THE JOURNAL OF INFECTIOUS DISEASES • VOL. 140, NO.6. • DECEMBER 1979 © 1979 by The University of Chicago. 0022-1899/79/4006-0021$00.75

Simultaneous Occurrence in the Same Serum of Hepatitis B Surface Antigen and Antibody to Hepatitis B Surface Antigen of Different Subtypes Anne-Marie Courouce-Pauty, Jacques Dronet, and Dieter Kleinknecht

From the Centre National de Transfusion Sanguine, Paris and Orsay-Courtaboeuf; and the Centre Hospitalier Intercommunal, Montreuil, France

Various authors have observed the simultaneous occurrence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in the same serum [1-5]. These observations have related the carriage of HBsAg of one subtype to the carriage of anti-HBs directed against a subspecificity that the host did not possess. In the first four cases observed, the combination of HBsAg and anti-HBs involved either the d/y system of determinants [6] or the w/r system [7]. We report here another case of the co-occurrence of HBsAg and anti-HBs; in this case, as in that reported by Koziol et al. [5], more restricted specificities were involved than in the first four cases observed-Le., the w(a) subdeterminants [8]. In the case we describe here, the HBsAg belonged to the ayw3 subtype, and the anti-HBs was selectively directed against the W2 specificity of the adw2 subtype.

and by CEP using two different reagents: HBsAg/ adw2 and HBsAg/ayw. The subtype of anti-HBs in serum samples was determined by CEP in tests and absorption experiments to ascertain which determinants blocked anti-HBs activity. A number of the HBsAg-containing sera used in tests involving CEP and all of the reagents used in the absorption experiments were tested and given international recognition during the workshop on HBsAg subtypes held in Paris in April 1975 [11]. The levels of serum glutamic-pyruvic transaminase (SGPT, also known as serum alanine aminotransferase) were determined by a colorimetric method [12], and the results were expressed in international milliunits (lmU). Case Report

Patient A, who was on maintenance hemodialysis, was first found to carry HBsAg during March 1974. Each subsequent month the level of SGPT was analyzed, the titer and subtype of HBsAg were determined by means of CEP, and serum was screened for anti-HBs by means of CEP and RIA. (The screening for anti-HBs was not done from March 1977 through August 1977.) Patient A was always dialyzed on the same unit, which was contaminated with HBsAg/ayw3 only, except during the period July 5, 1976-September 10, 1976, when he was treated in a unit contaminated with both HBsAg/adw2 (1 j 070) and HBsAg/ayw3 (87 0J0). From July 1976 until the end of October 1976, the patient received more than 10 units of packed red blood cells.

Materials and Methods

HBsAg was detected and subtyped by means of agar gel diffusion and counterelectrophoresis (CEP) as described previously [8]. Hepatitis B e antigen (HBeAg) was detected by agar gel diffusion [9]. Serum was screened for anti-HBs both by radioimmunoassay (RIA) in the liquid phase [10] using [12SI]HBsAg/ayw2 and [12SI]HBsAg/adw2 Received for publication February 27, 1979, and in revised form June 15, 1979. Please address requests for reprints to Dr. Anne-Marie Courouce-Pauty, Centre National de Transfusion Sanguine, 6, rue Alexandre Cabanel, 75739 Paris Cedex 15, France.

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The simultaneous occurrence of hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs) of different subtypes in the serum of a hemodialyzed 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*'

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Courouce-Pauty, Drouet, and Kleinknecht

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Figure 1. Serologic data for a patient undergoing maintenance hemodialysis. The solid line represents the titer of hepatitis B surface antigen (HBsAg) subtype aYW3; the dashed line represents the titer of antibody to subdeterminant W2 (anti-HBs W2); and the dotted line represents the level of serum glutamic-pyruvic transaminase (SOPT, also known as serum alanine aminotransferase). RIA = radioimmunoassay; CEP = counterelectrophoresis; mI.U. = international milliunits. The abscissa on the left refers to the concentration of SOPT, and that on the right shows the titer of antigen or antibody. Results

Biologic and immunologic data. The HBsAg found in the serum of patient A was of the ayw3 subtype, as was that in sera of all of the other carriers of HBsAg who were undergoing dialysis on the same unit. The titer of HBsAg as determined by CEP was 1:256, a value that has remained fairly constant through the present. HBeAg was detected when it was first tested for in February 1977, and its presence has continued. Through January 1977, no anti-HBs was detected by CEP or RIA. On February 9, 1977, anti-HBs was detected, but only by RIA. This antibody bound only with labeled HBsAgl adw 2 and not with HBsAg/ayw2. From September 1977 until the present, this anti-HBs was detected by CEP but only when HBsAg/adw2 was used as the reagent.

The titer of HBsAg was not significantly affected by the appearance of this anti-HBs. However, an increase in levels of SOPT (to 580 ImUI ml) that paralleled the appearance of anti-HBs was observed (figure 1). Each month prior to this increase, the patient's SOPT level had been either normal or subnormal. Subtyping of anti-HBs. Serum samples shown by CEP to contain anti-HBs were tested against 128 sera (and dilutions of these sera) containing HBsAg of 12 different subtypes: PI-PIO [11] and two new subtypes, ad(w4) q+ and adr q- [13]. The same technique was used for each subtype. The anti-HBs reacted positively with all of 23 sera containing HBsAg of the adw2 subtype, with both of two sera containing HBsAg of the ad(w4) q+ subtype, and with all of four sera containing HBsAg of the adyw subtype. The anti-HBs did not react

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Co-Occurrence ojHBsAg and Anti-HBs

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Discussion

The anti-HBs that appeared concurrently with HBsAg/ayw3 in the serum of the hemodialyzed patient studied here was devoid of anti-a activity. It also seemed to be devoid of anti-d activity since it recognized neither the adw4 nor the adr subtype and was not absorbed by these d subtypes. The anti-HBs seemed to be directed exclusively against the W2 subdeterminant of the adw subtype and not against the W2 subdeterminant of the ayw2 subtype. In fact, these two W2 subdeterminants apTable 1. Results of counterelectrophoresis of human serum containing a monospecific antibody to hepatitis B surface antigen (HBsAg), anti-w2*' against various HBsAg-containing sera. HBsAg subtype (no. of sera), designation aYWl (11), PI aYWl (23), P2 aYWl (16), P3 aYW4 (11), P4 ayr (3), P5 adw2* (23), P6 ad(w4) q+ (2) adw4 q- (10), P7 adr q+ (15), P8 adr q- (8) adyw (4), P9 adywr (2), PIO Total (128)

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NOTE. The anti-wl* serum studied was obtained from a patient who was a carrier of HBsAg/aywl'

peared to be similar but not identical [8]. Absorption experiments have already shown that the w2 subdeterminant of adw2 resembles the WI subdeterminant more than the W2 subdeterminant of the ayw2 subtype does. However, until now, no antibody to subdeterminant W2 was available, so that it has been characterized only on the basis of negative reactions with antibody to subdeterminants Wit W3, W4, and r and a positive reaction with antibody to x(g) [11, 14]. To differentiate between these two W2 subdeterminants, we have designated the W2 subdeterminant that is associated with the d subdeterminant as W2*. The fact that similar results are obtained with subtypes adyw and adw2* can be interpreted as indicating the presence of a subtype composed of a mixture of two HBsAg subtypes: ayw3 and adw2*. Similarly, the rare adywr subtype might be a mixture of subtypes ayw2 and adr, and the adw4 q+ subtype might be a mixture of subtypes adw2+and adw4. A more likely possibility is that the serologic data obtained with adw4q+ may be explained by postulating the presence of an intermediate HBsAg subtype, Le., between adw2 and adw4q-. Patient A had been infected with HBsAg/ayw3 for 33 months before he was exposed to another subtype of HBsAg, probably adw2*. Results of immunologic tests using the newly developed antiHBs as well as data concerning the possible source of exposure support our hypothesis. Biological signs of exposure to a second subtype were observed in the beginning of February 1977. There were two possible sources of this exposure. (1) The patient was repeatedly injected with units of packed red blood cells from July 1976 until the end of October 1976. (2) During most of this same period, hemodialysis was performed on a unit contaminated with both ayw3 (87070) and adw2* (13%). The patient is now being treated on the usual unit, where carriers of HBsAg have subtype ayw3 exclusively. However, neither of the above two possible sources of the new subtype could be proved to be the actual source.· If dialysis had been involved, the time between exposure to an antigen and the appearance of the corresponding antibody would have been at least five months. If transfusions had been responsible, the period before the appearance of the corresponding anti-HBs would have been only three months. Unfortunately, we were unable to rescreen the donors of the packed red blood cells for the presence of HBsAg.

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with all sera containing the following subtypes: aywI (11 sera), ayw2 (23), ayw3 (16), ayw4 (11), ayr (three), adw4 q- (10), adr q+ (15), adr q- (eight), and adywr (two) (table 1). Absorption experiments were performed with eight subtypes of HBs Ag-aywI' ayw2' aywJ, ayw4, adwl, adw4 q-, adr, and adyw-each adjusted to the same titer of antigen. Anti-HBs activity remained when 10 parts of patient A's serum were combined with one part of HBsAg of any of the four ayw subtypes, the adw4 q- subtype, or the adr subtype. However, antiHBs activity was absorbed with either HBsAg/adw2 or HBsAg/adyw. Absorption experiments could not be performed with the adw4 q+ subtype because all specimens had too low a titer of HBsAg.

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5.

6. 7.

8.

9.

10.

11. References 1. Tabor, E., Gerety, R. J., Smallwood, L. A., Barker, L. F. Induction of antibody to the "y" determinant of HBsAg in a chimpanzee carrier of HBsAg subtype "adw." J. Immunol. 117:2038-2040, 1976. 2. Sasaki, T., Ohkubo, Y., Yamashita, Y., Imai, M., Miyakawa, Y., Mayumi, M. Co-occurrence of hepatitis B surface antigen of a particular subtype and antibody to a

12.

13.

14.

heterologous subtypic specificity in the same serum. J. Immunol. 117:2258-2259, 1976. Le Bouvier, G. L., Capper, R. A., Williams, A. E., Pelletier, M., Katz, A. J. Concurrently circulating hepatitis B surface antigen and heterotypic anti-HBs antibody. J. Immunol. 117:2262-2264, 1976. Tabor, E., Gerety, R. J., Smallwood, L. A., Barker, L. F. Coincident hepatitis B surface antigen and antibodies of different subtypes in human serum. J. Immunol. 118: 369-370, 1977. Koziol, D. E., Alter, H. J., Kirchner, J. P., Holland, P. V. The development of HBsAg-positive hepatitis despite the previous existence of antibody to HBsAg. J. Immunol. 117:2260-2262, 1976. Le Bouvier, G. L. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675, 1971. Bancroft, W. H., Mundon, F. K., Russell, P. K. Detection of additional antigenic determinants of hepatitis B antigen. J. Immunol. 109:842-848, 1972. Courouce-Pauty, A. M., Soulier, J. P. Further data on HBs antigen subtypes-geographical distribution. Vox Sang. 27:533-549, 1974. Courouce-Pauty, A. M., Plalll;on, A. e-Antigen and antie in two categories of chronic carriers of hepatitis B surface antigen. Vox Sang. 34:231-238, 1978. Amouch, P. J., Drouet, J. Dosage radio-immunologique de l'antigene Australie. Nouvelle Revue Francaise d'Hematologie 11:656-668, 1971. Courouce-Pauty, A. M., Holland, P. V., Muller, J. V., Soulier, J. P. [ed.]. HBs antigen subtypes. Vol. 42. Bibliotheca Haematologica, Karger, Basel, 1976. Cabaud, S., Leeper, R., Wroblewski, F. Colorimetric measurement of serum glutamic-oxaloacetic transaminase. Am. J. Clin. Pathol. 26:1101-1105,1956. Courouce-Pauty, A. M., Lemaire, J. M., Roux, J. F. New hepatitis B surface antigen subtypes inside the ad category. Vox Sang. 35:304-308, 1978. Shorey, J. A new hepatitis B virus surface antigen. J. Infect. Dis. 133:1-6,1976.

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Whatever the explanation, the persistent carrier of HBsAg/ayw3 was exposed secondarily to a subtype of HBsAg-presumably adwz*-that was different from the one to which he had originally been exposed. An immune response occurred to one determinant of the new subtype that was not present in the HBsAg that the patient already carried. This immune response was a partial one, since the d determinant was not present in the first subtype of HBsAg. It appears that, in this case and probably in the similar cases described previously [1-4], there is not a complete immune response to all of the determinants present in the second subtype of HBsAg and absent in the original HBsAg. This response demonstrates a tolerance to the determinants of the original HBsAg and to some of the determinants of the second HBsAg. This situation has resulted in the formation of a truly monospecific anti-HBs antiserum. Thus we have been provided with a reagent that we were unable to obtain by absorption of animal antisera in vitro.

Courouce-Pauty, Drouet, and Kleinknecht

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

THE JOURNAL OF INFECTIOUS DISEASES • VOL. 140, NO.6. • DECEMBER 1979 © 1979 by The University of Chicago. 0022-1899/79/4006-0021$00.75 Simultaneous O...
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