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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Evaluation and Characterization of Neutralizing Antibodies against Viruses Relevant to Blood-derived Products

Principal Investigator: Pei Zhang, MD
Office / Division / Lab: OBRR / DH / LPD

General Overview

FDA establishes tests to determine the quality of products it regulates. One class of products that the Office of Blood Research and Review regulates is known as "specific immune globulins", which are concentrated solutions of antibodies directed against specific infectious microbes. Hepatitis C Immune Globulin Intravenous (HCIGIV) belongs to this class and is currently under development but not yet licensed for the treatment of hepatitis C virus infections. Like all other products in its class, HCIGIV will require a reliable potency test as part of the lot release testing that correlates with its clinical effectiveness. It is particularly urgent to have such a test in place during the development phase of the product. Institution of an appropriate potency assay during clinical trials allows collection of data that may demonstrate the correlation of the potency assay test results and clinical outcome in terms of efficacy. These data are then submitted for FDA review as part of a license application. The validity of these studies depends on accurate measuring and monitoring of the level of effective anti-hepatitis C virus antibodies in individuals who received this product in clinical trials.

We use a variety of techniques to characterize the neutralizing and non-neutralizing antibodies present in HCIGIV and use these antibodies to identify specifically where antibodies bind the virus. This information will help us to develop tests that FDA can use to ensure the quality of commercially produced HCIGIV.

In addition, our work is helping us to understand why much of the currently manufactured HCIGIV has proven to be ineffective; specifically, some antibodies present in these HCIGIV appear to interfere with other neutralizing antibodies that would otherwise effectively inactivate the virus.

Scientific Overview

Neutralizing antibodies directed against hepatitis C virus (HCV) are present in human immune globulins (Igs) made from anti-HCV-positive plasma. However, unlike hepatitis B Ig products, which have been approved by the FDA to prevent hepatitis B, hepatitis C Ig preparations when used as an investigational drug have been largely ineffective. The underlying mechanism for the poor efficacy is currently unknown.

Using a multidisciplinary approach including phage display, bioinformatics, and virus neutralization assays, we identified two important regions (Epitope I and Epitope II) on the E2 envelope glycoprotein of HCV. From our previous work, we know that Epitope I, which is highly conserved across all the genotypes of HCV, is an important neutralization site while Epitope II is involved in the interference of antibody binding to Epitope I. We hypothesized that the non-neutralizing antibodies in HCV-specific Igs interfere with the function of neutralizing antibodies. This interference might account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies.

We therefore characterized these epitopes further and identified the amino acid residues within the epitopes that are crucial for antibody binding. By changing the proportion of interfering to neutralizing antibodies in the plasma of HCV-infected patients, we were able to reveal otherwise undetectable, cross-genotype neutralizing activity. These data support the concept that viral evasion of antibody neutralization does not necessarily require mutations of the neutralization epitope. Instead, there appears to be an additional escape mechanism whereby a non-neutralizing antibody binding to the virus interferes with the binding of antibodies with neutralizing capability.

Further study of the mechanisms of antibody-mediated neutralization and interference should provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.


Vaccine 2010 Jun 7;28(25):4138-44
Hepatitis C virus with a naturally occurring single amino-acid substitution in the E2 envelope protein escapes neutralization by naturally-induced and vaccine-induced antibodies. Exit Disclaimer
Duan H, Struble E, Zhong L, Mihalik K, Major M, Zhang P, Feinstone S, Feigelstock D

Biochem Biophys Res Commun 2009 Dec 18;390(3):1056-60
Antibody-mediated Synergy and Interference in the Neutralization of SARS-CoV at an Epitope Cluster on the Spike Protein. Exit Disclaimer
Zhong L, Haynes L, Struble EB, Tamin A, Virata-Theimer ML, Zhang P

Proc Natl Acad Sci U S A 2009 May 5;106(18):7537-41
Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity. Exit Disclaimer
Zhang P, Zhong L, Struble EB, Watanabe H, Kachko A, Mihalik K, Virata-Theimer ML, Alter HJ, Feinstone S, Major M

Proc Natl Acad Sci U S A 2007 May 15;104(20):8449-54
Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma. Exit Disclaimer
Zhang P, Wu CG, Mihalik K, Virata-Theimer ML, Yu MY, Alter HJ, Feinstone SM

Proc Natl Acad Sci U S A 2006 Jun 13;103(24):9214-9
Neutralization epitope responsible for the hepatitis B virus subtype-specific protection in chimpanzees. Exit Disclaimer
Zhang P, Yu MY, Venable R, Alter HJ, Shih JW

Proc Natl Acad Sci U S A 2004 May 18;101(20):7705-10.
Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Exit Disclaimer
Yu MY, Bartosch B, Zhang P, Guo ZP, Renzi PM, Shen LM, Granier C, Feinstone SM, Cosset FL, Purcell RH



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