This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

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*

Human alphaherpesvirus 2 (HSV-2) is the main pathogen resulting human genital herpes, which poses a major threat to the socio-economic development, while there is no effective vaccine. In this study, we developed a novel lipopolyplex (LPP)-delivered mRNA vaccine expressing the HSV-2 envelope glycoprotein gD and evaluated its immunogenicity in mice. The mRNA vaccine was prepared from the genetically modified gD mRNA synthesized in vitro combined with the LPP delivery platform and it was named gD-ORI mRNA. The expression of gD antigen in the mRNA vaccine was validated in vitro by Western blotting and indirect immunofluorescence assay, then the immune responses induced by this mRNA vaccine in mice were evaluated. The immunization with gD mRNA alone induced strong humoral and cellular immune responses in mice. Robust and long-lasting gD-specific IgG antibodies were detected in the mouse serum after booster immunization with gD-ORI mRNA. The immunized mice exhibited a Th1/Th2 balanced IgG response and robust neutralizing antibodies against HSV-2, and a clear dose-response relationship was observed. The gD-specific IgG antibodies were maintained in mice for a long time, up to 18 weeks post-booster immunization. At the same time, multifunctional gD-specific CD4⁺ and CD8⁺ T cells in vaccinated mice were detected by intracellular cytokine staining (ICS). This novel gD-expressing mRNA vaccine delivered by LPP induces strong and long-lasting immune responses in mice post booster immunization and has a promising prospect for development and application. This study provides scientific evidence and reference for the development of a new mRNA vaccine for HSV-2.
Animals ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Mice ; Herpes Genitalis/immunology* ; RNA, Messenger/immunology* ; Female ; Mice, Inbred BALB C ; Antibodies, Viral/blood* ; mRNA Vaccines/immunology* ; Antibodies, Neutralizing/blood* ; Humans

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.
Humans ; Animals ; Swine ; Porcine respiratory and reproductive syndrome virus/genetics* ; Porcine Reproductive and Respiratory Syndrome/prevention & control* ; HEK293 Cells ; Viral Envelope Proteins/genetics* ; Antibodies, Viral ; Viral Vaccines/genetics* ; Recombinant Proteins ; Vaccines, Subunit

Adenoviridae ; genetics ; Ebolavirus ; genetics ; pathogenicity ; Gene Transfer Techniques ; Genetic Vectors ; therapeutic use ; Glycoproteins ; genetics ; HEK293 Cells ; Hemorrhagic Fever, Ebola ; genetics ; pathology ; virology ; Humans ; Inflammation ; genetics ; pathology ; virology ; Mucins ; genetics ; Transfection ; Viral Envelope Proteins ; genetics

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Humans ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Sf9 Cells ; Vaccination ; Vaccines, Virus-Like Particle ; administration & dosage ; genetics ; immunology ; Viral Envelope Proteins ; administration & dosage ; genetics ; immunology

Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus that can induce myelocytomas. A high-frequency mutation in gene envelope endows ALV-J with the potential for cross-species transmission. We wished to ascertain if the ALV-J can spread across species under selection pressure in susceptible and resistant hosts. First, we inoculated (in turn) two susceptible host birds (specific pathogen-free (SPF) chickens and turkeys). Then, we inoculated three resistant hosts (pheasants, quails and ducks) to detect the viral shedding, pathologic changes, and genetic evolution of different isolates. We found that pheasants and quails were infected under the selective pressure that accumulates stepwise in different hosts, and that ducks were not infected. Infection rates for SPF chickens and turkeys were 100% (16/16), whereas those for pheasants and quails were 37.5% (6/16) and 11.1% (3/27). Infected hosts showed immune tolerance, and inflammation and tissue damage could be seen in the liver, spleen, kidneys and cardiovascular system. Non-synonymous mutation and synonymous ratio (NS/S) analyses revealed the NS/S in hypervariable region (hr) 2 of pheasants and quails was 2.5. That finding suggested that mutation of isolates in pheasants and quails was induced by selective pressure from the resistant host, and that the hr2 region is a critical domain in cross-species transmission of ALV-J. Sequencing showed that ALV-J isolates from turkeys, pheasants and quails had moved away from the original virus, and were closer to the ALV-J prototype strain HPRS-103. However, the HPRS-103 strain cannot infect pheasants and quails, so further studies are needed.
Amino Acid Sequence ; Animals ; Avian Leukosis ; transmission ; virology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Chickens ; Ducks ; virology ; Galliformes ; virology ; Host Specificity ; Molecular Sequence Data ; Poultry Diseases ; transmission ; virology ; Quail ; virology ; Sequence Alignment ; Turkeys ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.
Animals ; Antibodies, Viral ; immunology ; Ebolavirus ; genetics ; immunology ; Female ; Gene Expression ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.
Coronavirus Infections/diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/*genetics/isolation & purification ; Nasopharynx/virology ; Open Reading Frames/genetics ; RNA, Viral/*analysis/metabolism ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics