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Aims: Human papillomavirus (HPV) L1, L2 and E7 proteins were used as target antigens for development of preventive and therapeutic vaccines. Moreover, linkage of antigens to heat shock proteins (HSPs) could enhance the potency of vaccines. Curcumin and nanocurcumin compounds were suggested as the chemopreventive and chemotherapeutic agents against cancer. In this study, two multiepitope DNA and peptide-based vaccine constructs (L1-L2-E7 and HSP70-L1-L2-E7) were used along with curcumin and nanocurcumin to evaluate immune responses, and protective/therapeutic effects in tumor mouse model., Main Methods: At first, the multiepitope L1-L2-E7 and HSP70-L1-L2-E7 fusion genes were subcloned in eukaryotic and prokaryotic expression vectors. The recombinant multiepitope peptides were generated in E. coli strain. Then, the cytotoxic effects of curcumin and nanocurcumin were evaluated on HEK-293 T non-cancerous and C3 cancerous cells. Finally, mice vaccination was performed using different regimens. Curcumin and nanocurcumin compounds were administered alone or along with different vaccine constructs., Key Findings: Our data indicated that the use of nanocurcumin along with the multiepitope HSP70-L1-L2-E7 vaccine construct could completely protect mice against HPV-related C3 tumor cells, and eradicate tumors in a therapeutic test. Furthermore, nanocurcumin showed higher protection than curcumin alone. Generally, curcumin and nanocurcumin compounds could reduce tumor growth synergistically with the multiepitope vaccine constructs, but they did not influence the immune responses in different regimens., Significance: These data demonstrated that the designed multiepitope vaccine constructs along with curcumin and nanocurcumin can be used as a promising method for HPV vaccine development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

CD8 + T cell immune response plays a critical role in the clearance of human papillomavirus (HPV)-infected cells. During the natural history of HPV infection, the E1 protein, an early-expressed helicase highly conserved among papillomaviruses, is involved in the replication of HPV genomes. We have previously shown, in a murine model, that immunization with HPV18 E1 protein combined with α -galactosylceramide elicits a specific CD8 + T cell response. We further proved those findings by analyzing whether CD8 + T cells from mice immunized with α -galactosylceramide plus HPV18 E1 protein could have a cytotoxic effect on cells expressing the carboxyl-terminal domain from the E1 proteins of other HPV types. Interestingly, CD8 + T cells raised against HPV18 E1 antigen presented cross-reactivity against the E1 protein from HPV53, 33, 16, and 31. Poor cross-reactivity was observed for HPV11, and none for HPV6. This outcome may be relevant for the design of broad-spectrum immune-protective agents against HPV infections.

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50-60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV + SCCOP have better overall and disease-specific survival than patients with HPV - SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV + SCCOP. Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV + oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR ., (©2018 American Association for Cancer Research.)

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., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Numerous types of human papillomaviruses (HPVs) have been identified, and the global burden of diseases associated with HPV infection is remarkable, especially in developing regions. Thus a low-cost broad-spectrum prophylactic vaccine is urgently needed. The N-terminal amino acid 17-36 of HPV 16 L2 protein is confirmed to be a major cross-neutralizing epitope (RG-1 epitope). Monomeric proteins containing RG-1 epitopes and scaffold proteins, such as bacterial thioredoxin or modified IgG1 Fc fragment and L2 epitope fusion protein, induced cross-neutralizing antibodies, arousing the possibility of the development of low-cost monomeric vaccine in bacterial expression system. Here we show that a novel immunogen-scaffold protein containing a lipidated triple-repeat HPV 16RG-1 epitope and a hFcγRI specific single-chain antibody fragment (H22scFv), named LpE3H22, elicited high titers of cross-neutralizing antibodies against a broad range of mucosal and cutaneous HPV types when adjuvanted with MF59 and poly I:C. LpE3H22 was produced in E. coli expression system. In contrast to three repeats of RG-1 epitope (E3) and unlipidated fusion protein E3H22, vaccination of LpE3H22 induced robust cross-neutralizing antibody responses in hFcγRI transgenic mice. Furthermore, the neutralizing antibody response induced by LpE3H22 was significantly weaker in WT mice than in the Tg mice. The cross-neutralizing antibodies induced by LpE3H22 sustained for at least 10months in Tg mice. Our results demonstrate that hFcγRI targeting and lipidation both contribute to the enhancement of immunogenicity of L2 antigen. Therefore, delivering the lipidated L2 antigen with H22scFv opens a new avenue for low-cost pan-HPV vaccine development., (Copyright © 2016. Published by Elsevier Ltd.)

HPV vaccines based on L1 virus-like particles (VLPs) provided a high degree of protection against HPVs infection. In this study, the codon optimized HPV16 L1 gene were sub-cloned into five procaryotic expression vectors (pET-28a, pET-32a, pGEX-4T-2, pE-sumo and pHSIE), and fused with different protein tags. No recombinant proteins were expressed in pET-28a-L1 and pHSIE-L1, and the proteins expressed by pET-32a-L1 plasmid with TRX-tag were in the form of inclusion body. Only SUMO-tagged and GST-tagged L1 proteins expressed by pE-Sumo-L1 or pGEX-4T-L1 were soluble. The yield of SUMO-L1 protein reached 260mg/L fermentation medium in shake flask. After SUMO tags were eliminated, a 90% purity of L1 proteins was generated by ion-exchange and Ni-NTA affinity chromatography. The purified HPV16 L1 protein self-assembled into virus-like particles (VLPs) and showed a haemagglutination activity. High titers specific and neutralizing antibodies were detected in HPV 16 L1VLPs vaccinated mice. Cytokines such as IFN-γ and IL-2 showed significant higher in VLPs vaccinated mice compared with negative control (p<0.05, p=0.055). Thus, the expression of recombinant HPV16 L1 VLPs in Escherichia coli was feasible, which could potentially be used for a VLP-based HPV vaccine., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.

Bovine papillomavirus (BPV) is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control). Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine.

Background: The protein products of the early genes E6 and E7 in high-risk HPV types 16 and 18 have been implicated in the oncogenic capability of these viruses. Therefore, these peptides represent attractive vaccine therapy targets., Methods: Thirty-two patients with advanced cervical cancer (HPV16 or 18 positive) were treated with HPV16 E6 (18-26) (Arm A) or HPV16 E7 (12-20) peptide (Arm B) pulsed on PBMCs in order to illicit immune response against the relevant peptide on both arms. These PBMCs were cultured for a short time (48 hours only) and in the presence of GM- CSF, accordingly, they were identified as "Pre-Immature Dentritic Cells"., Results: 51Cr release assay and ELISPOT demonstrated evidence of specific immune response against the relevant peptide in 10/16 (63%) evaluable patients in arm A and 7/12 (58%) in arm B. HPV16 E6 was found to be homologous to HPV18 E6 in both vivo and vitro. The median overall survival (OS) and progression free survival (PFS) for the full cohort was 10.0 and 3.5 months, respectively. There were no RECIST responses in any patient. The majority of toxicities were grade I and II., Conclusions: We demonstrated the feasibility and ability of Pre-Immature Dentritic Cells pulsed with HPV16 E6 (18-26) or HPV16 E7 (12-20) to induce a specific immune response against the relevant peptide despite the advanced disease of the cervical cancer patients treated on this trial. We believe that this observation deserves further investigations.

Plasmid DNA (pDNA) and small interfering RNAs (siRNAs) are very useful tools for the treatment of cancer. However, pDNA and siRNAs efficacy is restricted by their negative charge and susceptibility to degradation by endonucleases that prevent them penetrating tissue and cellular barriers such as the plasma and endolysosomal membranes. Viral vectors have some advantages but their use is largely limited by their immunogenicity. On the other hand, synthetic nanoparticles have advantage of being relatively non-immunogenic but their ability to deliver nucleic acids remains less efficient than their viral counterparts. The present study is focussed on the development and evaluation of biomimetic lipid nanocapsules (LNCs) functionalized with a L1 papillomavirus type-16 capsid-derived lipopeptide on their surface, for transfection of U87MG glioma cells and Caco-2 colorectal adenocarcinoma cells with pDNA or siRNAs. Since the L1-peptide has been described as a nuclear localization signal able to complex with nucleic acids and bind to heparan sulfate on the cell surface, the structure and function of L1-peptide bound to LNCs (L1-LNCs) were investigated. Although L1-LNCs were shown to complex with both pDNA and siRNAs, the pDNA-L1-LNC complexes showed only weak transfection efficiency. In contrast, siRNA-L1-LNC complexes appeared as effective repressors of targeted messengers., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) is the cause of most cases of cervical cancer. HPV type 58 (HPV58) is the second most frequent cause of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) in Asia and South / Central America, respectively. However, there is no vaccine against HPV58, although there are commercially available vaccines against HPV16 and 18. In this study, we produced HPV58 L1 protein from Saccharomyces cerevisiae, and investigated its immunogenicity. We first determined the optimum period of culture for obtaining HPV58 L1. We found that a considerable portion of the HPV58 L1 resulting from 48 h culture cannot be recovered by purification, while the HPV58 L1 resulting from 144 h culture is recovered efficiently: the yield of HPV58 L1 finally recovered from 144 h culture was 2.3 times higher than that from 48 h culture, although the production level of L1 protein from 144 h culture was lower than that from 48 h culture. These results indicate that the proportion of functional L1 protein from 144 h-cultured cells is significantly higher than that of 48 h-cultured cells. The HPV58 L1 purified from the 144 h culture was correctly assembled into structures similar to naturally occurring HPV virions. Immunization with the HPV58 L1 efficiently elicited anti-HPV58 neutralizing antibodies and antigen-specific CD4+ and CD8+ T cell proliferations, without the need for adjuvant. Our findings provide a convenient method for obtaining substantial amounts of highly immunogenic HPV58 L1 from S. cerevisiae.

CIA05 is a toll-like receptor (TLR) 4 agonist derived from an Escherichia coli lipopolysaccharide (LPS) mutant and has been shown to have potential as a vaccine adjuvant. In this study, we investigated the immunopotentiating activity of the adjuvant system CIA06, which is comprised of CIA05 and aluminum hydroxide (alum), when used with the human papillomavirus (HPV) L1 virus-like particles (VLPs) vaccine. BALB/c mice were immunized intramuscularly three times at 2-week intervals with HPV16 L1 VLPs alone or in the presence of various combinations of CIA05 and alum, and the immune responses were assessed. We found that the combination of CIA05 and alum at a ratio of 1:50 (designated CIA06B) yielded the highest immune response in terms of serum anti-HPV L1 VLP IgG antibody titers, splenocyte interferon (IFN)-γ secretion, and antigen-specific memory B cell responses. The immunogenicity of the CIA06B-adjuvanted HPV16/18 L1 VLP vaccine was compared with that of the currently licensed HPV vaccine Cervarix™. The CIA06B-adjuvanted vaccine was similar to Cervarix™ with regard to eliciting serum antigen-specific IgG antibodies and virus-neutralizing antibodies but more effective at inducing splenic cytokine production and memory B cells. We also observed that the antigen-specific IgG antibody titers, splenic IFN-γ secretion and memory B cells induced by the CIA06B-adjuvanted HPV vaccine remained high up to 24 weeks post-immunization. Based on these data, we concluded that CIA06B may have potential as an adjuvant in a potent prophylactic vaccine against HPV infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To determine whether immunotherapy with HPV6 L1 virus like particles (VLPs) without adjuvant (VLP immunotherapy) reduces recurrence of genital warts following destructive therapy., Trial Design: A randomized placebo controlled blinded study of treatment of recurrent genital warts amenable to destructive therapy, conducted independently in Australia and China., Methods: Patients received conventional destructive therapy of all evident warts together with intramuscular administration of 1 µg, 5 µg or 25 µg of VLP immunotherapy, or of placebo immunotherapy (0.9% NaCl), as immunotherapy at week 0 and week 4. Primary outcome, assessed at week 8, was recurrence of visible warts., Results: Of 33 protocol compliant Brisbane recipients of placebo immunotherapy, 11 were disease free at two months, and a further 9 demonstrated reduction of > 50% in total wart area. Wart area reduction following destructive treatment correlated with prior duration of disease. Among 102 protocol compliant Brisbane recipients of VLP immunotherapy, disease reduction was significantly greater than among the placebo immunotherapy (50% ± s.e.m. 7%) recipients for subjects receiving 5 µg or 25 µg of VLP immunotherapy/dose (71% ± s.e.m. 7%) but not for those receiving 1 µg VLP immunotherapy/dose (42% ± 7%). Of 52 protocol compliant placebo immunotherapy recipients in Wenzhou, 37 were disease free at two months, and a further 8 had > 50% disease reduction. Prior disease duration was much shorter in Wenzhou subject (8.1 ± 1.1 mo) than in Brisbane subjects (53.7 ± 5.5 mo). No significant reduction in mean wart area was observed for the 168 Wenzhou protocol compliant subjects who also received VLP immunotherapy., Conclusions: This study confirms the findings in a previous open label trial that administration of VLP immunotherapy may assist in clearance of recurrent genital warts in patients for whom destructive therapy is unsuccessful and that unsuccessful destructive therapy is more common with increasing prior disease duration.

Aim: To study the effect of chimeric E7 protein of human papillomavirus type 18 on activation of adaptive immunity in absence of adjuvant., Materials and Methods: Chimeric protein was genetically engineered and represents the protein molecule consisting of full-size E7 oncoprotein and heat-shock protein 70 (HSP70) of Mycobacterium tuberculosis in one polypeptide chain. Antibody titers as well as isotypes and subisotypes of immunoglobulins were measured by ELISA in sera of immunized animals., Results: It was shown that studied construction E7 (HPV-18)-HSP70 significantly increases titers of antibodies to E7 protein of HPV type 18 and have cross-reactive antigenic activity with E7 protein of HPV type 16. Immunization with chimeric protein resulted in increase of IgG1 and IgG2b levels and decrease of IgG2a and IgM levels., Conclusion: . Oncoprotein E7 included in chimeric construction with HSP70 could be used for further studies on development of therapeutic vaccine for treatment of cervical cancer and precancerous lesions. Skew of immune response to Th2 type after intraperitoneal administration of the studied construction points to necessity for control of immunity during such studies.

Fve is a fungal protein isolated from the golden needle mushroom Flammulina velutipes and has previously been reported to trigger immunological responses in both mouse and human lymphocytes. In this study, we evaluated the potential application of Fve as an adjuvant for tumour immunotherapy and examined the underlying mechanism(s). When the human papillomavirus (HPV)-16 E7 oncoprotein was used as a model antigen, mice coimmunized with HPV-16 E7 and Fve showed enhanced production of HPV-16 E7-specific antibodies as well as expansion of HPV-16 E7-specific interferon (IFN)-gamma-producing CD4(+) and CD8(+) T cells as compared with mice immunized with HPV-16 E7 alone. Tumour protection assays showed that 60% of mice coimmunized with HPV-16 E7 plus Fve, as compared with 20% of those immunized only with HPV-16 E7, remained tumour-free for up to 167 days after challenge with the tumour cells. Tumour therapeutic assays showed that HPV-16 E7 plus Fve treatment significantly prolonged the survival of tumour-bearing mice as compared with those treated only with HPV-16 E7. In vivo cell depletion and adoptive T-cell transfer assays showed that CD4(+) and CD8(+) T cells and IFN-gamma played critical roles in conferring the antitumour effects. Interestingly, Fve could stimulate the maturation of splenic dendritic cells in vivo and induce antigen-specific CD8(+) T-cell immune responses. In summary, Fve has potent adjuvant properties that enhance T helper type 1 antigen-specific humoral and cellular immune responses which confer strong antitumour effects. The use of Fve as an adjuvant could be an attractive alternative to the current vaccination strategy for cancer immunotherapy.

Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.

Background: Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility., Results: We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes., Conclusion: In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing prophylactic/therapeutic vaccines. The fact that they are produced in plants, may lower production costs considerably.

Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.

Human papilloma-virus (HPV) infection is the major cause of cervical cancer. HPV18 is the most prevalent high-risk HPV after type 16 that accounts for the largest number of cervical cancer cases worldwide. Currently, although prophylactic vaccines have been developed, there is still an urgent need to develop therapeutic HPV vaccines for targeting tumors post-infection. In this study, we utilize a novel multi-phase strategy for HPV18 antigen development with the goal of increasing anti-HPV18 cellular immunity. Our data show that this construct can induce strong cellular immune responses against HPV18 E6 and E7 antigens in a murine model. Moreover, when applied to rhesus monkeys, this construct is also able to elicit cellular immunity. These data suggest such DNA immunogens are candidates for further study in the eventual context of immunotherapy for HPV-associated cancers.

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD4+ Th1/Th2 and CD8+ T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity., ((c) 2008 Wiley-Liss, Inc.)

Genital human papillomavirus (HPV) infection is the primary cause of cervical cancer in women. Although the HPV recombinant L1 protein was recently licensed as an available vaccine, it has numerous shortcomings. New vaccination strategies should be considered. To enable the design of a prophylactic and therapeutic low-cost vaccine candidate, chimeric HPV16 L1DeltaC34E7N1-60 capsomeres were produced in Escherichia coli. The immune characteristics and potential prophylactic and therapeutic effects of these capsomeres were examined in C57BL/6 mice. Following protein purification and renaturation, the majority of the recombinant chimeric proteins (L1DeltaC34E7N1-60) assembled into capsomeres. These capsomeres were able to induce conformational and neutralizing antibodies against HPV virus-like particles and trigger cell-mediated specific immune responses against the L1 and E7 peptides. In vivo tumor challenge assays showed that mice immunized with the capsomeres were protected against a challenge with both C3 and TC-1 tumor cells. Furthermore, in vivo tumor rejection assays showed that capsomeres have therapeutic efficacy in mice following inoculation with C3 and TC-1 tumor cells. Chimeric capsomeres are capable of preventing and eliminating HPV16 infection. Therefore, our study has provided an economical vaccine candidate.

We are developing recombinant attenuated vesicular stomatitis virus (VSV) as a vaccine vector to generate humoral and cell-mediated immune responses. Here, we explore the use of VSV vaccines for cancer immunotherapy. Immunotherapy targeting high-risk human papillomavirus (HPV) lesions has the potential to benefit HPV-infected individuals and cervical cancer patients by generating cytotoxic T cells that kill tumor cells that express viral antigens. A single dose of VSV expressing the HPV type 16 (HPV16) E7 oncogene was used for therapeutic vaccination of mice bearing TC-1 syngeneic tumors, which express HPV16 E7. HPV16 E7-specific T cells were generated and displayed cytotoxic activity against the tumor cells. By 14 days postvaccination, average tumor volumes were 10-fold less in the vaccinated group than in mice that received the empty-vector VSV, and regression of preexisting tumors occurred in some cases. This antitumor effect was CD8 T-cell dependent. Our results demonstrate antitumor responses to HPV16 E7 and suggest that recombinant-VSV-based vaccination should be explored as a therapeutic strategy for cervical carcinoma and other HPV-associated cancers.

Human papillomaviruses have been recognized as the causative agent of anogenital cancer. In 2006, a commercial vaccine based on virus-like particles composed of the L1 major capsid protein of the papillomaviruses has been available. This vaccine induces virus-neutralizing antibody responses upon parenteral injection. Here we investigated the oral immunogenicity of different assembly forms of HPV 16 L1, that is: T7-VLPs, T1 particles and capsomeres. Our results show that all three assembly forms induce humoral and cellular immune responses after oral vaccination of mice. The anti-L1 antibodies were conformation-specific and showed neutralizing activity in a pseudovirion-based assay. We also investigated if adjuvants have an influence on the oral immunogenicity of the different L1 forms. For saponins we observed a significant toxicity if applied orally. Co-administration of either CpG DNA or Escherichia coli heat-labile enterotoxin LT(R192G) had no apparent enhancing effect on the production of anti-L1 antibodies. More pronounced was the effect of CpG administration on the long-term immunity as we observed a significantly stronger recall response 244 days after the first vaccination. Compared to capsomeres, VLPs induced stronger humoral immune responses while the CTL responses were induced at comparable levels. Finally, we were also able to induce neutralizing antibodies and L1-specific cytotoxic T-lymphocytes after oral administration of crude extracts of L1-expressing insect cells. In conclusion, all three assembly forms of the L1 protein are immunogenic when administered orally.

Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus-like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric virus-like particles (CVLP) consist of a carboxy-terminally truncated HPV16L1 protein fused to the amino-terminal part of the HPV16 E7 protein and self-assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1- and E7-specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo-controlled clinical trial has been conducted in 39 HPV16 mono-infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 mug or 250 mug) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty-six percent of the responders were also HPV16 DNA-negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine., ((c) 2007 Wiley-Liss, Inc.)

To determine the systemic cytokine pattern induced by vaccination with human papillomavirus (HPV) L1 virus-like particles (VLP), we analyzed 22 different cytokines in culture supernatants of L1 VLP-stimulated peripheral blood mononuclear cells from vaccine (n = 19) and placebo (n = 7) recipients at months 0 and 2 after vaccination, using a multiplex cytokine bead array. In vaccine recipients, incubation with L1 VLP in vitro led to a statistically significant increase in production of Th1 (granulocyte-macrophage colony-stimulating factor, interleukin-2 [IL-2], gamma interferon; P < 0.0007) and Th2 (IL-4, IL-5, IL-10, IL-13; P < 0.0017) cytokines and the chemokine IP-10 (P = 0.0021) at month 2 after immunization, compared to levels seen prior to vaccination. These responses were not seen in placebo recipients. Cytokine and neutralizing antibody responses to vaccination followed the same pattern, with the highest antibody responses seen for subjects with higher cytokine responses. Cytokine profiling studies using samples from efficacy trials may provide important information about discriminators of long-term protection against HPV.

Infection with human papillomavirus type 16 (HPV16) is strongly associated with a number of disease states, of which cervical and anal cancers represent the most drastic endpoints. Induction of T-cell-mediated immunity, particularly cytotoxic T lymphocytes (CTL), is important in eradication of HPV-induced lesions. Studies have shown that heat shock protein fusion proteins are capable of inducing potent antigen-specific CTL activity in experimental animal models. In addition, E7-expressing tumors in C57BL/6 mice can be eradicated by treatment with HspE7, an Hsp fusion protein composed of Mycobacterium bovis BCG Hsp65 linked to E7 protein of HPV16. More importantly, HspE7 has also displayed significant clinical benefit in phase II clinical trials for the immunotherapy of HPV-related diseases. To delineate the mechanisms underlying the therapeutic effects of HspE7, we investigated the capability of HspE7 to induce antigen-specific protective immunity. Here, we demonstrate that HspE7 primes potent E7-specific CD8(+) T cells with cytolytic and cytokine secretion activities. These CD8(+) T cells can differentiate into memory T cells with effector functions in the absence of CD4(+) T-cell help. The HspE7-induced memory CD8(+) T cells persist for at least 17 weeks and confer protection against E7-positive murine tumor cell challenge. These results indicate that HspE7 is a promising immunotherapeutic agent for treating HPV-related disease. Moreover, the ability of HspE7 to induce memory CD8(+) T cells in the absence of CD4(+) help indicates that HspE7 fusion protein may have activity in individuals with compromised CD4(+) functions, such as those with invasive cancer and/or human immunodeficiency virus infection.

Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.

Human papillomaviruses (HPVs) are the causative agents of several disease states, including genital warts and cervical cancer. There are around 500 million cases of genital warts per annum worldwide and around 450,000 cases of cervical cancer. Although HPV vaccines should eventually reduce the incidence of these diseases, new and effective treatments are still urgently required. The E2 (early) proteins from some HPV types induce growth arrest and apoptosis, and these proteins could be used as therapeutics for HPV-induced disease. A major obstacle to this approach concerns the delivery of the protein to HPV-transformed cells and/or HPV-infected cells in vivo. One possible solution is to use recombinant viruses to deliver E2. Another possible solution is to use purified E2 proteins or E2 fusion proteins. The herpes simplex virus VP22 protein is one of a small number of proteins that have been shown to cross the cell membrane with high efficiency. VP22-E2 fusion proteins produced in bacterial cells are able to enter mammalian cells and induce apoptosis. This suggests that VP22-E2 fusion proteins could be topically applied as a treatment for HPV-induced diseases, most probably post-surgery. In this review, we discuss this and other approaches to the topical delivery of selective therapeutic agents against HPV-associated conditions.

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.

Vaccination with human papillomavirus type 16 (HPV16) L1 virus-like particles (VLP) induces both high titer neutralizing IgG and protective immunity. Because protection from experimental infection by papillomavirus is mediated by neutralizing IgG, we sought the mechanisms that trigger humoral immunity to HPV16 L1 VLP. We find that HPV16 L1 VLP bind to murine B lymphocytes thereby inducing activation-induced cytidine deaminase expression and Ig class switch recombination to cause the generation of IgG. HPV16 L1 VLP also activate production of proinflammatory factors IFN-alpha, IL-6, MIP-1alpha, RANTES, and KC, up-regulate the expression of costimulatory molecules by naive B cells, and increase the B1 B cell subpopulation. These B cell responses to HPV16 L1 VLP are dependent upon MyD88. Although MyD88(-/-) B cells produce only mu transcript after exposure to HPV16 L1 VLP, MyD88(+/+) B cells express alpha, gamma, and mu Ig H chain and activation-induced cytidine deaminase transcripts. Notably, TLR4 mutant C3H/HeJ mice exhibited significantly reduced HPV16 VLP-specific IgG1, IgG2a, IgG2b, and IgG3 titers after vaccination as compared with the control C3H/HeOuJ mice. HPV16 L1 VLP directly activated class switch recombination and costimulatory molecule expression by B cells of C3H/HeOuJ mice but not C3H/HeJ mice. Thus HPV16 L1 VLP directly activate B cells to induce CD4(+) T cell independent humoral immune responses via TLR4- and MyD88-dependent signaling.

To establish an edible HPV16 vaccine, we constructed a recombinant HPV16 L1-expressing Schizosaccharomyces pombe yeast strain (HPV16L1 yeast). A preliminary study revealed that freeze-dried yeast cells could be delivered safely, and were digested in the mouse intestine. The freeze-dried HPV16 L1 yeast was administered orally as an edible vaccine, with or without the mucosal adjuvant heat-labile toxin LT (R192G), to 18 female BALB/c mice. After the third immunization, none of the mice that received the edible HPV16 vaccine showed specific antibody responses, whereas all of the positive controls that were administered intranasally with 5 microg of HPV16-virus-like particles (VLP) had serum IgG, and genital IgA and IgG that reacted with HPV16-VLP in enzyme-linked immunosorbent assays (ELISAs). When a suboptimal dose (1 microg) of HPV16-VLP was administered to all the mice, including the negative control mice, 50% of the mice that were pre-immunized with the edible HPV16 vaccine showed positive serum IgG responses, while none of the negative controls showed any response. Vaginal IgG and IgA antibodies were also elicited in 33 and 39%, respectively, of the mice that were given with the edible HPV16 vaccine and the intranasal boost. All of the antibodies reacted more strongly to intact HPV16-VLP than to denatured HPV16-L1 protein suggesting that the edible vaccine primes for antibody responses against conformation-dependent epitopes. The inclusion of adjuvant in the vaccine formulation marginally increased the genital IgA response (P=0.06). HPV16-L1 protein in the yeast might induce tolerance in the vaccinated animals that could be recovered by intranasal boosting with a suboptimal dose of HPV-VLP. This freeze-dried yeast system may be useful as an oral delivery of HPV 16 L1 protein.

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n=20) or placebo (n=4) before and after vaccination. 11 cytokines were measured: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient tool for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies.

A combination of different types of vaccines usually induces enhanced immune responses in comparison to immunization with single vaccines. The highest efficacy of a heterologous prime-boost strategy is mostly achieved after priming with a DNA vaccine and boosting with a recombinant virus or a protein vaccine. The aim of this study was to determine whether the combination of a DNA and cellular vaccine elicits stronger antitumor immune responses than vaccines used alone and to find out whether the efficacy of this combined immunization depends on the sequence in which the vaccines were applied. We utilized experimental vaccines that proved to be partially effective in protection against mouse tumor cells representing models of human papillomavirus-induced malignancies. The fusion gene Sig/E7GGG/LAMP-1, inoculated via a gene gun, was used for DNA immunization. As cellular vaccines, HPV16 E6/E7 and H-ras transformed B9 or 181 cells transduced with the gene coding for GM-CSF or IL-2, respectively, were applied. In both preventive and therapeutic immunizations, inoculation first with the DNA vaccine followed by application of a cellular vaccine induced the best protection from tumor growth. These results were confirmed by detection of immune reactions with in vitro tests. We failed to enhance immune reactions by utilization of an equivalent mix of B9 and 181 cells, but the addition of the second DNA-vaccine dose applied simultaneously with a cellular-vaccine boost moderately increased antitumor response. Our findings suggest the benefit of the heterologous prime-boost strategy based on combination of a DNA vaccine with a cellular vaccine and importance of sequence in which the vaccines are administered.

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.

We have previously shown that intradermal coadministration of DNA encoding Bcl-x(L), an antiapoptotic protein, with DNA encoding E7 antigen linked to the sorting signal of the lysosome-associated membrane protein type 1 (Sig/E7/LAMP-1) prolongs dendritic cell life and enhances antigen presentation through the MHC class I and II pathways. In the current study, we compared this approach with a conventional DNA prime-vaccinia boost protocol on the basis of their ability to generate antigen-specific CD8(+) memory T cells and longterm antitumor effects against an E7-expressing tumor. Mice primed and boosted with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA generated significantly higher numbers of E7-specific CD8(+) memory T cells and a better long-term protective antitumor effect compared with mice primed with Sig/E7/LAMP-1 DNA and boosted with Sig/E7/LAMP-1 vaccinia (Vac-Sig/E7/LAMP-1). Furthermore, coadministration of Sig/E7 /LAMP-1 DNA mixed with Bcl-x(L) DNA also generated higher avidity E7-specific CD8(+) T cells than did vaccination with Sig/E7/LAMP-1 DNA followed by a Vac-Sig/E7/LAMP-1 booster. Our results indicate that coadministration of a DNA vaccine employing intracellular targeting strategies and a DNA encoding antiapoptotic proteins may potentially generate a higher number of memory CD8(+) T cells and better long-term protective antitumor effects compared with the conventional DNA prime-vaccinia boost regimen.

Our goal in the present work was to characterize the multiple steps involved in overcoming the anergy that exists in tumor hosts to tumor-associated antigen (TAA). Our studies showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L vector resulted in secretion of the TAA/ecdCD40L protein for at least 10 days from infected cells. Binding of the TAA/ecdCD40L protein to dendritic cells (DCs) resulted in the induction of CCR-7 chemokine receptor expression and cytokine release. This was followed by migration of the DCs to regional lymph nodes. Tetramer staining, enzyme-linked immunospot (ELISPOT) assay, and cytotoxicity assay all showed that the Ad-sig-TAA/ecdCD40L vector increased the levels of splenic CD8(+) T cells specific for the 2 TAAs (human MUC1 [hMUC1] and HPV E7) tested. Vaccination with the Ad-sighMUC1/ecdCD40L vector suppressed the growth of hMUC1 antigen-positive tumor cells in 100% of the test mice that were previously anergic to the hMUC1 antigen. These data suggest that Ad-sig-TAA-ecd/ecdCD40L vector injections may be of value in treating the many epithelial malignancies in which TAA-like hMUC1 is overexpressed.

Infection by high-risk HPV (human papillomavirus) is supposed to be the primary cause of cervical cancer. The HPV E2 protein (E2) is a DNA-binding protein that regulates viral gene expression and is required for efficient viral replication. Overexpression of the E2 protein in cervical cancer cells can induce growth arrest and/or apoptotic cell death, suggesting that E2 might be useful in the treatment of this disease. In the present study, we show that VP22 (herpes simplex virus VP22 protein) can be used to deliver E2 to target cells. VP22-E2 fusion proteins induce apoptosis in transiently transfected HPV-transformed cervical carcinoma cell lines. However, VP22-E2 fusion proteins do not kill COS-7 cells, probably because these cells constitutively express the simian-virus-40 T antigen and this protein sequesters the tumour suppressor protein p53. When COS-7 cells producing VP22-E2 are seeded into cultures of HPV-transformed cells, VP22-E2 enters the non-producing cells and induces apoptosis. VP22-E2 proteins produced in bacterial cells can also enter cervical cancer cells and induce apoptosis in a dose-dependent manner. Our results suggest that local delivery of VP22-E2 fusion proteins could be used to treat cervical cancer and other HPV-associated diseases.

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., (Copyright 2004 National Science Council, ROC and S. Karger AG, Basel)

Objective: The objective of this study was to assess the safety and efficacy of a novel therapeutic, ZYC101a, for the treatment of women with histologically confirmed cervical intraepithelial neoplasia (CIN) 2/3. ZYC101a contains plasmid-DNA-encoding fragments derived from the E6 and E7 proteins of human papillomavirus (HPV) 16 and 18, and is formulated within small biodegradable microparticles., Methods: A multicenter, double-blind, randomized, placebo-controlled trial was conducted in a group of women with biopsy-confirmed CIN 2/3. Subjects were randomized to 3 intramuscular doses of either placebo or ZYC101a (100 or 200 microg). Six months after the first injection, subjects underwent cervical conization. The primary endpoint for this study was histologically confirmed resolution of CIN 2/3. A total of 161 subjects were randomized, dosed, and evaluated for safety. After central pathology review, 127 subjects were evaluable for efficacy., Results: The most common adverse events were related to the injection site, were mild to moderate, and did not worsen at later treatments. The proportion of subjects who resolved was higher in the ZYC101a groups compared to placebo (43% versus 27%), but the difference was not statistically significant (P =.12). In a prospectively defined population of women younger than 25 years (n = 43), resolution was significantly higher in the combined ZYC101a groups compared to placebo (70% versus 23%; P =.007). ZYC101a activity was not restricted to HPV-16-or HPV-18-positive lesions., Conclusions: ZYC101a was shown to be well tolerated in all patients and to promote the resolution of CIN 2/3 in women younger than 25 years., Level of Evidence: I

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-gamma effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as priming to virus without CTL induction is associated with inhibition. However, IL-10(-/-) mice, in contrast to IL-10(+/+) mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-gamma-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-x(L), prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-x(L) with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8(+) T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA showed the greatest increase in E7-specific CD8(+) T cells ( approximately 13-fold increase). This combination of strategies resulted in increased CD8(+) T cell functional avidity, an increased E7-specific CD4(+) Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-x(L) DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.

The role of natural killer T (NKT) cells in the immune response to tumor cells has been largely unexplored. As a model of adoptive tumor immunotherapy, cells from the draining lymph nodes of mice immunized with a tumor-specific or irrelevant antigen were transferred to naïve recipients with established tumor. Inhibition of early tumor growth (day 4) required the transfer of both CD8(+) and J alpha 18(+) (NKT) cells from immunized animals without regard to immunogen. In contrast, CD8(+) cells, but not J alpha 18(+) cells, were necessary for the inhibition of late tumor growth (day 8). Thus, the developing tumor changes in sensitivity to NKT-mediated events and the role for NKT cells cannot be replaced by the presence of tumor-specific cells during early tumor growth. This suggests that recruitment/activation of J alpha 18(+) NKT cells is an important consideration during the immune therapy of early stage tumors.

Human papillomavirus type 16 (HPV-16) L1- and E7-specific T cell responses were measured in 58 women with abnormal cervical cytology in a prospective study. On recruitment, patients responded most frequently and with the highest numbers of responding cells to the L1 region aa 311-345 and this response was significantly associated with the presence of cervical disease (P=0.041). Responses to the L1 peptide aa 281-295 were significantly higher in patients with CIN III than in those with HPV/CIN I or CIN II lesions (P=0.027). The E7 region aa 70-98 was the most immunogenic in patients with squamous intraepithelial lesions of the cervix (SIL) but the responses detected were not significantly higher than in patients without SIL. Following treatment, the T cell response profiles of patient groups did not change significantly. However, on analysis of the responses of individual patients with and without recurrent disease on follow-up, significant differences were found. Recurrence of disease was associated with T cell responses to the E7 region aa 70-98 at the patient's first clinic visit (P=0.017). Recurrence of disease was also accompanied by an increase in the total number of L1-specific short-term T cell lines (STLs) at follow-up, whereas absence of disease was accompanied by a decrease in L1-specific STLs. The data also suggested a possible link between E7 70-98-specific responses and acquisition of disease by patients who were previously disease-free. Further studies are warranted to determine whether this response could be useful as a marker of recurrent disease in some patients.

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.