Pseudorabies (PR) is an infectious disease caused by the pseudorabies virus (PRV), affecting various domesticated and wild animals. Since pigs are the only natural hosts of PRV, PR poses a serious threat to the pig farming industry. Currently, PR is primarily prevented through vaccination with inactivated vaccines or genetically modified attenuated live vaccines. Developing safe and effective genetically engineered vaccines would facilitate the eradication and control of PR. In this study, the PRV vaccine strain Bartha-K61 was used as the reference strain. The gB protein was expressed via the baculovirus-insect cell expression system. Non-denaturing gel electrophoresis confirmed that the gB protein could form a trimeric structure. The purified gB protein was used to immunize mice, and the immune effect was evaluated by a challenge test. The results showed that the gB antigen induced a strong immune response in mice, with the serum-neutralizing antibody titer above 1:70. The lymphocyte stimulation index reached more than 1.29, and the level of (interferon gamma, IFN-γ) release was higher than 100 pg/mL. After immunization, mice were challenged with the virus at a dose of 10⁴ TCID₅₀/mL, 200 μL per mouse, and the clinical protection rate was 100%. Immunohistochemistry, histopathological section, and tissue viral load results showed that the pathological damage and viral load in the gB-immunized group were significantly lower than those in the PBS group. In summary, the gB protein obtained in this study induced strong humoral and cellular immune responses in mice, laying a foundation for developing a recombinant gB protein subunit vaccine.
Animals ; Mice ; Baculoviridae/metabolism* ; Viral Envelope Proteins/biosynthesis* ; Herpesvirus 1, Suid/genetics* ; Pseudorabies/immunology* ; Swine ; Pseudorabies Vaccines/genetics* ; Antibodies, Viral/blood* ; Insecta/cytology* ; Mice, Inbred BALB C ; Female ; Viral Vaccines/immunology*

The global outbreak of monkeypox in 2022 has aroused widespread concern in public health. To date, the prevention and treatment of monkeypox has mainly relied on smallpox vaccines and drugs. This study aims to screen and obtain therapeutic antibodies with high affinity, neutralizing activity, and protective effects, and provide candidate molecules for the development of specific therapeutic antibodies against monkeypox. Therefore, humanized mice were immunized to screen for antibodies against the envelope protein of the mpox virus. Two M1R-specific antibodies, 12G5 and 12H6, were obtained, with the affinity of 0.095 nmol/L and 0.089 nmol/L, respectively. The 50% reduction of the plaque counts (PRNT₅₀) of 12G5 and 12H6 was (1.821±1.766) μg/mL and (17.605±2.383) μg/mL, respectively. The two antibodies targeted two binding epitopes of M1R. Moreover, 12H6 could protect 60% of mice from death following the vaccinia virus challenge. This study provides research materials for subsequent in-depth studies on the immunoprotection of mpox virus and potential therapeutic strategies.
Animals ; Mice ; Antibodies, Viral/immunology* ; Monkeypox virus/immunology* ; Mpox, Monkeypox/immunology* ; Antibodies, Neutralizing/immunology* ; Viral Envelope Proteins/immunology* ; Humans ; Antibodies, Monoclonal/biosynthesis* ; Female

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Blotting, Western ; Epitope Mapping ; Hybridomas ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Reticuloendotheliosis virus ; immunology ; Viral Envelope Proteins ; immunology

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.
Base Sequence ; Blotting, Western ; Coronavirus NL63, Human ; chemistry ; Fluorescent Antibody Technique, Indirect ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

In 1990, it was reported that the naked DNA encoding an antigen (so-called DNA vaccine) transduced directly into the muscle is able to induce immune responses just like antigen inoculation. Since then, a number of DNA vaccines against different diseases have been developed and shown to induce different levels of specific humoral and/or cell-mediated immunity. Efforts have been made to develop effective DNA vaccines against classical swine fever (CSF). This review covered the following aspects in the development and application of CSF DNA vaccines: construction and evaluation, application of adjuvants, combination with other vaccines and the existing problems and solutions.
Adjuvants, Immunologic ; pharmacology ; Animals ; Classical Swine Fever ; prevention & control ; Swine ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.
Animals ; HEK293 Cells ; Herpesvirus 1, Human ; immunology ; metabolism ; Humans ; Immunization ; Mice ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of postweaning multisystemic wasting syndrome (PMWS). Co-infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) can result in severe economic losses to the swine industry. In this study, we constructed the recombinant adenovirus rAd-Cap-GP5 expressing Cap of PCV2 and GP5 of PRRSV. And the expression of Cap and GP5 protein in the HEK-293 cells inoculated with rAd-Cap-GP5 were confirmed by immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. The immunogenicity of recombinant adenoviruses rAd-Cap-GP5 was examined in mice by vaccination with the recombinant adenovirus. The results showed that the mice could produce anti-PCV2 and PRRSV antibodies detected by indirect ELISA and virus neutralization assay. It indicated that rAd-Cap-GP5 could provide humoral immunity responses in mice. The recombinant adenovirus rAd-Cap-GP5 might be an attractive candidate vaccine for preventing the disease associated with PCV2 and PRRSV infection.
Adenoviridae ; genetics ; metabolism ; Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; prevention & control ; virology ; Circovirus ; genetics ; metabolism ; Immunization ; Mice ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immune-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Kidney ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics

To express the domain III gene of Japanese encephalitis virus (JEV) and to learn the possibility of developing the Dil protein as a subunit vaccine, we amplified the JEV DIII gene by PCR and constructed the expression plasmid pET-JE DIII by inserting JEV DIII gene into the prokaryotic expression vector pET-32a(+). The domain III protein of the attenuated strain SA14-14-2 was expressed as a thioredoxin (Trx) fusion protein, which was unique in forming a large fraction of the soluble recombinant protein. We immunized the rabbits and mice with the purified protein, tested the antigenicity and immunogenicity of JEV DIII protein by ELISA, Western blotting, plaque reduction test and observed the protective efficacy on challenged weanling mice with JEV. Rabbits immunized with the purified JEV DIII protein generated 1: 7 x 10(5) anti-JEV specific antibody titers. BALB/c mice immunized with the purified JEV E DIII protein generated 1: 8.2 x 10(4) anti-JEV specific antibody titers. And the neutralized antibody titer can reach 1:256, the survival rate of the immunized weanling mice was approximately 75%. Overall, this study highlighted that recombinant JEV E DIII protein delivered in mice and rabbits can generate high antibody titers against JEV, and protect some mice challenged with JEV. These studies can provide useful information for further developing the domain III recombinant protein as subunit vaccine against JEV.
Animals ; Antibodies, Viral ; blood ; Encephalitis Virus, Japanese ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Immunization ; Japanese Encephalitis Vaccines ; biosynthesis ; immunology ; Mice ; Mice, Inbred BALB C ; Protein Structure, Tertiary ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology