Virus Research, 17 (1990) 75-92
Elsevier VIRUS 00605
Recent advances in hepatitis A vaccine development Giinter Siegl 1 and Stanley M. Lemon * ’ Division of Virology, Institute for Medical Microbiology, University of Bern, Bern, Switzerland and ’ Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, North Carolina, U.S.A.
(Accepted 10 July 1990)
Clinical and epidemiologic background
of the liver
A virus (HAV). Its clinical course is characterized by a short phase of prodromal illness comprised of mostly general manifestations such as fatigue, malaise, fever, chills, and headache, followed by more specific symptoms of nausea, vomiting, and abdominal discomfort. In about two thirds of clinically overt cases, these symptoms are accompanied by the appearance of jaundice. The disease usually lasts 2 to 3 weeks but may be followed by a prolonged period of convalescence. Until recently, it was thought that only occasional patients experience a more protracted course with bimodal or even polyphasic elevation of liver enzymes. During the past few years, however, evidence has accumulated which suggests that a considerable number of affected individuals - in one report up to 20% of pediatric cases - may relapse and that relapses might be associated with reactivation of virus shedding (Chiriaco et al., 1986; SjGgren et al., 1987; Fagan et al., 1990). Nonetheless, persistent infections and chronic hepatitis A have not been documented. Severe, fulminant hepatitis A with a fatal outcome is rare. The overall mortality following infection is as low as O.l-0.5% (for references see Gust and Feinstone, 1988). HAV is shed in the feces early in the incubation period (which is usually 3-4 weeks in duration) and reaches a maximum just as symptoms begin to develop. Hence, the virus is efficiently transmitted under conditions of low hygienic standards. Contaminated drinking water and food, and shellfish harvested from estuaries or coastal regions polluted with human feces and unprocessed wastewater, serve as efficient vehicles for the virus. In addition, person to person spread among houseresulting
Correspondence to: G. Siegl, Institute for Clinical Microbiology and Immunology. Frohbergstrasse CH-9000 St. Gallen, Switzerland. 0168-1702/90/$03.50
0 1990 Elsevier Science Publishers
hold contacts, pre-school children in day-care centers, residents of institutions for the mentally retarded, male homosexuals and, with apparently increasing frequency, intravenous drug addicts contribute significantly to transmission of the disease (Mutton and Gust, 1984; Gust and Feinstone, 1988). Consistent with its mode of transmission, HAV is hyperendemic in undeveloped or developing countries where poor sanitary standards facilitate spread of the virus (Papaevangelou, 1984; Gust and Feinstone, 1988). In such an environment most children acquire infection in the first few years of life, when the infection is usually anicteric and frequently even asymptomatic. Thus, because infection leads to a long-lasting, protective immunity, clinically overt hepatitis A is usually not a prominent problem among the populations of such countries. However, as conditions of hygiene improve in such regions, the likelihood of encountering HAV infection is progressively delayed to adolescence and even to adulthood. At that age, infection results in clinical disease in the majority of persons. As a result, the improvement of hygienic standards in regions where HAV has been endemic may lead to unprecedented epidemics of hepatitis A. A prime example of such an outbreak was that which occurred in the Shanghai area in the People’s Republic of China, where over 300,000 cases of hepatitis A were recorded in the first quarter of 1988 as part of an epidemic linked to consumption of contaminated hairy clams (Yao, 1990). Hepatitis A occurring in the populations of highly developed countries of Western Europe is usually imported by individuals who have traveled to endemic areas, while the United States presents a somewhat intermediate picture between these two extremes. Given the above, the young, active and productive part of the adult population is usually that which suffers most from hepatitis A, independent of the level of sanitary standards. Despite its normally benign course, the marked morbidity of the disease in adults results in considerable economic losses which are estimated within the United States to exceed US$ 200,000,000 per year (S. Hadler, communication, The 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Texas, 1990). Hygienic measures alone will not suffice to control the occurrence of hepatitis A. Rather, well targeted, selective or even universal vaccination programs will be necessary to prevent epidemics in developing nations and sporadic disease in less endemic areas. The requirements for vaccination programs against hepatitis A in these two epidemiologic settings, however, are quite different. In endemic areas, universal vaccination programs will need to accompany continuing improvement of sanitary standards over a period of 10 to 20 years to remove the virus from the environment effectively. Hence, the vaccine has to be readily available at low cost, must be very safe and should be easily administered in a schedule consistent with current immunizations. In regions with a lower incidence of infection, on the other hand, specific high risk groups will form the initial targets for immunization. Business travelers and tourists going to endemic areas, military personnel and members of the Peace Corps also bound for those regions, pre-school children placed in day-care centers and possibly their adult contacts, residents of certain institutions, as well as male homosexuals and drug users constitute these groups. Under these more defined
and controllable conditions, relatively expensive vaccines and even elaborate multiple-shot immunization schemes may be acceptable.
of the virus
The potential usefulness of a hepatitis A vaccine was recognized as early as World War II when reportedly more than 20 million individuals (both military and civilian) may have experienced the disease (cf. Bancroft and Lemon, 1984). However, the causative virus was not identified in the feces of patients by Feinstone and coworkers until 1973. Subsequent characterization has revealed HAV to be a picornavirus (for references see Siegl, 1984, 1988; Gust and Feinstone, 1988). The small spherical, envelopeless particle has a diameter of approximately 28 nm, sediments at 156S, and bands around 1.33 g/ml in CsCl. Its genome is a linear, single-stranded RNA of messenger-sense polarity with a covalently-linked 5’ terminal protein and a 3’ terminal poly(A) tract. The HAV genome appears to follow the classical L434 org~izational and tr~slational strategy of picorna~ruses (Najarian et al., 1985; Cohen et al., 1987a). A total of 11 structural and nonstructural proteins are encoded by a single open reading frame resembling that described in detail for members of the four distinct picornavirus genera - enterovirus, rhinovirus, cardiovirus, aphthovirus. Three structural proteins, VP1 - VP3, ranging between 33 and 24 kDa, have been positively identified. A fourth capsid protein, VP4, only about 2.5 kDa in size, has been predicted from the nucleic acid sequence of the virus, and from similarities between the genomic organization of HAV and other well known picomaviruses, but has never been identified in virions. Although the post-translational processing and eventual fate of the far amino terminus of the expressed polyprotein remains poorly defined, a consensus myristylation signal at the seventh amino acid residue after the first methionine within the short, putative VP4 region suggests the possibility of an abbreviated leader (L) peptide (Palmenberg, 1989, 1990). HAV differs from all known picomaviruses in several respects and, thus, should be considered the prototype virus of a new, fifth genus (Ticehurst, 1986). The characteristics supporting this statement include its strong tropism for the liver, a low G + C content of only 38% a truncated VP4, with possibly a short leader peptide, as well as a very low overall nucleotide sequence relatedness to viruses in each of the other genera. Likewise, amino acid sequence relatedness is extraordinarily limited. It amounts at best to 28% among relatively conserved non-structural picornavirus proteins like 3C protease and 3D RNA-polymerase. In addition, HAV exceeds poliovirus in temperature stability by a full 2O”C, surviving heating to 60” C for a prolonged period of time (Siegl et al., 1984b). This enhanced thermal stability suggests significant differences in structural features of the HAV capsid that have yet to be identified. Finally, HAV replication in cell culture is quite unlike that of most other well characterized picornaviruses (cf. Siegl, 1988). Even under optimal conditions, its replication cycle is protracted and extends over 24 to 48 h. Replication-associated cytopathogenicity is a rare event seen only with carefully
selected virus variants, in only a few cell systems, and under closely defined culture conditions (Venuti et al., 1985; Anderson, 1987; Cromeans et al., 1987; Nasser and Metcalf, 1987). There is no shut down of host cell macromolecular synthesis. In general, persistent infection is readily established and progeny virus remains largely cell-associated (Siegl et al. 1984a). In some cases it may require 2 to 3 weeks of culture to obtain virus yields which even then are 100 to 10,000 fold lower than, for example, poliovirus yields under typical cell culture conditions. The replication characteristics of HAV present considerable obstacles to production and large scale use of hepatitis A vaccines. The outstanding stability of the virus at the structural and antigenic levels (for reference see Siegl, 1988), however, clearly facilitates such attempts. A wealth of data collected during almost half a century of hepatitis A research support the view that variant viruses with altered pathogenicity or antigenicity do not occur (cf. Gust and Feinstone, 1988). Whenever epidemiologic or clinical observations have suggested a change in the fundamental biological properties of the virus, as during the waterborne epidemic of hepatitis in Delhi in 1956, subsequent analysis (Wong et al., 1980) has revealed the involvement of an unrelated, yet also enterically transmitted viral agent of non-A, non-B hepatitis (which is now increasingly being described as hepatitis E virus). Antigenic stability and homogeneity of HAV may be reasonably inferred from the observation that administration of pooled human serum immunoglobulin confers protection against hepatitis A all over the world, independent of the geographical origin of the immunoglobulin preparation used. The conclusion that there is only one stable serotype of HAV, has been put to direct test by comparing the genetic and antigenic properties of HAV strains collected from various geographical and epidemiologic sources. Analysis of the genetic relatedness of such wild type viruses and cell culture-adapted strains by oligonucleotide mapping (Weitz and Siegl, 1985), nucleic acid hybridization, (Lemon et al., 1987), and nucleic acid sequencing (Cohen et al., 1987) indicated that nucleotide sequence diversity among strains amounts in general to not more than l-4% and, only occasionally, exceeds 10%. Using polymerase chain reaction (PCR) amplification of reverse transcribed cDNA and subsequent sequencing of short selected nucleic acid sequences within the region of the viral genome coding for proteins VP1/2A and VP3, Jansen and coworkers (1990) confirmed this remarkable genetic conservation for more than 30 HAV isolates. Similar results were reported by Robertson et al. (1990). However, several human HAV strains differing from the majority of human isolates at 16-24% of nucleotide base positions have been identified (Jansen et al., 1990). To the extent studied, none of these HAV isolates could be distinguished from the bulk of human HAV strains by cross-neutralization tests or radioimmunoassay with polyclonal antisera. In radioimmunoassay tests carried out with a panel of 18 monoclonal antibodies, only subtle antigenic differences between a genetically divergent virus strain recovered from a naturally infected owl monkey and a HAV strain representative of the great majority of human isolates could be detected (Brown et al., 1989). Cross-challenge experiments with these two viruses in owl monkeys revealed a high level of protection following infection with either strain.
More recently, however, antigenically variant viruses have been reportedly isolated from several non-human primate species. Such viruses have not been recovered from humans as yet (Nainan et al., 1990). It may be concluded that human HAV strains of diverse epidemiological and geographical origin share highly conserved antigenic and immunogenic determinants.
Immunity to hepatitis A virus Immunity to HAV is complex and only partly characterized. Given the prevalence of hepatitis A in many populations, the absence of well documented examples of viral persistence attests to the efficacy of host immune responses in clearing HAV infection. Remarkably, although many persons with human immunodeficiency virus infections are in high risk groups for hepatitis A, HAV has not emerged as a threat in this group of globally compromised individuals. The mechanisms by which HAV is eliminated from the liver remain poorly defined, but most likely include the induction of nonspecific killer cells (Kurane et al., 1985) as well as human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (Vallbracht et al., 1989). The molecular target(s) for such activated cells are unknown, but presumed to be virally encoded proteins presented on the surface of the infected cell. These cell-mediated responses to intrahepatic HAV infection may be responsible for much of the liver damage accompanying acute hepatitis A, as extensive viral replication precedes the onset of hepatocellular injury and replication of most HAV strains is noncytopathic in cell culture. Mononuclear cell infiltrates also figure prominently in the histopathology of acute type A hepatitis. Although the role played by interferons in the clearance of HAV from the liver is not clearly delineated, HAV replication appears to be quite sensitive to interferon-p (II?+) and IFN,. However, IFNp is not produced by infected fibroblasts and the evidence for IFN, production is conflicting. IFN,, on the other hand, is known to be produced by T-lymphocytes in response to acute hepatitis A and may play a major role in the pathogenesis of the disease (Maier et al., 1988). In addition to a direct antiviral effect, IFN, may promote the action of cytotoxic lymphocytes by inducing enhanced display of HLA class I antigens on the hepatocyte surface. Solid protection against symptomatic reinfection is provided by serum neutralizing antibodies to the virus. Such antibodies generally appear just prior to the onset of hepatocellular injury, and persist in high titers for many years following acute hepatitis A (Lemon and Binn, 1983). Both 19s (IgM) and 7S (IgG) immunoglobulin isotypes have been shown to neutralize HAV. Only low levels of serum neutralizing antibodies are required for a high degree of protection, as administration of as little as 2 ml of pooled human immune serum globulin provides protection from clinical hepatitis A for 3 months or longer. Such passively transferred antibody results in low levels of neutralizing antibody (mean titer about 1: 40 in a 50% radioimmunofocus reduction assay) (Stapleton et al., 1985) and is not detectable by commercial competitive inhibition immunoassays for anti-HAV. Accordingly,
candidate vaccines which do result in seroconversion in such assays may be considered likely to induce clinically significant protective immunity. Unlike poliomyelitis, where secretory immunity is thought to play a major if not dominant role in protection against infection, mucosal IgA antibody appears to be less important in immunity against hepatitis A. Not only is effective immunity achieved solely with passively transferred IgG, appreciable levels of HAV neutralizing activity are not found in either saliva or fecal suspensions collected following hepatitis A (Stapleton et al., 1990). The absence of an apparent secretory neutralizing antibody response to HAV infection may reflect an absence of substantial replication of HAV within the gut. In summary, the conventional view is that an effective hepatitis A vaccine need only induce significant and persisting serum levels of neutralizing anti-HAV antibody. It is fair to say, however, that the possible role of T-cell immunity in protection against disease due to HAV (indeed, almost any picornavirus) remains largely unexplored. Were it possible to prime an HAV-specific cytotoxic T-cell response, infection of the liver might be severely limited following subsequent exposure to HAV. At least in theory, such an aborted infection might elicit substantial and lasting humoral immunity to the virus, in the absence of clinically significant disease. The feasibility of this approach, while hampered by problems of identification and presentation of HLA class I-restricted T-cell epitopes, is suggested by recent experiments with lymphocytic choriomeningitis virus (Klavinskis et al., 1989). Although cytotoxic T lymphocytes have yet to be shown to have comparable importance in the hepatitis A system, it might be a mistake to ignore the T-cell response in evaluating various strategies for vaccine development.
The classical approach to a hepatitis A vaccine
One of the most important conclusions to be drawn from the properties of human HAV presented above is that a single strain of HAV used for development of an HAV vaccine should be expected to confer protection against infection in all parts of the world. The genetic and antigenic stability of the virus also suggest that such a vaccine would be useful over an extended period of time without need for additional adjustments against newly appearing variant hepatitis A viruses. Given the similarities between HAV and other picornaviruses, it is reasonable to consider technical approaches which have led to the successful development of attenuated “live” and inactivated “killed” poliovirus vaccines as the most promising practical approaches to development of a hepatitis A vaccine. This conclusion was drawn by Provost and Hilleman (1978) soon after the identification of HAV in clinical specimens, and a great number of attempts in this direction have since been made in various laboratories. Depending on the type of vaccine that is being developed, quite different technical problems have been encountered. All attempts, however, have suffered from the difficulties inherent in cultivation of HAV in the laboratory.
Attenuated u~ccjnes Attenuation of HAV has been shown to result from extended cell culture passage of virus and has been proposed as an approach to the development of attenuated vaccines (Provost et al., 1982; Provost et al., 1986b). Wild-type HAV, recovered from feces or liver of infected primates, replicates very slowly and usually only to low titers in cell cultures. With successive passage, however, the virus becomes progressively adapted to growth in cell culture, resulting in a shortening of the interval between inoculation of cultures and maximum virus yield, as well as increases in the final yield of virus. This apparently multi-step process also results in attenuation as evidenced by reductions in virus shedding and virus-induced liver injury in experimentally challenged chimpanzees, marmosets and, in limited experiments, man (Provost et al., 1982, 1986b; Karron et al., 1988). Extensively passaged virus appears even incapable of replication in vivo, as no clinical evidence of infection and no measurable antibody response has followed parenteral challenge of humans with 106.3 tissue culture infectious units of virus at the 31st cell culture passage level (Provost et al., 1986b). Susceptibility to cell culture passaged virus may vary among different primate species (chimpanzees, marmosets, and owl monkeys), however, making the outcome of administration to humans somewhat difficult to predict. Because some virus variants that are well adapted to growth in cell culture retain a nearly unaltered ability to induce disease in nonhuman primates (Lemon et al., 1990), attenuation and cell culture adaptation represent distinct although closely related phenotypic characteristics of HAV. The molecular genetics of either attribute remain poorly defined. The genomes of two independently isolated and cell culture-adapted variants of HM175 strain HAV have been completely sequenced. One of these variants (cell culture passage level 35, p35 HM175) is highly attenuated and contains within its sequence a total of 17 mutations from wild-type virus (excluding silent mutations within the open reading frame) (Cohen et al., 1987b). The other variant (~16 HM175) is considered to be virulent in primates and has only 14 mutations (Fig. 1) (Jansen et al., 1988). These two independent HM175 isolates share mutations at 7 common sites, located throughout the genome. However, the distribution of mutations suggests that alterations in viral proteins, and possibly, regions of the 5’ noncoding sequence, involved in RNA replication might play a central role in adaptation of virus to growth in cell culture, possibly by allowing more efficient utilization of host-specific cell factors participating in this process (Jansen et al., 1988; Ross et al., 1989). A greater understanding of the genetics of the cell culture replication competence and attenuation of the virus became possible with the successful construction of an infectious, geno~c-length cDNA clone derived from the attenuated ~3.5 HM175 virus (Cohen et al., 1987~). A similarly constructed genomic-length wild-type HM175 cDNA clone was not infectious as either DNA or RNA, probably reflecting both the intrinsically low replication competence of wild-type HAV in cell culture and low efficiency of transfection (Cohen et al., 1989). Replication competence can be conferred on the wild-type construct, however, by replacement of P2 sequence
82 G AUJ
PI6 ““u P35
Fig. 1. Mutations from wild-type virus present in cell culture adapted ~16 (virulent) and ~35 (attenuated) HAV genomes. Mutations common to both viruses are shown above, while those unique to each virus are shown below the respective genomes. Only mutations in non-coding regions (single letter nucleotide code) and nonsilent mutations in the open reading frame (single letter ammo acid code) are shown. Wild-type sequence is shown closer to genome, inside the ~16 or p35 sequence at each site. Noncoding regions (5’ and 3’) of the genome are depicted as a solid line, while the open reading frame is separated by the boxed regions (@ PI region, capsid proteins; D P2 and !?I P3 regions encoding nonstructural proteins).
with cDNA derived from p35 HM175 virus. This suggests that mutations in the PZregion of ~35 HM175 (specifi~~ly in proteins 23 and 2C) may be critical for cell culture adaptation. Nonetheless, additional studies which include analysis of p16/p35 HM175 chime& viruses (Day and Lemon, 1990) suggest that mutations in the 5’ noncoding region also significantly influence replication competence in cell culture. Each of the viable p35/wild type HM175 chimeras studied by Cohen et al. (1989) was found to have an attenuation phenotype. As all of these viruses contained p35 HM175 sequence within the P2 region, mutations in this region also may be important for attenuation as well as the ability to replicate effectively in cell culture. Further studies of this type are hindered, however, by the current nonavailability of an infectious clone representing virulent virus. The clinical success of candidate attenuated vaccines has been limited to date. Early trials of the Merck CR326 strain vaccine, during which virus at various cell culture passage levels was administered to human volunteers, suggested that immunogenicity (that is, “take rate” assessed by the production of antibody) and hepatovirulence were closely linked (Provost et al., 1982, 1986b). Thus, virus that had been passaged sufficiently to eliminate elevations in serum liver enzymes following parenteral administration was found to be poorly immunogenic and to induce relatively low levels of antibody. Moreover, antibody responses were significantly delayed following infection, raising substantial questions about the fate of the inoculated virus. It remains to be seen whether adjusting the vaccine dose, or selecting alternate cell culture passage levels, will improve ~mmunogenicity without comparably increasing reactogenicity. A number of fundamental questions remain unanswered with respect to the possible development of an attenuated HAV vaccine. First, how much (if any) elevation of liver enzymes may be safely tolerated in immunized individuals? Indeed, is it possible to obtain an antigenic mass in the liver (the only apparent
replication site for these attenuated viruses) sufficient for vigorous antibody induction, without also evoking a cytotoxic T cell response leading to clinically significant liver injury? Does long-term persistence of antibody following administration of such vaccine candidates suggest any possibility of virus persistence? What are the kinetics and magnitude of fecal virus shedding? Provost et al. (1983, 1986b) found that fecal shedding of virus in immunized chimpanzees occurred only at low levels, but the potential for transmission and reversion remains uncertain. Lastly, are other approaches to development of an attenuated vaccine possible? The Sabin poliovirus vaccine strains have been so successful because they are less neurotropic than most wild-type strains, but retain comparable ability to replicate in the gut (Sabin, 1985). In contrast, current attenuated HAV candidates appear to replicate very poorly in the liver, and do not replicate elsewhere. Are there extra-hepatic sites of HAV replication that could be exploited for development of new attenuated candidates with favorably altered cell tropism? Inactiuuted vaccines Successful development of an inactivated or “killed” HAV vaccine might help to circumvent several of the problems inherent to the use of a live attenuated vaccine. With an inactivated vaccine, the problem of hepatic injury due to viral replication in the vaccinee, potential reversion of the virus to wild-type virulence, and its excretion and transmission to unvaccinated contacts may be disregarded. Killed vaccines, however, predominantly stimulate the humoral, antibody side of the immune system and magnitude and speed of this response depend largely on the quantity and quality of the antigen injected. With HAV replication being slow and inefficient in most cell culture systems, the most prominent problem in development of an inactivated vaccine is to produce a sufficient quantity of hepatitis A antigen at reasonable costs. This situation is illustrated best when replication of poliovirus and of HAV is compared. Under optimal conditions, harvests of poliovirus can be prepared within one to several days and at titers in the range of lo9 to 1O’c TCID,,/ml. For HAV, on the contrary, presently available culture systems take at best one or, in the vast majority of systems, several weeks to replicate the virus to titers of about 10’ TCID=&nl. Higher titers can be obtained, but standardizing such conditions for large scale production of antigen appears to be difficult. Nevertheless, formalin inactivated HAV appears to be quite immunogenic and successful experimental inactivated HAV vaccines have been prepared by a number of laboratories and vaccine manufacturers both in rather crude and in more advanced, highly purified form. Development of inactivated HAV vaccines was pioneered by Provost and Hilleman (1978). These authors partly purified HAV from the liver of an infected marmoset, inactivated it with formalin and injected multiple doses of this preparation at two week intervals into marmosets. The animals developed a virus-specific antibody response which was shown to prevent hepatitis A following subsequent challenge with live, virulent HAV. With the advent of in vitro cultivation of the virus, viral antigen originating from various cell systems (cf. Gust and Feinstone,
1988) - LLC-MK2 cells (transformed African green monkey), BS-C-I (African green monkey kidney) and, preferably diploid human embryonic fibroblast cells (either secondary cultures or established lines like MRC-5) - have been used to formulate inactivated vaccines. Because HAV remains largely cell-associated in most culture systems, viral antigen is usually collected following lysis of cells with detergents and/or freezing and thawing. The generally low concentration of viral antigen in cell cultures and the relatively greater amount of cellular proteins in such harvests then requires a series of more or less sophisticated steps of concentration and purification (Provost et al., 1986a; Flehmig et al., 1987, 1989; Andre et al., 1990). For early experimental vaccines this merely involved differential centrifugation. For more advanced products now undergoing clinical investigation, purification schedules have also included precipitation, gradient centrifugation, filtration, and column chromatography on various substrates. Antigen obtained by these means is well characterized with respect to the particle type (generally containing empty as well as full particles) and, as shown by SDS-PAGE, is also quite pure. Inactivation of viable HAV has been attempted by treatment with formalin (1 : 2000 and 1: 4000 at temperatures of 35-37 o C) (Provost and Hilleman, 1978; Binn et al., 1986; Provost et al., 1986a; Flehmig et al., 1989; Andre et al., 1990) or with p propiolactone (0.05% at 4“ C) (Flehmig et al., 1987). Inactivation of HAV by formalin evidently proceeds at a rate similar to that observed for poliovirus (Salk and Salk, 1984) with up to a 106-fold reduction in infectivity being achieved within the first 24-48 h of treatment. As with other picomaviruses, however, low levels of residual infectious HAV, possibly in the form of aggregate-entrapped virus, could be demonstrated even after prolonged treatment. Filtration of antigen suspensions to remove such aggregates, and extension of inactivation for up to 12 to 20 days, have helped to overcome this problem without significantly reducing antigenic potency. The potential for residual infectivity remains somewhat of a worry, but can be offset by use of highly attenuated virus variants to produce inactivated vaccines (Calandra et al., 1990). Purified inactivated viral antigen has been used as vaccine either with or without adsorption onto aluminium hydroxide. Concentrations of protein per dose of vaccine have ranged between 1 and 7500 ng. Yet, because the purity of these products varies widely and antigenic mass has never been controlled against an international antigen reference reagent, the results of different animal immunization studies and of human vaccination trials are difficult to compare. Altogether, however, available results suggest that with the most advanced, highly purified and aluminum hydroxide-adsorbed vaccines, immunogenic antigen concentrations will most likely range between 100 and 300 ng/dose. Immunization schedules in mice, guniea pigs, goats, and monkeys have varied widely both in number and timing of doses, and routes of administration. In contrast, most vaccination studies in humans have relied on three doses given intramuscularly, at two to four week intervals or a schedule of 0, 1, and 6 months. Following a full schedule of three inoculations, seroconversion rates have been excellent in animals and man and have almost always approached 100%. Such
results were particularly evident when antibody titers were measured by neutralization tests or with radio- or e~yme-aged i~unoassays of increased sensitivity. Less sensitive, commercially available tests for anti-I-WV antibody, however, have generally failed to reveal an early and significant seroconversion. Titers of neutralizing antibodies varied greatly between 1: 80 to > 1: 6000 after successful vaccination; yet, in general they were in the range of 1: 320 to 1: 1280. This is noteworthy because, following injection of standard human immunoglobulin preparations, neutralizing antibody titers are as low as 1: 10-l : 40 (Stapleton et al., 1985). Yet, this amount of antibody has been proven to be effective in preventing clinical hepatitis A upon exposure to the virus. Vaccine-induced antibody has persisted without significant fall in titer over periods of up to 3 years. Booster injections 6 or 12 months after primary vaccination invariably resulted in a dramatic, rapid increase in circulating antibody even against the background of an originally low immune response. To date, no fewer than seven inactivated HAV vaccines have entered clinical trials. Published data on performance of most of these vaccines are scarce (Flehmig et al., 1989, 1990; And& et al., 1990; d’Hondt et al., 1990; Desmyter et al., 1990; Ellerbeck et al., 1990; Just et al., 1990; Sjiigren et al., 1990) and do not greatly exceed the information presented above. Extended clinical studies, however, are underway with several vaccines. Results of these studies, as made available thus far, are similar for each of these vaccines and underline the general usefulness and practicability of inactivated HAV vaccines. Specifically, data indicate that one dose of 100-300 ng of antigen with aluminium hydroxide as adjuvant produces seroconversion in at least 25 to 50% of recipients within a period of one month. Following a second dose given 4 weeks after the first, seroconversion occurs in 80 to 95% of vaccinees and after a final, third injection only occasional individuals fail to develop detectable neutralizing antibodies. In the absence of adjuvant, purified HAV antigen seems to have slightly reduced immunogenicity. As described above, vaccine-induced antibody titers remain detectable at a significant level over a period of at least three years and specific i~~ty to HAV can be easily stimulate by a single booster injection. This observation stresses the excellent i~unoge~city of form~n-inactivated HAV vaccines and suggests that, given the proper antigenic mass, a vaccine can be formulated for which two appropriately spaced (perhaps even only one) injections might regularly lead to a reasonably long-lasting and measurable antibody response against HAV. The true protective efficacy of this immunity in man, however, awaits evaluation as does the possibility that immunologic memory may provide truly long lasting protection. Lastly, despite the substantial immunogenicity of these prototype vaccines, it should be noted that production costs may prove to be quite high.
Molecular approaches to an HAV vaccine
The obstacles hindering development of safe and economically feasible HAV vaccines by conventional methods have prompted considerable interest in biotech-
nology oriented approaches to HAV vaccine development. The lack of significant antigenic variation and the relatively low levels of antibody required for adequate protection make HAV an attractive target for such efforts. However, current evidence suggests that the neutralization epitopes of HAV, like those of most other picornaviruses, are highly conformational and defined by highly ordered structural features of the virus capsid (Lemon and Ping, 1988). Polyclonal antisera with high neutralization titers generally do not react in Western blots of purified denaturated HAV. The conformational nature of the critical B cell epitopes has thus far defied attempts at meaningful expression of a subunit antigen. Some progress has been made, however, in defining the neutralization epitopes of HAV. Mutant viruses capable of escape from neutralizing murine monoclonal antibodies have been selected under antibody pressure (Stapleton and Lemon, 1987). Antibody resistance has been shown to result from reduced affinity of the antibody for the mutant virion. Sequencing of virion RNA has identified replacements of aspartic acid residue 70 of capsid protein VP3 or serine residue 102 of VP1 (Ping et al., 1988). The VP3 residue 70 mutants demonstrate high level, broad resistance to a large number of murine anti-HAV monoclonal antibodies, whereas the VP1 residue 102 mutant demonstrates more restricted resistance. Thus, these residues contribute to important neutralization epitopes of HAV. More recently, applying site-directed mutagenesis to infectious cDNA, Cox et al. (1990) have created synthetic escape mutants by replacement of VP3 residue 70 with serine, or the serine residue 114 of VP1 with glutamine. Computer-assisted alignments suggest that HAV residues 70 of VP3 and 102-114 of VP1 represent regions analogous to the j3B-pC loops of VP3 and VP1 of poliovirus and other picomavi~ses with known atone-resolution structures (Ping et al., 1988). These two loop structures are known to contribute to functionally separate antigenic sites in poliovirus types 1 and 3, and rhinovirus type 14. Thus HAV appears to share general structural features with these other, very distantly related picomaviruses. In the case of HAV, however, mutations at either VP3 residue 70 or VP1 residue 102 induce resistance to certain monoclonal antibodies, suggesting that these capsid protein loops are functionally related and contribute to the same antigenic site (Ping et al., 1988). This in turn suggests that these residues are more closely positioned within the HAV structure. An alternative but less likely possibility is that single amino acid substitutions in one loop region are capable of inducing changes in the affinity of antibodies normally binding at a second site that is quite distant on the virion surface. Only crystallographic studies will distinguish between these two possibilities. Synthetic peptides representing the PB-fiC loops of VP3 and VP1 of HAV show neither HAV antigenicity nor immunogenicity after coupling to carrier proteins (Lemon et al., 1989), consistent with other evidence that the antigenic sites of the native virus are entirely conformationally defined. Similarly, attempts to express antigen from recombinant cDNA in a variety of eucaryotic and procaryotic cells have generally not resulted in production of sig~ficant antigens (Ostermayr et al., 1987; Johnston et al., 1988). Priming of neutralizing antibody responses has been noted in animals immunized with recombinant VP3 and VP1 expressed in E. cob
(Johnston et al., 1988), but this most likely reflects HLA class II-restricted T helper cell induction rather than specific stimulation of antibody secreting B cells. Such responses are unlikely to be of much clinical significance. Attention has thus been focused on the possibility of expression of capsid proteins capable of assembling into immunogenic particles. This has been achieved in the poliovirus and foot and mouth virus systems (Clarke and Sangar, 1988). Such efforts with HAV are handicapped, however, by incomplete understanding of the cleavage sites at which the Pl polyprotein undergoes processing, as well as the proteases mediating such cleavages. Nonetheless, the insertion of the complete open reading frame of HAV into vaccinia virus under control of a late vaccinia promoter (with removal of the HAV 5’ noncoding region, and with translation beginning at the natural initiator AUG codon) has been reported to result in production of low levels of HAV antigen in vaccinia-infected cell cultures and induction of anti-HAV in vaccinated rabbits (Feng et al., 1989). While this result requires confirmation, it suggests that biotechnological approaches to HAV vaccine development may eventually pay off. An alternate approach to overcome the limitations posed by the conformational nature of the HAV antigenic site is the use of anti-idiotypic antibodies as immunogens. While this is an attractive strategy in theory, no data have yet been advanced to show that the approach is practically feasible with HAV. Finally, Emini et al. (1985) reported that a synthetic peptide representing residues 13 to 24 of HAV VP1 (coupled to a carrier protein) induced anti-HAV neutralizing antibodies in small animals. More recently, this same HAV peptide sequence has been inserted into the pB-/3C loop of VP1 of the Sabin type 1 poliovirus vaccine strain, and the resulting chimeric virus has been shown capable of eliciting very low levels of neutralizing (but not immunoprecipitating) antibodies to HAV in some but not all immunized animals (Lemon et al., in preparation). The analogous amino terminal domain of VP1 lies internally within the poliovirus capsid structure, in close proximity to the viral RNA, but is brought to the surface of the poliovirus capsid as part of a major conformational rearrangement following attachment of the virus to cellular receptors (Frick and Hogle, 1990). The weak anti-HAV neutralizing activities of antibodies raised against the comparable HAV VP1 amino terminal domain suggest that HAV might undergo a similar change in capsid conformation, but appear at present to be of little practical importance with respect to vaccine development.
Future directions The advances summarized above document a continued progress toward development of practical HAV vaccines. Most technical problems associated with the production of conventional, inactivated whole virus vaccines appear to have been resolved. Although there may be lingering doubts about the completeness of inactivation in large, production size batches, there is yet no clinical evidence to suggest that this will be a problem. Standardized methods must be developed to
measure antigen content, an important regulatory issue, and to monitor antibody responses. The major problem here is likely to be cost, a variable which is difficult for all but the manufacturers to predict. Also, duration of protective immunity, and whether there will be a need for periodic boosters, remain important unknowns. Acceptance of any HAV vaccine, no matter how low its cost, may be poor if it does not elicit longstanding immunity. Particularly in developing regions, an HAV vaccine will not be welcomed if it provides protection for only 5 to 10 years, and thus leaves a large percentage of immunized children susceptible to hepatitis A later in life. To succeed in the very places it is most needed, an HAV vaccine must provide longterm protection and should also be suitable for inclusion in the Expanded Programme of Immunization of the World Health Organization. Given these constraints, inactivated vaccines may remain limited to “boutique” status, used only among high risk populations in economically advantaged nations (including possibly children entering preschool day care). It is unlikely, given current progress, that recombinant DNA technology will be able to overcome these limitations in the near term, although the ultimate power of molecular approaches to expression of a useful immunogen should not be underestimated. Despite the fact that progress with live attenuated HAV vaccines has been relatively slow and beset with problems of poor immunogenicity or residual hepatovirulence, such vaccines may offer the greatest present hope for development of a product capable of inducing lifelong immunity following administration of a single dose. As suggested above, however, it may be necessary to attempt different approaches to attenuation of the virus, including perhaps the purposeful manipulation of the virus genome. In addition, valid markers of attenuation must be defined and genetic stability of the attenuation phenotype must be demonstrated. For such efforts to succeed, much more needs be learned of the basic pathobiology of hepatitis A, as well as the molecular genetics of HAV attenuation and virulence.
Acknowledgement Work on this subject in the laboratories of both authors part by Technical Services Agreements with the Programme ment of the World Health Organization.
has been supported in for Vaccine Develop-
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