THE JOURNAL OF INFECTIOUS DISEASES • VOL. 140, NO.4· OCTOBER 1979
© 1979 by The University of Chicago. 0022-1899/79/4004-0035$00.75
NEWS
From the National Institute of Allergy and Infectious Diseases Summary of an International Workshop on Hepatitis B Vaccines An international workshop on hepatitis B virus (HBV) vaccines brought together investigators who are actively engaged in the research and development of vaccines to prevent HBV infection. This report is a summary of that workshop.
In the absence of successful propagation of HBV in vitro, the plasma of asymptomatic humans who are chronic carriers of hepatitis B surface antigen (HBsAg) serves as the source material for all HBV vaccines at the present time. Plasma from persons with high titers of HBsAg can be obtained by repeated plasmapheresis. A second source of antigen is plasma obtained from units of blood found during routine screening to contain high titers of HBsAg. HBV coexists in varying ratios with HBsAg in the plasma of persons who are chronic carriers of HBsAg. Data suggest that the presence of circulating hepatitis B e antigen (HBeAg) correlates with higher HBV/HBsAg ratios than those ratios observed in the plasma of persons who lack HBeAg or who possess its antibody (anti-HBe). Another marker associated with high HBV/HBsAg ratios is specific HBV DNA polymerase. A World Health Organization recommendation has stated that "consideration should be given to selection of HBsAg-positive plasma that is devoid of HBV particles, DNA polymerase and HBeAg as the starting material from which vaccine is to be prepared" [1]. This recommendation was recently modified to permit the use of source material containing both HBeAg and HBV DNA polymerase, since the exclusion of HBsAg-positive plasma containing these markers would make large-scale proThe National Institute of Allergy and Infectious Diseases and the Bureau of Biologics of the Food and Drug Administration sponsored this workshop in Bethesda, Maryland, on January 18 and 19, 1979. Please address requests for reprints to Dr. Robert J. Gerety, Hepatitis Branch, Bureau of Biologics, Food and Drug Administration, Building 29, Room 311, 8800 Rockville Pike, Bethesda, Maryland 20205. 642
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Vaccine Source Material
duction of vaccine impossible [2]. Use of such source material has not been shown to pose a hazard to vaccine recipients. Weekly plasmapheresis of suitable persons with high titers of HBsAg is currently permitted in the United States to provide source material for HBV vaccines. Such plasmapheresis requires careful isolation of the selected HBsAg-positive donors and careful monitoring of their levels of serum aminotransferases to assure that their liver function remains stable while they undergo repeated plasmapheresis. Such plasmapheresis of selected HBsAg-positive individuals over prolonged periods could assure the availability of HBsAg with uniform antigenic specificity. The antigen specificity, however, appears at this time to be of little significance with regard to protection against HBV. In addition, it is likely that the need for HBsAg will exceed the amount available from such "pedigreed" donors when the demand for vaccine increases subsequent to its licensure. Even at the present time, another method of acquiring adequate source material for HBV vaccine is necessary, since plasmapheresis programs do not exist in many countries, including Japan. The Japanese Red Cross Transfusion Service collects five million units (250 mllunit) of blood annually, of which about 100,000 units contain HBsAg and are routinely discarded. It is estimated that 35,000 such units contain sufficient HBsAg for use in the preparation of HBV vaccine. Dr. R. Thomssen (University of Goettingen, Goettingen, West Germany) reported that about 50% of the HBsAg-positive, HBeAg-negative plasma units collected in West Germany contained sufficient HBsAg for vaccine manufacture. However, two to three times the amount of HBeAgnegative plasma as compared to HBeAg-positive plasma was required, since the titers of HBsAg were lower in this plasma. Although the enrichment process was expensive, Dr. Thomssen felt that the resultant highly purified vaccine justified the increased cost. Highly purified HBsAg preparations, however, can be derived from HBeAgpositive plasma, with the advantage of an increased
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Approaches to HBV Vaccine Production
The plasma of persons who are chronic carriers of HBsAg contains three morphologically distinct particles, the complex HBV (40-42 mm), tubular HBsAg particles (20 nm in width and variable in length), and 20- to 22-nm spherical HBsAg particles. The majority of investigators purified the antigenic 20- to 22-nm particles from the others because of their lack of infectivity and the relative ease with which they can be separated from HBV. An alternative approach to vaccine preparation included the use of HBsAg polypeptides derived from the 20- to 22-nm particles; these consist of two glycoproteins, one with a mol wt of 22,00023,000 and the other of 25,000-28,000. A third ap-
proach used a variety of less highly purified HBV antigens as vaccine. Methods for purifying the 20- to 22-nm spherical HBsAg particles include ultracentrifugation, fractional precipitation, and chromatography. Dr. J. Gerin (National Institute of Allergy and Infectious Diseases [NIAID], Bethesda, Md., and Georgetown University, Washington, D.C.) described the purification of two lots of vaccine in collaboration with Dr. R. Purcell (NIAID). Each consisted of 20- to 22-nm HBsAg particles purified by isopycnic and rate zonal ultracentrifugation. Dr. M. Hilleman (Merck, Sharp and Dohme, West Point, Pa.) described the preparation of 11 lots of HBV vaccine consisting of 20- to 22-nm HBsAg particles, all similarly prepared by ultracentrifugation followed by a series of final treatments including digestion with pepsin to remove host antigens. Dr. P. Maupas (Institute of Virology, Tours, France) described the purification of HBsAg by either affinity chromatography using both human and rabbit antibody to HBsAg (anti-HBs) or by a combination of treatment with polyethylene glycol followed by ultracentrifugation. Dr. V. Cabasso (Cutter Laboratories, Berkeley, Calif.), Dr. H. Brummelhuis (Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), Dr. T. Shikata (Nihon University School of Medicine, Tokyo, Japan), and Dr. K. Takahashi (Jichi Medical School, Tokyo, Japan) all have successfully purified HBsAg using affinity chromatography, ultracentrifugation, and/or gel chromatography with or without pepsin digestion. Dr. J. Skelly (London School of Hygiene and Tropical Medicine, London, United Kingdom) de-
Table 1. Source material for hepatitis B virus vaccines. Investigator Gerin-Purcell Hilleman Cabasso Okochi Thomssen Maupas Brummelhuis Takahashi
Starting material
HBsAg concentration"
HBsAg subtype specificities
Plasma Plasma Plasma Plasma Blood Plasma Serum Blood Plasma
~1:50
adw ayw adw + ayw adw + ayw adr + adw adw + ayw ayw adw adr
~1:50 ~1:128
1:1,024
t 8 J.Lg/ml ~1:4
2
~g/ml
t
HBeAg
Anti-HBe
+ + + + + + + + +
NOTE. HBsAg = hepatitis B surface antigen; HBeAg = hepatitis B e antigen; anti-HBe = antibody to hepatitis B e antigen. * Protein concentration (Lowry [3]) or HBsAg reciprocal end-point titration by counterelectrophoresis. t Unspecified.
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yield of vaccine. Table 1 summarizes the source material for HBV vaccines produced by a number of investigators and the markers of HBV present in these source materials. There was general agreement that actively acquired antibody to the group-specific determinant of HBsAg (anti-a) would provide adequate protection against infection by HBV of any HBsAg subtype. Data on cross-protection following HBV infections with one HBV subtype in chimpanzees were presented by Dr. E. Tabor (Bureau of Biologics, Bethesda, Md.). Nine chimpanzees that had recovered from HBV infection acquired six to 88 weeks earlier were reinoculated with HBV bearing HBsAg of a second subtype contained in inocula with known infectivity. Following a total of 12 challenges, no reinfections by HBV were detected as judged by serologic analyses of weekly serum samples obtained over a 12-month period.
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Methods to Inactivate Residual HBV in Vaccines
No method of HBsAg purification is capable of removing all HBV, and little quantitative data exist regarding the ability of agents or procedures to inactivate HBV. Heat treatments such as those that inactivate HBV in normal serum albumin (60 C for 10 hr) have been shown to be incapable of inactivating high titers of HBV in whole plasma. HBV in purified HBsAg preparations, however, would probably be more easily inactivated than HBV in either whole plasma or in plasma derivatives such as normal serum albumin. All HBV vaccines discussed here were treated with either heat, formalin, or a combination of both to inactivate residual HBV. Heat treatments ranged from 100 C for 90 sec to 60 C for 10 hr. Concentrations of formalin ranged from 1:4,000 to 1:1,000 and were applied for varying intervals. All appeared adequate to inactivate HBV in some purified preparations of HBsAg. Dr. Purcell presented data on inactivation of HBV in the HBV vaccines made by Drs. Gerin and Purcell. The purification procedure itself removed HBV infectivity when the starting plasma contained as much as 104 CID 50 (50% chimpanzee infectious doses) of HBV. However, a later preparation made from plasma containing 107 C1D5o of
HBV contained residual virus that was not inactivated by purification alone. Formalin did, however, remove the remaining infectivity. Subsequent experiments in collaboration with Drs. Tabor and R. Gerety (Bureau of Biologics) showed that treatment of whole serum containing 5 X 104 • 5 CID 5 0 of HBV with a 1:4,000 dilution of formalin at 37 C for 72 hr failed to inactivate all of the HBV. Despite the small likelihood of large amounts of infectious HBV in purified vaccines, each lot of hepatitis vaccine manufactured in the United States will be tested for safety in chimpanzees as discussed below, until the kinetics of inactivation of HBV are better understood. Dr. Shikata estimated that 10-100 CID 50 of HBV remained in each lot of purified HBsAg produced in his laboratory. Heating at 60 C for 10 hr was inadequate to inactivate this amount of infectivity, although it resulted in a decrease in concentration of HBV as judged by chimpanzee inoculation studies. Additional treatment with formalin (l :2,000) resulted in complete inactivation of the residual HBV. Dr. Shikata concluded that inactivation of residual HBV should combine both heat and formalin treatments. The acquisition of additional data regarding the effect of specific treatments in inactivating HBV is being given a high priority by the Bureau of Biologics. In Vitro and Animal Testing of Vaccines
Methods described for assessment of vaccine purity and uniformity include electron microscopic examination, protein electrophoresis, quantitative determination of HBsAg, and immunofluorescence blocking to assure the absence of potentially sensitizing liver-specific protein or hepatocyte membrane antigens. Establishing a correlation between the protein content of vaccine and the HBsAg content would help assure the consistency of HBV vaccines. Acquisition of an international reference HBsAg is being pursued by the Bureau of Biologics to assist in vaccine standardization. It was suggested that data be obtained to assess the role of other antigens of HBV distinct from HBsAg in inducing protection against HBV infection. The Bureau of Biologics is in the process of generating these data at the present time. Immunogenicity (potency) tests of vaccines in guinea pigs revealed that between 0.1 and 1.0 JAg of HBsAg induced anti-HBs responses in 50070 of
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scribed the purification of HBsAg by equilibrium and rate zonal centrifugation using Urografin" (Schering Chemicals Ltd., Burgess Hill, United Kingdom). The final material produced by each of these investigators consisted of 20- to 22-nm spherical HBsAg particles. Dr. F. Hollinger (Baylor University, Houston, Tex.) described his experience with an experimental HBsAg polypeptide consisting of two polypeptides (mol wt, 22,000 and 25,000) derived from 50070-55070 of the HBsAg found in the original plasma. Dr. Skelly fractionated HBsAg using Triton-XI00 detergent to produce two HBsAg polypeptides with mol wt 23,000 and 28,000. The theoretical advantages of polypeptide vaccines are the exclusion of genetic material of viral origin, the exclusion of donor-derived substances, and the possibility of eventual development of a synthetically produced HBV polypeptide vaccine entirely devoid of undesirable host or viral components. Further studies on the immunogenicity and stability of such vaccines are needed.
NIAIDNews
B core antigen] were used). Thus, the presence of infectious HBV can be masked by the administration of a large amount of vaccine antigen, a finding that suggests that safety tests which use large amounts of vaccine may yield invalid results. . Dr. Hilleman reported that 11 lots of HBV vaccine from Merck, Sharp and Dohme had been safety-tested in four chimpanzees each, with follow-up for six months in compliance with the guidelines established by the Bureau of Biologics. It would be difficult to obtain sufficient numbers of susceptible chimpanzees for such safety testing once large-scale vaccine production was begun. Alternatives to safety testing with chimpanzees are being sought. When specific data are available regarding the inactivation of HBV by a variety of procedures, such safety testing might be less important than it is at present. Dr. A. Prince (New York Blood Center, New York, N.Y.) reported testing a bivalent (adw/ayw) HBV vaccine from the NIAID in chimpanzees. Five chimpanzees received 10 iv doses (50 ug/dose), and two received a single iv dose of vaccine. Three of the five receiving 10 doses produced anti-HBs; the two receiving a single dose did not. None showed evidence of HBV infection. The possibility that infectious virus was masked in the test that used 10 doses has to be considered in this instance, according to Dr. Prince. Groups of four chimpanzees were inoculated sc with two doses one month apart of either 50,20, 10, or 5 Jlg of vaccine to assess immunogenicity of the vaccine. Of four chimpanzees immunized with 50 Jlg, three gave a rapid, strong anti-HBs response, and one gave a less strong response. Following challenge with 103 • s CID so of HBV, these chimpanzees did not develop HBV infection. Of four immunized with 20 Jlg of vaccine, three gave a strong, rapid antiHBs response and resisted challenge. The fourth gave a weak response later in time and was infected by the challenge inoculum. The same pattern was seen among four chimpanzees immunized with 10 Jlg of vaccine and among four immunized with 5 Jlg of vaccine. To determine the duration of immunity to HBV provided by this vaccine, four chimpanzees were challenged a second time at one year and four at two years. The four chimpanzees challenged at one year and three of four challenged at two years did not develop HBV infection; not all have been tested for anti-HBc, how-
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random strains of guinea pigs. Boiling the vaccine for 90 sec or boiling for 90 sec followed by heating for 10 hr at 60 C resulted in a marked diminution in HBsAg immunogenicity. Dr. Hilleman presented data indicating that guinea pigs, grivet monkeys, and chimpanzees responded too readily to a low dose of vaccine and for this reason were not useful for immunogenicity (potency) tests. He described a test for vaccine immunogenicity in mice that consisted of a single ip injection of graded doses of vaccine into fiveweek-old mice followed by a test for anti-HBs after one month. Data from this test indicated that 90%, 70070, or 10% of the mice seroconverted with anti-HBs following the injection of 50 Jlg, 10 ug, or 1 Jlg of vaccine, respectively. A correlation between immunogenicity of HBV vaccine in humans and mice will be sought from data generated in human clinical trials currently being conducted in the United States. The adequacy of the chimpanzee as a safety test animal for HBV vaccine was discussed. Dr. Gerety presented data indicating that using sensitive serologic techniques, it was possible to detect HBV infections in all chimpanzees inoculated in the collaborative studies conducted by the Bureau of Biologics, NIAID, and the Center for Disease Control (Atlanta, Ga.). No single serologic or clinical test, however, could detect HBV infection in all of the chimpanzees. During these collaborativestudies, no incubation period longer than six months was seen in chimpanzees. This observation forms the basis for the current guidelines of a sixmonth follow-up in chimpanzees during safety testing. Unless further developments occur to improve our understanding of the inactivation kinetics of HBV when treated with various agents, it will be required that every lot of vaccine have a safety test in chimpanzees prior to use in humans. Drs. Tabor and Gerety presented data indicating that safety tests in chimpanzees should be performed with single or small doses of vaccine, since very large doses can mask the detection of residual HBV. In each of two chimpanzees that received both 100 ml of vaccine (40 Jlg/ml) and one ml of a preparation containing 101 • S CID so of infectious HBV, there was no serologic evidence of HBV infection during 12 months of testing on weekly samples (radioimmunoassay tests for HBsAg, anti-HBs, and anti-HBc [antibody to the hepatitis
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of autoimmune responses in vaccine-recipient chimpanzees, including tests for antibody in nuclei, smooth muscle, and mitochondria, were negative. Dr. Cabasso presented data on intravenous safety tests in four chimpanzees; two received a single vaccine dose, and two received 10 doses of vaccine. There was no evidence of HBV infection in any of the four animals. Dr. Hilleman described efficacy testing in chimpanzees. Six chimpanzees resisted infection with 103 • s CID so of live HBV after immunization (despite the absence of detectable anti-HBs in one). Five unimmunized control chimpanzees were infected following inoculation with 103 • S C1Dso of HBV. Less immunogenicity of HBV vaccines was observed in humans than in chimpanzees and small laboratory animals. This finding led to the use of adjuvants for most of the vaccines discussed. Adjuvants included aluminum hydroxide, aluminum phosphate, and a synthetic material (carbopol 934P); the latter was used only in chimpanzees. Neutralization of the formaldehyde in the Merck, Sharp and Dohme vaccine with bisulfite prior to use was noted to decrease considerably the immunogenicity of this vaccine, whereas digestion with pepsin appeared to produce a vaccine that was rapidly cleared from the circulation with increased immunogenicity. This pepsintreated vaccine appeared to be stable for longer than 12 months as judged by its immunogenicity in mice, guinea pigs, and chimpanzees. Clinical Testing
Dr. Hilleman described four lots of HBV vaccine (40 /-lg/dose), which were safety tested first in chim-
panzees and then in humans. In 10 clinical studies, 350 seronegative and 164 seropositive recipients received HBV vaccine from these four lots. Seroconversions to anti-HBs were documented in 75070-80070 of the seronegative recipients after two doses of vaccine administered one month apart. Individuals with preexisting anti-HBs responded to a single dose of vaccine with a rapid increase in anti-HBs titer that in most cases declined after four to eight months. Dr. Maupas presented data on the active immunization of 294 staff members and 96 patients
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ever. Dr. Prince noted that among all of the immunized chimpanzees, 25070 responded less vigorously than their cohorts and were termed "hyporesponders." The reason for their failure to respond to vaccine is not clear at this time. Dr. Purcell performed intravenous safety tests of monovalent adw or ayw HBV vaccines from the NIAID in eight chimpanzees. Chimpanzees that received two doses of either vaccine produced anti-HBs, most within two weeks after the initial vaccination and all within two weeks after the second vaccination. Anti-HBs persisted at a high level for at least six months in every case. Each chimpanzee was subsequently challenged with HBV, and none developed HBV infection. Dr. Purcell pointed out that although the chimpanzee provides an excellent model for safety tests of residual HBV in vaccine, the usefulness of this animal for detecting non-A, non-B hepatitis virus that might contaminate HBV vaccines is less well defined at the present time. Although the suitability of the chimpanzee as an animal model for human non-A, non-B hepatitis agents has been documented, there is presently no known method to detect subclinical infections, and there is no certain serologic method for selecting susceptible chimpanzees. For precisely this reason, the Bureau of Biologics recommends that, whenever possible, chimpanzees used for safety testing of HBV vaccines be those which have never previously received human blood, blood products, or plasma derivatives. Dr. Shikata presented data from 18 chimpanzees that were used to study vaccine efficacy. One "hyporesponder" was described also here. Anti-HBs was detected in all chimpanzees by passive HA eight weeks after the second dose of vaccine. All 18 were protected when challenged with live HBV. Safety tests in chimpanzees that employed a single dose of vaccine administered iv with the recommended six-month follow-up had not been performed, however. Each chimpanzee in these studies received multiple doses of vaccine with the attendant possibility of masking the detectability of residual HBV. Dr. Brummelhuis presented data obtained in a study of two chimpanzees (safety testing), which received a single iv dose of vaccine. No HBV infections were observed. Studies of liver biopsy tissues and sera performed to assess the presence
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tients and 1,200 staff members at 25 U.S. hemodialysis centers. In the first trial, 800 of these patients and 700 of these staff members were found to be susceptible to HBV based on serologic analyses. A separate trial conducted by Dr. Szmuness and sponsored by Merck, Sharp and Dohme is currently in progress in 300 male homosexuals. Dr. S. Krugman (New York University School of Medicine, New York, N.Y.) described a clinical trial of immunogenicity of an alum-precipitated HBV vaccine made by Merck, Sharp and Dohme, which is being conducted in health care personnel in the United States. Of 55 volunteers, 73% developed antibody after three doses of vaccine (40 Jig/dose) to date. Tests to Assure Consistency, Safety, and Potency of Vaccine
In vitro tests for HBsAg, a test for protein content, and an in vivo potency test appear to be the minimum tests required to assure vaccine consistency. These tests might include counterelectrophoresis, protein analysis by the Lowry method [3], and mouse potency assay (if this test proves appropriate), respectively. The need for an international HBV vaccine reference has been assigned a high priority by the Bureau of Biologics for the purpose of standardizing vaccine potency and assuring consistency of vaccines. All other applicable in vitro and in vivo tests of safety, purity, and potency, including those required by the Code of Federal Regulations, also apply to HBV vaccines. Until seronegative chimpanzees are no longer available or until data on the kinetics of HBV inactivation by acceptable agents and methods are established, four chimpanzees are to be used to safety test each lot of HBV vaccine. Two of these test chimpanzees must receive one vaccine dose iv, and two must receive 10 doses iv; all should be followed with sensitive serologic tests and liver biopsy specimen analyses for six months. Adjuvants appear to be necessary and are likely to be employed for all HBV vaccines. Conclusion
Data presented at this workshop indicate that active immunization for the prevention and control of HBV infection is feasible with the use of
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in a hemodialysis unit in Tours, France, with use of a bivalent (adw/ayw) vaccine (5 Jig/dose). The vaccine safety test in chimpanzees on this material consisted of the injection of 10 vaccine doses with serologic follow-up for three months. This vaccine was administered in the human clinical trial as a monthly injection for three months with a booster at one year. Among patients and staff, 60% and 930,10 respectively, seroconverted with anti-HBs. While 32% of the unvaccinated group developed HBV infection, the finding that 16% of the vaccinees who failed to develop anti-HBs also developed HBV infections was disturbing; 11.4% remained chronically infected. These results suggest that residual HBV may have remained in this vaccine despite treatment with formalin (table 1). Dr. V. McAuliffe (NIAID) described safety and immunogenicity data obtained from 16 human recipients of the NIAID aqueous adw vaccine. Ten volunteers received one dose (50 Jig) of vaccine sc and were followed for six months. None showed evidence of HBV infection, a finding that confirmed the safety data generated in the earlier chimpanzee studies. Six additional volunteers were inoculated with two doses of vaccine given 28 days apart. Various booster regimens were explored in each of these volunteers. Although 67% developed anti-HBs within seven months, the slow and variable development of antibody made this aqueous vaccine impractical for use in further clinical studies. Five volunteers were inoculated with one dose (50 Jig) of the aqueous ayw vaccine and followed for six months as a safety test. One of these vaccinees developed non-A, non-B hepatitis, but it is unclear whether this infection was vaccine-associated. Dr. W. Szmuness (New York Blood Center) described a large-scale clinical trial of the Merck, Sharp and Dohme HBV vaccine and emphasized the necessity both for prior seroepidemiologic studies of the population to be vaccinated and for controlled, randomized, double-blind clinical trials with simultaneous enrollment of all participants. Dr. Szmuness discussed planned clinical efficacy trials in individuals at high risk for acquiring HBV infection from two groups, patients undergoing chronic hemodialysis and male homosexuals. Under the sponsorship of the NIAID, base-line seroepidemiologic studies have been completed or are in progress to identify those susceptible to HBV among 2,200 hemodialysis pa-
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purified HBsAg. This material can be manufactured and treated so that it is free of infectious HBV. Large-scale clinical trials designed to demonstrate the efficacy of at least one HBV vaccine will be underway in the United States by late fall 1979; answers regarding vaccine efficacy can be anticipated two to three years later. In the absence of in vitro cultivation of HBV, progress toward prophylaxis by immunization against HBV has been truly remarkable. ROBERT J. GERETY
References
1. W.H.O. Technical Report Series. No. 602, 1977. 2. W.H.O. Proposed Requirements for Hepatitis B Vaccine, Biological Substances. No. 31-79, p. 1239. 3. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951.
EDWARD TABOR
ROBERT FRANKLIN
H.
PURCELL
J.
TYERYAR
National Institute of Allergy and Infectious Diseases Bethesda, Maryland
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Bureau of Biologics Food and Drug Administration Bethesda, Maryland
© 1979 by The University of Chicago. 0022-1899/79/4004-0035$00.75
NEWS
From the National Institute of Allergy and Infectious Diseases Summary of an International Workshop on Hepatitis B Vaccines An international workshop on hepatitis B virus (HBV) vaccines brought together investigators who are actively engaged in the research and development of vaccines to prevent HBV infection. This report is a summary of that workshop.
In the absence of successful propagation of HBV in vitro, the plasma of asymptomatic humans who are chronic carriers of hepatitis B surface antigen (HBsAg) serves as the source material for all HBV vaccines at the present time. Plasma from persons with high titers of HBsAg can be obtained by repeated plasmapheresis. A second source of antigen is plasma obtained from units of blood found during routine screening to contain high titers of HBsAg. HBV coexists in varying ratios with HBsAg in the plasma of persons who are chronic carriers of HBsAg. Data suggest that the presence of circulating hepatitis B e antigen (HBeAg) correlates with higher HBV/HBsAg ratios than those ratios observed in the plasma of persons who lack HBeAg or who possess its antibody (anti-HBe). Another marker associated with high HBV/HBsAg ratios is specific HBV DNA polymerase. A World Health Organization recommendation has stated that "consideration should be given to selection of HBsAg-positive plasma that is devoid of HBV particles, DNA polymerase and HBeAg as the starting material from which vaccine is to be prepared" [1]. This recommendation was recently modified to permit the use of source material containing both HBeAg and HBV DNA polymerase, since the exclusion of HBsAg-positive plasma containing these markers would make large-scale proThe National Institute of Allergy and Infectious Diseases and the Bureau of Biologics of the Food and Drug Administration sponsored this workshop in Bethesda, Maryland, on January 18 and 19, 1979. Please address requests for reprints to Dr. Robert J. Gerety, Hepatitis Branch, Bureau of Biologics, Food and Drug Administration, Building 29, Room 311, 8800 Rockville Pike, Bethesda, Maryland 20205. 642
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Vaccine Source Material
duction of vaccine impossible [2]. Use of such source material has not been shown to pose a hazard to vaccine recipients. Weekly plasmapheresis of suitable persons with high titers of HBsAg is currently permitted in the United States to provide source material for HBV vaccines. Such plasmapheresis requires careful isolation of the selected HBsAg-positive donors and careful monitoring of their levels of serum aminotransferases to assure that their liver function remains stable while they undergo repeated plasmapheresis. Such plasmapheresis of selected HBsAg-positive individuals over prolonged periods could assure the availability of HBsAg with uniform antigenic specificity. The antigen specificity, however, appears at this time to be of little significance with regard to protection against HBV. In addition, it is likely that the need for HBsAg will exceed the amount available from such "pedigreed" donors when the demand for vaccine increases subsequent to its licensure. Even at the present time, another method of acquiring adequate source material for HBV vaccine is necessary, since plasmapheresis programs do not exist in many countries, including Japan. The Japanese Red Cross Transfusion Service collects five million units (250 mllunit) of blood annually, of which about 100,000 units contain HBsAg and are routinely discarded. It is estimated that 35,000 such units contain sufficient HBsAg for use in the preparation of HBV vaccine. Dr. R. Thomssen (University of Goettingen, Goettingen, West Germany) reported that about 50% of the HBsAg-positive, HBeAg-negative plasma units collected in West Germany contained sufficient HBsAg for vaccine manufacture. However, two to three times the amount of HBeAgnegative plasma as compared to HBeAg-positive plasma was required, since the titers of HBsAg were lower in this plasma. Although the enrichment process was expensive, Dr. Thomssen felt that the resultant highly purified vaccine justified the increased cost. Highly purified HBsAg preparations, however, can be derived from HBeAgpositive plasma, with the advantage of an increased
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643
Approaches to HBV Vaccine Production
The plasma of persons who are chronic carriers of HBsAg contains three morphologically distinct particles, the complex HBV (40-42 mm), tubular HBsAg particles (20 nm in width and variable in length), and 20- to 22-nm spherical HBsAg particles. The majority of investigators purified the antigenic 20- to 22-nm particles from the others because of their lack of infectivity and the relative ease with which they can be separated from HBV. An alternative approach to vaccine preparation included the use of HBsAg polypeptides derived from the 20- to 22-nm particles; these consist of two glycoproteins, one with a mol wt of 22,00023,000 and the other of 25,000-28,000. A third ap-
proach used a variety of less highly purified HBV antigens as vaccine. Methods for purifying the 20- to 22-nm spherical HBsAg particles include ultracentrifugation, fractional precipitation, and chromatography. Dr. J. Gerin (National Institute of Allergy and Infectious Diseases [NIAID], Bethesda, Md., and Georgetown University, Washington, D.C.) described the purification of two lots of vaccine in collaboration with Dr. R. Purcell (NIAID). Each consisted of 20- to 22-nm HBsAg particles purified by isopycnic and rate zonal ultracentrifugation. Dr. M. Hilleman (Merck, Sharp and Dohme, West Point, Pa.) described the preparation of 11 lots of HBV vaccine consisting of 20- to 22-nm HBsAg particles, all similarly prepared by ultracentrifugation followed by a series of final treatments including digestion with pepsin to remove host antigens. Dr. P. Maupas (Institute of Virology, Tours, France) described the purification of HBsAg by either affinity chromatography using both human and rabbit antibody to HBsAg (anti-HBs) or by a combination of treatment with polyethylene glycol followed by ultracentrifugation. Dr. V. Cabasso (Cutter Laboratories, Berkeley, Calif.), Dr. H. Brummelhuis (Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), Dr. T. Shikata (Nihon University School of Medicine, Tokyo, Japan), and Dr. K. Takahashi (Jichi Medical School, Tokyo, Japan) all have successfully purified HBsAg using affinity chromatography, ultracentrifugation, and/or gel chromatography with or without pepsin digestion. Dr. J. Skelly (London School of Hygiene and Tropical Medicine, London, United Kingdom) de-
Table 1. Source material for hepatitis B virus vaccines. Investigator Gerin-Purcell Hilleman Cabasso Okochi Thomssen Maupas Brummelhuis Takahashi
Starting material
HBsAg concentration"
HBsAg subtype specificities
Plasma Plasma Plasma Plasma Blood Plasma Serum Blood Plasma
~1:50
adw ayw adw + ayw adw + ayw adr + adw adw + ayw ayw adw adr
~1:50 ~1:128
1:1,024
t 8 J.Lg/ml ~1:4
2
~g/ml
t
HBeAg
Anti-HBe
+ + + + + + + + +
NOTE. HBsAg = hepatitis B surface antigen; HBeAg = hepatitis B e antigen; anti-HBe = antibody to hepatitis B e antigen. * Protein concentration (Lowry [3]) or HBsAg reciprocal end-point titration by counterelectrophoresis. t Unspecified.
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yield of vaccine. Table 1 summarizes the source material for HBV vaccines produced by a number of investigators and the markers of HBV present in these source materials. There was general agreement that actively acquired antibody to the group-specific determinant of HBsAg (anti-a) would provide adequate protection against infection by HBV of any HBsAg subtype. Data on cross-protection following HBV infections with one HBV subtype in chimpanzees were presented by Dr. E. Tabor (Bureau of Biologics, Bethesda, Md.). Nine chimpanzees that had recovered from HBV infection acquired six to 88 weeks earlier were reinoculated with HBV bearing HBsAg of a second subtype contained in inocula with known infectivity. Following a total of 12 challenges, no reinfections by HBV were detected as judged by serologic analyses of weekly serum samples obtained over a 12-month period.
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Methods to Inactivate Residual HBV in Vaccines
No method of HBsAg purification is capable of removing all HBV, and little quantitative data exist regarding the ability of agents or procedures to inactivate HBV. Heat treatments such as those that inactivate HBV in normal serum albumin (60 C for 10 hr) have been shown to be incapable of inactivating high titers of HBV in whole plasma. HBV in purified HBsAg preparations, however, would probably be more easily inactivated than HBV in either whole plasma or in plasma derivatives such as normal serum albumin. All HBV vaccines discussed here were treated with either heat, formalin, or a combination of both to inactivate residual HBV. Heat treatments ranged from 100 C for 90 sec to 60 C for 10 hr. Concentrations of formalin ranged from 1:4,000 to 1:1,000 and were applied for varying intervals. All appeared adequate to inactivate HBV in some purified preparations of HBsAg. Dr. Purcell presented data on inactivation of HBV in the HBV vaccines made by Drs. Gerin and Purcell. The purification procedure itself removed HBV infectivity when the starting plasma contained as much as 104 CID 50 (50% chimpanzee infectious doses) of HBV. However, a later preparation made from plasma containing 107 C1D5o of
HBV contained residual virus that was not inactivated by purification alone. Formalin did, however, remove the remaining infectivity. Subsequent experiments in collaboration with Drs. Tabor and R. Gerety (Bureau of Biologics) showed that treatment of whole serum containing 5 X 104 • 5 CID 5 0 of HBV with a 1:4,000 dilution of formalin at 37 C for 72 hr failed to inactivate all of the HBV. Despite the small likelihood of large amounts of infectious HBV in purified vaccines, each lot of hepatitis vaccine manufactured in the United States will be tested for safety in chimpanzees as discussed below, until the kinetics of inactivation of HBV are better understood. Dr. Shikata estimated that 10-100 CID 50 of HBV remained in each lot of purified HBsAg produced in his laboratory. Heating at 60 C for 10 hr was inadequate to inactivate this amount of infectivity, although it resulted in a decrease in concentration of HBV as judged by chimpanzee inoculation studies. Additional treatment with formalin (l :2,000) resulted in complete inactivation of the residual HBV. Dr. Shikata concluded that inactivation of residual HBV should combine both heat and formalin treatments. The acquisition of additional data regarding the effect of specific treatments in inactivating HBV is being given a high priority by the Bureau of Biologics. In Vitro and Animal Testing of Vaccines
Methods described for assessment of vaccine purity and uniformity include electron microscopic examination, protein electrophoresis, quantitative determination of HBsAg, and immunofluorescence blocking to assure the absence of potentially sensitizing liver-specific protein or hepatocyte membrane antigens. Establishing a correlation between the protein content of vaccine and the HBsAg content would help assure the consistency of HBV vaccines. Acquisition of an international reference HBsAg is being pursued by the Bureau of Biologics to assist in vaccine standardization. It was suggested that data be obtained to assess the role of other antigens of HBV distinct from HBsAg in inducing protection against HBV infection. The Bureau of Biologics is in the process of generating these data at the present time. Immunogenicity (potency) tests of vaccines in guinea pigs revealed that between 0.1 and 1.0 JAg of HBsAg induced anti-HBs responses in 50070 of
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scribed the purification of HBsAg by equilibrium and rate zonal centrifugation using Urografin" (Schering Chemicals Ltd., Burgess Hill, United Kingdom). The final material produced by each of these investigators consisted of 20- to 22-nm spherical HBsAg particles. Dr. F. Hollinger (Baylor University, Houston, Tex.) described his experience with an experimental HBsAg polypeptide consisting of two polypeptides (mol wt, 22,000 and 25,000) derived from 50070-55070 of the HBsAg found in the original plasma. Dr. Skelly fractionated HBsAg using Triton-XI00 detergent to produce two HBsAg polypeptides with mol wt 23,000 and 28,000. The theoretical advantages of polypeptide vaccines are the exclusion of genetic material of viral origin, the exclusion of donor-derived substances, and the possibility of eventual development of a synthetically produced HBV polypeptide vaccine entirely devoid of undesirable host or viral components. Further studies on the immunogenicity and stability of such vaccines are needed.
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B core antigen] were used). Thus, the presence of infectious HBV can be masked by the administration of a large amount of vaccine antigen, a finding that suggests that safety tests which use large amounts of vaccine may yield invalid results. . Dr. Hilleman reported that 11 lots of HBV vaccine from Merck, Sharp and Dohme had been safety-tested in four chimpanzees each, with follow-up for six months in compliance with the guidelines established by the Bureau of Biologics. It would be difficult to obtain sufficient numbers of susceptible chimpanzees for such safety testing once large-scale vaccine production was begun. Alternatives to safety testing with chimpanzees are being sought. When specific data are available regarding the inactivation of HBV by a variety of procedures, such safety testing might be less important than it is at present. Dr. A. Prince (New York Blood Center, New York, N.Y.) reported testing a bivalent (adw/ayw) HBV vaccine from the NIAID in chimpanzees. Five chimpanzees received 10 iv doses (50 ug/dose), and two received a single iv dose of vaccine. Three of the five receiving 10 doses produced anti-HBs; the two receiving a single dose did not. None showed evidence of HBV infection. The possibility that infectious virus was masked in the test that used 10 doses has to be considered in this instance, according to Dr. Prince. Groups of four chimpanzees were inoculated sc with two doses one month apart of either 50,20, 10, or 5 Jlg of vaccine to assess immunogenicity of the vaccine. Of four chimpanzees immunized with 50 Jlg, three gave a rapid, strong anti-HBs response, and one gave a less strong response. Following challenge with 103 • s CID so of HBV, these chimpanzees did not develop HBV infection. Of four immunized with 20 Jlg of vaccine, three gave a strong, rapid antiHBs response and resisted challenge. The fourth gave a weak response later in time and was infected by the challenge inoculum. The same pattern was seen among four chimpanzees immunized with 10 Jlg of vaccine and among four immunized with 5 Jlg of vaccine. To determine the duration of immunity to HBV provided by this vaccine, four chimpanzees were challenged a second time at one year and four at two years. The four chimpanzees challenged at one year and three of four challenged at two years did not develop HBV infection; not all have been tested for anti-HBc, how-
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random strains of guinea pigs. Boiling the vaccine for 90 sec or boiling for 90 sec followed by heating for 10 hr at 60 C resulted in a marked diminution in HBsAg immunogenicity. Dr. Hilleman presented data indicating that guinea pigs, grivet monkeys, and chimpanzees responded too readily to a low dose of vaccine and for this reason were not useful for immunogenicity (potency) tests. He described a test for vaccine immunogenicity in mice that consisted of a single ip injection of graded doses of vaccine into fiveweek-old mice followed by a test for anti-HBs after one month. Data from this test indicated that 90%, 70070, or 10% of the mice seroconverted with anti-HBs following the injection of 50 Jlg, 10 ug, or 1 Jlg of vaccine, respectively. A correlation between immunogenicity of HBV vaccine in humans and mice will be sought from data generated in human clinical trials currently being conducted in the United States. The adequacy of the chimpanzee as a safety test animal for HBV vaccine was discussed. Dr. Gerety presented data indicating that using sensitive serologic techniques, it was possible to detect HBV infections in all chimpanzees inoculated in the collaborative studies conducted by the Bureau of Biologics, NIAID, and the Center for Disease Control (Atlanta, Ga.). No single serologic or clinical test, however, could detect HBV infection in all of the chimpanzees. During these collaborativestudies, no incubation period longer than six months was seen in chimpanzees. This observation forms the basis for the current guidelines of a sixmonth follow-up in chimpanzees during safety testing. Unless further developments occur to improve our understanding of the inactivation kinetics of HBV when treated with various agents, it will be required that every lot of vaccine have a safety test in chimpanzees prior to use in humans. Drs. Tabor and Gerety presented data indicating that safety tests in chimpanzees should be performed with single or small doses of vaccine, since very large doses can mask the detection of residual HBV. In each of two chimpanzees that received both 100 ml of vaccine (40 Jlg/ml) and one ml of a preparation containing 101 • S CID so of infectious HBV, there was no serologic evidence of HBV infection during 12 months of testing on weekly samples (radioimmunoassay tests for HBsAg, anti-HBs, and anti-HBc [antibody to the hepatitis
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of autoimmune responses in vaccine-recipient chimpanzees, including tests for antibody in nuclei, smooth muscle, and mitochondria, were negative. Dr. Cabasso presented data on intravenous safety tests in four chimpanzees; two received a single vaccine dose, and two received 10 doses of vaccine. There was no evidence of HBV infection in any of the four animals. Dr. Hilleman described efficacy testing in chimpanzees. Six chimpanzees resisted infection with 103 • s CID so of live HBV after immunization (despite the absence of detectable anti-HBs in one). Five unimmunized control chimpanzees were infected following inoculation with 103 • S C1Dso of HBV. Less immunogenicity of HBV vaccines was observed in humans than in chimpanzees and small laboratory animals. This finding led to the use of adjuvants for most of the vaccines discussed. Adjuvants included aluminum hydroxide, aluminum phosphate, and a synthetic material (carbopol 934P); the latter was used only in chimpanzees. Neutralization of the formaldehyde in the Merck, Sharp and Dohme vaccine with bisulfite prior to use was noted to decrease considerably the immunogenicity of this vaccine, whereas digestion with pepsin appeared to produce a vaccine that was rapidly cleared from the circulation with increased immunogenicity. This pepsintreated vaccine appeared to be stable for longer than 12 months as judged by its immunogenicity in mice, guinea pigs, and chimpanzees. Clinical Testing
Dr. Hilleman described four lots of HBV vaccine (40 /-lg/dose), which were safety tested first in chim-
panzees and then in humans. In 10 clinical studies, 350 seronegative and 164 seropositive recipients received HBV vaccine from these four lots. Seroconversions to anti-HBs were documented in 75070-80070 of the seronegative recipients after two doses of vaccine administered one month apart. Individuals with preexisting anti-HBs responded to a single dose of vaccine with a rapid increase in anti-HBs titer that in most cases declined after four to eight months. Dr. Maupas presented data on the active immunization of 294 staff members and 96 patients
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ever. Dr. Prince noted that among all of the immunized chimpanzees, 25070 responded less vigorously than their cohorts and were termed "hyporesponders." The reason for their failure to respond to vaccine is not clear at this time. Dr. Purcell performed intravenous safety tests of monovalent adw or ayw HBV vaccines from the NIAID in eight chimpanzees. Chimpanzees that received two doses of either vaccine produced anti-HBs, most within two weeks after the initial vaccination and all within two weeks after the second vaccination. Anti-HBs persisted at a high level for at least six months in every case. Each chimpanzee was subsequently challenged with HBV, and none developed HBV infection. Dr. Purcell pointed out that although the chimpanzee provides an excellent model for safety tests of residual HBV in vaccine, the usefulness of this animal for detecting non-A, non-B hepatitis virus that might contaminate HBV vaccines is less well defined at the present time. Although the suitability of the chimpanzee as an animal model for human non-A, non-B hepatitis agents has been documented, there is presently no known method to detect subclinical infections, and there is no certain serologic method for selecting susceptible chimpanzees. For precisely this reason, the Bureau of Biologics recommends that, whenever possible, chimpanzees used for safety testing of HBV vaccines be those which have never previously received human blood, blood products, or plasma derivatives. Dr. Shikata presented data from 18 chimpanzees that were used to study vaccine efficacy. One "hyporesponder" was described also here. Anti-HBs was detected in all chimpanzees by passive HA eight weeks after the second dose of vaccine. All 18 were protected when challenged with live HBV. Safety tests in chimpanzees that employed a single dose of vaccine administered iv with the recommended six-month follow-up had not been performed, however. Each chimpanzee in these studies received multiple doses of vaccine with the attendant possibility of masking the detectability of residual HBV. Dr. Brummelhuis presented data obtained in a study of two chimpanzees (safety testing), which received a single iv dose of vaccine. No HBV infections were observed. Studies of liver biopsy tissues and sera performed to assess the presence
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tients and 1,200 staff members at 25 U.S. hemodialysis centers. In the first trial, 800 of these patients and 700 of these staff members were found to be susceptible to HBV based on serologic analyses. A separate trial conducted by Dr. Szmuness and sponsored by Merck, Sharp and Dohme is currently in progress in 300 male homosexuals. Dr. S. Krugman (New York University School of Medicine, New York, N.Y.) described a clinical trial of immunogenicity of an alum-precipitated HBV vaccine made by Merck, Sharp and Dohme, which is being conducted in health care personnel in the United States. Of 55 volunteers, 73% developed antibody after three doses of vaccine (40 Jig/dose) to date. Tests to Assure Consistency, Safety, and Potency of Vaccine
In vitro tests for HBsAg, a test for protein content, and an in vivo potency test appear to be the minimum tests required to assure vaccine consistency. These tests might include counterelectrophoresis, protein analysis by the Lowry method [3], and mouse potency assay (if this test proves appropriate), respectively. The need for an international HBV vaccine reference has been assigned a high priority by the Bureau of Biologics for the purpose of standardizing vaccine potency and assuring consistency of vaccines. All other applicable in vitro and in vivo tests of safety, purity, and potency, including those required by the Code of Federal Regulations, also apply to HBV vaccines. Until seronegative chimpanzees are no longer available or until data on the kinetics of HBV inactivation by acceptable agents and methods are established, four chimpanzees are to be used to safety test each lot of HBV vaccine. Two of these test chimpanzees must receive one vaccine dose iv, and two must receive 10 doses iv; all should be followed with sensitive serologic tests and liver biopsy specimen analyses for six months. Adjuvants appear to be necessary and are likely to be employed for all HBV vaccines. Conclusion
Data presented at this workshop indicate that active immunization for the prevention and control of HBV infection is feasible with the use of
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in a hemodialysis unit in Tours, France, with use of a bivalent (adw/ayw) vaccine (5 Jig/dose). The vaccine safety test in chimpanzees on this material consisted of the injection of 10 vaccine doses with serologic follow-up for three months. This vaccine was administered in the human clinical trial as a monthly injection for three months with a booster at one year. Among patients and staff, 60% and 930,10 respectively, seroconverted with anti-HBs. While 32% of the unvaccinated group developed HBV infection, the finding that 16% of the vaccinees who failed to develop anti-HBs also developed HBV infections was disturbing; 11.4% remained chronically infected. These results suggest that residual HBV may have remained in this vaccine despite treatment with formalin (table 1). Dr. V. McAuliffe (NIAID) described safety and immunogenicity data obtained from 16 human recipients of the NIAID aqueous adw vaccine. Ten volunteers received one dose (50 Jig) of vaccine sc and were followed for six months. None showed evidence of HBV infection, a finding that confirmed the safety data generated in the earlier chimpanzee studies. Six additional volunteers were inoculated with two doses of vaccine given 28 days apart. Various booster regimens were explored in each of these volunteers. Although 67% developed anti-HBs within seven months, the slow and variable development of antibody made this aqueous vaccine impractical for use in further clinical studies. Five volunteers were inoculated with one dose (50 Jig) of the aqueous ayw vaccine and followed for six months as a safety test. One of these vaccinees developed non-A, non-B hepatitis, but it is unclear whether this infection was vaccine-associated. Dr. W. Szmuness (New York Blood Center) described a large-scale clinical trial of the Merck, Sharp and Dohme HBV vaccine and emphasized the necessity both for prior seroepidemiologic studies of the population to be vaccinated and for controlled, randomized, double-blind clinical trials with simultaneous enrollment of all participants. Dr. Szmuness discussed planned clinical efficacy trials in individuals at high risk for acquiring HBV infection from two groups, patients undergoing chronic hemodialysis and male homosexuals. Under the sponsorship of the NIAID, base-line seroepidemiologic studies have been completed or are in progress to identify those susceptible to HBV among 2,200 hemodialysis pa-
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purified HBsAg. This material can be manufactured and treated so that it is free of infectious HBV. Large-scale clinical trials designed to demonstrate the efficacy of at least one HBV vaccine will be underway in the United States by late fall 1979; answers regarding vaccine efficacy can be anticipated two to three years later. In the absence of in vitro cultivation of HBV, progress toward prophylaxis by immunization against HBV has been truly remarkable. ROBERT J. GERETY
References
1. W.H.O. Technical Report Series. No. 602, 1977. 2. W.H.O. Proposed Requirements for Hepatitis B Vaccine, Biological Substances. No. 31-79, p. 1239. 3. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951.
EDWARD TABOR
ROBERT FRANKLIN
H.
PURCELL
J.
TYERYAR
National Institute of Allergy and Infectious Diseases Bethesda, Maryland
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Bureau of Biologics Food and Drug Administration Bethesda, Maryland
