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Seasonal influenza is an annually severe crisis for global public health, and an ideal influenza vaccine is expected to provide broad protection against constantly drifted strains. Compared to highly flexible hemagglutinin (HA), increasing data have demonstrated that neuraminidase (NA) might be a potential target against influenza variants. In the present study, a series of genetic algorithm-based mosaic NA were designed, and then cloned into recombinant DNA and replication-defective Vesicular Stomatitis Virus (VSV) vector as a novel influenza vaccine candidate. Our Results showed that DNA prime/VSV boost strategy elicited a robust NA-specific Th1-dominated immune response, but the traditional inactivated influenza vaccine elicited a Th2-dominated immune response. More importantly, the superior NA-specific immunity induced by our strategy could confer both a full protection against lethal homologous influenza challenge and a partial protection against heterologous influenza infection. These findings will provide insights on designing NA-based universal vaccine strategy against influenza variants., Competing Interests: Declaration of competing interest J.G. is employed by Shenzhen Kangtai Biological Products Co., Ltd. Other authors declare that the there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Human papillomavirus type 16 (HPV-16) infection is strongly associated with considerable parts of cervical, neck, and head cancers. Performed investigations have had moderate clinical success, so research to reach an efficient vaccine has been of great interest. In the present study, the immunization potential of a newly designed HPV-16 construct was evaluated in a mouse model., Results: Initially, a construct containing HPV-16 mutant (m) E6/E7 fusion gene was designed and antigen produced in two platforms (i.e., DNA vaccine and recombinant protein). Subsequently, the immunogenicity of these platforms was investigated in five mice) C57BL/6 (groups based on several administration strategies. Three mice groups were immunized recombinant protein, DNA vaccine, and a combination of them, and two other groups were negative controls. The peripheral blood mononuclear cells (PBMCs) proliferation, Interleukin-5 (IL-5) and interferon-γ (IFN-γ) cytokines, IgG1 and IgG2a antibody levels were measured. After two weeks, TC-1 tumor cells were injected into all mice groups, and subsequently further analysis of tumor growth and metastasis and mice survival were performed according to the schedule. Overall, the results obtained from in vitro immunology and tumor cells challenging assays indicated the potential of the mE6/E7 construct as an HPV16 therapeutic vaccine candidate. The results demonstrated a significant increase in IFN-γ cytokine (P value < 0.05) in the Protein/Protein (D) and DNA/Protein (E) groups. This finding was in agreement with in vivo assays. Control groups show a 10.5-fold increase (P value < 0.001) and (C) DNA/DNA group shows a 2.5-fold increase (P value < 0.01) in tumor growth compared to D and E groups. Also, a significant increase in survival of D and E (P value < 0.001) and C (P value < 0.01) groups were observed., Conclusions: So, according to the findings, the recombinant protein could induce stronger protection compared to the DNA vaccine form. Protein/Protein and DNA/Protein are promising administration strategies for presenting this construct to develop an HPV-16 therapeutic vaccine candidate., (© 2024. The Author(s).)

Vaccine manufacturing fosters the prevention, control, and eradication of infectious diseases. Recombinant DNA and in vitro (IVT) mRNA vaccine manufacturing technologies were enforced to combat the recent pandemic. Despite the impact of these technologies, there exists no scientific announcement that compares them. Digital Shadows are employed in this study to simulate each technology, investigating root cause deviations, technical merits, and liabilities, evaluating cost scenarios. Under this lens we provide an unbiased, advanced comparative technoeconomic study, one that determines which of these manufacturing platforms are suited for the two types of vaccines considered (monoclonal antibodies or antigens). We find recombinant DNA technology to exhibit higher Profitability Index due to lower capital and starting material requirements, pertaining to lower Minimum Selling Price per Dose values, delivering products of established quality. However, the potency of the mRNA, the streamlined and scalable synthetic processes involved and the raw material availability, facilitate faster market penetration and product flexibility, constituting these vaccines preferable whenever short product development cycles become a necessity., (© 2024. The Author(s).)

Toxoplasma gondii is an important protozoan pathogen, which can cause severe diseases in the newborns and immunocompromised individuals. Developing an effective vaccine against Toxoplasma infection is a critically important global health priority. Immunofluorescence staining analysis revealed that TgSAG2 and TgSRS2 are membrane associated and displayed on the surface of the parasite. Immunizations with pBud-SAG2, pBud-SRS2 and pBud-SAG2-SRS2 DNA vaccines significantly increased the production of specific IgG antibodies. Immunization with pBud-SAG2-SRS2 elicited cellular immune response with higher concentrations of IFN-γ and IL-4 compared to the control group. Antigen-specific lymphocyte proliferations in the pBud-SRS2 and pBud-SAG2-SRS2 groups were significantly higher compared to that in the control group. Furthermore, 30 % of mice immunized with pBud-SAG2-SRS2 survived after the challenge infection with virulent T. gondii RH tachyzoites. This study revealed that immunization with pBud-SAG2-SRS2 induced potent immune responses, and has the potential as a promising vaccine candidate for the control of T. gondii infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is an intracellular protozoan parasite that infects all nucleated cells except the red blood cells. Currently, nucleic acid vaccines are being widely investigated in Toxoplasma gondii control, and several nucleic acid vaccine candidate antigens have shown good protection in various studies. The aim of this study was to construct a nucleic acid vaccine with Toxoplasma gondii SRS29C as the target gene. We explored the nucleic acid vaccine with Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1 and evaluated its immunoprotective effect against Toxoplasma gondii. To amplify the gene fragment and clone it to the expression vector, the recombinant plasmid pEGFP-SRS29C was constructed by PCR. Eukaryotic cells were transfected with the plasmid, and the expression of the target protein was assessed using the Western blot method. The level of serum IgG was determined via ELISA, and the splenic lymphocyte proliferation ability was detected using the CCK-8 method. The percentages of CD4 + and CD8 + T cells were measured by flow cytometry. Mice were immunised three times with single-gene nucleic acid vaccine and combination vaccine. Splenic lymphocytokine expression was determined using ELISA kits. The mice's survival time was monitored and recorded during an in vivo insect assault experiment, and the vaccine's protective power was assessed. The outcomes showed that PCR-amplification of an SRS29C gene fragment was successful. The 4,733-bp vector fragment and the 1,119-bp target segment were both recognised by double digestion. Additionally, after transfection of the recombinant plasmid pEGFP-SRS29C, Western blot examination of the extracted protein revealed the presence of a target protein strip at 66 kDa. The test results demonstrated that the IgG content in the serum of the pEGFP-SRS29C group and the co-immunization group was significantly higher than that of the PBS group and the empty vector group. The IgG potency induced by the co-immunization group was higher than that of the pEGFP-SRS29C group and the pEGFP-SAG1 group, the number of splenic lymphocyte proliferation number was higher than that of the PBS group and the empty vector group. The CD4 + /CD8 + T ratio was higher than that of the PBS group and the empty vector group. The expression of IFN-γ and TNF-α in the splenocytes of the pEGFP-SRS29C group and the combined immunisation group was significantly higher following antigen stimulation. In the worm attack experiments, mice in the PBS and empty vector groups perished within 9 days of the worm attack, whereas mice in the pEGFP-SRS29C group survived for 18 days, mice in the pEGFP-SAG1 group survived for 21 days, and mice in the co-immunization group survived for 24 days. This demonstrates that the constructed Toxoplasma gondii nucleic acid vaccine pEGFP-SRS29C and the combined gene vaccine can induce mice to develop certain humoral and cellular immune responses, and enhance their ability to resist Toxoplasma gondii infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflicts of interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Middle East respiratory coronavirus (MERS-CoV) is a newly emergent, highly pathogenic coronavirus that is associated with 34% mortality rate. MERS-CoV remains listed as priority pathogen by the WHO. Since its discovery in 2012 and despite the efforts to develop coronaviruses vaccines to fight against SARS-CoV-2, there are currently no MERS-CoV vaccine that has been approved. Therefore, there is high demand to continue on the development of prophylactic vaccines against MERS-CoV. Current advancements in vaccine developments can be adapted for the development of improved MERS-CoV vaccines candidates. Nucleic acid-based vaccines, including pDNA and mRNA, are relatively new class of vaccine platforms. In this work, we developed pDNA and mRNA vaccine candidates expressing S.FL gene of MERS-CoV. Further, we synthesized a silane functionalized hierarchical aluminosilicate to encapsulate each vaccine candidates. We tested the nucleic acid vaccine candidates in mice and evaluated humoral antibodies response. Interestingly, we determined that the non-encapsulated, codon optimized S.FL pDNA vaccine candidate elicited the highest level of antibody responses against S.FL and S1 of MERS-CoV. Encapsulation of mRNA with nanoporous aluminosilicate increased the humoral antibody responses, whereas encapsulation of pDNA did not. These findings suggests that MERS-CoV S.FL pDNA vaccine candidate induced the highest level of humoral responses. This study will enhance further optimization of nanosilica as potential carrier for mRNA vaccines. In conclusion, this study suggests MERS-CoV pDNA vaccine candidate as a suitable vaccine platform for further pivotal preclinical testings.

Infections caused by the influenza virus lead to both epidemic and pandemic outbreaks in humans and animals. Owing to their rapid production, safety, and stability, DNA vaccines represent a promising avenue for eliciting immunity and thwarting viral infections. While DNA vaccines have demonstrated substantial efficacy in murine models, their effectiveness in larger animals remains subdued. This limitation may be addressed by augmenting the immunogenicity of DNA-based vaccines. In the investigation here, protein expression was enhanced via codon optimization and then mouse cytotoxic T-lymphocyte antigen 4 (CTLA-4) was harnessed as a modulatory adjunct to bind directly to antigen-presenting cells. Further, the study evaluated the immunogenicity of two variants of the hemagglutinin (HA) antigen, i.e. the full-length and the C-terminal deletion versions. The study findings revealed that the codon-optimized HA gene (pcHA) led to increased protein synthesis, as evidenced by elevated mRNA levels. Codon optimization also significantly bolstered both cellular and humoral immune responses. In cytokine assays, all plasmid constructs, particularly pCTLA4-cHA, induced robust interferon (IFN)-γ production, while interleukin (IL)-4 levels remained uniformly non-significant. Mice immunized with pcHA displayed an augmented presence of IFNγ + T-cells, underscoring the enhanced potency of the codon-optimized HA vaccine. Contrarily, CTLA-4-fused DNA vaccines did not significantly amplify the immune response.

Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae , drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae , but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

In this study, we aimed to evaluate the immunogenic profile of a chimeric DNA-based hepatitis C virus (HCV) vaccine candidate encoding the full-length viral core-E1-E2 (HCV-CE) fragment. The vaccine candidate was designed to uniformly express the HCV genotype 4 core-E1-E2 protein. The recombinant HCV-CE protein was bacterially expressed in C41 (DE3) cells, and then BALB/c mice were immunized with different combinations of DNA/DNA or DNA/protein prime/boost immunizations. The proper construction of our vaccine candidate was confirmed by specific amplification of the encoded fragments and basic local alignment search tool (BLAST) results of the nucleotide sequence, which revealed a high degree of similarity with several HCV serotypes/genotypes. The platform for bacterial expression was optimized to maximize the yield of the purified recombinant HCV-CE protein. The recombinant protein showed high specific antigenicity against the sera of HCV-infected patients according to the ELISA and western blot results. The predicted B- and T-cell epitopes showed high antigenic and interferon-γ (IFN-γ) induction potential, in addition to cross-genotype conservation and population coverage. The mice antisera further demonstrated a remarkable ability to capture 100% of the native viral antigens circulating in the sera of HCV patients, with no cross-reactivity detected in control sera. In conclusion, the proposed HCV vaccination strategy demonstrated promising potential regarding its safety, immunogenicity, and population coverage., (Copyright© by the Egyptian Association of Immunologists.)

Severe acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies (P < 0.0001), strong Th1-biased immune response as shown by IgG2a polarization (P < 0.01), increase in IFN-γ levels (P < 0.01), and increment in the ratio of IFN-γ secreting CD4 + (3.78-10.19%) and CD8 + (5.24-12.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID + EP routes conferred 90-100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36 h fermentation process of E. coli DH5α cells containing pcoSpikeD614G resulting in a wet cell weight of 242 g/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine., (© 2024. The Author(s).)

As of December 2022, 2603 laboratory-identified Middle East respiratory syndrome coronavirus (MERS-CoV) infections and 935 associated deaths, with a mortality rate of 36%, had been reported to the World Health Organization (WHO). However, there are still no vaccines for MERS-CoV, which makes the prevention and control of MERS-CoV difficult. In this study, we generated two DNA vaccine candidates by integrating MERS-CoV Spike (S) gene into a replicating Vaccinia Tian Tan (VTT) vector. Compared to homologous immunization with either vaccine, mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses. The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012, England1 and KNIH strains of MERS-CoV. Prime-Boost immunization also induced strong MERS-S specific T cells responses, with high memory and poly-functional (CD107a-IFN-γ-TNF-α) effector CD8 + T cells. In conclusion, the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S. This study not only provides a promising set of MERS-CoV vaccine candidates, but also proposes a heterologous sequential immunization strategy worthy of further development., Competing Interests: Conflict of interest Prof. Yiming Shao is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease caused by the SFTS virus (Dabie bandavirus), which has become a substantial risk to public health. No specific treatment is available now, that calls for an effective vaccine. Given this, we aimed to develop a multi-epitope DNA vaccine through the help of bioinformatics. The final DNA vaccine was inserted into a special plasmid vector pVAX1, consisting of CD8 + T cell epitopes, CD4 + T cell epitopes and B cell epitopes (six epitopes each) screened from four genome-encoded proteins--nuclear protein (NP), glycoprotein (GP), RNA-dependent RNA polymerase (RdRp), as well as nonstructural protein (NSs). To ascertain if the predicted structure would be stable and successful in preventing infection, an immunological simulation was run on it. In conclusion, we designed a multi-epitope DNA vaccine that is expected to be effective against Dabie bandavirus, but in vivo trials are needed to verify this claim., (© 2024. The Author(s).)

Vaccines play essential roles in the fight against the COVID-19 pandemic. The development and assessment of COVID-19 vaccines have generally focused on the induction and boosting of neutralizing antibodies targeting the SARS-CoV-2 spike (S) protein. Due to rapid and continuous variation in the S protein, such vaccines need to be regularly updated to match newly emerged dominant variants. T-cell vaccines that target MHC I- or II-restricted epitopes in both structural and non-structural viral proteins have the potential to induce broadly cross-protective and long-lasting responses. In this work, the entire proteome encoded by SARS-CoV-2 (Wuhan-hu-1) is subjected to immunoinformatics-based prediction of HLA-A*02:01-restricted epitopes. The immunogenicity of the predicted epitopes is evaluated using peripheral blood mononuclear cells from convalescent Wuhan-hu-1-infected patients. Furthermore, predicted epitopes that are conserved across major SARS-CoV-2 lineages and variants are used to construct DNA vaccines expressing multi-epitope polypeptides. Most importantly, two DNA vaccine constructs induce epitope-specific CD8 + T-cell responses in a mouse model of HLA-A*02:01 restriction and protect immunized mice from challenge with Wuhan-hu-1 virus after hACE2 transduction. These data provide candidate T-cell epitopes useful for the development of T-cell vaccines against SARS-CoV-2 and demonstrate a strategy for quick T-cell vaccine candidate development applicable to other emerging pathogens.

More than half of the world's population is infected with Helicobacter pylori (H. pylori), which may lead to chronic gastritis, peptic ulcers, and stomach cancer. LeoA, a conserved antigen of H. pylori, aids in preventing this infection by triggering specific CD3+ T-cell responses. In this study, recombinant plasmids containing the LeoA gene of H. pylori are created and conjugated with chitosan nanoparticle (CSNP) to immunize BALB/c mice against the H. pylori infection. We used the online Vaxign tool to analyze the genomes of five distinct strains of H. pylori, and we chose the outer membrane as a prospective vaccine candidate. Afterward, the proteins' immunogenicity was evaluated. The DNA vaccine was constructed and then encapsulated in CSNPs. The effectiveness of the vaccine's immunoprotective effects was evaluated in BALB/c mice. Purified activated splenic CD3+ T cells are used to test the anticancer effects in vitro. Nanovaccines had apparent spherical forms, were small (mean size, 150-250 nm), and positively charged (41.3 ± 3.11 mV). A consistently delayed release pattern and an entrapment efficiency (73.35 ± 3.48%) could be established. Compared to the non-encapsulated DNA vaccine, vaccinated BALB/c mice produced higher amounts of LeoA-specific IgG in plasma and TNF-α in splenocyte lysate. Moreover, BALB/c mice inoculated with nanovaccine demonstrated considerable immunity (87.5%) against the H. pylori challenge and reduced stomach injury and bacterial burdens in the stomach. The immunological state in individuals with GC with chronic infection with H. pylori is mimicked by the H. pylori DNA nanovaccines by inducing a shift from Th1 to Th2 in the response. In vitro human GC cell development is inhibited by activated CD3+ T lymphocytes. According to our findings, the H. pylori vaccine-activated CD3+ has potential immunotherapeutic benefits., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer., (© 2024. The Author(s).)

The swine industry across the globe is recently facing a devastating situation imparted by a highly contagious and deadly viral disease, African swine fever. The disease is caused by a DNA virus, the African swine fever virus (ASFV) of the genus Asfivirus. ASFV affects both wild boars and domestic pigs resulting in an acute form of hemorrhagic fever. Since the first report in 1921, the disease remains endemic in some of the African countries. However, the recent occurrence of ASF outbreaks in Asia led to a fresh and formidable challenge to the global swine production industry. Culling of the infected animals along with the implementation of strict sanitary measures remains the only options to control this devastating disease. Efforts to develop an effective and safe vaccine against ASF began as early as in the mid-1960s. Different approaches have been employed for the development of effective ASF vaccines including inactivated vaccines, subunit vaccines, DNA vaccines, virus-vectored vaccines, and live attenuated vaccines (LAVs). Inactivated vaccines are a non-feasible strategy against ASF due to their inability to generate a complete cellular immune response. However genetically engineered vaccines, such as subunit vaccines, DNA vaccines, and virus vector vaccines, represent tailored approaches with minimal adverse effects and enhanced safety profiles. As per the available data, gene deleted LAVs appear to be the most potential vaccine candidates. Currently, a gene deleted LAV (ASFV-G-∆I177L), developed in Vietnam, stands as the sole commercially available vaccine against ASF. The major barrier to the goal of developing an effective vaccine is the critical gaps in the knowledge of ASFV biology and the immune response induced by ASFV infection. The precise contribution of various hosts, vectors, and environmental factors in the virus transmission must also be investigated in depth to unravel the disease epidemiology. In this review, we mainly focus on the recent progress in vaccine development against ASF and the major gaps associated with it., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The most crucial disadvantage of DNA-based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV-1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single-cycle replicable (SCR) HIV-1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th-1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ-Nef and CD40L-Nef fusion DNA constructs show higher secretion of IFNγ and TNF-α from virion-infected lymphocytes than other groups. Therefore, the IFNγ-Nef and CD40L-Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV-1 vaccine., (© 2023 Wiley-VCH GmbH.)

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs., (© 2024. The Author(s).)

Importance: Toxoplasma gondii , an obligate intracellular eukaryotic parasite, can infect about one-third of the world's population. One vaccine, Toxovax, has been developed and licensed commercially; however, it is only used in the sheep industry to reduce the losses caused by congenital toxoplasmosis. Various other vaccine approaches have been explored, including excretory secretion antigen vaccines, subunit vaccines, epitope vaccines, and DNA vaccines. However, current research has not yet developed a safe and effective vaccine for T. gondii . Here, we generated an mRNA vaccine candidate against T. gondii . We investigated the efficacy of vaccination with a novel identified candidate, TGGT1&#95;278620, in a mouse infection model. We screened T. gondii -derived protective antigens at the genome-wide level, combined them with mRNA-lipid nanoparticle vaccine technology against T. gondii , and investigated immune-related factors and mechanisms. Our findings might contribute to developing vaccines for immunizing humans and animals against T. gondii ., Competing Interests: The authors declare no conflict of interest.

Background: Heterologous combinations in vaccine design are an effective approach to promote T cell activity and antiviral effects. The goal of this study was to compare the homologous and heterologous regimens targeting the Nef-Tat fusion antigen to develop a human immunodeficiency virus-1 (HIV-1) therapeutic vaccine candidate., Methods: At first, the DNA and protein constructs harboring HIV-1 Nef and the first exon of Tat as linked form (pcDNA- nef-tat and Nef-Tat protein) were prepared in large scale and high purity. The generation of the Nef-Tat protein was performed in the E. coli expression system using an IPTG inducer. Then, we evaluated and compared immune responses of homologous DNA prime/ DNA boost, homologous protein prime/ protein boost, and heterologous DNA prime/protein boost regimens in BALB/c mice. Finally, the ability of mice splenocytes to secret cytokines after exposure to single-cycle replicable (SCR) HIV-1 was compared between immunized and control groups in vitro ., Results: The nef-tat gene was successfully subcloned in eukaryotic pcDNA3.1 (-) and prokaryotic pET-24a (+) expression vectors. The recombinant Nef-Tat protein was generated in the E. coli Rosetta strain under optimized conditions as a clear band of ~ 35 kDa detected on SDS-PAGE. Moreover, transfection of pcDNA- nef-tat into HEK-293T cells was successfully performed using Lipofectamine 2000, as confirmed by western blotting. The immunization studies showed that heterologous DNA prime/protein boost regimen could significantly elicit the highest levels of Ig- G2a, IFN-γ, and Granzyme B in mice as compared to homologous DNA/DNA and protein/protein regimens. Moreover, the secretion of IFN-γ was higher in DNA/protein regimens than in DNA/DNA and protein/protein regimens after exposure of mice splenocytes to SCR HIV-1 in vitro ., Conclusion: The chimeric HIV-1 Nef-Tat antigen was highly immunogenic, especially when applied in a heterologous prime/ boost regimen. This regimen could direct immune response toward cellular immunity (Th1 and CTL activity) and increase IFN-γ secretion after virus exposure., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A plasmid production process has been established to manufacture plasmid DNA at a large scale in High-Quality grade. This is used as a starting material to produce mRNA vaccines for clinical trials. Recently, the World Health Organization (WHO) has released regulatory guidelines related to the quality, safety, and efficacy for DNA- as well as for mRNA-based vaccines. Following an extraordinary year of scientific, regulatory, and manufacturing developments, the scientific community today stands considerably better equipped to deal with urgent production requirements in large scale for nucleic acid-based vaccinations and therapies. Going forward, work needs to be done in better coordinating the supply and logistics of essential raw materials for biological manufacturing, especially under emergency conditions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load., Methods: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4 + and T CD8 + ) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen., Results: T CD4 + and T CD8 + lymphocytes cells stimulated by HBZ-peptides (HBZ 42-50 and HBZ 157-176 ) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8 + cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44 +High /CD62L -Low ), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8 + T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA., Conclusions: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine., (© 2023. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

Purpose: To observe the anti-caries effect of transgenic tomato anti-caries vaccine after immunization with SD rats by gavage and to explore its immunity mechanism initially., Methods: SD rats were used to establish an experimental caries model. The transgenic anti-caries tomatoes expressing the target protein were cultivated and identified. The SIgA and IgG contents of specific anti-PAcA in saliva and blood samples of SD rats were detected by ELISA. Then, the SD rats were sacrificed, the maxillary and mandibular bones were taken for Keyes dental caries score, and spleens were taken for the analysis of RNA-seq. Statistical analysis was performed with SPSS 18.0 software package., Results: The target protein concentration in the transgenic tomato anti-caries vaccine was 36.28 μg/mL. After vaccine immunization of SD rats, group D (8 mL/kg) produced the highest levels of specific SIgA and IgG antibodies at week 6 and was significantly different from the other groups(P＜0.05), and caries counting score was also significantly different than the other groups (P＜0.05). The spleen mRNA of SD rats in group D was extracted and sequenced by RNA-seq, and 40 genes with significant differences in mRNA expression were obtained(P-adjust＜0.05, |Fold Change|≥1.5). 26 genes were significantly upregulated, including IGFBP6 and COL15A1. The upregulated gene GO enrichment was enriched to humoral immune response, B-cell activation, and immunoglobulin receptor binding; KEGG enrichment was enriched to 56 signaling pathways, including PI3K-AKT and NF-κB, and F＜0.001. Fourteen genes were significantly downregulated, but the analysis of downregulated gene GO and KEGG enrichment was not statistically significant(F＞0.1)., Conclusions: Transgenic tomato anti-caries vaccine may reduce caries occurrence by upregulating the activation of PI3K-AKT signaling pathway mediated by IGFBP6 in SD rats.

As a non-viral transfection method, ultrasound and microbubble-induced sonoporation can achieve spatially targeted gene delivery with synergistic immunostimulatory effects. Here, we report for the first time the application of sonoporation for improving DNA vaccination performance. This study developed a new microbubble design with nanoscale DNA/PEI complexes loaded onto cationic microbubbles to attain significant increases in DNA-loading capacity (0.25 pg per microbubble) and in vitro transfection efficiency. Using live-cell imaging, we revealed the membrane perforation and cellular delivery characteristics of sonoporation. Using luciferase reporter gene for in vivo transfection, we showed that sonoporation increased the transfection efficiency by 40.9-fold when compared with intramuscular injection. Moreover, we comprehensively optimized the sonoporation protocol and further increased the transfection efficiency by 43.6-fold. Immunofluorescent staining results showed that sonoporation effectively activated the MHC-II + immune cells. Using a hepatitis B DNA vaccine, sonoporation induced significantly higher serum antibody levels when compared with intramuscular injection, and the antibodies sustained for 56 weeks. In addition, we recorded the longest reported expression period (400 days) of the sonoporation-delivered gene. Whole genome resequencing confirmed that the gene with stable expression existed in an extrachromosomal state without integration. Our results demonstrated the potential of sonoporation for efficient and safe DNA vaccination., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yaxin Hu reports financial support was provided by National Natural Science Foundation of China. Xin Chen reports financial support was provided by Shenzhen Basic Science Research., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Malaria affects ∼ ¼ billion people globally and requires the development of additional tools to aid in elimination efforts. The recently approved RTS,S/AS01 vaccine represents a positive step, however, the moderate efficacy necessitates the development of more efficacious vaccines. PfCSP is a key target antigen for pre-erythrocytic vaccines aimed at preventing Plasmodium falciparum malaria infections. Epitopes within the central repeat region and at the junction of the repeat and N-terminal domain are well documented as major protective B cell epitopes. On the other hand, a majority of antibodies against the epitopes in the C-terminal domain, have been shown to be non-protective against sporozoite challenge. The C-terminal domain, however, contains CD4 +  and CD8 +  T cell epitopes previously shown to be important for regulating immune responses. The present study was designed to further explore the immunomodulatory potential of the C-terminal domain using DNA vaccines encoding PfCSP with sequential C-terminal truncations following known T cell epitopes. Five DNA vaccines encoding different truncations of PfCSP within the C-terminal domain were administered via intramuscular route and in vivo electroporation for effective immunogenicity. Protection in mice was evaluated by challenge with transgenic P. berghei expressing PfCSP. In Balb/c mice, antibody responses and protective efficacy were both affected progressively with sequential deletion of C-terminal amino acid residues. Similar studies in C57Bl/6 mice revealed that immunizations with plasmids encoding truncated PfCSP showed partial protection from sporozoite challenge with no significant differences in antibody titers observed compared to full-length PfCSP DNA immunized mice. Further analysis revealed murine strain-specific differences in the recognition of specific epitopes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Recently, many efforts have been made to treat cancer using recombinant bacterial toxins and this strategy has been used in clinical trials of various cancers. Therapeutic DNA cancer vaccines are now considered as a promising strategy to activate the immune system against cancer. Cancer vaccines could induce specific and long-lasting immune responses against tumors. This study aimed to evaluate the antitumor potency of the SEB DNA vaccine as a new antitumor candidate against breast tumors in vivo. To determine the effect of the SEB construct on inhibiting tumor cell growth in vivo, the synthetic SEB gene, subsequent codon optimization, and embedding the cleavage sites were sub-cloned to an expression vector. Then, SEB construct, SEB, and PBS were injected into the mice. After being vaccinated, 4T1 cancer cells were injected subcutaneously into the right flank of mice. Then, the cytokine levels of IL-4 and IFN-γ were estimated by the ELISA method to evaluate the antitumor activity. The spleen lymphocyte proliferation, tumor size, and survival time were assessed. The concentration of IFN-γ in the SEB-Vac group showed a significant increase compared to other groups. The production of IL-4 in the group that received the DNA vaccine did not change significantly compared to the control group. The lymphocyte proliferation increased significantly in the mice group that received SEB construct than PBS control group (p < 0.001). While there was a meaningful decrease in tumor size (p < 0.001), a significant increase in tumor tissue necrosis (p < 0.01) and also in survival time of the animal model receiving the recombinant construct was observed. The designed SEB gene construct can be a new model vaccine for breast cancer because it effectively induces necrosis and produces specific immune responses. This structure does not hurt normal cells and is a safer treatment than chemotherapy and radiation therapy. Its slow and long-term release gently stimulates the immune system and cellular memory. It could be applied as a new model for inducing apoptosis and antitumor immunity to treat cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

A promising approach to the development of new means for preventing infection caused by tick-borne encephalitis virus can be DNA vaccines encoding polyepitope T-cell immunogens. A DNA vaccine pVAX-AG4-ub encoding an artificial polyepitope immunogen that includes cytotoxic and T-helper epitopes from the NS1, NS3, NS5, and E proteins of the tick-borne encephalitis virus has been obtained. The developed construct ensured the synthesis of the corresponding mRNAs in transfected eukaryotic cells. Immunization of mice with pVAX-AG4-ub induced the formation of a virus-specific T-cell response providing 50% protection from lethal infection with the virus., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease., Competing Interests: Both T.-C.W. and R.B.S.R. are co-founders of and have an equity ownership interest in Papivax LLC. Also, T.-C.W. and R.B.S.R. own Papivax Biotech Inc. stock options and are members of Papivax Biotech Inc.'s Scientific Advisory Board. Under a licensing agreement between Papivax Biotech Inc. and the Johns Hopkins University, the university and both T.-C.W. and R.B.S.R. are entitled to royalties on a technology described in this article. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret ( Mustela putorius furo )-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 10 5.0 TCID 50 . Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

Infectious Bursal Disease (IBD) is a common and contagious viral infection that significantly affects the poultry industry. This severely suppresses the immune system in chickens, thereby threating their health and well-being. Vaccination is the most effective strategy for preventing and controlling this infectious agent. The development of VP2-based DNA vaccines combined with biological adjuvants has recently received considerable attention due to their effectiveness in eliciting both humoral and cellular immune responses. In this study, we applied bioinformatics tools to design a fused bioadjuvant candidate vaccine from the full-length sequence of the VP2 protein of IBDV isolated in Iran using the antigenic epitope of chicken IL-2 (chiIL-2). Furthermore, to improve the antigenic epitope presentation and to maintain the three-dimensional structure of the chimeric gene construct, the P2A linker (L) was used to fuse the two fragments. Our in-silico analysis for the design of a candidate vaccine indicates that a continuous sequence of amino acid residues ranging from 105 to 129 in chiIL-2 is proposed as a B cell epitope by epitope prediction servers. The final 3D structure of the VP2-L-chiIL-2 105-129 was subjected to physicochemical property determination, molecular dynamic simulation, and antigenic site determination. The results of these analyses led to the development of a stable candidate vaccine that is non-allergenic and has the potential for antigenic surface display potential and adjuvant activity. Finally, it is necessary to investigate the immune response induced by our proposed vaccine in avian hosts. Notably, increasing the immunogenicity of DNA vaccines can be achieved by combining antigenic proteins with molecular adjuvants using the principle of rational vaccine design., Competing Interests: Declaration of competing interest All authors of this manuscript in order are Safoura Soleymani, Fatemeh Janati-fard, Mohammad Reza Housaindokht. Also, all authors of this paper agree with the content of the manuscript. None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. This paper (or closely related research) has not been published or accepted for publication, and, it is not under consideration in any other journal., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Crimean-Congo hemorrhagic fever (CCHF) is a World Health Organization prioritized disease because its broad distribution and severity of disease make it a global health threat. Despite advancements in preclinical vaccine development for CCHF virus (CCHFV), including multiple platforms targeting multiple antigens, a clear definition of the adaptive immune correlates of protection is lacking. Levels of neutralizing antibodies in vaccinated animal models do not necessarily correlate with protection, suggesting that cellular immunity, such as CD8 + T cells, might have an important role in protection in this model. Using a well-established IFN-I antibody blockade mouse model (IS) and a DNA-based vaccine encoding the CCHFV M-segment glycoprotein precursor, we investigated the role of humoral and T cell immunity in vaccine-mediated protection in mice genetically devoid of these immune compartments. We found that in the absence of the B-cell compartment (µMT knockout mice), protection provided by the vaccine was not reduced. In contrast, in the absence of CD8 + T cells (CD8 + knockout mice) the vaccine-mediated protection was significantly diminished. Importantly, humoral responses to the vaccine in CD8 + T-cell knockout mice were equivalent to wild-type mice. These findings indicated that CD8 + T-cell responses are necessary and sufficient to promote protection in mice vaccinated with the M-segment DNA vaccine. Identifying a crucial role of the cellular immunity to protect against CCHFV should help guide the development of CCHFV-targeting vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs&#95;0636/Rs&#95;0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs&#95;0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO-K1 cells. The results showed that Rs&#95;0636/Rs&#95;0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs&#95;0636/Rs&#95;0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Introduction: In humans, approximately 5% of all cancers are attributable to HPV infection. Prophylactic vaccines can inhibit viral migration and persistence. However, further studies are still required to develop such treatments. To achieve this goal, we designed a therapeutic HPV DNA vaccine encoding a construct of E6/E7/L1 and used NSP4 antigen as an adjuvant to assess the efficiency of this construct in generating antigen-specific antitumor immune responses., Materials and Methods: Sixty female C57BL/6 mice (6-8 weeks old) were purchased from the Institute Pasteur of Iran. Through a subcutaneous (s.c) injection of a suspension of 100 µl PBS containing 10 6 TC-1 cells/mouse in the back side, 30 of them became cancerous, while 30 of them were healthy control mice. To amplify E6/E7/L1-pcDNA3 and NSP4-pcDNA3, the competent cells of DH5α and to generate a tumor, TC-1 cell line was used. Mice were then immunized with the HPV DNA vaccine. Cell proliferation was assessed by MTT assay. Finally, cytokine responses (IL-4, IL-12, IFN- γ) were measured in the supernatant of mice spleen cells., Result: Mice receiving the NSP4/E6-E7-L1 vaccine had the highest stimulatory index compared to other groups, although it was not statistically significant. Interleukin 4/12 and IFN-γ production were significantly higher in E6-E7-L1 / NSP4 group and E6-E7-L1 group compared to other groups (P < 0.05). Among different groups, E6/E7/L1 + NSP4 group was able to slow down the tumor growth process, but it was not significant (p > 0.05). Among the aforementioned cytokines, IFN-γ and IL-12 are among the cytokines that stimulate the Th1 pathway and IL-4 cytokine stimulates the Th2 pathway and B lymphocytes., Conclusion: Our data revealed that the present vaccine can reduce tumor size, and cytokine measurement showed that it stimulates innate and acquired immune responses, thus it can be a therapeutic vaccine in the tumor-bearing mice model., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

The global demand for nucleic acid-based vaccines, including plasmid DNA (pDNA) and mRNA vaccines, needs efficient production platforms. However, conventional hosts for plasmid production have encountered challenges related to sequence integrity due to the presence of insertion sequences (ISs). In this study, we explored the potential of a genome-reduced Escherichia coli as a host for pDNA production. This strain had been constructed by removing approximately 23% of the genome which were unessential genes, including the genomic unstable elements. Moreover, the strain exhibits an elevated level of NADPH, a coenzyme known to increase plasmid production according to a mathematical model. We hypothesized that the combination of genome reduction and the abundance of NADPH would significantly enhance pDNA production capabilities. Remarkably, our results confirmed a three-fold increase in pDNA production compared to the widely employed DH5α strain. Furthermore, the genome-reduced strain exhibited heightened sensitivity to various antibiotics, bolstering its potential for large scale industrial pDNA production. These findings suggest the genome-reduced E. coli as an exciting candidate for revolutionizing the pDNA industry, offering unprecedented efficiency and productivity.

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., Competing Interests: Douglas G. McNeel has ownership interest, has received research support, and serves as consultant to Madison Vaccines, Inc. which has licensed intellectual property related to this content. IR has no relevant potential conflicts of interest., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b that showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 Mycobacterium tuberculosis (MTB) H 37 Rv-infected BALB/c mice were treated with phosphate-buffered saline, 10, 50, 100, and 200 μg ag85a/b DNA vaccine delivered by IM and EP three times at 2-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from three mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive polymerase chain reaction and the GEO database. After MTB infection, most of the upregulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine (such as Iapp, Scg2, Chga, Amy2a5), and most of the downregulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of the alveolar epithelial cell (such as Sftpc, Sftpd, Pdpn). Most of the abnormally upregulated or downregulated DE genes in the TB model group were recovered in the 100 and 200 μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway downregulated and the Rap1 signal pathway upregulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action targets and mechanisms of IM and EP are highly consistent. Tuberculosis infection causes rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB., (© 2023 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Chlamydia psittaci ( C. psittaci ), a zoonotic pathogen, poses a potential threat to public health security and the development of animal husbandry. Vaccine-based preventative measures for infectious diseases have a promising landscape. DNA vaccines, with many advantages, have become one of the dominant candidate strategies in preventing and controlling the chlamydial infection. Our previous study showed that CPSIT&#95;p7 protein is an effective candidate for a vaccine against C. psittaci . Thus, this study evaluated the protective immunity of pcDNA3.1(+)/CPSIT&#95;p7 against C. psittaci infection in BALB/c mice. We found that pcDNA3.1(+)/CPSIT&#95;p7 can induce strong humoral and cellular immune responses. The IFN-γ and IL-6 levels in the infected lungs of mice immunized with pcDNA3.1(+)/CPSIT&#95;p7 reduced substantially. In addition, the pcDNA3.1(+)/CPSIT&#95;p7 vaccine diminished pulmonary pathological lesions and reduced the C. psittaci load in the lungs of infected mice. It is worth noting that pcDNA3.1(+)/CPSIT&#95;p7 suppressed C. psittaci dissemination in BALB/c mice. In a word, these results demonstrate that the pcDNA3.1(+)/CPSIT&#95;p7 DNA vaccine has good immunogenicity and immunity protection effectiveness against C. psittaci infection in BALB/c mice, especially pulmonary infection, and provides essential practical experience and insights for the development of a DNA vaccine against chlamydial infection.

The coronavirus disease 2019 (COVID-19) pandemic has infected 673,010,496 patients and caused the death of 6,854,959 cases globally until today. Enormous efforts have been made to develop fundamentally different COVID-19 vaccine platforms. Nucleic acid-based vaccines consisting of mRNA and DNA vaccines (third-generation vaccines) have been promising in terms of rapid and convenient production and efficient provocation of immune responses against the COVID-19. Several DNA-based (ZyCoV-D, INO-4800, AG0302-COVID19, and GX-19N) and mRNA-based (BNT162b2, mRNA-1273, and ARCoV) approved vaccine platforms have been utilized for the COVID-19 prevention. mRNA vaccines are at the forefront of all platforms for COVID-19 prevention. However, these vaccines have lower stability, while DNA vaccines are needed with higher doses to stimulate the immune responses. Intracellular delivery of nucleic acid-based vaccines and their adverse events needs further research. Considering re-emergence of the COVID-19 variants of concern, vaccine reassessment and the development of polyvalent vaccines, or pan-coronavirus strategies, is essential for effective infection prevention., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hemorrhagic fever in humans and is mainly transmitted by ticks. There is no effective vaccine for Crimean-Congo hemorrhagic fever (CCHF) at present. We developed three DNA vaccines encoding CCHFV nucleocapsid protein (NP), glycoprotein N-terminal (Gn) and C-terminal (Gc) fused with lysosome-associated membrane protein 1 (LAMP1) and assessed their immunogenicity and protective efficacy in a human MHC (HLA-A11/DR1) transgenic mouse model. The mice that were vaccinated three times with pVAX-LAMP1-CCHFV-NP induced balanced Th1 and Th2 responses and could most effectively protect mice from CCHFV transcription and entry-competent virus-like particles (tecVLPs) infection. The mice vaccinated with pVAX-LAMP1-CCHFV-Gc mainly elicited specific anti-Gc and neutralizing antibodies and provided a certain protection from CCHFV tecVLPs infection, but the protective efficacy was less than that of pVAX-LAMP1-CCHFV-NP. The mice vaccinated with pVAX-LAMP1-CCHFV-Gn only elicited specific anti-Gn antibodies and could not provide sufficient protection from CCHFV tecVLPs infection. These results suggest that pVAX-LAMP1-CCHFV-NP would be a potential and powerful candidate vaccine for CCHFV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Zhang, Liu, Ye, Zhao, Zhang, Qiang, Zhang, Lei, Jiang, Cheng and Zhang.)

Toxoplasma gondii can infect a wide range of warm-blooded animals, causing a global toxoplasmosis zoonotic epidemic. Surface antigen 1 (SAG1) protein is expressed at the proliferative tachyzoite stage, whereas matrix antigen 1 (MAG1) is expressed at the bradyzoite and tachyzoite stages. These two proteins were found to perform protective roles in previous studies; however, their synergetic protective efficacy as a DNA vaccine against toxoplasmosis has not been clarified. In this study, we constructed recombinant pcDNA3.1( +)-TgMAG1 (pMAG1), pcDNA3.1( +)-TgSAG1 (pSAG1), and pcDNA3.1( +)-TgMAG1-TgSAG1 (pMAG1-SAG1) plasmids and administered them intramuscularly to immunize mice. The levels of anti-T. gondii IgG in serum and cytokines, such as Interleukin (IL)-4, IL-10, and Interferon (IFN)-γ, in splenocytes were measured using ELISA and the respective culture supernatants. Lethal doses of T. gondii (type I) RH strain tachyzoites were administered to immunized mice, and mortality was assessed. Conversely, mice infected with low doses of tachyzoites were monitored to determine their survival rates, and parasite burden analyses of the brains and livers were conducted. The bivalent TgMAG1 and TgSAG1 DNA vaccines exhibited excellent protective immunity against toxoplasmosis in mice, with higher serum IgG and splenocyte IFN-γ release levels, longer survival days, and reduced parasite burden in the brain and liver tissues (p < 0.05). These findings provide a new perspective for the development of T. gondii vaccines., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chronic hepatitis C can lead to cirrhosis and hepatocellular carcinoma. We studied the safety and immunogenicity of a novel therapeutic hepatitis C virus (HCV) genotype 1a/1b consensus DNA vaccine, INO-8000, encoding HCV NS3, NS4A, NS4B, and NS5A proteins alone or co-administered with DNA-encoding IL12 (INO-9012), a human cytokine that stimulates cellular immune function, in individuals with chronic hepatitis C. This was a phase I, multisite dose-escalation trial with an expansion cohort evaluating doses of 0, 0.3, 1.0, and 3.0 mg of INO-9012 (IL12 DNA) as an addition to 6.0 mg of (INO-8000; HCV DNA vaccine). Vaccines were administered by intramuscular injection followed by electroporation at study entry and at weeks 4, 12, and 24. HCV-specific CD4+ and CD8+ T-cell immune responses were measured by IFNγ ELISpot and flow cytometry-based assays. Transient, mild-to-moderate injection site reactions unrelated to IL12 DNA dose were common. Increases in HCV-specific IFNγ production occurred in 15/20 (75%) participants. Increases in the frequency of HCV-specific CD4+ and CD8+ T cells occurred at all dose levels, with the greatest increases seen at 1.0 mg of INO-9012. HCV-specific CD8+ and CD4+ T-cell activities increased in 16/18 (89%) and 14/17 (82%) participants with available data, respectively. The vaccine regimen was safe and induced HCV-specific CD4+ and CD8+ cellular immune responses of modest magnitude in most HCV-infected participants. The addition of 1.0 mg of IL12 DNA provided the best enhancement of immune responses. The vaccine regimen had little effect on controlling HCV viremia., Prevention Relevance: The administration of IL12 DNA along with a hepatitis C viral antigen DNA vaccine enhanced the HCV-specific immune responses induced by the vaccine in individuals with chronic hepatitis C, an important cause of hepatocellular carcinoma. IL12 could be an effective adjuvant in vaccines targeting HCV and other oncogenic viruses., (©2022 American Association for Cancer Research.)

SARS-CoV-2 has caused the COVID-19 pandemic, with over 673 million infections and 6.85 million deaths globally. Novel mRNA and viral-vectored vaccines were developed and licensed for global immunizations under emergency approval. They have demonstrated good safety and high protective efficacy against the SARS-CoV-2 Wuhan strain. However, the emergence of highly infectious and transmissible variants of concern (VOCs) such as Omicron was associated with considerable reductions in the protective efficacy of the current vaccines. The development of next-generation vaccines that could confer broad protection against both the SARS-CoV-2 Wuhan strain and VOCs is urgently needed. A bivalent mRNA vaccine encoding the Spike proteins of both the SARS-CoV-2 Wuhan strain and the Omicron variant has been constructed and approved by the US FDA. However, mRNA vaccines are associated with instability and require an extremely low temperature (-80 °C) for storage and transportation. They also require complex synthesis and multiple chromatographic purifications. Peptide-based next-generation vaccines could be developed by relying on in silico predictions to identify peptides specifying highly conserved B, CD4 + and CD8 + T cell epitopes to elicit broad and long-lasting immune protection. These epitopes were validated in animal models and in early phase clinical trials to demonstrate immunogenicity and safety. Next-generation peptide vaccine formulations could be developed to incorporate only naked peptides, but they are costly to synthesize and production would generate extensive chemical waste. Continual production of recombinant peptides specifying immunogenic B and T cell epitopes could be achieved in hosts such as E. coli or yeast. However, recombinant protein/peptide vaccines require purification before administration. The DNA vaccine might serve as the most effective next-generation vaccine for low-income countries, since it does not require an extremely low temperature for storage or need extensive chromatographic purification. The construction of recombinant plasmids carrying genes specifying highly conserved B and T cell epitopes meant that vaccine candidates representing highly conserved antigenic regions could be rapidly developed. Poor immunogenicity of DNA vaccines could be overcome by the incorporation of chemical or molecular adjuvants and the development of nanoparticles for effective delivery.

Introduction: A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants. The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid., Materials and Methods: An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated., Results: Mice immunization with rNS5B in a mixture with dnC60 at doses of 250 g/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 720 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60., Conclusions: Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus's intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50 % survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). The AcHERV-COVID19D induced higher neutralizing antibodies against the Delta variant than the prototype or Omicron variant. On the other hand, cellular immunity was similarly high for all three SARS-CoV2 viruses. Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100 % survival upon challenge with Delta and Omicron variants and 71.4 % survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. It has been shown that the severity of symptoms depends on the functioning of the host immune system. Although T. gondii infection typically does not lead to severe disease in healthy people and after infection, it induces a stable immunity, but it can contribute to severe and even lethal Toxoplasmosis in immunocompromised individuals (AIDS, bone marrow transplant and neoplasia). The antigens that have been proposed to be used in vaccine candidate in various studies include surface antigens and secretory excretions that have been synthesized and evaluated in different studies. In some studies, secretory antigens play an important role in stimulating the host immune response. Various antigens such as SAG, GRA, ROP, ROM, and MAG have been from different strains of T. gondii have been synthesized and their protective effects have been evaluated in animal models in different vaccine platforms including recombinant antigens, nanoparticles, and DNA vaccine. Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and Google Scholar were searched for articles published up to 2020.The current review article focuses on recent studies on the use and usefulness of recombinant antigens, nanoparticles, and DNA vaccines., (© 2022. The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland.)

A novel strategy that has the potential to solve the drawbacks of the present conventional vaccines is the development of DNA vaccines. DNA vaccines offer a versatile and adaptable platform for treating a wide variety of diseases, as immunization targets may be easily adjusted by altering the gene sequences encoded in the plasmid DNA delivered. Due to their ability to elicit both humoral and cellular immune responses, their stability, and the ease with which they may be produced, plasmid DNA vaccines are quickly becoming the vaccine of choice, they are frequently safer than conventional vaccinations. Despite the highly encouraging outcomes of ongoing clinical trials, these vaccines' immunogenicity is compromised by a few factors. The use of various vaccine delivery techniques, the use of various polymer-based carriers, and the use of adjuvants are some of the several approaches that might be examined to better the immunogenicity of DNA vaccines made from plasmids. These advancements taken together might allow plasmid DNA vaccines to be successfully used in clinical settings., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

DNA vaccine is a creative and promising method for cancer treatment. As part of cancer immunotherapy, one or more antigen-specific immune responses are triggered or strengthened using DNA vaccines for cancer immunotherapy, which convey one or more genes encoded by tumour antigens to the immune system. Vaccine efficacy may be greatly increased by new delivery routes, the incorporation of molecular active ingredients and immunomodulatory signals, the modification of prime-boost protocols, or the inhibition of immunological checkpoints. It is possible to overcome the self-tolerance of many tumour antigens by using a mix of adaptive immune system and vaccine design strategies to generate protective adaptive immune responses. Both preventative and therapeutic vaccinations are being developed using this technology in several clinical investigations on DNA cancer immunotherapy. This study examines the immunogenicity and efficacy of DNA vaccines for immunotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)