REVIEW OF DNA VACCINE APPROACHES AGAINST THE PARASITE TOXOPLASMA GONDII.

Toxoplasma gondii is an apicomplexan parasite that affects both humans and livestock. Transmitted to humans through ingestion, it is the second-leading cause of foodborne illness-related death. Currently, there exists no approved vaccine for humans or most livestock against the parasite. DNA vaccines, a type of subunit vaccine which uses segments of the pathogen's DNA to generate immunity, have shown varying degrees of experimental efficacy against infection caused by the parasite. This review compiles DNA vaccine efforts against Toxoplasma gondii, segmenting the analysis by parasite antigen, as well as a review of concomitant adjuvant usage. No single antigenic group was consistently more effective within in vivo trials relative to others.

DNA vaccination induced protective immunity against SARS CoV-2 infection in hamsterss.

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19.

Optimization of Zika DNA vaccine by delivery systems.

In our previous study, we designed and evaluated the efficacy of six DNA vaccine candidates based on the E protein of Zika virus (ZIKV). To optimize the DNA vaccine, we inoculated C57BL/6 and IFNAR1- mice with the vaccine candidate expressing tandem repeated ZIKV envelope domain III (ED III × 3) doses; 50 µg by intramuscular (IM), jet injection (JET), or electroporation (EP) routes. Results showed that vaccination by all routes induced humoral and cellular immunity. Among them, EP induced robust ZIKV E specific-total IgG and neutralizing antibodies, as well as T cell responses. Additionally, EP showed superior protective efficacy against the ZIKV Brazil strain compared to the IM and JET routes. Finally, in the dose optimization test of EP route, cellular immunity of 50 µg was induced a significant level than other dose groups. These results showed that the EP delivery system enhanced the potential immunogenicity and protective efficacy of DNA vaccines.

Epitope Analysis and Efficacy Evaluation of Phosphatase 2C (PP2C) DNA Vaccine Against Toxoplasma gondii Infection.

Toxoplasma gondii infects almost all warm-blooded animals and negatively affects the health of a wide range of these animals, including humans. Protein phosphatase 2C (PP2C) is a T. gondii protein secreted by rhoptry organelles during host cell invasion. However, very little is known about whether this protein can induce protective immunity against T. gondii. In this study, bioinformatics analysis of PP2C revealed some useful information in the context of anti-toxoplasmosis treatments and vaccine research. In addition, the PP2C gene was amplified, and a eukaryotic expression vector (pEGFP-PP2C) was successfully constructed to express PP2C. Finally, the constructed pEGFP-PP2C was injected into mice to evaluate whether it could induce immunoprotection. Compared with the control groups, we found that immunizations with the pEGFP-PP2C plasmid could elicit specific IgG antibodies and cytokines against T. gondii infection. The survival of mice immunized with the pEGFP-PP2C plasmid was significantly prolonged compared with that of the control group mice. Based on the ability of pEGFP-PP2C to induce specific immune responses against T. gondii, we propose that PP2C merits consideration as a potential vaccine candidate against toxoplasmosis.

The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (RNA and DNA) vaccines.

Nucleic acid (DNA and RNA) vaccines are among the most advanced vaccines for COVID-19 under development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of nucleic acid vaccines. This will facilitate the assessment by key stakeholders of potential safety issues and understanding of overall benefit-risk. The structured assessment provided by the template can also help improve communication and public acceptance of licensed nucleic acid vaccines.

Comparison of protective efficacy between two DNA vaccines encoding DnaK and GroEL against fish nocardiosis.

Fish nocardiosis is a chronic granulomatous bacterial disease mainly caused by three pathogenic bacteria, including Nocardia seriolae, N. asteroids and N. salmonicida. Molecular chaperone DnaK and GroEL were identified to be the common antigens of the three pathogenic Nocardia species in our previous studies. To evaluate the immune protective effect of two DNA vaccines encoding DnaK or GroEL against fish nocardiosis, hybrid snakehead were vaccinated and the immune responses induced by these two vaccines were comparatively analyzed. The results suggested it needed at least 7 d to transport DnaK or GroEL gene from injected muscle to head kidney, spleen and liver and stimulate host's immune system for later protection after immunization by DNA vaccines. Additionally, non-specific immunity parameters (serum lysozyme (LYZ), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP) and superoxide dismutase (SOD) activities), specific antibody (IgM) production and immune-related genes (MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα) were used to evaluate the immune responses induced in vaccinated hybrid snakehead. It proved that all the above-mentioned immune activities were significantly enhanced after immunization with these two DNA vaccines. The protective efficacy of pcDNA-DnaK and pcDNA-GroEL DNA vaccines, in terms of relative percentage survival (RPS), were 53.01% and 80.71% respectively. It demonstrated that these two DNA vaccines could increase the survival rate of hybrid snakehead against fish nocardiosis, albeit with variations in immunoprotective effects. Taken together, these results indicated that both pcDNA-DnaK and pcDNA-GroEL DNA vaccines could boost the innate, humoral and cellular immune response in hybrid snakehead and show highly protective efficacy against fish nocardiosis, suggesting that DnaK and GroEL were promising vaccine candidates. These findings will promote the development of DNA vaccines against fish nocardiosis in aquaculture.

Immuno-efficacy of DNA vaccines encoding PLP1 and ROP18 against experimental Toxoplasma gondii infection in mice.

We constructed a new plasmid pIRESneo/ROP18/PLP1 that was injected intramuscularly into Kunming mice to evaluate its immune efficacy. The immunized mice exhibited significantly increased serum IgG2a levels, lymphocyte counts and Th1-type cytokine (IL-2, IL-12 and IFN-γ) levels. Moreover, the immunized mice exhibited longer survival times (44.7 ± 2.1 days for ROP18/PLP1 and 47.2 ± 2.9 days for ROP18/PLP1 + IL-18) and lower brain cyst burden (68.9% for ROP18/PLP1 and 72.4% for ROP18/PLP1 + IL-18) than control mice after T. gondii challenge. Our results demonstrate that the multiple-gene DNA vaccine including both ROP18 and PLP1 elicits greater protection against T. gondii challenge and stronger immunogenicity than single-gene vaccines.

DNA Vaccines: Regulatory Considerations and Safety Aspects.

DNA vaccines have great potential as preventive or therapeutic vaccines against viral, bacterial, or parasitic diseases as well as cancer, and may also be used as gene therapy products. Although many human and veterinary DNA vaccines have been investigated in laboratory trials, only four of these have been approved for commercial use. In this paper an overview of the regulatory requirements for the development of DNA vaccines is given. The regulatory process in EU and USA is described. A discussion concerning the relevance of national regulations on gene technology is included. In addition the main safety concerns associated with DNA vaccines, relating to unwanted side effects in the vaccinated mammal or fish, are presented. Finally, the need for greater openness regarding the assessment information is discussed.

Efficacy of a potential DNA vaccine encoding Cryptosporidium baileyi rhomboid protein against homologous challenge in chickens.

The parasite Cryptosporidium baileyi can infect the larynx, trachea, bursa and cloaca of poultry, causing high mortality during severe infection and leading to substantial economic losses of the poultry industry. The rhomboid protein is very important in Cryptosporidium infection. In this study, a nucleic acid based vaccine candidate pEGFP-CbROM was constructed. After orally challenging with C. baileyi oocysts, the corresponding immune responses induced were analyzed and the immunoprotective effect evaluated in chickens. Obtained results revealed that this nucleic acid based vaccine could induce antibody responses and peripheral blood T lymphocytes proliferation significantly (P<0.05), while the peripheral blood B lymphocyte proliferation increased significantly (P<0.05) only at a high dose of 100µg of pEGFP-CbROM, compared with the PBS control group. After C. baileyi infection, the duration of oocysts shedding was shortened by 2days in the 100µg pEGFP-CbROM group, and the rate of reduction could reach to around 71.3%. While no significant difference in body weight gain was observed among the immunized groups (P>0.05), the differences between the immunized and the non-immunized groups were found to be significant (P<0.05). Our data provides a useful basis for further work in cryptosporidiosis prevention and treatment.

Protective efficacy of pVAX-RON5p against acute and chronic infections of Toxoplasma gondii in BALB/c mice.

Toxoplasma gondii can infect all the warm-blooded animals and humans and causes serious diseases especially in immuno-compromised patients and pregnant women. Rhoptry neck proteins (RONs) play an important role in the formation of moving junction, which mediates the invasion of this parasite. A recombinant plasmid pVAX-RON5p, which can express part of RON5 protein in the eukaryocyte, was generated and used to immune BALB/c mice for evaluating the protective efficacy against the acute and chronic infections of T. gondii. Both humoral and cellular immune responses were evoked in mice by pVAX-RON5p immunization, and a slightly prolonged survival period was detected in the immunized group (7.6 ± 3.31 days) compared to the blank control (4.9 ± 0.32 days) after acute T. gondii infection (P < 0.05). For chronic infection of T. gondii, the number of cysts in the brain of pVAX-RON5p-immunized mice decreased 25.8% compared to blank control (P < 0.05). Our data suggested that RON5p DNA vaccine can induce partial protective immunity against acute and chronic T. gondii infections.

Co-administration of interleukins 7 and 15 with DNA vaccine improves protective immunity against Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite, which can infect all warm-blooded animals including humans. Cytokines, including IL-15 and IL-7, play a critical role in the regulation of the homeostasis of naive and memory T cells. Co-administration the DNA vaccine with cytokines may improve its efficacy. IL-7 and IL-15 from splenic tissues of Kunming mice were cloned, and eukaryotic plasmid pVAX-IL-7-IL-15 was constructed. Kunming mice were administrated with DNA vaccine expressing T. gondii calcium-dependent protein kinase 1 (TgCDPK1), pVAX-CDPK1, in the presence or absence of IL-7 and IL-15 plasmids (pVAX-IL-7-IL-15), immune responses were analyzed including lymphoproliferative assay, cytokine and serum antibody measurements, flow cytometric surface markers on lymphocytes, and thus protective immunity against acute and chronic T. gondii infection was estimated. Mice injected with pVAX-CDPK1 supplemented with pVAX-IL-7-IL-15 showed higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2 as well as cell-mediated cytotoxic activity where stronger frequencies of IFN-γ secreting CD8+ and CD4+ T cells (CD8+/CD4+ IFN-γ+ T cells) compared to controls. Co-administration of pVAX-IL-7-IL-15 and pVAX-CDPK1 significantly (P < 0.05) increased survival time (18.07 ± 5.43 days) compared with pVAX-CDPK1 (14.13 ± 3.85 days) or pVAX-IL-7-IL-15 (11.73 ± 1.83 days) alone, and pVAX-IL-7-IL-15 + pVAX-CDPK1 significantly reduced the number of brain cysts (73.5%) in contrast to pVAX-CDPK1 (46.0%) or pVAX-IL-7-IL-15 alone (45.0%). Our results indicate that supplementation of DNA vaccine with IL-7 and IL-15 would facilitate specific humoral and cellular immune responses elicited by DNA vaccine against acute and chronic T. gondii infection in mice.

Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens.

Chimeric DNA vaccines encoding Eimeria tenella (E. tenella) surface antigen 5401 were constructed and their efficacies against E. tenella challenge were studied. The open reading frame (ORF) of 5401 was cloned into the prokaryotic expression vector pGEX-4T2 to express the recombinant protein and the expressed recombinant protein was identified by Western blot. The ORF of 5401 and chicken cytokine gene IFN-γ or IL-2 were cloned into the eukaryotic expression vector pVAX1 consecutively to construct DNA vaccines pVAX-5401-IFN-γ, pVAX-5401-IL-2 and pVAX-5401. The expression of aim genes in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Fourteen-day-old chickens were inoculated twice at an interval of 7 days with 100 µg of plasmids pVAX-5401, pVAX-5401-IFN-γ and pVAX-5401-IL-2 or 200 µg of recombinant 5401 protein by leg intramuscular injection, respectively. Seven days after the second inoculation, all chickens except the unchallenged control group were challenged orally with 5 × 10(4) sporulated oocysts of E. tenella. Seven days after challenge, all chickens were weighted and slaughtered to determine the effects of immunization. The results showed the recombinant protein was about 90 kDa and reacted with antiserum against soluble sporozoites. The animal experiment showed that all the DNA vaccines pVAX-5401, pVAX-5401-IFN-γ or pVAX-5401-IL-2 and the recombinant 5401 protein could obviously alleviate body weight loss and cecal lesions as compared with non-vaccinated challenged control and empty vector pVAX1control. Furthermore, pVAX-5401-IFN-γ or pVAX-5401-IL-2 induced anti-coccidial index (ACI) of 180.01 or 177.24 which were significantly higher than that of pVAX-5401. The results suggested that 5401 was an effective candidate antigen for vaccine. This finding also suggested that chicken IFN-γ or IL-2 could effectively improve the efficacies of DNA vaccines against avian coccidiosis.

Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

Safety and immunogenicity of a DNA vaccine encoding human calcium-activated chloride channel 1 (hCLCA1) in asthmatic mice.

BACKGROUND: Calcium-activated chloride channels (CLCAs) have been found to be preferentially expressed on the secretory epithelium. They may play a pivotal role in mucous overproduction by bronchial goblet cells in asthma. It has been reported that the inhibition of CLCAs with niflumic acid could relieve the symptoms of asthma. However, niflumic acid has serious adverse effects. DNA vaccination is considered to be a promising strategy to treat allergic diseases such as asthma and dust mite allergy. METHODS: We constructed a vaccine encoding human CLCA1 (hCLCA1) and evaluated its effects on promoting antibodies against hCLCA1 and the related preventive function in a mouse model of asthma. RESULTS: Our results reveal that the induced hCLCA1 antibodies can be detected in the first 2 weeks after immunization with hCLCA1 plasmids (hCLCA1-p) by intramuscular injection and augmented gradually in the following several weeks. The autoantibodies against hCLCA1 induced by the DNA vaccine bound to three segments of the mouse CLCA3 (mCLCA3) protein, including the amino terminal (PepN), the carboxyl terminal (PepC) and the middle of the protein (PepM). In our study, mice immunized with hCLCA1-p developed fewer pathological changes compared with other control groups, including a remarkable reduction in the air pressure-time index of the trachea, the number of eosinophils and mast cells in the bronchoalveolar lavage fluid and the mRNA level of MUC5AC in goblet cells. CONCLUSION: Taken together, our results suggest that a DNA vaccine encoding the CLCA protein may have potential as a useful pharmacotherapy for asthma in the future.

Biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken.

To evaluate the biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken, two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E at dose of 50 µg/chicken. At the 15 days post-injection, the tissue samples were collected, the total DNA was extracted, and the 3-1E gene was amplified by PCR. Genomic DNA was first purified away from free plasmid using gel electrophoresis, and then assayed for integrated plasmid using PCR amplification of the 3-1E gene. Simultaneously, the environmental dejection samples were collected, the total bacterial DNA was extracted and then transfer of the pcDNA3-1E gene was detected by PCR amplification of the 3-1E gene. Two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E with three dosage groups of 100 µg, 500 µg and 2500 µg/chicken for 14 days respectively, and with physiological saline at dose of 2500 µL/chicken as control group for acute toxicity test. A target band of 583 bp was obtained by PCR with chicken genomic DNA as template. If the chicken genomic DNA was purified, no target band could be obtained. It showed that the recombinant plasmid pcDNA3-1E existed in tissues, and no genomic integration of DNA plasmid was detected in the immunized chickens. No target band was found by PCR with environmental dejection bacteria genomic DNA as template. It showed that integration and transfer phenomenon did not exist in environment. The acute toxicity results showed the typical clinical symptoms did not occur in the inoculated chickens, the blood biochemical indices and viscera configuration were not affected significantly in the inoculated group and control group (P>0.05). The results showed that the plasmid pcDNA3-1E was safe and suitable for chicken clinical trials.

Alphavirus replicon vaccines.

The alphavirus replicon technology has been utilized for many years to develop vaccines for both veterinary and human applications. Many developments have been made to the replicon platform recently, resulting in improved safety and efficacy of replicon particle (RP) vaccines. This review provides a broad overview of the replicon technology and safety features of the system and discusses the current literature on RP and replicon-based vaccines.

Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.

The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model.

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.

Human serum amyloid P functions as a negative regulator of the innate and adaptive immune responses to DNA vaccines.

The utility of DNA vaccines has been limited by their failure to elicit sufficiently potent immune responses in many human applications, whereas DNA vaccinations in mice have been very successful. However, the underlying mechanisms remain unknown. We hypothesize that serum amyloid P component (SAP), which has a species-specific, DNA-binding ability, contributes to the differences between human and mice and then limits DNA vaccine's efficacy in vivo. In our study, DNA vaccine-induced adaptive immune responses were also significantly decreased in the human SAP (hSAP) transgenic mice. Using human promonocytic cell line THP-1-derived macrophages as a cell model, we found that cells incubated with a hSAP-DNA complex showed significant defects in innate immune activations, whereas mouse SAP had similar, albeit very weak, activities. hSAP also significantly inhibited the functions of two identified DNA sentinels, high-mobility group B protein 1 and antimicrobial peptide LL37, and redirected DNA update to FcRs leading to endocytosis and endosomal degradation. We also found that a chemical SAP inhibitor strongly recovered the suppressed innate immune responses to DNA in the presence of human serum and enhanced the immunogenicity of DNA vaccines in vivo. Our data indicated that SAP is a key negative regulator for innate immune responses to DNA and may be partly responsible for the insufficient immune responses after DNA vaccinations in humans. SAP suppression may be a novel strategy for improving efficacy of human DNA vaccines and requires further clinical investigations.

Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles.

BACKGROUND: Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming METHODS: GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants. RESULTS: Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM. CONCLUSIONS: MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine.