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. 2018 Sep 20;194(3):361–370. doi: 10.1111/cei.13202

Immunogenicity of meningococcal polysaccharide ACWY vaccine in primary immunized or revaccinated adults

C Ferlito 1,†, R Biselli 2,†, M S Cattaruzza 3, R Teloni 4, S Mariotti 4, E Tomao 5, G Salerno 1, M S Peragallo 6, P Lulli 1,‡, S Caporuscio 1, A Autore 7, G Bizzarro 7, V Germano 1, M I Biondo 1, A Picchianti Diamanti 1, S Salemi 1, R Nisini 4,†,✉, R D'Amelio 1,†,‡
PMCID: PMC6231002  PMID: 30099753

Summary

Meningococcal polysaccharide (Men‐Ps) vaccine immunogenicity following either primary immunization or revaccination in adults was evaluated. The study population consisted of subjects who have received tetravalent Men‐Ps vaccine once (group 1) or at least twice, with a 2–6 dose range (group 2). Human leucocyte antigen (HLA)‐typing was performed by polymerase chain reaction and specific immunoglobulin (Ig)G was measured by enzyme‐linked immunosorbent assay. Nine months post‐immunization, the percentages of individuals with levels of anti‐Men‐Ps IgG ≥ 2 µg/ml were comparable in both groups, with the exception of anti‐Men‐PsW135 IgG, which were significantly higher in group 2. The percentage of subjects doubling IgG levels at 9 months was significantly higher in group 1. The high baseline anti‐Men‐Ps antibody levels negatively influenced the response to revaccination, suggesting a feedback control of specific IgG. The calculated durability of anti‐Men‐Ps IgG was 2·5–4·5 years, depending on the Men‐Ps, following a single vaccine dose. No interference by other vaccinations nor HLA alleles association with immune response were observed. This study confirms that Men‐Ps vaccine in adults is immunogenic, even when administered repeatedly, and underlines the vaccine suitability for large‐scale adult immunization programmes that the higher costs of conjugate vaccines may limit in developing countries.

Keywords: antibodies, bacterial, vaccination

Introduction

Neisseria meningitidis is a Gram‐negative diplococcus, which may colonize the human nasopharynx and occasionally leads to invasive meningococcal disease (IMD) in selected individuals; that is, invasion of the bloodstream with septic shock and/or transition through the blood–brain barrier with consequent meningococcal meningitis 1. Approximately 500 000 annual cases of IMD occur worldwide, with an estimated lethality of 10% 2.

Based on the capsular polysaccharide (Ps) structure, Neisseria meningitidis can be classified into 13 serogroups (A, B, C, D, 29E, H, I, K, L, W135, X, Y, Z), six of which (A, B, C, W135, X, Y) are responsible for most IMD cases in humans. In particular, A, B, C, and W135 are the main serogroups responsible for epidemics, whereas X and Y are particularly responsible for endemic disease and occasional outbreaks 3.

Since the second half of the last century meningococcal (Men) capsular Ps vaccines [first monovalent, secondly bivalent and thirdly tetravalent (A, C, W135 and Y) ] have been available 4, 5. These vaccines have influenced the epidemiology of IMD favourably 1, 6, 7, but only in adolescents and adults, as infants aged < 18 months are almost unable to respond to these T cell‐independent Ps antigens 8. Consequently, conjugate protein‐Ps vaccines have been designed to overcome the lack of immunogenicity of Ps vaccines in infancy 9. Moreover, conjugate protein‐Ps vaccines, by transforming T cell‐independent into T cell‐dependent antigens, are believed to stimulate B cell memory effectively, thus increasing the specific immune response. In particular, it has been proposed that even in periodically revaccinated adults, the use of conjugate instead of plain Ps vaccines may be preferable to prevent the risk of depletion of the peripheral memory B cell pool 10. Furthermore, the response may even be influenced by concomitant administration of other vaccines 11.

In the current study, we analysed the tetravalent Men‐Ps vaccine immune response in two groups of military personnel. The first group was represented by students of military training schools, who received their first vaccination against N. meningitidis, and the second group by older soldiers frequently operating abroad who, over several years, received multiple vaccinations against N. meningitidis (range = 2–6). Comparative analysis of the two groups permitted evaluation of the consequences of revaccination in the model of T cell‐independent antigens. This study is part of a larger survey on the safety and immunogenicity of vaccinations in the Italian military; all the military participating in the project were typed for human leucocyte antigen (HLA) in order to analyse its possible influence on the immune response to vaccinations.

Materials and methods

Study population

From September 2012 to June 2014 two groups of military personnel were enrolled. Group 1 was represented by military students of training schools for both officers and non‐commissioned officers, who were vaccinated with Men‐Ps at enlistment. Group 2 was represented by the personnel of the ‘Lancieri di Montebello’ regiment, whose operational activity includes periodic missions to Lebanon. They were vaccinated with Men‐Ps at least twice (range = 2–6, mean = 3·6) at enlistment and revaccinated periodically for international missions. The minimum, maximum and mean intervals between multiple doses of tetravalent Men‐Ps in this group were 2, 10 and 5, respectively. The vaccination schedule was described previously 12. Briefly, depending on clinical history, a personalized vaccination schedule was defined, which could include the following vaccines for the two groups: tetanus/diphtheria for adults [Td, DifTetAll; Novartis Vaccines and Diagnostics (now GSK), Siena, Italy], inactivated polio (Imovax polio; Sanofi Pasteur MSD SpA, Roma, Italy), measles/mumps/rubella (MMR; Priorix‐GlaxoSmithKline SpA, Verona, Italy), varicella (Varivax; Sanofi Pasteur MSD SpA, Roma, Italy), tetravalent (A, C, W135,Y) Men‐Ps (Mencevax; Pzifer Srl, Latina, Italy), hepatitis A (HA; Epaxal‐Crucell Italy Srl, Baranzate, Italy) and hepatitis B (HB, Engerix B; GlaxoSmithKline SpA, Verona, Italy). Influenza (Fluad; Seqirus Srl, Siena, Italy) and typhoid (Vivotif; Berna‐PaxVax Ltd, Birmingham, UK) were only administered to group 2 subjects. Vaccines were generally administered on the same day (in different arms) but, in a few cases, up to 2 weeks apart.

The study was approved by the Ministry of Defence ethical committee in 2011 and was registered in clinicaltrials.gov in 2012 with the identifier NCT01807780. After informed consent, blood samples were collected at T0 (prevaccination), T1 and T2 (1 and 9 months post‐vaccination, respectively) and at T0 and T2 (before and after a 9‐month‐long international mission) in groups 1 and 2 subjects, respectively.

Specific anti‐Men‐PsA, C, W135, Y antibodies

Specific antibodies were analysed by enzyme‐linked immunosorbent assay (ELISA), in agreement with previously published studies 4, 13. Briefly, 96‐well plates were coated with 100 μl/well of A, C, W135, Y Men‐Ps, kindly donated by Novartis Vaccines (now GSK), Siena, Italy, at a concentration of 10 μg/ml in phosphate‐buffered saline (PBS) pH 8·2 and incubated at +4°C overnight. After three washes with PBS supplemented with 0·005% Tween 20 (TPBS), a blocking step of 100 μl/well of TPBS with bovine serum albumin (BSA) 3% was performed for 1 h at 37°C. After three more washes, 100 μl/well of the samples and control serum [kindly donated by Novartis Vaccines (now GSK), Siena, Italy] diluted in TPBS 1 : 200 were incubated for 2 h at 37°C. After three further washes, 100 μl/well of the alkaline phosphatase‐conjugated recognition anti‐human immunoglobulin (Ig)G anti‐serum diluted 1 : 1000 in TPBS were added for 1 h at 37°C. Finally, 100 μl/well of substrate (paranitrophenil phosphate/5 ml carbonate–bicarbonate) were added and left at room temperature in the dark. The reaction was blocked by NaOH 3M and the absorbance read at 405 nm. The concentration of Men‐Ps‐specific IgG in test sera was obtained by plotting the control serum dilutions (from 1 : 50 to 1 : 6400) and the corresponding IgG concentration values (μg/ml). As the titre of serum bactericidal antibodies is considered the gold standard for meningococcal seroprotection and IgG ELISA values are considered a surrogate of protection 14, we defined the IgG concentration ≥ 2 μg/ml as a ‘putative’ seroprotective level 11, 15, 16, 17, 18, 19. The seroconversion, defined as a twofold increase in IgG concentrations from T0 to T2, was considered a measure to identify subject responders to vaccination or booster doses 11.

HLA typing

DNA extraction from ethylenediamine tetraacetic acid (EDTA)‐treated whole blood was performed using a Qiagen kit (Qiagen Inc., Germantown, MD, USA) as per the manufacturer's protocol. HLA‐A,‐B,‐C loci, belonging to HLA class I, were genotyped at low molecular resolution (at allele group level) using polymerase chain reaction sequence‐specific oligonucleotide (PCR‐SSO) assays, based on the reverse‐hybridization principle (typing kits INNO‐LiPA® HLA‐A,‐B,‐C, Fujirebio Europe N.V., Ghent, Belgium). HLA‐DRB1 and ‐DQB1 loci, belonging to HLA class II, were typed by the PCR sequence‐specific primers (PCR‐SSP) method (micro SSP DNA typing kits; One Lambda Inc., Canoga Park, CA, USA).

HLA allele frequencies were estimated by direct counting in the study subjects. The alleles were identified assuming that HLA‐A,‐B,‐C,‐DRB1,‐DQB1 loci have no blanks. When a single allele was found, the individual was considered homozygous for that allele.

Statistical analysis

Geometric mean concentrations (GMCs) and their 95% confidence intervals (CI) were calculated to describe the IgG ELISA response for each group, pre‐ and post‐vaccination. Antibody concentration below the ELISA limit of detection were arbitrarily assigned a value of 0·001 μg/ml for analyses. All comparisons between groups were carried out from log‐transformed data, using a t‐test. Categorical variables were analysed by Yates'‐corrected, two‐tailed χ2 test. Correlation was explored by Spearman's test. P‐values ≤ 0·05 were considered significant. Statistical analysis was performed using the GraphPadPrism package version 5.0 (GraphPad Software Inc., San Diego, CA, USA). Two logistic regression analyses were carried out, the first using the putatively protected subjects at T2 as dependent variable (anti‐Men‐Ps IgG ≥ 2 µg/ml) and the second using the subjects defined as responders at T2 as dependent variable (anti‐Men‐Ps IgG at T2/anti‐Men‐Ps IgG at T0 ≥ 2), and in both as covariates ln(anti‐Men‐Ps IgG levels) at T0, age, number of Men‐Ps re‐vaccinations, and number of concomitant vaccinations. We used r software for the test (copyright 2004–2013; The R Foundation for Statistical Computing, Vienna, Austria).

The half‐life of vaccination‐induced antibodies was calculated with the following equation: log(Men‐Ps antibody concentration) = α + β* years + ε, where α represented mean log concentration at time of vaccination; β represented decay rate and ε represented the error term (β  =  [log(antibody concentration)‐α‐ε]/years 20.

Results

Study population

Group 1 consisted of 141 subjects vaccinated with Men‐Ps for the first time (mean age ± standard deviation = 20·7 ± 2·53, 119 males) and group 2 of 57 subjects vaccinated with Men‐Ps at least twice (range = 2–6, mean = 3·6) (mean age ± standard deviation = 30·83 ± 4·53, 52 males). The difference between the two groups was highly significant for age (P < 0.0001), but not for sex (Table 1). Table 1 also summarizes the number and type of vaccinations administered in addition to Men‐Ps: the number of concomitant received vaccinations between the two groups was significantly different.

Table 1.

Demographic/immunization characteristics and immune response of the two military groups

Characteristics Group 1 Group 2
n subjects 141 57
M/F (%) 119/22 (84/16) 52/5 (91/9)
Age (years) 20·7 ± 2·5# 30·8 ± 4·5
Men vaccinations; mean   ±   standard deviation 1# 3·61 ± 0·93
Concomitant vaccinations; mean   ±   standard deviation 2·18 ± 0·98# 3·17 ± 0·75
n (%) tetanus/diphtheria Vaccination 26 (18%) 11 (19%)
n (%) polio vaccination 44 (31%) 9 (16%)
n (%) measles/mumps/rubella vaccination 43 (30%) 1 (1·7%)
n (%) hepatitis A vaccination 49 (35%) 5 (22%)
n (%) influenza vaccination 0 57 (100%)
n (%) typhoid vaccination 0 39 (68%)
Geometric mean concentrations T0 T2 T0 T2
PsA 0·56 (0·47–0·66) 21·64 (16·4–28·5) 5·19 (3·35–8·05) 17·79 (11·5–27·6)
PsC 0·04 (0·02–0·07) 5·86 (3·9–8·76) 6·48 (3·7–11·3) 9·00 (5·3–15·3)
PsW135 0·39 (0·35–0·4) 2·30 (1·7–3·1) 4·72 (3·2–6·9) 7·24 (4·9–10·6)
PsY 0·34 (0·3–0·37) 3·98 (2·8–5·6) 2·46 (1·5–4·0) 4·86 (2·7–8·6)
Protected (≥ 2 μg/ml) T0 T2 T0 T2
PsA 11/141 (8%)## 128/141 (91%) 42/57 (74%) 51/57 (89%)
PsC 12/141 (8·5%)## 108/141 (76%) 41/57 (72%) 45/57 (79%)
PsW135 3/141 (2%)## 61/141 (43%)### 38/57 (66%) 46/57 (81%)
PsY 3/141 (2%)## 84/141 (59·5%) 32/57 (56%) 33/57 (58%)
Responders to vaccination (T2/T0 ≥ 2) T2 T2
PsA 134/141 (95%)* 37/57 (65%)
PsC 122/141 (86·5%)* 15/57 (26%)
PsW135 96/141 (68%)* 14/57 (24%)
PsY 102/141 (72%)** 25/57 (44%)
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#P < 0·0001 versus group 2 by Student's t–test; ##P < 0·0000001 versus group 2 T0; ###P = 0·00004 versus group 2 T2; *P < 0·0000001 and **P = 0·0003 versus group 2 by χ2 Yates' corrected, two‐tailed. M/F = male/female.

Antibody response to tetravalent Men‐Ps vaccine

GMCs and 95% CI at T0 and T2 in both groups are shown in Table 1 and Fig. 1. GMCs were higher in group 2 than in group 1 both at T0 (GMC T2/T0 ratio: PsA = 9·26; PsC = 162; PsW135  = 12·1 and PsY = 7·23) and at T2, with the exception of PsA (GMC T2/T0 ratio: PsA = 0·82; PsC = 1·53; PsW135  = 3·15 and PsY = 1·22).

Figure 1.

Figure 1

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Humoral response to tetravalent meningococcal Ps vaccine. Squares represent group 1 and triangles group 2 subjects. Geometric mean concentrations (ln) of Men‐Ps antibody are represented (black bars). *P‐value T0 versus T2 < 0·0001 (t‐test of natural logarithm); **P‐value T0 versus T2 = 0·01 (t‐test of natural logarithm).

At T0, 11 of 141 (8%) for PsA, 12 of 141 (8·5%) for PsC, three of 141 (2%) for PsW135 and three of 141 (2%) for PsY group 1 subjects had Ps‐specific IgG ≥ 2 μg/ml. A significantly higher number of group 2 subjects had Ps‐specific IgG ≥ 2 μg/ml, 42 of 57 (74%) for PsA, 41 of 57 (72%) for PsC, 38 of 57 (66%) for PsW135 and 32 of 57 (56%) for PsY (Table 1). The number of previous Men‐Ps vaccinations was correlated significantly and directly with the prevaccination anti‐Ps antibody levels for all Men‐Ps (Fig. 2).

Figure 2.

Figure 2

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Correlation between Ps‐specific antibodies concentration at T0 and number of previous Men‐vaccinations. Anti‐Men‐Ps immunoglobulin (Ig)G at T0 were divided by number of previous Men vaccines; the direct correlation between the two variables is represented by Spearman's coefficient (r). The dotted line represents the cut‐off for seroprotection.

At T2, subjects in group 1 with specific IgG ≥ 2 μg/ml increased up to 128 of 141 (91%) for PsA, 108 of 141 (76%) for PsC, 61 of 141 (43%) for PsW135 and 84 of 141 (59·5%) for PsY, whereas subjects with specific IgG ≥ 2 μg/ml were 51 of 57 (89%) for PsA, 45 of 57 (79%) for PsC, 46 of 57 (81%) for PsW135 and 33 of 57 (58%) for PsY in group 2 (Table 1). The percentages of subjects with specific IgG ≥ 2 μg/ml were similar in the two groups, with the exception of IgG anti‐PsW135, which were ≥ 2 μg/ml in a significantly higher percentage of subjects in group 2. The subjects doubling their baseline specific IgG levels were significantly higher in group 1 (responders to vaccination) than in group 2 [responders to booster(s)] for all Men‐Ps (Table 1).

The response to vaccination/booster of the whole population (groups 1 and 2) was analysed further by univariate analysis (Table 2) or logistic regression (Table 3) to investigate the population characteristics associated with baseline IgG ≥ 2 μg/ml or < 2 μg/ml.

Table 2.

Demographic/immunization characteristics and antibody response of the whole population of military personnel according to the prevaccination antibody levels (T0)

Anti‐PsA (T0) Anti‐PsC (T0) Anti‐PsW135 (T0) Anti‐PsY (T0)
Characteristics < 2 μg/ml ≥ 2 μg/ml < 2 μg/ml ≥ 2 μg/ml < 2 μg/ml ≥ 2 μg/ml < 2 μg/ml ≥ 2 μg/ml
n (%) subjects 145 (73%) 53 (27%) 145 (73%) 53 (27%) 157 (79%) 41 (21%) 163 (82%) 35 (18%)
n (%) female 22 (15%) 5 (6%) 23 (16%) 4 (7·5%) 25 (16%) 2 (5%) 27(16·5%) 0
Age (years) 21·8 ± 4·1 28·6 ± 6·1 21·9 ± 4·3 28·35  ±  6 21·9 ± 4·3 30·3 ± 5·2 22·8 ± 5·3 28·5 ± 4·9
Previous Men‐Ps vaccinations#; mean  ±  standard deviation 1·25 ± 0·8 3·1 ± 1·4 1·3 ± 0·9 3 ± 1·4 1·3 ± 0·8 3·6 ± 1·12 1·4 ± 0·9 3·6 ± 1·3
Concomitant vaccinations#; mean  ±  standard deviation 2·3 ± 1 3 ± 0·9 2·25 ± 1 3 ± 0·8 2·3 ± 1 3 ± 0·9‡ 2·3 ± 1 3·05 ± 0·8§
n (%) Tetanus/diphtheria vaccination 24 (16·5%) 13 (24·5%) 27 (19%) 10 (19%) 28 (18%) 9 (22·5%) 32 (20%) 5 (14%)
n (%) polio vaccination 42 (29%) 11 (21%) 41 (28%) 12 (23%) 48 (30%) 5 (12·5%) 47 (29%) 6 (17%)
n (%) measles/mumps/rubella vaccination 39 (27%) 5 (9%) 40 (28%) 4 (7·5%) 43 (27%) 1 (2·5%) 42 (26%) 2 (6%)
n (%) hepatitis A vaccination 44 (30%) 10 (18%) 41 (28%) 13 (9%) 50 (32%) 4 (10%) 49 (30%) 5 (14%)
n (%) influenza vaccination 15 (10%) 42 (79%) 16 (11%) 41 (77%) 19 (12%) 38 (95%) 25 (15%) 32 (91%)
n (%) typhoid vaccination 12 (8%) 27 (51) 12 (8%) 27 (51) 12 (8%) 27 (51) 17 (10%) 22 (63%)
n (%) responders T2 (T2/T0 ≥ 2) 134 (92%) 38 (72%)* 124 (85%) 13 (25%)** 103 (65%) 7 (17%)‡ 110 (67%) 17 (49%)#
n (%) protected T2 (≥ 2 μg/ml) 127 (88%) 52 (98%) 101 (70%) 52 (98%)** 68 (43%) 39 (95%)** 83 (51%) 34 (97%)**
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†

P < 0·0001; ‡ P = 0·0002 and § P = 0·001 versus the corresponding low baseline antibody levels by Student's t‐test. *P = 0·0003; **P < 0·0001; #P = 0·05453; P = 0·05; all versus the corresponding low baseline antibody levels by χ2Yates' corrected, two‐tailed.

Table 3.

Logistic regression analyses to investigate seroprotection and seroconversion

Seroprotected T2 (≥ 2 μg/ml) Responders to vaccination or booster T2
Anti‐PsA Anti‐PsC Anti‐PsW135 Anti‐PsY Anti‐PsA Anti‐PsC Anti‐PsW135 Anti‐PsY
OR P OR P OR P OR P OR P OR P OR P OR P
Ln Men‐Ps T0 ≥ 2 μg/ml (versus < 2 μg/ml) 3·04 0·001 1·22 0·002 3·03 0·001 2·86 0·000 0·75 0·073 0·72 0·001 0·68 0·048 0·79 0·111
Age ≥ 30 years (versus < 30) 1·12 0·913 1·25 0·704 0·52 0·304 1·07 0·912 0·74 0·591 1·39 0·592 0·28 0·026 0·37 0·046
Concomitant vaccinations ≥ 2 (versus 1) 0·75 0·682 0·66 0·382 0·60 0·185 0·51 0·095 1·74 0·482 0·91 0·871 0·25 0·004 0·48 0·100
Men vaccinations ≥2 (versus 1) 0·19 0·023 0·40 0·142 0·95 0·942 0·17 0·003 0·18 0·016 0·16 0·002 0·89 0·846 0·93 0·881
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Statistically significant values are shown in bold type. OR =  odds ratio.

Subjects with IgG ≥ 2 μg/ml at T0 showed significantly higher values for age, number of previous Men‐Ps vaccinations and number of concomitant vaccinations than subjects with IgG < 2 μg/ml at T0 (Table 2).

On one hand, individuals with IgG ≥ 2 μg/ml had a significantly higher risk than subjects with IgG < 2 μg/ml to be unable to double their baseline IgG levels at T2 for all Men‐Ps, with the exception of PsY. On the other hand, individuals with IgG ≥ 2 μg/ml had a significantly higher probability than subjects with IgG < 2 μg/ml at baseline to either reach or maintain a level of specific IgG ≥ 2 μg/ml (putative protection) at T2 (Table 2).

The logistic regression analysis (Table 3) confirmed the inverse association between (i) anti‐Men‐Ps IgG levels at T0 and response to PsC and PsW135, (ii) number of previous Men‐Ps vaccinations and response to PsA and PsC, (iii) age and response to PsW135 and PsY and (iv) a number of concomitant vaccinations ≥ 2 and response to PsW135. Moreover, the logistic regression analysis showed a significantly higher probability for individuals with IgG ≥ 2 μg/ml at T0 to be putatively seroprotected at T2 and a significantly lower probability for individuals with a number of previous meningococcal vaccinations ≥ 2 to be putatively seroprotected for PsA and PsY at T2 (Table 3).

The half‐lives of these antibodies could be calculated in group 1 subjects, as three time‐points were available. The calculated half‐lives of vaccine‐induced IgG were 1 year for anti‐PsA, 1·12 for anti‐PsC, 0·91 for anti‐W135 and 1·24 for anti‐PsY. The calculated mean duration of protection was 4·5 years for PsA, 3·5 years for PsC, 2·5 years for PsW135 and 4 years for PsY.

All the 198 subjects were genotyped for HLA‐A,‐B,‐C,‐DRB1 and ‐DQB1 loci at low resolution. Allele frequencies were compared among 82 individuals able and 18 unable to respond at T2 to all Men‐Ps by at least doubling the baseline antibody levels, after discarding individuals who provided contrasting responses to the different Men‐Ps. No differences were observed for the HLA‐A,‐B,‐C loci, HLA‐DRB1 and HLA‐DQB1 alleles.

Discussion

In this study, we measured the specific anti‐Ps response of adults receiving one (group 1) or at least two (group 2) doses of tetravalent Men‐Ps vaccine.

A relatively low percentage of group 1 subjects showed prevaccination antibodies ≥ 2 µg/ml for PsA and PsC (8 and 8.5%, respectively) and for PsW135 and PsY (2%). Considering that this group was never vaccinated previously, the presence of baseline Men‐Ps‐specific antibodies may only be the expression of natural immunization. Interestingly, the percentages of individuals with prevaccination antibodies to PsA and PsC in a similar population observed 27 years ago 5 were much higher (49 and 28%, respectively) than those observed in this study population, suggesting a current marked reduction of N. meningitidis A and C circulation among the Italian military, as a probable reflection of the Italian general population of the same age 21.

Previous Men‐Ps vaccinations are, instead, responsible for the high baseline antibody levels observed in group 2 individuals. In fact, a boosting effect on the immune response by exposure in Lebanon seems unlikely in the light of lack of evidence for a different epidemiology of N. meningitidis infection in Lebanon and Italy 22, 23. Previous vaccinations could have generated long‐living plasma cells 24 or have caused an antigen‐driven continuous stimulation, due to Men‐Ps persistence in the organism following vaccine administration 25, able to induce and maintain elevated baseline IgG levels. Ps vaccines have been reported previously 26, 27, 28, 29, 30, 31 to induce hyporesponsiveness when administered repeatedly, even though hyporesponsiveness was not observed following repeated pneumococcal Ps vaccine administration in older adults 32. Hyporesponsiveness has been ascribed to progressive reduction of the specific immune repertoire, due to serial stimulation of B cell clones by T cell‐independent antigens unable to activate memory cells 10. However, we have observed a ‘booster effect’ in group 2 (Figs 1 and 2) that seems to favour the possible role of memory B cells, generated and expanded by T cell‐independent type II Men‐Ps antigens 33. Although the frequency of responders (specific IgG at T2/T0 ≥ 2) to booster in group 2 was lower than the frequency of responders to vaccination in group 1, the percentage of individuals putatively protected (specific IgG ≥ 2 μg/ml) at T2 was similar in the two groups. These data suggest the possibility of a feedback control of pre‐existing specific antibodies on the response to Men‐Ps vaccinations. In light of this, the baseline high antibody levels were shown to condition the likelihood of increasing response to pneumococcal Ps negatively 34. Interestingly, an inverse correlation between baseline and post‐vaccination antibody levels has been reported after influenza 35 and meningococcal 36, 37 vaccinations. Pre‐existing antibody may interfere with Ps vaccination through different mechanisms, including the reduced availability of Ps antigens for epitope masking or competition with the B cell receptor and/or the possible formation of Ps antigen–antibody complexes with consequent antigen clearance or transduction of FcR‐mediated negative signals which, in turn, would influence the B cell function negatively in a feedback control of new antibody secretion 38. Furthermore, from the 1980s belief in the absolute T cell‐independence of Ps antigens has been questioned 39, 40, 41, and even more recently 42 also taking advantage of the systems biological approach 43. Our data favour the possibility that high prevaccination antibody levels may have a prevalent role over the B cell clone exhaustion as a cause for the reduced increase of anti‐Men‐PsC and PsW135 IgG, in addition to age, which may be responsible for hyporesponsiveness to PsW135 and PsY, as already observed with influenza vaccination 44. Moreover, when the performance of the tetravalent Men‐Ps was compared with the homologous conjugate vaccine, equivalent results at 1 month post‐vaccination 45 were observed. Furthermore, in a 2‐year post‐immunization survey it was shown that Men‐Ps induced a higher response to PsA and PsC 43 and, at least for Men‐PsC, a rate of putatively seroprotected individuals higher than the conjugate vaccine 46.

The high baseline anti‐Men‐Ps antibody levels in group 2 individuals correlated significantly with the number of received Men‐Ps boosters, and the direct significant association between high baseline antibody levels and post‐immunization putative protection supports the interpretation that repeated immunizations with Men‐Ps vaccine induce and maintain putative seroprotection in a high percentage of subjects.

The two study groups received different concomitant vaccinations in addition to Men‐PS and represented a valuable tool to identify possible interferences caused by concurrent vaccines. In fact, previous papers have reported the possible interference of simultaneous influenza vaccine 47, 48 or conjugate pneumococcal vaccine 49 on tetanus, diphtheria and acellular pertussis. We could not establish any specific vaccine interference, but we noted the association of reduced responsiveness to PsW135 with more than two vaccinations. In addition, no association between HLA and immune response to T cell‐independent Ps antigens was found.

Anti‐Men‐Ps IgG were measured in group 1 subjects at three time‐points, thus enabling calculation of the durability of vaccine‐induced protection 20. Results of these calculations indicated an approximate duration of seroprotection of 2·5–4·5 years, according to Men‐Ps type. This estimate is lower than the calculated durability by Elias et al. with another formula 50, but similar to the observed durability of anti‐Men‐PsC antibody by Patel et al. 46.

It is well known that infants are unresponsive to plain Ps vaccines, and this limitation led to the development of more expensive protein‐conjugate Ps vaccines, which are immunogenic in children aged less than 18 months because of their ability to elicit protein‐specific T cells able to provide help to Ps‐specific B lymphocytes. However, our data suggest that the low‐cost tetravalent Men‐Ps vaccine may still represent a resource for the prevention of meningococcal meningitis not only for troops, but also for adults living in endemic areas, often located in developing countries, where the higher cost of conjugate vaccine may limit large‐scale adult immunization programmes, thanks to its safety 12 and immunogenicity, strictly associated with its efficacy 6, 7.

Disclosures

All authors have no conflicts of interest to declare.

Author contributions

R. N., R. B., E. T. and R. D. conceived the study, designed the protocol and helped to draft the manuscript; A A., G. B., M. I. B. and V. G. organized and administered the vaccines; M. S.C., M.S. P. and G. S. performed statistical analysis; R. T., S. M., S. C., A. P. D. and C. F. analysed serum samples; P. L. performed human leucocyte antigen (HLA) typing; C. F., R. B., R. N., R. D., S. S. and A. S. critically interpreted anti‐Men‐Ps antibodies results. All authors read and approved the final manuscript.

Acknowledgements

We thank Francesco Simonetti, Gennaro Banchini, Egidio Fracasso, Luigi Morgera, Fernando Murru, Claudio De Angelis, Florigio Lista, Donato Pompeo De Cesare, Salvatore Scandale, Giuseppe De Lorenzo, Raffaele Vento, Angelo Di Pietro, Antonio Rossi, Federico Sabatini and Francesco Piccolo for their constant support and the military personnel who voluntarily agreed to participate in the study. The study was funded by the Italian Ministry of Defense (project grant application: 85D10000250001).

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