Clin. exp. Immunol. (1991) 83, 460-465
ADONIS
000991049100084A
Clonotypic analysis of human antibodies specific for Neisseria meningitidis polysaccharides A and C in adults S. LE MOLI, P. M. MATRICARDI, I. QUINTI*, T. STROFFOLINIt & R. D.'AMELIO Laboratory of Immunology, Department of Medicine, DASRS, Pratica di Mare, *Department of Clinical Immunology, University 'La Sapienza' of Rome, and tDepartment of Epidemiology, Istituto Superiore di Sanita', Rome, Italy (Acceptedfor publication 26 September 1990)
SUMMARY Serum antibodies to the capsular polysaccharides A and C (PSA and PSC) of N. meningitidis in healthy adults before and after vaccination with the sole polysaccharides were analysed by isoelectric focusing (IEF). Before vaccination, 49% and 28% had naturally acquired antibodies against PSA and PSC, respectively, whereas 18 days after vaccine administration 84% and 91%, respectively, showed a detectable spectrotypic pattern. Oligoclonality appeared to be the main feature of naturally acquired and vaccine-induced antibodies for both polysaccharides. In all subjects the anti-PSA response, showing dominant bands at the same pH position, was more homogeneous than anti-PSC one. Most subjects with naturally acquired antibodies (25 out of 38 for PSA and 20 out of 22 for PSC) showed a spectrotypic pattern after vaccination, similar to that observed before vaccination (any differences were just related to band intensity), suggesting that PSA and PSC are able to recruit the same B cell clones previously primed with a T-dependent form of the antigen, i.e. the whole bacterium. However, in one-third of subjects with naturally acquired anti-PSA antibodies, the appearance of new alkaline bands after vaccination was observed. Furthermore, in subjects with absence of detectable natural antibodies, the vaccine-induced antibody response started in correspondence of alkaline pH areas, subsequently extending to neutral and acidic areas. Therefore, it may be hypothesized that alkaline antibody-secreting B cell clones are the first to be recruited. The final spectrotype in these subjects was similar to that observed in subjects with naturally acquired antibodies. This observation, together with the above reported data, allow us to conclude that natural (T-dependent pathway) and vaccine (T-independent pathway) immunization induce the expression of the same antibody repertoire, for both meningococcal PSA and PSC. Keywords antibody clonotypes meningococcal polysaccharide A meningococcal polysaccharide C
Meningococcal polysaccharide A (PSA) and polysaccharide C (PSC) are structurally different: PSA is a linear homopolymer of (-1 6)mannosamine-6-phosphate residues O-acetylated at position 3; and PSC is a linear homopolymer of (-2-9)-linked sialic acid residues O-acetylated at position 7 and/or 8, but an O-acetyl negative form also exists (Egan, 1980). These two capsular subtypes of PSC, O-acetyl-positive and O-acetylnegative, contain common and distinct epitopes. In humans, natural antibodies against PSA are readily detectable and their titre increases with age. Conversely, natural antibodies against PSC appear later in life and their titre is generally lower (Gold, 1979). Vaccination with PSA seems to be effective also in children under 2 years of age in stimulating clinical protection (Gold et al., 1977; Frasch, 1983; Cadoz et al., 1985) more than vaccination with PSC (Gold, 1979). However, studies carried out on African children show a dramatic decline of response to PSA 3 years after vaccination (Reingold et al., 1985).
INTRODUCTION Neisseria meningitidis is a significant human pathogen that causes a variety of infections, most notably meningitis. Previous studies have shown that capsular polysaccharides, responsible for serogroup specificity, elicit the production of antibodies that are protective in humans (Gold, 1979; Frasch, 1983). However, the dynamics of the immune response against polysaccharide antigens have yet to be clarified. Problems such as unresponsiveness in children under 2 years of age (Smith et al., 1973; Gold et al., 1977; Borgono et al., 1978) or the possible role of T cells in this response (Baker et al., 1985; Muller & Apicella, 1988; Taylor & Bright, 1989), classically defined T independent (TI), as well as the diversity of the repertoire of human antibodies have yet to be defined. Correspondence: R. D'Amelio MD, Laboratory of Immunology, Department of Medicine, DASRS-00040 Pratica di Mare (RM), Italy.
460
Antibody spectrotypes to meningococcal PSA and PSC Because of these chemical and immunological differences between PSA and PSC, a comparative analysis of the heterogeneity of the antibody repertoire would contribute to clarify the immune response against polysaccharide antigens. This study was undertaken to evaluate the diversity of the repertoire of anti-PSA and anti-PSC antibodies, in a population of healthy adult men immunized with PSA + C vaccine. We used analytical isoelectric focusing (IEF), which has a level of sensitivity that easily copes with single clone products (Askonas, Williamson & Wright, 1970). Interesting comparative data on antibodies acquired through natural or vaccine immunization against these polysaccharide antigens have also been obtained. MATERIALS AND METHODS
Subjects and immunization One-hundred military recruits, aged 18-25 years, were vaccinated subcutaneously with a single dose of Menpovax A + C (Sclavo, Siena, Italy), containing 50 pg of each polysaccharide. This vaccine is commercially available in Italy and its protein content is less than 1%. Lipopolysaccharide (LPS) is present in traces as confirmed by its negative pyrogeneicity. Moreover, this vaccine contains only the O-acetylated form of PSC. Serum samples were obtained from each subject both before and 18 days after vaccination. Antibody level determination Antibody quantity determination was carried out using an ELISA. Flat-bottomed polystyrene microtest plates (Pro-bind, Falcon) were coated with 50 yg/ml meningococcal PSA or PSC (Merieux, France) in carbonate/bicarbonate buffer (0 05 M, pH 9-6) for 3 h at 370C and overnight at 40C. To minimize nonspecific absorbtion of serum protein to the plastic, the wells were incubated with a blocking solution consisting of 0-1% gelatine in phosphate-buffered saline (PBS) for 2 h at room temperature. After four washes with PBS containing 0 05% Tween 20 (PBST), the serum samples, optimally diluted (1/100) in PBS 0-1% gelatine, were added and incubated for 2 h at room temperature. After four washes with PBS-T, a 1/1000 in PBS-T dilution of alkaline phosphatase-conjugated goat F(ab')2 anti-human IgG (Tago) was added. After 30 sec of incubation at 37 C, the plates were washed three times with PBS-T and once with Tris-HCl (0-1 M, pH 8 6) and 100 p1 of p-nitrophenyl phosphate (Sigma p104-105), at I pg/ml in Tris-HCI (I M, pH 8-6), were added to each well. After 20 min, the reaction was stopped with 3 M NaOH. The OD was read at 405 nm by an automated photometer (Multiskan, Titertek). The test was standardized, also for the inter-assay variability, with a positive serum (kindly provided by Institute Merieux, Lyon, France) containing 37 Mg/ ml of anti-PSA IgG and 37 pg/ml of anti-PSC IgG. The results were expressed in pg/ml. The geometric mean concentrations of antibody and 95% confidence intervals for the geometric mean were calculated. In a group of 30 subjects, specific IgG subclasses were also measured by a four-step ELISA (Quinti et al., 1988). The conditions of antigen coating and serum sample incubations were the same used for IgG determination. Monoclonal antibody specific for human IgGI (SG-16), IgG2 (GOM-2), IgG3 (HP5060), IgG4 (SK-40) (Bio-Yeda, Rehovot, Israel) were
461
employed and the plates incubated for 2 h at 37°C. A goat antimouse immunoglobulin alkaline-phosphatase-conjugated was added for 1 h at 37°C and the reaction was revealed by adding the appropriate substrate (p-nitrophenyl phosphate). The OD was read after 30 min. Results were expressed as A OD, calculated for each subclass in each sample subtracting the control OD from the sample OD.
Labelling ofpolysaccharides Tyramine groups were covalently coupled directly to PSA and PSC by cyanogen bromide activation (Axen & Emback, 1977). Tyraminated PSA and PSC were then radioiodinated by the chloramine T method (Greenwood, Hunter & Glover, 1963) and tested by IEF against standard serum of Merieux (positive control) and against sera of healthy adult men with absence of specific antibodies (negative control), as detected by ELISA. The specificity of radiolabelled polysaccharide binding and IEF pattern was also tested. Cold meningococcal PSA or PSC were used to inhibit the binding of positive control sera with labelled meningococcal PSA or PSC. The inhibition test was also performed with a cold unrelated polysaccharide (Staphylococcus pneumoniae type 14 Ps). Agarose IEF analysis IEF was carried out as previously described (D'Amelio et al., 1989) with minor modifications. Antibodies to PSA and PSC were separated by IEF in agarose gel and visualized by autoradiography after labelling with '25I-PSA or '25I-PSC. Briefly, thin agarose gels (0 5 mm) containing 1% agarose IEF (Pharmacia), 10% sorbitol and 3% ampholyte (Pharmacia), pH 3 5-9-5, were formed by pouring agarose solution at 65°C into a pre-heated casting assembly where the gel solidified and bound firmly to Gel Bond (Pharmacia) film-subbed glass plates. Focusing was performed in horizontal slab gel on a Bio-Rad apparatus, cooled at 10°C by a Multitemp apparatus (LKB). The electrode strips contained 0-5 N sodium hydroxide (catholyte) or 0-5 M acetic acid (anolyte). Twenty microlitres of sera, diluted 1/4 in 0 9% NaCI were applied near the anode on paper sample strips. Standard serum from Merieux was utilized as positive control. Proteins were allowed to migrate and focus for about 90 min at constant power of 15 W. The pH gradient was measured by cutting pieces of gel every centimeter and placing them in 0 5 ml of H20. After focusing, the gels were treated according to Insel, Kittelberger & Anderson (1985), by incubation for I h in 21 % Na2SO4, followed by two I -h incubations in 18% Na2SO4, and then in 18% Na2SO4 with 1% bovine serum albumin (BSA) and 0-01 M phosphate buffer (PB) adjusted to pH 7. The gels were then overlaid with 1251-PSA or 251-PSC (2-5 x 107 ct/min) in 50 ml of 18% Na2SO4 with 1% BSA-PBS overnight. In the specificity assay the gel was incubated overnight with various dilutions of unlabelled meningococcal polysaccharide or unrelated one and washed before the incubation with radiolabelled polysaccharide. Gels were repeatedly washed with 18% Na2SO4 for 24 h, then fixed with 01% glutaraldehyde in 18% Na2SO4 for 1 h at room temperature. After further washes with PBS, followed by two 1-h washes in water, the gels were dehydrated by two 1-h incubations with 40% ethanol, air-dried, and autoradiographed (96 h with Kodak AR films).
S. Le Moli et al.
462
Table 1. Anti-meningococcal polysaccharide A and C antibody clonotypic analysis before and 18 days after vaccination in 77 healthy men
Subjects (%)
Clonotypic pattern Negative Monoclonal Oligoclonal Polyclonal
PSA
PSC
Day 0 Day 18
Day 0 Day 18
51 8 37 4
16 4 70 10
9 8 77 6
72 3 24 1
RESULTS
Specificity of IEF pattern In Fig. 1, the results of specificity assay on '25I-PSC binding are shown. As reported, cold PSC strongly inhibited the binding of specific antibodies with radiolabelled PSC. S. pneumoniae type 14 Ps was unable to block '251-PSC binding. The figure shows experiments in which the cold/labelled ratio was 50/1. Similar results were obtained also at different ratios (10/1, 100/1). Comparable patterns were obtained from the inhibition assays on '251-PSA binding (not shown). IEF pattern before vaccine administration Natural antibodies to PSA and PSC were detected by IEF in 38 out of 77 (49%) and in 22 out of 77 (28%) subjects, respectively, before immunization (Table 1). None had been vaccinated before this study or reported a meningococcal disease. Natural antibodies to PSA in the majority of sera ranged in a pH from 6 to 8 (Fig. 2 lanes I a, 2a, 3a, 7a, 8a). Conversely, anti-
5-0
PSC natural antibodies showed greater heterogeneity for pH position of bands. Oligoclonality was predominantly represented both for anti-PSA and for anti-PSC natural antibodies; few sera showed a monoclonal pattern and only four a polyclonal one (three of them only with PSA antigen). IEF pattern after vaccination (day 18) After immunization, specific antibodies to PSA and/or PSC were detectable by IEF in 84% and 91 % of subjects, respectively (Table 1). Oligoclonality was the main feature observed also at this time with both antigens (Table 1, Figs 2 and 3). With regard to anti-PSA response, subjects could be divided in three classes: (1) Subjects (25 out of 38) with natural antibodies before immunization showing a substantially unmodified spectrotype for number and pH distribution of bands, with a higher or unmodified intensity (Fig. 4, no. 4). (2) Those with natural antibodies before immunization whose spectrotype showed an addition of new bands (13 out of 38), all distributed in the alkaline zone (Fig. 2; nos 1-3, 6-8). (3) Those with no antibodies before immunization, who showed an oligoclonal pattern, covering a wide range of pH, from alkaline to acidic values (Fig 2, no. 5). Three subjects had only a mild response with a monoclonal pattern (Fig. 2, no. 4). The case of one subject with a paradoxic decrease of antiPSA IgG concentration (as detected by ELISA), with a modification of spectrotype from a polyclonal pattern, characterized by diffuse smears through the gel, to a monoclonal, mild positive one after immunization (Fig. 4, lanes la, lb and lc), should be noted. Compared with the anti-PSA response, the anti-PSC response (Fig. 3) showed a greater percentage of sera with no qualitative variation in the spectrotype (first class) (Fig. 3, no. 7) and only two subjects with addition of new bands to those already detectable before immunization (second class) (Fig. 3, no. 2). The great majority of subjects belonged to the third class, showing an entirely newly acquired spectrotype distributed from alkaline to acidic values of pH (Fig. 3, lanes I b, 3b, 4b, 5b, 6b). Only five subjects had a mild, monoclonal response, in the alkaline region (Fig. 3, lane lb).
IEF pattern at day 240 To investigate the qualitative evolution of anti-PSA and antiPSC response, IEF was performed in 20 sera 8 months after immunization. Almost all sera showed a marked increase in band intensity, but the number and the distribution of bands was substantially unchanged with respect to day 18 (Figs 4 and 5).
9-0
b
c
Fig. 1. Inhibition assay on 1251-PSC binding to positive control serum. Serum was focused, as described in Materials and Methods, in triplicate. Each replicate was pre-incubated with PBS-BSA, cold PSC or cold S. pneumoniae type 14 polysaccharide, and then incubated with the radiolabelled PSC. Lane a, sample pre-incubated with PBS-BSA; lane b, sample pre-incubated with cold PSC (cold/labelled ratio 50/1); lane c, sample pre-incubated with cold S. pneumoniae type 14 polysaccharide (ratio 50/1).
Comparison of ELISA and IEF We observed an overall good relationship between ELISA and IEF with regard to the percentage of seroconversion. Sera negative at IEF analysis had generally low or absent antipolysaccharide IgG levels (Table 2). No correlation was found, on the contrary, between specific IgG concentrations and spectrotype pattern complexity in individual sera (Figs 2-5,
legends). Specific IgG subclass evaluation on 30 subjects showed that the response was distributed among all four subclasses, with a prevalence of IgG1 to PSA and IgG2 to PSC (data not shown).
Antibody spectrotypes to meningococcal PSA and PSC
463 4
aw 6 7001I I i_-I_
9 0
i[
"
2a
lb
2b
3a
3b 4a-
4b
5c 5b
6a 6b
7a
8b
7b 80
Fig. 2. Spectrotypic pattern of anti-meningococcal PSA antibodies in sera from adult male subjects before and after vaccination with Menpovax A+C. Focused immunoglobulins were overlaid with '25I-PSA and autoradiographed. Numbers 1-8 refer to subjects. a, Sample before vaccination; b, sample 18 days after vaccination. Specific IgG titres (pg/ml) are given. Subject 1, a 0-9, b 6 9; subject 2, a
ADONIS
000991049100084A
Clonotypic analysis of human antibodies specific for Neisseria meningitidis polysaccharides A and C in adults S. LE MOLI, P. M. MATRICARDI, I. QUINTI*, T. STROFFOLINIt & R. D.'AMELIO Laboratory of Immunology, Department of Medicine, DASRS, Pratica di Mare, *Department of Clinical Immunology, University 'La Sapienza' of Rome, and tDepartment of Epidemiology, Istituto Superiore di Sanita', Rome, Italy (Acceptedfor publication 26 September 1990)
SUMMARY Serum antibodies to the capsular polysaccharides A and C (PSA and PSC) of N. meningitidis in healthy adults before and after vaccination with the sole polysaccharides were analysed by isoelectric focusing (IEF). Before vaccination, 49% and 28% had naturally acquired antibodies against PSA and PSC, respectively, whereas 18 days after vaccine administration 84% and 91%, respectively, showed a detectable spectrotypic pattern. Oligoclonality appeared to be the main feature of naturally acquired and vaccine-induced antibodies for both polysaccharides. In all subjects the anti-PSA response, showing dominant bands at the same pH position, was more homogeneous than anti-PSC one. Most subjects with naturally acquired antibodies (25 out of 38 for PSA and 20 out of 22 for PSC) showed a spectrotypic pattern after vaccination, similar to that observed before vaccination (any differences were just related to band intensity), suggesting that PSA and PSC are able to recruit the same B cell clones previously primed with a T-dependent form of the antigen, i.e. the whole bacterium. However, in one-third of subjects with naturally acquired anti-PSA antibodies, the appearance of new alkaline bands after vaccination was observed. Furthermore, in subjects with absence of detectable natural antibodies, the vaccine-induced antibody response started in correspondence of alkaline pH areas, subsequently extending to neutral and acidic areas. Therefore, it may be hypothesized that alkaline antibody-secreting B cell clones are the first to be recruited. The final spectrotype in these subjects was similar to that observed in subjects with naturally acquired antibodies. This observation, together with the above reported data, allow us to conclude that natural (T-dependent pathway) and vaccine (T-independent pathway) immunization induce the expression of the same antibody repertoire, for both meningococcal PSA and PSC. Keywords antibody clonotypes meningococcal polysaccharide A meningococcal polysaccharide C
Meningococcal polysaccharide A (PSA) and polysaccharide C (PSC) are structurally different: PSA is a linear homopolymer of (-1 6)mannosamine-6-phosphate residues O-acetylated at position 3; and PSC is a linear homopolymer of (-2-9)-linked sialic acid residues O-acetylated at position 7 and/or 8, but an O-acetyl negative form also exists (Egan, 1980). These two capsular subtypes of PSC, O-acetyl-positive and O-acetylnegative, contain common and distinct epitopes. In humans, natural antibodies against PSA are readily detectable and their titre increases with age. Conversely, natural antibodies against PSC appear later in life and their titre is generally lower (Gold, 1979). Vaccination with PSA seems to be effective also in children under 2 years of age in stimulating clinical protection (Gold et al., 1977; Frasch, 1983; Cadoz et al., 1985) more than vaccination with PSC (Gold, 1979). However, studies carried out on African children show a dramatic decline of response to PSA 3 years after vaccination (Reingold et al., 1985).
INTRODUCTION Neisseria meningitidis is a significant human pathogen that causes a variety of infections, most notably meningitis. Previous studies have shown that capsular polysaccharides, responsible for serogroup specificity, elicit the production of antibodies that are protective in humans (Gold, 1979; Frasch, 1983). However, the dynamics of the immune response against polysaccharide antigens have yet to be clarified. Problems such as unresponsiveness in children under 2 years of age (Smith et al., 1973; Gold et al., 1977; Borgono et al., 1978) or the possible role of T cells in this response (Baker et al., 1985; Muller & Apicella, 1988; Taylor & Bright, 1989), classically defined T independent (TI), as well as the diversity of the repertoire of human antibodies have yet to be defined. Correspondence: R. D'Amelio MD, Laboratory of Immunology, Department of Medicine, DASRS-00040 Pratica di Mare (RM), Italy.
460
Antibody spectrotypes to meningococcal PSA and PSC Because of these chemical and immunological differences between PSA and PSC, a comparative analysis of the heterogeneity of the antibody repertoire would contribute to clarify the immune response against polysaccharide antigens. This study was undertaken to evaluate the diversity of the repertoire of anti-PSA and anti-PSC antibodies, in a population of healthy adult men immunized with PSA + C vaccine. We used analytical isoelectric focusing (IEF), which has a level of sensitivity that easily copes with single clone products (Askonas, Williamson & Wright, 1970). Interesting comparative data on antibodies acquired through natural or vaccine immunization against these polysaccharide antigens have also been obtained. MATERIALS AND METHODS
Subjects and immunization One-hundred military recruits, aged 18-25 years, were vaccinated subcutaneously with a single dose of Menpovax A + C (Sclavo, Siena, Italy), containing 50 pg of each polysaccharide. This vaccine is commercially available in Italy and its protein content is less than 1%. Lipopolysaccharide (LPS) is present in traces as confirmed by its negative pyrogeneicity. Moreover, this vaccine contains only the O-acetylated form of PSC. Serum samples were obtained from each subject both before and 18 days after vaccination. Antibody level determination Antibody quantity determination was carried out using an ELISA. Flat-bottomed polystyrene microtest plates (Pro-bind, Falcon) were coated with 50 yg/ml meningococcal PSA or PSC (Merieux, France) in carbonate/bicarbonate buffer (0 05 M, pH 9-6) for 3 h at 370C and overnight at 40C. To minimize nonspecific absorbtion of serum protein to the plastic, the wells were incubated with a blocking solution consisting of 0-1% gelatine in phosphate-buffered saline (PBS) for 2 h at room temperature. After four washes with PBS containing 0 05% Tween 20 (PBST), the serum samples, optimally diluted (1/100) in PBS 0-1% gelatine, were added and incubated for 2 h at room temperature. After four washes with PBS-T, a 1/1000 in PBS-T dilution of alkaline phosphatase-conjugated goat F(ab')2 anti-human IgG (Tago) was added. After 30 sec of incubation at 37 C, the plates were washed three times with PBS-T and once with Tris-HCl (0-1 M, pH 8 6) and 100 p1 of p-nitrophenyl phosphate (Sigma p104-105), at I pg/ml in Tris-HCI (I M, pH 8-6), were added to each well. After 20 min, the reaction was stopped with 3 M NaOH. The OD was read at 405 nm by an automated photometer (Multiskan, Titertek). The test was standardized, also for the inter-assay variability, with a positive serum (kindly provided by Institute Merieux, Lyon, France) containing 37 Mg/ ml of anti-PSA IgG and 37 pg/ml of anti-PSC IgG. The results were expressed in pg/ml. The geometric mean concentrations of antibody and 95% confidence intervals for the geometric mean were calculated. In a group of 30 subjects, specific IgG subclasses were also measured by a four-step ELISA (Quinti et al., 1988). The conditions of antigen coating and serum sample incubations were the same used for IgG determination. Monoclonal antibody specific for human IgGI (SG-16), IgG2 (GOM-2), IgG3 (HP5060), IgG4 (SK-40) (Bio-Yeda, Rehovot, Israel) were
461
employed and the plates incubated for 2 h at 37°C. A goat antimouse immunoglobulin alkaline-phosphatase-conjugated was added for 1 h at 37°C and the reaction was revealed by adding the appropriate substrate (p-nitrophenyl phosphate). The OD was read after 30 min. Results were expressed as A OD, calculated for each subclass in each sample subtracting the control OD from the sample OD.
Labelling ofpolysaccharides Tyramine groups were covalently coupled directly to PSA and PSC by cyanogen bromide activation (Axen & Emback, 1977). Tyraminated PSA and PSC were then radioiodinated by the chloramine T method (Greenwood, Hunter & Glover, 1963) and tested by IEF against standard serum of Merieux (positive control) and against sera of healthy adult men with absence of specific antibodies (negative control), as detected by ELISA. The specificity of radiolabelled polysaccharide binding and IEF pattern was also tested. Cold meningococcal PSA or PSC were used to inhibit the binding of positive control sera with labelled meningococcal PSA or PSC. The inhibition test was also performed with a cold unrelated polysaccharide (Staphylococcus pneumoniae type 14 Ps). Agarose IEF analysis IEF was carried out as previously described (D'Amelio et al., 1989) with minor modifications. Antibodies to PSA and PSC were separated by IEF in agarose gel and visualized by autoradiography after labelling with '25I-PSA or '25I-PSC. Briefly, thin agarose gels (0 5 mm) containing 1% agarose IEF (Pharmacia), 10% sorbitol and 3% ampholyte (Pharmacia), pH 3 5-9-5, were formed by pouring agarose solution at 65°C into a pre-heated casting assembly where the gel solidified and bound firmly to Gel Bond (Pharmacia) film-subbed glass plates. Focusing was performed in horizontal slab gel on a Bio-Rad apparatus, cooled at 10°C by a Multitemp apparatus (LKB). The electrode strips contained 0-5 N sodium hydroxide (catholyte) or 0-5 M acetic acid (anolyte). Twenty microlitres of sera, diluted 1/4 in 0 9% NaCI were applied near the anode on paper sample strips. Standard serum from Merieux was utilized as positive control. Proteins were allowed to migrate and focus for about 90 min at constant power of 15 W. The pH gradient was measured by cutting pieces of gel every centimeter and placing them in 0 5 ml of H20. After focusing, the gels were treated according to Insel, Kittelberger & Anderson (1985), by incubation for I h in 21 % Na2SO4, followed by two I -h incubations in 18% Na2SO4, and then in 18% Na2SO4 with 1% bovine serum albumin (BSA) and 0-01 M phosphate buffer (PB) adjusted to pH 7. The gels were then overlaid with 1251-PSA or 251-PSC (2-5 x 107 ct/min) in 50 ml of 18% Na2SO4 with 1% BSA-PBS overnight. In the specificity assay the gel was incubated overnight with various dilutions of unlabelled meningococcal polysaccharide or unrelated one and washed before the incubation with radiolabelled polysaccharide. Gels were repeatedly washed with 18% Na2SO4 for 24 h, then fixed with 01% glutaraldehyde in 18% Na2SO4 for 1 h at room temperature. After further washes with PBS, followed by two 1-h washes in water, the gels were dehydrated by two 1-h incubations with 40% ethanol, air-dried, and autoradiographed (96 h with Kodak AR films).
S. Le Moli et al.
462
Table 1. Anti-meningococcal polysaccharide A and C antibody clonotypic analysis before and 18 days after vaccination in 77 healthy men
Subjects (%)
Clonotypic pattern Negative Monoclonal Oligoclonal Polyclonal
PSA
PSC
Day 0 Day 18
Day 0 Day 18
51 8 37 4
16 4 70 10
9 8 77 6
72 3 24 1
RESULTS
Specificity of IEF pattern In Fig. 1, the results of specificity assay on '25I-PSC binding are shown. As reported, cold PSC strongly inhibited the binding of specific antibodies with radiolabelled PSC. S. pneumoniae type 14 Ps was unable to block '251-PSC binding. The figure shows experiments in which the cold/labelled ratio was 50/1. Similar results were obtained also at different ratios (10/1, 100/1). Comparable patterns were obtained from the inhibition assays on '251-PSA binding (not shown). IEF pattern before vaccine administration Natural antibodies to PSA and PSC were detected by IEF in 38 out of 77 (49%) and in 22 out of 77 (28%) subjects, respectively, before immunization (Table 1). None had been vaccinated before this study or reported a meningococcal disease. Natural antibodies to PSA in the majority of sera ranged in a pH from 6 to 8 (Fig. 2 lanes I a, 2a, 3a, 7a, 8a). Conversely, anti-
5-0
PSC natural antibodies showed greater heterogeneity for pH position of bands. Oligoclonality was predominantly represented both for anti-PSA and for anti-PSC natural antibodies; few sera showed a monoclonal pattern and only four a polyclonal one (three of them only with PSA antigen). IEF pattern after vaccination (day 18) After immunization, specific antibodies to PSA and/or PSC were detectable by IEF in 84% and 91 % of subjects, respectively (Table 1). Oligoclonality was the main feature observed also at this time with both antigens (Table 1, Figs 2 and 3). With regard to anti-PSA response, subjects could be divided in three classes: (1) Subjects (25 out of 38) with natural antibodies before immunization showing a substantially unmodified spectrotype for number and pH distribution of bands, with a higher or unmodified intensity (Fig. 4, no. 4). (2) Those with natural antibodies before immunization whose spectrotype showed an addition of new bands (13 out of 38), all distributed in the alkaline zone (Fig. 2; nos 1-3, 6-8). (3) Those with no antibodies before immunization, who showed an oligoclonal pattern, covering a wide range of pH, from alkaline to acidic values (Fig 2, no. 5). Three subjects had only a mild response with a monoclonal pattern (Fig. 2, no. 4). The case of one subject with a paradoxic decrease of antiPSA IgG concentration (as detected by ELISA), with a modification of spectrotype from a polyclonal pattern, characterized by diffuse smears through the gel, to a monoclonal, mild positive one after immunization (Fig. 4, lanes la, lb and lc), should be noted. Compared with the anti-PSA response, the anti-PSC response (Fig. 3) showed a greater percentage of sera with no qualitative variation in the spectrotype (first class) (Fig. 3, no. 7) and only two subjects with addition of new bands to those already detectable before immunization (second class) (Fig. 3, no. 2). The great majority of subjects belonged to the third class, showing an entirely newly acquired spectrotype distributed from alkaline to acidic values of pH (Fig. 3, lanes I b, 3b, 4b, 5b, 6b). Only five subjects had a mild, monoclonal response, in the alkaline region (Fig. 3, lane lb).
IEF pattern at day 240 To investigate the qualitative evolution of anti-PSA and antiPSC response, IEF was performed in 20 sera 8 months after immunization. Almost all sera showed a marked increase in band intensity, but the number and the distribution of bands was substantially unchanged with respect to day 18 (Figs 4 and 5).
9-0
b
c
Fig. 1. Inhibition assay on 1251-PSC binding to positive control serum. Serum was focused, as described in Materials and Methods, in triplicate. Each replicate was pre-incubated with PBS-BSA, cold PSC or cold S. pneumoniae type 14 polysaccharide, and then incubated with the radiolabelled PSC. Lane a, sample pre-incubated with PBS-BSA; lane b, sample pre-incubated with cold PSC (cold/labelled ratio 50/1); lane c, sample pre-incubated with cold S. pneumoniae type 14 polysaccharide (ratio 50/1).
Comparison of ELISA and IEF We observed an overall good relationship between ELISA and IEF with regard to the percentage of seroconversion. Sera negative at IEF analysis had generally low or absent antipolysaccharide IgG levels (Table 2). No correlation was found, on the contrary, between specific IgG concentrations and spectrotype pattern complexity in individual sera (Figs 2-5,
legends). Specific IgG subclass evaluation on 30 subjects showed that the response was distributed among all four subclasses, with a prevalence of IgG1 to PSA and IgG2 to PSC (data not shown).
Antibody spectrotypes to meningococcal PSA and PSC
463 4
aw 6 7001I I i_-I_
9 0
i[
"
2a
lb
2b
3a
3b 4a-
4b
5c 5b
6a 6b
7a
8b
7b 80
Fig. 2. Spectrotypic pattern of anti-meningococcal PSA antibodies in sera from adult male subjects before and after vaccination with Menpovax A+C. Focused immunoglobulins were overlaid with '25I-PSA and autoradiographed. Numbers 1-8 refer to subjects. a, Sample before vaccination; b, sample 18 days after vaccination. Specific IgG titres (pg/ml) are given. Subject 1, a 0-9, b 6 9; subject 2, a
