Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein in mice and rhesus macaques

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.     

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.     

Immunogenicity of an HPV-16 L2 DNA vaccine

The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunized with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The L2 DNA vaccine elicited a non-neutralizing antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct.     

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.     

Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN,an HPV16 L2E7E6 fusion protein vaccine

TA-CIN is a vaccine that comprises the human papillomavirus (HPV) type 16 L2, E6 and E7 as a single fusion protein. In a mouse model, TA-CIN effectively prevented outgrowth of HPV16-positive tumour cells. To assess the safety and immunogenicity of TA-CIN, a dose escalating (26, 128, 533 micro g), double blind and placebo-controlled phase I study was conducted in 40 healthy volunteers. TA-CIN was administered without adjuvant by intramuscular injection on weeks 0, 4 and 8. No serious adverse events of the vaccination were reported during the study. Both IgG antibodies and proliferative responses against TA-CIN were elicited at all three doses. More importantly, T-cell immunity against the HPV16 E6 and E7 oncoproteins was detected by IFN gamma ELISPOT in 8/11 evaluable subjects vaccinated with the 533 micro g dose.     

A novel multi-epitope vaccine of HPV16 E5E6E7 oncoprotein delivered by HBc VLPs induced efficient prophylactic and therapeutic antitumor immunity in tumor mice model

Human papilloma virus type 16 (HPV16) is the most prevalent etiologic agent associated with cervical cancer, and its early proteins E5, E6 and E7 play important roles in cervical epithelium transformation to cervical intraepithelial neoplasia and even cervical cancer. Hence, these oncoproteins are ideal target antigens for developing immunotherapeutic vaccines against HPV-associated infection and cervical cancer. Currently, multi-epitope vaccines have been a promising strategy for immunotherapy for viral infection or cancers. In this study, the E5aa28-46, E6aa37-57 and E7aa26-57 peptides were selected and linked to form a novel multi-epitopes vaccine (E765m), which was inserted into the major immune dominant region (MIR) of hepatitis B virus core antigen (HBc) to construct a HBc-E765m chimeric virus-like particles (cVLPs). The immunogenicity and immunotherapeutic effect of the cVLPs vaccine was evaluated in immunized mice and a tumor-bearing mouse model. The results showed that HBc-E765m cVLPs elicited high E5-, E6- and E7- specific CTL and serum IgG antibody responses, and also relatively high levels of the cytokines IFN-γ, IL-4 and IL-5. More importantly, the cVLPs vaccine significant suppressed tumor growth in mice bearing E5-TC-1 tumors. Our findings provide strong evidence that this novel HBc-E765m cVLPs vaccine could be a candidate vaccine for specific immunotherapy in HPV16-associated cervical intraepithelial neoplasia or cervical cancer.     

A novel therapeutic fusion protein vaccine by two different families of heat shock proteins linked with HPV16 E7 generates potent antitumor immunity and antiangiogenesis

Human papillomaviruses (HPV), particularly HPV16, is considered a necessary cause of cervical and oral cancer. Thus, the development of a therapeutic vaccine against HPV is important for the control of cervical cancer. However, therapeutic vaccination has been limited by inadequate antigen-specific immune responses. Heat shock proteins (HSP), including calreticulin (CRT), HSP70 and gp96, have been shown to act as potent immunoadjuvant to enhance antigen-specific tumor immunity. Previous studies have shown that N domain CRT (NCRT) or C-terminal half of HSP70 (hsp) linked with HPV16 E7 are capable of inducing potent antigen-specific CTL activity in experimental animal models. Here we developed a recombinant NCRT/E7/hsp fusion protein to investigate the synergistic effects of NCRT and hsp for enhancing the potency of HPV16 E7 therapeutic vaccine and evaluated the immune responses induced by this fusion protein. Our results demonstrated that NCRT and hsp synergistically exhibited significant increases in E7-specific CD8(+) T cell responses and impressive antitumor effects against E7-expressing tumors. Furthermore, the NCRT/E7/hsp fusion protein also generates potent antiangiogenic effects. These results indicate that NCRT/E7/hsp fusion protein is a promising therapeutic vaccine for treatment of cervical cancer through a combination of antigen-specific immunotherapy and antiangiogenesis, with possible therapeutic potential in clinical settings.     

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.     

人乳头瘤病毒16E7 DNA疫苗微针给药效果研究

目的 探讨DNA疫苗微针给药方法的有效性.方法 构建pcDNA3.1-HPV16E7重组质粒,在体外透皮条件下,观察pcDNA-HPV16E7皮肤透过量,再利用微针给药方式免疫BALB/c小鼠,分三组(实验组、空载体对照组、阴性对照组),每组10只,每2周免疫1次,共3次,每次免疫剂量为200 μg/只,对照组参照实验组进行,最后1次免疫后2周采血,分别分离血清和淋巴细胞,用间接免疫荧光试验检测小鼠的体液免疫功能,用淋巴细胞转化试验检测小鼠的细胞免疫功能.结果 体外透皮试验显示DNA疫苗微针给药可以透过皮肤,而且透过量随着时间的延长而逐步增加,在第30小时时可以达到0.738 19 mg/cm2;通过微针给药方法DNA疫苗可以诱导小鼠产生特异性抗体,淋巴细胞转化试验显示:实验组(平均淋巴细胞转化率为47.25%)和阴性对照组(平均淋巴细胞转化率为30.00%)之间比较,χ2=12.903,P＜0.001,差异有显著统计学意义;空载体组(平均淋巴细胞转化率为43.00%)和阴性对照组之间比较,χ2=7.292,P=0.007,差异有显著统计学意义;实验组和空载体组之间比较,χ2=0.817,P=0.366,差异无统计学意义.结论 HPV16E7 DNA疫苗经微针给药可以透过皮肤吸收并诱导小鼠产生体液免疫和细胞免疫反应.     

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

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

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.     

Listeria-based HPV-16 E7 vaccines limit autochthonous tumor growth in a transgenic mouse model for HPV-16 transformed tumors

We have shown that Listeria-based cancer vaccines inhibit the growth of transplanted tumors in a transgenic mouse model of immune tolerance where HPV-16 E7 is expressed in the thyroid gland. In this study we determine whether these vaccines are able to inhibit autochthonous tumor growth in this animal model. Mice treated with Listeria vaccines expressing E7 had significantly smaller thyroid tumors than did mice treated with controls and possessed higher numbers of antigen-specific CD8(+) T cells within the spleens, tumors, and peripheral blood. This study shows that Listeria-based vaccines are able to slow autochthonous tumor growth and break immunological tolerance.     

Induction of protective and therapeutic antitumor immunity by a DNA vaccine with C3d as a molecular adjuvant

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer therapies is unclear. In this study, we have engineered a DNA vaccine that expresses extracellular region of murine VEGFR-2 (FLK1_265–2493) and 3 copies of C3d (C3d3), a component of complement as a molecular adjuvant, designed to increase antitumor immunity. VEGFR-2 has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during angiogenesis in the tumor vasculature. Immunization of mice with vector encoding FLK1_265–2493 alone generated only background levels of anti-VEGFR-2 antibodies and slight inhibitory effect on tumor growth. However, the addition of C3d3 to the vaccine construct significantly augmented the anti-VEGFR-2 humoral immune response and inhibited the tumor growth. The antitumor activity induced by vaccination with vector encoding FLK1_265–2493-C3d3 fusion protein was also demonstrated via growth inhibition of established tumors following passive transfer of immune serum from vaccinated mice. Our results suggest that vaccination with vector encoding FLK1_265–2493 with C3d3 as a molecular adjuvant induces adaptive humoral activity, which is directed against the murine VEGFR-2 and can significantly inhibit tumor growth, and that administration of C3d as a molecular adjuvant to increase antibodies levels to VEGFR-2 may provide an alternative treatment modality for cancer therapies.     

Enhancement of immunity to an Escherichia coli vaccine in mice orally inoculated with a fusion gene encoding porcine interleukin 4 and 6

Experiments were conducted to investigate the effect of a fusion gene of porcine IL-4 and IL-6 (PIL4/IL6) packaged with chitosan nanoparticles (CNPs) in terms of the development of a novel effective adjuvant. The IL4/PIL6 fusion gene was constructed and inserted into a eukaryotic expression vector. The plasmid was bound to CNP and then utilized to orally inoculate 21-day-old female Kunming mice that simultaneously received intramuscular injection of inactivated Escherichia coli vaccine. At 35 days post-vaccination, the mice were challenged by oral feeding with virulent O139: K88 strain EPEC E. coli bacteria. Compared with those of control mice, the content of immunoglobulins and specific antibodies to E. coli increased significantly in the sera of mice immunized with VPIL4/IL6-CNP (P<0.05). Furthermore, the levels of IL-2, IL-4 and IL-6 increased remarkably in the sera of immunized mice (P<0.05). After challenge, these immunological markers were elevated to different degrees in the mice immunized with the fusion gene construct (IL4/VPIL6-CNP) or individual plasmids (VPIL4+VPIL6-CNP). The immunized mice all survived the challenge and did not show any symptoms or lesion, whereas the VR1020-CNP control mice manifested obvious clinical symptoms and hemorrhagic lesions in the digestive tracts. These results demonstrated that VPIL4/IL6 entrapped with CNP is a novel promising adjuvant to promote specific immunity and resistance of animals against infectious pathogen.     

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.     

MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7

DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180–210 nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-γ (IFN-γ) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines.     

Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins

Cross-protection between different subtypes of influenza A virus has been attributed to heterosubtypic immunity (HSI). Although, HSI can occur in the absence of anti-HA or anti-NA antibodies, HSI seems to be mediated, in part, by cross-reactive antibodies. In this study, we examined the effects of a DNA vaccine expressing an influenza HA fused to three copies of murine C3d of complement (HA-mC3d(3)). HA-mC3d(3) elicited heterosubtypic immunity more efficiently than non-fused forms of HA and protected mice from lethal challenge of influenza with different subtypes. Plasmid encoding for various forms of HA were constructed from two influenza strains, A/Puerto Rico/8/34 (H1N1) or A/Aichi/2/68-x31 (H3N2). Vaccinated mice were analyzed for enhancement of anti-HA titers, affinity maturation of antibody, hemagglutinin-inhibition activity, and altered cytokine profiles. The HA-mC3d(3)-DNA vaccinated mice were protected from heterologous influenza challenge, even though sera from these mice had no viral-neutralizing activity against heterologous virus.     

Genetic variability of HPV-58 E6 and E7 genes in Southwest China

HPV accounts for most of incidence of cervical cancer. Genetic variations of E6 and E7 may be associated with the development of cervical cancer in specific geographic regions. HPV-58 has been found to be a relatively prevalent high-risk HPV among southwest Chinese women. To explore gene intratypic variations and polymorphisms of HPV-58 E6 and E7 genes originating in Southwest China, a total of 2000 scraped cell samples were collected for DNA extraction and HPV typing. Then, the E6 and E7 genes of HPV-58 (n = 22) were sequenced and compared to others submitted to GenBank, followed by an analysis of the diversity of secondary structure by DNASTAR software. Phylogenetic trees were then constructed by Neighbor-Joining and the Kimura 2-parameters methods, followed by an analysis of selection pressures acting on the E6/E7 genes by PAML software. 22 were HPV-58 positive among 215 high-risk types’ samples. The nucleotide variation rate of E6 was 86.36% (19/22) among the 22 HPV-58 E6 sequences studied. 4 single nucleotide changes were identified among the E6 sequences with 3/4 synonymous mutations (C187T, A260C, C307T) and 1/4 non-synonymous mutations (A388C, from Lys to Asn, in alpha helix). The most common mutations of E6 genes are the C307T and A388C. 8 single nucleotide changes were identified among the HPV-58 E7 sequences with 2/8 synonymous mutations (T726C, T744G) and 6/8 non-synonymous mutations (G599A, C632T, G694A, G760A, G761A, T803C). The nucleotide variation rate of E7 was 72.73% (17/22). The most common mutations of E7 genes are C632T, G694A, T744G, G760A (from Gly to Ser, in turn), G761A and T803C. The phylogenetic analyses demonstrate that all HPV-58 E6/E7 variants identified belonged to the Southeast Asia lineage. There was no evidence of positive selection in the sequence alignment of HPV-58 E6 and E7 genes.     

Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice

We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.     

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens

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.