Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

IMPORTANCE: The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. METHODS: We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. RESULTS: We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. CONCLUSIONS: These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

B cells require licensing by dendritic cells to serve as primary antigen-presenting cells for plasmid DNA.

DNA vaccines have been an attractive approach for treating cancer patients, however have demonstrated modest immunogenicity in human clinical trials. Dendritic cells (DCs) are known to cross-present DNA-encoded antigens expressed in bystander cells. However, we have previously reported that B cells, and not DCs, serve as primary antigen-presenting cells (APCs) following passive uptake of plasmid DNA. Here we sought to understand the requirements for B cells to present DNA-encoded antigens, to ultimately increase the immunogenicity of plasmid DNA vaccines. Using ovalbumin-specific OT-1 CD8+ T cells and isolated APC populations, we demonstrated that following passive uptake of plasmid DNA, B cells but not DC, can translate the encoded antigen. However, CD8 T cells were only activated by B cells when they were co-cultured with DCs. We found that a cell-cell contact is required between B cells and DCs. Using MHCI KO and re-purification studies, we demonstrated that B cells were the primary APCs and DCs serve to license this function. We further identified that the gene expression profiles of B cells that have been licensed by DCs, compared to the B cells that have not, are vastly different and have signatures similar to B cells activated with a TLR7/8 agonist. Our data demonstrate that B cells transcribe and translate antigens encoded by plasmid DNA following passive uptake, however require licensing by live DC to present antigen to CD8 T cells. Further study of the role of B cells as APCs will be important to improve the immunological efficacy of DNA vaccines.

Molecular characterization of hexokinase (HK) in Haemaphysalis longicornis and evaluation of HK protein- and DNA-based vaccines against adult ticks.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite, which transmits various pathogens to humans, livestock and wild animals. Hexokinase (HK) is a key regulatory enzyme of the glycolytic pathway in the organisms. However, little is known about hexokinase and its functions in ticks. RESULTS: The open reading frame of the H. longicornis HK (HlHK) gene was 1425 bp and encoded a protein of 474 amino acids, containing conserved domains for glucose, glucose 6-phosphate, and adenosine triphosphate. The expression of HlHK gene was detected at different developmental stages and in different tissues of unfed female ticks. Enzyme-linked immunosorbent assay revealed that both HK protein- and DNA-based vaccines increased the antibody levels of the immunized animals. A vaccination trail on rabbits against H. longicornis infestation indicated that the rHlHK protein and HlHK DNA vaccines reduced the number of attached female ticks by 9% and 12%, egg mass weight by 36% and 34%, and egg hatching rate by 41% and 17%, respectively. Overall, protein vaccination conferred 65.6% protection against adult female ticks, whereas the DNA vaccine conferred 51.8% protection. CONCLUSION: This is the first report of the molecular characterization of the HK protein and sequencing of the HK gene from H. longicornis. Positive results from vaccination trials on rabbits of the recombinant HK protein and HK DNA suggest that these novel anti-tick vaccines potentially can be used as viable tick control tools for the management of the Asian longhorned tick. Additionally, inhibition of glucose metabolism may be a new strategy for tick control. © 2023 Society of Chemical Industry.

Evaluation of anti-tick efficiency in rabbits induced by DNA vaccines encoding Haemaphysalis longicornis lipocalin homologue.

Haemaphysalis longicornis is an obligate haematophagous ectoparasite, transmitting a variety of pathogens, which brings great damage to human health and animal husbandry development. Lipocalins (LIP) are a family of proteins that transport small hydrophobic molecules and also involve in immune regulation, such as the regulation of cell homeostasis, inhibiting the host's inflammatory response and resisting the contractile responses in host blood vessels. Therefore, it is one of the candidate antigens for vaccines. Based on previous studies, we constructed the recombinant plasmid pcDNA3.1-HlLIP of LIP homologue from H. longicornis (HlLIP). ELISA results showed that rabbits immunized with pcDNA3.1-HlLIP produced higher anti-rHlLIP antibody levels compared with the pcDNA3.1 group, indicating that pcDNA3.1-HlLIP induced the humoral immune response of host. Adult H. longicornis infestation trial in rabbits demonstrated that the engorgement weight, oviposition and hatchability of H. longicornis fed on rabbits immunized with pcDNA3.1-HlLIP decreased by 7.07%, 14.30% and 11.70% respectively, compared with that of the pcDNA3.1 group. In brief, DNA vaccine of pcDNA3.1-HlLIP provided immune protection efficiency of 30% in rabbits. This study demonstrated that pcDNA3.1-HlLIP can partially protect rabbits against H. longicornis, and it is possible to develop a new candidate antigen against ticks.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Development of a Novel Multi-Epitope Vaccine Against Crimean-Congo Hemorrhagic Fever Virus: An Integrated Reverse Vaccinology, Vaccine Informatics and Biophysics Approach.

Crimean-Congo hemorrhagic fever (CCHF) is a highly severe and virulent viral disease of zoonotic origin, caused by a tick-born CCHF virus (CCHFV). The virus is endemic in many countries and has a mortality rate between 10% and 40%. As there is no licensed vaccine or therapeutic options available to treat CCHF, the present study was designed to focus on application of modern computational approaches to propose a multi-epitope vaccine (MEV) expressing antigenic determinants prioritized from the CCHFV genome. Integrated computational analyses revealed the presence of 9 immunodominant epitopes from Nucleoprotein (N), RNA dependent RNA polymerase (RdRp), Glycoprotein N (Gn/G2), and Glycoprotein C (Gc/G1). Together these epitopes were observed to cover 99.74% of the world populations. The epitopes demonstrated excellent binding affinity for the B- and T-cell reference set of alleles, the high antigenic potential, non-allergenic nature, excellent solubility, zero percent toxicity and interferon-gamma induction potential. The epitopes were engineered into an MEV through suitable linkers and adjuvating with an appropriate adjuvant molecule. The recombinant vaccine sequence revealed all favorable physicochemical properties allowing the ease of experimental analysis in vivo and in vitro. The vaccine 3D structure was established ab initio. Furthermore, the vaccine displayed excellent binding affinity for critical innate immune receptors: TLR2 (-14.33 kcal/mol) and TLR3 (-6.95 kcal/mol). Vaccine binding with these receptors was dynamically analyzed in terms of complex stability and interaction energetics. Finally, we speculate the vaccine sequence reported here has excellent potential to evoke protective and specific immune responses subject to evaluation of downstream experimental analysis.

COVID-19 vaccine: where are we now and where should we go?

INTRODUCTION: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has currently caused the pandemic with a high progressive speed and has been considered as the global public health crisis in 2020. This new member of the coronavirus family has created a potentially fatal disease, called coronavirus disease-2019 (COVID-19). Despite the continuous efforts of researchers to find effective vaccines and drugs for COVID-19, there is still no success in this matter. AREAS COVERED: Here, the literature regarding the COVID-19 vaccine candidates currently in the clinical trials, as well as main candidates in pre-clinical stages for development and research, were reviewed. These candidates have been developed under five different major platforms, including live-attenuated vaccine, mRNA-based vaccine, DNA vaccines, inactivated virus, and viral-vector-based vaccine. EXPERT OPINION: There are several limitations in the field of the rapid vaccine development against SARS-CoV-2, and other members of the coronavirus family such as SARS-CoV and MERS-CoV. The key challenges of designing an effective vaccine within a short time include finding the virulence ability of an emerging virus and potential antigen, choosing suitable experimental models and efficient route of administration, the immune-response study, designing the clinical trials, and determining the safety, as well as efficacy.

Understanding Pseudomonas aeruginosa-Host Interactions: The Ongoing Quest for an Efficacious Vaccine.

Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection.

Effective inhibition of tumor in vivo with a novel DNA vaccine targeting chimeric G250.

OBJECTIVE: To develop a promising approach for tumor immunotherapy with G250 antigen-based DNA vaccine and to investigate its anti-tumor response in mice with renal cell carcinoma. MATERIALS AND METHODS: G250 derived from human, monkey and mouse were prepared by PCR. The heterogeneous chimeric G250 gene was obtained by integrating different gene fragments of three species. Then, the chimeric G250 was inserted into a eukaryotic expression plasmid pVAX1-IRES-GM/B7 to obtain DNA vaccine (named pVAX1-tG250-GM/B7) which could express chimeric G250 antigen and immune adjuvants simultaneously. By transfecting into Cos7 cells, the expression of chimeric G250 antigen was tested using flow cytometry and immunofluorescence assay. The immunological response and protection against tumor were evaluated in vivo. RESULTS: Recombinant plasmid DNA vaccine was constructed successfully through identification of PCR and gene sequencing. The chimeric G250 antigen was well expressed in Cos7 cells. A strong immune response can be detected through ELISPOT and ELISA induced by pVAX1-tG250-GM/B7. The mice vaccinated with pVAX1-tG250-GM/B7, balb/c showed significant inhibition of tumor and a longer time of survival compared with control group. CONCLUSIONS: The experimental results of this study exhibited that the DNA vaccine based on heterogeneous chimeric antigen can produce efficient anti-tumor effect in vivo and they represent a promising strategy for tumor immunotherapy.

Current Status of COVID-19 (Pre)Clinical Vaccine Development.

The current COVID-19 pandemic has a tremendous impact on daily life world-wide. Despite the ability to dampen the spread of SARS-CoV-2, the causative agent of the diseases, through restrictive interventions, it is believed that only effective vaccines will provide sufficient control over the disease and revert societal live back to normal. At present, a double-digit number of efforts are devoted to the development of a vaccine against COVID-19. Here, we provide an overview of these (pre)clinical efforts and provide background information on the technologies behind these vaccines. In addition, we discuss potential hurdles that need to be addressed prior to mass scale clinical translation of successful vaccine candidates.

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2.

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7-8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.

Application of antigen presenting cell-targeted nanovaccine delivery system in rhabdovirus disease prophylactics using fish as a model organism.

BACKGROUND: Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease. MATERIALS AND METHODS: In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention. RESULTS: Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group. CONCLUSION: This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.

Engineering a "PEG-g-PEI/DNA nanoparticle-in- PLGA microsphere" hybrid controlled release system to enhance immunogenicity of DNA vaccine.

Controlled release strategies of DNA vaccine hold promise for the design of in vivo vaccination platforms, yet the formulation and sustained delivery still pose a substantial challenge. In this study, we developed a novel hybrid dual-particulate delivery system, nanoparticle-in-microsphere (NIM), to integrate the advantages of nano-sized polymer/DNA polyplex with the sustained-release microsphere for DNA vaccine delivery. The nano-sized cores, consisting of polyethylene glycol-graft-polyethylenimine (PEG-g-PEI)/DNA polyplexes, were formulated into PLGA microspheres using a solid-in-oil-in-water (S/O/W) emulsion. The PEG block was used as stabilizing excipient to make DNA soluble and stable in organic solvent to prevent the inactivation of DNA at aqueous-organic interface during encapsulation. The fashion of DNA in dry solid state greatly increased the encapsulation efficiency of DNA in NIMs. This new formulation exhibited a burst release less than 15% and then sustain release close to zero-order kinetics in physiological environment. In addition, the microspheres showed pH-sensitivity and degraded faster in lysosomal compartments, which contributed to the accelerated intracellular release kinetics of DNA. Finally, intramuscular injection of NIMs encoding HIV proteins elicited distinct humoral and cellular immune response in mice at low dose. These results thus may aid NIM-based vaccination towards more extensive clinical evaluations.

Dynamic profiles, biodistribution and integration evaluation after intramuscular/intravenous delivery of a novel therapeutic DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in vaccinated normal rodent.

BACKGROUND: The persistence, biodistribution, and risk of integration into the host genome of any new therapeutic DNA vaccine must be established in preclinical studies. We previously developed the DNA vaccine pcDNA-CCOL2A1 encoding chicken type II collagen (CCII) for the treatment of rheumatoid arthritis (RA). In the present study, we characterized its dynamic profile, biodistribution, and potential for genomic DNA integration in normal vaccinated rodent. RESULTS: A real-time quantitative PCR analysis (RT-qPCR) of animals administered a single dose of pcDNA-CCOL2A1 (300 µg/kg by intramuscular injection) showed that CCOL2A1 mRNA level in the blood peaked between 2 and 6 h post-immunization and then rapidly declined, and was undetectable between day 1-42. CCOL2A1 transcript was detected at the muscle injection site on days 3-14 post-immunization. Starting from day 14, the transcript was detected in the heart, liver, lung, and kidney but not in the spleen or thymus, and was expressed only in the lung on day 28. There was no CCOL2A1 mRNA present in the testes or ovaries at any time point. Non-invasive in vivo fluorescence imaging revealed CCII protein expression from 2 h up to day 10 and from 2 h up to day 35 after administration of pcDNA-CCOL2A1 via the intravenous and intramuscular routes, respectively; the protein had disappeared by day 42. Importantly, CCOL2A1 was not integrated into the host genome. CONCLUSIONS: These results indicate that pcDNA-CCOL2A1 vaccine is rapidly cleared within a short period of time and is therefore safe, and merits further development as a therapeutic vaccine for RA treatment.

DNA vaccine encoding OmpA and Pal from Acinetobacter baumannii efficiently protects mice against pulmonary infection.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen that causes serious infections in the lungs, blood, and brain in critically ill hospital patients, resulting in considerable mortality rates every year. Due to the rapid appearance of multi-drug resistance or even pan-drug resistance isolates, it is becoming more and more difficult to cure A. baumannii infection by traditional antibiotic treatment, alternative strategies are urgently required to combat A. baumannii infection. In this study, we developed a DNA vaccine encoding two antigens from A. baumannii, OmpA and Pal, and the immunogenicity and protective efficacy was further evaluated. The results showed that the DNA vaccine exhibited significant immune protective efficacy against acute A. baumannii infection in a mouse pneumonia model, and cross protective efficacy was observed when immunized mice were challenged with clinical strains of A. baumannii. DNA vaccine immunization induced high level of humoral response and a mixed Th1/Th2/Th17 cellular response, which protect against lethal bacterial challenges by decreased bacterial loads and pathology in the lungs, and reduced level of inflammatory cytokines expression and inflammatory cell infiltration in BALF. These results demonstrated that it is possible to prevent A. baumannii infection by DNA vaccine and both OmpA and Pal could be serve as promising candidate antigens.

Construction of Lactobacillus casei ghosts by Holin-mediated inactivation and the potential as a safe and effective vehicle for the delivery of DNA vaccines.

BACKGROUND: Bacterial ghosts (BGs) are empty bacterial cell envelopes generated by releasing the cellular contents. In this study, a phage infecting Lactobacillus casei ATCC 393 (L. casei 393) was isolated and designated Lcb. We aimed at using L. casei 393 as an antigen delivery system to express phage-derived holin for development of BGs. RESULTS: A gene fragment encoding holin of Lcb (hocb) was amplified by polymerase chain reaction (PCR). We used L. casei 393 as an antigen delivery system to construct the recombinant strain pPG-2-hocb/L. casei 393. Then the recombinants were induced to express hocb. The immunoreactive band corresponding to hocb was observed by western-blotting, demonstrating the efficiency and specificity of hocb expression in recombinants. The measurements of optical density at 600 nm (OD600) after induction showed that expression of hocb can be used to convert L. casei cells into BGs. TEM showed that the cytomembrane and cell walls of hocb expressing cells were partially disrupted, accompanied by the loss of cellular contents, whereas control cells did not show any morphological changes. SEM showed that lysis pores were distributed in the middle or at the poles of the cells. To examine where the plasmid DNA was associated, we analyzed the L. casei ghosts loading SYBR Green I labeled pCI-EGFP by confocal microscopy. The result demonstrated that the DNA interacted with the inside rather than with the outside surface of the BGs. To further analyze where the DNA were loaded, we stained BGs with MitoTracker Green FM and the loaded plasmids were detected using EGFP-specific Cy-3-labeled probes. Z-scan sections through the BGs revealed that pCI-EGFP (red) was located within the BGs (green), but not on the outside. Flow cytometry and qPCR showed that the DNA was loaded onto BGs effectively and stably. CONCLUSIONS: Our study constructed L. casei BGs by a novel method, which may be a promising technology for promoting the further application of DNA vaccine, providing experimental data to aid the development of other Gram-positive BGs.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.

Construction and protective immunogenicity of DNA vaccine pNMB0315 against Neisseria meningitidis serogroup B.

Neisseria meningitidis (N. meningitidis) is a major cause of meningitis and sepsis. Capsular polysaccharide­based vaccines against serogroups A, C, Y, and W135 are available; however, the development of a vaccine against N. meningitidis serogroup B (NMB) has been problematic. NMB0315 is an outer membrane protein of NMB that may be a virulence factor for N. meningitidis and a possible target for functional bactericidal antibodies. The present study aimed to develop a potent DNA vaccine against NMB by cloning the NMB0135 gene into the pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1(+)/NMB0315 (designated pNMB0315). pNMB0315 was transfected into eukaryotic COS­7 and RAW264.7 cells to express the recombinant (r)NMB0315 protein. Protective immunogenicity of the DNA vaccine was assessed in an in vivo mouse model. The levels of rNMB0315­specific immunoglobulin G (IgG), IgG1 and IgG2a antibodies in the pNMB0315­immunized group increased dramatically up to week 6 following the initial vaccination, and were significantly higher compared with the levels in the Control groups. The serum concentrations of interleukin­4 and interferon­Î³ were significantly higher in the pNMB0315­immunized group compared with the control groups. Following intraperitoneal challenge with a lethal dose of NMB strain MC58, the survival rate in the pNMB0315 + CpG group was 70% (14 out of 20 mice) at 14 days; by contrast, all mice in the control groups succumbed within 3 days. The serum bactericidal titers of the pNMB0315 + CpG group in vitro reached 1:128 following three immunizations. The results indicated that pNMB0315 may serve as a promising DNA vaccine against NMB.