Clinical evaluation of a group B meningococcal N-propionylated polysaccharide conjugate vaccine in adult, male volunteers

Vaccine 22 (2004) 1087–1096

Clinical evaluation of a group B meningococcal N-propionylated polysaccharide conjugate vaccine in adult, male volunteers Joëlle Bruge a,∗ , Nancy Bouveret-Le Cam a,1 , Bernard Danve a , Geneviève Rougon b,2 , Dominique Schulz a b

a Aventis Pasteur France, 1541 Avenue Marcel Mérieux, 69280 Marcy-l’Etoile, France Institut de Biologie du Développement (IBDM), UMR-CNRS, Case 907, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France

Received 6 December 2002; received in revised form 9 October 2003; accepted 15 October 2003

Abstract The safety and immunogenicity of a group B meningococcal vaccine, consisting of N-propionylated (NPr) B capsular polysaccharide conjugated to tetanus toxoid, was tested for the first time, in 17 healthy male volunteers aged between 18 and 40 years. Four escalating dosages of vaccine were tested and each was given as three intramuscular injections at 4-week intervals. The vaccine was well tolerated and induced only mild and transient, dose-dependent, injection–site reactions. One month after the last injection, there was no evidence of the production of autoantibodies or antibodies binding to PSA-NCAM. The vaccine induced an increase in the pre-existing titres of IgM specific to B polysaccharide and NPr B polysaccharide. Moreover, it induced IgG antibodies specific to NPr B polysaccharide, which were undetectable before vaccination. However, no functional activity of vaccine-induced antibodies was demonstrated in bactericidal assays, opsonophagocytic tests or passive protection tests. © 2003 Elsevier Ltd. All rights reserved. Keywords: Group B meningococcal vaccine; N-Propionylated capsular polysaccharide conjugate; Phase I clinical trial

1. Introduction Neisseria meningitidis causes a variety of human diseases worldwide but is most often associated with severe sepsis and rapid-onset meningitis [1]. The meningococcal disease mostly affects children and adolescents, the highest incidence being observed in children less than 1 year old, especially for group B. Even with optimal anti-microbial treatment, the case-fatality rate of meningococcal disease reaches 5% or more, and a similar proportion of patients sustain severe sequelae [1–3]. N. meningitidis is classified into 12 serogroups, based on the structure of the capsular polysaccharides [4]. Of the 12 serogroups, A, B and C are responsible for approximately 90% of cases of meningococcal disease. Serogroups B and C predominate in Europe and are isolated in sporadic cases, whereas serogroup A is responsible for epidemics in devel∗

Corresponding author. Tel.: +33-437-37-3728; fax: +33-437-37-3244. E-mail address: [email protected] (J. Bruge). 1 Present address: Programme for Appropriate Technology in Health (PATH), Meningitis Vaccine Project, 13 Chemin du Levant, 01210 Ferney-Voltaire, France. Fax: +33-450-28-0407. 2 Fax: +33-491-26-9748. 0264-410X/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.10.005

oping countries. With the recent increase in the incidence of infections with serogroup Y, this group is now as prevalent as serogroups B and C in some parts of the USA and there is an increasing number of reports of cases due to serogroup W-135 in Europe [5–10]. Currently available vaccines against N. meningitidis are mainly based on the capsular polysaccharides of serogroups A, C, Y and W-135, and therefore offer no protection against serogroup B meningococcal disease [11,12]. Indeed, there is no polysaccharide vaccine against serogroup B because the B polysaccharide is poorly immunogenic in man [13]. This may be due to conformational complexity [14,15] or internal esterification [15], but is most likely due to a structural homology between B polysaccharide and polysialic acid (PSA) chains (␣-2,8-linked N-acetylneuraminic acid) present in mammalian fetal and adult tissues (namely the polysialylated form of the neural cell adhesion molecule; PSA-NCAM) [16]. The relatively long chains of PSA (>12 residues) associated with fetal tissue become progressively shorter after birth except on particular cells, such as neurons in certain areas of the brain or natural killer (NK) cells [16–18]. PSA-NCAM is also present in adults during muscle regeneration under certain pathological conditions and in some tumours [18–22]. It has been suggested that the

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expression of long chains of PSA on these cells may result in immune tolerance to PSA and that immunisation with B polysaccharide might induce autoimmune reactions [16]. Attempts to generate immunogenic group B vaccines have therefore focused on outer membrane protein (OMP) vaccines, or vaccines containing chemically modified B polysaccharide conjugated with an appropriate carrier (i.e. conjugate vaccines). Most OMP vaccines tested in clinical trials are based on outer membrane vesicles (OMVs). OMV vaccines derived from wild-type isolates have an estimated efficacy of 51–83% [12,23–26]. Furthermore, these vaccines mainly induce antibodies directed at subcapsular antigens and are therefore limited by the variability of these antigens. Bactericidal activity, considered as a protection marker, was shown to be induced by an epidemic strain-specific vaccine, in 90% of infants, against the homologous strain. However, cross-reactive antibodies against heterologous strains were only induced in older children and adults [27]. Promising results were recently obtained with a recombinant hexavalent PorA–OMV vaccine (porin A is a class 1 OMP) [28,29]. Among the conjugate vaccines, a candidate group B vaccine has been developed, in which the N-acetyl groups of the sialic acid residues in the polysaccharide are replaced with N-propionyl groups. This N-propionylated (NPr) B polysaccharide is conjugated to tetanus toxoid. The conjugate vaccine induced in mice, IgG directed to NPr B polysaccharide, which were protective in vitro and in vivo [30,31]. However, attempts to improve the immunogenicity of the polysaccharide by chemical modification and conjugation could potentially lead to autoimmune reactions against PSA-NCAM [32,33]. Potential effects on fetal or postnatal development could be particularly significant because the vaccine would mainly be intended for infants under 1 year old (the age-group most frequently affected by the disease). Thus, the safety of such a conjugate vaccine must be investigated thoroughly. Here we present the results of a phase I clinical trial performed in adults in order to evaluate the short-term safety of an NPr B polysaccharide conjugate vaccine together with a preliminary assessment of its immunogenicity. 2. Materials and methods This study was an open, non-randomised, phase I trial in healthy male, adult volunteers. The trial was conducted after approval from the French Viral Safety Committee and the French Ethics Committee. After being informed, all volunteers gave written consent to participate. The trial was conducted at a phase I agreed centre, Institut Aster, Paris, France. 2.1. The vaccine The vaccine was prepared at Aventis Pasteur as follows. The group B polysaccharide was purified from culture

supernatants of N. meningitidis strain IM 2228 (Aventis Pasteur) according to the method of Gotschlich et al. [34]. The polysaccharide was heated with 2 M NaOH to remove N-acetyl groups, then N-propionylated by adding propionic anhydride. The NPr polysaccharide was oxidised then coupled to tetanus toxoid by reductive amination using an aliphatic molecule as a spacer arm (method derived from Jennings et al. [14]). The conjugate was purified on Sephacryl S300 (Pharmacia). Finally, the vaccine was adsorbed onto aluminium hydroxide immediately before administration. In the conjugate vaccine, the weight ratio of polysaccharide to protein and polysaccharide to aluminium was 0.31 and 0.1, respectively, whatever the dose administered. 2.2. Selection criteria Adult men between 18 and 40 years old were enrolled. Exclusion criteria were: a history of local or systemic allergy or asthma, a history of reactions to tetanus toxoid, severe chronic disease, acute disease with an elevated temperature in the 72 h before enrolment, major medical treatment, or a tetanus vaccination in the previous 5 years. 2.3. Immunisation procedures Volunteers received three intramuscular injections into the deltoid at 4-week intervals. Four escalating dosages (1 ␮g, 5 ␮g, 25 ␮g and 50 ␮g of polysaccharide) were tested using a sequential protocol: three volunteers were injected with 1 ␮g; three volunteers with 5 ␮g; five volunteers with 25 ␮g; and five volunteers with 50 ␮g. Aluminium (Al) content was equivalent to 10-fold the polysaccharide content and consequently varied from 10 to 500 ␮g Al per dose. 2.4. Safety evaluation During the three hours after each injection, participants were monitored by physical examination and interview for clinical symptoms such as anaphylactic reactions, rash, pruritus, respiratory manifestations, hypotension and malaise. Local reactions and general adverse events were evaluated by physical examination and interview at the visits on days 1, 2, 7, 14 and 28 after each vaccination, and were recorded between visits by the participants using diary cards. Prelisted local reactions included local pain, redness, induration, ecchymosis, abscesses, localised pruritus and eruption. Prelisted general reactions included oral temperature ≥38 ◦ C, asthenia, sweating, malaise, headache, dizziness, nausea, myalgia, arthralgia, rash and generalised pruritus. Subjective reactions were rated for severity: pain was rated using a three-point scale (1 = slight and well-tolerated, 2 = moderate and impeding movements, but not affecting activity, 3 = strong and preventing normal activity). Objective reactions were rated according to the diameter of the lesion (on a 14-point scale where one point corresponds to 0.5 cm).

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The investigator collected results on case report forms and had the opportunity to mention other adverse events not prelisted in the forms, in a specific section entitled “Others”. Standard extensive haematological (haemogram and coagulation parameters) and biochemical (in serum and urine) variables were tested before inclusion and on days 0, 14, 28, 42, 56, 70 and 84. Autoantibodies were tested at pre-inclusion and 28 days after each injection (see detailed description in Section 2.7). 2.5. Serological analyses Blood samples were collected before the first injection and 4 weeks after each injection (days 0, 28, 56 and 84) and sera were prepared for analyses. Anti-B polysaccharide and anti-NPr B polysaccharide IgG and IgM antibody titres were evaluated using ELISA. Polystyrene microplates (Dynatech) were coated with either B polysaccharide or NPr B polysaccharide complexed to human methylated albumin and, after a blocking step with bovine serum albumin, plates were incubated for 1 h with 2-fold serial dilutions of serum samples. Bound antibodies were then detected with peroxidase-labelled goat anti-human IgG or IgM. IgG subclasses of anti-NPr B polysaccharide were also measured using anti-human IgG1 , IgG2 , IgG3 and IgG4 mouse monoclonal antibodies conjugated to peroxidase. IgG and IgM were separated by size exclusion chromatography on Ultrogel (Biosepra) and by affinity separation on Hi-Trap protein G (Pharmacia). The capacity of vaccine-induced antibodies to bind to group B capsulated meningococcus was investigated using two techniques [35]. Firstly, the last serum sample from each participant receiving the highest dose of the vaccine was incubated with group B N. meningitidis strain H44/76 (1010 bacteria/ml) twice at 37 ◦ C for 30 min then at 4 ◦ C overnight. Unadsorbed antibodies were measured by ELISA. Secondly, flow-cytometric analysis using glutaraldehyde-inactivated bacteria (strain H44/76) was used on days and 84 blood samples from one participant. 2.6. Functional activity assessments Bactericidal activity of all samples was measured against group B N. meningitidis strains M986 and H44/76. Bacterial cells (100 colony forming units (CFU) per well) were incubated with 2-fold serial dilutions of heat-inactivated sera and human complement (from agammaglobulinaemic donor) at 37 ◦ C for 1 h (100 ␮l total volume per well) in 96-well microplates. Viable bacteria were counted after overnight incubation on agar plates at 37 ◦ C and compared to controls containing bacteria and complement only, to calculate the percentage of killing. Bactericidal titres were expressed as the reciprocal dilution, which provided 50% killing of the bacteria and had to increase from pre-vaccination baseline by at least four-fold to be

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considered significant [36]. With selected serum samples, bactericidal assays were also conducted using baby rabbit complement. Opsonophagocytic activity of blood samples taken before the first injection (day 0) and after the last injection was measured by incubating serum with group B N. meningitidis strain H44/76 (106 CFU per assay), human complement, and human polymorphonuclear leukocytes (PMN) (5 × 106 per assay) at 37 ◦ C for 1 h. Viable bacteria were counted and compared with controls without PMN. Serum samples taken before the first injection (day 0) and after the last injection (day 84) were assessed in passive protection experiments [37,38]. Each sample (0.1 ml) was administered subcutaneously to five infant rats (Wistar– Furth, Iffa-Credo, L’Arbresle, France), 18 h before an intraperitoneal challenge with group B N. meningitidis strain H44/76 (four times rat passaged). Buffered saline was used as a negative control and hyper-immune murine serum (B36) was used as a positive control. Five hours later, blood samples were taken from the rats and bacteraemia was determined by plate counts. 2.7. Autoantibody assays and PSA-NCAM cross-reactivity The presence of autoantibodies specific to the nucleus of human cell line HEP2 and smooth muscle (oesophageal sections) was measured in all blood samples (days 0, 28, 56 and 84) using an immunofluorescence technique. A medium positive control serum (with a titre of 400) was introduced in each series of analyses at the same dilutions as the test samples. Reactivity to PSA-NCAM was assessed in serum samples taken on days 0, 56 and 84. This was done by immunoprecipitation using embryonic and neonatal mouse brain extracts and immunofluorescence detection on live cells, respectively. Mouse material was used as more readily available than human tissue and as PSA-NCAM is expressed by all embryonic mammals in the same manner. For immunoprecipitation, PSA-NCAM was purified by affinity chromatography from detergent membrane extracts of embryonic mouse brains and radiolabelled with 125 I Na (Amersham) using the Iodogen technique. Human sera to be tested were incubated with protein A–Sepharose CL4B beads coated with rabbit anti-human IgM or IgG antibodies for 4 h at 4 ◦ C under shaking. After washing, the beads were incubated with 125 I PSA-NCAM for 14 h at 4 ◦ C under shaking. Complexes were then washed and mixed (v/v) with Laemmli buffer (Tris–HCl 0.125 M, SDS 4%, glycerol 20%, ␤-mercaptoethanol, bromophenol blue 0.001%) and heated for 4 min at 100◦ C. Immunoprecipitated proteins and antibodies were separated from protein A beads by centrifugation and supernatant subjected to 7% SDS–PAGE. Radiolabelled proteins were visualised by autoradiography. For immunofluorescence detection, AtT20 cells derived from a mouse anterior pituitary tumor and expressing

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PSA-NCAM [33,39] were used. Briefly, the AtT20 cells were seeded on glass coverslips and allowed to grow for 1 day or 2 days, respectively, before labelling. Live cells were incubated for 30 min at room temperature with the respective human sera 1/100 diluted. Bound antibodies were visualised with anti-human IgG or IgM antibodies conjugated to fluorescein. In the above experiments, the mouse monoclonal IgM anti-MenB obtained after immunisation with live group B meningococci, was used as a positive control 1/1000 diluted and an anti-NCAM rabbit anti-serum as well. Their characteristics have been published elsewhere [33]. 2.8. Statistical methods Due to the limited number of volunteers of such a Phase I study, descriptive statistics were used. Geometric mean titres (GMTs) in participants receiving the 25 ␮g or 50 ␮g doses (five per group) were compared using a paired sample t-test (α-level: 5%); comparisons of IgG and IgM ELISA titres were made between days 0 and 28, between days 28 and 56, and between days 56 and 84. 3. Results

reactions were reported and the greater their severity (Table 1). However, all injection–site reactions were mild and short-lived. Minor adverse events were reported in seven volunteers in the 3 h after injections (five and two volunteers in the groups receiving 25 and 50 ␮g dosage, respectively). These adverse events were: transient sore throat (three reports); transient headache (two reports); strained knee (one report); and jugulocarotid adenopathy from days 2 to 4 plus slight rhinorrhoea (one report). None of these adverse events was considered to be related to the study vaccine. The elevated ALT measured in the participant withdrawn from the study was likely due to a primary herpetic infection (HSV-1). It was first detected on day 14 and returned to normal levels by day 106. The biochemical variables in all other participants remained within the normal range throughout the trial. At all time points, sera from all 17 participants were negative, i.e. under the threshold value of 80, for autoantibodies directed against nucleus or smooth muscle. Immunofluorescence tests on cells expressing PSANCAM and immunoprecipitation of purified iodinated PSA-NCAM conducted on sera from volunteers receiving 25 ␮g or 50 ␮g, gave negative results whereas control antibodies directed against live meningococci or NCAM were strongly reactive in both tests.

3.1. Population 3.3. Serological analyses Of the 23 male volunteers enrolled, 17 were included and 16 completed the trial; one participant was withdrawn on day 56 because of elevated alanine aminotransferase (ALT) activity, and therefore received only two injections (1 ␮g). The mean age, weight and height of the participants were similar in each dosage group. The mean age of the 17 participants was 23.0 years (19.0–27.0 years), mean weight was 71.2 kg (61.0–83.6 kg) and mean height was 178 cm (160–188 cm). 3.2. Safety No serious adverse events were reported during the study. The higher the dosage of vaccine the more injection–site

All 17 participants had detectable IgM specific to NPr B polysaccharide and 16/17 had detectable IgM specific to B polysaccharide before immunisation. Vaccination resulted in increased IgM titres to B polysaccharide, mostly after the first injection in all but one volunteer. The increases in titres of IgM specific to NPr B polysaccharide (maximum 44-fold and 17 times in average) were significantly higher than the increases in IgM specific to B polysaccharide (maximum 11-fold and two-fold in average). The titres of IgM specific to NPr B polysaccharide were generally highest 1 month after the third vaccination (in 13/16 participants), whereas maximum titres of IgM specific for B polysaccharide were

Table 1 Number and maximum severity of injection–site reactions at any time during the trial Reaction

Pain Erythema Induration Ecchymosis Skin rash Pruritus

Number of reports (maximum severity)a 1 ␮g dosage group (n = 4)b

5 ␮g dosage group (n = 3)

25 ␮g dosage group (n = 5)

50 ␮g dosage group (n = 5)

1 (1)

4 (2) 1 (8)

5 1 1 1

8 3 3 2 2 1

1 (NA)

(2) (1) (1) (9)

(2) (12) (8) (1) (NA) (NA)

NA: method of grading severity not applicable to these reactions. a Severity of pain rated using a three-point scale (from 1 = slight and well-tolerated, to 3 = strong and preventing normal activity); severity of erythema, induration and ecchymosis rated using a 14-point scale where each point corresponds to a diameter of 0.5 cm. b n = 3 after day 56.

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IgM specific to NPr B polysaccharide

log(titer)

3

Day 2

0 28 56 84

1

(A)

1 µg

5 µg

25 µg

50 µg

IgM specific to B polysaccharide

Log(titer)

3

Day 2

0 28 56 84

1

(B)

1 µg

5 µg

25 µg

50 µg

IgG specific to NPr B polysaccharide

3

Day 0 28 56 84

Log(titer)

2

1

(C)

1 µg

5 µg

25 µg

50 µg

Fig. 1. Geometric mean titres in ELISA units (EU)/ml of IgM specific for NPr B (A) and polysaccharides (B) and IgG specific for NPr B polysaccharide (C).

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reached at various time points. The highest GMTs of IgM were in participants receiving the 25 ␮g or 50 ␮g dosages (Fig. 1A and B). The increase in the GMT of IgM specific to NPr B polysaccharide from days 0 to 28 was significant (P = 0.0023 and 0.027, in the 25 and 50 ␮g groups, respectively), whereas subsequent increases were not significant. No individuals had IgG specific to either the NPr B or B polysaccharide before immunisation. While no IgG specific to B polysaccharide was subsequently detected, IgG specific to NPr B polysaccharide were detected at one or more post-vaccination time point in 15/17 of the participants; both individuals with no antibody response had received the lowest dosage (1 ␮g) of the conjugate vaccine. The titres of IgG specific to NPr B polysaccharide were highest 1 month after the third dose (14/14), and the highest IgG GMTs were in men receiving the 25 ␮g or 50 ␮g dosages (Fig. 1C). The increase in the GMT of IgG specific to NPr B polysaccharide from days 0 to 28 was significant (P = 0.027 and 0.022, in the 25 and 50 ␮g groups, respectively), and in the highest dosage group (50 ␮g) a further significant increase in GMT was observed from days 28 to 56 (P = 0.0086). A further significant increase in GMT was observed from days 56 to 84 (P = 0.021 and 0.022, in the 25 and 50 ␮g groups, respectively). IgG subclass analysis of serum samples obtained on day 84 from the 10 individuals receiving either the 25 ␮g or 50 ␮g dosages of the vaccine demonstrated that the IgGs specific to NPr B polysaccharide were mainly IgG1 with lower levels of IgG2 and even lower levels of IgG3 (data not shown). Antibodies of the IgG4 subclass were not detected.

meningococcal strains. Vaccination did not significantly increase (i.e. a ≥4-fold increase is not reached) bactericidal titres (Table 2). Additional bactericidal assays using rabbit complement were conducted on serum samples obtained on days 0 and 84 from two individuals receiving the 25 ␮g dosage and the five individuals receiving the 50 ␮g dosage of the vaccine (Table 2). With this technique, all seven individuals had high pre-immunisation titres (1024–8192) and none had a significant increase in bactericidal titre on day 84. The separated IgG and IgM fractions from one individual (from the 25 ␮g dosage group) were assayed in the presence of rabbit complement. The pre-immunisation titres were high in the IgM fraction (8192) and undetectable in the IgG fraction (<4), but both titres were not significantly increased on day 84. No consistent increase in opsonophagocytic activity was detected in sera from patients given the higher dosages of the vaccine (25 and 50 ␮g) (Table 2). In most samples, the percentage survival of bacteria was slightly lower at day 84 than at day 0, but only one serum sample had a two-fold decrease (number 112). To investigate the lack of functionality of induced antibodies and confirm the more active role of IgM in the preimmune activity, the binding of different anti-polysaccharide antibody classes to the bacteria was measured on days 0 and 84. IgM bound to live group B N. meningitidis strain H44/76 cells to a greater extent than IgG (Table 2). The sera of one volunteer, were also studied using flow cytometry and showed a binding to meningococcus limited to IgM and identical in the pre- and post-immunisation samples. Functionality was further assessed by measuring passive protection conferred by pre- and post-immunisation sera from two volunteers who received the 25 ␮g dose in an infant rat model. Similar profiles of total or partial protection

3.4. Functional activity assessments When human complement was used in the bactericidal assays, all participants had no or low pre-immunisation bactericidal titres (from <4 to 16) against the two group B

Table 2 Bactericidal titres (using rabbit or human complement) against N. meningitidis strains M986 or H44/76, opsonophagocytic titres (using strain H44/76) and binding to live N. meningitidis strain H44/76 of sera from the five participants receiving the 50 ␮g dose of vaccine Participant number

Bactericidal titresa M986 strain

112 113 114 115 116

H44/76 strain

With rabbit complement

With human complement

With human complement

Day 0

Day 84

Day 0

Day 84

Day 0

Day 84

2048 8192 4096 4096 4096

4096 8192 4096 4096 4096

<4 <4 [4]c <4 [8]

<4 [4] [4] <4 <

<4 <4 [8] <4 <4

<4 [4] [8] <4 <4

Opsonophagocytic titres (% survival)

Binding to meningococcus (using the samples from day 84) (% adsorption)

Day 0

Day 84

NPr B IgGb

NPr B IgMb

B IgMb

50 20 42 85 52

27 18 33 76 48

14 12 18 28 45

92 71 73 91 91

100 100 100 100 100

a The validity of the results is ensured through the use of the required controls (i) without sera to check that the complement used has no intrinsic killing activity on bacteria and (ii) with heat-inactivated complement to check that sera have no activity at the two first serum dilutions tested, in the absence of complement. b Abbreviations: NPr B IgG: IgG specific to NPr B polysaccharide; NPr B IgM: IgM specific to NPr B polysaccharide; B IgM: IgM specific to B polysaccharide. c Square brackets [ ] denotes incomplete killing (between 50 and 90% killing).

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Table 3 Passive protection in the infant rat model (five rats per sample) Sample

Bacteraemia 5 h after challenge (×103 CFU/ml of blood)

Negative controlb Positive control

4500; 3400; 33000; 6800; 9800 <0.4c ; <0.4; <0.4; <0.4;

11500 <0.4

0/5 4/4

Pre-immunisation sera (day 0) Participant number 110 Participant number 111

3900; 500; 1400; 1100; 1 6100; 2300; <400d ; <4d ; <0.4

1380 <1760

1/5 2/5

Post-immunisation sera (day 84) Participant number 110 Participant number 111

90; 2700; 0.5; <0.4; <0.4 6000; 9100; 40; 600; <0.4

<558 <3148

3/5 1/5

Arithmetic mean (×103 CFU/ml)

Number of protected ratsa

a Protection is considered as total when counts are <0.4 × 103 CFU/ml, as partial when counts are between 0.4 × 103 and 10 × 103 CFU/ml. No protection when counts >10 × 103 CFU/ml. b The negative control group received buffered saline and the positive control rats received undiluted serum of mice that had been hyperimmunised with the conjugate (three injections in the presence of Freund’s adjuvant). c Below detection limit (0.4 × 103 CFU/ml). d Counting difficult due to plate contamination.

were observed before and after vaccination (3/10 and 4/10 animals, respectively) (Table 3).

4. Discussion Our results show that the group B meningococcal Npropionylated polysaccharide conjugate vaccine has a good short-term safety profile and is able to induce specific IgG and IgM. The increase in injection–site reactions seen with the increasing doses is most likely due to the presence of aluminium hydroxide (10 ␮g of aluminium in the lowest dosage of vaccine increasing up to 500 ␮g in the highest dosage). The systemic safety profile was good, in keeping with other highly purified polysaccharide vaccines [40]. Within the limits of sensitivity of the methods used, we observed an absence of detectable autoantibodies at the end of the trial (i.e. 4 weeks after dose 3). This is further supported by the absence of antibodies binding to PSA-NCAM, the only identified PSA carrier in mammals. This is in agreement with a similar study conducted on mice hyper-immunised with the same conjugate [39]. The response to the vaccine is directed mainly towards the modified (NPr) polysaccharide, thus lessening the possibility of autoimmune effects or effects on fetal development. Hyperimmunisation with the conjugate vaccine had no effect on reproduction, or on periand post-natal development, in a previous study conducted in cynomolgus monkeys [41]. The results of this volunteer study and of the previous study in cynomolgus monkeys suggest no major concerns over safety. However, the short duration of the human study and the small sample size of both these studies prevent us from definitively excluding the potential for induction of autoimmune reactions. Vaccination of adults with the conjugate increased IgG and IgM titres to the modified polysaccharide and also in-

creased IgM titres to the native polysaccharide. The greater increase of the anti-NPr IgM antibody titres compared with the anti-B IgM titres indicates induction of a humoral response in favour of the modified polysaccharide, in contrast to the anti-B IgM increase seen in response to natural infection [42–44]. The presence of anti-B IgM before vaccination suggests that the volunteers had been exposed to N. meningitidis or to other commensal organisms with similar antigens [45,46], and absence of pre-immune anti-B IgG may confirm that such exposure does not induce a persisting IgG response [47,48]. In addition, the absence of anti-B IgG after vaccination is consistent with the poor humoral response to this antigen in man. This may stem from immune tolerance, which is overcome by the modification of the polysaccharide leading to the induction of IgG specific to NPr B polysaccharide. The failure of the vaccine to induce functional antibodies may have a number of possible explanations. Factors worth considering include: the sensitivity of the tests used, the pre-existing titres in adults, the IgG subclass of the antibodies, their low avidity for the bacteria or their lack of fixing complement. The in vitro tests used (i.e. serum bactericidal assay, passive protection and opsonophagocytic tests) may not be sensitive enough to detect functional activity. The serum bactericidal assay cut-off was a titre of 4, but the lowest titre consistent with protection against group B meningococcal disease is not known. Titres <4 were associated with disease in early human studies [37], and a protective threshold titre of 8 in bactericidal assays with human complement and 128 with rabbit complement has been proposed for group C meningococci [49,50]. Pre-existing titres much higher than 128 were observed here, with rabbit complement, suggesting that participants were protected already before immunisation. In addition, we had previously demonstrated protection of animals even in the absence of detectable bactericidal

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activity in their sera using the active protection test described by Schryvers and Gonzalez [51]. In several experiments, immunisation of mice with the conjugate vaccine (with no adjuvant) induced low antibody titres and no detectable bactericidal activity, but protected mice against a high dose challenge (approximately 3000 LD50 ) with group B N. meningitidis (results not shown). These findings suggest that the bactericidal assay may underestimate the efficacy of vaccines since it is restricted to one aspect of the immune response and may not reflect fully the protective mechanisms involved in vivo. Also, the opsonophagocytosis assay and the passive protection model in infant rat, used in our present study may also have required higher concentrations of induced antibodies to demonstrate the functional activity of human antibodies. Some serum samples showed opsonophagocytic activity or partial passive protection, but these activities were not different in pre- and post-vaccination samples. Higher concentrations of vaccine-induced antibodies may be needed to convey complete protection or an increase in passive protection or opsonophagocytic responses. Previously, this vaccine was shown to confer passive protection in mice only with hyperimmune sera (from mice given three intraperitoneal injections with the vaccine and Freund’s adjuvant) [30,31]. Similarly, the positive control included in our test (a hyperimmune mouse serum, which has a bactericidal activity titre of 1024 with rabbit complement) conferred total protection to infant rats when injected undiluted or diluted three-fold, whereas a nine-fold dilution removed its ability to reduce bacteraemia in infant rats (results not shown). This threshold may not have been reached in human test samples despite their high bactericidal titres observed with rabbit complement. It is unlikely that the lack of functional activity of induced antibodies in our study is due to the absence of complement-fixing antibody because IgM and IgG1 fix very efficiently complement in humans and both these immunoglobulin classes were detected in immunised individuals. But, although the conjugate induced mostly IgG1 , it may not induce sufficient levels of this IgG subclass. Alternatively, the lack of function could be due to the induction of low-avidity antibodies by the vaccine [52]. It may be significant that very few vaccine-induced IgG antibodies bind to meningococcus cells. Just such a low-avidity antibody response to infection with N. meningitidis has been demonstrated in infants [53] and may explain the lack of bactericidal activity. Low avidity could stem from inappropriate specificity of induced antibodies because the modified polysaccharide used in the vaccine contains epitopes of insufficient similarity to important epitopes on the capsular polysaccharide. However, we have demonstrated that mouse monoclonal IgG2b antibodies strictly specific to NPr B polysaccharide are bactericidal [39]. Strategies that lead to the development of high-avidity antibody after immunisation (e.g. the use of strong adjuvants or new carriers, addition of cytokines) may therefore be key in the devel-

opment of effective serogroup B meningococcal vaccines [54]. Although tetanus toxoid is known as a good carrier, the nature of the carrier can influence the functional activity of a vaccine. For example, the N-propionylated form of the Escherichia coli K1 polysaccharide conjugated to tetanus toxoid or to a group B meningococcal porin [55] elicited similar high antibody levels in mice, but only the porin conjugate elicited bactericidal activity. In contrast to our results, using the same porin conjugate, Fusco et al. [55] observed significant increases (>4-fold) of bactericidal titres in non-human primates, irrespective of the often high pre-immune titres and of the source of complement. The highest increase was obtained 14 days after the first injection suggesting a booster of the pre-existing immunity. However, we cannot conclude whether these better results are due to the species or to the protein. In our study, bactericidal activity of IgM was higher than that of IgG when rabbit complement was used. The suggestion that, in human sera, the predominant bactericidal activity against group B meningococci is IgM directed against the B polysaccharide, and that rabbit complement enhances this activity, has been made before [56–58]. The high pre-immunisation bactericidal titre observed in adults with rabbit complement suggests that increases in bactericidal activity after vaccination can only be detected in na¨ıve individuals. Indeed, most adults are naturally protected against group B meningococcus, and the decreasing incidence of the disease with increasing age in children corresponds with an increase in IgM antibodies specific to group B polysaccharide. Anti-B IgM may therefore have an important role in the natural protection against group B meningococcus. Despite this, antibodies to the B polysaccharide do not appear to be bactericidal when human complement is used in bactericidal assays [43,57], and the biological relevance of bactericidal activity, especially that of IgM, in the presence of heterologous complement is uncertain. Finally, it is not clear how relevant the results from in vitro tests are to the in vivo situation. Protection could be better evaluated through the development of tests that reflect in vivo entire mechanisms involving both innate and specific host defences. In the absence of such tests, efforts should be made to identify new markers of protection. The most important protective mechanism for serogroups A and C appears to be complement-mediated lysis by bactericidal antibodies [37]. For serogroup B, the protective mechanisms are less well understood. Some authors emphasised the role of opsonic antibodies and cellular components and it has recently been suggested to use a combination of tests to provide valuable information on the potential protection induced by candidate vaccines [59–62]. In conclusion, the N-propionylated group B polysaccharide conjugated to tetanus toxoid was well tolerated and no evidence of autoantibodies induced by the vaccine was found within the duration of the study. The vaccine is immunogenic in adults, but the induced antibodies

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lack functional activity in vitro. Further investigations are needed to clarify if protection is induced and to confirm the safety of the conjugate vaccine over time and in larger cohorts, particularly in teenagers and children under 1 year of age who are the main target populations of the vaccine. However, prior to test the vaccine in infants, more extensive preclinical toxicological studies may be recommended (e.g. in juvenile animals) although our in vitro tests have demonstrated no cross-reactivity with self antigens of the antibodies induced 1 month after each injection.

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