Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis

OBJECTIVE: To explore the role of IL-21 in the pathogenesis of myasthenia gravis (MG) and its influence on the the class switch of anti-AChR antibodies. METHODS: Blood was taken from 26 patients and 18 healthy controls, and the expression of IL-21R mRNA in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR. The expression of IL-21R on B lymphocytes was measured by flow cytometry, while the concentrations of serum IL-21 and the levels of anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3) were tested by ELISA. RESULTS: The serum concentration of IL-21 in the MG group was higher than that in the control group (31.686±8.499 pg/mL, 15.147±6.366 pg/mL) and the difference was significant (P<0.01). IL-21R mRNA expressed on PBMCs in the MG group was higher than that in the control group (0.139±0.052, 0.101±0.022), and the difference was significant (P<0.05). There was no difference between ocular MG and generalized MG subgroup (P>0.05). Compared with the control group, the expression of IL-21R on B lymphocytes also increased in the MG group (P<0.05). In the anti-AChR-Ab positive MG group, the serum concentration of IL-21 showed positive correlation with anti-AChR-IgG(P<0.05),but no correlation with its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05). Expression of IL-21R mRNA in the PBMCs showed no correlation with the level of serum anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05); however the expression of IL-21R in B lymphocytes showed positive correlation with anti-AChR-IgG and it's isotype IgG(1) and IgG(3) (P<0.05,P<0.01,P<0.05), but no correlation with IgG(2) (P>0.05). CONCLUSION IL-21 might induce the class switch of anti-AChR antibodies to IgG(1) and IgG(3) isotype through IL-21R on B lymphocytes which promotes the pathogenesis of the MG.
Adolescent ; Adult ; Autoantibodies ; classification ; immunology ; Female ; Humans ; Immunoglobulin Class Switching ; immunology ; Immunoglobulin G ; classification ; Interleukins ; blood ; genetics ; Male ; Middle Aged ; Myasthenia Gravis ; blood ; immunology ; RNA, Messenger ; blood ; genetics ; Receptors, Cholinergic ; immunology ; Receptors, Interleukin-21 ; blood ; genetics ; Young Adult

BACKGROUNDThe adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.

METHODSMost circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.

RESULTSResponse of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.

CONCLUSIONPolyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.

AIDS Vaccines ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Female ; HIV Envelope Protein gp120 ; genetics ; immunology ; Humans ; Immunization ; Immunoglobulin G ; blood ; Phylogeny ; Rabbits ; Vaccines, DNA ; immunology

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.
Animals ; Hepatitis B Core Antigens ; genetics ; immunology ; Immunity, Cellular ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Vaccines, Virus-Like Particle ; genetics ; immunology

OBJECTIVETo investigate the anti hepatitis E virus (HEV) and HEV RNA in acute and convalescent sera of patients with NonA-E acute hepatitis.

METHODSThe serum samples were taken from 95 patients who were diagnosed as acute NonA-E hepatitis. Enzyme immunoassay (EIA) was used for detecting anti-HEV Immunoglobulin G (IgG, Genolable and Wantai EIA anti-HEV kits). RT-PCR amplification of HEV RNA was based on the open reading frame 2 region of HEV and the PCR products were sequenced.

RESULTSSera from 95 patients who were negative for anti-HEV in acute phase were followed up for 11-35 days to detect the anti-HEV antibody in recovery phase, 16/95 (16.84%) were positive for anti-HEV (wantai EIA anti-HEV kits). Ten (62.50%) were positive for HEV RNA in acute phase. Sequence analysis showed that 4 were HEV genotype. 6 were HEV genotype; 12/95 (12.50%) were positive for anti-HEV (Genolable EIA anti-HEV kits). Seven were positive for HEV RNA; 4 belonged to HEV genotype, 3 were HEV genotype.

CONCLUSIONIt is significant and necessary to detect anti HEV antibody and HEV RNA in patients with HEV infection during acute phase and convalesent phase.

Acute Disease ; Amino Acid Sequence ; Base Sequence ; Convalescence ; Genotype ; Hepatitis Antibodies ; blood ; Hepatitis E ; genetics ; immunology ; virology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Immunoglobulin G ; blood ; RNA, Viral ; blood ; genetics ; Sequence Homology ; Viral Proteins ; genetics

OBJECTIVETo understand antibody responses to and RNA sequences of Hantavirus in patients with hemorrhagic fever renal syndrome (HFRS) in Yantai areas and to demonstrate the type of the prevalent viruses caused HFRS.

METHODSSerum specimens collected at acute and convalescent stages from 90 patients with HFRS and IgM and IgG antibodies against Hantavirus were detected with ELISA, and cross plaque reduction neutralizing tests were performed to detect neutralizing antibody. Viral RNA was extracted from the patients? sera by using Trizol method and nested PCR was utilized to amplify the specific segments of the viral cDNA and the products of the PCR were TA cloned and then the nucleotide sequences were determined.

RESULTSThe IgM antibody was positive in 82.2% (88/107) of the patients while the IgG antibody was positive in 85.7% (66/77) of the patients. Both the serologic and sequence analyses demonstrated that the epidemic of HFRS in Yantai areas was caused by mixed types of Hantavirus. The prevalent strains of Hantavirus had higher homology with the strains isolated in Korea than with those isolated previously in China.

CONCLUSIONSThe serologic and sequencing analyses indicated that the epidemic of HFRS in Yantai areas was caused by mixed types of Hantavirus dominated by type SEO.

Antibodies, Viral ; blood ; Base Sequence ; China ; DNA, Viral ; analysis ; Disease Reservoirs ; Hantaan virus ; classification ; genetics ; immunology ; Hemorrhagic Fever with Renal Syndrome ; virology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Molecular Sequence Data ; Sequence Analysis, DNA ; Serotyping

OBJECTIVETo investigate the pathogenic factors of human cytomegalovirus (HCMV) infections.

METHODSTotally 36 serum samples were obtained from early pregnant woman and examined with ELISA for anti-HCMV antibody IgG and IgM. After artificial abortion,chorionic villus and decidua were also examined with polymerase chain reaction (PCR) for HCMV-DNA. When the results of PCR were positive, pathological changes of these chorionic villus and decidua were analyzed.

RESULTSThe results showed that only 10 samples were PCR positive while IgG and/or IgM antibody to HCMV was positive. After infection with HCMV, different changes occurred in chorionic villus and decidual trophoblastic cells placental villus were hyperplasic and decidua cells degenerated and necrotized followed by lymphocytes infiltration.

CONCLUSIONThese pathological changes may be one of pathogenic factors of HCMV.

Adult ; Antibodies, Viral ; blood ; Chorionic Villi ; pathology ; virology ; Cytomegalovirus ; genetics ; immunology ; isolation & purification ; Cytomegalovirus Infections ; pathology ; DNA, Viral ; analysis ; Decidua ; pathology ; virology ; Female ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Pregnancy ; Pregnancy Complications, Infectious ; pathology ; virology

To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.
Adult ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Immunoglobulin G ; blood ; immunology ; Infant ; Infant, Newborn ; Metapneumovirus ; genetics ; Middle Aged ; Plasmids ; genetics ; Protein Engineering ; Protein Subunits ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

The experiment was conducted to prepare chitosan nanoparticles (CNP), to entrap VRMIL-2 with CNP, the eukaryotic VR1020 expression plasmid containing murine IL-2 gene (mlL-2), and to investigate the expression in vivo and the regulatory effect of mIL-2 on immune-response and immuno-protection in mice inoculated muscularly with CNP entrapped VMIL-2 at 21 days old. The results showed that IgG, IgM and IgA contents increased to different degrees in the sera from the inoculated mice, which were remarkably higher than those of the controls inoculated VR1020 packed with CNP (P<0.05); so were the IL-2, IL-4 and IL-6 contents in the sera of the immunized mice. The number of white blood cells and lymphocytes significantly increased respectively in the vaccinated mice, compared with those of controls. These mice were orally challenged with virulent E. coli 35 days post-inoculation, and all the immune responses were significantly higher than those of the control except the number of neutrophils. The mice inoculated with VRMIL-2 survived healthily, while the mice of control group were ill with the evident lesions. Although there are no remarkable differences between the cellular and humoral immune indexes of mice inoculated with CNP-VRMIL-2 and nude VRMIL-2 (P>0.05), the dosage of CNP-VRMIL-2 is only one fifth of the VRMIL-2. These indicated that entrapment of mIL-2 gene with chitosan nanoparticles could remarkably enhance the expression of mIL-2 in vivo, and significantly raise the levels of cellular and humoral immune, and increase the resistance of mice against E. coli infection. The results suggested that chitosan nanoparticles and IL-2 gene could be used as an effective immunoenhancer to increase the immunity of animals against infection.
Adjuvants, Immunologic ; pharmacology ; Animals ; Chitosan ; pharmacology ; Escherichia coli Infections ; immunology ; prevention & control ; Female ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; pharmacology ; Mice ; Nanostructures