Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

A large number of studies have demonstrated that mRNA vaccine has been characterized as a technique with good safety, strong immunogenicity and high developmental potential, which makes it have broad prospects in immunotherapy. In recent years, the stability and in vivo delivery efficiency of mRNA vaccines have been largely addressed by the progresses in mRNA engineering and delivery innovation. And some mRNA vaccines are now clinical approved or in preclinical trials. Here, we summarize current knowledge on the research advances, technology, and application in major infectious diseases in humans and animals of mRNA vaccines, with the aim to provide a reference for improving the development of novel mRNA vaccines.
Animals ; Humans ; Communicable Diseases ; Vaccines, Synthetic/genetics* ; mRNA Vaccines

Cardiovascular Diseases/prevention & control* ; Humans ; RNA, Messenger ; Vaccines ; Vaccines, Synthetic/genetics* ; mRNA Vaccines

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.
Animals ; Blotting, Western ; Caliciviridae Infections/veterinary* ; Hemorrhagic Disease Virus, Rabbit/immunology* ; Rabbits ; Vaccines, Synthetic/immunology* ; Viral Structural Proteins/genetics*

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Animals ; Antibodies, Viral/blood ; *Chickens ; Chimera/genetics/immunology ; Coronavirus Infections/prevention & control/*veterinary/virology ; Female ; *Immunity, Innate ; Infectious bronchitis virus/genetics/*immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology ; Injections, Intramuscular/veterinary ; Mice ; Mice, Inbred BALB C ; Neuraminidase/genetics ; Poultry Diseases/*prevention & control/virology ; Recombinant Fusion Proteins/genetics/immunology ; Spike Glycoprotein, Coronavirus/genetics/*immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology ; Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology ; Viral Proteins/genetics

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.
Adenoviridae/genetics/immunology ; Administration, Intranasal ; Animals ; Capsid Proteins/*genetics/immunology ; Circoviridae Infections/*immunology ; Circovirus/*genetics/immunology ; Epitopes/genetics/immunology ; Female ; Immunity, Mucosal/immunology ; Immunoglobulin A/blood/immunology ; Immunoglobulin G/blood/immunology ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides/genetics ; Vaccines, Synthetic/genetics/immunology ; Viral Vaccines/administration & dosage/*genetics/immunology

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.
Adjuvants, Immunologic/genetics/*metabolism ; Administration, Intranasal ; Animals ; Antigens, Protozoan/genetics/*immunology ; BCG Vaccine/administration & dosage/*genetics ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Chickens ; Coccidiosis/*prevention & control ; Disease Models, Animal ; Drug Carriers/administration & dosage ; Eimeria tenella/genetics/*immunology ; Genetic Vectors ; Injections, Subcutaneous ; Interleukin-2/genetics/*metabolism ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Spleen/immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology

OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology