Bisheng Shi

Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

BACKGROUND On March 30, a novel influenza A subtype H7N9 virus (A/H7N9) was detected in patients with severe respiratory disease in eastern China. Virological factors associated with a poor clinical outcome for this virus remain unclear. We quantified the viral load and analysed antiviral resistance mutations in specimens from patients with A/H7N9. METHODS We studied 14 patients with A/H7N9 disease admitted to the Shanghai Public Health Clinical Centre (SPHCC), China, between April 4, and April 20, 2013, who were given antiviral treatment (oseltamivir or peramivir) for less than 2 days before admission. We investigated the viral load in throat, stool, serum, and urine specimens obtained sequentially from these patients. We also sequenced viral RNA from these specimens to study the mutations associated with resistance to neuraminidase inhibitors and their association with disease outcome. FINDINGS All patients developed pneumonia, seven of them required mechanical ventilation, and three of them further deteriorated to become dependent on extracorporeal membrane oxygenation (ECMO), two of whom died. Antiviral treatment was associated with a reduction of viral load in throat swab specimens in 11 surviving patients. Three patients with persistently high viral load in the throat in spite of antiviral therapy became ECMO dependent. An Arg292Lys mutation in the virus neuraminidase (NA) gene known to confer resistance to both zanamivir and oseltamivir was identified in two of these patients, both also received corticosteroid treatment. In one of them, wild-type sequence Arg292 was noted 2 days after start of antiviral treatment, and the resistant mutant Lys292 dominated 9 days after start of treatment. INTERPRETATION Reduction of viral load following antiviral treatment correlated with improved outcome. Emergence of NA Arg292Lys mutation in two patients who also received corticosteroid treatment led to treatment failure and a poor clinical outcome. The emergence of antiviral resistance in A/H7N9 viruses, especially in patients receiving corticosteroid therapy, is concerning, needs to be closely monitored, and considered in pandemic preparedness planning. FUNDING National Megaprojects of China for Infectious Diseases, Shanghai Municipal Health and Family Planning Commission, the National Key Basic Research Program of China, Ministry of Science and Technology, and National Natural Science Foundation of China.

HBsAg inhibits TLR9-mediated activation and IFN-α production in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), the professional producers of type I interferons (IFN-alpha/beta), play a pivotal role in innate and adaptive immune responses against viral infections. Although functional impairment of circulating pDCs in chronic hepatitis B (CHB) patients has been reported previously, the mechanism responsible for these defects remains unclear. We hypothesize that HBsAg circulating in high amounts during HBV infection may interact with pDC and contribute to pDC dysfunction. In support of this hypothesis we show that pDCs treated with HBsAg secreted much less IFN-alpha than control pDCs. Furthermore, suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-alpha secretion. HBsAg inhibited TLR9-mediated IRF-7 expression and nuclear translocation, which are important for induction of IFN-alpha gene transcription. HBsAg upregulated the SOCS-1 expression and bound to BDCA-2 receptors on the plasma membrane of pDCs, resulting in the inhibition of the IFN-alpha production. In conclusion, the above data suggested that HBsAg may directly interfere with the function of pDC through HBsAg-mediated upregulation of SOCS-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.

Hepatitis B Virus Surface Antigen Selectively Inhibits TLR2 Ligand–Induced IL-12 Production in Monocytes/Macrophages by Interfering with JNK Activation

It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK–MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

HBsAg inhibits IFN-α production in plasmacytoid dendritic cells through TNF-α and IL-10 induction in monocytes.

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.

Measles Virus Infection in Adults Induces Production of IL-10 and Is Associated with Increased CD4+CD25+ Regulatory T Cells

Despite steady progress in elimination of measles virus globally, measles infection still causes 500,000 annual deaths, mostly in developing countries where endemic measles strains still circulate. Many adults are infected every year in China, with symptoms more severe than those observed in children. In this study, we have used blood samples from adult measles patients in Shanghai and age-matched healthy controls to gain an understanding of the immune status of adult measles patients. IFN-α mRNA was reduced in patient PBMC compared with healthy controls. In contrast, gene expression and plasma production of IL-2, IL-10, and IFN-γ were elevated in patient blood. A similar cytokine profile was observed at early times when cultured PBMC were infected with a clinical isolate of measles virus. In contrast to previous studies in pediatric patients, we did not find a reduction in total CD4+ and CD8+ T cells in patient PBMC. Interestingly, we found that CD4+CD25+CD127low regulatory T cells were significantly increased in patient PBMC compared with controls. Using intracellular cytokine staining we also show that the measles virus induces IL-10-producing CD14+ and CD4+CD25+ cells in PBMC. Our results show that adult measles patients in the acute phase of the disease have a mixed Th1/Th2 type response, accompanied with severe immunosuppression of both innate and adaptive responses including suppression of type I IFN. Both regulatory T cells and plasma IL-10 may contribute to the immunosuppression.

Polarization of Monocytic Myeloid-Derived Suppressor Cells by Hepatitis B Surface Antigen Is Mediated via ERK/IL-6/STAT3 Signaling Feedback and Restrains the Activation of T Cells in Chronic Hepatitis B Virus Infection.

Chronic hepatitis B virus (HBV) infection is characterized by T cell tolerance to virus. Although inhibition of T cell responses by myeloid-derived suppressor cells (MDSCs) has been observed in patients with chronic hepatitis B (CHB), the mechanism for expansion of MDSCs remains ambiguous. In this study, a significant increased frequency of monocytic MDSCs (mMDSCs) was shown positively correlated to level of HBsAg in the patients with CHB. We further found hepatitis B surface Ag (HBsAg) efficiently promoted differentiation of mMDSCs in vitro, and monocytes in PBMCs performed as the progenitors. This required the activation of ERK/IL-6/STAT3 signaling feedback. Importantly, the mMDSCs polarized by HBsAg in vitro acquired the ability to suppress T cell activation. Additionally, treatment of all-trans retinoic acid, an MDSC-targeted drug, restored the proliferation and IFN-γ production by HBV-specific CD4+ and CD8+ T cells in PBMCs from patients with CHB and prevented increase of viral load in mouse model. In summary, HBsAg maintains HBV persistence and suppresses T cell responses by promoting differentiation of monocytes into mMDSCs. A therapy aimed at the abrogation of MDSCs may help to disrupt immune suppression in patients with CHB.

Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses.

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg+alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-gamma and IL-4, but neither IFN-gamma nor IL-4 was detected in the HBsAg+alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.

Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patientsfrom healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients’ plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for various maladies.

Inhibition of hepatitis B virus replication by cIAP2 involves accelerating the ubiquitin-proteasome-mediated destruction of polymerase.

ABSTRACT Cellular inhibitor of apoptosis protein 2 (cIAP2) is a potent suppressor of apoptotic cell death. We have shown previously that cIAP2 is involved in the tumor necrosis factor alpha (TNF-α)-induced anti-hepatitis B virus (HBV) response; however, the mechanism for this antiviral effect remains unclear. In the present study, we demonstrate that cIAP2 can significantly reduce the levels of HBV DNA replication intermediates but not the total viral RNA or core protein levels. Domain-mapping analysis revealed that the carboxy-terminal domains of cIAP2 were indispensable for this anti-HBV ability and that an E3 ligase-deficient mutant of cIAP2 (termed cIAP2*) completely lost its antiviral activity. We further identified HBV polymerase as the target of cIAP2. Overexpression of cIAP2 but not cIAP2* reduced polymerase protein levels, while cIAP2 knockdown increased polymerase expression. In addition, we observed that cIAP2 promoted the degradation of the viral polymerase through a proteasome-dependent pathway. Further experiments demonstrated that cIAP2 can bind to polymerase and promote its polyubiquitylation. Finally, we found that cIAP2 downregulated the encapsidation of HBV pregenomic RNA. Taken together, these data reveal a novel mechanism for the inhibition of HBV replication by cIAP2 via acceleration of the ubiquitin-proteasome-mediated decay of polymerase and reduction of the encapsidation of HBV pregenomic RNA, making this mechanism a novel strategy for HBV therapy.

Infection of inbred BALB/c and C57BL/6 and outbred Institute of Cancer Research mice with the emerging H7N9 avian influenza virus

A new avian-origin influenza virus A (H7N9) recently crossed the species barrier and infected humans; therefore, there is an urgent need to establish mammalian animal models for studying the pathogenic mechanism of this strain and the immunological response. In this study, we attempted to develop mouse models of H7N9 infection because mice are traditionally the most convenient models for studying influenza viruses. We showed that the novel A (H7N9) virus isolated from a patient could infect inbred BALB/c and C57BL/6 mice as well as outbred Institute of Cancer Research (ICR) mice. The amount of bodyweight lost showed differences at 7 days post infection (d.p.i.) (BALB/c mice 30%, C57BL/6 and ICR mice approximately 20%), and the lung indexes were increased both at 3 d.p.i. and at 7 d.p.i.. Immunohistochemistry demonstrated the existence of the H7N9 viruses in the lungs of the infected mice, and these findings were verified by quantitative real-time polymerase chain reaction (RT-PCR) and 50% tissue culture infectious dose (TCID50) detection at 3 d.p.i. and 7 d.p.i.. Histopathological changes occurred in the infected lungs, including pulmonary interstitial inflammatory lesions, pulmonary oedema and haemorrhages. Furthermore, because the most clinically severe cases were in elderly patients, we analysed the H7N9 infections in both young and old ICR mice. The old ICR mice showed more severe infections with more bodyweight lost and a higher lung index than the young ICR mice. Compared with the young ICR mice, the old mice showed a delayed clearance of the H7N9 virus and higher inflammation in the lungs. Thus, old ICR mice could partially mimic the more severe illness in elderly patients.