Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1998-09-03
2000-12-05
Wortman, Donna
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 795, 436513, 436518, 436820, C12Q 170
Patent
active
061564990
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention is in the field of hepatitis A virology. More specifically, the invention relates to methods for detecting antibodies to the hepatitis A virus 3C proteinase.
BACKGROUND OF THE INVENTION
Hepatitis A virus (HAV) is a picornavirus that causes acute liver disease in humans and some lower primates. As with other viruses of the picornavirus family, the genome of HAV contains three regions that encode the proteins of the virus. The structural proteins which form the viral capsid are encoded by the P1 region and the nonstructural proteins, which include viral proteinases, polymerase and other replication proteins are encoded by the P2 and P3 regions.
During a natural infection, the immune system of the infected individual produces antibodies to both the structural and nonstructural proteins (Stapleton et al (1995), J. Infect. Dis., 171 (Suppl. 1): S9-14). By comparison, an inactivated vaccine will induce antibodies to only the structural proteins and not to the nonstructural proteins (Robertson et al. (1993) J. Med. Virol., 40:76-82).
Currently marketed diagnostic assays used to determine HAV exposure detect antibodies to only the structural proteins. Thus, the current testing procedures cannot distinguish an individual with a natural infection from one who has been vaccinated. For diagnostic, safety and epidemiological reasons it is therefore important to develop an immunoassay which is capable of distinguishing whether antibodies were induced by vaccination or by an HAV infection. Previous studies using immunoprecipitation showed the production of antibodies to the entire P2 or P3 regions but not to the 3C proteinase specifically (Jia et al. (1992), J. Infec Dis., 165:273-280; Robertson et al. (1993), J. Med. Virol., 40:76-82).
SUMMARY OF INVENTION
The present invention relates to methods for detecting antibodies specific for the HAV 3C proteinase in biological samples. The methods are useful for diagnosis of infection and disease caused by HAV, for distinguishing a natural infection from vaccination with an inactivated vaccine, and for monitoring the efficacy of preventive agents such as vaccines.
DESCRIPTION OF FIGURES
FIGS. 1A-1F show anti-3C proteinase antibody response compared to seroconversion to the structural proteins (anti-HAV) and to alanine aminotransferase (ALT) levels in chimpanzees with hepatitis following inoculation with wild-type virus HM-175 (FIG. 1A) or SD-11 (FIGS. 1B and 1C), or in chimpanzees with an infection modified by injection of immune serum globulin (ISG) prior to inoculation with wild-type HM-175 (FIGS. 1D-1F). ALT levels are indicated as units/liter (U/L) on the right-hand vertical axis of FIGS. 1A-1F; OD levels for the anti-3C proteinase ELISA obtained using 1:20, 1:100 or 1:1000 dilutions of sera are indicated in the Figures as bars marked 20, 100 and 1000, respectively; and the horizontal bar labelled anti-HAV in each of FIGS. 1A-1F represents samples which were seropositive for HAV structural proteins (value of N/S>2.0 for antibodies to HAV structural proteins).
FIGS. 2A and 2B show anti-3C proteinase antibody response compared to seroconversion to the structural proteins (anti-HAV) and to isocitrate dehydrogenase (ICD) levels in tamarins infected with HAV 8Y (FIG. 2A) and HAV/7 (FIG. 2B). ICD levels are indicated as units/milliliter (U/ml) on the right-hand vertical axis of FIGS. 2A and 2B; OD levels for the anti-3C proteinase ELISA obtained using 1:50 (or 1:20), 1:100 or 1:1000 dilutions of sera are indicated in FIG. 2A as bars marked 50, 100 and 1000 respectively and in FIG. 2B as bars marked 20, 100 and 1000 respectively; and the horizontal bar labelled anti-HAV in FIGS. 2A and 2B represents samples which were seropositive for HAV structural proteins (value>2.0 for antibodies to HAV structural proteins).
FIGS. 3A-3F show IgG antibody response to 3C proteinase in chimpanzees inoculated with virulent HAV strain HM-175 and inactivated HAV vaccine (FIG. 3A); with virulent HAV strain SD-11 (FIG. 3B); with inactivated HAV
REFERENCES:
Robertson et al., Antibody Response to Nonstructural Proteins of Hepatitis A Virus Following Infection. Journal of Medical Virology 40:76-82, 1993.
Schultheiss et al., Clevage Specificity of Purified Recombinant Hepatitis A Virus 3C Proteinase on Natural Substrates, Journal of Virology 69(3):1727-1733, 1995.
Jokik et al., Eds, Zinsser Microbiology, 20th Ed., 1992 Appleton and Lange, Norwalk, p. 1040.
Emerson Suzanne U.
Morris Tina S.
Purcell Robert H.
Stewart Deneen
The United States of America as represented by the Department of
Wortman Donna
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