Attempt to prevent hepatitis by using specific anti-HBs immunoglobulin.

Sixteen batches of specific anti-HBs immunoglobulin have been prepared from December 1970 up to now. They were used in an attempt to prevent hepatitis B: (1) By a single injection, within seven days, in patients exposed by HBs Ag-positive blood transfusion: with a dose of 0.16 ml/kg, two hepatitis B cases were observed among 29 followed up patients out of 43 cases; with a dose of 0.5 ml/kg, no hepatitis was reported to us in the next 26 patients. (2) By a single injection, within seven days, in laboratory and hospital personnel accidentally contaminated. With a dose of 0.08 ml/kg administered to more than 1000 persons, an excellent protection was observed. (3) By injections repeated every five weeks during the first four months, then more widely spaced during the following months, in hemodialysis units staff. The attack rate of clinical hepatitis B was reduced from about 44 per cent to 0 per cent. No hepatitis case was observed in 90 persons protected from four to 26 months. In a control group (eight subjects) three hepatitis B cases occurred (37 per cent).

[The HBs antigen carriage in blood donors of French Polynesia A new antigenic sub-type (author's transl)]. / Le portage de l'antigène HBs chez les donneurs de sang de Polynesie françaisse. Un nouveau sous-type antigenique.

In the blood transfusion center of Papeete )Tahiti Island), a survey on HBs antigen has been made in 5 406 blood donors by reverse hemagglutination method. Its shown 2,53 p. 100 of carriers. In european donors recently arrived in the territory, the rate of carrier state is 0,27 p. 100 versus 3,01 in the autochtonous donors. From this point of view, the situation of Polynesia is better than that prevailing in some of the suroundings territories but worse than in developped countries. French Polynesia is too extended and too heterogenous to permit an extrapolation of our data to the whole territory. Anyway, the study of the geographical origin of the carriers, if compared to that of the donors, gives evidence that the prevalence of HBs antigen carriage is higher in remote archipelagos than in Tahiti. The research of subtypes of HBs antigen shown the presence of a new subtype adr q negative which seems very particular to South Pacific.. In french Polynesia it seems localized to some archipelagos. In addition, the frequence and preponderance of adw4 subtype in other archipelagos have been compiled. The data collected lead to ethnological hypothesis. More extensive epidemiological studies appear necessary to precise the frequency of HBs antigen carriage-state and the subtypes distribution in the various archipelagos of the territory.

e-Antigen and anti-e in two categories of chronic carriers of hepatitis B surface antigen.

Screening for e antigen and anti-e was performed in 517 HBs antigen chronic carriers, 329 blood donors and 188 hemodialyzed patients. e antigen was detected in 35 blood donors (10.6 percent): 88 percent had a high titre of HBsAg, and 80 percent had a distrubed liver function. No difference was noted regarding sex and age of the carrier. Anti-e was detected in 33.7 percent of the bood donors. A significant difference (p less than 0.01) was noted between males (29 percent) and females (46.6 percent). Anti-e was found more prevalent in young people between 21 and 34 than in older people (p less than 0.05). Anti-e was rarely found when the titre of HBsAg was low or high. 88 percent of the anti-e carriers had an intermediate titre from 1/1 to 1/32 by CEP. Both e Ag and anti-e were more prevalent in ad subtype than in ay subtype (p less than 0.02 for both). Inside the ad subtype, anti-e is less frequent in adr (6 percent) than in adw2 (42 percent) and in adw4 (57 percent); p less than 0.01. The difference between the prevalence of e Ag in adr (31 percent) and in adw2 (17 percent) is not significant but the healthy carriers with e antigen are more numerous in adr than in adw2 subtype. e antigen was detected in 53.7 percent of HBs Ag-hemodialyzed patients and anti-e in 3.7 percent of these patients. These results are neither correlated with the liver function nor with the state of chronic carrier (27 percent of e Ag in transient HBs Ag carrier).

[2000 chronic carriers of HBs antigen]. / Observations chez 2000 porteurs chroniques de l'antigène HBs.

By a study of 2000 asymptomatic carriers of the HBs antigen, the following information was obtained: their frequency is 3 times greater in men than in women and twice as great in young subjects as compared with subjects aged over 35 years. However an increase in this frequency is noted beyond 55 years. The titre of the HBs antigen is not related to age, except for low titres, (5.65% of carriers frequent beyond the age of 45 years. The ratio an/ay (major subtypes) is 2.6 in French subjects. It does not vary with age. It is 1.6 in subjects of Group 0. Normal liver function tests were observed in 78.5% of all carriers, (women 91.5%, men 73.4%) and in only 45.6% of those with a high HBs antigen titre.

Geographical distribution of HBs Ag in Africa.

The incidence of HBs Ag carrier is high in Africa. The HBs Ag subtypes vary in 5 mean areas of the Africa continent. The subtype ayw4(a3) is the most characteristic African subtype, being found with a striking frequency in West Africa (88.8%). In North Africa ayw2(a21) is predominant as in other mediterranean countries. In Saharan area mixed types of HBs Ag are found, intermediary between North and West Africa. In Central Africa y is predominantly associated with w2(a21) or with w4(a3). In east and South Africa ad is largely predominant and the most widespread subtype is adw2(a21). No adr, nor adw4 were found in Africa.

Further data on HBs subtypes: geographical distribution.

1123 HBs antigens from silent carriers of different parts of Europe, Africa and Asia were subtyped and classed in the eight categories previously described: a1y(w1), a2yl(w2), a2(3)y(w3), a3y(w4), ayr, a2ld(w2), a3d(w4) and adr: We have showed that w can be subdivided into the system of a subdeterminants, but for the present time, we do not know if r is a fifth subdeterminant of a. Subtyping was performed by CIEP and by ID using antisera from guinea pigs and goats immunized with selected HBs antigens. With these 8 categories it is possible to greatly increase our knowledge about the geographical distribution of HBs antigen: in Africa six areas can be distinguished: North Africa with a predominance of a2ly(w2) (75%) and with none a3y(w4); areas between North and West Africa where a2ly(w2) and a3y(4) represent respectively 62.8% and 25.7%; West Africa with a great predominance of a3y(w4) (84.5%); Central Africa with less a3y(w4) (53.4%), appearance of a2ld(w2) (8.7%) and presence of a2ly(w2) (37%); East Africa appears to be especially a2ld(w) (only 3 sera from Burundi); South Africa with a great predominance of a2ld(w2) (86%) and with ay subtype represented by a2(3)y(w3), very exceptional in the other parts of Africa. In France, The Netherlands, Hungary, a2ld(w) subtype is predominant, respectively 73.3%, 74% and 63%, a2(3)y(w3) is the main ay subtype in France and the only one in The Netherlands whereas in Hungary the same amount of a2ly(w) and a2(3)(w) is encountered. In Rumania, Italy and Greece, a great predominance of ay subtype is found (80%): a2ly(w2) is predominant in Rumania (66%) and Italy (as in North Africa) whereas a weak predominance of a2(3)y(w3) over a2ly(w2) is found in Greece. In the Antilles, 75% of a2ld(w2) are encountered. In the Far East, a great predominance of adr is found in Laos, Japan and Thailand (100% and 84% respectively) but no adr is encountered in Vietnam where 8 a1y(w1) and 1 ayr are found of of 11 HBs sera. This a1y(w1) subtype has also been found, although infrequently, in some countries of Africa. Among French people who have lived in Africa, many examples are found which show that the HBs subtype is related to the country and does not seen to depend on the host. Among the French who have been contaminated in Indochina all are carriers of adr HBsAg and none exhibits an a1y(w1) subtype.

[Culture of hepatitis virus B]. / Culture du virus B de l'hepatite

For the last 25 years, numerous attempts have been made to isolate the HBV agent responsible for hepatitis B by means of cultures 'in vitro'. We have undertaken longterm cultures of children's hepatic tissue (C.H.), conjunctive tissue (human adult H.A.F. and human embryonic fibroblasts H.E.F.) and KB cells; these were put in the presence of 7 sera HB + rich in Dane particles. These cells were trypsinized twice a week for almost 3 months and did not present any cytopathogenic effects. Electromicroscopy revealed, 15 days after infection, the presence of icosahedral particles (25 to 27 nm in diameter), free or in dense clusters, but more often empty (20 nm in diameter). These structures seemed to be made up of an assembly of capsomers approximately 5 nm in diameter, joined together in fours to form a ridge. Older cultures revealed clusters of icosahedrons some of which degenerated spontaneously; others were surrounded by proteinic structure having a fringed aspect. Certain rare particles of 35 to 45 nm in diameter are similar to full Dane particles. EID immunological results were positive in the case of sera of patients convalescent from hepatitis B, containing anti-HBc antibodies, on C.H. cells the 27th and 40th days, and negative with anti-HBs antibodies. By immunofluorescence we observed 12 to 20 days after infection of the cells, a clear fluorescence at first nuclear, then essentially cytoplasmic, by means of fluorescent anti-HBc sera of human or animal origin. With the fluorescent anti-HBs antibodies, the reaction is weak and solely cytoplasmic although in DRI, with H.E.F. and KB cells, we obtained from time to time weakly positive results in HBs. The relations between the morphological structures and the immunological results observed are discussed.

[Detection of HBs with latex tests]. / Dépistage de l'antigène HBs par les tests au latex

Since 1971, several inert particles have been tested and proposed for HBs antigen screening, none of them being really used in routine. An evaluation is realized and presented, pointing out the main characteristics and the practical problems. We report our experience on two types of latex, simultaneously conducted through manual techniques and through Groupamatic 360 equipments. For one of them, TG Antigex, sensitivity and specificity are very satisfying and several batches are presently tested in order to quantify the reproducibility and stability features of the reagent.

Distribution of HBsAg subtypes in the world.

HBsAg subtyping was performed in 5,337 sera from chronic carriers who originated from 54 different countries of the 5 continents. Nine subtypes were defined: ayw1, ayw2, ayw3, ayw4, ayr, adw2, adw4, adr q- and adr q+. The repartition of these subtypes, according to the country of origin of the carriers, enhances the previous results and supplies new data: ayw1 is prevalent only in Vietnam (51%); ayw2 in Mediterranean countries (73%); ayw3 in Greece and Yugoslavia (54%) along with ayw2 (41%); ayw4 in West Africa (82%) and Central Africa (42%) along with ayw2; ayr was only found in Vietnam (3.4%); adw2 is prevalent in North and Central Europe (70%), East and South Africa (95%), India (55%), along with ayw3 (35%) in northern South American (74%), and in the Antilles (82%); adw4 is widespread in French Polynesia (45%)--with a 100% frequency in the Marquesas archipelago--as well as in Argentina (42%); adr q--was found only in Ocean: French Polynesia (34%) with a 69% frequency in the Australes, New Caledonia (3 out of 3 HBsAg carriers); adr q+ is the prevalent subtype in South-East Asia if we exclude Vietnam (61%). These results show that a precise geographical distribution of HBsAg subtypes needs more than 'four main subtypes' generally used. Enlarging from 4 subtypes to 9 is a requirement for valuable epidemiologic studies, as well as for the specification of anti-HBs antibodies produced by hybrids or induced by synthetic peptide. The geographical distribution of these 9 HBsAg subtypes and the serological relationship between some determinants suggest a genetic recombination of viral DNA.

Simultaneous occurrence in the same serum of hepatitis B surface antigen and antibody to hepatitis B surface antigen of different subtypes.

The simultaneous occurrence of hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs) of different subtypes in the serum of a hemodiaylzed patient was studied. The w(a) subdeterminants were involved. The HBsAg belonged to the ayw3 subtype, and the anti-HBs exhibited monospecific anti-w2 activity. Both the HBsAg and the anti-HBs were detectable by counterelectrophoresis (CEP). The specificity of the antibody was demonstrated by CEP in tests against 128 sera containing HBsAg of 12 different subtypes and in absorption experiments with eight sera containing HBsAg of eight different subtypes, as well as by radioimmunoassay in the liquid phase. The monospecific antibody was selectively directed against the w2 subdeterminant of the adw2 subtype and was designated anti-w2.

New hepatitis B surface antigen subtypes inside the ad category.

In addition to the 10 HBs Ag subtypes already described, 2 new subtypes were defined by using the q determinant. Exceptions to the rule generally accepted were found in that the q determinant was only lacking in HBs Ag/adw4. These exceptions occurred in adw and adr categories. These 2 new subtypes are adw q positive and adr q negative. Out of 98 HBs Ag/adw4 from silent carries and patients from different parts of the world, mainly from France (79), 6 were found q positive. 3 out of these 6 cases came from Montpellier (South of France) and another 3 from Germany. The 92 other cases were found q negative. Further studies will be necessary to better know the location of this new subtype adw4 q positive, but it seems to be present only in certain parts of Europe. Out of 86 HBs Ag/adr from silent carriers from Asia (58), Oceania (17), France (10, most of them contaminated in Asia) and Réunion (1), 10 were found q negative. All these 10 cases were detected in Oceania, 2 out of 2 in carriers from New Caledonia and 8 out of 13 from French Polynesia. The new subtype adr q negative seems localized in Melanesia and Polynesia and absent from Asia. These 2 new markers of hepatitis B virus will allow better epidemiological and geographical studies.

[HBs antigen screening with Groupamatic. Inhibition of passive hemagglutination and latex test]. / Dépistage de l'antigène HBs sur les équipements Groupamatic. Inhibition de l'hémagglutination passive et test au latex.

Some agglutination tests for HBs Ag and anti-HBs detection may be easily automated. Reverse hemagglutination, needing a lengthy incubation and several steps to carry out is unfit for Groupamatic, but hemagglutination inhibition and latex agglutination may be used. Hemagglutination inhibition using selected human red cells (ORh+) coated with cesium chloride purified HBs Ag, has been now routinely used at the C.N.T.S. with visual reading for almost 2 years. Negative and positive controls are necessary. The anti-HBs serum utilized has to be checked daily to get sensitive detection. Every new batch of coated red cells (valid 4 to 6 weeks) is checked in microtiter plates in the laboratory, before being used on Groupamatic. Both positive and dubious results are systematically retested in RIA. We found only 0,5% of false positive. The sensibility of the test compared favourably with other third generation test as reverse hemagglutination. It was close to the results obtained with radio-immuno-assay or enzyme-immuno-assay and it was more sensitive than counter-immuno electrophoresis. 65,472 samples were tested in parallel: we were able to detect 97% of HBs Ag detected by RIA. False negative results were less than 1 per 10 000 and may be avoided by using citrated solution for the anti-HBs serum dilution (suppression of auto-agglutination of the red cells). Antigex TG (the most active batch of latex coated with guinea pig, anti-HBs antibody) made by Pfizer, gave good results on Groupamatic and automatic reading was possible. But more results are necessary using various batches, before to recommend such reagent.

Kinetics of HBs antigen in man.

The metabolism of HBs Antigen had been studied in three human volunteers. One had chronic hepatitis and two were "silent carriers". The HBs Antigen had been isolated and purified from the plasma of each of the three subjects and, after iodination, reinjected to the same donor. The parameters of plasma kinetics of 131I HBsAg have been analyzed according to a two compartmental model on the basis of the radioactivity of TCA precipitate (TP) and immunoprecipitate (IP). The fast initial volume of distribution was approximately equal in the three subjects (46.6 ml/kg). The metabolic clearance rate (MCR) of IP was the very same in two subjects but is four times higher in one of the silent carrier. The total renewal time (TRT) was about 3.3 days. Assuming that the HBs Antigen extraction was of the order of 65% the plasma HBs Antigen concentration per liter of plasma would be 12 and 53 mg/liter fot two silent carriers and 61 mg/liter for the patient with chronic hepatitis. The radioactive efflux from the model (calculated as IP.MCR multiplied by HBs Antigen concentration) was identical for the two silent carriers adn 50% higher in the patient with chronic hepatitis. This increase possibly reflects an increased synthesis of HBs Antigen in the patient with chronic hepatitis. The cumulative urinary radioactivity when added to the whole body counting demonstrated that radioactivity was excreted solely in the urine. The ratio of organ counting to precordium counting did not vary significantly with time in all subjects.