Zigang Shen

Cyclophilin A inhibits rotavirus replication by facilitating host IFN-I production

Rotavirus (RV) infection causes serious dehydrating diarrhoea in infants and newborn animals. Our previous study revealed that cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase (PPIase), could be temporarily upregulated in RV-infected MA104 cells in early stage of infection (unpublished data). To find out the possible roles of CYPA in RV infection, we overexpressed and silenced CYPA in various cell lines by gene transfection and shRNA. We found that transfection of CYPA significantly inhibited RV replication, while silencing the expression of CYPA significantly increased RV replication. Accordingly, overexpression of CYPA significantly increased IFN-β production; while silencing CYPA significantly reduced IFN-β production. This effect of CYPA on IFN-β production was independent of its PPIase activity. Moreover, IFN-β secreted by host cells in RV infection had a critical repressive effect on viral replication. Finally, we found that inhibiting JNK pathway by SP600125 and JNK siRNA abrogated the effect of CYPA on IFN-β transcription in RV-infected MA104 cells. Together, our data suggested that CYPA inhibited RV replication by facilitating host IFN-β production, which was independent on the PPIase activity of CYPA but dependent on the activation of JNK signaling pathway.

Protein prime-peptide boost as a new strategy induced an Eppin dominant B-cell epitope specific immune response and suppressed fertility.

To identify the neutralizing B-cell epitope of epididymal protease inhibitor (Eppin) and promote its immunogenicity, we designed four candidate B-cell epitopes peptide and employed a recombinant human Eppin (rhEppin) protein prime/peptide boost strategy to compare the immune responses and fertility inhibition effect with that of rhEppin or peptide alone vaccination. Our results indicate that priming with Eppin and boosting with different peptide showed similarly enhanced antibodies titers but different fertility inhibition effect, which coordinated with the motility inhibition of the antisera to human sperm. Thus we explored an alternative approach to induce dominant neutralizing B-cell epitope specific immune response and an ideal protocol for providing effective and long-term fertility inhibition of male mice.

Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted.

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4+ and CD8+ T cells, especially the CD4+CD25+Foxp3+ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.

Proteomic methods reveal cyclophilin a function as a host restriction factor against rotavirus infection

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl‐prolyl cis‐trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.

A novel dominant B-cell epitope of FSHR identified by molecular docking induced specific immune response and suppressed fertility

The follicle stimulating hormone (FSH) is of great importance in reproduction modulation of both sexes. The extracellular domain (ECD) of its receptor (FSHR) is crucial for FSH binding and subsequent signal transduction; therefore, it is the potential target for fertility control. To avoid unwanted side-effect when used as immunocontraceptive agent, the ECD was analysed by online prediction combined with molecular docking to identify the candidate B-cell epitopes. Four potential B-cell epitopes were identified and synthesised in tandem with Pan DR epitope. Then the epitope-based peptides were used to boost adult male mice following rhFSHR protein priming, thus to determine their immune responses and fertility inhibition capacity. Three of the four peptides showed suppressed fertility accompanied with small testis and lower serum testosterone level, which was consistent with absolutely lower sperm quantity and poor quality. Among the four epitope peptides, Pep2 displayed the lowest fertility rate of 26.67%, which was similar to that of rhFSHR homologously prime/boost mice (23.30 and 25.00%). Thus, we identified a novel immunodominant B-cell epitope by molecular docking and protein prime/peptide boost strategy.

Antiviral effects of cyclosporine A in neonatal mice with rotavirus-induced diarrhea.

Objectives: Because rotavirus gastroenteritis is associated with high morbidity and mortality especially in developing countries, it is necessary to develop antirotavirus drugs for the treatment of rotavirus infection. Previous studies have demonstrated that cyclosporin A (CsA) has antiviral properties against rotavirus. Its effect has not yet been evaluated against rotavirus diarrheal disease. The aim of this study was to assess the anti-rotavirus efficacy of CsA in neonatal mice after induction of rotavirus diarrhea. Methods: Suckling mice were inoculated with murine rotavirus. On the onset of diarrhea, mice were given different concentrations of CsA. To evaluate the effects of CsA on reduction of rotavirus diarrhea, diarrhea score, fecal virus shedding, and pathological lesion change in the small intestine, messenger RNA (mRNA) expression levels in the small intestine and spleen of mice were measured for type I interferon (IFN-&agr; and IFN-&bgr;), inflammation-related cytokines (interleukin [IL]-8, IL-10, IFN-&ggr;, and tumor necrosis factor-&agr;), and inflammatory signaling pathways (p38, c-Jun N-terminal kinase, activator protein-1, and nuclear factor-kappa B). Results: Among virus-inoculated and CsA-treated groups, a dose of 5 mg · kg−1 · day−1 of CsA inhibited diarrhea and improved fecal virus shedding and intestinal lesion changes. IFN-&bgr; mRNA expression was significantly increased in rotavirus-induced diarrhea mice treated with 5 mg · kg−1 · day−1 of CsA, whereas the mRNA expression levels of inflammation-related cytokines (IL-8, IL-10, IFN-&ggr;, and tumor necrosis factor-&agr;) and inflammatory signaling pathways (p38, c-Jun N-terminal kinase, activator protein-1, and nuclear factor-kappa B) were markedly decreased. Antiviral effects of CsA were dose dependent. Conclusions: CsA can inhibit rotavirus infection in neonatal mice through its antiviral properties. The mechanism for this may be through CsA suppression of inflammation by viral inhibition in animal models.

Astagalus Polysaccharide Attenuates Murine Colitis through Inhibiton of the NLRP3 Inflammasome.

Astragalus polysaccharide is an important bioactive component of Astragalus membranaceus, an herb used in traditional Chinese medicine for treating inflammatory bowel disease. The NOD-like receptor protein 3 inflammasome plays an important role in the pathogenesis of inflammatory bowel disease. However, little is known about the role of NOD-like receptor protein 3 inflammasome in Astragalus polysaccharide-treated mice with experimental colitis. For this study, we investigated the molecular mechanisms that underlie the treatment of inflammatory bowel disease by Astragalus polysaccharide. We show that Astragalus polysaccharide treatment reduces the disease activity index and histological injury scores compared to the colitis model group. Astragalus polysaccharide also effectively inhibited the expression of NOD-like receptor protein 3, apoptotic speck protein containing a c-terminal caspase recruitment domain, caspase-1, interleukin-18, and interleukin-1β, as shown by quantificational RT-PCR or the enzyme-linked immunosorbent assay. Furthermore, Astragalus polysaccharide treatments produced significant dose-dependent improvements in dextran sulfate sodium-induced experimental colitis. Our data provide the reliable evidence that Astragalus polysaccharide is able to exert a therapeutic effect in dextran sulfate sodium-induced colitis by inhibiting the activation of the NOD-like receptor protein 3 inflammasome, which acts to decrease the production of inflammatory factors such as interleukin-18 and interleukin-1β.

Induction of specific immune response and suppression of fertility by B-cell-epitope-based mimovirus vaccine

SPINLW1 (previously known as eppin (epididymal protease inhibitor)) is a target under intense scrutiny in the study of male contraceptive vaccines. B-cell-dominant epitopes are now recognized as key parts of the induction of humoral immune responses against target antigens. The generation of robust humoral responses in vivo has become a crucial problem in the development of modern vaccines. In this study, we developed a completely novel B-cell-dominant-epitope-based mimovirus vaccine, which is a kind of virus-size particulate antigen delivery system. The mimovirus successfully self-assembled from a cationic peptide containing a cell-penetrating peptide of TAT49-57 and a plasmid DNA encoding both three SPINLW1 (103-115) copies and adjuvant C3d3. The male mice were immunized with the epitope-based mimovirus vaccine, which resulted in a gradual elevation of specific serum IgG antibody levels. These reached a peak at week 4. Mating for the fertility assay showed that the mimovirus vaccine had accomplished a moderate fertility inhibition effect and investigation into the mechanism of action showed that it did so by interfering with the reproductive function of the sperm but that it did not damage the structures of the testes or cause serum testosterone to decline. Our results suggest an ideal protocol for suppressing fertility in mice by an engineered mimovirus vaccine.

A highly specific antibody response after protein prime-peptide boost immunization with Eppin/B-cell epitope in mice.

Eppin seems to be a promising target for developing immunocontraceptives for males. In an attempt to develop a safer vaccine, the immune response should be specifically directed according to its fertility inhibition mechanism. The mechanism of Eppin as an immunocontraceptive agent is now assumed to be the inhibitory effect of Eppin-specific antibody on sperm motility. Hence, to make Eppin a successful immunocontraceptive, avid and specific antibody responses have to be elicited. We had employed the inoculation modality of protein prime-peptide boost with rhEppin and epitope-based peptide, which has previously showed a satisfying result of fertility inhibition with minimal adverse effects. We here further explored the immunological features and efficiency of a protein prime-peptide boost strategy. The results of the present study showed that all the animals injected with rhEppin followed by epitope-based peptide boost, elicited enhanced specific anti-Eppin antibodies. The IgG subclasses of the antibodies generated by this regimen were primarily of IgG2b and IgG1 isotypes, and more importantly, the IgA level of epidydimis lavage was much higher, which correlated well with the results of cytokine profiles in spleen cell cultures. Furthermore, the results of peptide competition assays demonstrated that rhEppin alone prime-boost vaccination lead to a broader B-cell response while protein prime-epitope-peptide boosts directed the immune response mainly against the epitope. The results indicate that this immunization strategy may be advantageous in eliciting a highly specific humoral immune response.