Efficacy Testing for Hepatitis B-Type Virus: Questions
Questions for the SAP
1. If the Agency decides to replace the chimpanzee test used in testing the efficacy of disinfectants against Human Hepatitis B-type virus, what test methodologies could be used as a replacement. Two possibilities that have been proposed to the Agency are the Duck Hepatitis B Virus Test ( DHVT ) and the Morphological Alteration and Disintegration Test (MADT). Could one or both of these tests, or additional tests be used to test for efficacy against Human Hepatitis Virus B.
2. If a surrogate test system ( ie . the DHVT) is found to be acceptable for efficacy testing using Hepatitis Virus B, then would the results be sufficient to allow the registrant to make a label claim that the product was efficacious against Human Hepatitis Virus B, even though it was tested against a surrogate virus (ie. Duck Hepatitis Virus) and not the human virus.
Human Hepatitis B virus (HBV) is a serious viral pathogen in man that is highly contagious and is responsible for several deaths every year. Because of its importance as a human pathogen, there is a lot of interest in making label claims for efficacy of disinfectants against HBV. Currently the Agency requires a test using HBV and a chimpanzee as the model system to determine efficacy against this virus. Since the chimpanzee is a protected species, the continued use of this model is in question. This is especially true because there are other surrogate models, which do not use protected species, which can be used to replace the chimpanzee test.
The major advantage of the chimpanzee test system is that the human Hepatitis B virus itself is used in the test. It allows the disinfectant to be tested against the actual human virus. So if the Agency decides to use a model system using a surrogate virus, then the question of how do results using a surrogate virus relates to the human virus. In other words, will a disinfectant that in effective against a surrogate hepatitis virus also be effective against HBV.
A lot of research has been done which compares HBV with its surrogate viruses, ie. Duck hepatitis B virus (DHBV) (mason et al), ground squirrel hepatitis virus (GSHV), and woodchuck hepatitis B virus (WHV) (summers). A strong case has been made in the literature that these viruses are very closely related. They have been placed in the same virus taxonomic family, Hepadnaviridae, based on many of these similar characteristics. Some of their points of similarity are as follows (robinson):
- Blood-borne 42-47 nm in size enveloped spherical virions accompanied
by numerous viral forms without nucleocapsids.
- Relaxed circular virion DNA with length corresponding to 3000-3300
base pairs. Minus strand is greater than genome length and has
protein covalently attached at 5' end. Plus strand is a variable
length and has RNA at the 5' end.
- DNA sequence contains 3 or 4 open reading frames encoding surface
antigens, core proteins, P protein and, in mammalian viruses,
- Replication via reverse transcription of a greater-than genome
length RNA intermediate. Forms of replicating viral DNA include
single stranded DNA and covalently closed circular DNA.
- Viral replication takes place predominantly in the liver.
- Persistent infection with incomplete forms and usually complete
virions are found continuously in the blood.
- Infection associated with hepatitis type symptoms.
- Species specific.
|virions||42 nm spherical 27nm core p=1.24 in CsCl DNA polymerase activity||45 nm spherical 27 nm core cross-reactive withHBeAg (10%) p=1.225 in CsCl DNA polymerase activity||47 nm spherical approx. 30 nm core cross-reactive with HBeAg DNA polymerase activity||40-45 nm spherical 27 nm core (spikes) p=1.16 in CsCl DNA polymerase activity|
|genome||DNA, circular large single-stranded gap cohesive ends 3,182 base pairs||DNA, circular large single-stranded gap cohesive ends 3,308 base pairs some homology with HBV||DNA, circular large single-stranded gap cohesive ends 3,250-3,300 base pairs||DNA, circular large single-stranded gap cohesive ends approx. 3,000 base pairs|
|"Surface antigen" particles||"HBsAg" numerous in the blood 22nm spherical and filamentous forms p=1.19-1.20 in CsCl||"WHsAg" numerous in the blood 20-25 nm spherical and filamentous forms p=1.18 in CsCl weak cross-reaction with HBsAg (0.1-1%)||"GSHsAg" numerous in the blood 15-25 nm spherical and long filamentous forms p=1.18 in CsCl weak cross-reaction with HbsAg||"DHBsAg" numerous in the blood 40-60 nm spherical convoluted forms p=1.14 in CsCl|
|"natural" host||human||eastern woodchuck (Marmota monax monax)||Beechey ground squirrel (Spermonphilus beecheyi)||Pekin duck and occasionally other breeds (Anas domesticus)|
|distribution in selected populations||0.1-"20%" persistent infections||16-30% persistent infections||0-50% persistent infections||5-10% persistent infections|
|transmission||vertical horizontal||?||?||Egg transmitted|
|associated disease||healthy carriers acute, chronic forms of hepatitis hepato-cellular carcinoma||healthy carriers chronic forms of hepatitis hepato-cellular carcinoma||healthy carriers ?||healthy carriers acute, chronic forms of hepatitis (Marion et al) hepato-cellular carcinoma (Yokosuka et al)|
In addition, to the above morphological and structural similarities, the woodchuck, duck and ground squirrel models have been use to study various aspects of the HBV disease process. This has occurred because of the limited role the chimpanzee model can play as a model for studying viral replication or evaluating antiviral agents ( murray ). These include the following studies:
* The Pekin duck model demonstrated a good correlation of experimentally induced viremia with incidence and severity of hepatitis and was shown to be a simple, rapid, and relatively inexpensive model to study the relation of lesions to Hepatitis B family infection in nonprimates (Marion et al).
* The Duck hepatitis model was used for the study of hepadnavirus inactivation using the antiviral guanine nucleoside analog penciclovir. Synthesis of all viral replicative intermediates was inhibited by penciclovir.
* A woodchuck model was used to study increases in the frequency of chronic hepatitis infections in adult woodchucks caused by immunosuppression using cyclosporin .
* The susceptibility of DHV to sodium hypochlorite ( NaCOCl ) and sodium dichloroisocyanate ( NaDCC ) was compared to the susceptibility of HBV. The results indicated that the total inhibition in vitro of hepadnavirus DNA polymerase activity by chemical disinfectants is predictive of inactivation of infectivity in vivo (. Tsiquaye ).
In addition to the in vivo model systems that have been discussed above, there are in vitro assays described in the literature. These involve using hepatocytes from human, chimpanzee, woodchuck, or duck. The major drawback with these models is that the virus concentrations tend to be very low. However, a duck hepatocyte procedure was shown to be effective as a surrogate infectivity test, thereby minimizing the need for primates (Prince et al).
The main properties of the DHV DNA polymerase have been compared with those of HBV and WHV DNA polymerases . The polymerases were found to be very similar, with all being active under high salt conditions in the presence of a high magnesium concentration, and inhibition by the triphosphate derivatives of several nucleoside analogs appeared to be similar for all three viruses ( Fouriel et al).
An alternative test described in the literature is the Morphological Alteration and Disintegration Test ( MADT ). (Prince et al). It is an electron microcopy-based method which uses structural integrity of HBV virions that remain after contact with a disinfectant to determine it's efficacy. It is used in conjunction with an acceptable infectivity model., such as the chimpanzee test or duck hepatocyte test to predict efficacy. The MADT was used to determine the number of virions destroyed or morphologically altered by the test disinfectants. After disinfection, neutralization and purification through sucrose, the samples were adsorbed onto electron microscopy grids and stained. Destruction of substantial numbers of virions is evidence that infection is not possible. Similarly, the presence of ultrastructural changes in the outer viral envelope is evidence that infection that is dependent upon envelope-mediated attachment to the host receptor is not possible. The results indicated that if only 10% or less of the virion survive disinfectant treatment, then the virus is not capable of causing an infection.
Due to the protected status of the chimpanzee, the continued use of this test model is in doubt. Therefore, the Agency is investigating alternate test procedures for HBV efficacy testing. A number of alternate testing models are available in the literature. The major drawback of most of these procedures is that they use surrogate viruses and not Human Hepatitis Virus B. One alternate test does use HBV (MADT), but it is a noninfectivity assay that uses electron-microcopy and a companion infectivity test. Therefore, the Agency has decided to allow for an open public discussion on this subject area. We request that the SAP consider the continued use of chimpanzees as a model system. If this is not an acceptable option, then we would like the SAP to consider the alternate test systems, such as the duck hepatitis B virus model (Long), to determine if one or more would be an acceptable replacement for the chimpanzee test. In addition, if a test using a surrogate virus is found to be a reasonable alternative, then the issue of what will be allowable as a label claim needs to be discussed by the SAP. Can human hepatitis B virus efficacy claims be made based on results from a surrogate virus, such as duck hepatitis virus B.
1. Cote PJ, Korba BE, Steinberg H, Ramirez-Mejia C, Baldwin B, Hornbuckle WE, Tennant BC, Gerin JL. Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity. Journal of Immunology 1991,146 (9):3138-44.
2. Fourel I, Hantz O, Cova L, Allaudeen HS, Trepo C. Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases. Antiviral Research 1987, 8:189-199.
3. Lin E, Luscombe C, Wang YY, Shaw T, Locarnini S. The guanine nucleoside analog penciclovir is active against chronic duck hepatitis B virus infection in vivo. Antimicrobial Agents and Chemotherapy, 1996, 40(2):413-418.
4. Long Z, Sun C, White EM, Horowitz B, Sito AF. Hepatitis B viral Clearance studies using duck virus model. Brown F (ed): Virological Safety Aspects of Plasma Derivatives. Dev. Biol. Stand. Basel, Karger. 1993, 81:163-168.
5. Marion PL, Knight SS, Ho B, Guo YY, Robinson WS, Popper H. Liver disease associated with duck hepatitis B virus infection of domestic ducks. Proc Natl. Acad. Sci., USA. 1984, 81:898-902.
6. Murray SM, Frieman JS, Vickery K, Lim D, Cossart YE, Whiteley RK. Duck hepatitis B virus: a model to assess efficacy of disinfectants against hepadnavirus infectivity. Epidemiol. Infect., 1994, 106:435-443.
7. Mason WS, Seal G, Summers J. Virus of Pekin ducks with structural and biological Relatedness to human hepatitis B virus. Journal of Virology. 1980, 36(3):829-836.
8. Prince DL, Prince NH, Thraenhart O, Muchmore E, Bonder E, Puch J. Methodological approaches to disinfection of human hepatitis B virus. Journal of clinical Microbiology. 1993, 31(12):3296-3304.
9. Robinson WS. Hepadnaviridae and their replication. Virology, Second Edition, Fields BN, Knipe DM et al (eds), Raven Press Ltd., New York. 1990. Chapter 76, 2137-2169.
10. Summers J. Three recently described animal virus models for human hepatitis B virus. Hepatology 1981 1(2):179-183.
11. Tsiquaye KN, Barnard J. Chemical disinfection of duck hepatitis B virus: a model for inactivation of infectivity of hepatitis B virus. Journal of Antimicrobial Chemotherapy. 1993, 32:313-323.
12. Yokosuka O, Omata M, Zhou Y, Imazeki F, Okuda K. Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma. Proc. Natl. Acad.. Sci. USA 1985, 82:5180-5184.Scientific Advisory Panel (SAP) September 1997 Meeting: Efficacy Testing for Hepatitis B-type Virus