Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.

Monoclonal antibody against H1N1 influenza virus hemagglutinin cross reacts with hnRNPA1 and hnRNPA2/B1.

Following influenza A vaccination, certain individuals exhibit adverse reactions in the nervous system, which causes a problem with the safety of the influenza A vaccine. However, to the best of our knowledge, the underlying mechanism of this is unknown. The present study revealed that a monoclonal antibody (H1­84mAb) against the H1N1 influenza virus hemagglutinin (HA) protein cross­reacted with an antigen from brain tissue. Total brain tissue protein was immunoprecipitated with this cross­reactive antibody, and mass spectrometry revealed that the bound antigens were heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2/B1. Subsequently, the two proteins were expressed in bacteria and it was demonstrated that H1­84mAb bound to hnRNPA1 and hnRNPA2/B1. These two proteins were expressed in three segments and the cross­reactivity of H1­84mAb with the glycine (Gly)­rich domains of hnRNPA1 (195aa­320aa) and hnRNPA2/B1 (202aa­349aa) was determined using ELISA blocking experiments. It was concluded that the Gly­rich domains of these two proteins are heterophilic antigens that cross­react with influenza virus HA. The association between the heterophilic antigen Gly­rich domains and the safety of influenza A vaccines remains to be investigated.

[Identification of B cell epitopes on hemagglutinin of H1N1 influenza virus].

Objective To study B cell epitopes of the influenza virus hemagglutinin (HA) by computer simulation and ELISA blocking experiment, and to establish a new method for pathogen microbial epitope detection. Methods The 2009 H1N1 influenza virus inactivated vaccine was used as the immunogen, and the monoclonal antibodies (mAbs) were prepared by conventional hybridoma fusion and screening techniques. The characteristics of mAbs were identified by ELISA, hemagglutination inhibition (HI) and Western blot analysis. The obtained mAbs were used as a tool to predict the epitope of H1N1 influenza virus by ELISA blocking experiment combined with computer simulation method. Results Four mAbs against the HA antigen on H1N1 influenza virus were obtained, and HA epitopes were classified into two types by ELISA blocking experiment. The computer simulation revealed that the four antibodies could bind to two epitopes on HA. Conclusion The results of computer simulation and ELISA blocking experiment are consistent, and a new method for predicting the epitopes of other pathogenic microorganisms had been established.

Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin.

Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941-946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191-199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.

H1N1 influenza virus epitopes classified by monoclonal antibodies.

Epitopes serve an important role in influenza infection. It may be useful to screen universal influenza virus vaccines, analyzing the epitopes of multiple subtypes of the hemagglutinin (HA) protein. A total of 40 monoclonal antibodies (mAbs) previously obtained from flu virus HA antigens (development and characterization of 40 mAbs generated using H1N1 influenza virus split vaccines were previously published) were used to detect and classify mAbs into distinct flu virus sub-categories using the ELISA method. Following this, the common continuous amino acid sequences were identified by multiple sequence alignment analysis with the GenBank database and DNAMAN software, for use in predicting the epitopes of the HA protein. Synthesized peptides of these common sequences were prepared, and used to verify and determine the predicted linear epitopes through localization and distribution analyses. With these methods, nine HA linear epitopes distributed among different strains of influenza virus were identified, which included three from influenza A, four from 2009 H1N1 and seasonal influenza, and two from H1. The present study showed that considering a combination of the antigen-antibody reaction specificity, variation in the influenza virus HA protein and linear epitopes may present a useful approach for designing effective multi-epitope vaccines. Furthermore, the study aimed to clarify the cause and pathogenic mechanism of influenza virus HA-induced flu, and presents a novel idea for identifying the epitopes of other pathogenic microorganisms.

[Preparation and identification of monoclonal antibody against H1N1 influenza virus].

Objective To prepare the monoclonal antibody (mAb) against influenza A virus (H1N1) using purified viral particles as antigen and investigate the characterization of host cells infected with influenza virus utilizing the mAb. Methods A/PR/8 (H1N1) virus was cultured in embryonated chicken eggs and further purified by differential and density gradient centrifugation. The structure of viral particles was identified by transmission electron microcopy (TEM). Immunogenicity of purified virus was evaluated by Balb/c mice immunized with formalin-inactivated virus. Hybridomas secreting mAbs were established through a fusion of Sp2/0 myeloma cells and splenocytes from the mice immunized with the virus. The characteristics of mAb were identified by ELISA, immunofluorescence assay (IFA), Western blotting, hemagglutinin inhibition assay (HI) and microneutralization assay. The outside hemagglutinin (HA) on the plasma membrane of the host MDCK cells in which the viruses were propagated and the apoptosis of MDCK cells infected with the viruses were measured using flow cytometry. Cell-based ELISA was established using mAb specific to HA. Subsequently, the growth of virus was analyzed by cell-based ELISA. Results Transmission electron microscopy revealed that the physical structures of the purified virus were spherical, elliptical and extended threadlike. Serum IgG titer to influenza virus showed a progressive increase, and the IgG titer reached 106 after the immunization for 6 weeks. Six hybridoma clones secreting mAb specific to A/PR/8 were developed by hybridoma technology. The HI and neutralization activities of PR8-10 mAb were significantly higher than those of the other mAbs. HI and neutralization titers of PR8-10 mAb were 1:2048 and 1:640, respectively. IFA and Western blotting confirmed that PR8-10 mAb could recognize HA. Flow cytometry showed that PR8-10 mAb also recognized HA on the membrane of MDCK in which the viruses were replicated and virus infection induced the apoptosis of MDCK cells. Based on the previous test results that PR8-10 mAb was able to recognize HA on the membrane of the host cells in which the viruses were replicated, cell-based ELISA we established was good at analyzing the growth of virus in MDCK cells. Conclusion We obtained whole viral particles that were demonstrated to be able to stimulate the production of a high IgG titer in a mouse model with formalin-inactivated viral particles, and successfully prepared the mAb against H1N1 of high binding affinity and neutralization potency. HA-specific mAb can be used to analyze the characteristics of virus infection process and the effect of virus infection on the host cells as well.

Toll-like receptor 9 regulates melanogenesis through NF-κB activation.

Toll-like receptors play essential roles in the modulation of melanogenesis, which has been implicated in the pathogenesis of hyper- or hypopigmentation-related diseases. However, little is currently known regarding the role of TLR9 in human melanocytes. TLR9 recognizes unmethylated cytosine-phosphate-guanine motif-containing oligodeoxynucleotides, and cytosine-phosphate-guanine ODN2006 acts as an hTLR9 agonist. The aim of the present study was to investigate the effect of cytosine-phosphate-guanine ODN2006 on melanogenesis in the human melanocyte cells. MTT assay and enzyme-linked immunosorbent assay indicated that ODN2006 stimulation (0, 1, 5, 10 µM) dose-dependently reduced cell viability and promoted the production of TNF-α, IL-6, and IL-8 in PIG1 melanocytes. The mRNA and protein levels of PMEL and TYRosinase were elevated at 6 h, and then decreased 24 h later, but were significantly augmented 72 h later following ODN2006 stimulation; whereas, TLR9 expressions were time-dependently increased in PIG1 melanocytes. Moreover, ultraviolet B irradiation combined with ODN2006 stimulation induced much more significant enhancement of PMEL, TYRosinase, and TLR9 mRNA and protein after three days in PIG1 melanocytes, and the similar results were obtained using the primary human melanocytes. The expression of TLR9 protein was down-regulated by TLR9 siRNA transfection. ODN2006 had an additive effect on ultraviolet B-induced melanogenesis and PMEL expression, as well as NF-κB activation, which could be blocked by TLR9 knockdown, the NF-κB specific inhibitor PDTC, or the TBK1 inhibitor BX795. Collectively, we concluded that TLR9 regulates melanogenesis through NF-κB activation, suggesting that TLR9 may play a role in microbial-induced melanogenesis.

[Detection of serum autoantibodies against premelanosome protein 17 increases in the vitiligo patients].

OBJECTIVE: To detect the distribution of autoantibodies against premelanosome protein 17 (Pmel 17) in the sera of vitiligo patients with the recombinant Pmel 17 protein. METHODS: The cDNA encoding human Pmel 17 was amplified by RT-PCR from the primary melanocytes and was cloned into vector pMD19-T and subsequently the expression vector pGEX-4T-1. After being induced by IPTG, the recombinant Pmel 17 protein was purified by high-performance affinity chromatography. The presence of autoantibodies against Pmel 17 in the sera of vitiligo patients was determined by indirect ELISA coated with recombinant Pmel 17 protein. RESULTS: The recombinant Pmel 17 plasmids were right as we expected by DNA sequencing. The recombinant Pmel 17 protein was successfully expressed and purified. The indirect ELISA revealed that, as compared with healthy control group (0/100, 0%), positive rate of the serum autoantibodies in the progress vitiligo patients was 22% and positive rate of stable vitiligo patients was only 2%(2/100). The difference of positive rate between patients with progress and stable vitiligo was statistically significant. CONCLUSION: The recombinant Pmel 17 protein was successfully expressed. The autoantibodies against Pmel 17 is closely related to the severity of vitiligo.

Comparison of three methods for the detection of Epstein-Barr virus in Hodgkin's lymphoma in paraffin-embedded tissues.

The percentage rate of Epstein-Barr virus (EBV)-positive cases of Hodgkin's lymphoma (HL) ranges between 20 and 70% in various studies worldwide. To further explore the definite rate in China, three methods, including immunohistochemistry for EBV latent membrane protein 1 (LMP1), in situ hybridization (ISH) for EBV-encoded RNA (EBER)-1 and polymerase chain reaction (PCR) for EBV BamHI­W fragment, were employed to detect EBV in 59 cases of HL in China using paraffin-embedded tissue samples. Our results revealed that the PCR method presented the highest (44/59, 74.6%) detection rate among the three methods. The other two methods identified 66.1% (39/59, LMP1) and 67.8% (40/59, EBER1 ISH) EBV-positive results, respectively. Three samples were positive for LMP1 but negative when using EBER1 ISH, while another four samples were EBER1-positive but LMP1-negative. Of the four major histopathological subtypes of HL, the lymphocyte predominant (LR) subtype is the one most frequently associated with EBV, followed by the mixed cellularity (MC), nodular sclerosis (NS) and lymphocyte depletion (LD) subtypes. Our results also indicated the seldomly reported fact that EBV-positive cases in children were more numerous than those of adults with HL.

A novel noninvasive method to detect rejection after heart transplantation

Prompt and accurate detection of rejection prior to pathological changes after organ transplantation is vital for monitoring rejections. Although biopsy remains the current gold standard for rejection diagnosis, it is an invasive method and cannot be repeated daily. Thus, noninvasive monitoring methods are needed. In this study, by introducing an IL-2 neutralizing monoclonal antibody (IL-2 N-mAb) and immunosuppressants into the culture with the presence of specific stimulators and activated lymphocytes, an activated lymphocyte-specific assay (ALSA) system was established to detect the specific activated lymphocytes. This assay demonstrated that the suppression in the ALSA test was closely related to the existence of specific activated lymphocytes. The ALSA test was applied to 47 heart graft recipients and the proliferation of activated lymphocytes from all rejection recipients proven by endomyocardial biopsies was found to be inhibited by spleen cells from the corresponding donors, suggesting that this suppression could reflect the existence of activated lymphocytes against donor antigens, and thus the rejection of a heart graft. The sensitivity of the ALSA test in these 47 heart graft recipients was 100%; however, the specificity was only 37.5%. It was also demonstrated that IL-2 N-mAb was indispensible, and the proper culture time courses and concentrations of stimulators were essential for the ALSA test. This preliminary study with 47 grafts revealed that the ALSA test was a promising noninvasive tool, which could be used in vitro to assist with the diagnosis of rejection post-heart transplantation.

A novel noninvasive method to detect rejection after heart transplantation.

Prompt and accurate detection of rejection prior to pathological changes after organ transplantation is vital for monitoring rejections. Although biopsy remains the current gold standard for rejection diagnosis, it is an invasive method and cannot be repeated daily. Thus, noninvasive monitoring methods are needed. In this study, by introducing an IL-2 neutralizing monoclonal antibody (IL-2 N-mAb) and immunosuppressants into the culture with the presence of specific stimulators and activated lymphocytes, an activated lymphocyte-specific assay (ALSA) system was established to detect the specific activated lymphocytes. This assay demonstrated that the suppression in the ALSA test was closely related to the existence of specific activated lymphocytes. The ALSA test was applied to 47 heart graft recipients and the proliferation of activated lymphocytes from all rejection recipients proven by endomyocardial biopsies was found to be inhibited by spleen cells from the corresponding donors, suggesting that this suppression could reflect the existence of activated lymphocytes against donor antigens, and thus the rejection of a heart graft. The sensitivity of the ALSA test in these 47 heart graft recipients was 100%; however, the specificity was only 37.5%. It was also demonstrated that IL-2 N-mAb was indispensible, and the proper culture time courses and concentrations of stimulators were essential for the ALSA test. This preliminary study with 47 grafts revealed that the ALSA test was a promising noninvasive tool, which could be used in vitro to assist with the diagnosis of rejection post-heart transplantation.