Editorials Inactivated Hepatitis A Virus Vaccines
Approximately 10 yr after the licensure of the first hepatitis B vaccine, the stage is set for the introduction of a safe and effective hepatitis A vaccine. However, accustomed as they are to the chronic complications of persistent hepatitis B and hepatitis C infections, many hepatologists may question the need for a hepatitis A vaccine. After all, hepatitis A virus (HAV) infections are never truly persistent, and the virus is not associated with chronic hepatitis or the development of HCC. Nonetheless, although it is usually considered a benign disease, a total of 31,441 cases of acute hepatitis A were reported to the Centers for Disease Control during 1990. These cases undoubtedly represent only the tip of a much larger iceberg, and most are never seen by the liver specialist. In addition, hepatitis A does account for a small proportion of cases of fulminant hepatitis and is responsible for about 100 deaths annually in the United States. Thus the overall hepatitis A disease burden, including direct medical care costs and the indirect costs of time lost from work, is large. Finally, not to be overlooked is the fact that the exceptional stability of the HAV particle contributes to an impressive capacity for epidemic transmission and occasional dramatic disease outbreaks. Thus little doubt exists that a safe and effective vaccine for hepatitis A would be welcomed in many quarters. HAV is now classified within the genus Hepatovirus of the family Picornaviridae. It is a positive-strand RNA virus, with a 7.5-kb genome encapsidated in a naked protein shell that neither is glycosylated nor contains appreciable quantities of lipids. It is a very different virus from that of hepatitis B and has thus required a very different approach to vaccine development. As a picornavirus, HAV shares many structural and biological features in common with poliovirus. Thus it is not surprising that the strategies taken for HAV vaccine development have to a large extent paralleled those taken previously for the development of formalininactivated and live, attenuated poliovirus vaccines. Provost and Hilleman (1) first demonstrated the ability of HAV to replicate in cultured primate cells. Early work demonstrated that the adaptation of wild-type HAV to growth in cell culture, followed by successive passages in cell culture, led to reductions in the ability of the virus to induce acute hepatocellular injury in otherwise susceptible primates (2,3).Although
Address reprint requests to: Stanley M. Lemon, Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030. 31/1/37590
the clinical development of such candidate attenuated hepatitis A vaccines has progressed to the point of phase 1 human trials, the overall results have been relatively disappointing. These attenuated vaccine candidates appear to have a markedly reduced capacity to replicate in primate hosts, with the result that the antibody response after immunization of human volunteers has been relatively weak (4). Moreover, the attenuated candidate vaccine strains are not able to infect humans or higher primates after oral challenge, and they therefore do not possess one of the most attractive attributes of the oral, attenuated poliovirus vaccine developed by Albert Sabin. Nonetheless, the development of such vaccines continues to be pursued aggressively in China, where preliminary results (perhaps using virus seeds that are less attenuated) appear more encouraging (5). A second approach to the development of an HAV vaccine follows that taken by Jonas Salk in developing the formalin-inactivated poliovirus vaccine. Provost and Hilleman (6) first demonstrated that HAV particles purified from marmoset liver, inactivated with formalin and administered in a series of multiple injections to susceptible primates, were capable of inducing protection against virulent virus challenge. Thus with the development of cell culture systems for the propagation of HAV, it was natural to consider the development of formalin-inactivated, cell culture-derived vaccines for HAV. The demonstration by Binn and coworkers (7) that very small quantities of formalin-inactivated HAV were highly immunogenic in susceptible owl monkeys, leading after several doses to a high level of protection against challenge with virulent virus, provided further encouragement. Several prototype-inactivated HAV vaccines made from infected cell cultures were subsequently tested in humans (8,9).Although these vaccines were shown capable of inducing neutralizing antibodies after several injections, more recent efforts by commercial manufacturers (notably Merck, Sharp & Dohme and SmithKline Beecham) have led to the production of cell culture-derived purified vaccines with substantially higher viral antigen content (10-12). However, a major and continuing limitation to the development and manufacture of such vaccines is the relatively low yield of HAV that is achieved in most cell culture systems. What is the evidence that these inactivated HAV vaccines are effective, and what can be said of their safety? Elsewhere in this issue of Hepatology, Fujiyama and coworkers describe a comparison of the human serological response to an inactivated HAV vaccine produced in Japan to that after passive immunization with pooled human serum immunoglobulins (ISG).
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Vol. 15, No. 6, 1992
INACTIVATED HEPATITIS A VIRUS VACCINES
Whereas natural immunity to HAV results from a complicated interplay of humoral and cellular responses to this virus infection (131, it has been known for almost half a century that passive immunization with ISG leads to a high level of protection against symptomatic hepatitis A. The precise mechanism of protection afforded by the administration of ISG has never been clearly defined, but it is likely that antibodies present in ISG effectively neutralize circulating HAV particles, thereby either preventing HAV from reaching its target cells in the liver or reducing the level of a secondary HAV viremia with the result that substantially fewer hepatocytes ultimately become infected. It should be evident, then, that any hepatitis A vaccine that is capable of inducing serum virus neutralizing antibodies that are equal in titer and activity to those found after passive administration of ISG should provide at least an equivalent level of protection (14).Such is the argument made by Fujiyama et al. and the developers of other inactivated HAV vaccines. In assessing the antibody response after passive administration of ISG, it is necessary to use serological tests that are considerably more sensitive than the standard solid-phase commercial competitive inhibition immunoassays such as the Havab assay (Abbott Laboratories, North Chicago, IL). Generally speaking, the administration of ISG in standard doses (0.02 ml/kg) does not lead to seroconversion in such tests. However, as first demonstrated by Provost and coworkers (10)and again by Fujiyama et al., the sensitivity of the standard Havab assay may be enhanced approximately 10-fold by increasing the volume of serum tested by a factor of 10. This “modified Havab” test is capable of detecting anti-HAV at a level of approximately 10 mIU/ml (when compared with a World Health Organization reference anti-HAV reagent) and may be positive in the first few weeks after the administration of ISG. How does the antibody detected in such tests correlate with virus neutralization activity? In the last few years, a considerable amount of information has been generated concerning the antigenic structure of HAV (15). The relevant neutralization epitopes have been shown to be highly conformational, formed by the tertiary and quaternary interactions of the three major capsid proteins that comprise the outer shell of the virus. Antibodies detected in the Havab assay are thus directed against the complete HAV virion and empty capsids that do not contain viral RNA. These antibodies do not react with denatured capsid proteins, nor are significant levels of antibodies to the individual capsid proteins present in most human convalescent sera. This is the reason why attempts to develop recombinant hepatitis A vaccines have generally failed. Because the empty and complete HAV particles appear to have very similar if not identical antigenicities, the presence of antibody detected in the Havab and similar immunoassays correlates very closely with the presence of viral neutralizing activity. Viral neutralization tests, which measure the ability of a serum sample to reduce the infectivity of laboratory strains of HAV in cell culture, are potentially more
1195
sensitive than even the modified Havab assay (14, 16). These neutralization tests are technically very difficult and expensive, but they provide a very useful means of comparing the antibody responses after active and passive immunization against hepatitis A. Fujiyama et al. found that the neutralizing antibody response after two doses of an inactivated hepatitis A vaccine (given at 0 and 1 mo) was greater than that after passive administration of ISG. A third vaccine dose (at 6 or 12 mo) enhanced this response considerably and probably results in a longer duration of antibody persistence. Such serological comparisons, which are similar to previous observations made with the Merck and SmithKline vaccines, strongly suggest that the current generation of formalin-inactivated HAV vaccines should be highly effective. Although such theoretical comparisons of antibody responses make good sense, they are unlikely to satisfy some regulatory agencies. Thus clinical trials have been designed to assess the efficacy of these vaccines, and preliminary reports of these efficacy trials have recently been presented at international meetings. The design of such studies has been complicated by the sporadic nature of hepatitis A in well-developed countries and difficulties in identifying high-risk populations suitable for study. However, in an efficacy study completed recently in Monroe, New York, by Werzberger and associates (Personal communication), the Merck vaccine was shown to induce nearly complete protection against symptomatic hepatitis A within several weeks of the administration of a single dose. This clinical trial, which was carried out in children, demonstrated conclusively that only very low levels of serum-neutralizing antibody are required for high levels of clinical protection. In addition, this study demonstrated the feasibility of protecting travelers against hepatitis A with the administration of a single dose of vaccine. A large-scale study of the SmithKline inactivated HAV vaccine, involving more than 40,000 school-age children, is currently being conducted by Innis and coworkers in Thailand. A recent interim look at this study (Personal communication) has indicated a comparable level of efficacy several months after the administration of two vaccine doses. What level of protection either of these vaccines might afford against inapparent infection remains uncertain, but it is likely to be determined by the Thai study. It is too early to say whether any clinically significant differences exist between these two commercial vaccines. Although both of these efficacy studies have been carried out in children, no reason exists to suspect that the efficacy will be substantially different in adults, provided that equivalent levels of antibody are demonstrated after immunization. However, the duration of protection after immunization remains uncertain. This can be determined only by continued observations in the field, but it is likely that the levels of antibody developing after a complete series of three immunizations will lead to protection for at least 5 to 10 yr. These vaccines are also likely t o protect against all strains of HAV.
1196
LEMON
Although HAV strains can be separated into several epidemiologically and phylogenetically distinct genotypes, each differing from each other at up to 25% of nucleotide base positions within certain regions of the RNA genome of the virus (171, all human HAV strains studied thus far are very closely related, if not identical, antigenically (15). Thus immunization with a single vaccine strain should protect against hepatitis A in all regions of the world. What about safety? Because the current generation of inactivated hepatitis A vaccines are produced from virus strains that have been adapted to growth in cell culture, the seed viruses are substantially or completely attenuated even before inactivation (12). Nonetheless, the formalin-inactivation kinetics have been carefully determined for HAV, and more than 35 yr of experience in the manufacture of formalin-inactivated polio vaccines has provided the commercial know-how to safely manufacture inactivated hepatitis A vaccines. (Unlike the seed viruses used for hepatitis A vaccines, those used for the production of inactivated polio vaccine are usually fully virulent.) The side effects associated with the administration of inactivated hepatitis A vaccines in clinical trials have been minimal and generally similar to those associated with use of the hepatitis B vaccine. Finally, how can these new vaccines best be used t o reduce the overall hepatitis A disease burden? Although decreasing circulation of HAV during the last several decades has led to a high level of susceptibility among young adults within the United States, molecular evidence strongly suggests the continued circulation of an endemic HAV genotype (17). Several groups appear to be at particular risk of HAV infection. Child-care centers caring for infants and young children who are not yet toilet trained remain prime sites for HAV transmission, from which there occurs radial spread of HAV into the community. Hepatitis A remains a problem among institutionalized persons, and it has been increasingly associated with the illicit use of drugs within the United States as it has in Europe (18). In addition, during 1991 a considerable increase in hepatitis A infections was noted among urban, homosexual men in the United States, Canada and Australia (19). Continuing sporadic outbreaks of hepatitis A remain a major nuisance for public health officials in the United States. Such outbreaks often have been associated with infected food handlers working in the final stages of food preparation and distribution, but more recently the outbreaks have been associated with contamination of foodstuffs (e.g., lettuce and frozen strawberries) at their source. Travelers to poorly developed overseas destinations where hepatitis A is particularly endemic represent a prime target for immunization. Such travelers number in the millions when one considers both North America and Western Europe. Although ISG affords a high level of protection for travelers, concerns exist regarding its continuing use. First of all, the continuing decline in the prevalence of hepatitis A in many well-developed countries may be leading to gradual reductions in the titer of HAV antibodies present in the plasma pools from which
HEPATOLOGY
ISG is made. In accordance with this notion, Fujiyama et al. found that the levels of HAV antibody in ISG lots manufactured in Japan were significantly lower when plasma came from domestic (Japanese) versus foreign sources. In addition, supplies of ISG may occasionally be limited. The rapid mobilization of American military forces to an HAV endemic area, the Middle East, during Operation Desert Storm effectively depleted stocks of ISG available in the United States, leading the military to seek foreign supplies of this product. Thus one might expect formalin-inactivated HAV vaccine to be recommended first for adult travelers to overseas destinations where HAV is endemic, to illicit drug users and promiscuous homosexual men who are at increased risk for HAV infection, to clients of institutions for the developmentally disabled and t o prison inmates. In addition, it is not difficult to envision how immunization of seronegative food handlers could lead to a marked reduction in the frequency of foodborne outbreaks of HAV, along with their attendant costs of epidemiological investigation and implementation of control measures. Lastly, as information concerning the safety of these vaccines continues to accrue, it is likely that immunization of young children attending preschool day-care centers will ultimately become a policy objective. Control of HAV within such centers might result in significant reductions in the community-wide incidence of hepatitis A, based on previous observations of the use of ISG in day-care centers. In any discussion of hepatitis A vaccines, however, it cannot be forgotten that the greatest need for an effective and safe (and inexpensive) hepatitis A vaccine exists in developing countries undergoing the transition from a low to a higher level of public health sanitation. Transitional countries, such as China, have noted a substantial increase in the hepatitis A disease burden as the average age of infection shifts from the very young (who are often asymptomatic when infected with HAV) to the older child and young adult (who are usually symptomatic and frequently icteric). However, the use of inactivated HAV vaccines in developing countries raises several issues. First of all, the length of protection afforded by such vaccines must be measured in decades if they are to be truly effective in such countries. This might well be the case with inactivated hepatitis A vaccines given in a series of several doses, but the existence of clinically relevant immunological memory (with protection outlasting detectable serum antibodies) will need to be determined. Given the age-related nature of the severity of hepatitis A, it is important that a more susceptible older population is not created by immunizing the young (20). Second, vaccines should optimally prevent transmission of the virus to others. This is likely to be the case with inactivated hepatitis A vaccines, even if they do not produce effective levels of secretory immunity. Most if not all of the virus that is shed in the feces of infected persons is produced in the liver, and inactivated vaccines clearly limit the hepatic replication and hence fecal shedding of the virus. Finally, for a vaccine to be acceptable for use in
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INACTIVATED HEPATITIS A VIRUS VACCINES
developing countries, it must be very inexpensive. The SmithKline Beecham-inactivated HAV vaccine, which was recently approved for clinical use in Switzerland and Belgium, costs approximately $25 to $30/dose, a price comparable t o recombinant hepatitis B vaccine or human diploid cell rabies vaccine. The relatively high cost of the HAV vaccine reflects in part difficulties in producing adequate quantities of this virus in cell culture, and the usual development and product liability costs inherent in commercial vaccine manufacture today. Thus although the introduction of formalininactivated HAV vaccines will represent a substantial step forward in the control of hepatitis virus infections in well-developed and affluent societies of North America, Western Europe and Asia, the control of hepatitis A (like the control of hepatitis B) is likely to remain a major problem in less-advantaged societies for some time to come.
STANLEY M. LEMON,M.D. Department of Medicine The University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599-7030 REFERENCES 1. Provost PJ, Hilleman MR. Propagation of human hepatitis A virus in cell culture in uitro. Proc SOCExp Biol Med 1979;160:213-221. 2. Provost PJ, Banker FS, Giesa PA, McAleer WJ, Buynak EB, Hilleman MR. Progress toward a live, attenuated human hepatitis A vaccine. Proc SOCExp Biol Med 1982;170:8-14. 3. Provost PJ, Bishop RP, Gerety RJ,Hilleman MR, McAleer WJ, Scolnick EM, Stevens CE. New findings in live, attenuated hepatitis A vaccine development. J Med Virol 1986;20:165175. 4. Midthun K, Ellerbeck E, Gershman K, Calandra G, Krah D, McCaughtry M, Nalin D, et al. Safety and immunogenicity of a live attenuated hepatitis A virus vaccine in seronegative volunteers. J Infect Dis 1991;163:735-739. 5. Mao JS, Dong DX, Zhang SY, Zhang H Y , Chen NL, Huang HY, Xie RY, et al. Further studies of attenuated live hepatitis Avaccine (H2 strain) in humans. In: Hollinger FB, Lemon SM, Margolis HS, eds.
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Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991:llO-111. 6. Provost PJ, Hilleman MR. An inactivated hepatitis Avirus vaccine prepared from infected marmoset liver. Proc SOCExp Biol Med 1978;159:201-203. 7. Binn LN, Bancroft WH, Lemon SM, Marchwicki RH, LeDuc SW, Trahan CJ, Staley EC, et al. Preparation of a prototype inactivated hepatitis A virus vaccine from infected cell cultures. J Infect Dis 1986;153:749-756. 8. Flehmig B, Haage A, Pfisterer M. Immunogenicity of a hepatitis A virus vaccine. J Med Virol 1987;22:7-16. 9. Sjogren MH, Eckels KH, Binn LN, Dubois DR, Hoke CH, Burke DS, Bancroft WH. Safety and immunogenicity of an inactivated hepatitis Avaccine. In: Zuckerman AJ, ed. Viral hepatitis and liver disease. New York: Alan R. Liss, Inc., 1988:94-96. 10. Provost PJ, Hughes JV,Miller WJ, Giesa PA, Banker FS, Emini EA. An inactivated hepatitis A viral vaccine of cell culture origin. J Med Virol 1986;19:23-31. 11. Andre FE, Hepburn A, D’Hondt E. Inactivated candidate vaccines for hepatitis A. Prog Med Virol 1990;37:72-95. 12. Lewis JA, Armstrong ME, Larson VM,Emini EA, Midthun K, Ellerbeck E, Nalin D, et al. Use of a live attenuated hepatitis A vaccine to prepare a highly purified, formalin-inactivated hepatitis A vaccine. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991~94-97. 13. Lemon SM, Ping L-H, Day S,Cox E, Jansen R, Amphlett E, Sangar D. Immunobiology of hepatitis A virus. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991:20-24. 14. Stapleton JT, Jansen RW, Lemon SM. Neutralizing antibody to hepatitis A virus in immune serum globulin and in the sera of human recipients of immune serum globulin. Gastroenterology 1985;89:637-642. 15. Ping L-H, Lemon SM. Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies. J Virol 1992;66. 16. Lemon SM, Binn LN. Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983;148:1033-1039. 17. Robertson BH, Jansen RW, Khanna B, Totsuka A, Nainan OV, Siegl G, Widell A, et al. Genetic relatedness of hepatitis A virus strains recovered from different geographic regions [in press]. J Gen Virol 1992. 18. Centers for Disease Control. Hepatitis A among drug abusers. MMWR 1988;37:297-305. 19. Centers for Disease Control. Hepatitis A among homosexual men: United States, Canada, and Australia. MMWR 1992;41:155-164. 20. Kane MA. Prospects for the introduction of hepatitis A vaccine into public health use. Prog Med Virol 1990;37:96-100.
Approximately 10 yr after the licensure of the first hepatitis B vaccine, the stage is set for the introduction of a safe and effective hepatitis A vaccine. However, accustomed as they are to the chronic complications of persistent hepatitis B and hepatitis C infections, many hepatologists may question the need for a hepatitis A vaccine. After all, hepatitis A virus (HAV) infections are never truly persistent, and the virus is not associated with chronic hepatitis or the development of HCC. Nonetheless, although it is usually considered a benign disease, a total of 31,441 cases of acute hepatitis A were reported to the Centers for Disease Control during 1990. These cases undoubtedly represent only the tip of a much larger iceberg, and most are never seen by the liver specialist. In addition, hepatitis A does account for a small proportion of cases of fulminant hepatitis and is responsible for about 100 deaths annually in the United States. Thus the overall hepatitis A disease burden, including direct medical care costs and the indirect costs of time lost from work, is large. Finally, not to be overlooked is the fact that the exceptional stability of the HAV particle contributes to an impressive capacity for epidemic transmission and occasional dramatic disease outbreaks. Thus little doubt exists that a safe and effective vaccine for hepatitis A would be welcomed in many quarters. HAV is now classified within the genus Hepatovirus of the family Picornaviridae. It is a positive-strand RNA virus, with a 7.5-kb genome encapsidated in a naked protein shell that neither is glycosylated nor contains appreciable quantities of lipids. It is a very different virus from that of hepatitis B and has thus required a very different approach to vaccine development. As a picornavirus, HAV shares many structural and biological features in common with poliovirus. Thus it is not surprising that the strategies taken for HAV vaccine development have to a large extent paralleled those taken previously for the development of formalininactivated and live, attenuated poliovirus vaccines. Provost and Hilleman (1) first demonstrated the ability of HAV to replicate in cultured primate cells. Early work demonstrated that the adaptation of wild-type HAV to growth in cell culture, followed by successive passages in cell culture, led to reductions in the ability of the virus to induce acute hepatocellular injury in otherwise susceptible primates (2,3).Although
Address reprint requests to: Stanley M. Lemon, Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7030. 31/1/37590
the clinical development of such candidate attenuated hepatitis A vaccines has progressed to the point of phase 1 human trials, the overall results have been relatively disappointing. These attenuated vaccine candidates appear to have a markedly reduced capacity to replicate in primate hosts, with the result that the antibody response after immunization of human volunteers has been relatively weak (4). Moreover, the attenuated candidate vaccine strains are not able to infect humans or higher primates after oral challenge, and they therefore do not possess one of the most attractive attributes of the oral, attenuated poliovirus vaccine developed by Albert Sabin. Nonetheless, the development of such vaccines continues to be pursued aggressively in China, where preliminary results (perhaps using virus seeds that are less attenuated) appear more encouraging (5). A second approach to the development of an HAV vaccine follows that taken by Jonas Salk in developing the formalin-inactivated poliovirus vaccine. Provost and Hilleman (6) first demonstrated that HAV particles purified from marmoset liver, inactivated with formalin and administered in a series of multiple injections to susceptible primates, were capable of inducing protection against virulent virus challenge. Thus with the development of cell culture systems for the propagation of HAV, it was natural to consider the development of formalin-inactivated, cell culture-derived vaccines for HAV. The demonstration by Binn and coworkers (7) that very small quantities of formalin-inactivated HAV were highly immunogenic in susceptible owl monkeys, leading after several doses to a high level of protection against challenge with virulent virus, provided further encouragement. Several prototype-inactivated HAV vaccines made from infected cell cultures were subsequently tested in humans (8,9).Although these vaccines were shown capable of inducing neutralizing antibodies after several injections, more recent efforts by commercial manufacturers (notably Merck, Sharp & Dohme and SmithKline Beecham) have led to the production of cell culture-derived purified vaccines with substantially higher viral antigen content (10-12). However, a major and continuing limitation to the development and manufacture of such vaccines is the relatively low yield of HAV that is achieved in most cell culture systems. What is the evidence that these inactivated HAV vaccines are effective, and what can be said of their safety? Elsewhere in this issue of Hepatology, Fujiyama and coworkers describe a comparison of the human serological response to an inactivated HAV vaccine produced in Japan to that after passive immunization with pooled human serum immunoglobulins (ISG).
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Vol. 15, No. 6, 1992
INACTIVATED HEPATITIS A VIRUS VACCINES
Whereas natural immunity to HAV results from a complicated interplay of humoral and cellular responses to this virus infection (131, it has been known for almost half a century that passive immunization with ISG leads to a high level of protection against symptomatic hepatitis A. The precise mechanism of protection afforded by the administration of ISG has never been clearly defined, but it is likely that antibodies present in ISG effectively neutralize circulating HAV particles, thereby either preventing HAV from reaching its target cells in the liver or reducing the level of a secondary HAV viremia with the result that substantially fewer hepatocytes ultimately become infected. It should be evident, then, that any hepatitis A vaccine that is capable of inducing serum virus neutralizing antibodies that are equal in titer and activity to those found after passive administration of ISG should provide at least an equivalent level of protection (14).Such is the argument made by Fujiyama et al. and the developers of other inactivated HAV vaccines. In assessing the antibody response after passive administration of ISG, it is necessary to use serological tests that are considerably more sensitive than the standard solid-phase commercial competitive inhibition immunoassays such as the Havab assay (Abbott Laboratories, North Chicago, IL). Generally speaking, the administration of ISG in standard doses (0.02 ml/kg) does not lead to seroconversion in such tests. However, as first demonstrated by Provost and coworkers (10)and again by Fujiyama et al., the sensitivity of the standard Havab assay may be enhanced approximately 10-fold by increasing the volume of serum tested by a factor of 10. This “modified Havab” test is capable of detecting anti-HAV at a level of approximately 10 mIU/ml (when compared with a World Health Organization reference anti-HAV reagent) and may be positive in the first few weeks after the administration of ISG. How does the antibody detected in such tests correlate with virus neutralization activity? In the last few years, a considerable amount of information has been generated concerning the antigenic structure of HAV (15). The relevant neutralization epitopes have been shown to be highly conformational, formed by the tertiary and quaternary interactions of the three major capsid proteins that comprise the outer shell of the virus. Antibodies detected in the Havab assay are thus directed against the complete HAV virion and empty capsids that do not contain viral RNA. These antibodies do not react with denatured capsid proteins, nor are significant levels of antibodies to the individual capsid proteins present in most human convalescent sera. This is the reason why attempts to develop recombinant hepatitis A vaccines have generally failed. Because the empty and complete HAV particles appear to have very similar if not identical antigenicities, the presence of antibody detected in the Havab and similar immunoassays correlates very closely with the presence of viral neutralizing activity. Viral neutralization tests, which measure the ability of a serum sample to reduce the infectivity of laboratory strains of HAV in cell culture, are potentially more
1195
sensitive than even the modified Havab assay (14, 16). These neutralization tests are technically very difficult and expensive, but they provide a very useful means of comparing the antibody responses after active and passive immunization against hepatitis A. Fujiyama et al. found that the neutralizing antibody response after two doses of an inactivated hepatitis A vaccine (given at 0 and 1 mo) was greater than that after passive administration of ISG. A third vaccine dose (at 6 or 12 mo) enhanced this response considerably and probably results in a longer duration of antibody persistence. Such serological comparisons, which are similar to previous observations made with the Merck and SmithKline vaccines, strongly suggest that the current generation of formalin-inactivated HAV vaccines should be highly effective. Although such theoretical comparisons of antibody responses make good sense, they are unlikely to satisfy some regulatory agencies. Thus clinical trials have been designed to assess the efficacy of these vaccines, and preliminary reports of these efficacy trials have recently been presented at international meetings. The design of such studies has been complicated by the sporadic nature of hepatitis A in well-developed countries and difficulties in identifying high-risk populations suitable for study. However, in an efficacy study completed recently in Monroe, New York, by Werzberger and associates (Personal communication), the Merck vaccine was shown to induce nearly complete protection against symptomatic hepatitis A within several weeks of the administration of a single dose. This clinical trial, which was carried out in children, demonstrated conclusively that only very low levels of serum-neutralizing antibody are required for high levels of clinical protection. In addition, this study demonstrated the feasibility of protecting travelers against hepatitis A with the administration of a single dose of vaccine. A large-scale study of the SmithKline inactivated HAV vaccine, involving more than 40,000 school-age children, is currently being conducted by Innis and coworkers in Thailand. A recent interim look at this study (Personal communication) has indicated a comparable level of efficacy several months after the administration of two vaccine doses. What level of protection either of these vaccines might afford against inapparent infection remains uncertain, but it is likely to be determined by the Thai study. It is too early to say whether any clinically significant differences exist between these two commercial vaccines. Although both of these efficacy studies have been carried out in children, no reason exists to suspect that the efficacy will be substantially different in adults, provided that equivalent levels of antibody are demonstrated after immunization. However, the duration of protection after immunization remains uncertain. This can be determined only by continued observations in the field, but it is likely that the levels of antibody developing after a complete series of three immunizations will lead to protection for at least 5 to 10 yr. These vaccines are also likely t o protect against all strains of HAV.
1196
LEMON
Although HAV strains can be separated into several epidemiologically and phylogenetically distinct genotypes, each differing from each other at up to 25% of nucleotide base positions within certain regions of the RNA genome of the virus (171, all human HAV strains studied thus far are very closely related, if not identical, antigenically (15). Thus immunization with a single vaccine strain should protect against hepatitis A in all regions of the world. What about safety? Because the current generation of inactivated hepatitis A vaccines are produced from virus strains that have been adapted to growth in cell culture, the seed viruses are substantially or completely attenuated even before inactivation (12). Nonetheless, the formalin-inactivation kinetics have been carefully determined for HAV, and more than 35 yr of experience in the manufacture of formalin-inactivated polio vaccines has provided the commercial know-how to safely manufacture inactivated hepatitis A vaccines. (Unlike the seed viruses used for hepatitis A vaccines, those used for the production of inactivated polio vaccine are usually fully virulent.) The side effects associated with the administration of inactivated hepatitis A vaccines in clinical trials have been minimal and generally similar to those associated with use of the hepatitis B vaccine. Finally, how can these new vaccines best be used t o reduce the overall hepatitis A disease burden? Although decreasing circulation of HAV during the last several decades has led to a high level of susceptibility among young adults within the United States, molecular evidence strongly suggests the continued circulation of an endemic HAV genotype (17). Several groups appear to be at particular risk of HAV infection. Child-care centers caring for infants and young children who are not yet toilet trained remain prime sites for HAV transmission, from which there occurs radial spread of HAV into the community. Hepatitis A remains a problem among institutionalized persons, and it has been increasingly associated with the illicit use of drugs within the United States as it has in Europe (18). In addition, during 1991 a considerable increase in hepatitis A infections was noted among urban, homosexual men in the United States, Canada and Australia (19). Continuing sporadic outbreaks of hepatitis A remain a major nuisance for public health officials in the United States. Such outbreaks often have been associated with infected food handlers working in the final stages of food preparation and distribution, but more recently the outbreaks have been associated with contamination of foodstuffs (e.g., lettuce and frozen strawberries) at their source. Travelers to poorly developed overseas destinations where hepatitis A is particularly endemic represent a prime target for immunization. Such travelers number in the millions when one considers both North America and Western Europe. Although ISG affords a high level of protection for travelers, concerns exist regarding its continuing use. First of all, the continuing decline in the prevalence of hepatitis A in many well-developed countries may be leading to gradual reductions in the titer of HAV antibodies present in the plasma pools from which
HEPATOLOGY
ISG is made. In accordance with this notion, Fujiyama et al. found that the levels of HAV antibody in ISG lots manufactured in Japan were significantly lower when plasma came from domestic (Japanese) versus foreign sources. In addition, supplies of ISG may occasionally be limited. The rapid mobilization of American military forces to an HAV endemic area, the Middle East, during Operation Desert Storm effectively depleted stocks of ISG available in the United States, leading the military to seek foreign supplies of this product. Thus one might expect formalin-inactivated HAV vaccine to be recommended first for adult travelers to overseas destinations where HAV is endemic, to illicit drug users and promiscuous homosexual men who are at increased risk for HAV infection, to clients of institutions for the developmentally disabled and t o prison inmates. In addition, it is not difficult to envision how immunization of seronegative food handlers could lead to a marked reduction in the frequency of foodborne outbreaks of HAV, along with their attendant costs of epidemiological investigation and implementation of control measures. Lastly, as information concerning the safety of these vaccines continues to accrue, it is likely that immunization of young children attending preschool day-care centers will ultimately become a policy objective. Control of HAV within such centers might result in significant reductions in the community-wide incidence of hepatitis A, based on previous observations of the use of ISG in day-care centers. In any discussion of hepatitis A vaccines, however, it cannot be forgotten that the greatest need for an effective and safe (and inexpensive) hepatitis A vaccine exists in developing countries undergoing the transition from a low to a higher level of public health sanitation. Transitional countries, such as China, have noted a substantial increase in the hepatitis A disease burden as the average age of infection shifts from the very young (who are often asymptomatic when infected with HAV) to the older child and young adult (who are usually symptomatic and frequently icteric). However, the use of inactivated HAV vaccines in developing countries raises several issues. First of all, the length of protection afforded by such vaccines must be measured in decades if they are to be truly effective in such countries. This might well be the case with inactivated hepatitis A vaccines given in a series of several doses, but the existence of clinically relevant immunological memory (with protection outlasting detectable serum antibodies) will need to be determined. Given the age-related nature of the severity of hepatitis A, it is important that a more susceptible older population is not created by immunizing the young (20). Second, vaccines should optimally prevent transmission of the virus to others. This is likely to be the case with inactivated hepatitis A vaccines, even if they do not produce effective levels of secretory immunity. Most if not all of the virus that is shed in the feces of infected persons is produced in the liver, and inactivated vaccines clearly limit the hepatic replication and hence fecal shedding of the virus. Finally, for a vaccine to be acceptable for use in
Vol. 15, No. 6, 1992
INACTIVATED HEPATITIS A VIRUS VACCINES
developing countries, it must be very inexpensive. The SmithKline Beecham-inactivated HAV vaccine, which was recently approved for clinical use in Switzerland and Belgium, costs approximately $25 to $30/dose, a price comparable t o recombinant hepatitis B vaccine or human diploid cell rabies vaccine. The relatively high cost of the HAV vaccine reflects in part difficulties in producing adequate quantities of this virus in cell culture, and the usual development and product liability costs inherent in commercial vaccine manufacture today. Thus although the introduction of formalininactivated HAV vaccines will represent a substantial step forward in the control of hepatitis virus infections in well-developed and affluent societies of North America, Western Europe and Asia, the control of hepatitis A (like the control of hepatitis B) is likely to remain a major problem in less-advantaged societies for some time to come.
STANLEY M. LEMON,M.D. Department of Medicine The University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599-7030 REFERENCES 1. Provost PJ, Hilleman MR. Propagation of human hepatitis A virus in cell culture in uitro. Proc SOCExp Biol Med 1979;160:213-221. 2. Provost PJ, Banker FS, Giesa PA, McAleer WJ, Buynak EB, Hilleman MR. Progress toward a live, attenuated human hepatitis A vaccine. Proc SOCExp Biol Med 1982;170:8-14. 3. Provost PJ, Bishop RP, Gerety RJ,Hilleman MR, McAleer WJ, Scolnick EM, Stevens CE. New findings in live, attenuated hepatitis A vaccine development. J Med Virol 1986;20:165175. 4. Midthun K, Ellerbeck E, Gershman K, Calandra G, Krah D, McCaughtry M, Nalin D, et al. Safety and immunogenicity of a live attenuated hepatitis A virus vaccine in seronegative volunteers. J Infect Dis 1991;163:735-739. 5. Mao JS, Dong DX, Zhang SY, Zhang H Y , Chen NL, Huang HY, Xie RY, et al. Further studies of attenuated live hepatitis Avaccine (H2 strain) in humans. In: Hollinger FB, Lemon SM, Margolis HS, eds.
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Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991:llO-111. 6. Provost PJ, Hilleman MR. An inactivated hepatitis Avirus vaccine prepared from infected marmoset liver. Proc SOCExp Biol Med 1978;159:201-203. 7. Binn LN, Bancroft WH, Lemon SM, Marchwicki RH, LeDuc SW, Trahan CJ, Staley EC, et al. Preparation of a prototype inactivated hepatitis A virus vaccine from infected cell cultures. J Infect Dis 1986;153:749-756. 8. Flehmig B, Haage A, Pfisterer M. Immunogenicity of a hepatitis A virus vaccine. J Med Virol 1987;22:7-16. 9. Sjogren MH, Eckels KH, Binn LN, Dubois DR, Hoke CH, Burke DS, Bancroft WH. Safety and immunogenicity of an inactivated hepatitis Avaccine. In: Zuckerman AJ, ed. Viral hepatitis and liver disease. New York: Alan R. Liss, Inc., 1988:94-96. 10. Provost PJ, Hughes JV,Miller WJ, Giesa PA, Banker FS, Emini EA. An inactivated hepatitis A viral vaccine of cell culture origin. J Med Virol 1986;19:23-31. 11. Andre FE, Hepburn A, D’Hondt E. Inactivated candidate vaccines for hepatitis A. Prog Med Virol 1990;37:72-95. 12. Lewis JA, Armstrong ME, Larson VM,Emini EA, Midthun K, Ellerbeck E, Nalin D, et al. Use of a live attenuated hepatitis A vaccine to prepare a highly purified, formalin-inactivated hepatitis A vaccine. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991~94-97. 13. Lemon SM, Ping L-H, Day S,Cox E, Jansen R, Amphlett E, Sangar D. Immunobiology of hepatitis A virus. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams & Wilkins, 1991:20-24. 14. Stapleton JT, Jansen RW, Lemon SM. Neutralizing antibody to hepatitis A virus in immune serum globulin and in the sera of human recipients of immune serum globulin. Gastroenterology 1985;89:637-642. 15. Ping L-H, Lemon SM. Antigenic structure of human hepatitis A virus defined by analysis of escape mutants selected against murine monoclonal antibodies. J Virol 1992;66. 16. Lemon SM, Binn LN. Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983;148:1033-1039. 17. Robertson BH, Jansen RW, Khanna B, Totsuka A, Nainan OV, Siegl G, Widell A, et al. Genetic relatedness of hepatitis A virus strains recovered from different geographic regions [in press]. J Gen Virol 1992. 18. Centers for Disease Control. Hepatitis A among drug abusers. MMWR 1988;37:297-305. 19. Centers for Disease Control. Hepatitis A among homosexual men: United States, Canada, and Australia. MMWR 1992;41:155-164. 20. Kane MA. Prospects for the introduction of hepatitis A vaccine into public health use. Prog Med Virol 1990;37:96-100.
