On the design of national vaccination programmes.

The decision to include a vaccine in a national vaccination programme (or not) is usually evidence-based. Thereby, it is essential that the target disease causes a high burden of disease and that vaccination reduces this burden considerably. Furthermore, vaccination should be considered to be cost-effective by a government. Vaccines are usually administered according to standard vaccination schedules, which have been established on historical grounds. We argue and demonstrate with examples (meningococci C, Haemophilus influenzae, pneumococci and Bordetella pertussis) that adaptation of these standard vaccination schedules can be cost-saving and lead to better protection. To facilitate the improvement of vaccination programmes, a better understanding of protective immune responses (correlates of protection) and immunologic memory are required.

Economic evaluation of meningococcal serogroup C conjugate vaccination programmes in The Netherlands and its impact on decision-making.

The cost-effectiveness of one time vaccination of all persons aged 14 months to 18 years (catch-up programme) and of routine childhood immunisation at either ages 2 + 3 + 4 months, 5 + 6 months, or 14 months with a meningococcal C conjugate vaccine was estimated for The Netherlands, from a societal and a health care payer perspective. A decision analysis cohort model was employed (time horizon 77 years), direct and indirect costs (friction cost method) were considered and future costs and effects were discounted at 4%. The results showed that all vaccination options yield a substantial health gain and that the catch-up programme and routine vaccination at 14 months render favourable cost-effectiveness ratios: between about 13,200 and 17,700 per life year gained for the catch-up programme and between about 2200 and 2400 per life year gained for routine childhood vaccination at 14 months, depending on the perspective. In comparison to vaccination at 14 months, routine childhood vaccination during the first year of life is much less cost-effective: each additional life year gained costs approximately 147,000 (2 + 3 + 4 months) or 102,000 (5 + 6 months), from both perspectives. Additionally, inclusion of the likely herd immunity effect of the catch-up programme increases these incremental cost-effectiveness ratios. These results played a major role in the decision to add meningococcal C vaccination to the routine childhood immunisation schedule at 14 months and to implement a catch-up vaccination programme in The Netherlands in 2002.

PorA-specific differences in antibody avidity after vaccination with a hexavalent Men B outer membrane vesicle vaccine in toddlers and school children.

A clinical phase II trial with an experimental hexavalent outer membrane vesicle (OMV) vaccine (HexaMen) containing six different porin A (PorAs) was carried out in toddlers (2-3 years) and schoolchildren (7-8 years) in The Netherlands. HexaMen exists of two OMVs each containing three different PorA types. The serum bactericidal activity (SBA) after vaccination against the six PorAs was significantly different and was higher in toddlers than in schoolchildren. After vaccination the SBA against P1.5-2,10 was 4-6 times higher than against P1.7-2,4. The aim of this study was to test whether the differences in SBA could be explained by a difference in subtype-specific antibody avidity maturation. The avidity index (AI) of antibodies against three subtypes (PorA types P1.5-2,10; P1.12-1,13 and P1.7-2,4) was measured by ELISA and evaluated in relation to SBA. A significant avidity maturation for the 3 PorA subtypes was found. This maturation was most pronounced for P1.5-2,10 (mean AI = 72%), correlating with the highest SBA titres. Generally, the avidity titre correlated best with SBA. No differences in avidity indices against the three tested PorAs were found between toddlers and school children indicating that avidity maturation induced by this vaccine is not age-dependent.

Cross-reactivity of antibodies against PorA after vaccination with a meningococcal B outer membrane vesicle vaccine.

The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and PorA-identical patient isolates were compared as a target in SBA, to ensure that the vaccine strains are representative for patient isolates. Geometric mean titers (GMTs) in SBA against patient isolates with subtypes P1.5-2,10 and P1.5-1,2-2 after vaccination with HexaMen were generally lower than those against vaccine strains with the same subtype, although the percentage of vaccine responders (> or =4-fold increase in SBA after vaccination) was not affected. Using various P1.7-2,4 patient isolates, GMTs as well as the number of vaccine responders were higher than for the P1.7-2,4 vaccine strain, indicating that the use of the P1.7-2,4 vaccine strain may have underestimated the immunogenicity of this subtype in HexaMen. Secondly, the cross-reactivity of antibodies induced by MonoMen and HexaMen was studied using several patient isolates that differed from the vaccine subtypes by having minor antigenic variants of one variable region (VR), by having a completely different VR or by having a different combination of VRs. MonoMen induced P1.4-specific antibodies that were cross-reactive with P1.4 variants P1.4-1 and P1.4-3. HexaMen induced a broader cross-reactive antibody response against various patient isolates with one VR identical to a vaccine subtype or a combination of VRs included in HexaMen. Cross-reactivity, measured by a fourfold increase in SBA after vaccination, against these strains ranged from 23 to 92% depending on the subtype of the tested strain and was directed against both VR1 and VR2. The extended cross-reactivity of vaccinee sera induced by HexaMen against antigenic variants has important favorable implications for meningococcal B OMV vaccine coverage.

Antibody avidity and immunoglobulin G isotype distribution following immunization with a monovalent meningococcal B outer membrane vesicle vaccine.

The avidity maturation and immunoglobulin G (IgG) isotype distribution of antibodies after vaccination with a meningococcal B outer membrane vesicle (OMV) vaccine were evaluated as indicators of protective immunity. Pre- and postvaccination sera from 134 healthy toddlers (ages, 2 to 3 years) immunized with a monovalent meningococcal B OMV (serosubtype P1.7-2,4) vaccine adsorbed with AlPO(4) or Al(OH)(3) were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. The children were vaccinated three times with intervals of 3 to 6 weeks between vaccinations or twice with an interval of 6 to 10 weeks between vaccinations. A booster was given after 20 to 40 weeks. The avidity index (AI) of antibodies increased significantly during the primary series of vaccinations and after the booster was given. No differences in AIs were found when the results obtained with the two vaccination schedules or with the two adjuvants were compared. After vaccination, IgG1 was the predominant IgG isotype, followed by IgG3. No IgG2 or IgG4 was detected. There was a strong correlation between serum bactericidal activity (SBA) and ELISA titers (r = 0.85 [P < 0.0001] for total IgG, r = 0.83 for IgG1 [P < 0.0001], r = 0.82 for IgG3 [P < 0.0001], and r = 0.84 [P < 0.0001] for the avidity titer). When two subgroups with similar anti-OMV IgG levels were compared before and after the booster vaccination, the higher AI after the booster vaccination was associated with significantly increased SBA. We concluded that avidity maturation occurs after vaccination with a monovalent meningococcal B OMV vaccine, especially after boosting, as indicated by a significant increase in the AI. Vaccination with the monovalent OMV vaccine induced mainly IgG1 and IgG3 isotypes, which are considered to be most important for protection against meningococcal disease. An increase in the AI of antibodies is associated with increased SBA, independent of the level of specific IgG and the IgG isotype distribution. Measuring the AI and IgG isotype distribution of antibodies after vaccination can be a supplementary method for predicting protective immunity for evaluation in future phase III trials with meningococcal serogroup B vaccines.

Pneumococcal conjugate vaccines overcome splenic dependency of antibody response to pneumococcal polysaccharides.

Protection against infections with Streptococcus pneumoniae depends on the presence of antibodies against capsular polysaccharides that facilitate phagocytosis. Asplenic patients are at increased risk for pneumococcal infections, since both phagocytosis and the initiation of the antibody response to polysaccharides take place in the spleen. Therefore, vaccination with pneumococcal polysaccharide vaccines is recommended prior to splenectomy, which, as in the case of trauma, is not always feasible. We show that in rats, vaccination with a pneumococcal conjugate vaccine can induce good antibody responses even after splenectomy, particularly after a second dose. The spleen remains necessary for a fast, primary response to (blood-borne) polysaccharides, even when they are presented in a conjugated form. Coadministration of a conjugate vaccine with additional nonconjugated polysaccharides of other serotypes did not improve the response to the nonconjugated polysaccharides. We conclude that pneumococcal conjugate vaccines can be of value in protecting asplenic or hyposplenic patients against pneumococcal infections.

Serum bactericidal activity and isotype distribution of antibodies in toddlers and schoolchildren after vaccination with RIVM hexavalent PorA vesicle vaccine.

A clinical phase II trial with the RIVM hexavalent OMV vaccine containing six different PorAs was carried out in toddlers (2-3 years) and schoolchildren (7-8 years) in The Netherlands. Children were vaccinated three times (0, 2, 8 months). Sera after two and three vaccinations were analysed for serum bactericidal activity (SBA) and isotype distribution in whole cell enzyme linked immunosorbent assay (ELISA). The SBA after vaccination against the six PorAs was significantly different. We investigated whether the age specific and PorA specific differences in SBA titers correlated with differences in PorA specific IgG isotype distribution. The SBA titers were higher in toddlers compared with schoolchildren. After vaccination, IgG1 antibodies dominated the response followed by IgG3 antibodies. IgG2 levels were low, whereas IgG4 was not detected. Irrespective of PorA, IgG total and isotype specific titers after two and three vaccinations were significantly higher in toddlers than in schoolchildren. A weak correlation was found between IgG total or IgG1 and SBA. Although the immunogenicity of the six PorAs is very different, the isotype distribution was similar for all six tested PorAs. We conclude that the RIVM hexavalent PorA vesicle vaccine induces bactericidal antibodies mainly of the IgG1 and IgG3 isotypes that are considered to be most important for protection against disease. The isotype distribution of the response is not age-dependent.

Serological characterization.

Immunoassays employ a range of methods to detect and quantify antigens or antibodies and to study the composition of antigens. This chapter describes four useful immunoassays for serological characterization of antigens of Neisseria meningitidis: whole-cell enzyme-linked immunosorbent assay (ELISA); dot blot, colony blot, and immunoblot. Serological characterization of N. meningitidis antigens is valuable for epidemiological studies as well as for identifying immunologically important antigens in vaccine development (1,2). Typing of N. meningitidis is based on the immunological detection of specific epitopes on the outer-membrane proteins (OMP) and lipopolysaccharides (LPS) (3), and panels of monoclonal antibodies (MAbs) have been developed by several laboratories to refine the serological classification system (4-8). Differences in capsular polysaccharides determine the meningococcal serogroup, whereas the serotypes and serosubtypes are based on antigenic differences of the PorB OMP and PorA OMP, respectively (3). The PorA protein contains two variable loops (VR1 and VR2), each of which determine a dis- tinct set of serosubtypes. Thus the serosubtypes of an isolate can include two independent designations (9-11). Variation in the oligosaccharide moiety of the LPS determines the immunotype, and more than one epitope can be present in the same population of a single isolate (6,12). In the current typing scheme the classification is given as [serogroup]: [serotype]: [serosubtype]: [immunotype], e.g., B: 15:P1.7,16:L3,7,9.

Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.

In vivo antibody response and in vitro CTL activation induced by selected measles vaccine candidates, prepared with purified Quil A components.

Semipurified Quil A and purified Quil A were used to prepare well-characterized subunit vaccine candidates against measles. Variation in the relative amounts of the measles virus (MV) fusion (F) protein, Quil A-components and lipids did not influence induction of antibody responses in mice, but had a pronounced effect on the capacity to induce cytotoxic T cell (CTL) activity of a CD8(+) MV F-protein specific human T cell clone in vitro. A characteristic MV iscom preparation based on the combined use of HPLC-purified Quil A-components QA-3 and QA-22 (QA-3/22) efficiently induced CTL activity in vitro. Comparable results were obtained by mixing beta-propiolactone inactivated MV with iscom-matrix QA-3/22 or free QA-22. On the basis of the data presented it was concluded that these three preparations are interesting MV vaccine candidates for further evaluation in pre-clinical experiments in a primate model.

Immunogenicity studies with a genetically engineered hexavalent PorA and a wild-type meningococcal group B outer membrane vesicle vaccine in infant cynomolgus monkeys.

The immunogenicity of two meningococcal outer membrane vesicle (OMV) vaccines, namely the Norwegian wild-type OMV vaccine and the Dutch hexavalent PorA OMV vaccine, were examined in infant cynomolgus monkeys. For the first time, a wild-type- and a recombinant OMV vaccine were compared. Furthermore, the induction of memory and the persistence of circulating antibodies were measured. The Norwegian vaccine contained all four classes of major outer membrane proteins (OMP) and wild-type L3/L8 lipopolysaccharide (LPS). The Dutch vaccine consisted for 90% of class 1 OMPs, had low expression of class 4 and 5 OMP, and GalE LPS. Three infant monkeys were immunised with a human dose at the age of 1.5, 2.5 and 4.5 months. Two monkeys of each group received a fourth dose at the age of 11 months. In ELISA, both OMV vaccines were immunogenic and induced booster responses, particularly after the fourth immunisation. The Norwegian vaccine mostly induced sero-subtype P1.7,16 specific serum bactericidal antibodies (SBA), although some other SBA were induced as well. The antibody responses against P1.7,16, induced by the Norwegian vaccine, were generally higher than for the Dutch vaccine. However, the Dutch vaccine induced PorA specific SBA against all six sero-subtypes included in the vaccine showing differences in the magnitude of SBA responses to the various PorAs.

Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7-8 years of age.

To study the reactogenicity and immunogenicity of a hexavalent meningococcal outer-membrane-vesicle vaccine (OMV), two different dosages of this vaccine (7.5 and 15 microg of individual PorA proteins) consisting of vesicles expressing class 1 outer-membrane proteins (OMPs) of subtypes P1.7,16; P1.5,2; P1.19,15 and P1.5(c), 10; P1.12,13; P1.7(h),4 were administered to a group of 7-8 year (n=165) and a group of 2-3 year old children (n=172). Control groups of children with similar ages were vaccinated against hepatitis B. All participants received three injections. Pre- and postimmunisation sera were tested for bactericidal antibodies against six isogenic meningococcal vaccine strains expressing different PorA proteins. Antibody titres against OMP of the two different vesicles (PL16215 and PL10124) were measured by ELISA. The meningococcal hexavalent OMV vaccine was well tolerated. No statistically significant differences were seen between the high and low dose of hexavalent meningococcal OMV vaccine. The percentage of children showing a fourfold increase of bactericidal antibody titres against the specific serosubtype varied in toddlers from 28 to 98% and in older children from 16 to 100%. Both ELISA antibody titres and bactericidal activity showed the highest level in the youngest age-group.

A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols.

Streptococcus pneumoniae (pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.

Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response.

The immunogenicity of two types of Streptococcus pneumoniae capsular polysaccharide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with free PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. However, unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness.

The role of macrophages in the induction and regulation of immunity elicited by exogenous antigens.

Different delivery vehicles may target to different antigen presenting cells (APC) because of their composition, size and/or physical properties. In this study, we examined the priming of cytotoxic T lymphocyte (CTL) responses to a soluble exogenous protein in vivo, using various delivery vehicles. In addition, we determined the role of macrophages as APC in vivo for each of these delivery vehicles by comparing the induction of antigen-specific CTL and serum antibodies in normal and macrophage-depleted mice. Influenza A virus-derived virosomes, liposomes and monophosphoryl lipid A/squalene (MPLSQ) efficiently induced antigen-specific CTL as well as antibody responses, of which virosomes proved to be the most efficient inducers. In mice that were immunized with cell-associated antigen, strong CTL responses but no antigen-specific antibodies were detectable, while aluminium hydroxide and aluminium phosphate elicited antigen-specific antibodies but no CTL responses. Elimination of macrophages in vivo before immunization abrogated CTL responses induced with liposomes and MPL/SQ, but did not affect induction of antigen-specific CTL with virosomes or cell-associated antigen. Importantly, serum antibody levels were not altered after macrophage depletion, regardless of the delivery vehicle used, suggesting that in the absence of macrophages, other APC may phagocytose the exogenous antigens for major histocompatibility complex (MHC) class II processing and presentation. These results suggest that soluble exogenous antigens delivered in different carrier systems may be processed differently by different APC in vivo for MHC class I- or class II-restricted presentation.

Mucosal immune responses to pneumococcal polysaccharides: implications for vaccination.

Systemic vaccination does not effectively induce a protective immune response to Streptococcus pneumoniae infections in infants, elderly people and immunosuppressed patients, who are highly susceptible to this pathogen. As mucosal immune responses develop early in life and still function well in the elderly, mucosal vaccination (that is, by oral, intratracheal or intranasal routes) may be an alternative approach to current strategies.

Characteristics of immune responses to native and protein conjugated pneumococcal polysaccharide type 14.

The immune response to pneumococcal polysaccharide type 14 (PPS-14), induced by native PPS-14 was compared with the response induced by PPS-14 conjugated with CRM197 (PPS-14-CRM197). In our animal model, immunization with PPS-14-CRM197 gave a significant enhancement of anti-PPS-14 serum titres for IgM and IgG, but not for IgA. Also an increase in total number of anti-PPS-14 antibody-secreting cells was found. Using immunohistochemical techniques, a different distribution pattern of specific antibody-containing cells in spleen section after immunization with PPS-14-CRM197 was observed. Furthermore, a higher number of IFN-gamma producing cells was found after immunization with PPS-14-CRM197, as compared with immunization with PPS-14. This enhanced IFN-gamma production may be the cause for enhanced IgG response observed after immunization with PPS-14-CRM197. The specific immune response was less affected by splenectomy in animals immunized with PPS-14-CRM197 than with PPS-14. However, an age-related response to the native as well as the conjugated form of the PPS-14 was observed, since no effect of conjugation with CRM197 was seen in the onset of the immune response to PPS-14 in young animals. In conclusion, our results affirm the hypothesis that conjugation of polysaccharides changes the characteristics of the antigen towards a thymus-dependent antigen.