Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines.

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Enhanced effect of modified Zika virus E antigen on the immunogenicity of DNA vaccine.

It has been reported worldwide that the Zika virus (ZIKV) could be transmitted through placentas and sexual contact. ZIKV can also cause Guillain-Barre syndrome, microcephaly and neurological abnormalities. However, there are no approved vaccines available. We constructed six DNA vaccine candidates and tested the immunogenicity. Tandem repeated envelope domain Ⅲ (ED Ⅲ × 3) induced highly total IgG and neutralization antibody, as well as CD8+ T cell responses. Also, stem region-removed envelope (E ΔSTEM) elicited a robust production of IFN-γ in mice. To examine in vivo protection, we used mice treated with an IFNAR1 blocking antibody before and after the challenge. Vaccination with the two candidates led to a decline in the level of viral RNAs in organs. Moreover, the sera from the vaccinated mice did not enhance the infection of Dengue virus in K562 cells. These findings suggest the potential for the development of a novel ZIKV DNA vaccine.

Novel adeno­associated virus­based genetic vaccines encoding hepatitis C virus E2 glycoprotein elicit humoral immune responses in mice.

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct­acting antiviral agents (DAAs), with some 185 million individuals infected with HCV worldwide. There is an urgent need for an effective prophylactic HCV vaccine. In the present study, we constructed genetic vaccines based on novel recombinant adeno­associated viral (rAAV) vectors (AAV2/8 or AAV2/rh32.33) that express the envelope glycoprotein E2 from the HCV genotype 1b. Expression of HCV E2 protein in 293 cells was confirmed by western blot analysis. rAAV2/8.HCV E2 vaccine or rAAV2/rh32.33.HCV E2 vaccine was intramuscularly injected into C57BL/6 mice. HCV E2­specific antigen was produced, and long­lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen­specific antibody levels than the rAAV2/8 vaccine or AAV plasmid. Moreover, both AAV vaccines induced neutralizing antibodies against HCV genotypes 1a and 1b. Finally, it is worth mentioning that neutralizing antibody levels directed against AAV2/rh32.33 were lower than those against AAV2/8 in both mouse and human serum. These results demonstrate that AAV vectors, especially the AAVrh32.33, have particularly favorable immunogenicity for development into an effective HCV vaccine.

Neutralization of the Plasmodium-encoded MIF ortholog confers protective immunity against malaria infection.

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.

The current state of therapeutic and T cell-based vaccines against human papillomaviruses.

Human papillomavirus (HPV) is known to be a necessary factor for many gynecologic malignancies and is also associated with a subset of head and neck malignancies. This knowledge has created the opportunity to control these HPV-associated cancers through vaccination. However, despite the availability of prophylactic HPV vaccines, HPV infections remain extremely common worldwide. In addition, while prophylactic HPV vaccines have been effective in preventing infection, they are ineffective at clearing pre-existing HPV infections. Thus, there is an urgent need for therapeutic and T cell-based vaccines to treat existing HPV infections and HPV-associated lesions and cancers. Unlike prophylactic vaccines, which generate neutralizing antibodies, therapeutic, and T cell-based vaccines enhance cell-mediated immunity against HPV antigens. Our review will cover various therapeutic and T cell-based vaccines in development for the treatment of HPV-associated diseases. Furthermore, we review the strategies to enhance the efficacy of therapeutic vaccines and the latest clinical trials on therapeutic and T cell-based HPV vaccines.

Personalized cancer vaccines: Targeting the cancer mutanome.

The development of next generation sequencing technologies has revolutionized our understanding of how specific genetic events contribute to cancer initiation and progression. Dramatic improvements in instrument design and efficiency, combined with significant cost reductions has permitted a systematic analysis of the mutational landscape in a variety of cancer types. At the same time, a detailed map of the cancer mutanome in individual cancers offers a unique opportunity to develop personalized cancer vaccine strategies targeting neoantigens. Recent studies in both preclinical models and human cancer patients demonstrate that neoantigens (1) are important targets following checkpoint inhibition therapy, (2) have been identified as the target of adoptive T cell therapies, and (3) can be successfully targeted with personalized vaccines. Taken together, these observations provide strong rationale for the clinical translation of personalized cancer vaccines.

Inducing Humoral Immune Responses Against Regulatory T Cells by Foxp3-Fc(IgG) Fusion Protein.

The existence of a developed network of suppressory factors and cells against an immune response in different cancers has been proven; regulatory T cells are a typical issue. Therefore their depletion, elimination, or suppression has been assessed in different research studies that were not entirely successful. By applying an improved vaccine against regulatory T cells, we have evaluated the B cell response elicited by the vaccine in an experimental design. A previously described DNA vaccine and recombinant protein of Foxp3-Fc fusion were produced and used in the vaccination regimen. DNA construct and respective protein were injected into C57BL/6 mice. After 2 weeks, serum levels of IgG antibody and its subtypes against Foxp3 were investigated by ELISA. To produce recombinant Foxp3 for ELISA antigen coating, pET24a-Foxp3 vector was transformed into Escherichia coli strain BL21 as host cells. Afterward, protein was expressed and then purified using Ni-NTA agarose. SDS-PAGE and Western blot analysis were carried out to confirm protein expression. The expression analysis of Foxp3 was confirmed by SDS-PAGE followed by Western blot analysis. FOXP3-Fc DNA vaccine/fusion protein vaccination regimen could induce T helper-dependent humoral responses. Due to the effectiveness of Foxp3-Fc(IgG) in inducing humoral responses, it would be expected to be useful in developing vaccines in tumor therapies for the removal of regulatory T cells as a strategy for increasing the efficiency of other means of immunotherapy.

Microbial ribonucleases (RNases): production and application potential.

Ribonuclease (RNase) is hydrolytic enzyme that catalyzes the cleavage of phosphodiester bonds in RNA. RNases play an important role in the metabolism of cellular RNAs, such as mRNA and rRNA or tRNA maturation. Besides their cellular roles, RNases possess biological activity, cell stimulating properties, cytotoxicity and genotoxicity. Cytotoxic effect of particular microbial RNases was comparable to that of animal derived counterparts. In this respect, microbial RNases have a therapeutic potential as anti-tumor drugs. The significant development of DNA vaccines and the progress of gene therapy trials increased the need for RNases in downstream processes. In addition, RNases are used in different fields, such as food industry for single cell protein preparations, and in some molecular biological studies for the synthesis of specific nucleotides, identifying RNA metabolism and the relationship between protein structure and function. In some cases, the use of bovine or other animal-derived RNases have increased the difficulties due to the safety and regulatory issues. Microbial RNases have promising potential mainly for pharmaceutical purposes as well as downstream processing. Therefore, an effort has been given to determination of optimum fermentation conditions to maximize RNase production from different bacterial and fungal producers. Also immobilization or strain development experiments have been carried out.

Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy.

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293 T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293 T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.

Advances in host and vector development for the production of plasmid DNA vaccines.

Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

Transposon leads to contamination of clinical pDNA vaccine.

We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

[Construction of a prokaryotic expression vector of human tau multi-epitope peptide and immunogenicity of the expressed product].

OBJECTIVE: To construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product. METHODS: The coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein. RESULTS: A gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein. CONCLUSION: The constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.

[Construction of a replicative anti-tumor DNA vaccine PSCK-2PFcGB and its expression in vivo and in vitro].

OBJECTIVE: To construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro. METHODS: The plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin ß chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry. RESULTS: Nde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGß-CTP37 were also detected in the muscular tissues of the mice. CONCLUSION: A novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.

Avaliação do potencial protetor da vacina gênica pvaxapa- dmt-me no modelo experimental de tuberculose / Protective potential evaluation of genetic vaccines pvaxapa- dmt-me in the experimental model of tuberculosis

A tuberculose (TB) é responsável por dois milhões de mortes a cada ano, apesar daampla utilização da vacina BCG (Bacilo Calmette-Guérin). Embora essa vacina nãoproteja contra a TB pulmonar no adulto, protege contra as formas graves da TB nainfância. Para muitos autores, a imunização heteróloga (prime-boost) talvezseja uma das estratégias mais importantes e realistas para o controle da TB,principalmente nos países endêmicos. Muitos estudos têm demonstrado que aimunização com BCG seguido de reforço com vacina gênica (genes de M.tuberculosis) induz melhor proteção contra TB do que somente a vacina BCG. Alémdisso, a estratégia prime-boost utilizando microesferas biodegradáveis pareceser uma imunização promissora para estimular a resposta imune de longa duração.A glicoproteína APA foi identificada como importante antígeno imunodominante,induzindo níveis significativos de proteção contra M. tuberculosis. Assim, esteestudo teve como objetivo avaliar a eficácia protetora da imunização heteróloga emmodelo experimental de TB. Para isso, camundongos BALB/c foram imunizados porvia subcutânea com BCG seguido de um reforço por via intramuscular com aformulação vacinal contendo apa e DMT co-encapsuladosem microesferas (BCG/APA). Essa estratégia prime-boost foi eficiente na indução daresposta imune protetora à infecção por M. tuberculosis, caracterizada pela reduçãosignificativa do número de bacilos no pulmão destes animais na fase mais tardia dainfecção (70 dias após desafio). Além de ser significativamente mais imunogênciaque a vacina BCG, permitiu uma melhor preservação do parênquima pulmonar, comredução do número e tamanho dos granulomas, limitando a extensão da injúriatecidual induzida pela infecção e restringindo a inflamação no tecido alveolar. Apotencialização do efeito protetor da vacina BCG induzida pelo reforço com a vacinapVAXapa+DMT-Me sugere que a estratégia utilizada pode ser importante para aprevenção da TB...

Tuberculosis (TB) is responsible for two millions of deaths each year despite thewidespread use of BCG vaccine (Bacillus Calmette-Guérin). Although this vaccinedoes not protect against adult pulmonary TB, it is protective against severe forms ofchildhood TB. For many authors, heterologous prime-boost regimen is perhaps themost realistic strategy for future TB control, especially in endemic countries. Manystudies have demonstrated that BCG-prime DNA-(M. tuberculosis genes)-boosterinduce greater protection against TB than BCG alone. APA glycoprotein has beenidentified as major immunodominant antigen and induces significant levels ofprotection against M. tuberculosis. Moreover, prime-boost strategy by usingbiodegradable microspheres seems to be a promising immunization to stimulate longlasting immune response. Thus, this study had as aim to investigate the immuneprotection against M. tuberculosis challenge in mice based on BCG priming and DNAvaccine boosting. For that, BALB/c mice were inoculated subcutaneously with aheterologous BCG priming-DNA boosting immunization using pVAXapa and TDM coencapsulatedin microspheres (BCG/APA), intramuscularly. This prime-boost strategywas effective in inducing protective immune response against M. tuberculosisinfection, characterized by significant lower numbers of bacilli in the lungs of theseanimals, in the late phase of infection (70 days after infection). In addition to beingsignificantly more immunogenic than BCG, it allowed better preservation of the lungparenchyma and reduction in the number and size of granulomas, limiting the extentof lung injury induced by infection and inflammation in the alveolar tissue. Theimprovement of the protective effect of BCG vaccine mediated by pVAXapa boostersuggests that our strategy can be important in the treatment for prevention of TB...

Production of adenovirus vectors and their use as a delivery system for influenza vaccines.

IMPORTANCE OF THE FIELD: With the emergence of highly pathogenic avian influenza H5N1 viruses that have crossed species barriers and are responsible for lethal infections in humans in many countries, there is an urgent need for the development of effective vaccines which can be produced in large quantities at a short notice and confer broad protection against these H5N1 variants. In order to meet the potential global vaccine demand in a pandemic scenario, new vaccine-production strategies must be explored in addition to the currently used egg-based technology for seasonal influenza. AREAS COVERED IN THIS REVIEW: Adenovirus (Ad) based influenza vaccines represent an attractive alternative/supplement to the currently licensed egg-based influenza vaccines. Ad-based vaccines are relatively inexpensive to manufacture, and their production process does not require either chicken eggs or labor-intensive and time-consuming processes necessitating enhanced biosafety facilities. Most importantly, in a pandemic situation, this vaccine strategy could offer a stockpiling option to reduce the response time before a strain-matched vaccine could be developed. WHAT THE READER WILL GAIN: This review discusses Ad-vector technology and the current progress in the development of Ad-based influenza vaccines. TAKE HOME MESSAGE: Ad vector-based influenza vaccines for pandemic preparedness are under development to meet global vaccine demand.

[Progress in DNA vaccines against classical swine fever: a review].

In 1990, it was reported that the naked DNA encoding an antigen (so-called DNA vaccine) transduced directly into the muscle is able to induce immune responses just like antigen inoculation. Since then, a number of DNA vaccines against different diseases have been developed and shown to induce different levels of specific humoral and/or cell-mediated immunity. Efforts have been made to develop effective DNA vaccines against classical swine fever (CSF). This review covered the following aspects in the development and application of CSF DNA vaccines: construction and evaluation, application of adjuvants, combination with other vaccines and the existing problems and solutions.

Construction of an HBV DNA vaccine by fusion of the GM-CSF gene to the HBV-S gene and examination of its immune effects in normal and HBV-transgenic mice.

BACKGROUND: The hepatitis B virus (HBV) DNA vaccine can generate both HBsAg-specific humoral and cellular immune responses. The immune response can be improved by inclusion of an adjuvant, such as the cytokine GM-CSF which is known to be a very good adjuvant. METHODS: To investigate the ability of GM-CSF to enhance HBV-DNA vaccines, we constructed the plasmids by fusion of GM-CSF gene to the HBV-S gene. Normal and HBV-transgenic mice were then immunized with these plasmids. RESULTS: Our results show that pCDNA3.1-GM-CSF-S induced the most powerful HBsAg-specific humoral and cellular immune response, and that it was able to overcome the non-response to HBsAg in HBV-transgenic mice. In contrast, pCDNA3.1-S-GM-CSF was able to induce only a very poor immune response. CONCLUSIONS: When the HBV-S gene is fused to the GM-CSF gene, the immune effects of the HBV DNA vaccine both in normal and HBV-transgenic mice can be strengthened and HBV-DNA plasmids fused with GM-CSF may be useful for both preventative and therapeutic purposes.

Engineering superior DNA vaccines: MHC class I single chain trimers bypass antigen processing and enhance the immune response to low affinity antigens.

It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, beta(2)m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4(+) helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8(+) T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8(+) T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.