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

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

Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract

The Toll-like receptor 5 (TLR5) agonist flagellin is an effective adjuvant for vaccination. Recently, we demonstrated that the adaptive responses stimulated by intranasal administration of flagellin and antigen were linked to TLR5 signaling in the lung epithelium. The present study sought to identify the antigen presenting cells involved in this adjuvant activity. We first found that the lung dendritic cells captured antigen very efficiently in a process independent of TLR5. However, TLR5-mediated signaling specifically enhanced the maturation of lung dendritic cells. Afterward, the number of antigen-bound and activated conventional dendritic cells (both CD11b⁺ and CD103⁺) increased in the mediastinal lymph nodes in contrast to monocyte-derived dendritic cells. These data suggested that flagellin-activated lung conventional dendritic cells migrate to the draining lymph nodes. The lymph node dendritic cells, in particular CD11b⁺ cells, were essential for induction of CD4 T-cell response. Lastly, neutrophils and monocytes were recruited into the lungs by flagellin administration but did not contribute to the adjuvant activity. The functional activation of conventional dendritic cells was independent of direct TLR5 signaling, thereby supporting the contribution of maturation signals produced by flagellin-stimulated airway epithelium. In conclusion, our results demonstrated that indirect TLR5-dependent stimulation of airway conventional dendritic cells is essential to flagellin's mucosal adjuvant activity.     

Anti-glioma effect of intracranial vaccination with tumor cell lysate plus flagellin in mice

The adjuvant effects of flagellin on regulation of immune response have been proved; whether flagellin could assist tumor cell lysate (TCL) to enhance anti-glioma immunity remains to be investigated. This study tests a hypothesis that therapeuticly intracranial administration with flagellin plus TCL enhances the effects of specific immunotherapy on glioma in mice. In this study, GL261 cells were transferred into C57BL/6 mice and the GL261-bearing mice were subcutaneously or intracranially inoculated with flagellin plus TCL, flagellin, TCL or saline. Our results showed that prophylacticly subcutaneous administration with TCL and flagellin could induce potent cytotoxic T lymphocyte (CTL) and prolong the survival of GL261-bearing mice significantly, but therapeuticly subcutaneous administration failed to. However, therapeuticly intracranial administration of TCL plus flagellin could prolong the survival. Moreover, intracranial administration of flagellin could recruit CD4⁺ T cells and CD8⁺ T cells to brain tissues, induce proliferation of natural killer (NK) cells, CD4⁺ T cells and CD8⁺ T cells in peripheral blood mononuclear cells and induce to splenomegaly. The results suggested that flagellin could be acted as an efficient adjuvant for TCL based vaccine.     

Flagellin is a Th1 polarizing factor for human CD4+ T cells and induces protection in a murine neonatal vaccination model of rotavirus infection

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4⁺ T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4⁺ and CD8⁺ T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4⁺ T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4⁺ T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response.     

Dendritic cells stimulated with outer membrane protein A (OmpA) of Salmonella typhimurium generate effective anti-tumor immunity

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA (AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4⁺ T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8⁺ T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4⁺ and E7-specific CD8⁺ T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8⁺ effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.     

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

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

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

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

Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection

The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.     

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

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

IgA and IgG antibody responses following systemic immunization of cattle with native H7 flagellin differ in epitope recognition and capacity to neutralise TLR5 signalling

Systemic immunization of cattle with H7 flagellin results in induction of both H7-specific IgA and IgG antibodies but only partially protects against subsequent colonization with Escherichia coli O157:H7. Recent studies indicate that anti-flagellin antibodies directed against TLR5 binding domains located in the conserved N- and C-terminal domains of flagellin can neutralise TLR5 activation and impair vaccine efficacy. In the current study we determined whether systemic immunization of cattle with H7 flagellin induces antibodies capable of interfering with flagellin-mediated TLR5 activation. Both anti-H7 IgG1 and IgG2 but not IgA antibodies recognised epitopes within the conserved N- and C-terminal domains of H7 flagellin, and purified H7-specific IgG but not IgA was capable of inhibiting H7-mediated TLR5 activation in vitro. These results suggest that (i) IgA and IgG isotypes originated from different populations of B cells and (ii) systemically induced H7-specific IgG but not IgA may impair innate immune responses to E. coli O157:H7 via neutralisation of TLR5 activation and subsequently reduce vaccine efficacy.     

CD8 T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2^d-restricted CD8⁺ T cell-specific epitope (CS_280–288) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS_280–288 peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS_280–288 peptide. The results showed that CS_280–288-specific cytotoxic CD8⁺ T cells were primed when BALB/c mice were orally inoculated with the expressing the CS_280–288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS_280–288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8⁺ T cell responses without the need of a heterologous booster immunization. The CD8⁺ T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c⁺ dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.     

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

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

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

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

Tumor therapy in mice by using a tumor antigen linked to modulin peptides from Staphylococcus epidermidis

Staphylococcus epidermidis releases a complex of at least four peptides, termed phenol-soluble modulins (PSM), which stimulate macrophages to produce proinflammatory cytokines via activation of TLR2 signalling pathway. We demonstrated that covalent linkage of PSM peptides to an antigen facilitate its capture by dendritic cells and, in combination with different TLR ligands, can favour the in vivo induction of strong and persistent antigen-specific immune responses. Treatment of mice grafted with HPV16-E7-expressing tumor cells (TC-1) with poly(I:C) and a peptide containing αMod linked to the H-2D^b-restricted cytotoxic T-cell epitope E7(49–57) from HPV16-E7 protein allowed complete tumor regression in 100% of the animals. Surprisingly, this immunomodulatory property of modulin-derived peptides was TLR2 independent and partially dependent upon the EGF-receptor signalling pathway. Our results suggest that alpha or gamma modulin peptides may serve as a suitable antigen carrier for the development of anti-tumoral or anti-viral vaccines.     

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.     

Optimal induction of HPV DNA vaccine-induced CD8 T cell responses and therapeutic antitumor effect by antigen engineering and electroporation

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8⁺ T cell responses. When E6^Co and E7^Co were fused (E67^Co), CD8⁺ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8⁺ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67^Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8⁺ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67^Co further enhances HPV-specific CD8⁺ T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67^Co delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.     

Overlapping synthetic peptides encoding TPD52 as breast cancer vaccine in mice: Prolonged survival

Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8⁺) and Class II-restricted (CD4⁺) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles has hampered the application of this approach. We have tested overlapping synthetic peptides (OSP) representing a tumor antigen as a novel approach that bypasses the need for epitope mapping, since OSP contain all possible epitopes for both CD8⁺ and CD4⁺ T cells. Here we report that vaccination of inbred and outbred mice with OSP representing tumor protein D52 (TPD52-OSP), a potential tumor antigen target for immunotherapy against breast, prostate, and ovarian cancer, was safe and induced specific CD8⁺ and CD4⁺ T-cell responses, as demonstrated by development of specific cytotoxic T cell (CTL) activity, proliferative responses, interferon (IFN)-γ production and CD107a/b expression in all mice tested. In addition, TPD52-OSP-vaccinated BALB/c mice were challenged with TS/A breast carcinoma cells expressing endogenous TPD52; significant survival benefits were noted in vaccine recipients compared to unvaccinated controls (p < 0.001). Our proof-of-concept data demonstrate the safety and efficacy of peptide library-based cancer vaccines that obviates the need to identify epitopes or MHC backgrounds of the vaccinees. We show that an OSP vaccination approach can assist in the disruption of self-tolerance and conclude that our approach may hold promise for immunoprevention of early-stage cancers in a general population.     

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

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