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.     

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.     

Flagellin enhances tumor-specific CD8 T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model

Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs. We previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, has potent adjuvant activity in various vaccination models. In this study, we investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We used an E6/E7-expressing TC-1 carcinoma implantation animal model and tested TA-specific immunomodulation by FlaB. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specific antitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival. We noticed that TC-1 cells do not express Toll-like receptor 5 (TLR5) on their surface and the TLR5 signaling pathway in TC-1 cells was not responsible for the antitumor effect of FlaB. FlaB potentiated the CTL activity and Ag-specific IFN-γ production of CD8⁺ T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivo depletion of CD8⁺ T cells and in TLR5 knockout (KO) or MyD88 KO mice. These results suggest that flagellin could enhance TA-specific CD8⁺ CTL immune responses through TLR5 stimulation in cancer immunotherapy.     

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

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

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.     

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

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

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.     

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

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

Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun,biojector, and syringe

DNA vaccines have emerged as an attractive approach for antigen-specific cancer immunotherapy. We have previously linked Mycobacterium tuberculosis heat shock protein 70 (HSP70) to human papillomavirus type 16 (HPV-16) E7 in the context of a DNA vaccine. Vaccination with DNA encoding E7/HSP70 has generated a dramatic increase of E7-specific CD8+ T cell precursors and a strong antitumor effect against E7-expressing tumor (TC-1) in vaccinated mice. The success of our strategy has led to two phases I/II clinical trial proposals in patients with HPV-16 associated high-grade squamous intraepithelial lesion (HSIL) of the cervix and in patients with advanced HPV-associated head and neck squamous cell carcinoma (HNSCC). To translate our HPV DNA vaccines into the clinical domain, the efficacy of pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and of various routes of administrations were assessed in mice. Our results indicated that pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine administered via gene gun generated the highest number of E7-specific CD8+ T cells. In addition, DNA vaccination via gene gun required the least dose to generate similar or slightly better antitumor effects compared to needle intramuscular (i.m.) and biojector administrations. Thus, our data suggest that DNA vaccination via gene gun represents the most potent regimen for DNA administration.     

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

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

Generation and characterization of a preventive and therapeutic HPV DNA vaccine

Cervical cancer is one of the most common cancers in women worldwide. Persistent infection with human papillomavirus (HPV) is considered to be the etiological factor for cervical cancer. Therefore, an effective vaccine against HPV infections may lead to the control of cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infections as well as cell-mediated immunity to eliminate established infection or HPV-related disease. In the current study, we have generated a potential preventive and therapeutic HPV DNA vaccine using human calreticulin (CRT) linked to HPV16 early proteins, E6 and E7 and the late protein L2 (hCRTE6E7L2). We found that vaccination with hCRTE6E7L2 DNA vaccine induced a potent E6/E7-specific CD8+ T cell immune response, resulting in a significant therapeutic effect against E6/E7 expressing tumor cells. In addition, vaccination with hCRTE6E7L2 DNA generated significant L2-specific neutralizing antibody responses, protecting against pseudovirion infection. Thus, the hCRTE6E7L2 DNA vaccines are capable of generating potent preventive and therapeutic effects in vaccinated mice. Our data has significant clinical implications.     

Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model

Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8⁺ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNγ production and the ability to protect mice against a challenge with HPV16-E7⁺ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.     

Immunization of protein HPV16 E7 in fusion with mouse HSP70 inhibits the growth of TC-1 cells in tumor bearing mice

Human papillomavirus (HPV) 16 is the primary etiologic agent of cervical cancer. Most HPV16 therapeutic vaccines target E7 protein which is consistently expressed in tumor cells. In this study, we cloned mouse autologous heat shock protein 70 (mHSP70) gene from mouse liver cells and then expressed mHSP70 and fused HPV16 E7-mHSP70 (E7 at the N-terminus and mHSP70 at the C-terminus) proteins in E. coli. Then we investigated the inhibition of TC-1 cell growth by using the E7-expressing murine tumor cell line, TC-1, as a model of cervical cancer. In this model, mice were immunized with the fusion protein of E7-mHSP70 without any adjuvant. The results showed that prophylactic immunization of E7-mHSP70 protected mice against challenge with TC-1 cells. In addition, therapeutic immunization with E7-mHSP70 could inhibit TC-1 tumor growth on lungs. Our study demonstrated that immunization with E7-mHSP70 protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.     

An IL-15 adjuvant enhances the efficacy of a combined DNA vaccine against Brucella by increasing the CD8 cytotoxic T cell response

We investigated whether a combined DNA vaccine delivered together with the IL-15 gene (DNA-IL-15(+)) enhanced the immune response against Brucella abortus in mice. Mice vaccinated with DNA-IL-15(+) developed a robust humoral response; Brucella-specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. Splenocytes from DNA-IL-15(+)-vaccinated mice induced significantly higher levels of IFN-γ (P < 0.01) and CD8⁺ T cell response (P < 0.01), suggesting induction of a T-helper-1-dominated immune response. In a specific cytotoxic-T-lymphocyte activity assay, DNA-IL-15(+) immunization elicited mainly CD8⁺ T cells, which mediate cytotoxicity, but also CD4⁺ T cells. In vivo depletion of T cell subsets showed that the DNA-IL-15(+)-induced protection against Brucella infection is mediated predominantly by CD8⁺ T cells, although CD4⁺ T cells also contribute. These data indicate that plasmid-delivered IL-15 increases the efficacy of the Brucella DNA vaccine.     

Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii

The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of naïve mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8⁺ T-cells. A single immunization of naïve mice with recombinant YF17D resulted in robust production of IFN-γ by CD8⁺ T-cells and IFN-γ and IL-2 by CD4⁺ T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo.     

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

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

Cellular and humoral immunity following vaccination with two different PCV2 vaccines (containing PCV2a or PCV2a/PCV2b) and challenge with virulent PCV2d

Porcine Circovirus type 2 (PCV2) associated disease is one of the most economically important swine diseases worldwide. Vaccines reduce PCV2 disease by inducing humoral immunity (neutralizing antibodies) and cell-mediated immunity (CMI) but may be improved by optimizing the immune response they induce. This study evaluated immune responses to a trivalent inactivated Porcine Circovirus (PCV) Type 1-Type 2a chimera (cPCV2a), cPCV2b and Mycoplasma hyopneumoniae (MH) (an experimental serial of Fostera® Gold PCV MH, also marketed as Circomax® Myco) vaccine or a bivalent recombinant PCV2a baculovirus expressed ORF2 capsid plus MH vaccine (Circumvent® PCV-M G2). Treatment Groups (T) received two doses of placebo (T01), one full or two split doses of the trivalent vaccine (T02, T03) or two split doses of the bivalent vaccine (T04) where two doses were given, there was a three-week period between administrations. All pigs were challenged with a virulent field isolate of PCV2d. CMI was measured as PCV2-specific IFN-γ secreting cells in blood and lymph node. Humoral immunity was measured as PCV2 antibodies. Vaccine efficacy was determined as viremia and fecal shedding of virus. There was a robust antibody response in T02 and T04 post the second vaccination and all vaccinated groups post challenge. There was a robust PCV2-specific IFN-γ response following the 1^st dose in T02 and T03 and after the second dose in T02. T04 induced a low but detectable PCV2-specific IFN-γ response only after the 2^nd dose. Among lymph node cells (study day 52), there was a significantly higher PCV2-specific, IFN-γ response to replicase and PCV2d capsid peptides in T01, consistent with active viral replication in non-vaccinated pigs. The trivalent chimeric vaccine induced robust CMI and protective efficacy, following a one dose regimen or splitting the dose into two vaccine administrations.     

A novel therapeutic vaccine of GM-CSF/TNFα surface-modified RM-1 cells against the orthotopic prostatic cancer

A novel therapeutic vaccine against prostate cancer was developed by simultaneous immobilization of streptavidin-tagged bioactive GM-CSF and TNFα on the biotinylated surface of 30% ethanol-fixed RM-1 prostatic cancer cells. This study showed that the GM-CSF/TNFα-doubly surface-modified vaccine significantly extended the survival in the orthotopic model of RM-1 prostate cancer, and was superior to single GM-CSF- or TNFα-surface-modified vaccine. Moreover, the splenocytes from the GM-CSF/TNFα-vaccine-treated mice showed the most potent cytotoxicity on RM-1 cells and the highest production of RM-1-specific IFNγ. In addition, more CD4⁺ and CD8⁺ T cells infiltrated into the tumor sites in the GM-CSF/TNFα-vaccine-treated mice than in the GM-CSF- or TNFα-vaccine-treated mice. Therefore, our study demonstrated that the efficacy of RM-1 prostate cancer cell vaccine could be improved by conjugating both GM-CSF and TNFα simultaneously on the surface of cancer cells, and that this modification thus has a potential translational significance.     

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.     

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.