Erik D'Hondt

Clinical assessment of the safety and efficacy of an inactivated hepatitis A vaccine: rationale and summary of findings

The objectives for the clinical testing of the inactivated hepatitis A vaccine developed by SmithKline Beecham Biologicals are reviewed and the results obtained are summarized. The first studies were carried out in healthy young adult volunteers using pilot vaccine lots prepared from the CLF and HM175 strains of hepatitis A virus (HAV). It was established that the candidate vaccines were well-tolerated, caused no hypersensitivity reactions and elicited a strong antibody response. As the yield in production with the HM175 strain was higher it was preferred over the CLF strain for further development of a vaccine. A dose-range study with HM175 vaccine in adults showed that an antigen dose of 720 ELISA units (El.U) produced almost 100% seroconversion after one injection. This dose was therefore chosen as appropriate for adults. A dose of 360 El.U was used in children. To date, a total of 67 studies in 18 countries involving 47,145 subjects, including 20,586 control subjects, have been initiated. In these studies, 55,259 doses of HM175-derived hepatitis A vaccine have so far been administered. This extensive experience has shown that the vaccine is well-tolerated, causing essentially only mild, transient local reactions. Vaccine reactogenicity was assessed using symptom checklists, filled in by the volunteers or their parents, for a period of 4 days following vaccination. Blood samples were obtained at different times after vaccination to evaluate the immune response. Clinical studies with six different lots, manufactured using commercial-scale production methods, have been carried out on 2901 adults. These studies have shown that a seroconversion rate of 95.7% (1225/1280) is obtained one month after the first vaccine dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivated hepatitis A vaccine: active and passive immunoprophylaxis in chimpanzees

Studies of active and passive immunoprophylaxis were carried out in chimpanzees to determine whether a candidate hepatitis A virus (HAV) vaccine could stimulate antibody to HAV (anti-HAV) that was qualitatively similar to anti-HAV stimulated by natural infection. Normal immune globulin (Ig) was prepared from plasma obtained from human volunteers before and after vaccination with the HAV vaccine, and these preparations or commercially prepared Ig were administered to chimpanzees. Protective efficacy was compared to that obtained after vaccination of chimpanzees. As expected, pre-vaccination Ig did not protect chimpanzees against challenge with virulent hepatitis A. In contrast, chimpanzees were protected against hepatitis A by Ig prepared from volunteers who had received hepatitis A vaccine. The protection was qualitatively similar to that afforded by commercial normal Ig containing convalescent anti-HAV. The minimum protective dose of passively acquired anti-HAV was approximately the minimum dose detectable by serological means. This information will be useful in calculating minimum acceptable titres of anti-HAV in normal Ig. Whereas administration of Ig protected chimpanzees against hepatitis A pathology, it did not protect them from infection with HAV. Thus, these chimpanzees were protected by classical passive-active immunoprophylaxis. In contrast, chimpanzees actively immunized with HAV vaccine were apparently protected against both hepatitis A pathology and HAV infection. The mechanism of this complete protection is unknown but may simply represent the higher titre of anti-HAV in the vaccinated chimpanzees, compared to the passively protected animals.

Inactivated hepatitis A vaccine: long-term antibody persistence

During the clinical development of safe, well tolerated and immunogenic vaccines against hepatitis A the persistence of protective antibodies was estimated, based on relatively short observation periods of 18 months to 3 years. We report here on longterm persistence of antibodies in volunteers who participated in one of the early clinical trials on inactivated hepatitis A candidate vaccines. In a randomized trial three groups of altogether 110 healthy adults, initially hepatitis A virus (HAV) seronegative persons were vaccinated with an inactivated hepatitis A vaccine according to the schedule 0-1-2-12 months. One group received 180 ELISA units, one group 360, and one 720 ELISA units per dose. Blood samples were taken prior to the first vaccination and at months 1, 2, 3, 4, 6, 12, 13, 18, 24, 36 and 84. The decrease of antibodies was characterized by two disappearance rates: a rapidly decreasing component and a slower decreasing one becoming predominant ca 12 months after booster vaccination. The disappearance of antibodies could be described by a two-component model which holds for t > or = 13 months. The estimated disappearance rates for the slow component (annual decrease) was found to be 11 and 13% for the 180 and 360 El. U groups, respectively (the 720 El. U group showed no decline, which was probably due to the small sample size). The estimated persistence of antibodies within protective range varied between 24 and 47 years depending on individual titres reached at month 13 and vaccination dose.

Persistence of vaccine-induced antibody to hepatitis A virus

A level of 10 mIU hepatitis A antibodies/ml as measured by ELISA is believed to be the minimal protective concentration. If this level is considered, the mean persistence of vaccine induced antibodies is approximately 10-11 years after booster dose, 6-7 years if only the primary doses are given and 5-6 years if the minimal individual titre is taken into account.

Hepatitis A in the US Army: epidemiology and vaccine development

Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers.

Clinical and laboratory observations following oral or intramuscular administration of a live attenuated hepatitis A vaccine candidate.

Clinical observations made after immunising volunteers with a live attenuated hepatitis A vaccine are described. The candidate vaccine was prepared with the HM175 strain of hepatitis A virus and shown to be safe, immunogenic and efficacious in experimental animals. When the candidate vaccine was tested by oral administration in humans at increasing doses--10(4), 10(5), 10(6) and 10(7) median tissue culture infective doses (TCID50)--an antibody response was not observed at any dose. Volunteers who received similar doses by the intramuscular route developed antibody to hepatitis A three weeks after immunization with 10(6) or 10(7) TCID50. The antibody response was sustained for the 12 weeks of the observation period. All volunteers remained healthy with normal results from liver tests throughout the monitoring period. Further clinical observations of this product are in progress.

Effect of virus strain and antigen dose on immunogenicity and reactogenicity of an inactivated hepatitis A vaccine

A randomized double-blind comparison of five killed hepatitis A vaccine preparations was carried out with eligible medical student and staff volunteers. Vaccines were prepared in M RC-5 cells and formalin-inactivated. Three monthly injections of 1 ml in the deltoid muscle were given. Group A received the CLF strain at a dose of 360 ELISA units (El.U) in 0.5 mg aluminium hydroxide (n = 35). The other groups received the HM175 strain as follows: 180 El.U in 1 mg aluminium hydroxide (to group B, n = 42), 360 El.U in 0.5 mg aluminium hydroxide (to group C, n = 40), 360 El.U in 1 mg aluminium hydroxide (to group D, n = 39) and 720 El.U in 1 mg aluminium hydroxide (to group E, n = 43). The geometric mean anti-HAV concentration (GMC) measured in mIV/ml by an ELISA method one month after each injection were: group A, 223, 480, 1635; group B, 123, 221, 649; group C, 185, 365, 1085; group D, 144, 323, 1076; group E, 229, 646, 2521. At month 6, the GMC had fallen by approximately 20%. Seroconversion as measured by ELISA was 100% in groups A and E after one injection, and 100% in all groups after three injections; after two injections, only one subject in group C was still negative. The dose effect with HM175 vaccine was significant. There was a good correlation between ELISA and neutralization (radioimmunofocus inhibition test) titres. One month after the second dose, all subjects in groups A and E had both hepatitis A virus immunoglobulin M (HAV IgM) geometric mean titre, (GMT > 5000) and IgG (GMT > 25,000) as measured by a sensitive terminal dilution ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible approaches to develop vaccines against hepatitis A

More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates. Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates. Several live vaccine candidates obtained through serial passages have been evaluated in volunteers. Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates. These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles. The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins. Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits. On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge. There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus. Chimeric polio-HAV VP1 viruses have been constructed. These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes. More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported.

Laboratory tests and reference reagents employed in studies of inactivated hepatitis A vaccine

Procedures to evaluate inactivated hepatitis A vaccines in volunteers have been examined. Solid-phase immunoassays were standardized with reference preparations and have been tested to measure antibody response to immunization and antigen content of vaccines. Following immunization, there was a good correlation between antibody response, determined with commercial immunoassays, and neutralization titres, as measured by the radioimmunofocus inhibition test. However, at lower titres of neutralizing antibody, the commercial immunoassay often yielded negative results. To improve the sensitivity of the immunoassay, the serum volume was increased. A fourfold increase of test serum resulted in greater sensitivity, increasing from 54 to 94%, while retaining 100% specificity. Further increases in the volume of test serum resulted in a loss of specificity. In a comparison of neutralization tests, similar titres of postvaccination sera were obtained by using the HM175/18f cytopathic strain of hepatitis A virus in a plaque reduction assay or the HM175 parental virus in the radioimmunofocus inhibition test. Use of the cytopathic virus obviates the need for radioactively labelled serum and reduces the time taken to conduct neutralization tests. The current laboratory procedures can meet the needs of large field trials of inactivated hepatitis A vaccines.