To investigate the adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in Flaviviridae virus DNA vaccines. After DNA immunization, the antibody levels of serum from mice were detected by ELISA and indirect immunofluorescence assay. Co-immunization of GM-CSF suppressed the immune responses induced by DV1 and DV2 candidate vaccines whereas enhanced the immune response induced by HCV C and E1 DNA vaccines. As genetic adjuvant for DNA vaccines, GM-CSF might display complex diversity on the immune responses: an augmentation or suppression due to different immunogens. Therefore, GM-CSF should be used with some cautions in clinic.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; DNA, Viral ; administration & dosage ; genetics ; immunology ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; administration & dosage ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Adult ; Aged ; Alcohol Drinking ; China ; ethnology ; Fatty Liver, Alcoholic ; etiology ; Female ; Humans ; Male ; Middle Aged ; Surveys and Questionnaires

BACKGROUNDDiabetic macular edema (DME) is a common manifestation of diabetic retinopathy (DR) that forms the main cause of central visual impairment. This study aimed to compare the efficacy and safety of a single intravitreal injection of bevacizumab alone versus bevacizumab combined with triamcinolone acetonide in eyes with diabetic macular edema (DME).

METHODSA total of 40 eyes in 40 Chinese patients (22 male and 18 female) diagnosed with diabetic macular edema were enrolled in this prospective, randomized, consecutive study. Among them, 21 patients in group 1 were treated with intravitreal injection of bevacizumab (1.25 mg/0.05 ml), and the other 19 patients in group 2 accepted intravitreal bavacizumab (1.25 mg/0.05 ml) combined with triamcinolone acetonide (2 mg/0.05 ml). All patients were examined at baseline and followed up at 4, 6 and 12 weeks after the injection. Changes in mean best correct visual acuity (BCVA) using ETDRS chart, central retina thickness (CRT) measured by optical coherence tomography (OCT), and intraocular pressure (IOP) were focused on.

RESULTSIn group 1, mean BCVA improved from (41.76 ± 15.59) letters (baseline) to (56.24 ± 18.56) letters, (52.57 ± 12.31) letters and (48.41 ± 17.90) letters at 4, 6 and 12 weeks post-injection, respectively (P = 0.004, P = 0.011 and P = 0.026, respectively). Mean CRT decreased from (525.76 ± 184.10) µm (baseline) to (270.33 ± 202.67)µm, (303.12 ± 168.43) µm and (402.26 ± 196.21) µm, respectively (P = 0.009, P = 0.016 and P = 0.030, respectively). In group 2, mean BCVA improved from (39.89 ± 12.27) letters (baseline) to (55.31 ± 19.27) letters, (51.25 ± 13.48) letters and (46.97 ± 16.23) letters at 4, 6 and 12 weeks after injection, respectively (P = 0.003, P = 0.010 and P = 0.027, respectively). Mean CRT decreased from (554.50 ± 169.05) µm (baseline) to (292.76 ± 196.05) µm, (323.46 ± 164.05) µm and (426.38 ± 169.05) µm, respectively (P = 0.009, P = 0.014 and P = 0.028, respectively). However, there was no significant difference between these two groups with regard to mean BCVA (F = 1.602, P = 0.216) and CRT (F = 0.412, P = 0.526). At 12 weeks after the injection, 11 of the patients in group 1 and nine patients in group 2 appeared recurrent macular edema and needed repeat injections. There was one patient in group 2 appeared transient intraocular pressure increases.

CONCLUSIONSIntravitreal injection of bevacizumab combined with/without triamcinolone acetonide had a beneficial effect on DME. However, the significant effect was not permanent. Our results showed that no significant differences were detected between intravitreal bevacizumab combined with/without triamcinolone acetonide for the eyes with diabetic macular edema in Chinese patients.

Adult ; Aged ; Antibodies, Monoclonal ; administration & dosage ; therapeutic use ; Antibodies, Monoclonal, Humanized ; Bevacizumab ; Diabetic Retinopathy ; drug therapy ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Intravitreal Injections ; Macular Edema ; drug therapy ; Male ; Middle Aged ; Treatment Outcome ; Triamcinolone Acetonide ; administration & dosage ; therapeutic use

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.
Antibodies, Monoclonal ; genetics ; immunology ; Antibodies, Neutralizing ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; HEK293 Cells ; HIV Antibodies ; genetics ; immunology ; HIV Envelope Protein gp120 ; genetics ; immunology ; HIV Infections ; immunology ; virology ; HIV-1 ; genetics ; immunology ; isolation & purification ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library ; Transfection

Chronic hepatitis B virus (HBV) infection is a primary cause for liver cancer. And the main challenge of curing hepatitis B is the elimination of the stable covalently closed circular DNA (cccDNA) of the viral genome. The formation of HBV cccDNA requires the filling of single-stranded region and the ligation of nicks in relaxed circular DNA (rcDNA) strands. Previously, our group reported that proliferating cell nuclear antigen (PCNA) was involved in the formation of HBV cccDNA. However, the underlying mechanism of the conversion of HBV rcDNA to cccDNA is poorly understood. In the present study, we aim to explore the mechanism by which PCNA contributes to the conversion of HBV rcDNA to cccDNA. Our data showed that PCNA was involved in the process of HBV rcDNA repair. The knockout of PCNA by the CRISPR/Cas9 system remarkably blocked the conversion of HBV rcDNA to cccDNA, while the ectopic expression of PCNA could effectively rescue the event (P<0. 001). Knockout of PCNA significantly slowed down the conversion kinetics of HBV rcDNA to cccDNA (P<0. 01). Mechanically, the DNA binding domain of PCNA was required for the process of HBV rcDNA repair to cccDNA (P<0. 01). Thus, we conclude that PCNA confers the conversion of HBV rcDNA to cccDNA by its DNA binding domain. Clinically, PCNA might serve as a novel target for antiviral therapy.

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.
Blood Group Antigens ; genetics ; Caliciviridae Infections ; blood ; complications ; virology ; Child, Preschool ; China ; Cross-Sectional Studies ; Diarrhea ; blood ; etiology ; virology ; Feces ; virology ; Gastroenteritis ; blood ; virology ; Genotype ; Humans ; Infant ; Norovirus ; physiology