Objective To construct a library of human-derived anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) single-chain variable fragments (scFv) and screen for broad-spectrum neutralizing antibodies to identify candidate molecules for the development of diagnostic and therapeutic agents. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of patients who had recovered from novel coronavirus infection. Total RNA was extracted from these PBMCs and reverse transcribed into cDNA, which was used as a template for constructing a human anti-SARS-CoV-2 scFv library. Phage display technology was used to screen for scFv antibodies specific to the SARS-CoV-2 S protein. Full-length IgG antibodies were synthesized through sequence analysis and human IgG expression, and their binding capacity and neutralizing activity against SARS-CoV-2 were evaluated. Results A human-derived scFv antibody library against SARS-CoV-2 with a capacity of 1.56×10⁷ CFU was successfully constructed. Two specific scFv antibodies were screened from this library and expressed as full-length IgG antibodies (IgG-A10 and IgG-G6). IgG-A10 exhibited strong neutralizing activity against both the original SARS-CoV-2 strain (WT) and the XBB subvariant of the Omicron variant. However, the neutralizing activity of this antibody against the JN.1 sub lineage of the Omicron BA.2.86 variant was moderate. Conclusion This study has successfully constructed a human anti-SARS-CoV-2 scFv antibody library from the peripheral blood of recovered patients, and screened and expressed anti-SARS-CoV-2 IgG antibodies with neutralizing activity, laying a foundation for the prevention, diagnosis, and treatment of SARS-CoV-2 infection.
Humans ; Single-Chain Antibodies/genetics* ; SARS-CoV-2/immunology* ; COVID-19/immunology* ; Immunoglobulin G/genetics* ; Antibodies, Viral/genetics* ; Peptide Library ; Spike Glycoprotein, Coronavirus/immunology* ; Antibodies, Neutralizing/immunology* ; Leukocytes, Mononuclear/immunology* ; Broadly Neutralizing Antibodies/immunology*

Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

BACKGROUND: Epstein-Barr (EB) virus infection stimulates the production of vascular endothelial growth factor (VEGF), which contributes to the progression of angiogenesis. Angiogenesis plays an important role in the development of atherosclerosis. Since serum anti-early antigen EB virus IgG (EBV EA-IgG) titer is a sign of active EB virus infection, EBV EA-IgG titer could be associated with atherosclerosis. The number of minor (T) alleles in VEGF polymorphism rs3025039 has been reported to be inversely associated with serum VEGF concentration, suggesting that rs3025039 might have a strong influence on the association between EBV EA-IgG titer and atherosclerosis. By focusing on the role of VEGF in the development of atherosclerosis, this study aimed to investigate the association between active EB virus infection and atherosclerosis. METHODS: A cross-sectional study of 2,661 older Japanese individuals aged 60-89 years who participated in annual health check-ups during 2017-2019 was conducted. Logistic regression was used to evaluate the association between EBV EA-IgG titer and atherosclerosis in relation to rs3025039 genotype. The influence of rs3025039 (T) allele carrier status on the association between EBV EA-IgG titer and atherosclerosis was also evaluated by using logistic regression. RESULTS: Among rs3025039 CC-homozygotes, with the lowest EBV EA-IgG titer tertile as the reference, the multivariable odds ratio (95% confidence interval) was 1.11 (0.82, 1.50) for the medium tertile and 1.07 (0.78, 1.47) for the high tertile. Among rs3025039 (T) allele carriers, the corresponding values were 1.44 (0.88, 2.36) and 1.88 (1.15, 3.05), respectively. There was a significant interaction between rs3025039 (T) allele carrier status and the association between EBV EA-IgG titer and atherosclerosis (adjusted p = 0.0497). CONCLUSION EBV EA-IgG titer was significantly positively associated with atherosclerosis only among participants who are genetically less likely to have progressive angiogenesis. An angiogenesis-related genetic factor was revealed as a determinant of the association between EBV EA-IgG titer and atherosclerosis. These findings introduce a novel concept that could explain the association between viral infection and atherosclerosis.
Humans ; Aged ; Male ; Middle Aged ; Female ; Japan/epidemiology* ; Atherosclerosis/virology* ; Aged, 80 and over ; Vascular Endothelial Growth Factor A/genetics* ; Epstein-Barr Virus Infections/virology* ; Polymorphism, Single Nucleotide ; Cross-Sectional Studies ; Herpesvirus 4, Human ; Antigens, Viral/immunology* ; Antibodies, Viral/blood* ; Immunoglobulin G/blood* ; Genotype ; East Asian People

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*

This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.
Animals ; Enzyme-Linked Immunosorbent Assay/methods* ; Swine ; Antibodies, Viral/blood* ; Recombinant Proteins/genetics* ; Deltacoronavirus/isolation & purification* ; Coronavirus Infections/virology* ; Swine Diseases/diagnosis* ; Coronavirus Nucleocapsid Proteins ; Sensitivity and Specificity

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

Vaccination is the most effective measure for reducing and preventing influenza and related complications. In this study, we analyzed the mutation trend and the antigen dominant site changes of the amino acid sequence of hemagglutinin subunit 1 (HA1) of human influenza A virus (IAV) in the northern hemisphere from 2012 to 2022. According to the HA1 sequences of A/Darwin/6/2021 (H3N2) and A/Wisconsin/588/2019 (H1N1) recommended by the World Health Organization in the 2022 influenza season in northern hemisphere, we employed the mosaic algorithm to design three Mosaic-HA1 antigens through stepwise substitution. Mosaic-HA1 was expressed and purified in 293F cells and then mixed with the alum adjuvant at a volume ratio of 1:1. The mixture was used to immunize BALB/c mice, and the immunogenicity was evaluated. Enzyme-linked immunosorbent assay showed that Mosaic-HA1 induced the production of IgG targeting two types of HA1, the specific IgG titers for binding to H3 protein and H1 protein reached 10⁵ and 10³ respectively. The challenge test showed that Mosaic-HA1 protected mice from H3N2 or H1N1. This study designs the vaccines by recombination of major antigenic sites in different subtypes of IAV, giving new insights into the development of multivalent subunit vaccines against influenza.
Animals ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A Virus, H3N2 Subtype/genetics* ; Mice, Inbred BALB C ; Mice ; Influenza Vaccines/genetics* ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Humans ; Antibodies, Viral/blood* ; Antigens, Viral/genetics* ; Immunoglobulin G/immunology* ; Female ; Orthomyxoviridae Infections/prevention & control* ; HEK293 Cells

This study developed ferritin-based nanoparticles carrying the African swine fever virus (ASFV) p30 protein and evaluated their immunogenicity, aiming to provide an experimental basis for the research on nanoparticle vaccines against ASFV. Initially, the gene sequences encoding the p30 protein and SpyTag were fused and inserted into the pCold-I vector to create the pCold-p30 plasmid. The gene sequences encoding SpyCatcher and ferritin were fused and then inserted into the pET-28a(+) vector to produce the pET-F-np plasmid. Both plasmids were expressed in Escherichia coli upon induction. Subsequently, the affinity chromatography-purified p30 protein was conjugated with ferritin in vitro, and the p30-ferritin (F-p30) nanoparticles were purified by size-exclusion chromatography. The morphology and structural integrity of F-p30 nanoparticles were examined by a particle size analyzer and transmission electron microscopy. Mice were immunized with F-p30 nanoparticles, and the humoral and cellular immune responses were assessed. The results showed that F-p30 nanoparticles were successfully prepared, with the particle size of approximately 20 nm. F-p30 nanoparticles were efficiently internalized by bone marrow-derived dendritic cells (BMDCs) cells in vitro. Compared with the p30 protein alone, F-p30 nanoparticles induced elevated levels of specific antibodies and cytokines in mice and stimulated the proliferation of follicular helper T cell (T_FH) and germinal center B cell (GCB) in lymph nodes as well as CD4⁺ and CD8⁺ T cells in the spleen. In conclusion, we successfully prepared F-p30 nanoparticles which significantly enhanced the immunogenicity of p30 protein, giving insights into the development of vaccines against ASFV.
Animals ; Nanoparticles/chemistry* ; Mice ; African Swine Fever Virus/genetics* ; Ferritins/chemistry* ; Swine ; Viral Vaccines/genetics* ; African Swine Fever/immunology* ; Mice, Inbred BALB C ; Viral Proteins/genetics* ; Escherichia coli/metabolism* ; Dendritic Cells/immunology* ; Immunogenicity, Vaccine ; Antibodies, Viral/blood* ; Female ; Capsid Proteins/genetics*

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.
Cricetinae ; Humans ; Animals ; Mice ; Immunoglobulin M/genetics* ; Antibodies, Viral ; CHO Cells ; Cricetulus ; Hybridomas ; Recombinant Fusion Proteins