CA1155764A - Detection of non-a, non-b hepatitis associated antigen - Google Patents
Detection of non-a, non-b hepatitis associated antigenInfo
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- CA1155764A CA1155764A CA000413076A CA413076A CA1155764A CA 1155764 A CA1155764 A CA 1155764A CA 000413076 A CA000413076 A CA 000413076A CA 413076 A CA413076 A CA 413076A CA 1155764 A CA1155764 A CA 1155764A
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Abstract
TITLE: DETECTION OF NON-A, NON-B HEPATITIS
ASSOCIATED ANTIGEN
ABSTRACT OF THE DISCLOSURE
A vaccine effective against non-A, non-B hepatitis injection in mammals comprising an inactive antigen or an inactivated immunologically-active polypeptide isolated from specimens of blood serum, tissue or a cell culture of a donor mammal known to be injected with non-A, non-B hepatitis.
ASSOCIATED ANTIGEN
ABSTRACT OF THE DISCLOSURE
A vaccine effective against non-A, non-B hepatitis injection in mammals comprising an inactive antigen or an inactivated immunologically-active polypeptide isolated from specimens of blood serum, tissue or a cell culture of a donor mammal known to be injected with non-A, non-B hepatitis.
Description
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,~
1~5764
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,~
1~5764
- 2 -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.
`` ` 1155764
-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.
`` ` 1155764
- 3 -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.
- 3a -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 ~ . ~
- 3b -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, 1 1557B`4 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.
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)
- 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.
- 3a -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 ~ . ~
- 3b -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, 1 1557B`4 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.
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-~-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~.
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
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-~-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~.
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
- 7 - 1155764 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.
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.
Serum was tested from 54 normal blood donors.
None had the non-A, non-B hepatitis associated antigen.
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.
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 g 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 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.
~ .
~ , .
~-
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.
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.
Serum was tested from 54 normal blood donors.
None had the non-A, non-B hepatitis associated antigen.
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.
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 g 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 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.
~ .
~ , .
~-
Claims (26)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. 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 polypeptide 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.
- 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 polypeptide 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.
2. 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.
- 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.
3. The method of claim 2 wherein the antigen is purified before being inactivated by the technique of:
- fractional precipitation;
- solubilization;
- gel filtration;
- molecular sieving;
- affinity, adsorption, or ion-exchange chromatography;
- density gradient centrifugation;
- electrophoresis; or - countercurrent distribution.
- fractional precipitation;
- solubilization;
- gel filtration;
- molecular sieving;
- affinity, adsorption, or ion-exchange chromatography;
- density gradient centrifugation;
- electrophoresis; or - countercurrent distribution.
4. The method of claim 3 wherein the antigen is further purified by at least one of:
- alterations in pH;
- chemical treatments; and - enzyme treatments.
- alterations in pH;
- chemical treatments; and - enzyme treatments.
5. The method of claim 2 wherein the antigen is inacti-vated by:
- heat treatment; or - treatment with formalin.
- heat treatment; or - treatment with formalin.
6. A method of preparation for a vaccine effective against non-A, non-B hepatitis infection in mammals comprising:
- isolating the 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;
- preliminarily purifying the antigen;
- separating immunologically active-polypeptides from the antigen by:
- detergent treatment, - limited hydrolysis, or - reduction; and - inactivating said immunologically-active polypeptide.
- isolating the 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;
- preliminarily purifying the antigen;
- separating immunologically active-polypeptides from the antigen by:
- detergent treatment, - limited hydrolysis, or - reduction; and - inactivating said immunologically-active polypeptide.
7. The method of claim 6 wherein the immunologically-active polypeptide is purified before being inactivated by the technique of:
- fractional precipitation;
- solubilization;
- gel filtration;
- molecular sieving;
- affinity, adsorption, or ion-exchange chromatography;
- density gradient centrifugation;
- electrophoresis; or - countercurrent distribution.
- fractional precipitation;
- solubilization;
- gel filtration;
- molecular sieving;
- affinity, adsorption, or ion-exchange chromatography;
- density gradient centrifugation;
- electrophoresis; or - countercurrent distribution.
8. The method of claim 6 wherein the immunologically active-polypeptide is further purified by at least one of:
- alterations in pH;
- chemical treatments; and - enzyme treatments.
- alterations in pH;
- chemical treatments; and - enzyme treatments.
9. The method of claim 6 wherein an immunologically active-polypeptide is inactivated by at least one of:
- heat treatment; or - treatment with formalin.
- heat treatment; or - treatment with formalin.
10. The method of claim 2 wherein the donor mammal is a chimpanzee.
11. The method of claim 2 wherein the donor mammal is a human being.
12. The method of claim 6 wherein the donor mammal is a chimpanzee.
13. The method of claim 6 wherein the donor mammal is a human being.
14. A vaccine effective against non-A, non-B hepatitis infection in mammals comprising an inactive antigen or an inacti-vated immunologically-active polypeptide isolated from specimens of blood serum, tissue or a cell culture of a donor mammal known to be infected with non-A, non-B hepatitis whenever prepared by the method of claim 1 or an obvious equivalent thereof.
15. A vaccine effective against non-A, non-B hepatitis infection in mammals comprising an inactive antigen isolated from a specimen of blood serum, tissue, or a cell culture of a donor mammal known to be infected with non-A, non-B hepatitis whenever prepared by the method of claim 2 or an obvious equivalent there-of.
16. A vaccine effective against non-A, non-B hepatitis infection in mammals comprising an inactivated immunologically-active polypeptide isolated from specimens of blood serum, tissue, or a cell culture of a donor mammal known to be infected with non-A, non-B hepatitis whenever prepared by the method of claim 6 or an obvious equivalent thereof.
17. A vaccine as claimed in claim 13 wherein the antigen is purified before being inactivated whenever prepared by the method of claim 3 or an obvious equivalent thereof.
18. A vaccine as claimed in claim 15 wherein the antigen is further purified whenever prepared by the method of claim 4 or an obvious equivalent thereof.
19. A vaccine as claimed in claim 15 wherein the antigen is inactivated whenever prepared by the method of claim 5 or an obvious equivalent thereof.
20. A vaccine as claimed in claim 16 wherein the immuno-logically-active polypeptide is purified before being inactivated whenever prepared by the method of claim 7 or an obvious equivalent thereof.
21. A vaccine as claimed in claim 16 wherein the immunologically-active polypeptide is further purified whenever prepared by the method of claim 8 or an obvious equivalent thereof.
22. A vaccine as claimed in claim 16 wherein the immunologically-active polypeptide is inactivated whenever prepared by the method of claim 9 or an obvious equivalent thereof.
23. A vaccine as claimed in claim 15 wherein the donor mammal is a chimpanzee whenever prepared by the method of claim 10 or an obvious equivalent thereof.
24. A vaccine as claimed in claim 15 wherein the donor mammal is a human being whenever prepared by the method of claim 11 or an obvious equivalent thereof.
25. A vaccine as claimed in claim 16 wherein the donor mammal is a chimpanzee whenever prepared by the method of claim 12 or an obvious equivalent thereof.
26. A vaccine as claimed in claim 16 wherein the donor mammal is a human being whenever prepared by the method of claim 13 or an obvious equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000413076A CA1155764A (en) | 1979-05-21 | 1982-10-07 | Detection of non-a, non-b hepatitis associated antigen |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40,921 | 1979-05-21 | ||
| US06/040,921 US4356164A (en) | 1979-05-21 | 1979-05-21 | Detection of non-A, non-B hepatitis associated antigen |
| CA000352378A CA1147647A (en) | 1979-05-21 | 1980-05-21 | Detection of non-a, non-b hepatitis associated antigen |
| CA000413076A CA1155764A (en) | 1979-05-21 | 1982-10-07 | Detection of non-a, non-b hepatitis associated antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1155764A true CA1155764A (en) | 1983-10-25 |
Family
ID=27166688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000413076A Expired CA1155764A (en) | 1979-05-21 | 1982-10-07 | Detection of non-a, non-b hepatitis associated antigen |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1155764A (en) |
-
1982
- 1982-10-07 CA CA000413076A patent/CA1155764A/en not_active Expired
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