Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

Immunogenicity of recombinant fowl-pox virus co-expressing structural protein precursor P1-2A and proteinase 3C of FMDV

Two recombinant plasmids, pUTA2P1 and pUTAL3CP1, were constructed by inserting structural pro-tein precursor P1-2A and proteinase 3C of foot-and-mouth disease virus (FMDV) into fowl-pox virus (FPV) recombi-nant vectors pUTA-2 and pUTA-16-LacZ respectively, and two recombinant FPVs (vUTA2P1 and vUTAL3CP1) screened by the RT-PCR, IFA assay and Western blotting assay were obtained successfully. Mice injected respectively with rFPVs were induced high level specific anti-FMDV an-tibodies, increasing of T subtypes, and higher cytotoxicities of splenocytes than those of control groups. These results indicated that a new method was used to construct a poten-tial candidate vaccine of FMDV.

Genetic and pathogenic characterization of new infectious bronchitis virus strains in the GVI-1 and GI-19 lineages isolated in central China

Avian infectious bronchitis(IB)is a highly contagious infectious disease caused by infectious bronchitis virus(IBV),which is prevalent in many countries worldwide and causes serious harm to the poultry industry.At present,many commercial IBV vaccines have been used for the prevention and control of IB;however,IB outbreaks occur frequently.In this study,two new strains of IBV,SX/2106 and SX/2204,were isolated from two flocks which were immunized with IBV H120 vaccine in central China.Phylogenetic and recombination analysis indicated that SX/2106,which was clustered into the GI-19 lineage,may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine.Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage,which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine.The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120.Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death,and H120 immunization could not provide effective protection against the two IBV isolates.It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously,with a mortality rate up to 60%.Considering the continuous mutation and recombination of the IBV genome to produce new variant strains,it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics.

Identification and characterization of genotype A and D recombinant hepatitis B virus from Indian chronic HBV isolates

AIM： To confirm the presence of recombination, fulllength hepatitis B virus （HBV） from chronic patients was sequenced and analyzed. METHODS： Full-length HBV genomes from 12 patients were amplified and sequenced in an automated sequencer. Phylogenetic analysis was carried out on full-length, Core and preS2/Surface regions using MEGA software. SimPIot Boot Scanning and amino acid sequence analysis were performed for confirmation of recombination. RESULTS： Eight patients were infected with genotype D strain; one patient with genotype A and three patients had genotype A and D recombination; two of them had cirrhosis and one had hepatocellular carcinoma. Phylogenetic analysis of core and preS2/surface regions separately showed evidence of genotype A and D recombination. The breakpoints of recombination were found to be at the start of preS2 and at the endof surface coding regions. CONCLUSION： We identified and characterized recombinant A and D genotype HBV in hepatitis B surface antigen （HBsAg）-positive patients.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Rabies Virus Neutralizing Activity,Safety,and Immunogenicity of Recombinant Human Rabies Antibody Compared with Human Rabies Immunoglobulin in Healthy Adults

Objective Preliminary assessment of rabies virus neutralizing activity,safety and immunogenicity of a recombinant human rabies antibody(NM57)compared with human rabies immunoglobulin(HRIG)in Chinese healthy adults.Methods Subjects were randomly(1:1:1)allocated to Groups A(20 IU/kg NM57),B(40 IU/kg NM57),or C(20 IU/kg HRIG).One injection was given on the day of enrollment.Blood samples were collected on days-7 to 0(pre-injection),3,7,14,28,and 42.Adverse events(AEs)and serious AEs(SAEs)were recorded over a period of 42 days after injection.Results All 60 subjects developed detectable rabies virus neutralizing antibodies(RVNAs)(>0.05 IU/mL)on days 3,7,14,28,and 42.The RVNA levels peaked on day 3 in all three groups,with a geometric mean concentration(GMC)of 0.2139 IU/mL in Group A,0.3660 IU/mL in Group B,and0.1994 IU/mL in Group C.At each follow-up point,the GMC in Group B was significantly higher than that in Groups A and C.The areas under the antibody concentration curve over 0-14 days and 0-42 days in Group B were significantly larger than those in Groups A and C.Fifteen AEs were reported.Except for one grade 2 myalgia in Group C,the other 14 were all grade 1.No SAEs were observed.Conclusion The rabies virus neutralizing activity of 40 IU/kg NM57 was superior to that of 20 IU/kg NM57 and 20 IU/kg HRIG,and the rabies virus neutralizing activity of 20 IU/kg NM57 and 20 IU/kg HRIG were similar.Safety was comparable between NM57 and HRIG.

Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA

Cyclin dependent kinases （cdks） play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus （rAAV） encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA （rAAV-EGFP-U6-cdc2-siRNA） was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Construction of Recombinant Fowlpox Virus(rFpv) Expressing HIV-1 Gag-pol Protein

The induction of human immunodeficiency virus（HIV）-specific T-cell response is generally considered as critical to the development of effective immunity to HIV type 1 （ HIV-1 ）. Recombinant Avipoxvirus vectors are used widely for vaccination against HIV-1, where the induction of a cytotoxic CD8 ＋ T-cell（CTL） response seems to be an important component of protective immunity. A recombinant fowlpox virus（rFPV/Gag-pol） expressing the Gag-pol protein of HIV was constructed and characterized. The specific expression protein in CEF cells infected by recombinant fowlpox and the specific antibody in the sera of mice immunized with rFPV were analyzed via Western-blot.

Immune Efficacy of a Recombinant Fowlpox Virus Co-Expressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.

Recombinant adeno-associated virus delivered human thioredoxin-PR39 prevents hypoxia-induced apoptosis of ECV304 cells

Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus （rAAAV）, AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein （GFP） gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast （CEF） or chicken embryonic liver （CEL） cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek＇s disease virus （MDV）, avian adenovirus, chicken embryo lethal orphan （CELO） virus or infectious bursal disease virus （IBDV）. Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein （gfp） expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV- mediated transgene expression could be enhanced by super infection with the helper viruses.

Protection by Recombinant Newcastle Disease Viruses (NDV) Expressing the Glycoprotein (G) of Avian Metapneumovirus (aMPV) Subtype A or B against Challenge with Virulent NDV and aMPV

Avian metapneumovirus (aMPV) and Newcastle disease virus (NDV) are threatening avian pathogens that can cause serious respiratory diseases in poultry worldwide. Vaccination, combined with strict biosecurity practices, has been the recommendation for controlling these diseases in the field. In the present study, we generated NDV LaSota vaccine strain-based recombinant viruses expressing the glycoprotein (G) of aMPV, subtype A or B, using reverse genetics technology. These recombinant viruses, rLS/aMPV-A G and rLS/aMPV-B G, were characterized in cell cultures and evaluated in turkeys as bivalent, next-generation vaccines. The results showed that these recombinant vaccine candi-dates were slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. The expression of the aMPV G protein in recombinant virus-infected cells was detected by immunofluorescence. Vaccination of turkeys with rLS/aMPV-A G or rLS/aMPV-B G conferred complete protection against velogenic NDV, CA02 strain challenge and partial protection against homologous patho-genic aMPV challenge. These results suggest that the LaSota recombinant virus is a safe and effective vaccine vector and expression of the G protein alone is not sufficient to provide full protection against aMPV-A or -B infections. Ex-pression of other aMPV-A or -B virus immunogenic protein(s) individually or in conjunction with the G protein may be necessary to induce stronger and more protective immunity against aMPV diseases.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

Multiplication of the Recombinant Strain Re-7 of Avian Influenza Virus Subtype H5 in MDCK Cells

This study was conducted to explore the multiplication pattern of the recombinant strain Re-7 of avian influenza virus subtype H5 in Madin Darby Canine Kidney （MDCK） cells and to determine the optimal multiplicity of infection （MOI） and the optimal time for virus harvest. The recombinant strain Re-7 was inoculated at different MOIs into MDCK cells grown in serum-free medium in 100 L bioreactors for replication. Then, the hemagglutination（HA） titer, 50% tissue culture infectious dose （TCID50） and 50% embryo infectious dose （EID50） of culture medium were measured once every 12 h from 24 h after virus inoculation to determine the optimal MOI. After that, virus was inoculated at the optimal MOI determined above into MDCK cells for large-scale virus replication to determine the optimal time for virus harvest. The results showed that the optimal MOI was 10 2, and the optimal time for virus harvest was 60 h after inoculation. Under these conditions, the HA titer, TCIDso per 1 mL and EIDso per 0.1 mL were increased to 1：102 4, 10^7.33 and 10^6.83, respectively. This study provides relatively stable parameters for large-scale production of the recombinant strain Re-7 of avian influenza virus subtype H5.