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Design of a vaccine to immunize neonates against GBS infections through a durable maternal immune response

Final Report Summary - DEVANI (Design of a vaccine to immunize neonates against GBS infections through a durable maternal immune response)

Executive summary

Direct vaccination cannot prevent diseases that affect neonates during the first days of life. Group B Streptococcus (GBS) is the major cause of neonatal septicaemia and meningitis affecting up to one in every thousand births. In the absence of intrapartum antimicrobial prophylaxis over 80 % of cases occur in the first 7 days after birth and the remaining 20 % of disease occurs between 8-90 days after birth. Previous studies indicated that high titres of serotype-specific maternal antibody to GBS capsular polysaccharides (CPS) correlate with reduced risk of disease in neonates, suggesting that maternal immunisation may be an effective strategy for delivery of a protective immune response to the child in this early period. In addition, preclinical studies with mice indicate that maternal immunisation with vaccines containing CPS conjugates and/or protective pilus protein antigens confer protection against neonatal challenge with virulent GBS. The overall objective of the DEVANI project was to design immunisation strategies capable of inducing strong durable and placentally transferable protective immune responses against GBS in women in order to confer protection to the newborn:

- using mouse models appropriate antigen combinations, adjuvant and delivery systems capable of inducing a long lasting protective response were identified;
- a Europe-wide study of serotype distribution of GBS disease in Europe was conducted in order to identify the main serotypes causing disease in order to select the appropriate CPS to include in the vaccine;
- a study of the variation in pilus protein types in strains causing disease in Europe in order to assess the importance of inclusion of these antigens in the vaccine;
- a study to assess the role of maternal antibodies against CPS and pilus proteins in conferring protection to the newborn and if possible to define the level of antibody required to confer protection.

During 3.5 year, more than 25 000 pregnancies were followed in 8 countries in Europe. Some 155 cases of neonatal GBS disease were identified. The strains of GBS causing disease from each case were isolated and characterised for CPS serotype, CPS genotype, pilus type and by multi-locus sequence typing. These strains were compared with proximate 1000 control strains isolated from women who delivered healthy babies. In parallel, serum was collected from the mothers of each sick baby and from the mothers of mothers of healthy babies as controls. Analysis of specific antibody levels against CPS and pili proteins were determined for each sample and the levels in mothers of cases were compared to the levels of mothers who delivered healthy babies. The results from these studies will impact the vaccine capable of inducing a long lasting immune response in pre-pregnant women, who will protect their newborns from the risk of invasive disease by GBS.

Project context and objectives

The objective was the identification of candidate formulation(s) of CPS conjugates and recombinant proteins, relevant immunisation schedules, potential adjuvants and correlates of protection for a vaccine to be used in women to confer protection against invasive GBS disease in the newborn.

In order to achieve this goal, the project addressed four major questions:

i. What adjuvants and immunisation schedules give the highest levels and most durable protective antibody responses?
ii. What are the major diseases causing serotypes circulating in Europe?
iii. What percentage of strains expresses at least one of the pilus types?
iv. What levels of maternal IgG concentrations are required in order to confer protection to the child?

The studies performed will give important information on the correlation between serotype and pilus type. This is important to assess the best combination of components and the likely coverage to be achieved.

Objective 1: Identify the best candidate formulation(s), adjuvants and immunisation schedules

Baseline
In human and most mouse studies, the polysaccharide has been conjugated to tetanus toxoid as carrier protein. Prior data from Novartis scientists have demonstrated the natural non-toxic mutant of diphtheria toxin (Crm197) used in other commercial vaccines is an effective carrier for GBS polysaccharides. Crm197 has been extensively used as a carrier protein in commercial vaccines against other diseases. For these reasons, Crm197 is the carrier of choice. Mouse studies of pilus antigens as vaccine candidates have demonstrated that these proteins, in the presence of Freunds adjuvant or alum adjuvant confer protection. Freunds adjuvant cannot be used in humans and, although alum adjuvant has shown efficacy in mouse models, it is not known at this point whether other adjuvants may improve immunologic responses to the antigens. New vaccine candidate protein antigens and adjuvants were evaluated in comparison with conjugated GBS polysaccharides and pilus proteins in mouse models. For late onset disease where no models presently exist, attempts were made to develop a new model of GBS meningitis by intracranial injection. These attempts failed since analysis of infected mice brains revealed encephalitis rather than meningitis and therefore does not reflect the human disease. However, a novel model of neonatal infection by intravaginal colonisation of the mother that reflects the natural transmission of disease in humans better was developed.

Objective 2: Identify major GBS serotypes causing disease in newborns

Baseline
GBS strain characteristics
The major diseases causing strains in Europe are not well defined and there are no data available on the distribution of pilus types among European strains. Over 1000 strains were collected and analysed for serotype, capsule genotype, pilus type and multi-locus sequence type. The serotype distribution in Europe was found to be similar to that in the USA. However, there was a clear difference between the distributions of serotypes causing disease compared to those colonising mothers of healthy babies.

Specific objective 3: Molecular studies: To assess by polymerase chain reaction (PCR) the distribution of the genes encoding pilus protein antigens identified by Novartis VD scientists among strains from cases of disease and from carriers. The relationship between presence of pili genes and CPSs was studied by multi-locus sequence typing (MLST). Complete genome sequencing was performed on a number of GBS strains in order to assess the technology for its capacity to efficiently enhance and replace PCR and MLST technologies for future studies.

Strains isolated from infected babies and from the mothers of healthy controls were analysed by pilus gene specific PCR and MLST. Also, the genes coding for the capsule biosynthesis were analysed using a multiplex PCR assay capable of distinguishing between all serotypes. The genetic structure of the population of circulating strains based on these characteristics was determined.

Specific objective 4: To establish the level of maternal antibody titres against CPS and candidate protein antigens which correlate with resistance to GBS disease in the newborn
Maternal serum from cases and controls has been assayed using enzyme-linked immuno assays (ELISA) Tests for the quantity, specificity, and avidity of specific anti-GBS IgG antibodies (Ia, IB, III and V).

GBS serology in Europe
There were no systematic studies on the prevalence of serum antibodies against CPS in Europe although two large studies have been done in the United States (US). From these studies, the amount of maternal antibody that correlates with protection of the newborn has been defined for serotypes Ia and III but not for any other serotype. There are no data on correlates of protection for serotypes circulating in Europe. There are no studies at all on the prevalence of antibodies against the protective pilus antigens in women in any region. Using ELISA developed during the project, sera from the mothers of infected babies were compared with those from mothers of healthy babies for the levels of antibodies against CPS and pilus antigens. A clear correlation between high titres of maternal anti-CPS antibodies and reduced risk of neonatal disease was demonstrated for three serotypes. No significant correlation for antibodies to pilus proteins was detected. The levels might have been too low in the population generally. A secondary objective was to harmonise and standardise GBS typing methods for enhanced surveillance of neonatal / paediatric GBS infections in Europe and to support networking and cooperation. Quality schemes for GBS characterisation amongst European centres were established and standard sets of protocols were prepared and agreed upon. Standardised methods for screening were established in all participating countries. All data obtained were collected and recorded in a web database. Safety of data was regulated according to the European Directive of 24 October 1995 (95/46/CE) regarding protection and circulation of personal data.

Project results

Sample collection

Within the DEVANI project, beneficiaries have characterised GBS strains, circulating over a wide area of Western Europe. After harmonising the protocol the participating beneficiaries obtained ethical approvals and started collecting. 192 strains from neonatal infections and 1089 from pregnant women with the relative sera were received. 155 isolates from neonatal infections could be accepted as fitting the case definition. 453 sera from pregnant women GBS negative have been collected. Among neonatal infection the most frequent serotype was serotype III (60 %), followed by Ia (16.7 %) and V (13 %). The high percentage of serotype III isolates was even more pronounced when dividing the isolates per type of infection (EOD versus LOD). While the percentages of different serotypes among EOD isolates ware more similar to the distribution observed among carriers, more than 75 % of isolates from LOD belonged to serotype III. This observation confirms the serotype distribution observed in the USA and points to a higher virulence of serotype III GBS strains. All tools necessary for the final analysis were developed, tested and used for final processing. The descriptive analysis and comparisons between populations and countries were disseminated within core members of the consortium to prepare conclusions and publications. The amount of data collected will be further analysed over the coming months.

Sample analysis

To harmonise methods for GBS serological and molecular characterisation in Europe, three multicentre EQA studies were undertaken. The first EQA distribution comprised 14 GBS isolates, the second distribution 15 and the third distribution 12. The isolates were coded prior to dispatch to blind the participants to their identity. Replicates of each isolate were prepared on Columbia blood agar (CBA) slopes and then sent by courier. The first EQA distribution was dispatched in September 2008, the second in February 2009 and the third in September 2009. Participants were asked to test a panel of 14 coded GBS isolates using their current serotyping and genotyping methods for the first EQA distribution. For the second EQA distribution, each centre was asked to serotype 15 GBS isolates using the standardised latex agglutination method based on the Strep-B-Latex kit. Only the eight beneficiaries within the DEVANI consortium took part in the third distribution which comprised testing a panel of 12 coded GBS isolates by the standardised Strep-B-Latex method, using the same batch of reagents (Lot La-1-4) in all participating centres. Participants were asked to genotype the strains using a novel multiplex PCR method developed by the DEVANI beneficiary from Italy. This method was not published at the time of this distribution and thus the protocol could not be disseminated to other non-DEVANI centres. From these studies, we were able to produce standardised protocols for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method using the same batch of validated reagents (Lot La-1-4) and a novel multiplex PCR assay which detects all 10 GBS capsular gene types (Ia to IX). These standardised methods have been published in Journal of Clinical Microbiology (JCM) and were used by DEVANI laboratories within a multicentre GBS clinical study as part of the DEVANI programme. Comparative tests of optimised GBS MLST analysis have been conducted. Samples have been investigated for MLST type and for the presence of pili. A total number of 767 GBS isolates was tested by MLST at the time of the DEVANI report. Some 66 STs were found, belonging to 9 clonal complexes (CC). Among these nine CCs, five are prevailing: CC1, CC10/12, CC17, CC19, CC23 - covering 92 % of GBS population tested (see WP4).

ELISA assays for detection of maternal antibodies to types Ia, Ib, III and V of the CPS have been developed and validated and the sera collected from the pregnant women have been analysed. ELISA assays for maternal antibodies to pilus antigens have been developed and used to test the serum from pregnant women. Only two cases of neonatal disease caused by serotype 1b were found. This is not sufficient to make conclusions. Statistically significant differences were found between the serum titres from mothers with infected babies compared to mothers of healthy babies for serotypes Ia and III. Although there were sufficient cases of serotype V, no statistically significant differences were found between disease and control groups. All sera were tested for antibodies against three pili antigens. There were significant differences in titres between colonised mothers compared to non-colonised mothers but no significant differences were found between cases and controls. Complete genome sequencing was performed on 101 GBS strains to assess the technology for its capacity to efficiently enhance and replace PCR and MLST technologies for future studies. Algorithms were developed to extract MLST and capsule genotype from the data. Analysis showed that this approach can replace other methods while generating a large amount of information. Genetic analysis of capsule biosynthesis genes of isolates that are non-typeable by serological means allowed assignment of these strains to the appropriate serotype.

Animal models

A mouse model of maternal immunisation and neonatal challenge with virulent GBS has been used to test formulations of candidate antigens. CPS conjugates and protein antigens have been tested in various combinations in the presence or absence of different adjuvants. The meningitis model was eventually abandoned because it was too artificial and too far from human neonatal infection. An intravaginal model of pregnant mice, infected one or three days before delivery, was developed for GBS strains, able to induce LD50 and 100 in newborn mice shortly after delivery. The causes of neonatal mortality were not evaluated. This model was used to test a vaccine preparation by using heath killed bacteria in FIA since it showed a significant protection in newborns of vaccinated mothers. This model can be used to evaluate the efficacy of new vaccine preparations.

Training and dissemination activities

Beneficiaries organised a number of trainings and workshops in different countries.

Progress of the work within the WPs

DEVANI achieved all the main objectives planned for the lifetime of the project.

WP 1. Preclinical studies and animal models.
Partners involved: 1 and 8.

The main objective was to identify appropriate vaccine formulations and immunisation schedules that can induce long lasting protective immunity. A secondary objective was to develop a model of GBS meningitis in mice to be used for further studies of virulence and immune response to different serotypes.

Appropriate vaccine formulations and immunisation

Novartis Vaccines is committed to the development of a vaccine against GBS infections. Three GBS protein antigens have been selected for vaccine development GBS80, GBS67 and SAN1518. The ability of each antigen to confer protection has been evaluated in vivo using the active maternal immunisation / neonatal challenge the mouse model of sepsis. Combination studies using the three selected protein components have been conducted. The trivalent protein vaccine was tested in the sepsis mouse model. The vaccine was able to confer protection against a panel of eight challenge strains representing the pathogenic GBS serotypes Ia, Ib, II, III, V, VIII. Conjugates of CPS type Ia, Ib, and III with CRM197 carrier have been prepared and shown to confer protection in the same sepsis mouse model reported above in different formulations (aluminium hydroxide, MF59 and no adjuvant). In Novartis VD, a combination of antigens containing the proteins GBS80 and GBS67 and the glycoconjugates CRM-Ia, CRM-Ib and CRM-III has been tested in three different formulations: aluminium hydroxide, MF59 and no adjuvant (PBS). The vaccine was able to confer protection in presence of adjuvants against five GBS strains chosen to selectively assess the contribution of each vaccine component. In the absence of adjuvant, the proteins were poorly immunogenic and unable to confer protection against neonatal infections. The antibody titres elicited by the components of the vaccine CRM-Ia, CRM-Ib, CRM-III, GBS67, GBS80 have been evaluated up to 29 weeks after the first immunisation. The antibody titres were found to be stable up to week 27 when an additional boost resulted in a strong increase of the antibody titres against vaccine components. Formulations capable of inducing a long lasting protective response have been established. The immune response could be boosted by a further immunisation after six months indicating that good immunological memory had been achieved. Optimisation of these formulations is ongoing.

GBS meningitis in mice

The purpose of the work of NCIPD was to set up a mouse model of GBS meningitis which reflects LOD and use it to test vaccine preparations against GBS neonatal meningitis. Initially was planned to use intracranial infection of the animals following a model published for pneumococci. The experimental work demonstrated that the intracranial route of infection is an artificial model that does not correspond to the pathogenesis of neonatal meningitis in humans. CD1 outbred mice were used (Harlan Lab s.r.l. Italy). Six different GBS strains to infect the mice supplied by Istituto Superiore di Sanita were used: type III reference strain NCTC 11080, type III clinical isolate 6373, mouse-adapted type III clinical isolate 6373; type V reference strain NCTC 10/84, type V clinical isolate 6789, mouse-adapted type V clinical isolate 6789. The intracranial infection of adult mice allowed to define the lethal dose (LD50) for the following strains: GBS type III mouse-adapted isolate 6373 at day 2, GBS type V reference strain at day 1, GBS type V clinical isolate 6789 at day 1, GBS type V mouse-adapted isolate 6789 at day 2. The histological analysis revealed that the animals had developed encephalitis, but not meningitis. By the intracranial infection of two days old newborn mice the mortality rate for GBS type III clinical isolate 6373 was determined. In this case, the histological analysis of the brain revealed encephalitis. The infection of pups led to death within 72 hours. GBS CFU in blood of the infected newborns were high, thus it was not possible to assess whether the animals died from brain infection or from the concomitant sepsis.

Intranasal infection: Three day old mice were given one dose GBS suspension (GBS type III reference/type III mouse-adapted / GBS type V reference/type V mouse-adapted strains - 10 animals per strain). 1-day old mice were given GBS suspension 1x103/5μl (GBS type III reference / type III mouse-adapted/GBS type V reference / type V mouse-adapted strains - 10 animals per strain). Two doses per day in two consecutive days were administered. For oral direct infection, the bacterial suspension of each GBS strain (type III reference, type III and V clinical strains approximately 1 x 106 CFU / 5 l / mouse) was administered directly into the mouth of 10 newborns of 1 day old. For oral indirect infection, 13 infant mice / GBS strain (type III reference, type III and V clinical strains) were used, fasted for 2 hours before infection. Approximately 2.3 x 105 CFU in 10l were used for infection. The bacterial suspension was brushed on the skin of the mother at the udder. The survival of newborns was followed for 10 days. The intra-auricular infection has been performed on 10 mice of 10 days old of matched sex. GBS suspensions (type III reference, type III and V clinical strains) have been administered at 1.5 x 107 CFU in 3l in each ear. The survival of newborns was followed for 10 days. No mortality was observed for any route of infection used.

An intravaginal challenge model was developed for pregnant mice 24 hour before delivery with a single dose of GBS type III reference, type III clinical and type V clinical isolates. The rate of survival of the newborns was above 80 %. For this reason, pregnant mice were intravaginally inoculated with six doses of 5x107/50μl of each strain: GBS type III reference, type III mouse-adapted, type V reference, type V mouse-adapted strains at days -3, -2, -1 with two doses per day. LD50 for the neonates was defined for GBS type III mouse-adapted strain on the 42nd hour and for GBS type V mouse-adapted strain on the 16th hour. Using the intravaginal model to test the efficacy of a vaccine preparation of heat killed whole cell bacteria GBS type III reference / type III mouse-adapted / type V reference / type V mouse-adapted strains was performed. Vaccination was done by s.c. injection of 107 CFU for priming (day 0) and 105 CFU for two boostings (days +7 and +14) in FIA. Then inoculation of the vaccinated and unvaccinated mothers followed as described. The vaccine conferred protection of newborns of vaccinated mothers for all strains but with statistical significance for type V reference (p < 0,0001; survival = +50 %), type V mouse-adapted (p < 0,0001; survival= +20 %), type III mouse-adapted (p < 0,0044; survival= +18 %) strains (Kaplan-Meier estimator).

Deliverables

D1.1: Inducing long lasting protective response (month 18)

Immunisations with various combinations of antigens have been shown to confer maternal protection to mice and the duration of the response determined.

D1.2: Model of neonatal meningitis (month 24)

- A model of meningitis could not be developed by intranasal or by intracranial route of infection.
- The intracranial infection of newborns does not work as an animal model for meningitis because it causes encephalitis and concomitant sepsis. The reason for death of animals within 72 hours is unclear.
- The intracranial infection of adult mice caused encephalitis, not meningitis. These experiments made it possible to define DL50 for GBS type III mouse-adapted strain, GBS type V reference, type V clinical, and type V mouse-adapted strains.
- A model of EOD was developed by intravaginal infection of pregnant mice with both GBS type III adapted and GBS type V adapted strains. The bacterial suspensions were given in six doses.

D1.3: Vaccine candidates tested (month 42)

The intravaginal model was used to test the efficacy of vaccine preparation - heat killed whole cell bacteria GBS type III reference/ mouse-adapted, type V reference/mouse-adapted strains. The vaccine conferred statistically significant protection of the pups of mothers immunised with GBS type V reference, type V mouse-adapted and type III mouse-adapted strains.

W 2: Strain and sera collection, material management.
Partners involved 2, 3, 4, 5, 7, 8, 9 and 10

The main objectives were to collect, store and distribute strains and serum samples from pregnant women (PW) and neonatal infections (NI) from participating project partners. A lower number of isolates from NI was collected than expected due to the fact that not all collected isolates (192) could be included in the study because of a lack of adherence to the case definition. The expected numbers of samples was exceeded for isolates from PW. The expected target was also reached for sera from pregnant-GBS negative women.

Serotyping of isolated strains

S. agalactiae is differentiated into 10 serotypes, termed Ia, Ib, and II to IX, based on the CPSs. The most common phenotypic method for serotyping is latex agglutination by specific antibodies. But this method may fail to type isolates because of low expression of CPS and it is highly dependent on the quality of the antibodies. Therefore, molecular capsular gene typing techniques are attractive. We compared the latex agglutination with three different PCR-based molecular capsular gene typing methods and furthermore performed molecular typing on all GBS isolates from all beneficiaries of the DEVANI project that were non-typable by serological techniques. A total of 281 S. agalactiae strains were included. The strains were collected from NIs (30 strains), other non-sterile sites from neonates, and from PW. The strains were collected in Denmark (113 strains), Italy (23 strains), United Kingdom (30 strains), Spain (24 strains), Germany (38 strains), the Czech Republic (9 strains), Bulgaria (37 strains), and Belgium (7 strains). A total of 77 of the strains were selected because they were problematic in serotyping by latex agglutination. All strains were typed by latex agglutination using the commercial kit Strep-B-Latex (Statens Serum Institut, Denmark). The isolates were also typed by the one set multiplex PCR described and the two set mulitiplex PCR. The molecular capsular gene types (except for types II and VII for which no specific PCR is available) were confirmed by the serotype-specific PCRs.

Latex agglutination assigned 206 strains to a serotype, 73 were non-typeable, and 2 showed auto-agglutination. For the molecular capsular gene typing 277 strains were unambiguously assigned to a type, showing full agreement between the PCR methods used. Four strains showed an aberrant pattern of amplicons in the PCR, and long-range PCR using primers flanking the cps operon demonstrated that this was caused by insertions / deletions in the region. Among the four aberrant strains one was assigned type IX by latex agglutination and type Ib by the other two molecular capsular gene typing methods, whereas the other three strains were non-typeable by latex agglutination and one of these was assigned type Ia and two were non-typeable by the other two molecular PCR methods. For the 205 strains that were typeable by both latex agglutination and molecular capsular gene typing 192 strains (93 %) showed agreement between the two methods. The 13 strains that showed disagreement were assigned to serotypes V, VIII, or IX by latex agglutination and to Ia, Ib, II, III, IV, or V by molecular capsular gene typing. In a few cases, aberrant profiles were seen in the two multiplex PCR- based methods. In the Poyart et al., multiplex PCR serotype IX strains amplify a PCR product of the same size as serotype VII strains. In the Imperi et al., multiplex PCR serotypes Ib and IV could be misidentified as type Ia because the middle bands were sometimes very weak or absent. Overall there is agreement between serotyping by latex agglutination compared to molecular capsular gene typing provided that serotyping is performed by a skilled person with experience with this technique. However, the results suggest that there is a particular problem with the serotype IX and V latex reagents used for agglutination test. With few exceptions, the molecular capsular gene typing methods give accurate identification of strains. A combination of the multiplex PCRs described by Imperi et al and Poyart et al provides a convenient and reliable assay for typing of GBS. The remaining problem is that it is unclear to what extent non-serotypable strains express CPS. This question is currently under investigation by sequence analysis of the capsular biosynthesis gen locus and by FACS analysis. Due to the discrepancies found for cps identification most of the beneficiaries also used the PCR multiplex method set up by beneficiary 2 and shared during the EQA exercises. Results of serotyping by agglutination and by molecular methods for strains from PW and NI are shown below.

Molecular characterisation of isolates shifted the frequency of serotype Ia and II (higher frequency than suggested) and of serotype IX (lower frequency). Among strains from cases of NI, there was no discrepancy between agglutination serotype and molecular serotype. This is likely due to the invasive isolates being more heavily encapsulated and hence presenting less problems of agglutination ambiguity or operator error. The most common serotypes amongst isolates from PW (III, Ia, and V) were also the most common amongst NI strains, with a large prevalence of serotype III among the latter. By grouping for type of infection (EOD or LOD) it was clear that EOD causing strains correlated more with the serotype distribution found among PW, whilst more than 70 % of LOD infections were caused by serotype III strains. The numbers of EOD and LOD reported by each country does not reflect the incidence of the disease.

Deliverables

D2.1 Collection of clinical (month 30)
GBS from neonatal invasive infection (155 isolates) and from colonised mothers (1089 isolates) were collected.

D2.2 Collection of sera (month 30)
Sera from mothers of diseased children, from carriers and from uncolonised mothers were collected.

D2.3 Knowledge of serotypes causing neonatal disease in Europe (month 42):
The number of isolates from EOD versus LOD was about 1:1. This does not reflect the situation in Europe as not all countries could access both neonatal and paediatric wards. Serotype III was the most common among both EOD and LOD-causing strains. EOD serotypes showed a wider distribution comparable to that of PW, while the majority of LOD isolates were mainly serotype III.

D 2.4 Knowledge of GBS infection in Europe (month 42)
Data on the incidence of GBS infection are analysed through data from the database. A preliminary evaluation indicates an incidence of infection ranging 0,2 to 0,97 per 1000 live births, with a ratio EOD to LOD of circa 1:1.

D 2.5 Knowledge of GBS carriage (month 42)
Data on GBS carriage among PW are still under analysis. A preliminary evaluation indicates a carriage rate ranging 10,6 to 30 %. Data however have to be corrected by considering the GBS screening coverage in each country.

D 2.6 Knowledge of serotypes circulating in carriers in Europe (month 42)
Serotype distribution among carriers indicated serotype III, followed by Ia and V, as the most common among PW.

WP 3: Serological studies.
Partners involved: 1

Summary of progress

The major objective is to obtain information on maternal antibody levels against GBS CPSs and pili proteins in mothers of babies with invasive GBS disease compared to healthy mothers. The final aim is establishing which levels of antibodies are respectively associated to diseased neonates or to colonised children for all the serotypes Ia, Ib, III and V. The distribution of antibodies against the pili proteins in cases and controls will be assayed. Highly reliable ELISA assays for measuring specific antibody against the CPS antigens and the pilus proteins were developed. The ELISA assays were set up and validated through a series of experiments. An existing pool of human sera was used as reference standard. IgG titres were calculated by using the reference line assay. Quantified human sera against pili proteins are not available yet. Standard sera for each protein antigen were established by pooling sera of highly responsive subjects against each antigen. Total IgG titres were calculated by using the reference line assay method and results were expressed as arbitrary ELISA units/ml (EU/ml). The developed assays were then used to assay the DEVANI collected sera. All the samples received have been already assayed for the presence of antibodies against the CPSs Ia, Ib, III and V and three pili protein antigens.

Level of antibodies in healthy population

All 1668 samples have been assayed for the presence of antibodies against each of the CPSs type Ia, Ib, III and V. The DEVANI serum collection is constituted by three groups of subjects: pregnant women colonised by GBS (PW+), pregnant women not colonised (PW-) and pregnant women who had neonate with GBS infection (NI). The analysis of sera from healthy non-colonised PW (464 subjects) revealed the presence of specific IgG against each of the four GBS CPSs tested. The GMT titres measured against type V CPS were 5 to 30 times higher than those measured against the other 3 polysaccharides. Sera from healthy colonised PW revealed the presence of IgG specific for the CPS of the respective colonising strains. Colonised PW exhibited GMT titres significantly higher than those measured in sera of non-colonised women in all cases (p = 0,0001 Mann-Whitney test). These results suggest that the colonisation status, with a strain expressing a specific CPS, may affect maternal antibody levels, against that polysaccharide.

Level of antibodies against pili proteins

We set up three ELISA assays for antibody titration against the following GBS pili proteins: GBS 80 (backbone protein of Pilus 1, BP-1), GBS67 (ancillary protein 1 of Pilus 2a, AP1-2a), and GBS1523 (backbone protein of Pilus 2b, BP-2b). IgG titres were expressed as arbitrary EU/ml, due to the lack of reference standard. All samples have been assayed for the presence of antibodies against each of the pilus protein antigens. The analysis of sera from healthy non-colonised PW revealed the presence of specific IgG against pili proteins. Specific IgG against pili proteins were also found in the sera of colonised PW. GMT titres, against each pilus protein, in colonised PW were significantly higher than those measured in sera of non-colonised PW (p < 0.005 Mann-Whitney test). Results may indicate that pili are expressed in vivo and are able to induce antibody response in colonised subjects. The analysis of antibody titres anti-pili proteins displayed by healthy colonised PW and by mothers of GBS infected babies was carried out taking into account which pili were expressed by the colonising GBS strain. All strains were assayed for the presence of pili genes and for the expression of the corresponding pili on their surface, respectively by PCR and by FACS. By gathering subjects carrying strains expressing one specific pilus in the group of healthy colonised women (PW) and mothers of GBS infected babies (NI), it was possible to observe how the distribution of antibody titres against that pilus varies between women carrying strains expressing the pilus (FACS+) and women carrying strains not expressing the pilus (FACS-). Significative differences were observed between subjects carrying FACS positive and negative strains for each pilus protein in the group of carrier women. In the group of mothers of GBS infected babies a significant differences was observed only for the pilus protein GBS1523. In this case, it seems that the colonisation status, with a strain expressing a specific surface antigen, may affect maternal antibody levels against that antigen.

Protective levels of antibodies

We recorded only two NIs caused by serotype Ib GBS strains. These are not sufficient for our study. It was clear that colonised PW exhibited GMT titres significantly higher than those measured in sera of non-colonised. It was also evident for serotype Ia and III cases that GMT titres in colonised PW were significantly higher than those measured in sera of mothers of infected babies, both EOD and LOD. These results confirmed previous findings that the probability of developing EOD declined with increasing maternal levels of anti-GBS Ia IgG. (Lin, Philips et al., 2001). Similar results were shown for the level of maternal IgG anti-GBS-III required to protect newborns from EOD (Lin, Weisman et al., 2004). In these two studies, the authors were able to estimate the level of maternal IgG required to protect newborns from EOD.

Deliverables

D3.1 Knowledge of level of antibodies (month 39)
We set up and validated four ELISA assays for antibody titration against GBS Ia, Ib, III and V CPSs. All 1666 samples have been assayed for the presence of antibodies against each of the CPSs type Ia, Ib, III and V.

D3.2 Knowledge of level of antibodies against pili proteins (month 39)
We set up and validated three ELISA assays for antibody titration against GBS pili proteins GBS 67, 80 and 1523. All samples have been assayed for the presence of antibodies against each of the protein antigens.

D3.3 Knowledge of protective levels of antibodies (month 42)
Only two cases of neonatal disease caused by serotype 1b were found. This is not sufficient to draw conclusions. Clear statistically significant differences were found between the serum titres from mothers with infected babies compared to mothers of healthy babies for serotypes Ia and III. No statistically significant differences were found between disease and control groups in serotype V.

WP 4 Genetic studies
Partners involved: 6, 2, 7, 9 and 10

Perform studies for the assessment of:

- distribution and the possible relationship between pili protein genes and CPSs among different GBS serotypes including serologically untypeable (D4.1);
- circulating clones responsible for neonatal GBS invasive disease and comparison with clones in carriers (D4.2);
- possible relationship between the presence of pili protein genes and the MLST types (D4.3).

Pili genes of all isolates received were characterised. The most striking result was that a majority of isolates from NI showed the PI-1+2b gene pattern, while the most common pattern among PW was PI-1+2a.

A rather strict association could be observed between certain serotypes and pili gene pattern: in serotype Ia, 79 % of isolates carried the PI-2a gene, whereas in serotypes II, IV and V, the isolates carried the PI-1+2a genes in 73 %, 81.5 % and 70 % respectively. Pili genes PI-1+2a and PI-1+2b were carried at comparable level among PW strains belonging to serotype III. This ratio was inverted in isolates from NIs, where the same pili genes among serotype III (the most common serotype) strains were present respectively in 15 % and 80 % of isolates. Beneficiary 6 performed MLST for 203 GBS isolates: from BE - 47 isolates, from BG - 38 isolates, from CZ - 118 isolates. Four beneficiaries performed MLST of their isolates and sent the MLST results to beneficiary 6: 189 isolates - beneficiary 7, 77 isolates (beneficiary 9, 102 isolates) beneficiary 3, 111 isolates - beneficiary 2, 99 isolates - beneficiary 5. The total number of GBS isolates investigated by MLST at the time of the DEVANI report was 781.

Three GBS categories were investigated:

- isolates from NI - 121
- isolates from mothers of ill newborns (PW-NI) - 14
- isolates from colonised pregnant women (PW) - 646.

The isolates from mothers of ill newborns (PW-NI) showed the same MLST patterns as the isolates from corresponding neonatal infection and they were excluded from the analysis. A total number of 767 GBS isolates was tested by MLST (at the time of the DEVANI report). 66 STs were found, belonging to 9 CC. Among these nine CCs five are prevailing: CC1, CC10/12, CC17, CC19, CC23 - covering 92 % of GBS population tested.

Results for pili gene patterns and MLST types were available for 600 PW and 90 NI isolates, in total 690 GBS isolates. In both populations an association between pili gene patterns and CC was found as follows:

- PI-1+2a and CC cc19, cc1, cc10/12
- PI-2a and clonal complex cc23
- PI-1+2b and clonal complex cc17.

The most frequent pili gene pattern in PW population was PI-1+2a, belonging mainly to cc19 (39 %), cc1 (30 %) and cc10/12 (20 %). The second most common was PI-2a, belonging mainly to cc23 (64 %), the third was PI-1+2b, belonging mainly to cc17 (83 %). The most frequent pili gene pattern in NI population was PI-1+2b, belonging mainly to cc17 (92 %). The second most common was PI-1+2a, belonging mainly to cc19 (43 %) and cc1 (40 %), the third was PI-2a, belonging mainly to cc23.

High-throughput next generation sequencing

Towards the end of the project, the feasibility of using Illumina sequencing technology to acquire genotypic serotype, pilus type and MLST was assessed. The complete genome sequence of 97 strains from the DEVANI collection was determined and computer methods were devised to extract the various genotypic features. The data extracted from the complete sequences matched closely the data derived from the individual assays. Minor differences were detected between the datasets for MLST probably due to rare sequence errors. We conclude that complete genome sequencing will become the preferred method for analysing CPS genotype, MLST and pilus type in populations and will additionally generate more information. Discussions have begun to determine the complete genome sequence of a further 400 - 500 of the DEVANI isolates.

Deliverables

D4.1 Assessment of the distribution and the possible relationship between pili protein genes and CPSs (month 42)
- Some 969 GBS strains from NI or PW have been characterised for pili genes pattern.
- There appears to be a tight, but not exclusive, correlation between a given serotype and its pili genes.
- Pili PI-1+2b is the most common among isolates from infections, which is not linked to the prevalence of serotype III among NI strains, as the same serotype in strains from PW carries more frequently a different gene pattern (PI-1+2a).
- Serologically non-typable strains did not differ in their pili gene patterns once the serotype was identified by molecular methods.

D4.2 Description of circulating clones responsible of neonatal GBS invasive disease (month 42)
- A total number of 767 GBS isolates from NI or PW has been characterised by MLST. A total of 66 STs were found, belonging to 9 CC.
- Among these nine CCs five are prevailing: CC1, CC10/12, CC17, CC19, CC23. Covering 92 % of the GBS population tested.
- The PW population is more heterogeneous compared to the NI population, which could be caused by different sample size.
- In 646 PW GBS isolates nine CC were found: cc1, cc4, cc7, cc10/12, cc17, cc19, cc22, cc23 and cc26. The most frequent was cc19 (23 %) followed by cc1 and cc23 (both 20 %).
- In 121 NI GBS isolates six CC were found: cc1, cc7, cc10/12, cc17, cc19 and cc23. The most frequent was cc17 (43 %) followed by cc19 and cc23 (both 14 %).
- No significant differences between participating countries in occurrence of CC were noticed.

D4.3 Possible relationship between presence of pili protein genes and MLST types (month 42)
-Pili gene patterns and MLST types were available for 600 PW isolates and 90 NI isolates, in total 690 GBS isolates.
- In both populations an association between pili gene patterns and CC was found as follows:

i. PI-1+2a and CC cc19, cc1, cc10/12
ii. PI-2a and clonal complex cc23
iii. PI-1+2b and clonal complex cc17.

WP 5. Data management
Partners involved: 4 and 1

A web-based database was developed and has been made available to all partners. Safety of data is regulated according to the European Directive of 24 October 1995 (95/46/CE) regarding protection and circulation of personal data.

Task 1: Case report forms (achieved)

Inventory of relevant data to be collected and types of results being produced was done within the first months of the project. During this first year, CRF's were finalised and became available to investigators and DEVANI beneficiaries for online completion in the secured web-based DEVANI platform or in print.

Task 2: Database generation (achieved)

- For data capture and management, the solution of an integrated, secured e-platform available to all participants involved in the project.
- The central database environment is fully secured, guaranteeing confidentiality, security and integrity of data.
- In November 2008, the website address was attributed: http://www.devani-project.eu/
- A DEVANI study eCRF user guide has been edited online for all users.
- A comprehensive validation check programme is part of the electronic platform and verifies the data.
- Automated validation programmes have identified missing data, out-of-range data, and other data inconsistencies and queries were electronically addressed to investigators.
- All actions in the database are traced and audit trails are accessible.

Task 3: Statistical analysis (main results achieved and more refined analysis ongoing)

Tools were developed to access the progress of the data collection of cases and controls and to observe the level of completion of the CRFs by the different beneficiaries were developed for the DEVANI e-platform. Since 2009, online reports and graphics were produced and updated daily. For the final statistical analysis of all the collected data and laboratory results, a well-experienced biostatistician was assigned in December 2010 to prepare the analysis, to perform a quality control and to execute the analysis of data. A statistical analysis plan (SAP) has been written and was submitted for comments and additional requests. All statistical analysis has been done using the SAS / STAT software. The expected final schedule was presented in December 2010. For clinical data collected by the investigators and capsular serotypes, a first extraction of data was done in January 2011, followed by a quality control of data and feedback. Further extraction for clinical data was done in March 2011. New extraction, quality control and statistical analysis were done by the end of the project.

Additional task: Traceability of GBS isolates and sera

To assure traceability of GBS isolates and sera during their long-term preservation and during their transfer, bar-coded pre-printed special labels were designed and provided to the eight investigators' sites.

Deliverables

D5.1 Access to database (from month 4)
Achieved
The mean objective was fulfilled a few months behind schedule but without compromising the efforts of other WPs. A web-based database was developed and is available to all partners.

D5.2 Synthesis and analysis of available data (month 12 and 24)
Achieved
The use of an e-platform provides an accurate and reliable tool for data collection. Tools are also available to produce files ready for the quality control of collected data and for the final statistical analysis.

D5.3 Final synthesis and statistical analysis (month 42)
Ongoing for more refined analysis but main results achieved
All tools necessary for the final analysis have been developed, tested successfully and used for processing by the end of the period project. The amount of data collected is enough to be further analysed in the next months.

WP 6. Method standardisation and external quality control (EQA)
Partners involved: 3, 2, 4, 5, 6, 7, 8, 9 and 10
Questionnaire on current laboratory policies for GBS characterisation

In 2008, a questionnaire-based surveillance was undertaken among eight DEVANI and six non-DEVANI participating laboratories from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis and typing. Results from this questionnaire have been incorporated in the EQA manuscript on 'International external quality assurance for laboratory diagnosis and typing of Streptococcus agalactiae (group B streptococci)' which has been published published (Afshar et al., 2011 and J Clin Microbiol) and is available online (see http://jcm.asm.org/cgi/reprint/49/4/1475 for details).

International external quality assessment scheme for GBS
First EQA distribution

The first EQA panel of 16 GBS strains for serotyping was distributed on 8 September 2008, to eight DEVANI participating centres and six non-DEVANI participating centres from the following countries: Canada, Denmark, France, Greece, Israel and Norway. These non-DEVANI participants were self-funded. For the first EQA study, all 14 centres (from 13 countries) submitted results and tested the entire panel. The majority of centres (12 out of 14) used serology-based methods for GBS serotyping; 10 used the 'Strep-B-latex' method and four used PCR-based methods. All centres were able to correctly identify the intended serotype of the duplicate strains and the three type strains. The majority of centres (11 out of 14) scored > 80 %. Results for two of the strains suggested that these two strains may be of a mixed serotype population. This was investigated and it was concluded that these two strains should be omitted from the study. A standard GBS serotyping protocol based on the Strep-B-latex kit (SSI, Denmark) was produced after the completion of the first EQA study. This standard protocol was approved by the DEVANI members in December 2008.

Second EQA distribution

The second panel comprised of 17 GBS strains and was distributed in February 2009 to 14 participants from 13 countries (8 DEVANI and 6 non-DEVANI). This EQA panel was tested by participating centres using the standardised 'Strep-B-latex' protocol and where possible the panel was tested using the multiplex PCR protocol as described.

Third EQA distribution

Only the eight beneficiaries within the DEVANI consortium took part in the third distribution which comprised testing a panel of 12 coded GBS isolates by the standardised Strep-B-Latex method, using the same batch of reagents (Lot La-1-4) in all participating centres. Participants were asked to genotype the strains using a novel multiplex PCR method developed by the DEVANI beneficiary from Italy. All DEVANI centres achieved fully concordant results for the entire panel using the standardised Strep-B-Latex method. Improvements in the performance of participating centres could also have been due to the fact that these laboratories had received training prior to the study. The recently developed multiplex PCR assay for GBS capsular typing was shown to be an easy, robust and highly discriminatory method as it used a single PCR reaction and could differentiate GBS strains that were serologically non-typeable into 10 known GBS capsular types (Ia - IX). This method was and adopted by DEVANI as the standard molecular method for GBS capsular typing. A draft manuscript was circulated to all participants before it was submitted to the JCM for publication.

Molecular characterisation of GBS

Preliminary work on optimising and evaluating a VNTR (variable-number tandem-repeat) molecular typing method was carried out. Primer sets targeting 10 specific VNTR loci have been tested against all GBS type strains and three GBS reference strains: A909 Ia/c, NEM316 III and 2603 V/R. Due to lack of time and other priorities this work has not been completed. This was an additional complimentary study.

Deliverables

D6.1 Publication questionnaire (month 12)
The results of the questionnaire undertaken in 2008 have been incorporated in the EQA manuscript on 'International external quality assurance for laboratory diagnosis and typing of Streptococcus agalactiae (group B streptococci)' which has been published and is available online (see http://jcm.asm.org/cgi/reprint/49/4/1475 for details).

D6.2 Harmonisation of methods for GBS serological and molecular characterisation in Europe (month 12)
To harmonise methods for GBS serological and molecular characterisation in Europe three studies were undertaken. From these multicentre EQA studies, we were able to produce standardised protocols for serological and molecular typing of GBS strains based on the Strep-B-latex agglutination method using the same batch of validated reagents and a novel multiplex PCR assay which detects all 10 GBS capsular gene types (Ia to IX).

D6.3 Guidelines for GBS microbiological diagnosis (month 18)
We were able to produce standardised protocols for serological and molecular typing of GBS strains based on the Strep-B-latex agglutination method and a novel multiplex PCR assay (Imperi et al., 2010) which detects all 10 GBS capsular gene types.

D6.4 Publication of data (month 36)
A manuscript on 'International external quality assurance for laboratory diagnosis and typing of Streptococcus agalactiae (group B streptococci)' was submitted to the JCM on 23 November 2010. This paper has been published (Afshar et al, J Clin Microbiol).

WP 7. Training and dissemination
Partners involved 10, 2, 3 and 1

Training activities

Training activities proposed by DEVANI have been aimed at improving knowledge and formation of scientists. DEVANI would like to promote the training of young female scientist and of scientists from eastern Europe.

Dissemination activities

The aim of the dissemination activities is to raise awareness of the DEVANI Seventh Framework Programme (FP7) funded project and raising knowledge on GBS disease and current efforts to prevent it. Main activities:

- presented at XVII Lancefield Symposium on Streptococci and Streptococcal diseases;
- presented at the XXXVII AMCLI Meeting;
- one-day symposium on 'Current advances in the diagnosis, management and treatment of neonatal group B streptococcal infections';
- final workshop 'An update on diagnosis, management and treatment of neonatal group B streptococcal infections';
- project meetings every six months;
- joint publication 'International external quality assurance for laboratory diagnosis and typing of Streptococcus agalactiae (group B streptococci)';
- illustrated on http://www.devaniproject.org

Deliverables

D7.1. Planning and organisation
Successful training initiatives have been organised during the project.

D7.2 Reporting and evaluation (months 12, 24, 36 and 42)
The final complete document reports the final evaluation of training initiatives.

D.7.3 Specialist symposium (month 18)
The first European workshop on 'Current insight into group B streptococcal diseases' was held in order to provide attendees with updates in the field of GBS diseases.

D7.4 Midterm plan (month 18)
Summarises the concrete actions of the DEVANI consortium for the protection, exploitation and dissemination of results of the FP7 project and was updated at the end of the project.

D7.5 Final plan (month 42)
Summarises all the concrete actions of the DEVANI consortium for the protection, exploitation and dissemination of the results of the FP7 project.

Potential impact:
The DEVANI project addresses the priorities of EU policies on public health as stated in the Decision No 1786/2002/EC of the European Parliament and the Council of 23 September 2002. The mission of the public health programme is actively promoting health protection and disease prevention measures. Novartis VD has recently entered the area of human vaccines by the acquisition of the vaccines arm of the American company Chiron Corporation. The development of vaccines requires research efforts of the pharmaceutical industry and also collaboration with clinical researchers and epidemiologists based in hospitals and other clinical settings. This project has brought together the appropriate expertise that will contribute to the development of a novel vaccine thus contributing to the objective of FP7 to improve the competitiveness of the European-based pharmaceutical industry (see http://ec.europa.eu/enterprise/phabiocom/p9.htm for details).

The most important impact will be on the development of European capability in reducing diseases which are currently untreatable. This project has exploited new knowledge in vaccinology to address neonatal sepsis and meningitis for which there is no vaccine or effective cure. Antibiotic prophylaxis of all culture positive women is not universally accepted in Europe due to perceived risks of increased antibiotic resistance and allergic responses to penicillin. The project took a translational approach using studies of natural human immunity to validate preclinical data obtained in animal models of infection and disease. Both CPS conjugate antigens and recombinant protein antigens have been shown to confer protection against GBS challenge in the newborns of immunised female mice. The studies have determined which CPS serotypes are most relevant to disease in Europe and also what levels of maternal antibody are required to ensure protection of the newborn.

By comparing natural immune responses to the recombinant proteins in mothers of healthy babies and mothers of babies with GBS disease, it has been possible to confirm the potential of these antigens as vaccine candidates. The consortium of DEVANI has focusing the skills of the individual participants towards the development of new vaccine to GBS. The consortium has integrated different approaches and disciplines and provided the necessary critical mass to test and compare different scientific ideas with the aim of delivering an efficacious GBS vaccine. DEVANI has become a benchmark work for future maternal vaccination to protect infants, using a comprehensive approach to standardise the preclinical procedures while taking further the development of the novel promising Novartis VD GBS vaccine.

Preclinical studies of formulations, adjuvants and immunisation strategies helped in the design of an efficacious vaccine. The mouse model is well adapted for these types of studies and has been used to test a number of formulations of candidate antigens. CPS conjugates and protein antigens have been tested in various combinations in the presence or absence of different adjuvants. Both CPS and pilus protein antigens have been shown to confer protection against neonatal disease. Alum antigen has been shown to increase levels of protection and to prolong protective immunity for more than 12 months in female mice. A preliminary evaluation indicates a carriage rate across Europe ranging from 10.6 to 30 %. This data is in line with epidemiologic studies from the US, so the risk of GBS disease in newborns in Europe is similar to that of other developed countries.

Quality assessment studies allowed definition of standardised protocols for serological and molecular typing. Results of the study showed that the protocol developed by the consortium was robust and repeatable over all centres. The protocol has been made available to the scientific community and to help in future studies. A multiplex PCR assay which detects all 10 GBS capsular gene types (Ia to IX) has been developed. This assay was found to be robust and highly accurate. Within the DEVANI project beneficiaries have characterised GBS strains, circulating over a wide area of Western Europe. The most common serotypes amongst isolates from PW carriers were also the most common amongst strains from NIs. By grouping for type of infection it was clear that strains causing early onset disease correlated more with the serotype distribution found among PW carriers, whilst more than 70 % of late onset disease infections were caused by serotype III strains.

There is a tight correlation between a given serotype and its pili genes. Pili PI-1+2b is most common among isolates from infections, which is not linked to the prevalence of serotype III among NI strains, as the same serotype in strains from PW carries more frequently a different gene pattern. Serologically non-typable strains did not differ in their pili gene patterns once the serotype was identified by molecular methods. MLST was performed on the majority of GBS isolates. In 646 GBS isolates from PW, nine CC were found. No significant differences between participating countries in occurrence of CC were found. 1668 samples have been assayed for the presence of antibodies against each of the major circulating CPSs, type Ia, Ib, III and V. Sera from healthy colonised PW revealed the presence of variable levels of IgG specific for the CPS of the respective colonising strains. Colonised PW exhibited geometric mean titres significantly higher than those measured in sera of non-colonised women in all cases.

This suggests that the colonisation status impacts the maternal antibody levels. Mothers of babies infected with serotype Ia and III had statistically significant lower levels of specific antibodies compared to mothers of healthy babies. All sera were tested for antibodies against three pili antigens. There were significant differences in titres between colonised mothers compared to non-colonised mothers but no significant differences between cases and controls. The levels of specific antibodies were low compared to the levels of antibodies induced in mice that confer protection. But the fact that specific antibodies were detected indicates that the pilus proteins are expressed by bacterium during colonisation and therefore the pilus antigens may still represent valid vaccine candidates. This is the first extensive European-wide study of GBS disease and collaboration.

Project website: http://www.devaniproject.org
The DEVANI project on HPACI website: http://www.hpa.org.uk/web/HPAweb&Page&HPAwebAutoListName/Page/1216711958769

Contacts:

WP1: Preclinical tests and animal models
John Telford (B1)/ Antoaneta Decheva (B8)
WP2: Strains and sera collection, material management
Lucilla Baldassarri (B2)

WP3: Serological studies
Domenico Maione (B1)

WP4: Genetic studies
Pavla Krizova (B6)

WP5: Data management
Pierrette Melin (B4)

WP6: Method standardisation and external quality control (EQA)
Androulla Efstratiou and Baharak Afshar (B3)

WP7: Training and dissemination
Aldo Tagliabue (B10)

WP8: Management
Aldo Tagliabue (B10).