Establishment of human clones producing neutralizing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus by EBV immortalization of immune CD22⁺ B cells.

We aimed to establish Human cell lines producing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus (HCV). Two protocols for EBV immortalization of CD22⁺ cells separated from HCV positive patients were used; 1) Immortalization with 100% EBV only, 2) immortalization by 30% EBV and CPG2006. Immortalization was checked microscopically and verified by screening the culture supernatant for antibody production using dot blot and ELISA analysis. ELISA plates were coated by HCV E1/E2 derived from cell lysate transfected by plasmid expressed HCV E1/E2. Also we tested the reactivity of human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315-319) and two peptides derived from HCV E2 (a.a 412-419) and (a.a 517-530). Neutralization activity was measured using H77C HCV retroviral pseudoparticles (HCVpp). Fifteen clones secreting human immunoglobulin G against HCV E1/E2 protein were isolated. Results of ELISA plates coated with HCV peptides showed that one antibody was binding to E2 peptide (a.a 517-530), and two antibodies binding to HCV E2 peptide (a.a 412-419). The three generated antibodies showed extremely neutralization activity against HCV pp. The three human antibodies were IgG3 and IgG2. These antibodies may be useful for passive immunotherapy of HCV infection.

Enteric disease surveillance under the AFHSC-GEIS: current efforts, landscape analysis and vision forward.

The mission of the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) is to support global public health and to counter infectious disease threats to the United States Armed Forces, including newly identified agents or those increasing in incidence. Enteric diseases are a growing threat to U.S. forces, which must be ready to deploy to austere environments where the risk of exposure to enteropathogens may be significant and where routine prevention efforts may be impractical. In this report, the authors review the recent activities of AFHSC-GEIS partner laboratories in regards to enteric disease surveillance, prevention and response. Each partner identified recent accomplishments, including support for regional networks. AFHSC/GEIS partners also completed a Strengths, Weaknesses, Opportunities and Threats (SWOT) survey as part of a landscape analysis of global enteric surveillance efforts. The current strengths of this network include excellent laboratory infrastructure, equipment and personnel that provide the opportunity for high-quality epidemiological studies and test platforms for point-of-care diagnostics. Weaknesses include inconsistent guidance and a splintered reporting system that hampers the comparison of data across regions or longitudinally. The newly chartered Enterics Surveillance Steering Committee (ESSC) is intended to provide clear mission guidance, a structured project review process, and central data management and analysis in support of rationally directed enteric disease surveillance efforts.

Detection of gyrA mutation among clinical isolates of Campylobacter jejuni isolated in Egypt by MAMA-PCR.

INTRODUCTION: Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. Fluoroquinolones such as ciprofloxacin are the antibiotics of choice for treatment. An increase in the frequency of ciprofloxacin-resistant Campylobacter has been reported globally due to a single base mutation (C-257 to T) in codon 86 of the quinolone resistance determining region (QRDR) of the gyrA gene altering the amino acid sequence from threonine at position 86 to isoleucine (Thr-86 to Ile). METHODOLOGY: Campylobacter spp (n = 118) were selected from a collection of Egyptian isolates spanning 1998 to 2005. The presence of C. jejuni gyrA gene was confirmed in each isolate by a PCR assay amplifying 368 bp portion of the gyrA gene. C to T alteration was detected by the mismatch amplification mutation assay MAMA PCR. The MIC of nalidixic acid (NA) and ciprofloxacin (CIP) was determined by E-test. RESULTS: C. jejuni gyrA gene was detected in 100 of the Campylobacter spp studied; the other 18 isolates were found to be Campylobacter coli by lpxA PCR. The mutation was detected in 89 C. jejuni resistant isolates with MIC values (NA; 8 - >256 µg/ml) and (CIP; 4 - >32 µg/ml). The other 11 sensitive C. jejuni isolates with MIC values (NA; 0.38 - 3 µg/ml) and (CIP; 0.03 - 0.125 µg/ml) were not amplified by the MAMA primers. There was 100% congruence with MAMA PCR, MIC results and gyrA gene sequence analysis. CONCLUSIONS: In Egypt the main mechanism for resistance to fluoroquinolones is an alteration in the gyrA QRDR. MAMA PCR provides an economical and rapid means for screening fluoroquinolone resistance.

Introductory evaluation of an oral, killed whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Egyptian infants.

BACKGROUND: We conducted the first trial to assess the safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli plus cholera toxin B-subunit vaccine in children <2 years old. METHODS: Three doses of vaccine or killed E. coli K-12 control were given at 2-week intervals to 64 Egyptian infants, 6 to 18 months old, in a randomized, double blind manner. Adverse events were monitored for 3 days after each dose. Blood was collected before immunization and 7 to 10 days after each dose to assess vaccine-specific serologic responses. RESULTS: There was no statistically significant intergroup difference in the percentage of subjects reporting the primary safety endpoint (diarrhea or vomiting) after the first (31%, vaccine; 30%, control) or third (14%, vaccine; 18%, control) dose, whereas there was a trend toward greater reporting in the vaccine group after Dose 2 (36%, vaccine; 18%, control; P = 0.052). The percentage of children showing IgA seroconversion after any dose was higher in the vaccine than the control group for recombinant cholera toxin B-subunit (97% vs. 46%), colonization factor antigen I (61% vs. 18%) and coli surface antigen 4 (39% vs. 4%) (P < 0.001 for each comparison). IgG seroconversion rates in the vaccine and control groups were 97 and 21% to recombinant cholera toxin B-subunit (P < 0.001), 64 and 29% for colonization factor antigen I (P < 0.01), 53 and 21% for coli surface antigen 2 (P < 0.05) and 58 and 4% for coli surface antigen 4 (P < 0.001), respectively. The third vaccine dose was followed by augmented IgG antitoxin titers. CONCLUSION: The oral enterotoxigenic E. coli vaccine was safe and immunogenic in this setting in Egyptian infants.