TRP-2 / gp100 DNA vaccine and PD-1 checkpoint blockade combination for the treatment of intracranial tumors.

Intracranial tumors present a significant therapeutic challenge due to their physiological location. Immunotherapy presents an attractive method for targeting these intracranial tumors due to relatively low toxicity and tumor specificity. Here we show that SCIB1, a TRP-2 and gp100 directed ImmunoBody® DNA vaccine, generates a strong TRP-2 specific immune response, as demonstrated by the high number of TRP2-specific IFNγ spots produced and the detection of a significant number of pentamer positive T cells in the spleen of vaccinated mice. Furthermore, vaccine-induced T cells were able to recognize and kill B16HHDII/DR1 cells after a short in vitro culture. Having found that glioblastoma multiforme (GBM) expresses significant levels of PD-L1 and IDO1, with PD-L1 correlating with poorer survival in patients with the mesenchymal subtype of GBM, we decided to combine SCIB1 ImmunoBody® with PD-1 immune checkpoint blockade to treat mice harboring intracranial tumors expressing TRP-2 and gp100. Time-to-death was significantly prolonged, and this correlated with increased CD4+ and CD8+ T cell infiltration in the tissue microenvironment (TME). However, in addition to PD-L1 and IDO, the GBM TME was found to contain a significant number of immunoregulatory T (Treg) cell-associated transcripts, and the presence of such cells is likely to significantly affect clinical outcome unless also tackled.

Nanoparticle-Mediated Mucosal Vaccination: Harnessing Nucleic Acids for Immune Enhancement.

Recent advancements in in vitro transcribed mRNA (IVT-mRNA) vaccine manufacturing have attracted considerable interest as advanced methods for combating viral infections. The respiratory mucosa is a primary target for pathogen attack, but traditional intramuscular vaccines are not effective in generating protective ion mucosal surfaces. Mucosal immunization can induce both systemic and mucosal immunity by effectively eliminating microorganisms before their growth and development. However, there are several biological and physical obstacles to the administration of genetic payloads, such as IVT-mRNA and DNA, to the pulmonary and nasal mucosa. Nucleic acid vaccine nanocarriers should effectively protect and load genetic payloads to overcome barriers i.e., biological and physical, at the mucosal sites. This may aid in the transfection of specific antigens, epithelial cells, and incorporation of adjuvants. In this review, we address strategies for delivering genetic payloads, such as nucleic acid vaccines, that have been studied in the past and their potential applications.

Immunogenicity study of a Novel DNA-Based HCV vaccine candidate.

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.

Combined Proteomic and Metabolomic Analysis Reveals Comprehensive Regulation of Somatostatin DNA Vaccine in Goats.

Somatostatin (SS) plays crucial regulatory roles in animal growth and reproduction by affecting the synthesis and secretion of growth hormone (GH). However, the mechanism by which SS regulates growth and development in goats is still unclear. In order to investigate the regulatory networks of the hypothalamus and pituitary in goats affected by SS DNA vaccines, in this study, we used a previously established oral attenuated Salmonella typhimurium SS DNA vaccine, X9241 (ptCS/2SS-asd), to treat wethers. We analyzed the protein changes in hypothalamic and pituitary tissues using a TMT-based proteomics approach. Additionally, we examined the metabolic profiles of the serum of control and immunized wethers through untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS). Key signaling pathways were identified based on differentially expressed metabolites (DEMs) and differentially expressed proteins (DEPs). Furthermore, the effect of critical DEPs on signaling pathways was confirmed through Western blotting (WB) experiments, which elucidated the mechanism of active SS immunization in wethers. A proteomics analysis revealed that the expression of 58 proteins in the hypothalamus and 124 in the pituitary gland was significantly altered following SS vaccine treatment (fold change > 1.2 or < 0.83, p < 0.05). In the hypothalamus, many DEPs were associated with gene ontology (GO) terms related to neuronal signaling. In contrast, most DEPs were associated with metabolic pathways. In the pituitary gland, the DEPs were largely related to immune and nutrient metabolism functions, with significant enrichment in KEGG pathways, particularly those involving the metabolic pathway, sphingolipid signaling, and the cGMP-PKG signaling pathway. A metabolomic analysis further showed that active SS immunization in wethers led to significant alterations in seven serum metabolites. Notably, the sphingolipid signaling pathway, secondary bile acid synthesis, sphingolipid metabolism, and lysine synthesis were significantly disrupted. SS vaccines induced marked changes in hypothalamic-pituitary proteins in wethers, facilitating alterations in their growth processes. This study not only provides insights into the mechanism of the SS gene in regulating GH secretion in wethers but also establishes a basis for hormone immunoregulation technology to enhance livestock production performance.

Nucleic acid cancer vaccines targeting tumor related angiogenesis. Could mRNA vaccines constitute a game changer?

Tumor related angiogenesis is an attractive target in cancer therapeutic research due to its crucial role in tumor growth, invasion, and metastasis. Different agents were developed aiming to inhibit this process; however they had limited success. Cancer vaccines could be a promising tool in anti-cancer/anti-angiogenic therapy. Cancer vaccines aim to initiate an immune response against cancer cells upon presentation of tumor antigens which hopefully will result in the eradication of disease and prevention of its recurrence by inducing an efficient and long-lasting immune response. Different vaccine constructs have been developed to achieve this and they could include either protein-based or nucleic acid-based vaccines. Nucleic acid vaccines are simple and relatively easy to produce, with high efficiency and safety, thus prompting a high interest in the field. Different DNA vaccines have been developed to target crucial regulators of tumor angiogenesis. Most of them were successful in pre-clinical studies, mostly when used in combination with other therapeutics, but had limited success in the clinic. Apparently, different tumor evasion mechanisms and reduced immunogenicity still limit the potential of these vaccines and there is plenty of room for improvement. Nowadays, mRNA cancer vaccines are making remarkable progress due to improvements in the manufacturing technology and represent a powerful potential alternative. Apart from their efficiency, mRNA vaccines are simple and cheap to produce, can encompass multiple targets simultaneously, and can be quickly transferred from bench to bedside. mRNA vaccines have already accomplished amazing results in cancer clinical trials, thus ensuring a bright future in the field, although no anti-angiogenic mRNA vaccines have been described yet. This review aims to describe recent advances in anti-angiogenic DNA vaccine therapy and to provide perspectives for use of revolutionary approaches such are mRNA vaccines for anti-angiogenic treatments.

Evaluation of protective immunity induced by a DNA vaccine encoding SAG2 and SRS2 against Toxoplasma gondii infection in mice.

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.

Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

Peptide-TLR7/8a-Coordinated DNA Vaccines Elicit Enhanced Immune Responses against Infectious Diseases.

DNA vaccines represent an innovative approach for the immunization of diverse diseases. However, their clinical trial outcomes are constrained by suboptimal transfection efficiency and immunogenicity. In this work, we present a universal methodology involving the codelivery of Toll-like receptor 7/8 agonists (TLR7/8a) and antigen gene using TLR7/8a-conjugated peptide-coated poly(ß-amino ester) (PBAE) nanoparticles (NPs) to augment delivery efficiency and immune response. Peptide-TLR7/8a-coated PBAE NPs exhibit advantageous biophysical attributes, encompassing diminutive particle dimensions, nearly neutral ζ potential, and stability in the physiological environment. This synergistic approach not only ameliorates the stability of plasmid DNA (pDNA) and gene delivery efficacy but also facilitates subsequent antigen production. Furthermore, under optimal formulation conditions, the TLR7/8a-conjugated peptide coated PBAE NPs exhibit a potent capacity to induce robust immune responses. Collectively, this nanoparticulate gene delivery system demonstrates heightened transfection efficacy, stability, biodegradability, immunostimulatory effect, and low toxicity, making it a promising platform for the clinical advancement of DNA vaccines.

Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production.

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.

PI3Kγ inhibition combined with DNA vaccination unleashes a B-cell-dependent antitumor immunity that hampers pancreatic cancer.

Phosphoinositide-3-kinase γ (PI3Kγ) plays a critical role in pancreatic ductal adenocarcinoma (PDA) by driving the recruitment of myeloid-derived suppressor cells (MDSC) into tumor tissues, leading to tumor growth and metastasis. MDSC also impair the efficacy of immunotherapy. In this study we verify the hypothesis that MDSC targeting, via PI3Kγ inhibition, synergizes with α-enolase (ENO1) DNA vaccination in counteracting tumor growth.Mice that received ENO1 vaccination followed by PI3Kγ inhibition had significantly smaller tumors compared to those treated with ENO1 alone or the control group, and correlated with i) increased circulating anti-ENO1 specific IgG and IFNγ secretion by T cells, ii) increased tumor infiltration of CD8+ T cells and M1-like macrophages, as well as up-modulation of T cell activation and M1-like related transcripts, iii) decreased infiltration of Treg FoxP3+ T cells, endothelial cells and pericytes, and down-modulation of the stromal compartment and T cell exhaustion gene transcription, iv) reduction of mature and neo-formed vessels, v) increased follicular helper T cell activation and vi) increased "antigen spreading", as many other tumor-associated antigens were recognized by IgG2c "cytotoxic" antibodies. PDA mouse models genetically devoid of PI3Kγ showed an increased survival and a pattern of transcripts in the tumor area similar to that of pharmacologically-inhibited PI3Kγ-proficient mice. Notably, tumor reduction was abrogated in ENO1 + PI3Kγ inhibition-treated mice in which B cells were depleted.These data highlight a novel role of PI3Kγ in B cell-dependent immunity, suggesting that PI3Kγ depletion strengthens the anti-tumor response elicited by the ENO1 DNA vaccine.

Proteomic analysis of plasma in healthy adults receiving recombinant vaccinia virus provides novel insights into HIV-1 neutralizing antibodies.

Human immunodeficiency virus (HIV) infection is still a global public health issue, and the development of an effective prophylactic vaccine inducing potent neutralizing antibodies remains a significant challenge. This study aims to explore the inflammation-related proteins associated with the neutralizing antibodies induced by the DNA/rTV vaccine. In this study, we employed the Olink chip to analyze the inflammation-related proteins in plasma in healthy individuals receiving HIV candidate vaccine (DNA priming and recombinant vaccinia virus rTV boosting) and compared the differences between neutralizing antibody-positive (nab + ) and -negative(nab-) groups. We identified 25 differentially expressed factors and conducted enrichment and correlation analysis on them. Our results revealed that significant expression differences in artemin (ARTN) and C-C motif chemokine ligand 23 (CCL23) between nab+ and -nab- groups. Notably, the expression of CCL23 was negatively corelated to the ID50 of neutralizing antibodies and the intensity of the CD4+ T cell responses. This study enriches our understanding of the immune picture induced by the DNA/rTV vaccine, and provides insights for future HIV vaccine development.

Module-combinatorial design and screening of multifunctional polymers based on polyaspartic acid for DNA delivery.

It is crucial to develop non-viral gene vectors that can efficiently and safely transfect plasmid DNA into cells. Low transfection efficiency and high cytotoxicity of cationic polymers hinder their application as gene carriers. Modification of cationic polymers has emerged as an attractive strategy for efficient and safe nucleic acids delivery. In this study, a simple and rapid method is developed to synthesize a series of multifunctional polymers by utilizing biodegradable polyaspartic acid as the backbone and modifying it with three modules. This one-component polymer possesses capabilities for nucleic acid condensation, cellular uptake, and endosomal escape. Polymers containing imidazole, triazole, or pyridine group exhibited promising transfection activity. Substituted with dodecylamine or 2-hexyldecan-1-amine enhance cellular uptake and subsequent transfection. Furthermore, the influence of ionizable amine side chains on gene delivery is investigated. Two optimal polymers, combined with the avian encephalomyelitis virus (AEV) plasmid vaccine, induced robust specific antibody responses and cellular immune responses in mice and chickens. Through module-combination design and screening of polyaspartamide polymers, this study presents a paradigm for the development of gene delivery vectors.

Safety and Immunogenicity of a DNA Vaccine With Subtype C gp120 Protein Adjuvanted With MF59 or AS01B: A Phase 1/2a HIV-1 Vaccine Trial.

BACKGROUND: An effective vaccine is required to end the HIV pandemic. We evaluated the safety and immunogenicity of a DNA (DNA-HIV-PT123) vaccine with low- or high-dose bivalent (TV1.C and 1086.C glycoprotein 120) subtype C envelope protein combinations, adjuvanted with MF59 or AS01B. METHODS: HIV Vaccine Trials Network (HVTN)108 was a randomized, placebo-controlled, double-blind, phase 1/2a trial conducted in the United States and South Africa. HIV-negative adults were randomly assigned to 1 of 7 intervention arms or placebo to assess DNA prime with DNA/protein/adjuvant boosts, DNA/protein/adjuvant co-administration, and low-dose protein/adjuvant regimens. HVTN111 trial participants who received an identical regimen were also included. Outcomes included safety and immunogenicity 2 weeks and 6 months after final vaccination. RESULTS: From June 2016 to July 2018, 400 participants were enrolled (N = 334 HVTN108, N = 66 HVTN111); 370 received vaccine and 30 received placebo. There were 48 grade 3 and 3 grade 4 reactogenicity events among 39/400 (9.8%) participants, and 32 mild/moderate-related adverse events in 23/400 (5.8%) participants. All intervention groups demonstrated high IgG response rates (>89%) and high magnitudes to HIV-1 Env gp120 and gp140 proteins; response rates for AS01B-adjuvanted groups approached 100%. V1V2 IgG magnitude, Fc-mediated functions, IgG3 Env response rates, and CD4+ T-cell response magnitudes and rates were higher in the AS01B-adjuvanted groups. The AS01B-adjuvanted low-dose protein elicited greater IgG responses than the higher protein dose. CONCLUSIONS: The vaccine regimens were generally well tolerated. Co-administration of DNA with AS01B-adjuvanted bivalent Env gp120 elicited the strongest humoral responses; AS01B-adjuvanted regimens elicited stronger CD4+ T-cell responses, justifying further evaluation.ClinicalTrials.gov registration: NCT02915016, registered 26 September 2016.

SWIFT clustering analysis of intracellular cytokine staining flow cytometry data of the HVTN 105 vaccine trial reveals high frequencies of HIV-specific CD4+ T cell responses and associations with humoral responses.

Introduction: The HVTN 105 vaccine clinical trial tested four combinations of two immunogens - the DNA vaccine DNA-HIV-PT123, and the protein vaccine AIDSVAX B/E. All combinations induced substantial antibody and CD4+ T cell responses in many participants. We have now re-examined the intracellular cytokine staining flow cytometry data using the high-resolution SWIFT clustering algorithm, which is very effective for enumerating rare populations such as antigen-responsive T cells, and also determined correlations between the antibody and T cell responses. Methods: Flow cytometry samples across all the analysis batches were registered using the swiftReg registration tool, which reduces batch variation without compromising biological variation. Registered data were clustered using the SWIFT algorithm, and cluster template competition was used to identify clusters of antigen-responsive T cells and to separate these from constitutive cytokine producing cell clusters. Results: Registration strongly reduced batch variation among batches analyzed across several months. This in-depth clustering analysis identified a greater proportion of responders than the original analysis. A subset of antigen-responsive clusters producing IL-21 was identified. The cytokine patterns in each vaccine group were related to the type of vaccine - protein antigens tended to induce more cells producing IL-2 but not IFN-γ, whereas DNA vaccines tended to induce more IL-2+ IFN-γ+ CD4 T cells. Several significant correlations were identified between specific antibody responses and antigen-responsive T cell clusters. The best correlations were not necessarily observed with the strongest antibody or T cell responses. Conclusion: In the complex HVTN105 dataset, alternative analysis methods increased sensitivity of the detection of antigen-specific T cells; increased the number of identified vaccine responders; identified a small IL-21-producing T cell population; and demonstrated significant correlations between specific T cell populations and serum antibody responses. Multiple analysis strategies may be valuable for extracting the most information from large, complex studies.

Sequence of androgen receptor-targeted vaccination with androgen deprivation therapy affects anti-prostate tumor efficacy.

RATIONALE: Androgen deprivation therapy (ADT) is the primary treatment for recurrent and metastatic prostate cancer. In addition to direct antitumor effects, ADT has immunomodulatory effects such as promoting T-cell infiltration and enhancing antigen processing/presentation. Previous studies in our laboratory have demonstrated that ADT also leads to increased expression of the androgen receptor (AR) and increased recognition of prostate tumor cells by AR-specific CD8+T cells. We have also demonstrated that ADT combined with a DNA vaccine encoding the AR significantly slowed tumor growth and improved the survival of prostate tumor-bearing mice. The current study aimed to investigate the impact of the timing and sequencing of ADT with vaccination on the tumor immune microenvironment in murine prostate cancer models to further increase the antitumor efficacy of vaccines. METHODS: Male FVB mice implanted with Myc-CaP tumor cells, or male C57BL/6 mice implanted with TRAMP-C1 prostate tumor cells, were treated with a DNA vaccine encoding AR (pTVG-AR) and ADT. The sequence of administration was evaluated for its effect on tumor growth, and tumor-infiltrating immune populations were characterized. RESULTS: Vaccination prior to ADT (pTVG-AR → ADT) significantly enhanced antitumor responses and survival. This was associated with increased tumor infiltration by CD4+ and CD8+ T cells, including AR-specific CD8+T cells. Depletion of CD8+T cells prior to ADT significantly worsened overall survival. Following ADT treatment, however, Gr1+ myeloid-derived suppressor cells (MDSCs) increased, and this was associated with fewer infiltrating T cells and reduced tumor growth. Inhibiting Gr1+MDSCs recruitment, either by using a CXCR2 antagonist or by cycling androgen deprivation with testosterone replacement, improved antitumor responses and overall survival. CONCLUSION: Vaccination prior to ADT significantly improved antitumor responses, mediated in part by increased infiltration of CD8+T cells following ADT. Targeting MDSC recruitment following ADT further enhanced antitumor responses. These findings suggest logical directions for future clinical trials to improve the efficacy of prostate cancer vaccines.

Enhanced effectiveness in preventing Nocardia seriolae infection utilizing heterologous prime-boost approach in orange-spotted grouper Epinephelus coioides.

Persistent nocardiosis has prompted exploration of the effectiveness of heterologous approaches to prevent severe infections. We have previously reported the efficacy of a nucleic acid vaccine in protecting groupers from highly virulent Nocardia seriolae infections. Ongoing research has involved the supplementation of recombinant cholesterol oxidase (rCho) proteins through immunization with a DNA vaccine to enhance the protective capacity of orange-spotted groupers. Recombinant rCho protein exhibited a maturity and biological structure comparable to that expressed in N. seriolae, as confirmed by Western blot immunodetection assays. The immune responses observed in vaccinated groupers were significantly higher than those observed in single-type homologous vaccinations, DNA or recombinant proteins alone (pcD:Cho and rCho/rCho), especially cell-mediated immune and mucosal immune responses. Moreover, the reduction in N. seriolae occurrence in internal organs, such as the head, kidney, and spleen, was consistent with the vaccine's efficacy, which increased from approximately 71.4 % to an undetermined higher percentage through heterologous vaccination strategies of 85.7 %. This study underscores the potential of Cho as a novel vaccine candidate and a heterologous approach for combating chronic infections such as nocardiosis.

Construction of Toxoplasma gondii SRS29C nucleic acid vaccine and comparative immunoprotective study of an SRS29C and SAG1 combination.

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.

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

A comprehensive comparison of DNA and RNA vaccines.

Nucleic acid technology has revolutionized vaccine development, enabling rapid design and production of RNA and DNA vaccines for prevention and treatment of diseases. The successful deployment of mRNA and plasmid DNA vaccines against COVID-19 has further validated the technology. At present, mRNA platform is prevailing due to its higher efficacy, while DNA platform is undergoing rapid evolution because it possesses unique advantages that can potentially overcome the problems associated with the mRNA platform. To help understand the recent performances of the two vaccine platforms and recognize their clinical potentials in the future, this review compares the advantages and drawbacks of mRNA and DNA vaccines that are currently known in the literature, in terms of development timeline, financial cost, ease of distribution, efficacy, safety, and regulatory approval of products. Additionally, the review discusses the ongoing clinical trials, strategies for improvement, and alternative designs of RNA and DNA platforms for vaccination.

DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice.

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.