Enhancement of DNA vaccine potency through linkage of antigen gene to ER chaperone molecules,ER-60, tapasin,and calnexin

DNA vaccines have emerged as an attractive approach for generating antigen-specific immunotherapy. Strategies that enhance antigen presentation may potentially be used to enhance DNA vaccine potency. Previous experiments showed that chimeric DNA vaccines utilizing endoplasmic reticulum (ER) chaperone molecules, such as Calreticulin (CRT), linked to an antigen were capable of generating antigen-specific CD8⁺ T cell immune responses in vaccinated mice. In this study, we tested DNA vaccines encoding the ER chaperone molecules ER-60, tapasin (Tap), or calnexin (Cal), linked to human papillomavirus type 16 (HPV-16) E7 for their abilities to generate E7-specific T cell-mediated immune responses and antitumor effects in vaccinated mice. Our results demonstrated that vaccination with DNA encoding any of these chaperone molecules linked to E7 led to a significant increase in the frequency of E7-specific CD8⁺ T cell precursors and generated stronger antitumor effects against an E7-expressing tumor in vaccinated mice compared to vaccination with wild-type E7 DNA. Our data suggest that DNA vaccines employing these ER chaperone molecules linked to antigen may enhance antigen-specific CD8⁺ T cell immune responses, resulting in a significantly more potent DNA vaccine.     

Liposomal delivery of p- ialB and p- omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge

Background

We have previously demonstrated protective efficacy against B. melitensis using formulations of naked DNA vaccines encoding genes ialB and omp25. The present study was undertaken to further understand the immune response generated by the protective vaccination regimens and to evaluate cationic liposome adsorption as a delivery method to improve vaccine utility.

Methods

The protective efficacy and immunogenicity of vaccines delivered as four doses of naked DNA, a single dose of naked DNA or a single dose of DNA surface adsorbed to cationic liposomes were compared using the BALB/c murine infection model of B. melitensis. Antigen-specific T cells and antibody responses were compared between the various formulations.

Results

The four dose vaccination strategy was confirmed to be protective against B. melitensis challenge. The immune response elicited by the various vaccines was found to be dependent upon both the antigen and the delivery strategy, with the IalB antigen favouring CD4+ T cell priming and Omp25 antigen favouring CD8+. Delivery of the p- ialB construct as a lipoplex improved antibody generation in comparison to the equivalent quantity of naked DNA. Delivery of p- omp25 as a lipoplex altered the profile of responsive T cells from CD8+ to CD4+ dominated. Under these conditions neither candidate delivered by single dose naked DNA or lipoplex vaccination methods was able to produce a robust protective effect.

Conclusions

Delivery of the p- omp25 and p- ialB DNA vaccine candidates as a lipoplex was able to enhance antibody production and effect CD4+ T cell priming, but was insufficient to promote protection from a single dose of either vaccine. The enhancement of immunogenicity by lipoplex delivery is a promising step toward improving the practicality of these two candidate vaccines, and suggests that this lipoplex formulation may be of value in situations where improvements to CD4+ responses are required. However, in the case of Brucella vaccine development it is suggested that further modifications to the candidate vaccines and delivery strategies will be required in order to deliver sustained protection.     

Antigen-specific CD8+ T cells induced by the ubiquitin fusion degradation pathway

We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8⁺ T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8⁺ T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8⁺ T cells.     

Vascular disrupting agent DMXAA enhances the antitumor effects generated by therapeutic HPV DNA vaccines

Antigen-specific immunotherapy using DNA vaccines has emerged as an attractive approach for the control of tumors. Another novel cancer therapy involves the employment of the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In the current study, we aimed to test the combination of DMXAA treatment with human papillomavirus type 16 (HPV-16) E7 DNA vaccination to enhance the antitumor effects and E7-specific CD8+ T cell immune responses in treated mice. We determined that treatment with DMXAA generates significant therapeutic effects against TC-1 tumors but does not enhance the antigen-specific immune responses in tumor bearing mice. We then found that combination of DMXAA treatment with E7 DNA vaccination generates potent antitumor effects and E7-specific CD8+ T cell immune responses in the splenocytes of tumor bearing mice. Furthermore, the DMXAA-mediated enhancement or suppression of E7-specific CD8+ T cell immune responses generated by CRT/E7 DNA vaccination was found to be dependent on the time of administration of DMXAA and was also applicable to other antigen-specific vaccines. In addition, we determined that inducible nitric oxide synthase (iNOS) plays a role in the immune suppression caused by DMXAA administration before DNA vaccination. Our study has significant implications for future clinical translation.     

Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects

The use of DNA vaccines for generating antigen-specific CD8+ T cell responses has been well established. However, little is known about the quantitative and qualitative aspects of CD8+ T cell responses and protective immunity generated after repeated DNA vaccinations. We used human papillomavirus (HPV) type-16 E7 as a model tumor antigen in an E7-expressing tumor model, TC-1, to assess the influence of the frequency of DNA vaccinations on E7-specific immunological and antitumor responses. Mice were vaccinated with 1–4 inoculations of pcDNA3-E7 DNA. Immunological assays and tumor protection experiments were performed to assess the effect of repeated E7 DNA vaccination on E7-specific T cells and E7-expressing tumors. Our results demonstrated that mice receiving an increased number of E7 DNA vaccinations exhibited higher E7-specific CTL activity, a rapid expansion of E7-specific IFN--secreting CD8+ T cells upon stimulation with E7 antigen, and a stronger antitumor effect against an E7-expressing tumor. Furthermore, we found that increasing the number of E7 DNA vaccinations followed by vaccinia booster enhanced the functional avidity of E7-specific CD8+ T cells. Our data suggest that quantitative and qualitative characteristics of antigen-specific CD8+ T cell responses and the ensuing protective antitumor effect can be influenced by the frequency of DNA vaccinations. These results have important clinical implications for the use of naked DNA vaccines in cancer immunotherapy.     

Concomitant gemcitabine therapy negatively affects DC vaccine-induced CD8+ T-cell and B-cell responses but improves clinical efficacy in a murine pancreatic carcinoma model

Background

Multiple studies have shown that dendritic cell (DC)-based vaccines can induce antitumor immunity. Previously, we reported that gemcitabine enhances the efficacy of DC vaccination in a mouse model of pancreatic carcinoma. The present study aimed at investigating the influence of gemcitabine on vaccine-induced anti-tumoral immune responses in a syngeneic pancreatic cancer model.

Materials and methods

Subcutaneous or orthotopic pancreatic tumors were induced in C57BL/6 mice using Panc02 cells expressing the model antigen OVA. Bone marrow-derived DC were loaded with soluble OVA protein (OVA-DC). Animals received gemcitabine twice weekly. OVA-specific CD8⁺ T-cells and antibody titers were monitored by FACS analysis and ELISA, respectively.

Results

Gemcitabine enhanced clinical efficacy of the OVA-DC vaccine. Interestingly, gemcitabine significantly suppressed the vaccine-induced frequency of antigen-specific CD8⁺ T-cells and antibody titers. DC migration to draining lymph nodes and antigen cross-presentation were unaffected. Despite reduced numbers of tumor-reactive T-cells in peripheral blood, in vivo cytotoxicity assays revealed that cytotoxic T-cell (CTL)-mediated killing was preserved. In vitro assays revealed sensitization of tumor cells to CTL-mediated lysis by gemcitabine. In addition, gemcitabine facilitated recruitment of CD8⁺ T-cells into tumors in DC-vaccinated mice. T- and B-cell suppression by gemcitabine could be avoided by starting chemotherapy after two cycles of DC vaccination.

Conclusions

Gemcitabine enhances therapeutic efficacy of DC vaccination despite its negative influence on vaccine-induced T-cell proliferation. Quantitative analysis of tumor-reactive T-cells in peripheral blood may thus not predict vaccination success in the setting of concomitant chemotherapy.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Immunogenicity of Bivalent Human Papillomavirus DNA Vaccine Using Human Endogenous Retrovirus Envelope-Coated Baculoviral Vectors in Mice and Pigs

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.     

TriVax-HPV: an improved peptide-based therapeutic vaccination strategy against human papillomavirus-induced cancers

Background

Therapeutic vaccines for cancer are an attractive alternative to conventional therapies, since the later result in serious adverse effects and in most cases are not effective against advanced disease. Human papillomavirus (HPV) is responsible for several malignancies such as cervical carcinoma. Vaccines targeting oncogenic viral proteins like HPV16-E6 and HPV16-E7 are ideal candidates to elicit strong immune responses without generating autoimmunity because: (1) these products are not expressed in normal cells and (2) their expression is required to maintain the malignant phenotype. Our group has developed peptide vaccination strategy called TriVax, which is effective in generating vast numbers of antigen-specific T cells in mice capable of persisting for long time periods.

Materials and methods

We have used two HPV-induced mouse cancer models (TC-1 and C3.43) to evaluate the immunogenicity and therapeutic efficacy of TriVax prepared with the immunodominant CD8 T-cell epitope HPV16-E7_49-57, mixed with poly-IC adjuvant and costimulatory anti-CD40 antibodies.

Results

TriVax using HPV16-E7_49-57 induced large and persistent T-cell responses that were therapeutically effective against established HPV16-E7 expressing tumors. In most cases, TriVax was successful in attaining complete rejections of 6–11-day established tumors. In addition, TriVax induced long-term immunological memory, which prevented tumor recurrences. The anti-tumor effects of TriVax were independent of NK and CD4 T cells and, surprisingly, did not rely to a great extent on type-I or type-II interferon.

Conclusions

These findings indicate that the TriVax strategy is an appealing immunotherapeutic approach for the treatment of established viral-induced tumors. We believe that these studies may help to launch more effective and less invasive therapeutic vaccines for HPV-mediated malignancies.     

Plasmid DNA vaccine encoding prostatic acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.     

Treatment with Imiquimod enhances antitumor immunity induced by therapeutic HPV DNA vaccination

Background

There is an urgent need to develop new innovative therapies for the control of advanced cancer. The combination of antigen-specific immunotherapy with the employment of immunomodulatory agents has emerged as a potentially plausible approach for the control of advanced cancer.

Methods

In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) with the TLR7 agonist imiquimod for their ability to generate E7-specific immune responses and antitumor effects in tumor-bearing mice.

Results

We observed that treatment with CRT/E7 DNA in combination with imiquimod leads to an enhancement in the E7-specific CD8+ T cell immune responses and a decrease in the number of myeloid-derived suppressor cells in the tumor microenvironment of tumor-bearing mice. Furthermore, treatment with CRT/E7 DNA in combination with imiquimod leads to significantly improved antitumor effects and prolonged survival in treated mice. In addition, treatment with imiquimod led to increased number of NK1.1+ cells and F4/80+ cells in the tumor microenvironment. Macrophages and NK1.1+ cells were found to play an important role in the antitumor effects mediated by treatment with CRT/E7 DNA in combination with imiquimod.

Conclusions

Thus, our data suggests that the combination of therapeutic HPV DNA vaccination with topical treatment with the TLR7 agonist imiquimod enhances the antitumor immunity induced by DNA vaccination. The current study has significant implications for future clinical translation.

     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

Skin vaccination against cervical cancer associated human papillomavirus with a novel micro-projection array in a mouse model

Background

Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer.

Methodology/Principal Findings

Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point.

Conclusions/Significance

Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.     

Bicistronic DNA Vaccines Simultaneously Encoding HIV,HSV and HPV Antigens Promote CD8+ T Cell Responses and Protective Immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

A comparison and critical analysis of preclinical anticancer vaccination strategies

Anticancer vaccines have been extensively studied in animal models and in clinical trials. While vaccination can lead to tumor protection in numerous murine models, objective tumor regressions after anticancer vaccination in clinical trials have been rare. B16 is a poorly immunogenic murine melanoma that has been extensively used in anticancer vaccination experiments. Because B16 has been widely used, different vaccination strategies can be compared. We reviewed the results obtained when B16 was treated with five common vaccine types: recombinant viral vaccines, DNA vaccines, dendritic cell vaccines, whole-tumor vaccines, and peptide vaccines. We also reviewed the results obtained when B16 was treated with vaccines combined with adoptive transfer of tumor antigen-specific T cells. We found several characteristics of vaccination regimens that were associated with antitumor efficacy. Many vaccines that incorporated xenogeneic antigens exhibited more potent anticancer activity than vaccines that were identical except that they incorporated the syngeneic version of the same antigen. Interleukin-2 enhanced the antitumor efficacy of several vaccines. Finally, several effective regimens generated large numbers of tumor antigen-specific CD8(+) T cells. Identification of vaccine characteristics that are associated with antitumor efficacy may aid in the development of more effective anticancer vaccination strategies.     

Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity

The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.     

Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation

Objectives

Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.

Methods

Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11−88 of 8 different HPV types (11−88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.

Results

Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.

Discussion

Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.     

Enhanced DNA vaccine potency by mannosylated lipoplex after intraperitoneal administration

BACKGROUND: Here we describe a novel DNA vaccine formulation that can enhance cytotoxic T lymphocyte (CTL) activity through efficient gene delivery to dendritic cells (DCs) by mannose receptor-mediated endocytosis. METHODS: Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. Mannosylated cationic liposomes (Man-liposomes) were prepared using cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiomannosylethyl)amino]butyl}formamide (Man-C4-Chol) with cationic lipid. The potency of the mannosylated liposome/pCMV-OVA complex (Man-lipoplex) was evaluated by measuring OVA mRNA in CD11c+ cells, CTL activity, and the OVA-specific anti-tumor effect after in vivo administration. RESULTS: An in vitro study using DC2.4 cells demonstrated that Man-liposomes could transfect pCMV-OVA more efficiently than cationic liposomes via mannose receptor-mediated endocytosis. In vivo studies revealed that the Man-lipoplex exhibited higher OVA mRNA expression in CD11c+ cells in the spleen and peritoneal cavity and provided a stronger OVA-specific CTL response than intraperitoneal (i.p.) administration of the conventional lipoplex and intramuscular (i.m.) administration of naked pCMV-OVA, the standard protocol for DNA vaccination. Pre-immunization with the Man-lipoplex provided much better OVA-specific anti-tumor effect than naked pCMV-OVA via the i.m. route. CONCLUSIONS: These results suggested that in vivo active targeting of DNA vaccine to DCs with Man-lipoplex might prove useful for the rational design of DNA vaccine.