Mei-ying W. Yu

Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma

Neutralizing antibodies directed against hepatitis C virus (HCV) are present in Igs made from anti-HCV-positive plasma. However, these HCV-specific Igs are largely ineffective in vivo. The mechanism for the poor effectiveness is currently unknown. We hypothesize that the presence of nonneutralizing antibodies in HCV-specific Igs interferes with the function of neutralizing antibodies, resulting in the reduction or blockage of their effect. In the present study, we identified at least two epitopes at amino acid residues 412–419 (epitope I) and 434–446 (epitope II), located downstream of the hypervariable region I within the HCV E2 protein. We demonstrated that epitope I, but not epitope II, was implicated in HCV neutralization and that binding of a nonneutralizing antibody to epitope II completely disrupted virus neutralization mediated by antibody binding at epitope I. The dynamic interaction between nonneutralizing and neutralizing antibodies may thus play a key role in determining the outcomes of HCV infection. Further exploration of this interplay should lead to a better understanding of the mechanisms of neutralization and immune escape and may indicate pathways for the manufacture of an effective HCV-specific Ig product for immune prophylaxis of HCV infection.

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

Detection and characterization of hepatitis C virus RNA in immune globulins

BACKGROUND: Hepatitis C virus (HCV) RNA was measured in immune globulins and its chemical and physical properties were characterized.

Quantification of hepatitis B virus genomes and infectivity in human serum samples

BACKGROUND: Hepatitis B virus (HBV) infections are still a major health issue, with approximately 350 million people chronically infected with HBV worldwide. Information about the minimum copy number of HBV genomes required for infection would be useful as a reference for drug and vaccine development; for monitoring HBV patients during treatment; for screening of blood, organ, and tissue donors; and for regulating nucleic acid amplification assays for HBV.

Parvovirus B19 infection transmitted by transfusion of red blood cells confirmed by molecular analysis of linked donor and recipient samples.

BACKGROUND: Extremely high viremic levels of parvovirus B19 (B19V) can be found in acutely infected, but asymptomatic donors. However, reports of transmission by single‐donor blood components are rare. In this prospective study, paired donor‐recipient samples were used to investigate the transfusion risk.

Parvovirus B19 DNA in Factor VIII concentrates: effects of manufacturing procedures and B19 screening by nucleic acid testing.

BACKGROUND: Parvovirus B19 (B19) is a common contaminant, especially in coagulation factors. Because of B19 transmission by pooled plasma, solvent/detergent treated in 1999, some fractionators initiated minipool nucleic acid testing (NAT) to limit the B19 load in manufacturing pools. In this study, the extent of B19 DNA contamination in commercial Factor VIII concentrates, that is, antihemophilic factor (human) (AHF), manufactured before and after B19 NAT screening was implemented, was determined.

International collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti‐A and anti‐B in normal intravenous immunoglobulin products

Background and Objectives  The aim of the study was to evaluate, in an international collaboration, three lyophilized intravenous immunoglobulin (IVIG) preparations for their suitability to standardize and control haemagglutination testing for anti‐A and anti‐B in IVIG products.

Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

International collaborative study to evaluate a candidate reference preparation to define an appropriate specified limit of anti-D in intravenous immunoglobulin products

Background and Objectives  The aim of the study was to evaluate a lyophilized intravenous immunoglobulin (IVIG) preparation containing anti‐D (02/228; nominal reciprocal titre of 8) for its suitability to define the maximum limit of anti‐D in IVIG products when used in a proposed reference method of direct haemagglutination of papain‐treated erythrocytes, in an international collaborative study.

HCV core antigen as an alternative to NAT to detect HCV viremia

Decay-accelerating factor (DAF) is a complement regulatory protein composed of four short-consensus repeats (SCRs), a serine-threonine-proline–rich region, and a glycosylphosphatidylinositol anchor. A series of singleamino-acid changes exist on DAF that are responsible for the Cromer blood group antigens including Tc/Tc/Tc, Es, and WES/WES on SCR1; Dr on SCR3; and Cr and UMC on SCR4. The most recently discovered Cromer antigen, GUTI, is also located on SCR4 and was found in the Mapuche Indians of Chile. Accordingly, we extended the original studies by testing populations having Native American backgrounds. We tested 122 individuals (61 Mexican Americans and 61 Choctaw Indians) for the GUTI polymorphism. The criteria used to select the Mexican Americans were that all four grandparents of each test subject had to be from Mexico. Mexicans are a triethnic group having Caucasian (Spaniard), Native American, and African admixture. Choctaw Indian ancestry was established through CODIS and by use of US legal documents as previously described. Ninety percent of the Oklahoma Choctaw are of full-blooded quantum. By use of procedures similar to those reported by Storry and colleagues, we employed DAFSCR4F and DAFSCR4R primers to amplify genomic DNA by PCR. The RFLP analysis was performed on the amplified samples using the TaiI enzyme for digestion and a 2 percent agarose gel containing ethidium bromide. A known GUTI+ heterozygous DNA sample, provided by M. Reid at the New York Blood Center, was used as a positive control. No GUTI+ samples were found in our 122 samples compared to 6 of 114 (5.3%) of the Mapuche Indians. Although both the Choctaw and the Mapuche are part of the Amerindian migration from Asia, the Mapuche are considered to be an isolate especially from a linguistic point (M. Foster, personal communication). Thus, we conclude that the high frequency of the GUTI antigen in the Mapuche may be due to the founder effect and that GUTI may not generally be associated with Native American Indian ethnicity. Nevertheless, further studies of South American Indians may help clarify its origins. Joann M. Moulds, PhD e-mail: moulds@drexel.edu Thomas R. Drames Bolaji Thomas, PhD Department of Microbiology and Immunology Drexel University College of Medicine 2900 Queen Lane, Room G44 Philadelphia, PA 19129-1096 REFERENCES