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Early appearance of neutralizing antibodies after vaccination with an inactivated hepatitis A vaccine
Journalof infection (1997) 35, 37 40
Early Appearance of Neutralizing Antibodies after Vaccination with an Inactivated Hepatitis A Vaccine B. Flehmig 1, H. Staedele 1, C. Xueref 2, E. Vidor 2, J. Zuckerman 3 and A. Zuckerman 4 ~Hygiene-Institut der Universitdt T~ibingen, Abteilung ffdr Medizinische Virologie und Epidemiologie der Viruskrankheiten, Germany, 2pasteur Merieux Serums & Vaccins, Marcy l'Etoile, France, 3Academic Unit of Travel Medicine and Vaccines, Royal Free Hospital School of Medicine, London, U.K. and 4WHO Collaborating Centre for Reference and Research on Viral Diseases, Royal Free Hospital School of Medicine, London, U.K. Sera from 30 subjects vaccinated with the Pasteur Merieux Serums & Vaccins (PM) inactivated hepatitis A vaccine, and from 30 subjects vaccinated with the Smithkline Beecham (SB) inactivated hepatitis A vaccine, were tested in two laboratories in order to provide comparative data on neutralizing activities of vaccine-induced antibodies. Sera were also evaluated by a modified radioimmnnoassay (mRIA) and results were compared to neutralization assays results. Neutralizing antibody titres provided by the two laboratories correlated well (coefficient or correlation 0.42, P< 0.001). Neutralizing antibodies were detected after vaccination with both vaccines, and the kinetics of neutralizing antibody were the same with both vaccines. The titres gradually increased between the second week after the first dose and the post-booster dose (week 28). A strong booster effect of the booster vaccine dose on neutralizing titres was observed. Significantly higher neutralizing antibody titres with the PM vaccine were observed as early immune respose on week 2 titres on both series of results. Vaccine-induced neutralizing antibody titres and vaccine-induced antibody mRIA titres correlated well (coefficient of correlation 0.82 and 0.72, respectively, P
Introduction Neutralizing antibody against hepatitis A virus (HAV) is believed to be the most important element of an effective i m m u n e response against HAV 1 and has been evidenced in the sera of h u m a n recipients of i m m u n e plasma globulins. 2 The validation of the efficacy of pooled n o r m a l h u m a n i m m u n e globulin against hepatitis A disease 3 has proved the usefulness of the m e a s u r e m e n t of neutralizing antibody as a valuable tool for the evaluation of the potential efficiacy of inactivated hepatitis A vaccines. Assays have been developed to measure neutralizing antibody against HAV.4-c' The first is a quantitative radioi m m u n o f o c u s inhibition test (RIFIT) based on the i m m u n e autoradiographic detection of loci of viral replication u n d e r an agarose overlay, and the second is an antigenreduction neutralization assay (HAVARNA) based on the i m m u n o d e t e c t i o n of HAV antigen produced in cell culture. HAV strains used in these assays are different (HM 175, CR326F and GBM), and there is evidence of immunological cross-protection between all HAV strains.7 The presence of neutralizing antibodies after vaccination
Address correspondence to B. Flehmig. Accepted for publication 19 November 1996. 0163-4453/97/040037+04 $12.00/0
with inactivated hepatitis A vaccines has been demonstrated as early as 2 weeks after the first vaccine dose with an increase after additional inoculations. 8'9 The correlation between vaccine-induced neutralizing antibody titres and vaccine-induced antibody titres measured by conventional assays (enzyme-linked i m m u n o s o r b e n t assay (ELISA) and r a d i o i m m u n o assay (RIA)) has been validated several times, 1°'11 indicating that results provided by conventional assays can also be used to predict the efficacy of inactivated hepatitis A vaccines. We describe here the results of neutralizing antibodies using a n e w inactivated hepatitis A vaccine produced by Pasteur Merieux Serums & Vaccins (PM), w h i c h had been compared to a n o t h e r licensed inactivated hepatitis A vaccine produced by Smithkline Beecham (SB) by a controlled clinical trial.
Material and Methods Vaccines The PM vaccine (AVAXIMTM) was derived from a strain of hepatitis A virus (GBM) isolated in Germany and studied extensively. 12 After viral propagation and amplification, a filtered harvest obtained by cellular lysis © 1997 The British Society for the Study of Infection
B. Flehmig et aL
38
was purified by chromatography and concentrated by tangential ultrafiltration. Purified HAV was inactivated by formaldehyde at a dilution of 1:4000 for 14 days at 37 °C with constant agitation. The bulk vaccine antigen was filtered at 0.22 gin, adjusted for antigen content (160 ELISA units/dose) and adsorbed to 0.5 mg aluminium hydroxide with 2-phenoxyethanol added as preservative. The vaccine was formulated in 0.5 ml doses and was administered as one injection of one vaccine dose for primary immunization. A booster injection was given 6 months after the primary immunization. The preparation and characteristics of the control SB vaccine (HAVRIXTM) have been described elsewhere. 13 This vaccine was formulated in 1 ml doses and contained 720 ELISA units of HAV antigen derived from strain HM175 (as both manufacturers used different in-house antigen reference materials and different in-house ELISA reagents leading to different ELISA units, vaccine antigen potency comparison was not possible between both products). The vaccine was administered as two injections of one vaccine dose at 1-month intervals for primary immunization and a booster injection was given 6 months after primary immunization, because at the time this trial was conducted, this was the only vaccine licensed on the market and consequently available for such comparison. Both vaccines were given intramuscularly into the deltoid region.
Ser a A multicentre European comparative study between the PM vaccine and the SB vaccine as control was carried out in 840 healthy adults volunteers, and the principal results have been published elsewhere. 14 Serum samples (from the U.K. centre) collected before, at week 2 (postdose 1), at week 8 (post-dose 1 for the PM and post-dose 2 for the control vaccine), at week 24 (booster) and at week 28 (post-booster) from 30 subjects selected at random from each group were tested for neutralizing antibodies. Selection was made in order to have two comparable groups in terms of sex ratio (15 females and 15 males in each group) and m e a n age.
Neutralization assays Two assays were performed on blinded sera. The first one was a simple HAV antigen-reduction neutralization assay developed on the basis of routine techniques used for polio virus antibodies neutralizing assay and similar to an assay described previously. 5 Briefly, serial dilutions of
sera (1/5 to 1/10 240) were incubated with 102 Tissue Culture Infective Doseso (TICD~0) of the HAV GBM strain (4 h at 37 °C and 1 night at 5 °C). Samples were then incubated on h u m a n diploid cells (MRCs) in 96-well plates for 21 days at 37 °C in a 5% CO2 atmosphere. The HAV measurement was done by ELISA on cells lysed by freezing and thawing and with appropriate controls. The neutralizing antibody titre was defined as the reciprocal of the highest serum dilution yielding an HAV growth inhibition in 50% of the wells. The World Health Organization (WHO) reference immune globin was used as positive control and its neutralization titre was from 1/ 74 000 to 1/28 000. The results were expressed in milfi International Units (mIU) per ml by comparison with this reference, and, by convention, titres below the detection cut-off (between 10 and 20 according to the series) were considered equal to 10 mIU/ml for calculating the geometric m e a n titre (GMT). The second assay was also a simple HAV antigen-reduction neutralization assay developed in the laboratory of B. Flehmig. 6 Briefly, serial dilutions in minimal Eagle medium (MEM) with 7% fetal calf serum (FCS) of heat-inactivated sera (1/10 to 1/ 25 600) were incubated with 10 TCIDso of HAV GBM strain (4 h at 37 °C). The samples were then incubated on MRC5 cells in 96-wefi plates for 21 days at 37 °C in 5% CO2. HAV measurement was carried out by ELISA on lysed cells with appropriate controls. The neutralizing antibody titre was defined as the reciprocal of the highest serum dilution yielding 50% growth inhibition. Paul Ehrlich Institut reference immune globulin was used as positive control. The results were expressed in mIU/ml by comparison with this reference, and, by convention, titres below the detection cut-off (10) were considered equal to 5 mIU/ml for calculation of the GMT.
Modified radio-immunoassay (mRIA) Sera were titrated blindly by a modified radioimmunoassay (mRIA) (HAVAB, Abbott Laboratories, N. Chicago, IL, U.S.A.) in an independent laboratory (Bioanalytical Research Corporation, Ghent, Belgium). The conventional RIA, which allows a detection cut-off around 90 mlU/ml for specific antibodies when the results are expressed in comparison with a reference curve generated from the WHO reference HAV i m m u n e globulin, was modified to measure lower levels of anti-HAV. This modification of the procedure was used as described previously 15 and consisted of mixing 100 gl of test serum (instead of 10 I~1) with 100 pl of ~25I anti-HAV (instead of 200 ~tl). This modification made it possible to reduce the detection cut-off to 7 - 1 0 mlU/ml.
Hepatitis A Vaccine Neutralizing Antibodies
39
Table i. Seroneutralizing antibodies after vaccination with the PM or the SB inactivated hepatitis A vaccines (serological analysis performed by HAV antigen-reduction neutralization assay in two different laboratories). Week 0 dose 1
Lab, 1
Lab, 2
Week 2
Week 8
No.
PM 30
SB 30
PM 30
SB 30
PM 30
after dose 2 SB 30
GMT (mfU/ml) 95% CI range GMT (mIU/ml) 95% ci range
7.0 6-8 5-15 5.5 5-6 5-17
6.6 6-7 5-10 5.3 5-6 5-14
43.5* 23-82 5-1920 43.4* 32-59 11-387
13,2 9-20 5-640 12.4 10-16 5-51
84.3 46-155 10-2560 103.7 74-145 21-545
60.4 33-110 10 2 5 6 0 117.5 80-172 17-1175
Week 24 booster
Week 28
PM 30
SB 30
PM 30
SB 30
100.8 52 194 10-3840 146.1 109-197 28-1586
83.3 46-152 5-1920 157.3 98-252 5-765
3183 2064-4909 4 8 0 - 2 0 480 3256.7 2275-4662 369 2 5 2 5 0
2308 1459-3653 120 30 720 3268 2222-4805 289-20200
* Statistically significantly different from SB, P<0.01.
100
Results
.
10 000
The demography of subjects There were 60 subjects (30 females and 30 males) enrolled in this study. Thirty of the subjects were vaccinated with the PM vaccine (15 females and 15 males), and 30 with the control vaccine (15 females and 15 males). Their mean age was 24.7 years (range 18-40) for the PM group and 25.1 years (range 17-38) for the control group.
~ . ~,~* ~
~tt~% *" ~*~
looo
100
,
lO r [ [lilll]
, = ,,,iJil
i i iiiill
10 000
o
1000
.-.v,l!,:ai
1
.,it.e
i I r IIIIIll
Results obtained with sera for neutralizing antibodies are presented in Table I. In both series of results from the two laboratories the appearance of neutralizing antibodies was earlier with the PM vaccine compared to the control vaccine. Seroneutralizing antibodies titres were higher at week 2 with the PM vaccine compared to the control vaccine (P
i i ,,HH[
100 000
10
Neutralization antibodies
I i lliliil
10 100 1000 10 000 100 000 Seroneutralization titre (Lab. 2)
Correlation between neutralization assays When the results provided from both laboratories on the same sera were compared, a good correlation was observed (coefficient correlation R = 0.42, P
000
.....
I I IIIIIll
I I IIIIIll
I I IIINli
I
10 100 1000 10 000 S e r o n e u t r a l i z a t i o n t i t r e (Lab. 1)
II
100 000
Figure 1. Correlations between mRIA HAVAB titres (mIU/ml) and seroneutralization titres (mIU/ml) obtained with HAV antigen-reduction neutralisation assays from two different laboratories (Lab. 1 (PM) and Lab. 2 (B. Flehmig), in 300 sera from subjects vaccinated with the PM or SB inactivated hepatitis A vaccines (sera collected before and at weeks 2, 8, 24 and 28 after vaccination).
Discussion Correlation between neutralization assags and mRIA assays Each series of results were compared with the mRIA tires and results are plotted on Fig. 1. The correlation coefficients were 0.82 and 0.79 (P
Results obtained by both laboratories confirmed the appearance of neutralizing antibodies as soon as 2 weeks after the first dose of both inactivated hepatitis A vaccines. As the PM vaccine was given with only one dose for primary immunization, compared to the SB vaccine
40
B. Flehmig e t al.
which was given in two doses, the comparison of results should take into account this difference in the immunization schedule. Nevertheless, the levels of neutralizing antibodies were comparable in both groups except at week 2, where the PM vaccine induced higher levels. These results were also in accord with those obtained using mRIA on the same sera.J4 Hepatitis A is a worldwide infection and is a substantial health risk for a huge population. About 1.3 million patients suffer each year from acute disease. A significant n u m b e r of patients have a severe infection associated with up to 2% mortality in adults. In industrialized countries the most c o m m o n way to contract infection with HAV is through travel to developing countries and hyperendemic areas. The number of persons travelling from developed to developing countries is about 25 million per year, and most of these travellers are not protected against HAV because the prevalence of antibodies to HAV has been decreasing dramatically during the past 1 0 - 2 0 years. The most effective way of preventing infectious diseases is vaccination, and for hepatitis A this has been shown to be highly effective. 1~'17 Because HAV causes not only high morbidity but also significant mortality in adults, the WHO recommend immunization against hepatitis A. The vaccines based on cell culture and inactivated hepatitis A virus have been developed and shown to be immunogenic and safe. A matter of greatest importance is the induction of a rapid i m m u n e response after vaccination, because travellers often decide at a late state to take advantage of prevention. A vaccine inducing early neutralizing antibodies is therefore of great value. In this study we have demonstrated that a new cell culture based inactivated hepatitis A vaccine induces very early, significantly higher neutralizing antibodies compared to the first licensed formulation of the SB vaccine. These results, which correlated well with those obtained by mRIA, indicate the potential efficacy of this new vaccine.
Acknowledgements The authors are grateful to the investigators and volunteers who participated in the study, and to Nadege Picat for the testing of antibodies by seroneutralization assays.
References 1 Lemon SM. Hepatitis A virus: current concepts of the molecular virology, immunobiology, and approaches to vaccine development. Rev Med ViroZ 1992; 2: 73-87. 2 Stapleton JT, Jansen RW, Lemon SM, Neutralizing antibody to hepatitis A virus in i m m u n e serum globulin and in the sera of h u m a n recipients of i m m u n e serum globulin, Gastroenterology 1985; 89: 6 3 7 - 6 4 2 . 3 Lerman Y, Shohat T, Ashkenazi S, Almog R, Heering SL, Shemer J, Efficacy of different doses of i m m u n e serum globulin in the prevention of hepatitis A: a three-year prospective study. Cli Infect Dis 1993; 17: 4 1 1 - 4 1 4 . 4 Lemon SM, Bin LN, Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983; 148: 1 0 3 3 - 1 0 3 9 . 5 Krah DL, Amin RD, Nalin DR, Provost PJ. a simple antigen-reduction assay for the m e a s u r e m e n t of neutralizing antibodies to hepatitis A virus. J Infect Dis 1991; 163: 6 3 4 - 6 3 7 . 6 Flehmig B. Zahn J, VaIlbracht A. Levels of neutralizing and binding antibodies to Hepatitis A virus after onset of icterus: a comparison. f Infect Dis 1984; 150: 461. 7 Lemon SM, Jansen RW, Brown EA. Genetic, antigenic and biological differences between strains of hepatitis A virus. Vaccine 1992; 10: $40-$44. 8 Ellerbeck EF, Lewis JA, Nalin DR et al. Safety profile and immunogenicity of an inactivated vacine derived from an attenuated strain of hepatitis A. Vaccine 1992; 10: 6 6 8 - 6 7 2 . 9 Wiedermann G, Ambrosch F, Kollaritsch H e t al. Safety and immunogenicity of an inactivated hepatitis A candidate vaccine in healthy adult volunteers. Vaccine 1990; 8 : 5 8 1 584. 10 Delem AD, Safary A, D e n a m u r F, Hauser R Hondt d'E. Characterization of the i m m u n e response of volunteers vaccinated with a killed vaccine against hepatitis A. Vaccine 1993; 11: 4 7 9 ~ t 8 4 . 11 Binn LN, Macarthy PO, Marchwicki RH et al. Laboratory tests and reference reagents employed in studies of inactivated hepatitis A vaccine. Vaccine 1992; 10: $102-S105. 12 Heinricy U, Stierhof YD, Pfisterer M, Flehmig B. Properties of a hepatitis A virus candidate vaccine strain. ] Gen VJrol 1987; 68: 2487-2493. 13 Peetermans J. Production, quality control and characterization of an inactivated Hepatitis A vaccine. Vaccine 1992; 10: $ 9 9 - $ 1 0 1 . 14 Goilav C, Zuckerman JN, Lafrenz M et al. Immunogenicity and safety of a n e w inactivated hepatitis A vaccine in a comparative study. J Med Virol 1995; 46: 2 8 7 - 2 9 2 . 15 Miller WJ, Clark W. Hurni WM, Kuter B, Schofield T, Nalin DR. Sensitive assays for hepatitis A antibodies. ] Med Virol 1993; 41: 201-204. 16 Werzberger A, Mensch BM Kuter B e t al. A controlled trial of a formalin-inactivated hepatitis A vaccine in healthy children. N Engl ] Med 1992; 327: 4 5 3 - 4 5 7 . 17 Innis BL, Snitbhan R, Kunasol P.et al. Protection against hepatitis A by a n inactivated vaccine. JAJ~/IA 1994; 271: 1 3 2 8 - 1 3 3 4 .
Early Appearance of Neutralizing Antibodies after Vaccination with an Inactivated Hepatitis A Vaccine B. Flehmig 1, H. Staedele 1, C. Xueref 2, E. Vidor 2, J. Zuckerman 3 and A. Zuckerman 4 ~Hygiene-Institut der Universitdt T~ibingen, Abteilung ffdr Medizinische Virologie und Epidemiologie der Viruskrankheiten, Germany, 2pasteur Merieux Serums & Vaccins, Marcy l'Etoile, France, 3Academic Unit of Travel Medicine and Vaccines, Royal Free Hospital School of Medicine, London, U.K. and 4WHO Collaborating Centre for Reference and Research on Viral Diseases, Royal Free Hospital School of Medicine, London, U.K. Sera from 30 subjects vaccinated with the Pasteur Merieux Serums & Vaccins (PM) inactivated hepatitis A vaccine, and from 30 subjects vaccinated with the Smithkline Beecham (SB) inactivated hepatitis A vaccine, were tested in two laboratories in order to provide comparative data on neutralizing activities of vaccine-induced antibodies. Sera were also evaluated by a modified radioimmnnoassay (mRIA) and results were compared to neutralization assays results. Neutralizing antibody titres provided by the two laboratories correlated well (coefficient or correlation 0.42, P< 0.001). Neutralizing antibodies were detected after vaccination with both vaccines, and the kinetics of neutralizing antibody were the same with both vaccines. The titres gradually increased between the second week after the first dose and the post-booster dose (week 28). A strong booster effect of the booster vaccine dose on neutralizing titres was observed. Significantly higher neutralizing antibody titres with the PM vaccine were observed as early immune respose on week 2 titres on both series of results. Vaccine-induced neutralizing antibody titres and vaccine-induced antibody mRIA titres correlated well (coefficient of correlation 0.82 and 0.72, respectively, P
Introduction Neutralizing antibody against hepatitis A virus (HAV) is believed to be the most important element of an effective i m m u n e response against HAV 1 and has been evidenced in the sera of h u m a n recipients of i m m u n e plasma globulins. 2 The validation of the efficacy of pooled n o r m a l h u m a n i m m u n e globulin against hepatitis A disease 3 has proved the usefulness of the m e a s u r e m e n t of neutralizing antibody as a valuable tool for the evaluation of the potential efficiacy of inactivated hepatitis A vaccines. Assays have been developed to measure neutralizing antibody against HAV.4-c' The first is a quantitative radioi m m u n o f o c u s inhibition test (RIFIT) based on the i m m u n e autoradiographic detection of loci of viral replication u n d e r an agarose overlay, and the second is an antigenreduction neutralization assay (HAVARNA) based on the i m m u n o d e t e c t i o n of HAV antigen produced in cell culture. HAV strains used in these assays are different (HM 175, CR326F and GBM), and there is evidence of immunological cross-protection between all HAV strains.7 The presence of neutralizing antibodies after vaccination
Address correspondence to B. Flehmig. Accepted for publication 19 November 1996. 0163-4453/97/040037+04 $12.00/0
with inactivated hepatitis A vaccines has been demonstrated as early as 2 weeks after the first vaccine dose with an increase after additional inoculations. 8'9 The correlation between vaccine-induced neutralizing antibody titres and vaccine-induced antibody titres measured by conventional assays (enzyme-linked i m m u n o s o r b e n t assay (ELISA) and r a d i o i m m u n o assay (RIA)) has been validated several times, 1°'11 indicating that results provided by conventional assays can also be used to predict the efficacy of inactivated hepatitis A vaccines. We describe here the results of neutralizing antibodies using a n e w inactivated hepatitis A vaccine produced by Pasteur Merieux Serums & Vaccins (PM), w h i c h had been compared to a n o t h e r licensed inactivated hepatitis A vaccine produced by Smithkline Beecham (SB) by a controlled clinical trial.
Material and Methods Vaccines The PM vaccine (AVAXIMTM) was derived from a strain of hepatitis A virus (GBM) isolated in Germany and studied extensively. 12 After viral propagation and amplification, a filtered harvest obtained by cellular lysis © 1997 The British Society for the Study of Infection
B. Flehmig et aL
38
was purified by chromatography and concentrated by tangential ultrafiltration. Purified HAV was inactivated by formaldehyde at a dilution of 1:4000 for 14 days at 37 °C with constant agitation. The bulk vaccine antigen was filtered at 0.22 gin, adjusted for antigen content (160 ELISA units/dose) and adsorbed to 0.5 mg aluminium hydroxide with 2-phenoxyethanol added as preservative. The vaccine was formulated in 0.5 ml doses and was administered as one injection of one vaccine dose for primary immunization. A booster injection was given 6 months after the primary immunization. The preparation and characteristics of the control SB vaccine (HAVRIXTM) have been described elsewhere. 13 This vaccine was formulated in 1 ml doses and contained 720 ELISA units of HAV antigen derived from strain HM175 (as both manufacturers used different in-house antigen reference materials and different in-house ELISA reagents leading to different ELISA units, vaccine antigen potency comparison was not possible between both products). The vaccine was administered as two injections of one vaccine dose at 1-month intervals for primary immunization and a booster injection was given 6 months after primary immunization, because at the time this trial was conducted, this was the only vaccine licensed on the market and consequently available for such comparison. Both vaccines were given intramuscularly into the deltoid region.
Ser a A multicentre European comparative study between the PM vaccine and the SB vaccine as control was carried out in 840 healthy adults volunteers, and the principal results have been published elsewhere. 14 Serum samples (from the U.K. centre) collected before, at week 2 (postdose 1), at week 8 (post-dose 1 for the PM and post-dose 2 for the control vaccine), at week 24 (booster) and at week 28 (post-booster) from 30 subjects selected at random from each group were tested for neutralizing antibodies. Selection was made in order to have two comparable groups in terms of sex ratio (15 females and 15 males in each group) and m e a n age.
Neutralization assays Two assays were performed on blinded sera. The first one was a simple HAV antigen-reduction neutralization assay developed on the basis of routine techniques used for polio virus antibodies neutralizing assay and similar to an assay described previously. 5 Briefly, serial dilutions of
sera (1/5 to 1/10 240) were incubated with 102 Tissue Culture Infective Doseso (TICD~0) of the HAV GBM strain (4 h at 37 °C and 1 night at 5 °C). Samples were then incubated on h u m a n diploid cells (MRCs) in 96-well plates for 21 days at 37 °C in a 5% CO2 atmosphere. The HAV measurement was done by ELISA on cells lysed by freezing and thawing and with appropriate controls. The neutralizing antibody titre was defined as the reciprocal of the highest serum dilution yielding an HAV growth inhibition in 50% of the wells. The World Health Organization (WHO) reference immune globin was used as positive control and its neutralization titre was from 1/ 74 000 to 1/28 000. The results were expressed in milfi International Units (mIU) per ml by comparison with this reference, and, by convention, titres below the detection cut-off (between 10 and 20 according to the series) were considered equal to 10 mIU/ml for calculating the geometric m e a n titre (GMT). The second assay was also a simple HAV antigen-reduction neutralization assay developed in the laboratory of B. Flehmig. 6 Briefly, serial dilutions in minimal Eagle medium (MEM) with 7% fetal calf serum (FCS) of heat-inactivated sera (1/10 to 1/ 25 600) were incubated with 10 TCIDso of HAV GBM strain (4 h at 37 °C). The samples were then incubated on MRC5 cells in 96-wefi plates for 21 days at 37 °C in 5% CO2. HAV measurement was carried out by ELISA on lysed cells with appropriate controls. The neutralizing antibody titre was defined as the reciprocal of the highest serum dilution yielding 50% growth inhibition. Paul Ehrlich Institut reference immune globulin was used as positive control. The results were expressed in mIU/ml by comparison with this reference, and, by convention, titres below the detection cut-off (10) were considered equal to 5 mIU/ml for calculation of the GMT.
Modified radio-immunoassay (mRIA) Sera were titrated blindly by a modified radioimmunoassay (mRIA) (HAVAB, Abbott Laboratories, N. Chicago, IL, U.S.A.) in an independent laboratory (Bioanalytical Research Corporation, Ghent, Belgium). The conventional RIA, which allows a detection cut-off around 90 mlU/ml for specific antibodies when the results are expressed in comparison with a reference curve generated from the WHO reference HAV i m m u n e globulin, was modified to measure lower levels of anti-HAV. This modification of the procedure was used as described previously 15 and consisted of mixing 100 gl of test serum (instead of 10 I~1) with 100 pl of ~25I anti-HAV (instead of 200 ~tl). This modification made it possible to reduce the detection cut-off to 7 - 1 0 mlU/ml.
Hepatitis A Vaccine Neutralizing Antibodies
39
Table i. Seroneutralizing antibodies after vaccination with the PM or the SB inactivated hepatitis A vaccines (serological analysis performed by HAV antigen-reduction neutralization assay in two different laboratories). Week 0 dose 1
Lab, 1
Lab, 2
Week 2
Week 8
No.
PM 30
SB 30
PM 30
SB 30
PM 30
after dose 2 SB 30
GMT (mfU/ml) 95% CI range GMT (mIU/ml) 95% ci range
7.0 6-8 5-15 5.5 5-6 5-17
6.6 6-7 5-10 5.3 5-6 5-14
43.5* 23-82 5-1920 43.4* 32-59 11-387
13,2 9-20 5-640 12.4 10-16 5-51
84.3 46-155 10-2560 103.7 74-145 21-545
60.4 33-110 10 2 5 6 0 117.5 80-172 17-1175
Week 24 booster
Week 28
PM 30
SB 30
PM 30
SB 30
100.8 52 194 10-3840 146.1 109-197 28-1586
83.3 46-152 5-1920 157.3 98-252 5-765
3183 2064-4909 4 8 0 - 2 0 480 3256.7 2275-4662 369 2 5 2 5 0
2308 1459-3653 120 30 720 3268 2222-4805 289-20200
* Statistically significantly different from SB, P<0.01.
100
Results
.
10 000
The demography of subjects There were 60 subjects (30 females and 30 males) enrolled in this study. Thirty of the subjects were vaccinated with the PM vaccine (15 females and 15 males), and 30 with the control vaccine (15 females and 15 males). Their mean age was 24.7 years (range 18-40) for the PM group and 25.1 years (range 17-38) for the control group.
~ . ~,~* ~
~tt~% *" ~*~
looo
100
,
lO r [ [lilll]
, = ,,,iJil
i i iiiill
10 000
o
1000
.-.v,l!,:ai
1
.,it.e
i I r IIIIIll
Results obtained with sera for neutralizing antibodies are presented in Table I. In both series of results from the two laboratories the appearance of neutralizing antibodies was earlier with the PM vaccine compared to the control vaccine. Seroneutralizing antibodies titres were higher at week 2 with the PM vaccine compared to the control vaccine (P
i i ,,HH[
100 000
10
Neutralization antibodies
I i lliliil
10 100 1000 10 000 100 000 Seroneutralization titre (Lab. 2)
Correlation between neutralization assays When the results provided from both laboratories on the same sera were compared, a good correlation was observed (coefficient correlation R = 0.42, P
000
.....
I I IIIIIll
I I IIIIIll
I I IIINli
I
10 100 1000 10 000 S e r o n e u t r a l i z a t i o n t i t r e (Lab. 1)
II
100 000
Figure 1. Correlations between mRIA HAVAB titres (mIU/ml) and seroneutralization titres (mIU/ml) obtained with HAV antigen-reduction neutralisation assays from two different laboratories (Lab. 1 (PM) and Lab. 2 (B. Flehmig), in 300 sera from subjects vaccinated with the PM or SB inactivated hepatitis A vaccines (sera collected before and at weeks 2, 8, 24 and 28 after vaccination).
Discussion Correlation between neutralization assags and mRIA assays Each series of results were compared with the mRIA tires and results are plotted on Fig. 1. The correlation coefficients were 0.82 and 0.79 (P
Results obtained by both laboratories confirmed the appearance of neutralizing antibodies as soon as 2 weeks after the first dose of both inactivated hepatitis A vaccines. As the PM vaccine was given with only one dose for primary immunization, compared to the SB vaccine
40
B. Flehmig e t al.
which was given in two doses, the comparison of results should take into account this difference in the immunization schedule. Nevertheless, the levels of neutralizing antibodies were comparable in both groups except at week 2, where the PM vaccine induced higher levels. These results were also in accord with those obtained using mRIA on the same sera.J4 Hepatitis A is a worldwide infection and is a substantial health risk for a huge population. About 1.3 million patients suffer each year from acute disease. A significant n u m b e r of patients have a severe infection associated with up to 2% mortality in adults. In industrialized countries the most c o m m o n way to contract infection with HAV is through travel to developing countries and hyperendemic areas. The number of persons travelling from developed to developing countries is about 25 million per year, and most of these travellers are not protected against HAV because the prevalence of antibodies to HAV has been decreasing dramatically during the past 1 0 - 2 0 years. The most effective way of preventing infectious diseases is vaccination, and for hepatitis A this has been shown to be highly effective. 1~'17 Because HAV causes not only high morbidity but also significant mortality in adults, the WHO recommend immunization against hepatitis A. The vaccines based on cell culture and inactivated hepatitis A virus have been developed and shown to be immunogenic and safe. A matter of greatest importance is the induction of a rapid i m m u n e response after vaccination, because travellers often decide at a late state to take advantage of prevention. A vaccine inducing early neutralizing antibodies is therefore of great value. In this study we have demonstrated that a new cell culture based inactivated hepatitis A vaccine induces very early, significantly higher neutralizing antibodies compared to the first licensed formulation of the SB vaccine. These results, which correlated well with those obtained by mRIA, indicate the potential efficacy of this new vaccine.
Acknowledgements The authors are grateful to the investigators and volunteers who participated in the study, and to Nadege Picat for the testing of antibodies by seroneutralization assays.
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