Objective: To investigate the role of CD4⁺CD25⁺regulatory cell (CD4⁺CD25⁺Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4⁺CD25⁺Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4⁺CD25⁺Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4⁺CD25⁺Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4⁺CD25⁺Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4⁺CD25⁺Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4⁺CD25⁺Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4⁺CD25⁺Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.
Animals ; Rats ; Forkhead Transcription Factors ; Myelin P0 Protein ; Rats, Sprague-Dawley ; T-Lymphocytes, Regulatory ; CD4 Antigens/immunology* ; Interleukin-2 Receptor alpha Subunit/immunology*

Objective: To study the non/hypo-response to hepatitis B vaccination in HIV-infected patients, identify the influencing factors and provide evidence for the development of hepatitis B prevention and control strategies and measures for special population. Methods: On the basis of the randomized controlled trial of 20 µg hepatitis B vaccine immunization at 0-1-6 month, 0-1-2-6 month and 60 µg hepatitis B vaccine immunization at 0-1-2-6 month, the HIV-infected patients who completed one-month follow-up after the full course vaccination were selected as study subjects. Quantification of antibody to hepatitis B surface antigen (anti-HBs) in serum samples was performed by using chemiluminescent microparticle immunoassay (CMIA) and demographic characteristics, disease history, HIV infection and treatment status of the study subjects were collected. Statistical analysis was conducted by χ² test, t test, unconditional logistic regression and interaction analyses. Results: The non/hypo-response rates to hepatitis B vaccination were 34.65% (35/101), 24.49% (24/98) and 10.99% (10/91) in 20 µg group at 0-1-6 month or 0-1-2-6 month and 60 µg group at 0-1-2-6 month (P<0.001), respectively. Logistic regression analysis showed that after controlling for confounding factors, the risk for non/hypo-response was 0.22 times higher in HIV-infected patients receiving 60 µg hepatitis B vaccine at 0-1-2-6 month than in patients receiving 20 µg hepatitis B vaccine at 0-1-6 month (95%CI: 0.10-0.50), the risk for non/hypo-response was higher in men than in women (OR=3.65, 95%CI: 1.88-7.07), and the risk for non/hypo-response was 2.64 times higher in those without hepatitis B vaccination history than in those with hepatitis B vaccination history (95%CI: 1.10-6.32). Moreover, there were multiplicative interactions between immunization schedule and gender (OR=2.49, 95%CI: 1.24-5.00). Conclusion: The non/hypo-response rate to hepatitis B vaccination was significantly lower in HIV-infected patients receiving 60 µg hepatitis B vaccine at 0-1-2-6 month than in those receiving 20 µg hepatitis B vaccine at 0-1-6 month and 0-1-2-6 month. Gender, vaccination schedule and history of hepatitis B vaccination were the influencing factors of the non/hypo-response to hepatitis B vaccination. There was a multiplicative interaction between vaccination schedule and gender, and men receiving 20 µg hepatitis B vaccines had a higher risk for non/hypo-response to hepatitis B vaccination.
Female ; Follow-Up Studies ; HIV Infections/immunology* ; Hepatitis B/prevention & control* ; Hepatitis B Antibodies ; Hepatitis B Surface Antigens ; Hepatitis B Vaccines/administration & dosage* ; Humans ; Immunization Schedule ; Male

Objectives: To evaluate the immunogenicity of Methods: Protein extracts from Results: Immunization with Conclusion This is the advanced study to investigate the immunogenicity of
Animals ; Antibodies, Bacterial/immunology* ; Antigens, Bacterial/immunology* ; Bacterial Proteins/immunology* ; Cross Reactions ; Cytokines/immunology* ; Female ; Genome, Bacterial ; Immunoglobulin G/immunology* ; Immunoglobulin M/immunology* ; Macrophages/immunology* ; Mice, Inbred BALB C ; Mycobacterium avium Complex/immunology* ; Mycobacterium tuberculosis/immunology* ; Tuberculosis Vaccines/administration & dosage* ; Whole Genome Sequencing

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.
Animals ; Hepatitis B Core Antigens ; genetics ; immunology ; Immunity, Cellular ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Vaccines, Virus-Like Particle ; genetics ; immunology

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; Immunogenicity, Vaccine ; Mice ; Mice, Inbred BALB C ; Vaccines, Subunit ; immunology ; Viral Vaccines ; immunology

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Monoclonal, Humanized ; immunology ; Antigens ; Epitopes ; metabolism ; Protein Domains ; immunology ; Receptors, Antigen ; chemistry ; immunology ; Sharks

OBJECTIVE: To investigate the effect of chidamide on the killing activity of NK (Natural killer cell, NK) cells targeting K562 cells and its related mechanism. METHODS: K562 cells were pretreated with chidamide at different concentrations and cocultured with NK cells at different effect-target ratios. The killing effect of chidamide on K562 cells by NK cells, the expression of natural killer group 2 member D (NKG2D) ligands and apoptosis rate of K562 cells were detected by flow cytometry. RESULTS: The killing sensitivity of NK cells to K562 cells could be enhanced by chidamide. The expression of ULBP2 on K562 cell surface could be up-regulate, however, the expression of ULBP1 and MICA/MICB showed no statistically difference as compared with control group. Chidamide showed no obvious cytotoxicity to K562 cells. CONCLUSION Chidamide can significantly improve killing efficiency of NK cells on K562 cells, which may be related to the up-regulation of ULBP2 expression.
Aminopyridines ; Benzamides ; GPI-Linked Proteins ; Histocompatibility Antigens Class I ; Humans ; Intercellular Signaling Peptides and Proteins ; K562 Cells ; Killer Cells, Natural ; immunology ; NK Cell Lectin-Like Receptor Subfamily K

OBJECTIVE: To retrospectively analyze the identification results of irregular antibodies, to clarify the distribution features and to explore the relation of alloantibodies and autoantibodies with the immunized history of patients and disease kinds. METHODS: 49 820 patients who applied for red blood transfusion during Sep 1st 2017 to Sep 1st 2018 were selected. All the specimens were screened for the antibody by microcolumn gel antiglobulin technique, which then were identified for irregular antibody. RESULTS: Antibodies were found in 861 (1.73%) of all 49 820 transfused samples. The alloimmunization history of the patients with antibodies was significantly different between male and female (χ=18.54，P＜0.01). The alloantibody was the most common, accounting for 59.50% in all of the antibodies. Warm autoantibody, anti-E, anti-M, anti-cE and anti-Ce accounted for 68.5% of the antibodies. The blood group of Rh, MNS and Lewis were responsible for 92.40% of alloantibody, especially anti-E accounted for the largest percentage(38.60%) of alloantibody. Patients with alloantiboies experienced much more the alloimmunization and transfusion history (χ=20.13，P＜0.01；χ=5.40，P＜0.05) . The distribution of auto and alloantibody was very significantly different among the ddifferent isease (χ=51.8，P＜0.01), Hematopathy, solid tumor and osteoarthropathy were often associated with alloantibody, otherwise, autoantibodies often occurred in hematopathy and autoimmune disease. CONCLUSION The most important factor that results in antibody-screening positive is alloantibody, in which anti-E antibody from Rh blood group system in most common.
Antibodies ; immunology ; Blood Group Antigens ; Blood Transfusion ; Erythrocytes ; Female ; Humans ; Isoantibodies ; Male ; Retrospective Studies

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*

OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (=5). In the former two groups, was inoculated intranasal administration to establish mouse models of pulmonary infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation ( < 0.05) and reduced chlamydia load in the lung tissue ( < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells ( < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages ( < 0.05) and Th1 cells ( < 0.05). CONCLUSIONS Inhibition of CD96 alleviates pneumonia in -infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.
Animals ; Antigens, CD ; metabolism ; Chlamydia Infections ; complications ; immunology ; physiopathology ; Chlamydia muridarum ; Interferon-gamma ; genetics ; metabolism ; Killer Cells, Natural ; metabolism ; Lung Injury ; etiology ; genetics ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL