EPA-600/1-77-049
October 1977
Environmental Health Effects Research Series
IDENTIFICATION, ISOLATION AND
CHARACTERIZATION OF THE INFECTIOUS
HEPATITIS (HEPATITIS A) AGENT
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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RESEARCH REPORTING SERIES
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EPA-600/1-77-049
October 1977
IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF THE
INFECTIOUS HEPATITIS (HEPATITIS A) AGENT
by
William T. Hall
Electro-Nucleonics Laboratories, Inc.
Bethesda, Maryland 20014
Grant No. R-804003
Project Officer
Norman A. Clarke
Laboratory Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay between the components of our physical
environment—air, water, and land. In Cincinnati, the Environmental
Research Center possesses this multidisciplinary focus through programs
engaged in
studies on the effects of environmental contaminants on
man and the biosphere, and
a search for ways to prevent contamination and to recycle
valuable resources.
The Health Effects Research Laboratory conducts studies to identify
environmental contaminants singly or in combination, discern their
relationships, and to detect, define, and quantify their health and
economic effects utilizing appropriate clinical, epidemiological,
toxicological, and socio-economic assessment methodologies.
Enteric viruses are an environmental contaminant that are found in
the feces of infected individuals and in sewage. The virus(es) of infect-
ious hepatitis (Hepatitis A) is a member of the enteric virus group and is
the one member that has been conclusively shown to be transmitted via
water. The virus(es) have proven very difficult to characterize in the
laboratory and this study was designed to combine the techniques of
electron microscopy, ultra centrifugation, column chromatography, tissue
culture and serology to isolate, identify and characterize the causative
agent(s).
R. J. Garner
Director
Health Effects Research Laboratory
ill1
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ABSTRACT
This research program has had the overall objective of combining
the techniques of electron microscopy, ultracentrifugation, column
chromatography, tissue culture and serology to identify, isolate and
characterize the infectious hepatitis (hepatitis A) etiologic agent,
to propagate it in cell cultures and to develop in vitro immunodiagnostic
assays capable of detecting the presence of the hepatitis A virus and
its antibody both in the patient and the environment.
Using program results, it was possible to relate this agent morpho-
logically and serologically to those isolated from other, geographically
separated hepatitis A epidemics. Employing hepatitis A antigen detected
by radioimmunoassay screening and isolated from stool specimens collected
from patients with either clinical or subclinical hepatitis, a radio-
immunoassay and an immune adherence hemagglutination assay were developed
for the detection of antibody to hepatitis A (anti-HA). Using these
assays, the protective effect of circulating anti-HA against reinfection
and the prophylactic effect of commercial immune serum globulin contain-
ing anti-HA were established. Likewise, a survey of a commercial plasma
donor population was conducted to obtain information on the distribution
of anti-HA among the population as a whole. It was found that approxi-
mately 37% of the tested donors had anti-HA, the frequency varying
directly with age.
All attempts to propagate the hepatitis A virus in tissue culture
have been unsuccessful.
This report was submitted in fulfillment of Grant No. R-804003
by Electro-Nucleonics Laboratories, Inc. under the sponsorship of the
U.S. Environmental Protection Agency. This report covers the period
1 December 1974 to 30 June 1977, and work was completed as of 30 June
1977.
iv
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CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Abbreviations and Symbols viii
Acknowledgments ix
1. Introduction 1
2. Conclusions 3
3. Materials and Methods 5
Populations tested 5
Panels and standards 5
Assay procedures 7
Immune electron microscopy 7
Radio irnmunoas say ;.. 7
Immune adherence hemagglutination 10
Density gradient ultracentrifugation 11
4. Results 12
Characterization of hepatitis A antigen 12
Distribution of hepatitis A antigen 16
Distribution of antibody to hepatitis A 18
References 34
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FIGURES
Number Page
Electron micrograph showing an immune complex of
27 nm HAAg particles and anti-HA (X 150,000) 13
Electron micrograph of a fecal extract containing
large concentration of 23 nm virus
particles (X 120,000) 15
Isopycnic banding of a human post-acute phase fecal
suspension in a CsCl density gradient 17
Electron micrograph of complexed 27 nm particles
banded at 1.41 g/cm3 -in CsCl (human stool) (X 150,000) . . 18
Typical pattern of HAAg excretion in a marmoset
experimentally infected with hepatitis A (GBG) virus ... 19
Inapparent hepatitis A in a marmoset experimentally
infected with hepatitis A virus 20
Pattern of HAAg excretion, SGPT levels and anti-HA
development during the course of clinical hepatitis A
in an experimentally infected marmoset 31
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TABLES
Number Page
1 Stool Sample Collection from Lynchburg Training
School and Hospital 12
2 Serologic Relationship of 27 nm Lynchburg Fecal Antigen
to Previously Reported HAAg from Geographically
Widespread Sources 14
3 Frequency of Fecal Samples RIA-reactive for the Presence of
HAAg relative to Type of Disease and SGPT Elevation. ... 16
4 Frequency of Antibody to Hepatitis A and Type of Disease
Among Patients in Ten Wards at Lynchburg Training School
and Hospital Which Were Exposed to Hepatitis A Antigen . . 22
5 Frequency of Hepatitis A and Antibody to Hepatitis A
Antigen by Age and Sex Among 385 Patients at Lynchburg
Training School and Hospital 23
6 Protective Effect of Antibody to Hepatitis A Existing Prior
to the 1970-71 Epidemic at Lynchburg 24
7 Effectiveness of Immune Serum Globulin in Preventing
Hepatitis A Infection as Related to Number of
Patients at Risk 26
8 Comparison of IAHA, IBM and RIA Titers of Anti-HA in
Human Serial Serum Samples 27
9 Comparison of RIA and IAHA Titers of Antibody to HA
in Human Sera 28
10 Comparison of IAHA, IEM and RIA Titers of Anti-HA in
Marmoset Serial Serum Samples 29
11 Frequency of Antibody to Hepatitis A Antigen Among a Group
of Regular Plasma Donors at Plasma Alliance According
to Age, Race and Sex 32
12 Frequency of Antibody to Hepatitis A Antigen According to
Age and Titer Among a Group of Plasma Donors at
Plasma Alliance 33
vii
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ABBREVIATIONS AND SYMBOLS
anti-HA
cpm
EDTA
g/cm3
GVB
HAAg (HAV)
hr
IAHA
125I anti-HA
IEM
IgG
ISG
KPO4
UCi
yi; yg
ml; mg
min
MS-1
MS- 2
PBS-FCS
RIA
SGPT
w/v
P/N
— antibody to hepatitis A
— counts per minute
— ethylenediaminetetraacetic acid
— grams per cubic centimeter
— gelatin veronal buffer
-- hepatitis A antigen (hepatitis A virus)
— hour
-- immune adherence hemagglutination assay
— antibody to hepatitis A radioactively labelled
with 125-iodine
— immune electron microscopy
— gamma globulin
— immune serum globulin
— potassium phosphate ion
— microCurie
— microliter (0.001 ml); microgram (0.01 g)
— milliliter; milligram
— minute
— viral hepatitis type A
— viral hepatitis type B
— ammonium sulfate
-- phosphate buffered saline - fetal calf serum
— radioimmunoassay
— serum glutamic pyruvic transaminase
— weight to volump
— positive cpm T negative control cpm
viii
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ACKNOWLEDGMENTS
The cooperation of the scientific staff of Electro-Nucleonics
Laboratories, Inc. is gratefully acknowledged. I am particularly indebted
to Drs. Dan Zimmerman and George Knight, Mr. Frank Mundon, Ms. Donna
Brandt and Mrs. Diana Greco for their active participation in many phases
of this research project.
None of the work reported herein could have been done without the
aid of Dr. David Madden, National Institute of Neurological and Communica-
tive Disorders and Stroke, who provided me with the original Lynchburg
material and without the encouragement and support of Dr. Norman A.
Clarke, Health Effects Research Laboratory, U.S. Environmental Protection
Agency.
Finally, I acknowledge the generous contribution of various research
reagents'by Drs. W. Bancroft and D. Gibson (Walter Reed Army Institute of
Research), Drs. D. Bradley and J. Maynard (Center for Disease Control,
Phoenix, Arizona), Drs. F. Deinhardt and D. Peterson (Rush-Presbyterian-
St. Luke's Hospital, Chicago, Illinois), Drs. W. Miller and W. McAleer
(Merck Institute for Therapeutic Research, West Point, Pennsylvania),
Dr. S. Krugman (New York University School of Medicine), Dr. J. Moor-Jan-
kowski (Laboratory of Experimental Medicine and Surgery in Primates,
Sterling Forest, New York) and Dr. W. Schultz (U.S. Naval Research
Institute).
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SECTION I
INTRODUCTION
Two forms of hepatitis have been described on the basis of clinical,
epidemiological and iiranunological features (1). Infectious hepatitis,
or viral hepatitis type A (MS-1), has an average incubation period of
about 30 days and is generally spread by direct contact, e.g. fecal-oral
transmission, or by fecal contamination of drinking water or food.
Serum hepatitis, or hepatitis type B (MS-2), on the other hand, has an
average incubation period of about 90 days and is usually, though not
necessarily, transmitted through parenteral routes (2-4). Following
the initial report of Blumberg et al. (5) of the existence of the
Australia antigen, a definite link was established by several investiga-
tors between serum hepatitis and this antigen. Subsequently much
detailed information has been published on morphological (6-10), bio-
physical (11-13), biochemical (14) and immunological (15, 16) character-
istics of the hepatitis B antigen (HBAg).
This has not been true, however, of the hepatitis A antigen (HAAg).
Since the report of Feinstone et al. (17), several investigators have
confirmed the role of a 27 nm particle in the etiology of hepatitis A
(18-20) and some morphological and biophysical studies have appeared
(21-26), but the replicative cycle, distribution, biochemistry and
immunological complexity of the virus are still relatively unknown.
Because of the general unavailability of HAAg in quantities necessary
for detailed research, much of this information must remain unknown
until reliable, affordable and reproducible in vitro and/or in vivo
hosts (chimpanzees and certain marmoset species) favor the development of
in vitro techniques, all of which, thus far, have been found unsuccessful.
Although few deaths occur as a direct result of infectious hepatitis,
over 250,000 cases have been reported in the United States during the
past 5 years (27). This figure is thought to represent about 10% of
the actual cases (28), which result in enormous financial costs due to
lost work time, medical expenses and general debilitation. In 1973,
the World Health Organization speculated that approximately 10% of the
people swimming in the Mediterranean Sea that year contracted hepatitis.
Because, according to published scientific literature, HAAg has
generally been found only in stools of icteric hepatitis patients, and
then only during a very restricted period of less than two weeks prior
to peak aminotransferase elevation (20, 23), the virus has usually
disappeared before actual diagnosis of the disease. Thus an adequately
thorough and controlled sample population is rare and valuable for
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detailed studies.
Such a population has been available to us from the epidemic of
hepatitis A that occurred in 1970 at the Lynchburg (Va.) Training School
and Hospital (29). This epidemic produced within five months approxi-
mately 565 cases of hepatitis A in a population of 3600 mentally re-
tarded. From these, special attention was given to a group of 473
severely retarded patients, 57% of whom contracted clinical hepatitis A.
Between June 1970 and February 1971, several thousand serum samples
were collected, mostly from the special study group. Blood was drawn
from these 473 patients at least bi-weekly during the period of their
illness and several times during a 6-month follow up. Infrequent or
single serum specimens were collected from 727 other patients in the
institution during the epidemic. Thus, serial specimens were accumulated
of pre-infaction, acute and convalescent sera, along with negative con-
trols. In addition, approximately 450 stool samples were obtained.
Serum glutamic pyruvic transaminase (SGPT) determinations were made on
all the sera at the time when the samples were originally collected.
Since January 1, 1975, we have been examining the Lynchburg serum
and fecal samples:
1. to identify, isolate and characterize the infectious agent
(HAAg) involved in this hepatitis A epidemic;
2. to relate it morphologically and serologically to the causative
agent of similar geographically isolated hepatitis A epidemics;
3. to determine its pattern of occurrence within a patient during
the course of disease;
4. to determine its distribution throughout the general population,
as indicated by the frequency of antibody to hepatitis A
(anti-HA);
5. to determine the course of development of anti-HA and its
protective effect against further infection by HAAg;
6. to determine the prophylactic effect of commercial immune serum
globulin in treating a population exposed to HAAg;
7. to develop in vitro diagnostic immunoassays for the detection
of hepatitis A antigen and antibody.
This report contains a description of the specific investigations
carried out between January 1, 1975 and June 30, 1977 in an effort to
attain these objectives.
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SECTION 2
CONCLUSIONS
Investigations supported by Grant No. R-804003 have enabled us to
accomplish the following:
1. show the morphological and serological similarity of the
Lynchburg hepatitis agent to MS-1 (1), the Joliet agent (30),
the Phoenix agent (22), the Costa Rican (and marmoset) agent
(18, 24) and German agent (31);
2. develop a microtiter radioimmunoassay for the detection of
hepatitis A antigen (32);
3. establish the reliability of RIA screening of fecal samples
for HAAg by comparing this technique with immune electron micro-
scopy (32);
4. adapt an immune adherence hemagglutination assay for the detec-
tion of antibody to hepatitis A utilizing fecal HAAg prepared
without the need for involved centrifugal and chromatographic
purification (33);
5. survey sera from the most heavily exposed and infected Lynchburg
wards for distribution of anti-HA and thus demonstrate the
protective effect of naturally occurring antibody against
reinfection (34);
6. show the protective effect of immune serum globulin, provided
it was administered prior to exposure to the virus (35);
7. show that anicteric as well as icteric patients can excrete
antigen in their stools (35);
8. show that HAAg excretion can begin soon after exposure and
continue beyond peak transaminase elevation (36);
9. demonstrate the existence in experimental animals (marmoset)
of an asymptomatic "antigen carrier" and suggest the possibility
of such a carrier among human populations (37);
10. demonstrate the feasibility of a surveillance program in institu-
tions for the mentally retarded, whereby the presence or absence
of anti-HA could be known by regular monitoring of each patient's
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serum. Such a program could provide a better means of prevent-
ing hepatitis epidemics in such institutions and would greatly
aid in proper prophylaxis in emergency situations (35);
11. suggest minimum standards for commercial immune serum globulin
destined for use in the treatment or prevention of hepatitis A.
This is now possible with the existence of assays for anti-HA
(38).
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SECTION 3
MATERIALS AND METHODS
POPULATIONS TESTED
Human
From April to September 1970, 375 clinical and 190 sub-clinical
cases of hepatitis A were recorded among 3600 patients at the Lynchburg
(Virginia) Training School and Hospital, an institution for the mentally
retarded (29). Between June 1970 and February 1971, several thousand
serum samples were collected, mostly from a special study group of 473
severely retarded patients, of whom 57% contracted clinically apparent
hepatitis A. Blood was drawn from these 473 patients bi-weekly during
the period of their illness and several times during a six month follow
up. Infrequent or single serum samples were collected from 727 other
patients in the institution during the epidemic. Thus, there were
accumulated serial specimens of pre-infection, acute and convalescent
sera, along with negative controls. In addition, several stool specimens
were also obtained from patients in the ten wards most seriously affected
by the epidemic. Since stools were collected randomly over the course
of the epidemic both from patients with and without the disease, it
turned out that the collection contained specimens from all stages of
infection, as well as several negative controls.
Marmoset
Six adult white-lipped marmosets (Saguinus fuscicollis, S. nigri-
collis) were inoculated intramuscularly with 0.5 ml of a 6.6% (w/v)
human fecal suspension containing hepatitis A virus, as demonstrated by
immune electron microscopy. The original fecal sample was collected by
G.G. Frosner (31) in Gomaringen, Germany, and sent to several labora-
tories for IBM identification. Finally it was sent to Drs. F. Deinhardt
and D. Peterson (Rush-Presbyterian-St. Luke's Hospital, Chicago, Illinois)
for infectivity studies in marmosets. Prior to the commencement of the
experiment, several fecal and serum samples were collected from each
animal to serve as negative controls. From the day of inoculation, stools
were collected daily, serum weekly for transaminase levels (SGOT/SGPT)
and liver biopsies bi-weekly for pathology over a period of several
months.
PANELS AND STANDARDS
Several investigators provided panels known to contain hepatitis A
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antigen or antibody, which served as controls and/or standards in the
development of, and subsequently the day to day use of, various immuno-
assays for the detection of HAAg and anti-HA.
Antibody to Hepatitis A
Paired Sera—
Human—Dr. S. Krugman (New York University School of Medicine)
provided 10 sets of paired sera collected from patients at Willowbrook
Hospital who had experienced clinically diagnosed MS-1 type hepatitis (1).
These sera had been titered for anti-HA by Dr. M. Hilleman (Merck
Institute for Therapeutic Research) using marmoset antigen in an immune
adherence hemagglutination assay (39).
—Dr. D. Gibson (Walter Reed Armed Forces Institute of Pathology)
provided paired sera from an experimentally infected volunteer from the
Joliet Prison hepatitis study (30).
Marmoset—Drs. F. Deinhardt and D. Peterson (Rush-Presbyterian-St.
Luke's Hospital, Chicago, Illinois) provided 6 sets of paired marmoset
sera collected prior to inoculation of GBG (31) hepatitis A antigen and
during convalescence from the induced clinically apparent disease.
Chimpanzee—Dr. J. Moor-Jankowski (Laboratory of Experimental Medi-
cine and Surgery in Primates, Sterling Forest, New York) provided several
sets of paired sera collected from chimpanzees during the course of
experimental induction of clinical hepatitis A in these animals.
—Research Resources Branch of the National Institute of
Allergy and Infectious Diseases provided paired sera which were distri-
buted by this agency as standards.
Unpaired Sera—
ENLI Standards—We have obtained by plasmapheresis several units of
convalescent plasma from a patient who developed hepatitis A during the
epidemic at San Diego Naval Base (20). These units have been assayed
by several laboratories, using various test procedures, and found to have
a very high titer of anti-HA. This source has served as the major
supply of gamma globulin used for IEM and RIA in these studies.
Individual small samples of human convalescent sera have been
supplied by several investigators, including Dr. D. Bradley (Center for
Disease Control, Phoenix, Arizona), Dr. W. Schultz (U.S. Naval Medical
Research Institute) and Dr. W. McAleer (Merck Institute for Therapeutic
Research).
Hepatitis A Antigen
Human—
Drs. D. Gibson and W. Bancroft (Walter Reed) provided fecal extracts
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prepared from a Joliet volunteer experimentally given MS-1 antigen (30).
These fecal samples had been studied by IBM for the presence of HAAg (17).
Marmoset—
Dr. W. McAleer (Merck Institute for Therapeutic Research) provided
a small amount of marmoset liver HAAg (18).
The major source of antigen used to generate data from the assays
reported in these pages came from stools collected at Lynchburg.
ASSAY PROCEDURES
Immune Electron Microscopy
Detection of Hepatitis A Antigen—
Paired sera, collected from a patient antecedent to and subsequent
to hepatitis A infection were used as negative control and source of
specific antibody to hepatitis A antigen (anti-HA). The assay was
performed in the following way. To 0.9 ml of a 5% fecal suspension, 0.1
ml of a 1:10 or 1:100 dilution of these sera was added and the mixture
was incubated overnight at room temperature. The suspension was centri-
fuged at 40,000 rpm in a Ti-50 rotor for 30 minutes and the resultant
pellet resuspended in 0.1 ml deionized water. A drop of the suspension
was placed on a 400 mesh carbon-coated grid, allowed to stand for two
minutes and then drawn off. Thereafter the residue was fixed with 3%
glutaraldehyde and stained with 3% phosphotungstic acid. The grids were
examined in the electron microscope for the presence of HAV antigen-
anti-HA antibody complexes. Five representative grid squares from each
of three grids were examined before a suspension was judged positive or
negative. Care must be taken to compare results of paired sera, since
stool suspensions and sera contain a large variety of particulate matter.
Detection of Antibody to Hepatitis A—
The same procedure was followed as for the detection of HAAg, except
that the fecal sample was a known positive and the serum (plasma) sample
(diluted 1:100) was the unknown. The presence of HAV antigen-antibody
complexes indicated that the serum contained anti-HA.
Radioimmunoassay
Detection of Hepatitis A Antigen—
The test is a solid phase "sandwich" type radioimmunoassay in which
gamma globulin with high anti-HA activity is immobilized by adsorption
to polyvinyl microtiter "U" plates (Cooke Engineering Co., Alexandria,
Va.). The second element of the "sandwich" is the antigen contained
in the test-sample and the third element is the 125I-anti-HA gamma
globulin, which is added after the sample has been removed from the
reaction well. The non-reacted radioactive label is removed and the
immobilized, reacted 125I-anti-HA is then determined. Specifically, the
test is prepared and performed in the following manner:
One hundred microliters (approximately 80 ug/ml) of chromatographi-
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cally purified irranunoglobulin from a serum with high anti-HA activity
was placed in a series of microtiter wells. (A convalescent serum
1:1000 in PBS was also satisfactory.) The plate was covered with
Parafilm and incubated at 4°C overnight in a moist chamber. Thereafter
the IgG was removed and the wells rinsed with 0.01M PBS containing 2%
fetal calf serum. The final rinse was left in the wells for 15-20
minutes. A 50 yl sample of the test materials (usually a 2-5% stool
suspension) was added to each of two wells (all samples were run in
duplicate) and incubated in a moist chamber at 45°C. Different sample-
incubation times were used (2 hr, 4 hr, overnight), depending on conven-
ience and level of sensitivity required. After the test samples were
removed from the wells, the wells were washed (3X) with PBS. Then 75 yl
of -1- I-labelled, chromatqgraphically purified IgG (specific'activity,
8-12 yCi/yg) containing high anti-HA activity was added to each well.
The plates were incubated at 45°C for 2 hrs in a moist chamber, washed
-1 -^ C
(5X) with PBS to remove unbound I-antibody, the wells cut out and
counted in a gamma counter for 1 min. Five samples known to be negative
for HAV were run as negative controls. Any sample which was three times
the mean radioactive counts per minute of these negative samples was
considered reactive, that is, positive for HAV.
Detection of Antibody to Hepatitis A—
Direct Test—Sera to be tested for the presence of anti-HA were
first diluted 1:1000 in 0.01M PBS. Then 100 yl of each serum was added
to two microtiter wells and incubated overnight in a moist chamber at
25 C. The diluted antiserum was removed and the wells washed (3X) with
PBS-FCS. In each well was placed 50 yl of a standard HAV-positive stool
suspension. (The stool dilution factor was determined as that necessary
to yield 2000-4000 cpm when assayed with a standard convalescent serum
and with the -'•"I-anti-HA used on the particular occasion.) The stool
suspension was incubated for 2 hrs (or 18 hrs) at 45°C and washed (3X)
with PBS. Then 75 yl 125I-anti-HA (150,000 cpm) was added to each well
and incubated for 2 hrs at 45°C. The radiolabelled antibody was removed,
the wells washed (5X) with PBS, cut out and counted on a gamma counter
for 1 min. Five wells coated with sera (1:1000) known to be nonreactive
for anti-HA and 2 wells coated with the serum (1:1000) source of the
12^I-anti-HA were used as negative and positive controls. A serum speci-
men was considered to contain anti-HA if the ratio of its cpm to the
mean cpm of the negative controls was greater than or equal to three.
Indirect Test—A "blocking test" was also used to detect the presence
of anti-HA in serum/plasma specimens. For this test, the wells were
not coated with the unknown serum, as in the direct assay, but with the
standard anti-HA serum (1:1000). Coating of the wells and rinsing were
done in the usual manner. Then 75 yl of a standard HAV-positive fecal
suspension (approximately 2000 cpm) was added to the coated wells and
incubated for 1 hr at 45°C. Prior to this addition, the fecal antigen
had been incubated at room temperature for 15-20 min with an equal
volume of the unknown serum (1:10 in PBS) in an uncoated well. This step
permitted the "blocking" of the standard antigen if anti-HA was present
in the unknown serum, with the subsequent reduction of radioactive cpm
-------
from the incubation of 75 yl 125I-anti-HA for 1 hr at 45°C. The positive
and negative controls in this assay were known paired sera, collected
before and after infection with hepatitis A. Antibody to hepatitis A
was considered to be present in any serum sample which reduced the net
cpm of the positive control by more than thirty percent. The net cpm of •
the positive control was determined as the cpm of the positive control
minus the mean cpm of the negative control.
Mercaptoethanol-RIA-Test—This assay was developed in an effort to
distinguish anti-HA (IgM)from anti-HA (IgG). The results are discussed
in the appropriate section of the text. Sera were treated with mercapto-
ethanol under conditions known to convert the pentameric 19S IgM to the
monomeric bivalent IS IgM subunit. To 25 yl of 0.2M 2-mercaptoethanol
(HSCH2 CH20H) in PBS (0.15M NaCl, 0.01M KP04, pH 7.5) was added 25 yl of
specimen £ .rum. The mixture was incubated in a 37°C water bath for 2 hrs.
Then 200 yl of PBS containing 0.03M iodoacetamide was added and incubated
at 37°C for 15 min. With this solution as a 1:10 dilution of the original
serum, a final 1:1000 dilution was made of each serum to be tested.
Wells were coated in the standard fashion and the remaining steps of the
assay carried out as described above for the "direct test."
Preparation of anti-HA IgG from Whole Serum—
Plasma known to contain high anti-HA activity was clotted by adding
2 units of thrombin per milliliter plasma. The mixture was incubated at
37°C for 1 hr and then kept at 4°C for 18 hrs. The clot was separated
from the serum by centrifugation in a GSA rotor at 6000 rpm for 1 hr.
The entire procedure was repeated on the recovered serum.
To purify the gamma globulin, the serum was first diluted (1:1) with
cold distilled water. Then 28.2 g solid (NH4)2SC>4 was slowly added to
100 ml of diluted serum with constant stirring for approximately 1.5 hrs.
To separate out the formed precipitate, the serum solution was centrifuged
in a GSA rotor at 10,000 rpm for 60 min, the supernatant poured off and
the pellet resuspended in 0.01M borate buffer (pH 8.0). A glass rod was
used to resuspend the IgG to about 20% of the starting volume of serum.
The dissolved precipitate was dialyzed for 18 hrs against 2 changes
(6 liters each) of 0.01M KP04 (pH 7.5). The IgG solution was removed
from the dialysis bag and clarified at 25,000 rpm for 30 min in a Spinco
30 rotor. The pelleted precipitate was discarded and the supernatant
saved for coating microtiter wells for RIA and for further purification
on a DEAE column to provide IgG for iodination with 125i.
To prepare the chromatographic column, 100 to 150 g DE52 (Whatman,
H. Reeve Angel, Pistcataway, N.J.) was washed 5 times with 4 liters of
0.1M KP04 (pH 7.5). The DEAE-cellulose was then washed 2X with deionized
water and finally 6X with 0.01M KP04 (pH 7.5). The washed DEAE-cellulose
was poured into a column 2.5 x 50 cm and then rewashed with 1500 ml of
the same buffer.
The IgG enriched solution (described above) was applied to the column,
which was then washed by 0.01M KP04 (pH 7.5) buffer. Fractions were
-------
collected and all those with &280 >1-0 were combined.
Preparation of 125I-IgG (anti-HA)—
IgG was purified from convalescent sera, known to have a high anti-HA
activity, by the method of Sober and Peterson (40), as modified above, and
stored between -20°C and -70°C at a concentration of approximately 8 mg/ml.
Just prior to iodination, the IgG was diluted to 2.0 mg/ml with l.OM
KPC>4 (pH 7.5) and clarified by centrifugation at 10,000 x g for 5 min.
The iodination procedure was essentially that of Hunter and Greenwood
(41). 4.0 mci (10-20 yl) of 125I in 0.1N NaOH was added to 20 yl of
Chloramine T (1.0 mg/ml in l.OM KPC>4, pH 7.5). The reaction was continu-
ously stirred in a conical microreaction vessel. 100 yl of IgG (2.0 mg/ml)
was added and the reaction terminated after 15 sec by the addition of
50 yl of sodium metabisulfite (4.0 mg/ml) and 500 yl potassium iodide
(10 mg/ml).
The iodinated IgG was separated from other components by Biogel
Chromatography. The iodinated sample was applied to a Biogel A 0.5m
column (1.5 cm x 95 cm) and eluted at a flow rate of 10 ml/hr. The
elution buffer was 0.2% BSA, 0.1% NaN3, 0.5% KI, 0.1M Tris-Cl (pH, 7.1).
Fractions of 1.3 ml were collected from the column and the iodinated IgG
was located by counting the radioactivity in 10 yl aliquots from the
collected fractions.
Immune Adherence Hemagglutination Assay
Procedure—
The IAHA used was that described by Miller et al. (39). All test
sera assayed were inactivated at 56°C for 30 min and the assay was per-
formed using Cooke Microtiter "U" bottom plates. 50 yl of heat inacti-
vated sample was placed in well #1 of duplicate microtiter lines and
serially diluted out, using 25 yl blanks of gelatin veronal buffer (GVB)
and Cooke tube manual diluters. Standard HAAg at appropriate dilution
(4 IA units) was added to one line and GVB was added to the duplicate
line as an anticomplement control. The plate was mixed on a Thomas
vibro mixer (10 sec) and incubated at 37°C for 1 hr. Guinea pig comple-
ment (1:100) was added to all wells (25 yl), the plate shaken for 10
sees and incubated at 37°C for 40 min. Then 25 yl dithiothreitol (3 mg/ml)
in 0.4M EDTA-GVB was added to each well, mixed for 10 sec and allowed to
stand at room temperature for 3 hrs to allow a pattern of agglutination
to develop. Only those wells with a 4+ agglutination were considered
to be positive. A control antibody and antigen was assayed each time
the test was run.
Preparation of Standard Antigen—
Using veal infusion broth adjusted to pH 8.5 with borate buffer con-
taining 0.05% polyethylene oxide, a 5% fecal suspension was prepared
from a Lynchburg stool specimen shown by IBM and RIA to contain a large
amount of HAAg. The raw stool was weighed in a small plastic bag and the
10
-------
appropriate volume of buffered broth added. It was then homogenized for
2-3 min in a "Stomacher 70" (provided by Cooke Laboratory Products,
Alexandria, Va.). The suspension was centrifuged at 1000 x g for 10 min,
the supernatant removed and recentrifuged at 10,000 x g for 10 min. The
above process was repeated and the ensuing second extract kept separately
as a source of HAAg. The fecal pellet was discarded and the supernatant
from the first extract mixed (1:1) with 50% acid washed kaolin in 0.85%
saline. The kaolin had been previously washed several times with dis-
tilled water until centrifugation at 1000 x g for 5 min left a clear
supernatant above the kaolin pellet. The kaolin-stool suspension mixture
was incubated in a 37°C water bath for 30 min with frequent vortexing.
Then it was centrifuged at 2000 rpm in a GLC-2 for 5 min, the supernatant
transferred to a clean tube and respun at 3000 rpm for 10 min. A dilution
series of this antigen was run against an antibody control to determine
the antigen dilution containing four IAHA units/25 pi.
Density Gradient Ultracentrifugation
Four to eight milliliters of a 5% stool suspension was layered onto
a 30 ml CsCl linear gradient (from 1.15 gin/cm-* to 1.50 gm/cm3) in a
buffer composed of 1.5% normal goat serum, 0.001M Tris (pH 7.2), 0.1%
sodium azide and 0.4% bovine serum albumin. The gradients were centri-
fuged at 45,000 rpm for 16 hrs in a Spinco Ti-60 rotor and fractionated
into 1 ml fractions using an ISCO Model D Density Gradient Fractionator.
The gradient samples were dialyzed overnight against PBS (0.01M sodium
phosphate, pH 7.2, and 0.15M NaCl) with one change of buffer. The
fractions were assayed for hepatitis A by RIA and/or IBM and combined
as desired.
Alternatively, HAAg in 10% fecal extracts was clarified by passage
through a Sepharose 2B column prior to density gradient centrifugation.
This was accomplished in the following way:
The fecal sample was first centrifuged at 20,000 rpm for 10 min in
a Beckman Ti-50 rotor to remove all debris with a sedimentation coeffi-
cient greater than 1500S. Then 5.0 ml of the clarified supernatant was
chromatographed on a 2 x 100 cm Sepharose 2B column at a flow rate of
6 ml/hr. Fractions of 5 ml each were collected and monitored for optical
density at 280 nm and by RIA for the presence of HAAg.
11
-------
SECTION 4
RESULTS
CHARACTERIZATION OF HEPATITIS A ANTIGEN
A total of 451 5% stool suspensions collected from 292 ambulatory
patients resident in 8 wards at the Lynchburg Hospital and Training
School during an outbreak of hepatitis A were examined by immune electron
microscopy (IEM) and radioimmunoassay (RIA) for the presence of hepatitis
A antigen. Of the 292 patients, 69 were diagnosed as having clinical
disease (i.e., fever, malaise, diarrhea, dark urine, and jaundice; bili-
rubinemia and elevated serum glutamic pyruvic transaminase, SGPT), 54 as
subclinical (SGPT weekly serum levels rose above 50 units) and 169 as
asymptomatic (i.e., SGPT serum levels remained below 50 units). Asympto-
matic patients were not necessarily considered to be uninfected. A
summary of the source of the Lynchburg stool samples is given in Table 1.
TABLE 1. STOOL SAMPLE COLLECTION FROM LYNCHBURG
TRAINING SCHOOL AND HOSPITAL
Patients
Clinical Subclinical Asymptomatic Total
Stool Samples
Collected 90 91 270 451
No. of Patients 69 54 169 292
No. of Wards Studied (4A,4B,4C,5A,5C,29A,25A,25B) 8
Morphology and Serology
Thirty-eight (38) Lynchburg stool suspensions were shown to contain
27 nm electron dense virus-like particles which formed antigen-antibody
complexes (Figure 1) in the presence of antibody found in convalescent
sera which was not present in the corresponding acute or pre-bleed sera
taken from the same source. This was true in sera from human patients
naturally exposed to hepatitis A as well as those exposed experimentally;
it was also true in paired sera from experimentally infected marmosets
and chimpanzees. Morphologically, the particles were identical to those
reported by others in published studies on hepatitis A. The serologic
similarity of the Lynchburg 27 nm virus-like particles to those isolated
12
-------
from other hepatitis A outbreaks is summarized in Table 2. In all cases,
antibody elicited in the patient in response to the viral infection
reacted with the HAAg recovered from each geographically separated
source to form easily identified, characteristic complexes (Figure 1).
Figure 1. Electron micrograph showing an immune
complex of 27 nm HAAg particles and
anti-HA (X 150,000).
In addition to the 27 nm HAAg, a virus-like particle with a diameter
of 23 nm was also found, sometimes in high concentrations (Figure 2).
However, this particle did not show a consistent pattern by IBM of com-
plexes (or non-complexes) based on the anticipated presence or absence
of anti-HA in paired sera, and thus was not considered to be the hepatitis
A virus.
Buoyant Density
The buoyant density of HAAg in human and marmoset stools was deter-
mined by isopycnic banding in preformed CsCl density gradients. IEM
and RIA were performed on fractions of the gradients to detect the
presence of the antigen.
Both human and marmoset feces known by RIA and IEM to have contained
HAAg were subjected to CsCl gradients for buoyant density determinations
of the contained virus.
13
-------
TABLE 2. SEROLOGIC RELATIONSHIP OF 27 nm LYNCHBURG FECAL ANTIGEN TO PREVIOUSLY
REPORTED HAAg FROM GEOGRAPHICALLY WIDESPREAD SOURCES
Serum Samples
Lynchburg (pre)
Lynchburg (conv)
Joliet (pre)°
Joliet (conv)
Phoenix (pre )
Phoenix (conv)
San Diego (pre)
San Diego (conv) e
Chimpanzee (pre)
Chimpanzee (convr
Marmoset (pre)
cr
Marmoset (conv)
2% Stool Suspension
Marmoset
Lynchburg San Diego Germany Liver
_
+ + + +
- NT NT
+ + NT NT
NT NT NT NT
+ + + +
NT NT NT NT
+ + + +
_
+ + + +
_
+ + + +
MS-1 Jolietc
Pre(FJl-2-4) Acute (FJ-1-2-5)
-
+
-
+
NT NT
+
NT NT
*
-
+
-
-
pre = Specimen collected prior to transaminase elevation
conv = Specimen collected during convalescent stage
+ = Antibody-coated 27 nm particles seen by EM
= Antibody-coated 27 nm particles not seen by EM
NT = Sample not tested
Reagents were kindly supplied by the following: (a) W. Schultz and
J.A. Routenberg, (b) G. Froesner, (c) D. Gibson, F. Top and W. Bancroft,
(d) D. Bradley and J.E. Maynard, (e) W. Schultz, (f) Research Resources
Branch (NIAID) and (g) F. Deinhardt and D. Peterson
-------
Figure 2. Electron micrograph of fecal extract containing large concentration of 23 nm virus particles
(X 120,000}.
-------
The human stool extract was collected 7-10 days after peak SGPT
elevation from an 8 year old female patient with sub-clinical hepatitis A.
Fractions collected from the density gradient contained peaks of radio-
activity at 1.27, 1.33 and 1.41 g/cm3 (Figure 3). Each of these peaks
contained 27 ran particles when examined by IEM. Figure 4 shows a complex
of 27 nm particles banded in CsCl at 1.41 g/cm3. The major peak of virus
from the human stool was at 1.33 g/cm3, while the only RIA peak of acti-
vity from the marmoset feces was at this same density (p = 1.33-1.35
g/cm3).
The marmoset stool extracts were taken from the same animal at days
14, 22 and 35 post inoculum of GBG antigen. The animal developed signs
of clinical hepatitis by day 36, as indicated by elevated SGPT and liver
biopsy. RIA examination of fecal samples collected for 80 consecutive
days post inoculum showed 3 periods of antigen excretion. Stools from
the peak day of each of these periods were put on gradients for antigen
density determination.
DISTRIBUTION OF HEPATITIS A ANTIGEN
Relation to Type of Hepatitis A
Of the 451 fecal samples examined, 38 were reactive by RIA for the
presence of HAAg. Of these, 9 were from patients with clinical hepatitis,
25 from sub-clinical and 4 from asymptomatic. Although published data
have reported the recovery of fecal HAAg only from stools collected prior
to peak transaminase elevation, 12 Lynchburg stools collected at or after
SGPT peak contained HAAg. The richest source of antigen was found in
a stool collected from an 8 year old female with sub-clinical hepatitis
approximately 10 days after maximum SGPT elevation. The frequency of
anti-positive stools in relation to the type of hepatitis disease and
level of SGPT is summarized in Table 3.
TABLE 3. FREQUENCY OF FECAL SAMPLE RIA-REACTIVE FOR
THE PRESENCE OF HAAg RELATIVE TO TYPE OF
DISEASE AND SGPT ELEVATION
SGPT Elevation
Disease State
Clinical
Sub-clinical
Asymptomatic
Pre-Peak
3
19
Peak
2
0
Post-Peak
4
6
"Normal" Total
9
25
4 4
Virus Excretion Patterns in Experimental Animals (Marmosets)
Since the exact day of infection could not be determined in any of
16
-------
(N
O
D.
O
I
<
CC
20
18
16
14
12
10
8
6
4
1.5
1.3
1.2
O
c
O
m
z
CO
H
CO
1,1
-L—' 1.0
10 12 14 16 18 20 22 24 26 28 30 32 34
GRADIENT FRACTIONS
Figure 3. Isopycnic banding of a human post-acute phase fecal suspension in a
CsCl density gradient.
-------
Figure 4. Electron micrograph of complexed 27 nm
particles banded at 1.41 g/cm3 in CsCl
(human stool) (X 150,000).
the Lynchburg patients and the number of stool samples collected per
patient was very limited, 6 marmosets were inoculated with HAAg to study
the pattern of virus excretion. In this group, 4/6 marmosets developed
acute hepatitis 29-43 days post inoculation. Clinical symptoms existed
for 1-3 weeks. Depending upon the animal, HAAg was detectable in the
feces by RIA from 8-48 days post inoculation and the excretion period of
10-20 days generally preceded maximal SGPT elevations by 6-21 days. A
typical pattern of antigen excretion is shown in Figure 5.
Two animals which did not develop abnormal transaminase levels
(or morphologic liver changes) had inapparent HAAg infections as demon-
strated by antigen fecal excretion (5-19 days post inoculum) and develop-
ment of anti-HA by day 36 post inoculum (Figure 6). Selected fecal
specimens from the HAAg excretion periods in animals with acute or
inapparent disease were examined by IBM and revealed typical 27 nm
particles.
DISTRIBUTION OF ANTIBODY TO HEPATITIS A
It has long been recognized that mentally retarded patients living in
institutions are highly susceptible to viral hepatitis. Attack rates of
1
-------
40960 r
Liver Biopsy
• = positive
0 = negative
DAYS
Figure 5. Typical pattern of HAAg excretion in a marmoset experimentally infected with
hepatitis A (GBG) virus.
-------
r
40960
< 10240
> 2560
x
640
.
30
25
20-
I 5
:_
150
100
.
.
Liver Biopsy
• = positive
C = negative
10
20
30
D
40
50
60
DAYS
70~
80
Figure 6. Inapparent hepatitis A in a marmoset experimentally infected with hepatitis A virus.
-------
acute viral hepatitis during epidemics in such institutions have reached
high levels. Approximately 16% (565/3600) of the entire institutional-
ized complement of Lynchburg Training School and Hospital and 57%
(269/473) of the most severely retarded patients contracted clinical
hepatitis in the epidemic described by Matthew et al. (29) and reported
herein. With the discovery of a source of fecal HAAg (during the RIA
screening of the Lynchburg stool samples), it became possible to develop
both RIA and IAHA assays for the detection of antibody to hepatitis A
and thus to obtain some insight into the level of exposure to this anti-
gen experienced by the patients over the course of time and into the level
of protection against reinfection offered by preexisting antibody.
Frequency of Anti-HA at Lynchburg
In Table 4 are summarized the results of RIA and IAHA tests for the
presence of anti-HA among patients in the 10 wards housing the most
severely mentally retarded. Of the 397 patients, sera were not available
from 12. Thus the determinations were gathered from 385 patients. Of
these, 41.6% (160) possessed anti-HA. The remaining 225 were without
antibody and thus supposed to be at high risk. This assumption was
borne out by the fact that 78.7% (177/225) contracted hepatitis A during
the epidemic.
Hepatitis A has long been considered by epidemiologists (42) to be
primarily a disease of children. An analysis of the distribution of
anti-HA among Lynchburg patients according to age and sex supports this
opinion. Only 5% (4/81) of the patients under 15 years of age had anti-
body to HAAg prior to the epidemic, while 34.3% (59/172) had anti-HA in
the 15-29 year old age group and 72.4% (76/105) in the 30-49 year old
group. Eighty-one (81) percent of the patients over 50 years of age had
developed detectable levels of.circulating antibody. These results
appear in tabular form in Table 5.
Protective Effect of Natural Antibody (Active Immunization)
The existence of specific antibody to HAAg was previously assumed
on the basis of apparent immunity to experimental reinfection (1, 43).
Direct serological determinations made at Lynchburg established the
factual basis of this assumption. As previously mentioned, 41.6% of
the patients in the 10 wards studied had anti-HA prior to the hepatitis
epidemic. None of this group showed any signs or symptoms of hepatitis A
during the period of observation (1970-71), nor any clinical signs of
hepatitis subsequently. This is in marked contrast to those patients at
risk, i.e., lacking antibody: 77.9% (60/77) of those under 15 years of
age, 91% (103/113) among the 15-29 year olds and 45% (13/29) among the
30-49 year olds developed hepatitis. All of this group now possess
detectable circulating antibody to hepatitis A. These data are summarized
in Table 6.
Protective Effect of Immune Serum Globulin (ISG)
During the first 10 weeks of the epidemic, commercial immune serum
21
-------
NJ
ro
TABLE 4. FREQUENCY OF ANTIBODY TO HEPATITIS A AND TYPE OF DISEASE AMONG PATIENTS IN
TWO WARDS AT LYNCHBURG TRAINING SCHOOL AND HOSPITAL WHICH WERE EXPOSED TO
HEPATITIS A ANTIGEN
Ward
4A
4B
4C
5A
5B
5C
8B
25A
25B
29A
No. of
Patients
36
34
46
42
51
46
56
24
24
38
Age
(Range)
13-28
13-34
15-35
20-65
19-63
20-65
6-20
6-22
6-22
14-41
397*
Hepatitis A Disease
Clinical Sub-clinical
14
6
12
12
1
5
45
4
12
4
115
7
13
10
3
1
4
7
4
7
6
62
State
Asymptomatic
15
15
24
27
49
37
4
16
5
28
220
Anti-HA(+)
14
11
20
25
38
30
3
1
4
14
160
HBsAg (+)
8
5
4
4
4
2
2
4
5
8
46
*Includes 12 for which data were unavailable
Hepatitis A Positive 177/385 (46%)
Anti-HA Positive 160/385 (41.6%)
Anti-HA Negative 225/385 (58.4%)
Disease Pos. _ 177
Anti-HA Neg. ~ 225
= 78.7%
-------
TABLE 5. FREQUENCY OF HEPATITIS A AND ANTIBODY TO HEPATITIS A ANTIGEN BY AGE AND SEX
AMONG 385 PATIENTS AT LYNCHBURG TRAINING SCHOOL AND HOSPITAL
N>
Age
(Yrs)
0-4
5-14
15-29
30-49
>50
Total
Patients Tested
Males
1
5
124
97
26
253
Females
0
76
48
8
0
132
Anti-HA (+)
Males
0
0
(36.3%) 45
(72.2%) 70
(80.8%) 21
(53.8%) 136
Females
0
4 (5.3%)
14 (29.2%)
6 (75%)
0
24 (18.2%)
Hepatitis A Disease
Males
1
(80%) 4
(58.9%) 73
(13.4%) 13
0
(40%) 91
Females
0
56 (73.7%
30 (62.5%
0
0
86 (65.2%
-------
TABLE 6. PROTECTIVE EFFECT OF ANTIBODY TO HEPATITIS A
EXISTING PRIOR TO THE 1970-71 EPIDEMIC AT
LYNCHBURG
Age
(Yrs)
0-4
5-14
15-29
30-49
50
Patients
Anti-HA(+) Disease (+)
0
4
59
76
21
0
0
0
0
0
Patients
Anti-HA(-) Disease (+)
1
77
113
29
5
1
60
103
13
0
Totals
160
225
177
globulin (ISG) was widely administered without apparent protective effect
(29). Therefore, a controlled study was undertaken the better to evalu-
ate the effectiveness of ISG.
At the end of the study indications were that ISG prophylaxis was
effective, if given at the appropriate time. However the distribution of
preexisting antibody in the test population, the level of protection
afforded by this antibody and the number of patients actually at risk was
unknown. With the development of in vitro diagnostic techniques (de-
scribed in this report) it was possible to reexamine the sera collected
from patients in the two wards used for the controlled experiment.
In these wards, ISG was given six and eight days respectively prior
to the appearance of the first clinical case of hepatitis A. The members
of the wards - all ambulatory patients - were divided alphabetically into
three groups: one received no ISG; a second received a dose of 0.02 ml/lb
body weight and a third received 0.08 ml/lb. In the latter two groups
a maximum of 10 ml ISG was given. The dose was repeated thirty days
later. Serum samples were collected prior to initial ISG administration
(pre-bleed) and 2, 3, 4, 5, 6, 8, 10, 12, 14 and 17 weeks thereafter.
They have since been collected annually, where possible.
The two wards were composed of 60 patients; 23 received no ISG, 21
received 0.02 ml/lb body weight and 16 received 0.08 ml/lb. In the first
group - those receiving no ISG - 19 of the 23 patients were shown to be
at risk. Of these, 12 developed either clinical (8 patients) or sub-clin-
ical (4 patients) hepatitis. On the other hand, 4 of 25 patients at risk
in the two groups receiving ISG developed hepatitis, of which three cases
were sub-clinical. Two of these four had their SGPT elevation on the
very day ISG was given, one seven days post and the fourth fourteen days
after inoculation. This would suggest that all four were infected prior
24
-------
to the administration of ISG. A summary of the results is shown in
Table 7.
Comparative Sensitivity of Assays to Detect Anti-HA
As described in Materials and Methods, several assays exist for the
detection of antibody to hepatitis A. They include RIA (44, 45), IBM
(17) and IAHA (39). From published data (17, 39, 46, 47), it was
apparent that all of these assays were capable of detecting circulating
antibody present in plasma collected during convalescence from the
disease. The more important information from a diagnostic point of view
was the comparative sensitivity of the various assays in detecting early
antibody, that is, the IgM arising immediately after exposure to HAAg (48).
To this end, two populations were studied by three assay methods.
In the first experiment, 10 serum samples from each of 15 Lynchburg
patients who had experienced clinical hepatitis A were assayed by IBM,
RIA and IAHA for the presence of anti-HA. The first serum sample was
collected in each case at least 3 weeks before peak SGPT elevation and
the last sample in each series was collected approximately 2 years after
peak SGPT. Table 8 contains the data from one of these patients and is
essentially the same as the other 14 series. The times are presented as
weeks before and after peak transaminase levels were reached. The RIA
(P/N) ratio was determined as the quotient of the cpm of each serum
sample divided by the cpm of the first sample of the respective series.
A P/N value >3.0 was considered to be positive. In every series, a
positive RIA and IBM assay indicating the presence of anti-HA was obtained
in sera collected at SGPT peak elevation, and generally by one week prior
to the peak. IAHA titers were not positive until 3-4 weeks later. RIA
and IAHA results on serial serum samples of five patients are summarized
in Table 9 to demonstrate the greater sensitivity of RIA in detecting
early antibody.
Course of Development of Anti-HA
Because the actual day of exposure to HAAg could not be determined
with certainty among the patients at Lynchburg, an experiment was per-
formed on six adult white-lipped marmosets to obtain more precise data
on the course of development of anti-HA (a description of the procedures
is detailed in Materials and Methods).
As in the human study, the IEM and RIA assays detected the presence
of antibody prior to SGPT elevation, while the IAHA required at least an
additional two weeks - a time when the SGPT level was returning to near
normal. Detectable levels of circulating antibody were present in the
marmosets within 28 days after the injection of HAV. Table 10 shows a-
comparison of IAHA, IEM and RIA titers of anti-HA in a typical series of
marmoset serum samples.
Both the human and marmoset serial serum samples were treated with
mercaptoethanol at a concentration sufficient to disrupt IgM. This
approach was used in an effort to develop a system to distinguish IgM from
25
-------
K>
TABLE 7. EFFECTIVENESS OF IMMUNE SERUM GLOBULIN IN PREVENTING HEPATITIS A INFECTION
AS RELATED TO NUMBER OF PATIENTS AT RISK
ISG
Dose No. in
ml/lb Group
0.0 23
0.02 21
0.08 16
No. with Hepatitis
Clinical Subclinical Total
8
1
0
4 12
3 4
0 0
No. of patients % of HAAb(-)
without patients
HAAb infected
19 63% (12/19)
15 26.6% (4/15)
10 0% (0/10)
A Chi-square (1 degree of freedom) test was applied to the
attack rates of those with and those without ISG.
..2 _
- 11.183
P = <0.005
-------
TABLE 8. COMPARISON OF IAHA, IBM AND RIA TITERS OF ANTI-HAAg IN HUMAN SERIAL SERUM SAMPLES
Sample
No.
2012
2723
3179
3913
4163
4243
4580
4618
5348
6346
Date
8/10/70
8/26/70
9/2/70
9/9/70
9/18/70
9/23/70
10/8/70
11/3/70
12/9/70
11/6/72
SGPT3
18
25
2800
330
90
37
15
18
15
18
IAHA
<20
<20
<20
<20
<20
<20
1600
1600
3200
3200
IEM°
0
1-2+
2-3+
2-3+
2-3+
3-4+
3-4+
3-4+
3-4+
3-4+
RIA (cpm)
80
242
1156
1091
1112
1074
1282
1220
1186
1202
P/Nd
1
3
14
13
13
13
16
15
14
15
.0
.0
.4
.6
.9
.4
.0
.2
.8
.0
(a)
(b)
SGPT expressed in International units.
IAHA titer expressed as reciprocal of
final
dilution giving
4+ agglutination.
(c) IEM expressed as relative amount of antibody complexed to
antigen particles.
(d) P/N = ratio of cpm of particular serum divided by the cpm of
the first sample in the series.
-------
TABLE 9. COMPARISON OF RIA AND IAHA TITERS OF ANTIBODY TO HAAg IN HUMAN SERA.
N>
OO
PATIENT 1
Date
8/10/70
8/28
9/2
9/9
9/18
9/23
10/8
11/3
2/10/71
RIAa
133
126
127
754C
1320
740
915
1514
1805
IAHAD
<20
<20
<20
<20
<20
<20
400
3200
6400
PATIENT 2
RIA
136
688
1009°
1132
762
906
1154
1565
1519
IAHA
<20
<20
<20
<20
<20
400
3200
6400
>12800
PATIENT 3
RIA
145
669
863C
626
793
835
948
ND
1456
IAHA
<20
<20
<20
<20
<20
<20
1600
3200
>12800
PATIENT 4
RIA
• 159
148
697
830°
956
935
1161
2140
1505
IAHA
<20
<20
<20
<20
<20
<20
800
6400
6400
PATIENT 5
RIA
80
242
1156°
1091
1112
1074
1282
904
980
IAHA
<20
<20
<20
<20
<20
<20
1600
1600
1600
(a) RIA values expressed as counts per minute.
(b) IAHA titer expressed as reciprocal of final dilution given 4+ agglutination.
(c) SGPT peak.
-------
TABLE 10. COMPARISON OF IAHA, IBM AND RIA TITERS OF ANTI-HAAg IN MARMOSET SERIAL SERUM SAMPLES
vo
Sample No.
C9581
HAAg Inoculum
C7493
C9008
C4087
C3956
C1332
C2661
C0608
C5571
C3873
C6287
C1402
Date
2/3/76
3/24/76
4/1/76
4/8/76
4/15/76
4/22/76
4/29/76
5/6/76
5/13/76
5/20/76
5/27/76
6/3/76
6/10/76
SGPT
12
12
8
12
30
148
18
10
26
52
55
50
IAHA
<20
-
<20
<20
<20
<20
<20
640
640
1280
2560
5120
5120
IEM RIA(cpm)
844
-
823
812
4- 1276
+ 2838
+ 3805
+ 5708
+ 5867
+ 5662
+ 5777
+ 6185
+ 7453
P/N
1.0
-
1.0
1.0
1.5
3.4
4.6
6.8
7.0
6.8
6.9
7.4
8.9
-------
IgG antibody response. In theory, the mercaptoethanol would inactivate
the IgM and thus reduce the radioactivity of the serum samples collected
during the early stages of the disease, at a time when the IgM-anti-HA
would be present in greater concentration than IgG-anti-HA. Figure 7
shows that the actual result was opposite from expected. The mercapto-
ethanol enhanced the determination of the IgM response, probably by
rupturing the pentameric 19s IgM to its monomeric moieties, thus providing
more antigenic sites for attachment of the I-anti-HA.
Figure 7 also serves to summarize the studies on marmosets experi-
mentally infected with HAAg. In general, HAV excretion occurred before
SGPT elevation, though, in this case, antigen was still found in the stools
at the time of highest transaminase levels. Circulating anti-HA detect-
able by RIA was already present in the marmoset by the time abnormal SGPT
levels were recorded. Measurable IAHA anti-HA titers lagged behind RIA
titers by several days and early RIA titers (thought to be of the IgM
class) were rendered more detectable by mercaptoethanol treatment.
Frequency of Anti-HA in General Population
During the course of this research grant, two reports appeared on
the variation found from lot to lot in several commercial sources of
immune serum globulin (39, 49). Data from several authors suggested
that about 47% of the adult population of the United States have anti-HA
(34, 50, 51, 52) thus at the same time being immune to further hepatitis
infection and potentially an excellent source of ISG for hepatitis pro-
phylaxis. Therefore, we determined the frequency of anti-HA in a commer-
cial plasma donor population and the titer (IAHA) of circulating antibody.
Antibody to hepatitis A antigen at an IAHA titer of 1:10 or greater
was detected in 91 of 245 (37%) regular plasma donors tested. Ninety of
these positive samples were also reactive at a dilution of 1:100.
Table 11 contains a summary of the frequency of anti-HA in this population
according to age, sex and race. There was a statistically significant
(P <0.01) increase in the frequency of anti-HA between the 18-29 year old
group (30%) and the 30-49 year old group (57%). A similar statistically
significant difference was found among Lynchburg patients for these
respective age groups (34% vs. 72%). There was no significant difference
between the frequency of anti-HA in whites and blacks nor in the frequency
of anti-HA between the white sexes. There was not a sufficient number of
black females in the donor population for statistical comparison.
The frequency of anti-HA in this population according to IAHA titer
is summarized in Table 12. Approximately 32% (29/91) of the positive
donors, or 12% (29/245) of all the donors had an anti-HA titer of at
least 1:1000. While the 30-40 year old group contained only 21% (51/245)
of the donor population tested, 48% (14/29) of the high titered plasma
donors were in this group.
30
-------
_
I
CL
O
IS)
200 -
150 -
100 -
50 -
Figure 7.
28 35 42
DAYS POST INOCULATION
Pattern of HAAg excretion, SGPT levels and anti-HA development during the course of
clinical hepatitis A in an experimentally infected marmoset.
0
X
-------
TABLE 11. FREQUENCY OF ANTIBODY TO HEPATITIS A ANTIGEN AMONG A GROUP OF REGULAR
PLASMA DONORS AT PLASMA ALLIANCE ACCORDING TO AGE, RACE AND SEX
CO
fO
Age
(Yrs)
18-29
30-49
>50
Total
White
Male
*
27/89
13/21
4/6
44/116
(30)
(62)
(67)
(38)
Female
18/68
14/22
1/1
33/91
(26)
(64)
(100)
(36)
Male
11/29
1/5
0/0
12/34
Black
(38)
(20)
(0)
(35)
Female
1/1
1/3
0/0
2/4
(100)
(33)
(0)
(50)
Total
57/187
29/51
5/7
91/245
(30)
(57)
(71)
(37)
* No. of donors positive for anti-HA
Total number of donors
( ) Percent of donors positive for anti-HA for respective group.
-------
TABLE 12. FREQUENCY OF ANTIBODY TO HEPATITIS A ANTIGEN ACCORDING
TO AGE AND TITER AMONG A GROUP OF PLASMA DONORS AT
PLASMA ALLIANCE
Reciprocal of IAHA Titer
Age 10
*
18-29 1
30-49
>50
Total 1
100
41
15
5
61
1000
7
8
15
2000
1
2
3
3000
5
3
8
6000
2
1
3
Total
57
29
5
91
* No. of samples positive for anti-HA at the indicated
dilution, but negative at the next higher dilution.
-------
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38
-------
TECHNICAL REPORT DATA
(Please read lustructions on the reverse before completing)
.. REPORT NO.
EPA-600/1-77-049
2.
4. TITLE AND SUBTITLE
Identification, Isolation and Characterization of
the Infectious Hepatitis (Hepatitis A) Agent
7. AUTHOR(S)
William T. Hall
9. PERFORMING ORGANIZATION NAME M
Electro-Nucleonics Laborati
4809 Auburn Avenue
Bethesda, Maryland 20014
JD ADDRESS
Dries, Inc.
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory - Gin., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268
15. SUPPLEMENTARY NOTES
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
October 1977 issuing date
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1CA046
11. CONTRACT/GRANT NO.
R-804003
13. TYPE OF REPORT AND PERIOD COVERED
Final ^r^nt RepTt-12/1/74 -
14. SPONSORING AGENCY CODE' Ll-inl-i-i
EPA/ 600/10
16. ABSTRACT ^his research program was initiated with the overall objecti
the techniques of electron microscopy, ultracentrifugation, column chr
tissue culture and serology to identify, isolate and characterize the
of infectious hepatitis, to propagate it in cell cultures and to devel
immunodiagnostic assays capable of detecting the presen.ce of the hepat
its antibody both in the patient and the environment.
Through the program, it was possible morphologically and serologica
agent to those isolated from other geographically separated hepatitis
Employing hepatitis A antigen detected by RIA screening and isolated f
collected from patients with both clinical and subclinical hepatitis,
and an immune adherence hemagglutination assay were developed for the
body to hepatitis A. Using these assays, the protective effect of cii
against reinfection and the prophylactic effect of commercial ISC cont
were established. Likewise, a survey of a commercial plasma donor poj
conducted to obtain information on the distribution of anti-HA among t
a whole. It was found that approximately 37% of the tested donors hac
frequency varying directly with age.
All attempts to propagate the hepatitis A virus in tissue culture I
ful.
17.
3. DESCRIPTORS
Infectious hepatitis
Serolog.y
18 DISTRIBUTION STATEMENT
Release to public.
KEY WORDS AND DOCUMENT ANALYSIS
b.lDENTIFIERS/OPEN ENDED TERMS
on Electron microscopy
Hepatitis A virus
Immunodiagnostic assay
Charac ter iza t ion
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (This page)
...1 Unclassified
ve of combining
omatography,
etiologic agent
op in vitro
itis A virus and
illy to relate thi
A epidemics.
rom stool specime
a radioimmunoassa
detection of anti
•culating anti-HA
:aining anti-HA
mlation was
:he population as
1 anti-HA with the
lave been unsucces
c. COSATI ricld/Group
68G
21. NO. OF PAGES
49
22. PRICE
IS
>™~
1
EPA Form 2220-1 (9-73)
39
$• U.S. GOVERNMENT PRINTING OFFICE: 1977- 767-140/6592
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