Specific antibody response of mice after immunization with COS-7 cell derived avian influenza virus (H5N1) recombinant proteins

To develop avian influenza H5N1 recombinant protein, the hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural (NS1) of avian influenza H5N1 isolates from Thailand were engineered to be expressed in prokaryotic (E. coli) and mammalian cell (COS-7) system. The plasmid pBAD-His and pSec-His were used as vectors for these inserted genes. Mice immunized with purified recombinant proteins at concentration 50–250 μg intramuscularly with Alum adjuvant at week 0, week 2, and week 3 showed a good immunogenicity measured by ELISA and neutralization assay. The HA and NS recombinant proteins produced in COS-7 cells can induce specific antibody titer detected by neutralization assay significantly higher than corresponding recombinant proteins produced in E. coli system. The antibody produced in immunized mice could neutralize heterologous avian influenza virus determined by micro-neutralization assay. This study shows that avian influenza virus H5N1 recombinant proteins produced in mammalian cell system were able to induce neutralizing antibody response

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation

Labcode 11- HIV neutralization Protocol PBMC Neutralization assay

This protocol describes a HIV neutralisation assay based on reduction in virus infectivity following exposure to monoclonal reagents. Virus infection in donor PHA-stimulated peripheral blood mononuclear cells is assessed by quantitative RT-PCR in culture supernatants two days after infection

p24 Antigen Detection Assay Modified with a Booster Step for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection

We modified a p24 antigen enzyme-linked immunosorbent assay as a method for diagnosis and monitoring of human immunodeficiency virus type 1 (HIV-1) subtype E infection. This modified assay is based on the use of preheated immune complex dissociation combined with a booster step using a regular Vironostika HIV-1 p24 antigen assay (bioMerieux) to decrease the lower limit of p24 antigen detection from 10 pg/ml (lower limit achievable when using a regular p24 antigen assay) to 0.5 pg/ml (100 virions/ml) by the new method. The correlation between the values obtained by the HIV-1 RNA (Amplicor HIV-1 Monitor) assay and the p24 antigen assay modified with a booster step antigen assay in 160 frozen plasma samples with known viral load and 80 blind fresh plasma samples by Spearman rank were 0.671 (R(2) = 0.450; P < 0.01) and 0.782 (R(2) = 0.612; P < 0.01). During antiretroviral treatment, the change of p24 antigen level at ≥0.5 log correlated well with the level of HIV-1 in plasma. In order to improve the early diagnosis of HIV-1 infection in 121 infants born to HIV-1-infected mothers, a heat-denatured plasma p24 antigen assay modified with a booster step was compared with DNA-PCR and HIV RNA (nucleic acid sequence-based amplification) assays. The sensitivity of the antigen test modified with a booster step was similar to that of the HIV-1 RNA (NASBA QL) assay and better than that of the DNA-PCR assay (100 versus 61.90%) for subjects 1 to 2 months old. The overall results from this study might renew interest in p24 antigen detection as an easily affordable alternative method for diagnosis of HIV-1 infection and monitoring of disease progression in developing countries

Epidemiological, Clinical and Virological Characteristics of Influenza B Virus from Patients at the Hospital Tertiary Care Units in Bangkok during 2011-2014

<div><p>Influenza B virus, which causes acute respiratory infections, has increased in prevalence in recent years. Based on the nucleotide sequence of the hemagglutinin (HA) gene, influenza B virus can be divided into two lineages, Victoria and Yamagata, that co-circulate during the influenza season. However, analysis of the potential association between the clinical and virological characteristic and the lineage of influenza B viruses isolated in Thailand was lacking. To investigate influenza B virus genetically and determine its neuraminidase (NA) inhibitor susceptibility phenotype, a total of 6920 nasopharyngeal-wash samples were collected from patients with influenza-like illness between the years 2011 and 2014 and were screened for influenza B virus by real-time PCR. Of these samples, 3.1% (216/6920) were confirmed to contain influenza B viruses, and 110 of these influenza viruses were randomly selected for nucleotide sequence analysis of the HA and NA genes. Phylogenetic analysis of the HA sequences showed clustering into various clades: Yamagata clade 3 (11/110, 10%), Yamagata clade 2 (71/110, 64.5%), and Victoria clade 1 (28/110, 25.5%). The analysis of clinical characteristic demonstrated that the Victoria lineage was significantly associated with the duration of hospitalization, number of deceased cases, pneumonia, secondary bacterial infection and underlying disease. When combined with phylogenetic analysis of the NA sequences, four samples showed viruses with reassortant sequences between the Victoria and Yamagata lineages. Statistical analysis of the clinical outcomes and demographic data for the reassortant strains did not differ from those of the other strains in circulation. Oseltamivir-resistant influenza B viruses were not detected. Our findings indicated the co-circulation of the Victoria and Yamagata lineages over the past four cold seasons in Bangkok. We also demonstrated differences in the clinical symptoms between these lineages.</p></div

Amino acid substitutions in the antigenic sites of isolated influenza B viruses.

<p>Amino acid substitutions in the antigenic sites of isolated influenza B viruses.</p

Case of Microcephaly after Congenital Infection with Asian Lineage Zika Virus, Thailand

We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly