The ongoing pandemic of Coronavirus disease 19 (COVID-19) is caused by a newly discovered β Coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). How long the adaptive immunity triggered by SARS-CoV-2 can last is of critical clinical relevance in assessing the probability of second infection and efficacy of vaccination. Here we examined, using ELISA, the IgG antibodies in serum specimens collected from 17 COVID-19 patients at 6-7 months after diagnosis and the results were compared to those from cases investigated 2 weeks to 2 months post-infection. All samples were positive for IgGs against the S- and N-proteins of SARS-CoV-2. Notably, 14 samples available at 6-7 months post-infection all showed significant neutralizing activities in a pseudovirus assay, with no difference in blocking the cell-entry of the 614D and 614G variants of SARS-CoV-2. Furthermore, in 10 blood samples from cases at 6-7 months post-infection used for memory T-cell tests, we found that interferon γ-producing CD4
Adaptive Immunity/physiology* ; Adult ; Aged ; Antibodies, Neutralizing/blood* ; COVID-19/immunology* ; Cohort Studies ; Female ; Humans ; Immunoglobulin G/blood* ; Male ; Middle Aged ; SARS-CoV-2/immunology* ; T-Lymphocytes/physiology* ; Time Factors ; Viral Proteins/immunology*

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.

METHODSLuciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.

RESULTSThe shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.

CONCLUSIONChemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.

Adenoviruses, Human ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Callithrix ; Cell Line ; Humans ; Immunity, Humoral ; Luciferases ; Plasmids

Although it is well known that pneumococcal conjugate vaccines provide cross-protection against some vaccine-related serotypes, these mechanisms are still unclear. This study was performed to investigate the role of cross-protective IgM antibodies against vaccine-related serotypes 6A, 6C, and 19A induced in children aged 12-23 months after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). We obtained serum samples from 18 Korean children aged 12-23 months after a PCV7 booster immunization. The serum IgG and IgM concentrations of serotypes 6B and 19F were measured by enzyme-linked immunosorbent assay (ELISA) in serum. The opsonic indices (OIs) against vaccine serotypes 6B and 19F and vaccine-related serotypes 6A, 6C, and 19A were determined by an opsonophagocytic killing assay (OPA) in IgM-depleted and control serum. Both IgG and IgM antibodies in ELISA and opsonic indices in OPA against serotypes 6B and 19F were demonstrated in the immune serum. IgM depletion decreased the OIs against vaccine serotypes 6B (geometric means of OIs (GMIs) of 3,009 vs. 1,396, 38% reduction) and 19F (1,117 vs. 750, 36% reduction). In addition, IgM depletion markedly decreased the OIs against vaccine-related serotypes 6A (GMIs of 961 vs. 329, 70% reduction), 6C (432 vs. 185, 72% reduction), and 19A (301 vs. 166, 58% reduction). The booster immunization PCV7 induced protective antibodies in the form of both IgG and IgM isotypes. IgM antibodies contributed to eliciting cross-protection against vaccine-related serotypes as well as against vaccine serotypes.
Antibodies, Bacterial/blood ; Antibodies, Neutralizing/blood ; Enzyme-Linked Immunosorbent Assay ; Heptavalent Pneumococcal Conjugate Vaccine/*immunology ; Humans ; Immunoglobulin M/*blood ; Infant ; Pneumococcal Infections/*prevention & control ; Pneumococcal Vaccines/*immunology ; Serogroup ; Streptococcus pneumoniae/immunology

The rapid mutation and widely spread of duck hepatitis A virus (DHAV) lead to the vast economic loss of the duck industry. To prepare and evaluate bivalent inactivated vaccine laboratory products of DHAV, 6 strains were screened from 201 DHAV-1 strains and 38 DHAV-3 strains by using serotype epidemiological analysis in most of the duck factory. Vaccine candidate strains were selected by ELD50 and LD50 tests in the 6 strains. Continuously passaged, the 5th passaged duck embryos bodies grinding fluid was selected as vaccine virus seeds. The virus seeds were treated with formaldehyde and water in oil in water (W/O/W) emulsions, making into three batches of two bivalent inactivated vaccine laboratory products. The safety test, antibody neutralization test, challenged protection and cross immune protection experiment suggested that the vaccines possessed good safety, and neutralizing antibodies were detected at 7th day and the challenged protection rate reached 90% to 100% at the 14th and 21st day. Moreover, immune duration of ducklings lasted more than five weeks. However, cross-immunity protection experiments with DHAV-SH and DHAV-FS only had 20%-30%. The two bivalent inactivated vaccine laboratory products of duck viral hepatitis were effective and reliable, providing a new method as well as a new product for DHAV prevention and control.
Animals ; Antibodies, Neutralizing ; blood ; Ducks ; virology ; Hepatitis Virus, Duck ; Hepatitis, Viral, Animal ; prevention & control ; virology ; Neutralization Tests ; Picornaviridae Infections ; prevention & control ; veterinary ; Poultry Diseases ; prevention & control ; virology ; Vaccines, Inactivated ; immunology ; Viral Hepatitis Vaccines ; immunology

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2>AAV3=AAVLK03>AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤35, 36-50, and ≥51 years old). People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.
Adult ; Aged ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Body Constitution ; Dependovirus ; classification ; immunology ; Female ; Genetic Vectors ; Humans ; Liver ; virology ; Male ; Middle Aged ; Serogroup

OBJECTIVETo clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODSVP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTSVP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONSVP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Enterovirus A, Human ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Immunoglobulin M ; blood ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo understand the genotypic characteristics and the neutralizing antibody levels of Japanese encephalitis virus (JEV) and Japanese encephalitis (JE) in both vector mosquitoes and in healthy people of Zhejiang province.

METHODSVirus was isolated from mosquitos sampled from the Monitoring Stations located in Xianju county during 2012 to 2013. Phylogenetic and homological studies were carried out on the E gene. A total of 1 263 blood specimens from 642 healthy people were collected before and after the seasons of JE epidemics. JEV neutralizing antibody was detected by the micro-neutralization test.

RESULTSTwenty-five JEV strains were isolated from a total of 11 650 mosquitoes. The identity of nucleotide appeared as 87.8%-99.7% both from 2012 to 2013 and from 1982 to 2010 while as 87.7%-88.0% with vaccine strain SA14-14-2, in Zhejiang. The phylogeny tree of E gene indicated that the newly isolated virus belonged to genotype I but no mutation of amino acid sequence coding conformational epitope was identified in the envelop protein. Both positive rates and the geometric mean titer (GMT) of neutralizing antibody in healthy people were 31.5%-42.0% and 1 : 2.56-1 : 3.53 in Xianju county, during 2012 and 2013, respectively. Both of the two positive rates (χ(2)≤1.76, P > 0.05) and the two GMTs (u≤0.64, P > 0.5) for antibodies pre or post the epidemic season did not show significant differences.

CONCLUSIONJEV isolated in Xianju during 2012 and 2013 belonged to genotype I. The positive rates of JEV neutralizing antibody from healthy people in Xianju were less than 42.0%, which showed no significant differendes pre or post JE epidemic season.

Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; China ; Culicidae ; virology ; Disease Vectors ; Encephalitis Virus, Japanese ; genetics ; immunology ; isolation & purification ; Encephalitis, Japanese ; virology ; Epitopes ; Genotype ; Humans ; Neutralization Tests ; Phylogeny

We aimed to compare the immune response induced by natural infection with 2009 pandemic influenza A/H1N1 (pH1N1) virus and by monovalent pH1N1 vaccination in children and adolescents. This cross-sectional clinical study was conducted at 3 hospitals in Korea from February to May 2010. A total of 266 healthy subjects aged from 6 months to 18 yr were tested for the presence of the antibody against pH1N1 using hemagglutination inhibition (HI) test. Information about pH1N1 vaccination and laboratory-confirmed pH1N1 infection history was obtained. The overall rate of HI titers of > or = 1:40 against pH1N1 was 38.7%, and the geometric mean titer (GMT) was 20.5. Immunogenicity of pH1N1 vaccination only was reflected by a 41.1% of seroprotection rate and a GMT of 22.5. Immunogenicity of natural infection only was reflected by a 61.0% of seroprotection rate and a GMT of 40.0. GMT was significantly higher in the subjects of natural infection group than in the subjects of pH1N1 vaccination group (P < 0.001). The immune responses induced by natural pH1N1 infection exceed those induced by pH1N1 vaccinations.
Adolescent ; Antibodies, Neutralizing/blood ; Antibody Formation ; Child ; Child, Preschool ; Cross-Sectional Studies ; Hemagglutination Inhibition Tests ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype/*immunology/metabolism ; Influenza, Human/epidemiology/*immunology/prevention & control ; Pandemics ; Vaccination

Treatment with interferon beta (IFN-beta) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-beta are associated with a loss of IFN-beta bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-beta in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-beta medications by ELISA. The NAbs against IFN-beta were measured in BAb-positive MS patients receiving IFN-beta using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-beta treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-beta. In conclusion, the three IFN-beta preparations have different degrees of immunogenicity.
Adolescent ; Adult ; Antibodies/*blood/immunology ; Antibodies, Neutralizing/*blood/immunology ; Cross Reactions ; DNA, Complementary/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interferon-beta/*immunology/therapeutic use ; Male ; Middle Aged ; Multiple Sclerosis/drug therapy/*immunology ; Myxovirus Resistance Proteins/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult