Altered gut microbiota is associated with the formation of occult hepatitis B virus infection.

Hepatitis B virus (HBV), a common blood transmission pathogen worldwide, can lead to viral hepatitis, cirrhosis, liver cancer, and other liver diseases. In particular, occult hepatitis B virus infection (OBI) may be caused by an immune response leading to suppressed virus replication. Gut microbiota can change the immunity status of the human body and, therefore, affect the replication of HBV. Thus, to identify whether there are differences in gut microbiota between HBV carriers and OBI carriers, we collected fecal samples from 18 HBV carriers, 24 OBI blood donors, and also 20 healthy blood donors as negative control. After 16S sequencing, we found that the abundance of Faecalibacterium was significantly reduced in samples from OBI blood donors compared with those from healthy blood donors. Compared with samples from HBV carriers, the samples from OBI blood donors had a significantly increased abundance of Subdoligranulum, which might stimulate immune activation, thus inhibiting HBV replication and contributing to the formation of occult infection. Our findings revealed the potential role of gut microbiota in the formation of OBI and further provided a novel strategy for the treatment of HBV infection.IMPORTANCEOccult hepatitis B virus infection (OBI) is a special form of hepatitis B virus infection with hepatitis B surface antigen (HBsAg) positive and hepatitis B virus (HBV) DNA negative. Gut microbiota may contribute to the immune response leading to suppressed virus replication and, thus, participates in the development of OBI. The study on gut microbiota of OBI blood donors provides novel data considerably advancing our understanding of the immune mechanism for the determination of occult hepatitis B virus infection, which is helpful for improving the strategy of the treatment of HBV infection.

Adenoviruses vectored hepatitis C virus vaccine cocktails induce broadly specific immune responses against multi-genotypic HCV in mice.

BACKGROUND: Hepatitis C virus (HCV) vaccines are an urgent need to prevent hepatitis C and its further progression of hepatocellular carcinoma. Since the promising T cell based chimpanzee adenovirus and modified vaccinia virus Ankara vectorial HCV vaccines were failed in clinical phase II trial, the vaccine designs to improve protection efficacy in combination of cellular and humoral immunity have been hypothesized against multi-genotypic HCV. METHODS: Eight HCV vaccine strains were constructed with two novel adenovirus vectors (Sad23L and Ad49L) encoding E1E2 or NS3-5B proteins of HCV genotype (Gt) 1b and 6a isolates, covering 80 % HCV strains prevalent in south China and south-east Asia. Eight HCV vaccine strains were grouped into Sad23L-based vaccine cocktail-1 and Ad49L-based vaccine cocktail-2 for vaccinating mice, respectively. RESULTS: The immunogenicity of a single dose of 107-1010 PFU HCV individual vaccines was evaluated in mice, showing weak specific antibody to E1 and E2 protein but a dose-dependent T cell response to E1E2/NS3-5B peptides, which could be significantly enhanced by boosting with an alternative vector vaccine carrying homologous antigen. Prime-boost vaccinations with vaccine cocktail-1 and cocktail-2 induced significantly higher cross-reactive antibody and stronger T cell responses to HCV Gt-1b/6a. The high frequency of intrasplenic and intrahepatic NS31629-1637 CD8+ T cell responses were identified, in which the high proportion of TRM and TEM cells might play an important role against HCV infection in liver. CONCLUSIONS: Prime-boost regimens with HCV vaccine cocktails elicited the broad cross-reactive antibody and robust T cell responses against multi-genotypic HCV in mice.

Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population.

Hepatitis B virus (HBV) genotype C is a prevalent HBV genotype in the Chinese population. Although genotype C shows higher sequence heterogeneity and more severe liver disease than other genotypes, its pathogenesis and immunological traits are not yet fully elucidated. In this study, we first established and chemically synthesized the consensus sequence based on representative 138 full-length HBV genotype C genomes from the Chinese population. The pHBV1.3C plasmid system, containing a 1.3-fold full-length HBV genotype C consensus sequence, was constructed for subsequent validation. Next, we performed functional assays to investigate the replicative competence of pHBV1.3C in vitro through the transient transfection of HepG2 and Huh7 cells and validated the in vivo function via a hydrodynamic injection to BALB/c recipient mice. The in vitro investigation revealed that the extracellular HBV DNA and intracellular replicative intermediate (i.e., pregenomic RNA, pgRNA) were apparently measurable at 48 h, and the HBsAg and HBcAg were still positive in hepatoma cells at 96 h. We also found that HBsAg and HBeAg accumulated at the extracellular and intracellular levels in a time-dependent manner. The in vivo validation demonstrated that pHBV1.3C plasmids induced HBV viremia, triggered morphological changes and HBsAg- or HBcAg- positivity of hepatocytes, and ultimately caused inflammatory infiltration and focal or piecemeal necrosis in the livers of the murine recipients. HBV protein (HBsAg) colocalized with CD8+ T cells or CD4+ T cells in the liver. F4/80+ Kupffer cells were abundantly recruited around the altered murine hepatocytes. Taken together, our results indicate that the synthetic consensus sequence of HBV genotype C is replication-competent in vitro and in vivo. This genotype C consensus genome supports the full HBV life cycle, which is conducive to studying its pathogenesis and immune response, screening novel antiviral agents, and further optimizing testing and therapeutics.

Occult Hepatitis B Virus Infection and Liver Fibrosis in Chinese Patients.

BACKGROUND: The impact of hepatitis B surface antigen (HBsAg)-negative/hepatitis B virus (HBV) DNA-positive occult HBV infection (OBI) on the severity of liver fibrosis remains unclear. METHODS: A total of 1772 patients negative for HBsAg but positive for antibody to hepatitis B core antigen (HBcAg), stratified by the presence or absence of OBI, were selected for long-term carriage leading to elevation of ≥2 of 4 liver fibrosis indexes-hyaluronic acid (HA), laminin, type III procollagen peptide (PCIII), and type IV collagen (CIV)-at testing in a Chinese hospital. Patients were tested for serum viral load, HBV markers, and histopathological changes in liver biopsy specimens. RESULTS: OBI was identified in 148 patients with liver fibrosis (8.4%), who had significantly higher levels of HA, laminin, PCIII, and CIV than 1624 fibrotic patients without OBI (P < .05). In 36 patients with OBI who underwent liver biopsy, significant correlations were observed between OBI viral load and serum HA levels (P = .01), PCIII levels (P = .01), and pathological histological activity index (HAI) scores (P < .001), respectively; HAI scores and PCIII levels (P = .04); HBcAg immunohistochemical scores and HA levels (P < .001); and HBcAg immunohistochemical scores and PCIII levels (P = .03). Positive fluorescent in situ hybridization results were significantly more frequent in patients with OBIs (80.6% vs 37.5% in those without OBIs). Among patients with OBIs, HBcAg was detected in the liver tissue in 52.8% and HBsAg in 5.6%. CONCLUSIONS: OBI status appears to be associated with liver fibrosis severity.

Mechanism and intervention of murine transfusion-related acute lung injury caused by anti-CD36 antibodies.

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab-mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab-mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab')2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab')2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab')2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.

Current Status of and Global Trends in Platelet Transfusion Refractoriness From 2004 to 2021: A Bibliometric Analysis.

Platelet transfusion refractoriness (PTR) is common in patients with hematology and oncology and is becoming an important barrier in the treatment of thrombocytopenia and hemorrhage. Bibliometrics is an effective method for identifying existing research achievements, important breakthroughs, current research hotspots, and future development trends in any given field. In recent years, research on PTR has received increasing attention, but a bibliometric analysis of this field has not yet been reported. In this study, we applied bibliometrics to analyze the existing literature on PTR research over the past 17 years. On November 1, 2021, we began a publications analysis of PTR research using the Science Citation Index Expanded of the Web of Science Core Collection with collection dates from 2004 to 2021. This research aimed to summarize the state of PTR research using Bibliometrix to identify connections between different elements (i.e., authors, institutions, countries, journals, references, and keywords) using VOS viewer analyses to visualize key topics and trends in PTR research using Cite Space and gCLUTO. The results of all 310 studies showed that the annual number of publications focused on PTR is steadily increasing, with the United States of America and Japan making significant contributions. We noted that the research group led by Dr. Sherrill J. Slichter was prominent in this field, while Estcourt Lise may become the most influential newcomer. Transfusion was the most popular journal, and Blood was the most cited journal. Using various analyses, including co-cited analysis, historiography analysis, citation burst analysis, and factorial analysis, we pointed out and discussed contributing publications. According to occurrence analysis, co-word biclustering analysis, landform map, thematic evolution, and thematic map, we believe that "activation," "p-selection," "CD36 deficiency," "gene-frequencies," "CD109," "HPA-1," and "beta (3) integrin" may become new trends in PTR research. The outcome of our bibliometric analyses has, for the first time, revealed profound insights into the current state and trends in PTR research. The systematic analysis provided by our study clearly demonstrates the field's significant advancements to all researchers who are interested in a quick and comprehensive introduction to the field.

Incidence of anti-D alloimmunization in D-negative individuals receiving D-positive red blood cell transfusion: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: The transfusion of D-negative red blood cells (RBCs) to D-negative patients has been widely adopted to prevent anti-D alloimmunization, especially in women of childbearing age. Still, transfusion of D-positive RBCs to D-negative recipients is occasionally inevitable in practice, and the resulting incidence of anti-D in different D-negative groups of patients has not been well summarized. MATERIALS AND METHODS: We searched the relevant literature using PubMed, Cochrane Library, and Embase databases from inception date to 30 September 2021. We looked for studies of anti-D occurring in D-negative recipients who received D-positive RBC transfusions. The anti-D incidence was summarized with 95% confidence intervals (CIs). Data with similar characteristics were combined using a random-effects model. RESULTS: About 42 studies (2226 cases), which found anti-D, the exact volume of D-positive RBC transfused, and the follow-up time for anti-D detection, met the inclusion criteria. The pooled anti-D incidence was 64% (95% CI, range 55%-74%) in volunteers receiving small volumes of D-positive RBCs, 84% (95% CI, 74%-94%) in those receiving whole units, 26% (95% CI, 19%-32%) in mixed patients, 12% (95% CI, 8%-16%) in oncology patients, 27% (95% CI, 13%-40%) in trauma patients, 4% (95% CI, 0%-8%) in immune-compromised transplant patients, and 6% (95% CI, 1%-39%) in those with AIDS. CONCLUSION: Compared with the high frequency of anti-D in healthy D-negative volunteers given D-positive RBCs, we found a lower rate of anti-D immunization in various D-negative patients and almost none in transplant and AIDS patients.

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5'UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3'UTR by constructing an intragenotypic recombinant using 5'UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3'UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5'UTR-NS5A and NS5B-3'UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml-1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Brucella Outer Membrane Lipoproteins 19 and 16 Differentially Induce Interleukin-18 Response or Pyroptosis in Human Monocytic Cells.

BACKGROUND: Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1). METHODS: Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively. RESULTS: Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1ß response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. CONCLUSIONS: Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.

Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates.

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 109 PFU Sad23L-nCoV-S, followed by boosting with 5 × 109 PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 103.16 anti-S, 102.75 anti-RBD binding antibody and 102.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 101.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/106 cells), IL-2 (334 SFCs/106 cells) and intracellular IFN-γ in CD4+/CD8+ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

Prevalence and evolutionary analyses of human T-cell lymphotropic virus in Guangdong province, China: Transcontinental and Japanese subtype lineages dominate the prevalence.

To systematically characterize the prevalence and evolution of human T-cell lymphotropic virus (HTLV) infection among voluntary blood donors (BDs) in Guangdong province, China. A three-year survey for HTLV epidemiology among BDs was performed in Guangdong during 2016-2018. Anti-HTLV-1/2 was screened by ELISA and ECLIA, and subsequently confirmed by western blot (WB) and nucleic acid testing (NAT). The prevalence of HTLV in donors from different cities was calculated. The identified HTLV-positive cases were phylogenetically genotyped and analyzed in a Bayesian phylogenetic framework. Among 3,262,271 BDs, 59 were confirmed positive for HTLV-1 (1.81 per 100,000) and no HTLV-2 infection was found. The prevalence of HTLV-1 varied significantly among 21 cities in Guangdong province, China. The highest prevalence was found in donors from Shanwei (13.94 per 100,000), which is a coastal city in eastern Guangdong. Viral genomic sequences genotyped from 55 HTLV-1 carriers showed that 39 were transcontinental subtype and 16 were Japanese subtype. Specially, 13 out of 39 transcontinental subtype sequences were characterized with L55P mutation and 21 out of 55 sequences were characterized with L19F mutation in viral gp46 protein. The L55P mutation seemed be specific to eastern Asia since it only presented in the sequences from Japan, mainland China, and Taiwan. Phylogenetic analysis of gp46 gene shows that HTLV-1a may have been introduced to Guangdong through four different introduction events and formed major transmission clusters: clades I(13,602 years ago), II(16, 010 years ago), III(15,639 years ago) and IV(16,517 years ago). In general, Guangdong is considered to be a low-prevalence region for HTLV-1 infection, but the prevalence is significantly higher in Shanwei city. Transcontinental and Japanese subtype lineages dominate the prevalence in Guangdong. In terms of blood safety, HTLV antibody screening for first-time blood donors can effectively reduce the risk of HTLV transmission.

Role of core protein mutations in the development of occult HBV infection.

BACKGROUND & AIMS: Occult HBV infection (OBI) is associated with transfusion-transmitted HBV infection and hepatocellular carcinoma. Studies on OBI genesis have concentrated on mutations in the S region and the regulatory elements. Herein, we aimed to determine the role of mutations in the core region on OBIs. METHODS: An OBI strain (SZA) carrying 9 amino acid (aa) substitutions in the core protein/capsid (Cp) was selected by sequence alignment and Western blot analysis from 26 genotype B OBI samples to extensively explore the impact of Cp mutations on viral antigen production in vitro and in vivo. RESULTS: A large panel of 30 Cp replicons were generated by a replication-competent pHBV1.3 carrying SZA or wild-type (WT) Cp in a 1.3-fold over-length of HBV genome, in which the various Cp mutants were individually introduced by repairing site mutations of SZA-Cp or creating site mutations of WT-Cp by site-directed mutagenesis. The expression of HBcAg, HBeAg, and HBsAg and viral RNA was quantified from individual SZA and WT Cp mutant replicons in transfected Huh7 cells or infected mice, respectively. An analysis of the effect of Cp mutants on intracellular or extracellular viral protein production indicated that the W62R mutation in Cp had a critical impact on the reduction of HBcAg and HBeAg production during HBV replication, whereas P50H and/or S74G mutations played a limited role in influencing viral protein production invivo. CONCLUSIONS: W62R and its combination mutations in HBV Cp might massively affect HBcAg and HBeAg production during viral replication, which, in turn, might contribute to the occurrence of OBI. LAY SUMMARY: Occult hepatitis B virus infections (OBIs) have been found to be associated with amino acid mutations in the S region of the HBV, but the role of mutations in the core protein (Cp) remains unclear. In this study, an OBI strain (SZA) carrying 9 amino acid substitutions in Cp has been examined comprehensively in vitro and in vivo. The W62R mutation in Cp majorly reduces HBcAg and HBeAg production during HBV replication, potentially contributing to the occurrence of OBI.

Marmoset Viral Hepatic Inflammation Induced by Hepatitis C Virus Core Protein via IL-32.

Common marmosets infected with GB virus-B (GBV-B) chimeras containing hepatitis C virus (HCV) core and envelope proteins (CE1E2p7) developed more severe hepatitis than those infected with HCV envelope proteins (E1E2p7), suggesting that HCV core protein might be involved in the pathogenesis of viral hepatitis. The potential role of HCV core in hepatic inflammation was investigated. Six individual cDNA libraries of liver tissues from HCV CE1E2p7 or E1E2p7 chimera-infected marmosets (three animals per group) were constructed and sequenced. By differential expression gene analysis, 30 of 632 mRNA transcripts were correlated with the immune system process, which might be associated with hepatitis. A protein-protein interaction network was constituted by STRING database based on these 30 differentially expressed genes (DEGs), showing that IL-32 might play a central regulatory role in HCV core-related hepatitis. To investigate the effect of HCV core protein on IL-32 production, HCV core expressing and mock constructs were transfected into Huh7 cells. IL-32 mRNA and secretion protein were detected at significantly higher levels in cells expressing HCV core protein than in those without HCV core expression (P < 0.01 and P < 0.001, respectively). By KEGG enrichment analysis and using the specific signaling pathway inhibitor LY294002 for inhibition of PI3K, IL-32 expression was significantly reduced (P < 0.001). In conclusion, HCV core protein induces an increase of IL-32 expression via the PI3K pathway in hepatic cells, which played a major role in development of HCV-related severe hepatitis.

Novel hepatitis B virus surface antigen mutations associated with occult genotype B hepatitis B virus infection affect HBsAg detection.

The causative factors of occult hepatitis B infection are complicated and not yet been fully elucidated. Mutations in hepatitis B virus (HBV) S gene are one of the factors may contributing to occult infection. In this study, 89 blood donors with genotype B occult HBV infection were investigated. Fifty-seven hepatitis B surface antigen (HBsAg)-positive/HBV DNA-positive blood donors served as control group for comparison. Occult HBV-related mutations with a high incidence (P < .05) in the S gene were identified. To further verify these occult infection-related mutations, a conservative full-gene expression vector of HBV B genotype (pHBV1.3B) was constructed. Then, the mutant plasmids on the basis of pHBV1.3B were constructed and transfected into HepG2 cells. Extracellular as well as intracellular HBsAg was analysed by electrochemical luminescence and cellular immunohistochemistry. Ten occult infection-related mutations (E2G, Q101R, K122R, M133T, D144E, G145R, V168A, S174N, L175S and I226S) were significantly more frequent in the occult infection group (P < .05). Five of the ten mutations (E2G, D144E, G145R, V168A and S174N) strongly decreased extracellular HBsAg level (P < .05) in the transfection system. Notably, the E2G mutation had the most significant impact on the ratio of extracellular HBsAg (3.8% vs pHBV1.3B) and intracellular HBsAg (239.3% vs pHBV1.3B) (P < .05), and the fluorescence density of E2G mutant HBsAg was significantly higher than that of pHBV1.3B (P < .0001). Hence, ten mutations were associated with genotype B occult HBV infection; E2G and V168A were novel mutations which we confirmed significantly affect HBsAg detection. E2G might cause HBsAg secretion impairment that results in intracellular accumulation and a decrease in HBsAg secretion.

A high infectious simian adenovirus type 23 vector based vaccine efficiently protects common marmosets against Zika virus infection.

Zika virus (ZIKV) has spread in many countries or territories causing severe neurologic complications with potential fatal outcomes. The small primate common marmosets are susceptible to ZIKV, mimicking key features of human infection. Here, a novel simian adenovirus type 23 vector-based vaccine expressing ZIKV pre-membrane-envelope proteins (Sad23L-prM-E) was produced in high infectious titer. Due to determination of immunogenicity in mice, a single-dose of 3×108 PFU Sad23L-prM-E vaccine was intramuscularly inoculated to marmosets. This vaccine raised antibody titers of 104.07 E-specific and 103.13 neutralizing antibody (NAb), as well as robust specific IFN-γ secreting T-cell response (1,219 SFCs/106 cells) to E peptides. The vaccinated marmosets, upon challenge with a high dose of ZIKV (105 PFU) six weeks post prime immunization, reduced viremia by more than 100 folds, and the low level of detectable viral RNA (<103 copies/ml) in blood, saliva, urine and feces was promptly eliminated when the secondary NAb (titer >103.66) and T-cell response (>726 SFCs/106 PBMCs) were acquired 1-2 weeks post exposure to ZIKV, while non-vaccinated control marmosets developed long-term high titer of ZIKV (105.73 copies/ml) (P<0.05). No significant pathological lesions were observed in marmoset tissues. Sad23L-prM-E vaccine was detectable in spleen, liver and PBMCs at least 4 months post challenge. In conclusion, a prime immunization with Sad23L-prM-E vaccine was able to protect marmosets against ZIKV infection when exposed to a high dose of ZIKV. This Sad23L-prM-E vaccine is a promising vaccine candidate for prevention of ZIKV infection in humans.

Occurrence of occult hepatitis B virus infection associated with envelope protein mutations according to anti-HBs carriage in blood donors.

OBJECTIVES: Occult hepatitis B virus infection (OBI) carries a risk of hepatitis B virus (HBV) transmission and hepatocellular carcinoma. As previous studies have had a limited sample size, the characteristics of OBI with genotype B and C (OBIB and OBIC) mutations relating to hepatitis B surface antibody (anti-HBs) elicited by vaccination or a limited host immune response to HBV have not been fully explored. METHODS: In this study, the occurrence of OBIB or OBIC strains associated with envelope protein (pre-S/S) amino acid substitutions obtained from 99 blood donors stratified according to anti-HBs carriage were characterized extensively. RESULTS: According to the presence of anti-HBs within each genotype, the number and frequency of substitution sites specific for anti-HBs(-) OBIB were higher than those specific for anti-HBs(+) OBIB strains (67 vs 31; 117 vs 41), but the reverse pattern was found in OBIC strains (3 vs 24; 3 vs 26). Mutations pre-s1T68I and sQ129R/L were found uniquely in 15-25% of anti-HBs(+) OBIB carriers and mutation pre-s1A54E was found preferentially in anti-HBs(+) OBIC, while 17 substitutions were found preferentially in 11-38% of anti-HBs(-) OBIB strains. In the major hydrophilic region (MHR) region, mutations sS167 in OBIB, sT118 in OBIC, and sA166 in both genotypes were possibly immune-induced escape mutation sites. CONCLUSIONS: Several mutations in pre-S/S of OBI appeared to be associated with carrier anti-HBs pressure, which might be risk factors for potential reactivation of viruses under anti-HBs selection in OBI carriers.

Seroprevalence of Human Adenovirus Type 5 Neutralizing Antibody in Common Marmosets Determined by a New Set of Two Assays.

Preexisting neutralizing antibody (NAb) against human adenovirus serotype 5 (AdHu5) can reduce the immunogenicity of AdHu5 vector-based vaccine, thus inhibiting the host's immune response and utility of other homologous vectors. Common marmoset (Callithrix jacchus), a small new world primate, has attracted considerable attention for its potential as a preclinical research model of vaccine development. However, the prevalence of anti-AdHu5 NAb activity in common marmosets bred in China remains unknown. A recombinant adenovirus expressing luciferase and Zs Green reporter genes were constructed to detect NAb against rAdHu5 by flow cytometry (FCM) and chemiluminescence (CL) assay. Five of 25 marmosets (20%) presented AdHu5 NAb detectable by FCM. Four animals had low titer (1/16), while the fifth one reached 1/64. While by CL assay, 7 of 25 (28%) marmosets were anti-AdHu5 NAb positive. Four animals, two of whom were negative by FCM, also had low titer NAb (1/16), suggesting assay discrepancy at low levels. Two marmosets, 1/32 titer by CL, were at 1/16 by FCM. A single animal showed a high titer with both assays (1/128 and 1/64 by CL and FCM, respectively). The CL method was simpler, more sensitive, accurate, and stable. The low prevalence of preexisting anti-AdHu5 NAb in marmosets provides important background information on the feasibility and applicability of using marmosets as a preclinical research model for vaccine development.

Hepatitis C virus 3b strains in injection drug users in Guangdong Province, China, may have originated in Yunnan Province.

The distribution of hepatitis C virus (HCV) genotypes/subtypes varies among different populations. Here, we investigated HCV infection and its genotype distribution in injection drug users (IDUs) in Guangdong Province of China. A total of 318 IDUs from two prisons were recruited. The genotypes/subtypes of HCV in IDUs were determined by phylogenetic analysis using E1 and/or NS5B gene sequences. Our previous data on blood donors (BDs) with no history of drug use were used as control population data for comparison. Our results showed that the prevalence of HCV 3b (20.9% vs. 3.6%, P = 3.4E-9) and 6a (57.0% vs. 39.8%, P = 1.2E-5) was higher in IDUs than in BDs. In contrast, the prevalence of HCV 1b (43.4% vs. 5.6%, P = 9.8E-23) in BDs was higher than in IDUs. Phylogeographic analysis indicated that HCV 3b migrated from Yunnan to Guangdong Province and became endemic, with further transmission to other regions of China. The trend of HCV 3b dissemination in China in IDUs requires further attention, and a strategy for prevention and therapy is needed.

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5'UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5'UTR and NS3-3'UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5'UTR and 3'UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated "CH6acc"), releasing HCV of 104.3-104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.

Finasteride Enhances the Generation of Human Myeloid-Derived Suppressor Cells by Up-Regulating the COX2/PGE2 Pathway.

Myeloid-derived suppressor cells (MDSCs) have been known to be a key factor in the regulation of the immune system under numerous conditions such as tumors, infections, autoimmune diseases, and transplantations. In contrast to the proposed deleterious role of MDSCs in tumors and infections, MDSCs with their suppressive function are now proved to have the beneficial potential of suppressing the autoimmune response and promoting tolerance to transplantation. Therefore, the expansion of MDSCs could be a promising therapeutic strategy for many diseases. In this study, we aimed to identify FDA-approved drugs that could aid in the expansion of functional MDSCs. We performed a high-throughput screening (HTS) of FDA-approved drugs based on the in vitro human MDSC-differentiation system and identified finasteride (FIN) to have the best potency to aid the generation of human MDSCs. The FIN-induced MDSCs were quite similar to monocytic MDSCs with regard to their surface phenotype, morphology, immunosuppressive function, and related gene expression. Next, we aimed to determine the mechanism of action of FIN and found that FIN induced the expansion of MDSCs through up-regulation of the COX2/PGE2 pathway by enhancing the activity of COX2 promoter. In addition, the administration of indomethacin (IND), a COX2 inhibitor, abrogated the effect of FIN. Based on these results, we suggested that FIN could find applications in the future in the expansion of MDSCs. Further development of FIN-like compounds could be a novel strategy for generating functional MDSCs for immunosuppressive therapies in various immune disorder conditions.