E7-specific CTL.     

Modification of HPV 16 E7 genes: correlation between the level of protein expression and CTL response after immunization of C57BL/6 mice

Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of E7-specific cytotoxic T cells [Liu WJ, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, et al. Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 2002;301:43]. Here, we sought to verify the hypothesis that there is a direct correlation between the level of protein expression and immunogenicity in mice. We generated HPV 16 E7 expression plasmids where the genes were inserted either as authentic sequence (wt) or after optimizing the codons for use in mammalian cells (opt). For enhancement of translation of the E7 gene a 5' Kozak sequence (K) was added. Transfection experiments revealed the strength of expression in the order of E7opt+K, E7opt-K, E7wt+K and E7wt-K. After immunization of C57/B6 mice we observed an equally strong CD8+T-cell response with the E7opt plasmids (+ or -K), followed by the E7wt+K and E7wt-K DNAs. The same difference in efficiency was obtained in tumor protection experiments. Regression of pre-existing tumors and CTL activity was observed only with the E7opt+K plasmid. From these data, we conclude that the level of protein expression correlates with the efficiency of CTL response and hence testing by transfection of cells in culture may allow a pre-selection of expression plasmids prior to DNA immunization.     

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.     

Efficiency of HPV 16 L1/E7 DNA immunization: influence of cellular localization and capsid assembly

Infections by human papillomaviruses (HPV) are the major cause of uterine cancer in women worldwide. Aiming to develop a combined prophylactic and therapeutic vaccine we have previously demonstrated immunogenicity of chimeric virus-like particles consisting of a C-terminally truncated HPV 16 L1 capsid protein fused to an E7 portion. Here we show that genetic vaccination with a corresponding DNA was inefficient in the induction of a L1-specific prophylactic immune response. DNA immunization with C-terminally truncated HPV 16 L1 genes of different lengths revealed that only short deletions (L1(1-498)) were tolerated for eliciting a humoral immune response against viral capsids. This correlates with the observation that the C-terminal sequences are critical for nuclear localization, capsomere and capsid assembly. However, only the ability of L1 protein to form capsomeres or capsids showed a direct influence on the outcome of the immune response. C-terminal insertion of 60 amino acids of E7 was tolerated in fusion constructs, whereas insertion of full-length E7(1-98) or shuffled E7 (149 aa) completely abolished the humoral immune response. The L1(1-498)/E7(1-60) fusion construct not only induced L1-specific antibodies but also L1- and E7-specific CTL responses after DNA vaccination.     

Immunotherapy with CTL peptide and VSSP eradicated established human papillomavirus (HPV) type 16 E7-expressing tumors

Peptide-based vaccines aimed at the induction of effective T-cell responses against established tumors have not been successful in clinic and require the use of new adjuvants. One of those is a new adjuvant in which gangliosides are incorporated into the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). In a preclinical model of human papillomavirus HPV16-induced cervical cancer we show that vaccination with HPV 16 E7 derived minimal CTL peptide and VSSP protects mice against tumor challenge, induces regression of established tumors and produces E7-specific CD8⁺ T-cell responses.     

Therapeutic efficacy of a human papillomavirus type 16 E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a preclinical mouse model for HPV-associated disease

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8⁺ T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.     

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting functional tumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable.     

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.     

Boosting with recombinant vaccinia increases HPV-16 E7-specific T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines

We have previously linked the sorting signals of the lysosome-associated membrane protein-1 (LAMP-1) to HPV-16 E7 antigen, creating a chimera, Sig/E7/LAMP-1. We found that both Sig/E7/LAMP-1-containing recombinant vaccinia virus (Vac-Sig/E7/LAMP-1) and Sig/E7/LAMP-1 DNA can generate strong antitumor immunity. To determine whether combination of Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 can further enhance immune responses, sequential vaccination with Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 was given. We found that priming with Sig/E7/LAMP-1 DNA and boosting with Vac-Sig/E7/LAMP-1 generated the strongest E7-specific CD8(+) T cell responses. Our results encourage the use of the DNA prime/vaccinia booster regimen in future clinical trials.     

The efficacy of a DNA vaccine containing inserted and replicated regions of the E7 gene for treatment of HPV-16 induced tumors

A majority of cervical cancers are associated with Human Papillomavirus (HPV)-16. A DNA vaccine (E7IR) was designed for prophylactic and therapeutic treatment of HPV-16+ tumors containing two repeats of the E7 gene to inactivate transformation and duplicate available epitopes. Mice were vaccinated then tumor challenged, or challenged and then immunized and monitored for tumor volume and survival. Splenocytes were utilized for in vivo CTL assays. The E7IR vaccine demonstrated decreased tumor volume and enhanced survival in prophylactic and therapeutic experiments and improved CTL-mediated lysis. The E7IR vaccine shows promise in prevention of tumor formation and elimination of established tumors.     

Enhanced immunogenicity of HPV16E7 accompanied by Gp96 as an adjuvant in two vaccination strategies

Human papillomavirus, particularly type 16 (HPV16) is present in more than 99% of cervical cancers. E7 is the major oncogenic protein produced in cervical cancer-associated HPV16. An efficient vaccine against viral infection requires induction of strong humoral and cellular responses against viral proteins. Heat shock proteins (HSPs) like Gp96 have been described as potent tumor vaccines in animal models and are currently studied in human clinical trials. In this study, we investigated the utility of HPV16 E7 along with Gp96 as an adjuvant in C57BL/6 mice model. We compared the level of humoral and cellular immune responses by E7+Gp96 co-injection as DNA/DNA and prime-boost (DNA/protein) immunization strategies. In prime-boost immunization strategies, we first immunized C57BL/6 mice with the complete open-reading frame of E7 and Gp96 (pcDNA-E7 and pcDNA-Gp96) and then boosted with rE7, rNT-gp96 (N-terminal extension of Gp96) and rCT-gp96 (C-terminal extension of Gp96) mixed with Montanide 720 in different formulations. The humoral immune responses against rE7 and the different truncated forms of rGp96 suggested a mixed Th1/Th2 response with high intensity toward Th2. Assessment of lymphoproliferative and cytokine responses against rE7 and the different fragments of Gp96, showed that DNA vaccination including E7 and Gp96 induced Th1 response. We concluded that co-delivery of naked DNA E7+Gp96 plasmid was immunologically more effective than E7 alone. Our study demonstrated that co-delivery of E7+Gp96 as DNA/DNA and E7+CT-gp96 as DNA/protein could be an effective approach to induce E7-specific immune responses as a potential vaccine candidate for cervical cancer.     

Efficient induction of T helper 1 CD4+ T-cell responses to hepatitis C virus core and E2 by a DNA prime-adenovirus boost

Hepatitis C virus (HCV) is an important causative agent of liver disease, but currently there is no available prophylactic vaccine against HCV infection. Here, we investigated the HCV E2- and core-specific T-cell responses induced by DNA (D) and/or recombinant adenovirus (A) vaccines. In single (D versus A) or double immunizations (D-D versus A-A), the recombinant adenovirus vaccines induced higher levels of IFN-gamma secreting T-cell response and cytotoxic T lymphocytes (CTL) response than the DNA vaccines. However, a heterologous (D-A) regimen elicited the highest level of T helper 1 (Th1) CD4(+) T-cell responses. Furthermore, three E2-specific CTL epitopes were mapped using a peptide pool spanning the E2 protein sequence (a.a. 384-713) in BALB/c mice, and one of these (E2 405-414: SGPSQKIQLV) was shown to be immunodominant. Interestingly, no significant differences were found in the repertoire of E2-specific T-cell responses or in the immunodominance hierarchy of the three epitopes induced by D-D, D-A, A-A, and A-D, indicating that the breadth and hierarchy of T-cell responses is independent of these different vaccination regimens. In conclusion, the heterologous DNA prime-recombinant adenovirus boost regimen described offers an efficient promising strategy for the development of an effective T-cell-based HCV vaccine.     

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.     

Generation of anti-tumor immunity using mammalian heat shock protein 70 DNA vaccines for cancer immunotherapy

In this study, we explored the protective anti-tumor potency of mouse (self) Hsp70 or Hsp110-based DNA vaccination approach targeting a tumor-associated antigen, human papilloma virus (HPV) type 16 E7 protein. Linkage of E7 to the N-terminus of the mouse Hsp70 not only elicits an E7-specific cytotoxic T cell (CTL) response, but also protects mice against challenge with E7 expressing tumors. CD8+ T-cells are crucial in both priming and effector phases for the induction of tumor immunity, whereas CD4+ T-cells and NK cells do not appear to play a major role. Furthermore, the ATP-binding domain deletion mutant Hsp70(382-641), when fused to E7, was immunologically effective, suggesting that the peptide-binding region, not the ATPase domain of Hsp70, is required for the vaccine activity of the E7-Hsp70 DNA. This study demonstrates that autologous Hsp70 is highly potent in enhancing antigen-specific immune responses. Functional domain mapping and orientation of the E7 and Hsp70 in the fusion gene may have clinical implications for the design and optimization of Hsp70-based DNA vaccines.