The enterotoxigenic Escherichia coli (ETEC) infection is a major factor restricting the development of animal husbandry. However, the abuse of antibiotics will lead to the antibiotic residues and emergence of antibiotic-resistant bacteria. The existing vaccines face challenges in stimulating intestinal immunity, demonstrating limited prevention effects. Therefore, it is indispensable to develop a new vaccine that is safe and suitable as a feed additive to activate intestinal immunity. This study constructed yeast-cell microcapsules (YCM) carrying the DNA vaccine against ETEC by genetic engineering. Furthermore, animal experiments were carried out to explore the regulatory effects of feeding YCM on the intestinal immune system and intestinal microbiota. Saccharomyces cerevisiae was selected as the oral delivery vehicle (microcapsules) of the DNA vaccine. The codon-optimized nucleic acid sequence of K88, the main antigen of mammal-derived ETEC, was synthesized, and the yeast shuttle vector containing the corresponding DNA vaccine expression cassette was constructed by DNA recombination. The recombinant strain of YCM was prepared by transforming JMY1. Additionally, the characteristics of the YCM strain and its feasibility as an oral vaccine were comprehensively evaluated by the fluorescence reporter assay, gastrointestinal fluid tolerance assay, intestinal epithelial cell adhesion assay, intestinal retention assessment, antiserum detection, and intestinal microbiota detection. The experimental results showed that the DNA vaccine expression cassette was expressed in mammals, and the recombinant strain of YCM could tolerate up to 8 hours of gastrointestinal fluid digestion and had good adhesion to intestinal epithelial cells. The results of mouse feeding experiments indicated that the recombinant strain of YCM could stay in the intestinal tract for at least two weeks, and the DNA vaccine expression cassette carried by YCM entered the intestinal immune system and triggered an immune response to induce the production of specific antibodies. Moreover, feeding YCM recombinant bacteria also improved the abundance of gut microbiota in mice, demonstrating a positive effect in regulating intestinal flora. In summary, we prepared the recombinant strain of YCM carrying the DNA vaccine against ETEC and comprehensively evaluated its characteristics and feasibility as an oral vaccine. Feeding the recombinant YCM could induce specific immune responses and regulate intestinal microbiota. The findings provide a reference for the immunoprevention of ETEC-related animal diseases.
Animals ; Enterotoxigenic Escherichia coli/genetics* ; Saccharomyces cerevisiae/metabolism* ; Vaccines, DNA/genetics* ; Mice ; Escherichia coli Infections/immunology* ; Escherichia coli Vaccines/genetics* ; Capsules ; Mice, Inbred BALB C ; Female

Foot-and-mouth disease (FMD) is one of the major animal infectious diseases in the world. All cloven-hoofed animals are susceptible to FMD. Vaccination is still the first choice for the prevention and control of FMD. mRNA vaccines can be rapidly designed, synthesized, and produced on a large scale in vitro, and they can induce effective protective immune responses, demonstrating the advantages of rapid development, easy preparation, and low biosafety risks. The design of untranslated regions is a key to enhancing the expression and efficacy of mRNA vaccines. In order to generate an efficient FMD mRNA vaccine, we designed three FMD P12A3C expression vectors with different untranslated regions and synthesized corresponding mRNAs. By comparing expression efficiency of these vectors and their mRNAs at different time points and in different cell lines, we found that the mRNA P12A3C-UTR3 had the best expression and universality. This study laid a foundation for the development of mRNA vaccines against FMD and provided a theoretical basis for the optimal sequence design of efficient mRNA.
Foot-and-Mouth Disease Virus/genetics* ; Animals ; RNA, Messenger/biosynthesis* ; Foot-and-Mouth Disease/immunology* ; Antigens, Viral/biosynthesis* ; Viral Vaccines/biosynthesis* ; Genetic Vectors/genetics* ; Cell Line ; Vaccines, DNA/immunology*

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.
Animals ; Antibodies, Viral/blood ; Cercopithecus aethiops ; Chickens ; *HN Protein/genetics/immunology ; Immunogenicity, Vaccine/*immunology ; Newcastle Disease/immunology ; Newcastle disease virus/enzymology/*genetics/immunology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/genetics/*immunology ; Vaccines, Inactivated/immunology ; Vero Cells ; *Viral Fusion Proteins/genetics/immunology ; Viral Vaccines/genetics/*immunology/*standards

Cervical cancer is the fourth most lethal women's cancer worldwide. Current treatments against cervical cancer include surgery, radiotherapy, chemotherapy, and anti-angiogenic agents. However, despite the various treatments utilized for the treatment of cervical cancer, its disease burden remains a global issue. Persistent infection of human papillomavirus (HPV) has been identified as an essential step of pathogenesis of cervical cancer and many other cancers, and nation-wide HPV screening as well as preventative HPV vaccination program have been introduced globally. However, even though the commercially available prophylactic HPV vaccines, Gardasil (Merck) and Cervarix (GlaxoSmithKline), are effective in blocking the entry of HPV into the epithelium of cervix through generation of HPV-specific neutralizing antibodies, they cannot eliminate the pre-existing HPV infection. For these reason, other immunotherapeutic options against HPV-associated diseases, including therapeutic vaccines, have been continuously explored. Therapeutic HPV vaccines enhance cell-mediated immunity targeting HPV E6 and E7 antigens by modulating primarily dendritic cells and cytotoxic T lymphocyte. Our review will cover various therapeutic vaccines in development for the treatment of HPV-associated lesions and cancers. Furthermore, we will discuss the potential of immune checkpoint inhibitors that have recently been adopted and tested for their treatment efficacy against HPV-induced cervical cancer.
Dendritic Cells/immunology ; Female ; Genetic Vectors ; Humans ; *Immunotherapy ; Papillomavirus Infections/*complications/therapy ; Papillomavirus Vaccines/therapeutic use ; *Translational Medical Research ; Uterine Cervical Neoplasms/*therapy ; Vaccines, DNA/therapeutic use ; Vaccines, Subunit/therapeutic use

OBJECTIVETo understand the distributions of DNA and neutralizing antibodies of human papillomavirus (HPV)16, 18 in 18-45 year-old women.

METHODSTotally, 1494 women were enrolled through multistage random sampling in Funing, Jiangsu province. Cervical exfoliated cells were collected from them for HPV DNA testing, and serum samples were taken from them for the detection of HPV16, 18 neutralizing antibodies by using pseudovirion-based neutralization assay(PBNA).

RESULTSAmong the 1494 women, 28(1.9%) and 188(12.6%) were positive for DNA and neutralizing antibody of HPV16 respectively, and 15(1.0%) and 60(4.0%) were positive for DNA and neutralizing antibody of HPV18, respectively. There were no significant differences in the detection rates of DNA and neutralizing antibody of HPV16, 18 among different age groups. About 16.7% of the women were infected with HPV16, 18, or both.

CONCLUSIONIn Funing county of Jiangsu province, most women aged 18-45 years has no immunity to HPV16 and 18, indicating that they are appropriate targets for HPV 16/18 vaccination.

Adolescent ; Adult ; Antibodies, Neutralizing ; isolation & purification ; Antibodies, Viral ; isolation & purification ; China ; DNA, Viral ; isolation & purification ; Female ; Human papillomavirus 16 ; immunology ; Human papillomavirus 18 ; immunology ; Humans ; Middle Aged ; Papillomavirus Infections ; prevention & control ; Papillomavirus Vaccines ; Young Adult

Therapeutic HIV vaccine was considered as a hopeful curative method for AIDS patients. However, there is still no suitable HIV animal model for vaccine study since the difference in the immune system between human and animals. To evaluate the therapeutic effect of combined immunization strategy with multiple vector vaccines in macaque models. Plasmid DNA, recombinant Ad5 and MVA vaccines which expressing SIV gag and env genes were constructed. Sequential and repeated immune strategy were applied to immunize mice with these three vaccines. Cellular immune responses in mice immunized with these three vaccines were measured by ELISPOT test in vitro and CTL assay in vivo. The results were analyzed and compared with different antigen combination, order of vaccines and intervals to choose a suitable immunization strategy for macaque immunization in future. It indicated that strong SIV-Gag/Env-specific cellular immune responses were induced by these three vector vaccines. It laid a foundation for evaluating the therapeutic effect of combined immunization strategy with multiple vector vaccines in SIV infected macaque models.
AIDS Vaccines ; administration & dosage ; genetics ; immunology ; Adenoviridae ; genetics ; metabolism ; Animals ; Antibodies, Viral ; immunology ; Female ; Gene Products, env ; administration & dosage ; genetics ; immunology ; Gene Products, gag ; administration & dosage ; genetics ; immunology ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Simian Immunodeficiency Virus ; genetics ; immunology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

OBJECTIVETo investigate the humoral and cellular immune responses induced by MUC1-2VNTR DNA vaccine in multiple myeloma (MM) tumor-bearing mice.

METHODSIn vitro, multiple myeloma cells were transfected by plasmid pcDNA3.1-2VNTR/myc-hisB with Lipofectamine2000. The above-mentioned mouse myeloma cells were inoculated subcutaneously into female BALB/c mice for establishing tumor-bearing animal models. These female BALB/c mice were immunized with pcDNA-2VNTR/myc-hisB or pcDNA/myc-hisB. The cytotoxic T lymphocyte (CTL) activity was detected by the LDH method and the spleen lymphocyte proliferation activity was detected by CCK-8 method.

RESULTSAfter immunization of BALB/c tumor-bearing mice with recombinant plasmid for 25 days, the tumor mass (0.5605 ± 0.2065 g) was significantly lighter than that in the empty plasmid control group (1.521 ± 0.6985 g) (P < 0.01) and the control group (1.5315 ± 0.5425 g) (P < 0.01). The difference of tumor mass was not statislically significant between empty plasmid control group (1.521 ± 0.6985 g) and the control group (1.5315 ± 0.5425 g) (P > 0.05). The CTL and NK cell activity was significantly higher in the group of intramuscular injection with recombinant plasmid than that in control group. The spleen lymphocyte proliferation was statistically significantly increased after being immunized with recombinant plasmid pcDNA3.1-2VNTR/myc-hisB, compared with empty vector (P < 0.01). The results showed that MUC1-2VNTR gene immunization could induce anti-tumor effect in MM tumor-bearing mice.

CONCLUSIONMUC1-2VNTR DNA immunization can elicit both humoral and cellular tumor specific immune response to multiple myeloma in MM tumor-bearing mice. It suggested that the MUC1-2VNTR DNA vaccine may be a potential treatment measure for patients with MM.

Animals ; Cancer Vaccines ; therapeutic use ; Female ; Genetic Vectors ; Humans ; Immunization ; Killer Cells, Natural ; immunology ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Minisatellite Repeats ; Mucin-2 ; genetics ; Multiple Myeloma ; immunology ; therapy ; Neoplasm Transplantation ; Plasmids ; Spleen ; cytology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Vaccines, DNA ; therapeutic use

BACKGROUND: Streptococcus pneumoniae causes life-threatening infections such as meningitis, pneumonia, and febrile bacteremia, particularly in young children. The increasing number of drug-resistant isolates has highlighted the necessity for intervening and controlling disease. To achieve this, information is needed on serotype distribution and patterns of antibiotic resistance in children. METHODS: All cases of invasive pneumococcal disease (IPD) in children aged less than 15 yr recorded at King Khalid University Hospital, King Saud University, Riyadh, Saudi Arabia, were reviewed for serotyping and antibiotic susceptibility. Isolates were collected from 78 consecutive patients with IPD between 2009 and 2012. All collected isolates were subjected to serotyping by co-agglutination, sequential multiplex PCR, and single PCR sequetyping as previously described. RESULTS: The most frequently isolated IPD serotypes were 23F, 6B, 19F, 18C, 4, 14, and 19A, which are listed in decreasing order and cover 77% of total isolates. The serotype coverage for the pneumococcal conjugate vaccine (PCV)7, PCV10, and PCV13 was 77%, 81%, and 90%, respectively. Results from sequential multiplex PCR agreed with co-agglutination results. All serotypes could not be correctly identified using single PCR sequetyping. Minimum inhibitory concentration showed that 50 (64%) isolates were susceptible to penicillin, whereas 70 (90%) isolates were susceptible to cefotaxime. CONCLUSIONS: The most common pneumococcal serotypes occur with frequencies similar to those found in countries where the PCV has been introduced. The most common serotypes in this study are included in the PCVs. Addition of 23A and 15 to the vaccine would improve the PCV performance in IPD prevention.
Adolescent ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Cefotaxime/pharmacology ; Child ; Child, Preschool ; DNA, Bacterial/analysis ; Humans ; Infant ; Meningitis/*diagnosis/microbiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Penicillins/pharmacology ; Pneumococcal Vaccines/immunology ; Pneumonia/*diagnosis/microbiology ; Protein Tyrosine Phosphatases/genetics ; Retrospective Studies ; Saudi Arabia ; Serotyping ; Streptococcus pneumoniae/*drug effects/genetics

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-gamma expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-gamma production than the other inoculated pigs (p < 0.05).
Animals ; Cell Line ; Escherichia coli/genetics ; Haplorhini ; Immunity, Cellular ; Interleukin-2/genetics/*metabolism ; Interleukin-4/genetics/*metabolism ; Neutralization Tests/veterinary ; Plasmids ; Porcine Reproductive and Respiratory Syndrome/*prevention & control ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/metabolism ; Swine ; Vaccines, DNA/immunology ; Viral Envelope Proteins/*genetics/metabolism ; Viral Vaccines/*immunology