Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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This application is a division of Applicants' copending
patentaFplication No. 352,378, filed May 21, 1980.
The present invention is concerned with the discovery of the
existence of a non-A, non-B hepatitis associated antigen and this
invention is also concerned with the use of this antigen to
identify infectious blood donors and to prepare a vaccine. This
latter aspect is claimed in the aforementiGned patent application.
It is realized that in the time span after discovery of the exist-
ence by the present inventors, there appeared an article by
Shirachi et al, "Hepatitis 'C' Antigen in Non-A, Non-B Post-
Transfusion Hepatitis," The Lancet, October 21, 1978, pages 853-
856.
In recent studies non-A,non-B hepatitis has been found to
occur in 10% of transfused patients in the United States, resulting
in about 200,000 cases per year. Fatalities from non-A, non-B
hepatitis in the United States probably number around 1,000 per
year among transfusion-related cases.
PRIOR ART STATEMENT
Shirachi et al, The Lancet, October 21,1978, pages 853-856.
Tabor et al, Viral Hepatitis, eds. G.N. Vyas et al, The
Franklin Institute Press, Philadelphia, 1978, pages 419-421.
Tabor et al, The Lancet, March 4, 1978, pages 463-466.
Tabor et al, Gastroenterology, 76:680-684, 1979.
Gocke et al, The Journal of Immunology, 104(4): 1031-1032,
~pril 1970.
,.,~i,~
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THE NON-A, NO~-B ANTIGEN AND ITS ANTIBODY
-
Many cases of acute and chronic hepatitis which
do not result ~rom infection by either hepatitis A virus
(HAV! or hepatitis B virus (HB~), are called "non-A, n~n-B
hepatitis." and now account for 89% of cases of post-
transrusion hepatitis in t~e United States. The ~resence
Or a transmissible agent in this disease has been
demonstrated by its transmission to chimpanzees by the
inoculation of serum from humans chronically infected with
non-A, non-B hepatitis, and by serial passage to additional
~himpanzees. Recently an antigen-antibody system detected
by counterelectrophoresis (~EP) was described in humans
with post-transfusion non-A, non-B hepatitis (Shirachi,
et al, supra). In the present invention is reported
antigen which is detectable by CEP in the serum Or chim-
panzees during the acute phase of experimentally induced
human non-A, non-B hepatitis, an antibody which appears
during convalescence, and the detection of this antigen-
antibody system in human~ wlth non-A, non-B hepatitis.
Thus in the aforementioned parent application, there is
claimed an immunologic test method for the detection of a
non-A, non-B hepatitis antigen in mammals comprising reacting
a blood serum sample from the mammal to be tested with an
antibody derived from specimens of blood serum, tissue
or a cell culture taken from a donor mammal known to have
a non-A, non-B hepatitis infection, utilizing one of the
following methods to detect the presence of an immuno-
precipitin which would indicate the presence of the non-~,
non-B hepatitis antigen:
a3 counterelectrophoresis;
b) radioimmunoassay techniques;
c) agar gel diffusion techniques;
d) passive hemagqlutination techniques;
e) latex agglutination;
f) complement fixation; or
g) enzyme linked immunosorbent.
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This invention provides a method of
preparation for a vaccine effective against non-A, non-B hepatitis
infection in mammals comprising:
- isolating an antigen associated with non-A, non-B
hepatitiS from specimens of blood serum, tissue
or a cell culture taken from a donor mammal known
to be infected by non-A, non-B hepatitis; and
- inactivating said antigen; or
when an inactivated immunologically-active polypep-
tide is required,
- preliminarily purifying the antigen prior to
inactivation;
- separating the immunologically-active polypeptides
from the antigen by:
- detergent treatment,
- limited hydrolysis, or
- reduction; and
- inactivating said immunologically-active polypeptide.
In another aspect the invention provides a vaccine
e~fective against non-A, non-B hepatitis infection comprising the
inactive antigen or the inactivated immunologically-active poly-
peptide.
The activity of the antigen has been shown in counter-
electrophoresis (CEP) as well as in a solid phase radioimmunoassay.
Additionally, human tests showed antigen activity up
to 1-5 years after transfusion in the donor. The tests enable
blood banks to identify blood donors whose blood may transmit
non-A, non-B hepatitis to recipients and eliminate the use of
their blood for transfusion. This results in a decrease in the
incidence ofthis disease. The test is also used to diagnose non-A,
non-B hepatitis in patients.
An antigen was detected by counterelectrophoresis in
serum samples from six of seven chimpanzees during the acute phase
of experimentally induced non-A, non-B hepatitis using antiserum
from a chimpanzee convalescent from human non-A, non-B hepatitis.
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This antigen could not be detected prior to the transfusion
in 35 pre-inoculation serum samples from these chimpanzees,
or in 94 weekly bleedings from three chimpanzees with
hepatitis A and three chimpanzees with hepatitis B.
three chlmpanzees ~ith hepatitis A and three chlmpanzees
with hepatitis B.
The antigen was also detected in each of two
serum samples obtalned ,rom a human with chronic hepatitis
whose blood had transmitted non-A, non-B hepatitis to a
nurse by accidental needlestick and to chimpanzees by
experimental inoculation. In addition, the antigen was
detected in serum obtained retrospectively from 11 Or 31
former blood donors whose blood had transmitted post-
trans~uslon non-A, non-B hepatitis several years previously
to recipients of a single unit of their blood.
Antibody to this antigen was detected in conYa-
lescent serum samples from all seven chimpanzee~ studied,
ln convalescent serum from the nurse infected by accidental
needlectick, and in serum rrom a hemodialysis patient
convalescent from non-A, non-B hepatltis.
COUNTERELE~ROPHORESIS
Counterelectrophoresis (CEP) whlch may be also
descrlbed as lmmunoelectrosmophoresis (IEOP) or lmmuno-
electrodiffusion (IED) or countercurrentelectrophoresis ls
utllized as follows.
Sera stored at -20C were tested by CEP uslng 1%
agarose (Indubiose A37, L'industrie Biologique Francaise,
Gennevilliers, France) in barbital buffer, pH 8.6, poured
onto 3.5 x 12.5 cm glass plates (16 ml per plate). Melted
agarose (16 ml) was poured onto a lantern slide. When it
had cooled, two rows of holes were punched lnto the agarose.
Antibody was placed in one row of hole~ and samples to be
tested were added to the other row. When testing for
* Trade Mark
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antibody, antigen was added to one row and samples in the
other row. The lantern slide was placed in a CEP chamber.
Paper wicks were used to connect each side of the slide to
each of two pools of barbital buffer, pH 8.6. An electric
s current was passed across the plate, 35 milliamps per plate,
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fo- one hour. Immunoprecipitin lines were read after 1, 24,
and 48 hours Or storage in a moist chamber at room temper-
ature. When the test sample was positive, a precipitin
line was seen between the rows, using the naked eye with
the aid of an electric lamp.
RADIOIri~lJIlOASSAY _(RIA~
Antibody to the non-A, non-B hepatiti-~ ~as puri-
~ied by precipitating it rrom serum using 30, a~onium
sulrate. This purified antibody was labeled with radio-
acti~e iodine using the chloramine-T method. Unpurified
antibody was coated on ~lastic beads. The coated beads
were placed in wells of a plastic plate. Samples to be
tested for antigen were added to each well. After 18 hours
incubatior., the excess sample (other than any antigen
which was then attached to the bead) was washed away. The
~adio-labeled purif~ed antibody was then added to the wells
and incubated for three hours; the excess has washed away.
~he amount ~f radioactivity adhering to the beads was
counted in gamma counter. Positive results were identi~ied
by the detection of radioactivity on the beads, in comparison
to negati~e samples. The presence o~ antibody was determined
by adding the sample to be tested to a known antigen-positive
serum, and then, following incubation for one hour, testing
~he mixture for antigen. The presence Or antibody was
~dentified by the decrease in radioacti~e counts compare~
~o the result obtained using the antigen alone.
In addition to C~P and RI~ used to detect antigen
~nd antibodyj alternate immunolo~ical ~lethcds may be used
zo detect the antigen including aga- gel dif~usion, passive
:~emagglutination, latex agglutination, com~lement ~ixation,
~nd enzymes linked i~.uno-sorbent a~say.
T~E ANTIGEN
An abbreviated or capsuli~ed desc-iption Or pur:i-
:~ication for the associated antigen anQ ac i~e slb;nits i.s
~u.marized as follows.
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The non-A, non-B hepatitis associated antigen
was purified rrom serum (or tissue and cell cultures when
the agent is ~ropogated) by selection from the follcwing
techniques:
(1) Fracticnal (selective) precipitation or
solubilization
(2) Gel filtration, molecular sieving
(3) Chromatographic techniques (affinity,
adsorption or ion-exchange chromatography)
(4) Density gradient centrifugation
(5) Electrophoresis including isotachophoresis
and isoelectric focusing
(6) Countercurrent distribution
Further purification treatments include altera-
tions in pH, chemical treatments and enzy~e treat~ents.
Subunits
Immunologically active subunit~ of the non-h,
non-B hepatitis associated antigen have been prepared
following preliminary purification of the antigen by z
selection from the following:
(1) detergent treatment
(2) limited hydrolysis
(3) reduction
Immunologically active polypeptides have been sepa-
rated here by procedures outlined above.
Development of In Vitro ~ests
By lnducing antibody specific for the n~n-A,
non-~ associated antigen in suitable animal species; '.e.,
chirpanzees, or selecting human sera containing these
3 anti~odies, immunologic tests to detect the antigan
(such as Agar gel diffuslon, counterelectrophoresis,
complement rixation, passive hemagglutination, raii~-
i~inoassay or enzyme-linked i~muno-sorbent assay! :~a!e
~en ~eveloped and used tc (1) detect persons trZ~c i ti-~-
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non~A, non-B hepatitis and (2) identify sources of antigen
ror _ Yitro tests and vaccine production.
~accine
A direct use Or purified antigen or immunologi-
cally act-ve subunits inactivated by either heat, formalin
or both m~y be conventionally utilized as a vaccine.
The table below shows a su~.~ary of clinical
testin~.
TABLE 1
Non-A, Non-B
Patients Tested Antigen Antibody
54 Norm~l volunteer blood 0 Not tested
donors
3 Humans with chronic non-A, 3
15non-B hepatitis who trans-
mitted the dlsease to
h~mans and chimpanzees
31 Blood donors who transmitted 11 5
non-A, non-B hepatitis one
. to fc~r years previously
2012 Humans with non-A, non-B 8 Not tested
hepatitis (weekly samples)
2 Humans who recovered from 0 2
non-~., non-B hepatitis
152 Hemo~hiliac patients Not tested 59
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EXAMPLE l
Serum samples were obtained from three humans
with chronic non-A, non-B henatitis. Blood from human #l
h~d caused non-A, non-B hepatitis in a nurse who acciden-
tally cut herself on a piece of glass contaminated with
his blood. Humans #2 and #3 had donated blood, and their
blood had caused non-A, non-B hepatitis in recipients.
Serum rrom all th~ee (hum~ns #1, #2, and #3) was inocllated
into chimpanzees and caused non-A, non-~ hepatitis in the
chi~pa~lzees. The non-A, non-B hepatitis associated antigen
was found -n the blood of all three humans.
EXAMPLE 2
Serum samples were obtained from 31 blood donors
whose blood had caused non-A, non-B hepatitis in p~tients
who had been transfused with a single unit of their blood
(and no other blood) one to four years previously. The
non-A, non-B hepatitis associated antigen was detecte~ in
ll Or these donors.
EXAMPLE 3
Serum was tested from 54 normal blood donors.
None had the non-A, non-B hepatitis associated antigen.
EXAMPLE 4
Flve of the 31 implicated blood àonors (con~er
Exa~ple 2) had antibody to the non-A, non-E associate
antigen, but no detectable antigen. The antibody in ~hese
cases indicated the presence of a diffe~ent state Or
Aisease and was also an indication that in some cases
heir blood woulc transmit the disease, as it had done
previously.
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EXA~I~LE 5
Chimpanzee Studies
Weekly serum samples from seven chimpanzees
beginnin~ four weeks before inoculation with human non-A,
non-B ~.epatitis were tested. The inoculation and course
of infe^tion in thes~ chimpa~zees are described in the
three ~abor et al ar~ic]es n~ted in the Prior Art State~,ent,
supra. ~ach chimpanzee was infected by intravenous
inoculation of seru~ from one of three humans chronically
inrected ~ith with non-A, non-B hepatitis. Chimpanzees
#922, ~:930, #911, ~916, and #946 were infected by inocula-
tion with Inoc~lum I, or with acute phase serum from a
chimpanzee infected by Inoculum I (Inoculum I passage).
Chimpanzee ~918 was infected by Inoculum II and #919 by
Inoculu~ III. A convalescent serum from each chimpanzee
was used as antibody in CEP against that chimpanzee's
own weekly ser~m sar.~les; in three chimpanzees (#922, ~918,
#919), the convalescent serum was obtained arter two
intravenous inoculations with infectious serum. In addition,
convalescent serum from chir..panzee #922 was used to test
all chim~anzee seru~. samples studied.
Results. The ant~gen was detected in the sera o~
six of seven chimpar.~ees du~in~ non-A, non-B h-epatitis.
In ger.~ral, the antigen was detected durin~ the ti~.e of
elevated aminotrans~erase l~vels but without a strict
correl~tion with hic,opatho o.~,ic changes in liver biopsy
speci,.~r.s. Ch~mpan-ee ~922 (Inoculu~ I) had ele~ated
a~.ino~ans~erase levels from Week 2 to 1~ and had anti~en
detec.^-;cle at Weeks 4-9 ana at Week 15. ~ impanzee ~9
3Q (Inoc~' ~m T ~ had el~vated ~minotransferase levels from
l~eek ^~ ~o 23 and hai antigen detectabl~ at ~ieeks 2-8
(includ-nr two ser~. sam~les shown to transmit non-A,
no~--3 a-~atitis to eY.perimenta'ly inoculated chimpanzees'
an~ a~ .;ee'~ 18. Ch. mpanzeê ~C l (Inoculu.. I pa~sa5e~
had e~-v~ed amino.-ansfer~ae levels fror. ..eek 5 to 21
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and had antigen detectable at Weeks 19 and 20. Chimpanzee
~946 (Inoculum I passage) had ele~ated aminotransferase
le~els from Week 3 to 11 and had antigen detectable at
Weeks 9, 10, 12, and 16. Chimpanzee #918 (Inoculum II)
had elevated aminotransferase levels from Week 4 to 20 and
had antigen detectable at Weeks 6, 11, 14, and 15.
Chim~anzee #9lg (Inoculum III) had elevated an.inotrans-
ferase levels from Week 3 to 20 and had antigen detectable
at Week 3. The antigen could not be detected in seru~
samples from Chimpanzee #916 (Inoculum I passage).
The antigen could not be detected in any of 35
pre-inoculation serum samples from these chimpanzees,
nor could it be detected in 28 weekly bleedings from three
chimpanzees during experimentally induced hepat1tis A
or in 66 weekly bleedings from three chim~anze~s durlng
15 - experimentally induced hepatitis B.
Antibody was detected in convalescent serum
samples from all seven chimpanzees. Antibody was detected
in every serum sample from chimpanzee #922 beginning with
Week 28 after inoculation, 13 weeks after the disappearance
of antigen and the return of aminotransferase levels to
near normal values. Antibody remained detectable until
longer than 19 months after inoculation. Titrations
performed on selected serum samples from chimpanzee ~922
before and after a second intravenous exposure to a non-A,
non-~ hepatitis inoculum (Inoculum III), revealed a
four-fold increase in antibody titer. An~onium sulfate
precipitation and DEAE cellulose chromatography revealed
the antibody to be in the 7S (IgG) fraction.
E~ANPLE 6
Human serum used as antibody ~n CEP included
convalescent serum from the nurse (Human #l) ~ho had re-
covered from non-A, non-3 hepatitis 4 years earlier after
the needle~ick xposure ~o ~noculunl I and convalescent
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serum rrom a multiply-transrused hemodialysis patient
with a history Or non-A, non-B hepatitis (Hum~an ~2).
~ esults. The antibody was detected in con~ales-
cent serum from Human #1 and Human #2. Antibody was not
detected in any Or two serum samples rrom the patient with
chronic non-A, non-B hepatitis whose serum became Inoculum I.
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