THE JOURNAL OF INFECTIOUS DISEASES • VOL. 133, NO.1· © 1976 by the University of Chicago. All rights reserved.
JANUARY 1976
MAJOR ARTICLES
A New Hepatitis B Vims Surface Antigen James Shorey
From the Department 0/ Internal Medicine, Dallas Veterans Administration Hospital and Southwestern Medical School, Dallas, Texas
Understanding of the epidemiology of hepatitis B virus (HBV) has been greatly advanced by the ability to distinguish serologic subtypes of this agent. At present, the most firmly established hepatitis B surface antigens (HB s Ag), which appear to be specified by the virus and not by the infected host, include the group-reactive a antigenes) apparently shared by all types of HBV and the mutually exclusive d/y [1] and w/r [2] antigen pairs. On the basis of these determinants, four different subtypes of HBV can be identified: adw, adr, ayw, and ayr. Since subtype r viruses are found almost exclusively in the Far East, for practical purposes only adw vs. ayw discrimination is Received for publication January 17, 1975, and in revised form September 8, 1975. Results of this study were reported, in part, at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, October 29, 1974. This investigation was supported by research funds of the U.S. Veterans Administration. Dr. Shorey is a clinical investigator of the Veterans Administration. I gratefully acknowledge the expert technical assistance of Ms. Kathleen Fitzgibbons and the aid of Ms. Dorothy Bass in preparation of the manuscript. The critical review of the manuscript by Dr. Burton Combes is greatly appreciated. Please address requests for reprints to Dr. James Shorey, Liver Unit, Dallas Veterans Administration Hospital, Dallas, Texas 75216.
available for epidemiologic studies in western countries. In the present report, evidence is presented for the existence of a new HB s Ag determinant, g (originally designated as [1] in a preliminary communication [3]). This new surface marker should be useful in the characterization of HBV. Materials and Methods
Serum specimens. HB s Ag-positive blood donors. Specimens were generously provided by Mr. R. Y. Dodd (American National Red Cross, Bethesda, Md.), Dr. Martin Goldfield (New Jersey State Health Department, Trenton, N.J.), and Drs. Ruth Guy and James Wheeler (Dallas, Tex.). Acute viral hepatitis. Specimens were obtained from persons treated at Parkland Memorial Hospital, Dallas, Tex. Other specimens. Sera subtyped for wand r were provided by Drs. W. H. Bancroft and G. Irwin (Walter Reed Army Institute of Research, Washington, D.C.). Specimens previously subtyped for a subdeterminants [4] were provided by Prof. J. Soulier (Centre National de Transfusion Sanguine, Paris, France). HB s Ag-positive specimens from patients undergoing hemodialysis, renal transplant recipients, and a nurse with acute hepatitis who worked in a transplant unit were
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on September 11, 2015
A new determinant of hepatitis B surface antigen (HB s Ag), g, which is distinct from the previously identified determinants a, d, y, w, and r, was studied. This new antigen tended to be associated with the d antigen; it was found in 97 % of ad specimens and in 15 % of ay specimens. With few exceptions, the atypical subtypes, adg- and ayg+, were identified in sera from blood donors, but not in sera from patients with acute viral hepatitis; this finding possibly reflects a reduced tendency of these viral strains to evoke a host immune response. The ability to identify g represents a refinement of HB s Ag serotyping that may be of value in the study of the epidemiology of hepatitis B virus.
Shorey
2
Results
Serum specimens subtyped for wand r, Single specimens, typed at Walter Reed Army Instit~te for Research as adw and adr, were both g+, while an ayw specimen was g-. Thus g was differentiated from wand r. Serum specimens typed for a subdeterminants. Serum specimens of Soulier's types a.yw, a21yw, a23yw, a:-lYw, a2dw, aadw, and adr were subtyped for d, y, and g by RIA (table 1). The a-yw serum could be distinguished from the a21yw serum in that the former gave a weakly positive reaction in the d assay. (This d reactivity was also much weaker than that found in our two adyg+ donor sera, which were strongly reactive in both the d and the y assay.) No distinction between a23yw and aaYw could be demonstrated by the subtyping assays employed. Blood donors and patients with acute hepatitis. A total of 238 sera from blood donors and 105 sera from patients with acute hepatitis were tested in the d, y, and g assays; results are shown in table 2. The majority of the patients with acute hepatitis had been infected with virus of type adg+ or ayg-. In contrast, nearly one-fourth of the type ay donor sera were g+, and five of 140 a~. sera were g-. In addition, two sera reacted positively in both the d and the y assay, and each serum was also g t . The prevalence of the atypical subtypes, adg: and ayg+, considered as a combined group, was significantly greater among blood donors than among patients with hepatitis. Hemodialysis/renal transplant patients. HB s Ag-positive specimens of serum from 11 hemodialysis/renal transplant patients were all subtype Table 1. Results of radioimmunoassays (RI~) for determinants d, y, and g in specimens previously typed for a subdeterminants [4]. Specimen no. 1
2 3 4
5 6
a subdeterminant
Type by RIA
a1yw a 21yw a 23yw aaYw
ayg+* ayg+ ayg- t ayg-t
~~
aadw
*+ adg-
* This specimen was also weakly reactive in the d assay, a result distinguishing it from the a 21yw specimen. t These two specimens could not be distinguished by RIA.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on September 11, 2015
typed for Dr. Marc LaForce (Veterans Administration Hospital, Denver, Colo.). BB s Ag subtyping assays. A double antibody radioimmunoassay (RIA) for HB s Ag described previously [5] was modified for determination of HB s Ag subtypes by use of subtype-specific antisera and radiolabeled purified HB s Ag of the appropriate subtype (Electronuc1eonics, Bethesda, Md. ). The monospecific antisera were prepared by incubation of human (hemophiliac) serum, previously shown to contain subtype-specific antibody, with an excess volume of serum containing HB s Ag of the appropriate subtype (three volumes of absorbent to one volume of antiserum) at 4 C for four days to remove all but the desired antibody. Initially, both antiserum to d and antiserum to y were prepared from a single hemophiliac serum (W6) by incubation with ayand ad-absorbent sera, respectively. Using these type-specific antisera, the d assay was then established with 125I-Iabeled ad, and the y assay with labeled ay, When 40 specimens from HB s Agpositive blood donors that had been previously subtyped by the American Red Cross were tested, there was complete agreement for the y determinant; however, results of the d assay differed from the Red Cross typing data in that seven of 22 ay specimens also reacted positively in the d assay, while three specimens labeled ad by the Red Cross were d - by our RIA. On further study of the W6 antiserum, it was found that binding activity for 1251-labeled ad could be .removed by certain absorbent sera containing ay (subsequently recognized as type ayg+) but that the binding activity persisted after incubation with some ad(g-) specimens. A new antibody to the d determinant was then prepared from serum of another hemophiliac donor, and a d assay with this antibody correctly identified the Red Cross ad specimens. The original d assay was recognized as a test for a new HB s Ag determinant, designated g. Thus the seven subtype y specimens also identified as d + in the original assay were in reality ayg+, and the three subtype d sera missed by the first assay were adg: . Three monospecific antisera have thus been prepared for these studies: antisera to y and to g from serum W6 and antiserum to d from a separate hemophiliac serum specimen. An example of a typing experiment using the d, y, and g assays is shown in figure 1.
A New Hepatitis B Virus Surface Antigen
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JANUARY 1976
MAJOR ARTICLES
A New Hepatitis B Vims Surface Antigen James Shorey
From the Department 0/ Internal Medicine, Dallas Veterans Administration Hospital and Southwestern Medical School, Dallas, Texas
Understanding of the epidemiology of hepatitis B virus (HBV) has been greatly advanced by the ability to distinguish serologic subtypes of this agent. At present, the most firmly established hepatitis B surface antigens (HB s Ag), which appear to be specified by the virus and not by the infected host, include the group-reactive a antigenes) apparently shared by all types of HBV and the mutually exclusive d/y [1] and w/r [2] antigen pairs. On the basis of these determinants, four different subtypes of HBV can be identified: adw, adr, ayw, and ayr. Since subtype r viruses are found almost exclusively in the Far East, for practical purposes only adw vs. ayw discrimination is Received for publication January 17, 1975, and in revised form September 8, 1975. Results of this study were reported, in part, at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, October 29, 1974. This investigation was supported by research funds of the U.S. Veterans Administration. Dr. Shorey is a clinical investigator of the Veterans Administration. I gratefully acknowledge the expert technical assistance of Ms. Kathleen Fitzgibbons and the aid of Ms. Dorothy Bass in preparation of the manuscript. The critical review of the manuscript by Dr. Burton Combes is greatly appreciated. Please address requests for reprints to Dr. James Shorey, Liver Unit, Dallas Veterans Administration Hospital, Dallas, Texas 75216.
available for epidemiologic studies in western countries. In the present report, evidence is presented for the existence of a new HB s Ag determinant, g (originally designated as [1] in a preliminary communication [3]). This new surface marker should be useful in the characterization of HBV. Materials and Methods
Serum specimens. HB s Ag-positive blood donors. Specimens were generously provided by Mr. R. Y. Dodd (American National Red Cross, Bethesda, Md.), Dr. Martin Goldfield (New Jersey State Health Department, Trenton, N.J.), and Drs. Ruth Guy and James Wheeler (Dallas, Tex.). Acute viral hepatitis. Specimens were obtained from persons treated at Parkland Memorial Hospital, Dallas, Tex. Other specimens. Sera subtyped for wand r were provided by Drs. W. H. Bancroft and G. Irwin (Walter Reed Army Institute of Research, Washington, D.C.). Specimens previously subtyped for a subdeterminants [4] were provided by Prof. J. Soulier (Centre National de Transfusion Sanguine, Paris, France). HB s Ag-positive specimens from patients undergoing hemodialysis, renal transplant recipients, and a nurse with acute hepatitis who worked in a transplant unit were
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on September 11, 2015
A new determinant of hepatitis B surface antigen (HB s Ag), g, which is distinct from the previously identified determinants a, d, y, w, and r, was studied. This new antigen tended to be associated with the d antigen; it was found in 97 % of ad specimens and in 15 % of ay specimens. With few exceptions, the atypical subtypes, adg- and ayg+, were identified in sera from blood donors, but not in sera from patients with acute viral hepatitis; this finding possibly reflects a reduced tendency of these viral strains to evoke a host immune response. The ability to identify g represents a refinement of HB s Ag serotyping that may be of value in the study of the epidemiology of hepatitis B virus.
Shorey
2
Results
Serum specimens subtyped for wand r, Single specimens, typed at Walter Reed Army Instit~te for Research as adw and adr, were both g+, while an ayw specimen was g-. Thus g was differentiated from wand r. Serum specimens typed for a subdeterminants. Serum specimens of Soulier's types a.yw, a21yw, a23yw, a:-lYw, a2dw, aadw, and adr were subtyped for d, y, and g by RIA (table 1). The a-yw serum could be distinguished from the a21yw serum in that the former gave a weakly positive reaction in the d assay. (This d reactivity was also much weaker than that found in our two adyg+ donor sera, which were strongly reactive in both the d and the y assay.) No distinction between a23yw and aaYw could be demonstrated by the subtyping assays employed. Blood donors and patients with acute hepatitis. A total of 238 sera from blood donors and 105 sera from patients with acute hepatitis were tested in the d, y, and g assays; results are shown in table 2. The majority of the patients with acute hepatitis had been infected with virus of type adg+ or ayg-. In contrast, nearly one-fourth of the type ay donor sera were g+, and five of 140 a~. sera were g-. In addition, two sera reacted positively in both the d and the y assay, and each serum was also g t . The prevalence of the atypical subtypes, adg: and ayg+, considered as a combined group, was significantly greater among blood donors than among patients with hepatitis. Hemodialysis/renal transplant patients. HB s Ag-positive specimens of serum from 11 hemodialysis/renal transplant patients were all subtype Table 1. Results of radioimmunoassays (RI~) for determinants d, y, and g in specimens previously typed for a subdeterminants [4]. Specimen no. 1
2 3 4
5 6
a subdeterminant
Type by RIA
a1yw a 21yw a 23yw aaYw
ayg+* ayg+ ayg- t ayg-t
~~
aadw
*+ adg-
* This specimen was also weakly reactive in the d assay, a result distinguishing it from the a 21yw specimen. t These two specimens could not be distinguished by RIA.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on September 11, 2015
typed for Dr. Marc LaForce (Veterans Administration Hospital, Denver, Colo.). BB s Ag subtyping assays. A double antibody radioimmunoassay (RIA) for HB s Ag described previously [5] was modified for determination of HB s Ag subtypes by use of subtype-specific antisera and radiolabeled purified HB s Ag of the appropriate subtype (Electronuc1eonics, Bethesda, Md. ). The monospecific antisera were prepared by incubation of human (hemophiliac) serum, previously shown to contain subtype-specific antibody, with an excess volume of serum containing HB s Ag of the appropriate subtype (three volumes of absorbent to one volume of antiserum) at 4 C for four days to remove all but the desired antibody. Initially, both antiserum to d and antiserum to y were prepared from a single hemophiliac serum (W6) by incubation with ayand ad-absorbent sera, respectively. Using these type-specific antisera, the d assay was then established with 125I-Iabeled ad, and the y assay with labeled ay, When 40 specimens from HB s Agpositive blood donors that had been previously subtyped by the American Red Cross were tested, there was complete agreement for the y determinant; however, results of the d assay differed from the Red Cross typing data in that seven of 22 ay specimens also reacted positively in the d assay, while three specimens labeled ad by the Red Cross were d - by our RIA. On further study of the W6 antiserum, it was found that binding activity for 1251-labeled ad could be .removed by certain absorbent sera containing ay (subsequently recognized as type ayg+) but that the binding activity persisted after incubation with some ad(g-) specimens. A new antibody to the d determinant was then prepared from serum of another hemophiliac donor, and a d assay with this antibody correctly identified the Red Cross ad specimens. The original d assay was recognized as a test for a new HB s Ag determinant, designated g. Thus the seven subtype y specimens also identified as d + in the original assay were in reality ayg+, and the three subtype d sera missed by the first assay were adg: . Three monospecific antisera have thus been prepared for these studies: antisera to y and to g from serum W6 and antiserum to d from a separate hemophiliac serum specimen. An example of a typing experiment using the d, y, and g assays is shown in figure 1.
A New Hepatitis B Virus Surface Antigen
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