The heterophilicic epitopes in conserved HA regions of human and avian influenza viruses can produce antibodies that bound to kidney tissue.

Influenza virus infection can cause kidney damage. However, the link between influenza infection and disease is still unclear. The purpose of this study was to analyze the relationship between heterophilic epitopes on H5N1 hemagglutinin (HA) and disease. The monoclonal antibody (mAb) against H5N1 was prepared, mAbs binding to human kidney tissue were screened, and the reactivities of mAbs with five different subtypes of influenza virus were detected. Design and synthesize the peptides according to the common amino acid sequence of these antigens, and analyze the distribution of the epitope on the crystal structure of HA. Immunological methods were used to detect whether the heterophilic epitopes could induce the production of antibodies that cross-react with kidney tissue. The results showed that H5-30 mA b binding to human kidney tissue recognized the heterophilic epitope 191-LVLWGIHHP-199 on the head of HA. The key amino acid were V192, L193, W194 and I196, which were highly conserved in human and avian influenza virus HA. The heterophilic epitope could induce mice to produce different mAbs binding to kidney tissue. Such heterophilic antibodies were also detected in the serum of the patients. It can provide materials for the mechanism of renal diseases caused by influenza virus infection.

Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance.

BACKGROUND: Immature dendritic cells (imDCs) play an important role in the induction of donor-specific transplant immunotolerance. However, these cells have limitations, such as rapid maturation and a short lifespan in vivo. In previous studies, induced pluripotent stem cells (iPSCs) differentiated into imDCs, and sinomenine (SN) was used to inhibit the maturation of imDCs. AIM: To study the capacity of SN to maintain iPSC-derived imDCs (SN-iPSCs-imDCs) in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance. METHODS: In this study, mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN (iPSCs-imDCs and SN-iPSCs-imDCs). The imDC-related surface markers, endocytotic capacity of fluorescein isothiocyanate-Dextran and apoptosis were analyzed by flow cytometry. The effects of iPSCs-imDCs and SN-iPSCs-imDCs on T-cell stimulatory function, and regulatory T (Treg) cell proliferative function in vitro were analyzed by mixed lymphocyte reaction. Cytokine expression was detected by ELISA. The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting. The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice. Statistical evaluation of graft survival was performed using Kaplan-Meier curves. RESULTS: Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained, and their biological characteristics and ability to induce immunotolerance were compared. SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs. Reduced major histocompatibility complex II expression, worse T-cell stimulatory function, higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs (P < 0.05). The levels of interleukin (IL)-2, IL-12, interferon-γ in SN-iPSCs-imDCs were lower than those in iPSCs-imDCs, whereas IL-10 and transforming growth factor-ß levels were higher (P < 0.05). The apoptosis rate of these cells was significantly higher (P < 0.05), and the expression levels of cleaved caspase3, Bax and cleaved poly(ADP-ribose) polymerase were higher after treatment with lipopolysaccharides, but Bcl-2 was reduced. In Balb/c mice recipients immunized with iPSCs-imDCs or SN-iPSCs-imDCs 7 d before skin grafting, the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+ T-cell proliferation (P < 0.05) and a higher capacity to induce CD4+CD25+FoxP3+ Treg cell proliferation in the spleen (P < 0.05). The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern. CONCLUSION: This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.

Identification and analysis of B cell epitopes of hemagglutinin of H1N1 influenza virus.

The frequent variation of influenza virus hemagglutinin (HA) antigen is the main cause of influenza pandemic. Therefore, the study of B cell epitopes of HA is of great significance in the prevention and control of influenza virus. In this study, the split vaccine of 2009 H1N1 influenza virus was used as immunogen, and the monoclonal antibodies (mAbs) were prepared by conventional hybridoma fusion and screening techniques. The characteristics of mAbs were identified by ELISA method, Western-blot test and hemagglutination inhibition test (HI). Using the obtained mAbs as a tool, the B cell epitopes of HA were predicted by ELISA blocking test, sandwich ELISA method and computer simulation method. Finally, four mAbs against HA antigen of H1N1 influenza virus were obtained. The results of ELISA and computer prediction showed that there were at least two types of epitopes on HA of influenza virus. The results of this study complemented the existing methods for predicting HA epitopes, and also provided a new method for predicting other pathogenic microorganisms.

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.

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.