Immunogenicity in adult males of a Neisseria meningitidis group B vaccine composed of polysaccharide complexed with outer membrane proteins M. Robert Lifely *LI,Susan C. Roberts*, Wilma M. Shepherd*, Jane Esdaile*, Zhen Wang*, Ann Cleverly *, Adel A. Aulaqi ,~ and Carlos M o r e n o ~ Twenty five adult male volunteers were 9iven a vaccine composed of the capsular B polysaccharide non-covalently complexed to serotype 6 outer membrane proteins (OMP) oJ Neisseria meningitidis. Subjects were divided into three dose 9roups receivin9 50, 100 or 150 P9 vaccine in aluminium hydroxide in each of three injections spaced 4 weeks apart. Systemic signs~symptoms considered clinically significant were recorded on 6% (4/70) o f occasions and were succeeded by withdrawal of two volunteers from the study. Local injection site reactions, mostly mild to moderate, were reported after all vaccinations with one such reaction leadin9 to a third volunteer withdrawing from the study. Geometric mean anti-B responses before immunization and 1 week after the third immunization (9 weeks) were 3.60 and 7.12 #9 ml-I in the 50 #9 9roup ( p < 0.05), 2.05 and 12.19 #9 ml-1 in the 100 #9 group (p < 0.001), and 3.68 and 14.20 P9 ml- 1 in the 150 #9 9roup (p < 0.001). The anti-B response was predominantly o f the I y M isotype and persistence above prevaccination levels was evident for at least 12 months. Anti-type 6 0 M P responses were also evidenced with 9eometric mean multiplicative increases over prevaccination levels at 9 weeks and 6 months o f 7.8 and 4.2 for the 50 #9 9roup, 11.6 and 5.6 for the 100 #9 9roup and6.8 and 3A for the 150 It9 9roup. The bulk o f this response was o f the IgG isotype. Passive protection of mice was achieved with both pre- and post-vaccination (9 weeks; 100 and 150 It9 9roups) pools of sera. Protection was abolished by prior adsorption of sera with B polysaccharide. Keywords:Neisseria men&#itidis; polysaccharide; outer membrane protein
INTRODUCTION The incidence of meningococcal disease has been steadily increasing in England and Wales since 1984, with no sign of abatement to the end of 1988 ~. Group B strains have predominated and account for about 60% of infections 1'2. The capsular polysaccharide of Neisseria meningitidis group B is known to be poorly immunogenic in animals 3 and humans 4 when given as the purified polymer, and non-covalent complexes of group B polysaccharide and N. meningitidis outer membrane proteins (OMPs) have given variable results in volunteer studies, with some investigations showing two to fivefold geometric mean increases in the antibody response to both the B polysaccharide and the OMPs with *Department of Experimental Immunobiology, Wellcome Biotech, and Departments of tClinical Research and **Scientific Computing Statistics, Wellcome Foundation Ltd, Langley Court, Beckenham, Kent BR3 3BS, UK..~Medical Advisory Services, Wellcome International Trading Ltd, Berkhamsted, Herts HP4 2DY, UK. ~MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Road, London W12 0HS, UK. tqTowhom correspondence should be addressed. (Received 4 April 1990; revised 19 July 1990; accepted 19 July 1990) 0264-410X/91/010060-07 ~(;; 1991 Butterworth-Heinemann Ltd
60 Vaccine, Vol. 9, January 1991
polysaccharide to protein ratios between 1:3 and 3:15, whereas others have been unable to detect any antibody increase to the B polysaccharide component 6. Subsequently, the immunogenicity of the B polysaccharide in animals was shown to be associated with the degree of binding of the polymer to OMPs in different preparations 7. Reasons for the generally poor immunogenicity of the group B polysaccharide have been proposed 8 10. We believe that it is caused by tolerance towards structurally similar host tissue components, namely the short chains (2-4 residues) of ~(2 ~ 8)-linked sialic acid on sialogangliosides and sialoproteins 3'9'1° and evidence has accumulated to suggest that antibodies to B polysaccharide are, therefore, directed against conformational determinants 11,12. Finne et al. s suggested that immunological tolerance to the B polysaccharide may be due to cross-reactive brain polysialoprotein, which is present in fetal tissue but diminishes rapidly after birth, and that breakdown of natural tolerance caused by an 'artificial' vaccine may initiate an autoimmune response. However, there is an impressive body of literature which fails to support this proposition 3'9'1°'13 ~5. Moreover, anti-B antibodies are naturally present in the majority of the population following, presumably, exposure to environ-
Immunogenic Neisseria meningitidis group B vaccine: M.R. Lifely et al.
mental antigens, or following vaccination or infection, without ill-effects, and those who advocate a role for the polysialoprotein in tolerance have been unable to explain how such a state of tolerance would be maintained without persistence of the toleragen. A non-covalent complex with a high degree of binding of the B polysaccharide to OMPs from N. meningitidis but low in lipopolysaccharide and nucleic acid content was prepared. The fluid vaccine was immunogenic and protective in animals 3 and induced a secondary response to the B polysaccharide when administered with aluminium hydroxide 16. Animal studies showed that aluminium ions, which may have been responsible for stabilization of the three-dimensional structure of the B polysaccharide non-covalently complexed to OMPs, resulted in an enhanced anti-polysaccharide response ~7. Our purpose was to assess, in a small, single-blind dose-response study, the clinical reactivity and immunogenicity of this meningococcal group B vaccine when administered to adult humans, with aluminium hydroxide. A male trial population was selected to obviate any possible problems arising from induction, in females, of antibodies with potential to cross-react with fetal antigens 8. SUBJECTS A N D M E T H O D S
Vaccine The vaccine (Lot MB6-334) is a non-covalent complex of the B polysaccharide and OMPs from N. meningitidis group B, type 6 (Wellcome CN No. 7622) selected for its chemical stability and immunogenicity in mice 7. Preparation and characterization of the vaccine has been described previously3. Briefly, the B polysaccharide and OMPs were coextracted from culture supernatants with cetavlon. Non-complexed B polysaccharide and OMPs, together with low molecular weight materials and nucleic acids, were removed from the high molecular weight (> 2 x 107 kDa) aggregates of polysaccharide and protein by gel filtration on Sepharose CL-2B. The high molecular weight aggregates were sterile filtered (0.22/~m) in the presence of 1% deoxycholate (pH 1I), precipitated with ethanol to remove the detergent, freeze-dried in 0.01 M sodium phosphate buffer (pH 7.2) containing 5% lactose, and stored at -20°C. Chemical characterization showed that, of the constituents measured, the vaccine contained B polysaccharide (54%), protein (45%), DNA (1.02%), phosphorus (0.32%) and deoxycholate (
INTRODUCTION The incidence of meningococcal disease has been steadily increasing in England and Wales since 1984, with no sign of abatement to the end of 1988 ~. Group B strains have predominated and account for about 60% of infections 1'2. The capsular polysaccharide of Neisseria meningitidis group B is known to be poorly immunogenic in animals 3 and humans 4 when given as the purified polymer, and non-covalent complexes of group B polysaccharide and N. meningitidis outer membrane proteins (OMPs) have given variable results in volunteer studies, with some investigations showing two to fivefold geometric mean increases in the antibody response to both the B polysaccharide and the OMPs with *Department of Experimental Immunobiology, Wellcome Biotech, and Departments of tClinical Research and **Scientific Computing Statistics, Wellcome Foundation Ltd, Langley Court, Beckenham, Kent BR3 3BS, UK..~Medical Advisory Services, Wellcome International Trading Ltd, Berkhamsted, Herts HP4 2DY, UK. ~MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Road, London W12 0HS, UK. tqTowhom correspondence should be addressed. (Received 4 April 1990; revised 19 July 1990; accepted 19 July 1990) 0264-410X/91/010060-07 ~(;; 1991 Butterworth-Heinemann Ltd
60 Vaccine, Vol. 9, January 1991
polysaccharide to protein ratios between 1:3 and 3:15, whereas others have been unable to detect any antibody increase to the B polysaccharide component 6. Subsequently, the immunogenicity of the B polysaccharide in animals was shown to be associated with the degree of binding of the polymer to OMPs in different preparations 7. Reasons for the generally poor immunogenicity of the group B polysaccharide have been proposed 8 10. We believe that it is caused by tolerance towards structurally similar host tissue components, namely the short chains (2-4 residues) of ~(2 ~ 8)-linked sialic acid on sialogangliosides and sialoproteins 3'9'1° and evidence has accumulated to suggest that antibodies to B polysaccharide are, therefore, directed against conformational determinants 11,12. Finne et al. s suggested that immunological tolerance to the B polysaccharide may be due to cross-reactive brain polysialoprotein, which is present in fetal tissue but diminishes rapidly after birth, and that breakdown of natural tolerance caused by an 'artificial' vaccine may initiate an autoimmune response. However, there is an impressive body of literature which fails to support this proposition 3'9'1°'13 ~5. Moreover, anti-B antibodies are naturally present in the majority of the population following, presumably, exposure to environ-
Immunogenic Neisseria meningitidis group B vaccine: M.R. Lifely et al.
mental antigens, or following vaccination or infection, without ill-effects, and those who advocate a role for the polysialoprotein in tolerance have been unable to explain how such a state of tolerance would be maintained without persistence of the toleragen. A non-covalent complex with a high degree of binding of the B polysaccharide to OMPs from N. meningitidis but low in lipopolysaccharide and nucleic acid content was prepared. The fluid vaccine was immunogenic and protective in animals 3 and induced a secondary response to the B polysaccharide when administered with aluminium hydroxide 16. Animal studies showed that aluminium ions, which may have been responsible for stabilization of the three-dimensional structure of the B polysaccharide non-covalently complexed to OMPs, resulted in an enhanced anti-polysaccharide response ~7. Our purpose was to assess, in a small, single-blind dose-response study, the clinical reactivity and immunogenicity of this meningococcal group B vaccine when administered to adult humans, with aluminium hydroxide. A male trial population was selected to obviate any possible problems arising from induction, in females, of antibodies with potential to cross-react with fetal antigens 8. SUBJECTS A N D M E T H O D S
Vaccine The vaccine (Lot MB6-334) is a non-covalent complex of the B polysaccharide and OMPs from N. meningitidis group B, type 6 (Wellcome CN No. 7622) selected for its chemical stability and immunogenicity in mice 7. Preparation and characterization of the vaccine has been described previously3. Briefly, the B polysaccharide and OMPs were coextracted from culture supernatants with cetavlon. Non-complexed B polysaccharide and OMPs, together with low molecular weight materials and nucleic acids, were removed from the high molecular weight (> 2 x 107 kDa) aggregates of polysaccharide and protein by gel filtration on Sepharose CL-2B. The high molecular weight aggregates were sterile filtered (0.22/~m) in the presence of 1% deoxycholate (pH 1I), precipitated with ethanol to remove the detergent, freeze-dried in 0.01 M sodium phosphate buffer (pH 7.2) containing 5% lactose, and stored at -20°C. Chemical characterization showed that, of the constituents measured, the vaccine contained B polysaccharide (54%), protein (45%), DNA (1.02%), phosphorus (0.32%) and deoxycholate (
