To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

Objective: To understand the disease progression and human leukocyte antigen (HLA) gene polymorphism of HIV-infected persons without disease progress for long term, also known as long-term non-progressors (LTNPs), in Henan province. Methods: A retrospective study was conducted in 48 LTNPs with complete detection and follow-up information during 2011-2016 in Henan. Changes of CD(4)(+)T cells counts (CD(4)) and viral load (VL) during follow-up period were discussed. Polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) was used for the analyses of HLA-A, HLA-B and HLA-DRB1 alleles between LTNPs and healthy controls. Results: From 2011 to 2016, forty-eight LTNPs showed a decrease of the quartile (P(25)-P(75)) of CD(4) from 601.00 (488.50-708.72)/μl to 494.00 (367.00-672.00)/μl, and the difference was significant (P<0.05). The increase of the quartile (P(25)-P(75)) of log(10)VL from 3.40 (2.87-3.97) to 3.48 (2.60-4.37), but the difference was not significant (P>0.05). HLA polymorphism analysis revealed that HLA-B*13:02 and HLA-B*40:06 were more common in LTNPs (P<0.05), while HLA-B*46:01 and HLA-DRB1*09:01 were more common in healthy controls (P<0.05). Conclusions: The CD(4) of LTNPs in Henan showed a downward trend year by year. HLA-B*13:02 and B*40:06 might be associated with delayed disease progression for HIV infected persons in Henan.
Adult ; Alleles ; Asian People/genetics* ; China ; Disease Progression ; Female ; HIV ; HIV Infections/virology* ; HIV-1/immunology* ; HLA-B Antigens/genetics* ; Humans ; Middle Aged ; Polymorphism, Genetic ; Retrospective Studies ; Viral Load

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

OBJECTIVETo study the clinical manifestation, pathologic features and immunophenotype of histiocytic necrotizing lymphadenitis (HNL).

METHODSThe clinicopathologic data of 84 patients with HNL from 2005 to 2014 were retrospectively studied. Immunohistochemical staining using EliVision method for CD20, PAX5, CD3, CD45RO, CD4, CD8, CD56, CD68, CD123, granzyme-B, TIA1 and MPO was carried out. In-situ hybridization for Epstein-Barr virus RNA was performed on archival lymph node biopsy tissue.

RESULTSImmunohistochemical study showed that the lesional cells were predominantly histiocytes (CD68+), plasmacytoid dendritic cells (CD123+) and T lymphocytes (CD3+ and CD45RO+). Clusters of CD68-positive cells with strong and diffuse MPO expression were identified. T lymphocytes with CD4 and CD8 positivity were noted. CD56+ natural killer cells and CD20+/PAX5 B cells were rare. Apoptosis-related markers, including TIA1 and granzyme B were expressed by T lymphocytes and histiocytes in lymph nodes of HNL. In-situ hybridization for Epstein-Barr virus RNA was positive in only 10.0% of the cases.

CONCLUSIONSHNL shows no specific clinical and laboratory findings. Recognition of the characteristic histopathologic changes in lymph node biopsy of HNL is the key to correct diagnosis. Immunohistochemical study using a panel of markers, including CD3, CD4, CD8, MPO, CD123, granzyme-B and TIA1, is helpful in the differential diagnosis of HNL.

Antigens, CD ; analysis ; Biomarkers ; Dendritic Cells ; pathology ; Diagnosis, Differential ; Granzymes ; analysis ; Herpesvirus 4, Human ; genetics ; Histiocytes ; pathology ; Histiocytic Necrotizing Lymphadenitis ; complications ; immunology ; pathology ; virology ; Humans ; Immunohistochemistry ; Immunophenotyping ; In Situ Hybridization ; methods ; Lymph Nodes ; RNA, Viral ; analysis ; Retrospective Studies ; T-Lymphocytes ; immunology ; pathology

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.
Animals ; Antibodies, Viral/blood ; Antigens, Viral/genetics/*immunology ; Body Weight ; Cross Protection/*immunology ; Disease Models, Animal ; Epitopes, T-Lymphocyte/genetics/immunology ; Female ; Influenza A Virus, H3N2 Subtype/genetics/*immunology ; Influenza Vaccines/*immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections/*immunology/mortality/pathology/prevention & control ; Peptides/genetics/*immunology ; Random Allocation ; Survival Analysis ; Vaccines, Synthetic/immunology ; Virus Replication

BACKGROUND/AIMS: Quantification of hepatitis B surface antigen (HBsAg) is an emerging serologic test and may be useful for identifying treatment strategies for chronic hepatitis B (CHB). This study aimed to evaluate HBsAg titers during the natural course of CHB and identify correlations between HBsAg titers and hepatitis B virus (HBV) DNA concentrations across different CHB phases measured using an immunoradiometric assay (IRMA). METHODS: CHB phases were defined on the basis of HBV DNA concentrations, the presence of hepatitis B e antigen/antibody (HBeAg/Ab) and serum alanine aminotransferase levels. Serum HBsAg titers and paired HBV DNA concentrations in the different phases of CHB were compared using 627 serum samples. RESULTS: Mean HBsAg titers were significantly higher in the immunotolerant (IT) phase and immunoreactive (IR) HBeAg-positive phase than in the low-replicative (LR) and HBeAg-negative CHB (ENH) states. The correlation between HBsAg titers and HBV DNA concentrations was modest in the IT (n=36, r=0.804, p<0.001) and IR (n=48, r=0.773, p<0.001) phases, and it was poor in the LR state (n=116, r=0.289, p=0.002); however, no significant correlation was observed in the ENH state (n=67, r=0.146, p=0.237) or in the oral nucleos(t)ide analogue-treated group (n=267). CONCLUSIONS: HBsAg quantification using IRMA might be useful for discriminating different CHB phases and different stages of chronic liver disease.
Adult ; Alanine Transaminase/blood ; Biomarkers/blood ; DNA, Viral/*blood ; Disease Progression ; Female ; Hepatitis B Surface Antigens/*blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*genetics/immunology ; Hepatitis B, Chronic/*immunology ; Humans ; *Immunoradiometric Assay ; Male ; Middle Aged ; Seoul ; Viral Load ; Virus Replication

Group-A rotaviruses are recognized as the most common cause of acute diarrhea. Phylogenetic analyses of the VP7 genes of rotaviruses circulating in Nanjing (China) could aid in the development of rotavirus vaccines. A total of 908 stool specimens were collected from patients suffering from acute diarrhea in Nanjing between October 2012 and December 2013, and were tested further for rotaviruses. Fifty rotavirus isolates selected randomly were typed by reverse transcription-polymerase chain reaction using serotype-specific primers for G genotyping. VP7 genes of 19 G9 strains were sequenced for further genetic characterization. Among the 908 stool specimens examined during the surveillance period, 103 (11.34%) were rotavirus-positive. G9 was the most predominant genotype (78.0%), followed by G2, G1 and G3. Sequence and phylogenetic analyses of the VP7 genes of serotype G9 rotaviruses revealed these strains to comprise two lineages (G9-VI, G9-III) and to be dominated by the G9-VI lineage (which belonged to a unique subcluster of Japanese and Chinese G9 strains). Amino-acid sequences of the four antigenic regions (A, B, C or F) were variant among a portion of strains, which may have contributed to the prevalence of G9 rotaviruses in this area.
Adult ; Amino Acid Sequence ; Antigens, Viral ; chemistry ; genetics ; Capsid Proteins ; chemistry ; genetics ; China ; Evolution, Molecular ; Humans ; Infant ; Molecular Sequence Data ; Mutation ; Phylogeny ; Rotavirus ; genetics ; immunology ; physiology ; Serogroup

OBJECTIVETo study the clinicopathologic features of Hodgkin lymphoma (HL) occurring in northern China, association with Epstein-Barr virus (EBV) infection and concordance between EBV protein immunohistochemistry (IHC) and in-situ hybridization (ISH).

METHODSTwo hundred and thirty-five cases were collected and their HE and IHC slides were reviewed to confirm the diagnosis and sort of HLs. All cases were performed with IHC staining for LMP-1 protein and ISH of EBV-encoded RNAs (EBER) was done in 101 cases to detect the existence of EBV.

RESULTSThe incidence peak was between age 25 and 35 years, followed by another peak between age 56 to 60 years. There were 135 males and 100 females. The tumor involved lymph nodes in 217 cases, and extranodal sites in 18 cases. There were 3 cases of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and 232 cases of classical Hodgkin lymphoma. All tumors were stained for CD30, CD20, CD3. CD30 was expressed in 227 cases (96.6%), CD20 was expressed in 53 cases (22.5%) with different level of intensity. CD3 was expressed only in 1 case (0.4%). CD15 staining was performed in 224 cases and 117 (52.2%) cases were positive. PAX-5 were performed in 213 cases and 160 (75.1%) cases showed weak to moderate expressions. Two hundred and thirty-five cases were immunohistochemically stained with LMP1 and 72 (30.6%) cases were positive. Meanwhile, EBER ISH were applied in 101 cases, and 40 cases (39.6%) were found positive. LMP1 was expressed in 30 cases among those EBER-positive cases, while LMP1 was only detected in 5 cases of the EBER-negative cases. There was no statistically significantce between LMP1 IHC and EBER ISH by pared chi-square test (P = 0.3), the overall concordance rate was 85.2%.

CONCLUSIONSThere was a bimodal age distribution in our group of HL cases from the northern part of China, with slight male predominance and mainly nodal involvement. Nodular sclerosis (NS) and mixed cellularity (MC) were major histologic subtypes. When it was compared with the EBER ISH method in detection EBV infection of HL, the more economical and convenient LMP1 IHC showed both high degree of consistency and overall concordance rate.

Adult ; Age Distribution ; Antigens, CD ; analysis ; China ; epidemiology ; Epstein-Barr Virus Infections ; complications ; epidemiology ; Female ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Hodgkin Disease ; immunology ; pathology ; virology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Incidence ; Male ; Middle Aged ; RNA, Viral ; analysis ; Sex Distribution

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

BACKGROUND/AIMS: The efficacy of tenofovir disoproxil fumarate (TDF) for the treatment of chronic hepatitis B (CHB) patients following prior treatment failure with multiple nucleos(t)ide analogues (NAs) is not well defined, especially in Asian populations. In this study we investigated the efficacy and safety of TDF rescue therapy in CHB patients after multiple NA treatment failure. METHODS: The study retrospectively analyzed 52 CHB patients who experienced failure with two or more NAs and who were switched to regimens containing TDF. The efficacy and safety assessments included hepatitis B virus (HBV) DNA undetectability, hepatitis B envelop antigen (HBeAg) seroclearance, alanine transaminase (ALT) normalization and changes in serum creatinine and phosphorus levels. RESULTS: The mean HBV DNA level at baseline was 5.4 +/- 1.76 log10 IU/mL. At a median duration of 34.5 months of TDF treatment, the cumulative probabilities of achieving complete virological response (CVR) were 25.0%, 51.8%, 74.2%, and 96.7% at 6, 12, 24, and 48 months, respectively. HBeAg seroclearance occurred in seven of 48 patients (14.6%). ALT levels were normalized in 27 of 31 patients (87.1%) with elevated ALT at baseline. Lower levels of HBV DNA at baseline were significantly associated with increased CVR rates (p < 0.001). However, CVR rates did not differ between TDF monotherapy or combination therapy with other NAs, and were not affected by mutations associated with resistance to NAs. No significant adverse events were observed. CONCLUSIONS: TDF is an efficient and safe rescue therapy for CHB patients after treatment failure with multiple NAs.
Adenine/adverse effects/*analogs & derivatives/therapeutic use ; Adult ; Aged ; Alanine Transaminase/blood ; Antiviral Agents/adverse effects/*therapeutic use ; Biological Markers/blood ; Creatinine/blood ; DNA, Viral/blood ; Drug Resistance, Viral/genetics ; Drug Substitution ; Female ; Genotype ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*drug effects/genetics/immunology/pathogenicity ; Hepatitis B, Chronic/blood/diagnosis/*drug therapy ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Mutation ; Phosphorous Acids/adverse effects/*therapeutic use ; Phosphorus/blood ; Retrospective Studies ; Time Factors ; Treatment Failure ; Viral Load ; Young Adult