Final Report Summary - CHILD-INNOVAC (Nasal vaccination against respiratory infections in young children) Respiratory infections are today still among the first causes of death in the world. In addition to mortality, the morbidity of respiratory infections poses an important economic and social burden. Among respiratory infections, pertussis or whooping cough remains one of the leading causes of morbidity and mortality, despite wide vaccination coverage with efficacious vaccines. With 300 000 pertussis-linked global annual deaths and approximately 40 million cases per year, whooping cough is in fact the least well-controlled vaccine-preventable disease. These facts illustrate the shortcomings of current vaccination strategies. Current whole-cell or acellular vaccines require at least three doses given one or two months apart to be optimally protective, and vaccination usually starts at two months of age. This implies that infants are not optimally protected before the age of six months, the age group with the highest susceptibility. One strategy to solve this problem would be to vaccinate newborns, possibly at birth. However, this may be difficult because of the immaturity of the newborn immune system. Previous studies have shown that infection with the whooping cough agent Bordetella pertussis very early in life nevertheless leads to strong immune responses. We have therefore developed an attenuated B. pertussis strain, named BPZE1, to be delivered as a nasal live vaccine in order to mimic as much as possible natural infection without causing disease. The objectives of the CHILD-INNOVAC project were to: (i) obtain as much pre-clinical efficacy and safety data on BPZE1; (ii) to improve our knowledge on T and B cell responses to pertussis infection and vaccination; (iii) to evaluate the effect of BPZE1 and its recombinant derivatives on heterologous infections, using respiratory syncytial virus (RSV); and (iv) to prepare clinical lots of BPZE1 and perform a first-in-man, placebo-controlled, double-blind phase I safety trial in adult volunteers, as a first step to further clinical development. The preclinical studies have shown that BPZE1 is safe in adult and neonatal mice, as well as in immunodeficient mice, such as IFN-?R-/-, SCID, Myd88-/- and µMT mice. It does not cause death, disseminated disease, lung inflammation and weight loss. In addition, it was genetically stable for at least one year of continuous passaging in vitro and in vivo. These findings have been essential do downgrade BPZE1 from biosafety level 2 to biosafety level 1 in several European countries, which was a prerequisite for its clinical development. A single nasal administration of BPZE1 to neonatal mice induced rapid and strong protection, which lasted for at least one year and could already be observed 3 days after vaccination. On human dendritic cells, BPZE1 induced a strong Th1/Th17 response in vitro. Study protocols using up-to-date technologies for the evaluation of T and B cell responses in infected or vaccinated children have been harmonised between several countries, and more than 400 children (roughly ¼ infected and ¾ vaccinated) were enrolled in 3 different countries. This has allowed us to obtain a wealth of information on B and T cell effector and memory responses to infection and vaccination in children. One of the most exciting observations made in this project is the protective effect of BPZE1 against non-related respiratory viruses, such as RSV. Vaccination with BPZE1 protected mice against weigh loss induced by RSV, which could be correlated with the induction of IL-10 and regulatory T cells, while maintaining the Th1 and Th17 responses. Recombinant BPZE1 strains producing protective RSV epitopes were then constructed in order to combine this non-specific protective effect with antigen-specific protection. Finally through a series of very intense interactions between beneficiaries and successful technological transfer, several GMP-grade lots of BPZE1 have been produced, and a phase I trial has been performed on 48 healthy male adults, divided into 4 groups (placebo, low dose, intermediate dose and high dose). The vaccine was found to be safe and able to colonise the respiratory tract. Colonisation was dose dependent, and a strong correlation between colonisation and immunogenicity was found. These findings open the way to further clinical development and product optimisation. Last but not least, the project coordination led by Inserm and Inserm-Transfert (IT) secured intellectual property generated from the project (one patent was filled with the partner National University of Ireland, Maynooth, Ireland, on Vaccine applications for prophylaxis or treatment of an allergen-driven airway pathology). The consortium also explored various dissemination / commercial exploitation strategies for further development of the BPZE1 vaccine platform beyond the end of the European Commission (EC) contract duration. IT is currently developing various scenarios and opportunities in order to maximise the dissemination / exploitation chances of the generated foreground, notably via licensing procurement to major pharmas or targeted biotech companies. Project context and objectives:The CHILD-INNOVAC project aimed at developing novel, nasal vaccines against two major respiratory pathogens, Bordetella pertussis and respiratory syncytial virus (RSV), which are among the most frequent and severe respiratory pathogens in young children. Both have a world-wide distribution and pose significant direct burdens on health systems in Europe, in neighboring developing countries and in migrant communities.Although efficacious vaccines against pertussis are widely used, roughly 40 million cases and 200 000 - 400 000 pertussis-linked deaths are recorded annually, mostly in infants aged 0-6 months, who have not yet received the three-doses vaccine regimen required for efficient protection against pertussis. Hence, pertussis vaccines are needed that induce individual protection soon after birth to protect the youngest and most vulnerable children. RSV is the first cause of bronchiolitis in infants and wheezy lower respiratory tract illness in early childhood. Virtually all children are infected by the age of three, and no vaccine is yet available against RSV. There is now a consensus that mucosally delivered live vaccines are strong candidates for future RSV vaccine development.One of the major objectives of CHILD-INNOVAC is to provide an innovative strategy to induce protective immunity against respiratory pathogens early in life, possibly at birth, with novel, single-dose nasal vaccines, based on B. pertussis BPZE1. The ultimate aim is to protect infants in the most vulnerable age group (< 6 months), before the administration of the full regimen of the currently available pertussis vaccines which could then act as a booster vaccine. Since B. pertussis can accommodate large foreign DNA inserts, the potential of BPZE1 as a multivalent vaccine vector is enormous. CHILD-INNOVAC goal was first to provide a proof of principle in mice using the RSV model. However, it is likely that recombinant BPZE1 derivatives can be designed to target other respiratory infections, both viral and bacterial. The development of such recombinant multivalent nasal vaccines may thus have a major impact. The overall project was based on integrating knowledge on the human B and T cell responses to infection and vaccination, with a special emphasis on memory, and on the vaccine strain characteristics. At this end, CHILD-INNOVAC consisted in the preparation and execution of first-in-man phase I trial (in adult volunteers) and the construction of novel, recombinant multivalent vaccine strains for future development. The research, development and innovative activities in the CHILD-INNOVAC project were focused on:1) protective immune responses induced by live attenuated B. pertussis BPZE1 in mouse models;2) human immune responses induced in vitro by BPZE1 using a dendritic cell (DC) model;3) safety assessment of BPZE1 in mouse models;4) evaluation of genetic and biological stability of BPZE1;5) construction of recombinant BPZE1 strains producing protective RSV antigens and their evaluation in mouse models;6) characterisation of the immune responses in children either naturally infected with B. pertussis or vaccinated with whole cell or acellular vaccine, with particular emphasis on memory and standardisation of B / T cell memory tests;7) preparation of clinical lots of the B. pertussis BPZE1 vaccine strain under GMP conditions;8) evaluation of safety and assessment of immunogenicity of the clinical lots of B. pertussis BPZE1 in adult humans.CHILD-INNOVAC consortium brought together 10 partners, including 2 private companies and 8 laboratories, from 7 Member States of the European Union (EU). The project was organised in five work packages (WP): the first one was dedicated to the management of the consortium; the others were for the development of the scientific aspects of the project. WP2: Preclinical studies on B. pertussis BPZE1CHILD-INNOVAC has served to obtain further information on B and T cell responses, including memory responses in children infected with B. pertussis or vaccinated with current pertussis vaccines. It also allowed us to show that the novel live attenuated pertussis vaccine BPZE1 is safe, even in severely immunocompromisad mice, yet able to induce quick (within three days) and long-lasting (for at least up to one year) protection. The excellent safety profile has allowed BPZE1 to be down-graded from biosafety level 2 to biosafety level 1, a pre-requisite for clinical trials in humans. The vaccine strain also has bystander protective effects against asthma and virus-induced weight loss and death and protects against other Bordetella species. The genetic stability of BPZE1 has been shown by continuous passaging both in vitro and in vivo through serial infections in mice. WP3: Immune responses to B. pertussis antigens in childrenIn order to evaluate the duration, as well as strength and diversity of immune responses induced by pertussis vaccination or infection, we selected and optimisad the following techniques: flow cytometry based assay for detection of B. pertussis specific memory T cells after in vitro stimulation of peripheral blood mononuclear cells (PBMC) and an Elispot assay for the detection of antigen-specific antibody-secreting cells after polyclonal stimulation of PBMC for detection of memory B cells. We harmonisad as much as possible between the different partners of WP3, but inevitable differences remained. We used a multiplex immunoassay for simultaneous determination of serum antibodies to B. pertussis. Although the intention was to stick with the enrolment plan, there were some deviations because we encountered difficulties in obtaining particular subgroups of children, notably infected children and older children who received whole cell vaccines. Using our techniques, we were able to detect memory responses even long time after vaccination or infection and more detailed statistical analysis is ongoing.WP4: Construction and evaluation of recombinant B. pertussis / RSVThe finding that BPZE1 provides non-specific protection against viral infection by innate mechanisms is important; not only is BPZE1 safe, but it may have broad protective efficacy against a range of respiratory infections and assist in maturation of mucosal responses.These results were intriguing in that the protective effects correlate with IL-10 production and an increase in regulatory T-cells, and also with IL-17 production. Moreover, marked protective effects could be demonstrated in neonatal mice, linking the effect to cells producing IL-17 (CD4 and NK cells). A particularly exciting and positive outcome of the WP is that similar mechanisms appear to operate in both the mouse model and in the human cells studied in vitro and that a recombinant BPZE1 making immunogenic viral protein was finally made in the last months of the project. This will be tested further in the future. Finally, several recombinant BPZE1 derivatives were constructed, and one of them was shown to induce strong immune responses against the RSV G protein.WP5: GMP production of vaccine lot to be used in phase I clinical trials on B. pertussis BPZE1During the four years project, WP5 has taken the genetically modified Bordetella pertussis strain BPZE1 from preclinical studies in mice to a phase I clinical trial in men. Methods for manufacturing a nasal vaccine for human use were developed according to good manufacturing practice (GMP). Preclinical testing of the vaccine in mice and rabbits were performed according to good laboratory practice (GLP). Finally a phase 1, single centre, dose-escalating, placebo-controlled study of BPZE1 was conducted according to good clinical practice (GCP). 48 healthy adult male volunteers were given a single intranasal dose of the vaccine and followed up for 5-6 months after vaccination with regard to safety, immunogenicity and the ability of the vaccine strain to colonise the nasopharyngeal mucosa.The BPZE1 vaccine strain was safe in the doses used for nasal administration. Colonisation of the nasopharyngeal mucosa was dose-related and the immune responses were strongly correlated to the colonisation.This opens the way for further product and clinical development, especially for improvement of the formulation and the method for application and optimisation of the dose to enhance vaccine take as determined by the rate of colonisation and the development of immune responses.Project results:WP 1: Coordination and management of the consortiumThe main objectives were to:- set up an effective management framework for the CHILD-INNOVAC consortium;- ensure the usual and contractual administrative tasks (financial, reporting, organisation of (annual, council, strategic) meetings; - manage safety issues, legal, ethical and intellectual commercial / property-related issues, and gender equality promotion;- coordinate the dissemination of knowledge (communication activities) inside and outside the network.The CHILD-INNOVAC coordination and management WP took care of the usual and contractual administrative tasks (financial, reporting, organisation of consortium meetings, management of intellectual property, etc.). The project management team also focused on monitoring complex aspects related to the network's specific properties, notably some changes that emerged in the CHILD-INNOVAC consortium and work programme and the maintenance of the information hub (website). The management team acted as the interface between CHILD-INNOVAC partners and EC was in regular contact with EC Scientific Officer, and relayed problems and questions to the EC as soon as they occurred. The team was involved in the organisation of a variety of meetings, promotional materials (leaflet, poster, newsletter, etc.), and the maintenance of the project's website.WP2: Preclinical studies on B. pertussis BPZE1Several lines of research have been carried out in this pre-clinical WP with the general objective to gather a maximum of pre-clinical data on safety, immunogenicity and protective efficacy of the novel live attenuated nasal anti-pertussis vaccine candidate BPZE1. This information was essential for BPZE1 to be down-graded from biosafety level 2 to biosafety level 1 in several European countries and to proceed with this vaccine candidate to clinical development and a first-in-man phase I safety trial in healthy human volunteers. The various tasks included: 1) evaluation of protective immunity induced in adult, infant and neonatal mice and the study of the protective mechanisms induced by BPZE1;2) evaluation of the longevity of BPZE1-induced protection in mice;3) in vitro analyses of human immune responses to BPZE1;4) establishing the safety profile of BPZE1 in mouse models;5) determining the genetic and biological stability of BPZE1.1) BPZE1-induced protective immunityThree types of mice have been used and vaccinated with a single nasal dose of BPZE1: six to eight-weeks-old adult mice, three-weeks-old infant mice and one-week-old neonates. When the mice were challenged 2 months after a single nasal administration of 106 colony-forming units of BPZE1, total bacterial clearance was observed one week after intra-nasal challenge with virulent B. pertussis, regardless of the age of the mice at the time of vaccination. In neonatal mice, this protection was significantly better than that induced by the administration of two doses of commercially available acellular pertussis vaccine given intraperitoneally given at a three to four weeks interval. Similar observations were made when the mice were challenged by aerosol instead of nasal drops. BPZE1 dose-range experiments have been carried out between 102 and 107 colony-forming units, and a strong correlation between the dose and protective efficacy was found up to 106 colony-forming units, the dose that was estimated to be the optimal one. At none of the doses any inflammatory reactions were observed and the vaccine was very well tolerated at all doses. BPZE1 induced a strong Th-1 type response in neonatal as well as in adult mice, with a predominance of IFN-gamma production and antibodies to B. pertussis antigens predominantly of the IgG2a class. Interestingly, although commercial acellular vaccines induce essentially a Th-2 type response, after priming with BPZE1 the Th-1 type response is maintained upon vaccination with acellular vaccine. Both antibodies and T cells were found to contribute to BPZE1-induced protection against B. pertussis challenge, as passive transfer of antiserum or spleen cells from BPZE1-vaccinated mice, but not from naïve mice could transfer protection in SCID mice. Among the spleen cells the CD4+ T cells, but not the CD8+ T cells were the major protective effector cells in these transfer experiments. Interestingly, BPZE1 also protects mice against infection by Bordetella parapertussis and Bordetella bronchiseptica, for which currently no vaccine is available. However, in this case, protection could only be transferred by BPZE1-induced T cells, and not by antisera. In addition, BPZE1 protected against high dose lethal challenge with B. bronchiseptica, which was found to be mediated by CD4+CD25+FoxP3+ regulatory T cells. 2) Longevity of BPZE1-induced protectionAdult, infant and neonatal mice were vaccinated nasally with a single dose of BPZE1 and then challenged either 3, 6, 9 or 12 months after vaccination with virulent B. pertussis. The protection levels were compared over time with those induced by two intraperitoneal administrations of commercial acellular pertussis vaccine. Whereas immunity induced by the acellular vaccine started to wane six months after vaccination, protection was still complete one year after nasal vaccination with BPZE1. When serum antibody titers to B. pertussis antigens were measured, long lived antibody responses were found in both groups of mice, those that were vaccinated with BPZE1 and those that had received acellular pertussis vaccine. However only the antibodies induced by BPZE1 were able to transfer protection to SCID mice one year after vaccination. Similarly, only T cells harvested one year after vaccination with BPZE1 but not with acellular pertussis vaccine were able to transfer protection to SCID mice. In addition to the longevity of BPZE1-induced protection, it was also of interest to investigate how quickly BPZE1 is able to induce protection against virulent B. pertussis challenge. Mice were therefore challenged intra-nasally one, two, three and four weeks after a single nasal administration of BPZE1. Compared to the non-vaccinated mice, a significant reduction (by approximately two orders of magnitude) in colony-forming units one week after challenge was already seen when the mice were challenged one week after immunisation. Mice were then co-infected with virulent B. pertussis and BPZE1, and, thanks to a specific marker introduced in the virulent strain, the colonisation profile of the virulent organism was followed and compared to that of the mice infected only with the virulent strain. These experiments allowed us to detect BPZE1-induced protection as soon as three days after vaccination, a time not sufficient for the adaptive immune response to kick in, suggesting that early protection by BPZE1 is mediated by innate immunity. This was confirmed by the observation that BPZE1 also induced early protection in SCID co-infection experiments. TLR2- and TLR4-deficient mice were then used to determine that the early protection induced by BPZE1 was at least partially due to TLR4 signalling. The use of MyD88- and TRIF-deficient mice further indicated that BPZE1-induced protection depends on the MyD88 signalling pathway. 3) In vitro analyses of human immune responses to BPZE1Incubation of human monocyte-derived dendritic cells with BPZE1 induces maturation of these cells and confers the ability of these cells to migrate to the lymph nodes, where naïve T cells can be activated and adaptive immunity can be triggered. When activated by BPZE1, these monocyte-derived dendritic cells can also efficiently present antigens to T lymphocytes and can produce elevated levels of cytokines, especially of the Th-1 and Th-17 type. In this model BPZE1 thus behaves in many ways similar to the virulent parent strain, but both strains differ with respect to the ability to inhibit dendritic cell migration, which is lost in BPZE1. BPZE1-treated monocyte-derived dendritic cells also induced functional suppressor T cell activity. However, these cells do not produce the typical phenotypic markers of regulatory T cells (CD4+CD25+FoxP3+). IL-10 levels were elevated after treatment with BPZE1 but not TGF-ß. However, treatment with anti-IL-10 antibodies (or anti-TGF-ß) did not abolish the suppressor activity. In contrast, the prevention of cell-to-cell contact in transwell experiments almost entirely eliminated the suppressor activity of these cells. 4) Safety of BPZE1 in mouse modelsA major issue in the development of live attenuated vaccines is safety, including for immuno-compromised individuals. Initial safety data indicated that BPZE1, unlike its virulent parental strain, did not induce inflammation in the lungs of adult mice infected by the nasal route, although it is able to colonise the mouse respiratory tract nearly as long as the virulent strain. Furthermore, in contrast to virulent bacteria, BPZE1 did not impact on the breathing capacity of the mice, as investigated by plethysmography. Similar observations were made in infant mice. When neonatal mice were infected with 106 colony-forming units of virulent B. pertussis, death occurred in about 50 % of the mice. In contrast, all mice survived when infected with BPZE1. Several immuno-deficient mice were then used to assess safety. Infection of IFN-gamma receptor-deficient mice with virulent B. pertussis results in extrapulmonary dissemination, and bacteria can readily be isolated from the spleen of the infected mice. In contrast, when the mice were infected with BPZE1, no extrapulmonary dissemination was observed. Similar results were obtained in µMT and in SCID mice. When MyD88- / - mice were infected with 104 colony-forming units of virulent B. pertussis, all mice died within one to two weeks after infection. The examination of the lungs of these mice showed evidence of severe lung inflammation. In contrast, when even 100-fold higher colony-forming units of BPZE1 were used to infect MyD88- / - mice, no mortality occurred, and no lung inflammation was observed. Furthermore, these mice did not show any weight loss or sign of sickness. The effect of BPZE1 on experimental asthma was also investigated. These experiments were carried out in order to determine whether infection with BPZE1 would exacerbate allergic airway inflammation, as was described for virulent B. pertussis. Mice were therefore infected with either BPZE1 or its virulent parental strain and then sensitisad to ovalbumin. After repeated intra-nasal challenge with ovalbumin, airway pathology, local inflammation and ovalbumin-specific immune responses were evaluated. In contrast to virulent B. pertussis, BPZE1 did not exacerbate airway pathology. Instead it appeared to protect against experimental allergic asthma. BPZE1 administration resulted in reduced eosinophil influx, in reduced levels of ovalbumin-driven IL-4, IL-5 and IL-13 and IgE, but an increased level of IFN-gamma. Finally, the effect of BPZE1 administration was tested on the outcome of lethal viral infection. From a safety standpoint it is important to determine whether nasal administration of BPZE1 has a negative impact on unrelated respiratory infections. This was tested in an influenza A virus model. Infection of mice with mouse-adapted H3N2 or H1N1 influenza virus leads to death within a few days after infection. Administration of BPZE1 before viral challenge did not lead to death at earlier time points. Instead, it resulted in significant protection against influenza virus-induced mortality. It also protected against weight loss. However, vaccination with BPZE1 had no impact on the viral load and did thus not appear to protect against viral infection and replication, and there was no antigenic T or B cell cross-reactivity between BPZE1 and the influenza viruses. Instead, BPZE1 protected against virus-induced lung inflammation and pneumonia. It decreased neutrophil infiltration and suppressed the induction of the major inflammatory mediators and dampened the virus-induced cytokine storm. Only live BPZE1 showed this protective effect. Killed BPZE1 did not protect against influenza virus-induced pneumonia and death. Interestingly, this protective effect could be boosted by a second administration of BPZE1. Whereas a single administration of BPZE1 provided partial, yet still significant protection, a second dose given four weeks after the first dose increased the protection to 100 % against H3N2 virus-induced mortality. 5) Genetic and biological stability of BPZE1In order to propose live attenuated BPZE1 as a broadly used vaccine at the global level, potential reversion to virulence has to be addressed. This was done by passaging BPZE1 by continuous in vitro cultivation and in vivo through serial passages in mice. At several time points over a total of 3 years, the isolated bacteria were analyzed for the presence of all introduced attenuating mutations, and the absence of other gene rearrangements by microarray studies. There was no evidence of reversion at any time tested. Also, the passaged bacteria maintained the protective efficacy as efficiently as the original BPZE1 strain. This work has implied the development of novel molecular tools to detect potential reversion of the point mutations in the pertussis toxin gene at a sensitivity of 10-6. These tools have then been implemented in the quality control protocols for BPZE1 lot releases. In addition, the biological stability was investigated by suspending BPZE1 at different concentrations in different saline-based buffers. The suspensions have then be stored at -80 °C, -20 °C, +4 °C, room temperature and + 37 °C, and bacterial viability has been evaluated over time. Although the BPZE1 suspensions appeared to be stable at -80 °C, viability was quickly lost at room temperature and at +37 °C. Storage at intermediate temperatures gave intermediate viability levels. Nevertheless, viability at room temperature was good enough for product development. WP3: Immune responses to B. pertussis antigens in childrenThe main objective of WP3 was to evaluate the strength, diversity and duration of human immune responses to B. pertussis antigens in children naturally infected with B. pertussis or vaccinated with whole cell or acellular vaccines. To achieve this, the first objective of WP3 was to select, optimise and harmonise techniques that allow the evaluation of B and T cell memory. We decided that the B cell assay would be developed by partners 10 RIVM and 12 VWS-NVI, and the T cell memory assay by partners 3 ULB and 4 ISS, according to the previous experience of the partners in this field. In between the yearly CHILD-INNOVAC consortium meetings, we organised meetings in Brussels that were limited to WP3 partners, in order to discuss and solve problems raised during the optimisation process. The optimisation of the in vitro characterisation of antigen-specific memory B cells, performed by partner 10 RIVM and 12 VWS-NVI, included comparison of starting material (peripheral blood mononuclear cells (PBMC) or isolated B cells), comparison of fresh and frozen samples, evaluation of the need to add cytokines during in vitro culture, and evaluation of the amount of antigen required for stimulation. The protocol was then transferred to partners 3 ULB and 4 ISS, and lab visits between all partners assured good implementation of the protocol in all laboratories. Some inevitable differences between labs remained, among which the type of the plate reader. Therefore the absolute numbers of spots, i.e. antibody-secreting cells, may not be comparable, but the standardisation will still allow good comparison of results between different partners. Partners 3 ULB and 4 ISS were in charge of the optimisation and harmonisation of the techniques used for the in vitro characterisation of antigen-specific memory T cells. This included the comparison of starting material (PBMC or whole blood), comparison of fresh and frozen samples, evaluation of cytokine kinetics after stimulation and decision to continue with a long term assay (five days of culture), optimisation of eight-colour flow cytometry (antibody-fluorochrome combinations, dead cell marker, selection of memory markers, intracellular staining protocol). Partner 3 ULB organised a one week lab visit in order to share experience and discuss about the problems that raised. Finally, one protocol circulated between partners, in which differences (when inevitable) are clearly defined. Among these differences are the starting material (two partners work on fresh samples, two on frozen), the type of flow cytometer six versus eight colours) and the staining procedure. However, the same antigens were used by all partners, provided by partner 4 for FHA and PT and by partner 10 for PRN, and all partners use the same software for analysis of flow cytometry data (Flowjo). As ELISA tests are antigen- and serum-consuming as well as time consuming, partner 10 proposed the use of an alternative method (multiplex immune-assay) to measure IgG antibodies. Partner 10 RIVM performed all the serological tests for partners 3, 4, 10 and 12, so that the results can be compared. The different partners started the inclusion of children and the standardised techniques for detection of T and B cell immune responses were applied to collected blood samples. All partners obtained the agreement of the local ethics committee for the enrolment of children who had whooping cough or who were vaccinated against pertussis. Information material and informed consent circulated between partners of WP3 for harmonisation. As during the last years there have been changes in the vaccine schedule or the type of pertussis vaccine that is administered in all the involved countries (Belgium, Italy and the Netherlands), this allows the recruitment of children from different age groups that received different vaccines, or that were infected in their first year of life. Inclusion of children was performed without any problem for those vaccinated with an acellular pertussis vaccine. However, we encountered some difficulties to obtain enough children in specific subgroups. This was the case for infected children, because we needed to respect the privacy law and the inclusion rate was lower than expected. Another problem that we encountered, especially by partner 3 ULB, is that children did not always receive the vaccine that they theoretically would, according to their age, meaning that there were many deviations from the vaccine recommended at that time. Finally, the following groups of children were included by the different partners: - Partner 3 ULB included 20 children who were infected early in life and 148 vaccinated children. Within this last group, 13 children had a confirmed whole cell vaccine, 43 the acellular vaccine Infanrix and 41 the acellular vaccine Tetravac. For the remaining vaccinated children we either couldn?t confirm the vaccine they received or either they were excluded because there was a deviation from the vaccination scheme. - Partner 4 ISS included 10 infected children for a follow-up study, but for the second blood sampling the number reduced to 5. Therefore, five more children were recruited. 104 children aged 5-7 years and vaccinated with an acellular vaccine were sampled, from which 38 received Infanrix and 66 Hexavac.- Partner 10 RIVM included 45 whole cell vaccinated children of 4, 6 and 9 years old, as well as 15 4-year old children who received the acellular vaccine Infanrix.-Partner 12 VWS-NVI included 76 children infected early in life, and the age at blood sampling was 0 to 14 years.If sample size allowed it, both memory T cell and B immune responses were determined, as well as antigen-specific antibodies in the serum. Using the techniques optimised in our WP, we were able to detect B and T cell immune responses, even longtime after vaccination or infection. Generally speaking, responses to FHA were higher than those induced by PT, which is in accordance with the specificity to B. pertussis. More detailed analysis will be presented in manuscripts by different partners regarding their own cohorts of children. We are also evaluating the possibility of a common publication on Infanrix-vaccinated three to four years old children as all partners have included children that meet these criteria. WP4: Construction and evaluation of recombinant B. pertussis / RSVThis WP was restricted in its initial success by the problems in generating immunogenic recombinant BPZE1 expressing RSV proteins. However, important and novel findings regarding the non-specific effects of BPZE1 were demonstrated and mechanisms investigated. These results were intriguing in that not only did the protective effects correlate with IL-10 production and an increase in regulatory T-cells, but that IL-17 production appears integral. Moreover, protective effects could be demonstrated in neonatal mice, proving IL-17 to be crucial and linking the effect to defined cell populations producing IL-17 (CD4 and NK cells).Work conducted under WP4 is to demonstrate: 1. that BPZE1 inoculated directly via the nose and distributed and throughout the respiratory tract, appears perfectly safe in mice; 2. BPZE1 has non-specific protective effects against secondary viral challenge and induces mucosal maturation following nasal administration; 3. parallel innate immune effects have been demonstrated human cell lines, strengthening our conviction that the beneficial effect seen in mice may also operate in humans; 4. new insights into the mechanisms of protection (by innate triggering and interleukin 17) will have important implications for the induction of broadly protective mucosal responses;5. the exciting breakthrough made in the final closing phase of the grant was that it is possible to generate an immunogenic recombinant expressing part of the major surface glycoprotein G of RSV that induces strong antibody responses in mice. This opens new fields of research which will be continued beyond the duration of the CHILD-INNOVAC grant. WP5: GMP production of vaccine lot to be used in phase I clinical trials on B. pertussis BPZE1The overall tasks of WP5 was to carry out a phase I clinical trial of the genetically modified strain of Bordetella pertussis, BPZE1, after due applications with approvals from the Swedish Medical Products Agency and Ethics Committee, according to Good Clinical Practice and in accordance with the recommendations guiding physicians in biomedical research involving human subjects and to summarise the study results and present a study report to the Medical Products Agency and to the consortium. First, intense collaborative interactions between partners have allowed transferring the technology to GMP production and the establishment of a master cell bank and working cell banks, to establish lot release criteria and develop methods for quality assurance and quality control. Several pre-clinical and three clinical lots were produced and tested for genetic stability and potency in the mouse model. All lots were produced in totally synthetic media. Toxicology studies were outsourced. Drug product stability studies were performed, which indicated that for this study BPZE1 is best stored and transported at temperatures below - 60 °C in saline with 5 % sucrose. All steps were executed to the satisfaction of the sponsor (INSERM) and the Swedish Medical Product Agency, which has allowed us to proceed with the phase I study in Sweden. The primary objective of the clinical trial was to assess the general safety and local tolerability in the respiratory tract of a single ascending dose of the genetically modified B. pertussis strain. The secondary objectives were to see if the modified B. pertussis strain has the ability to colonise the human respiratory tract and to assess the B and T cell immune responses to 5 different B. pertussis antigens: pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae and whole Bordetella pertussis cell lysates.The study design for the phase I clinical trial of BPZE1 was presented for and discussed with the European Medical Agency, the Swedish Medical Product Agency and the INSERM advisory committee before the final protocol was finalised.Applications to the regional ethical committee and the Swedish Medical Product Agency were submitted in April 2010. The documentation (approximately 5500 pages) contained besides the study protocol informed consent form, subject diary, patient card, results of toxicity testing, investigator's brochure, gene modified organisms - environmental risk assessment, gene modified organisms - history of BPZE1, gene modified organisms - summary in Swedish, toxicity testing certificates, good manufacturing practice certificates. Many of the documents were available both in Swedish and English. The whole dossier is stored on the CHILD-INNOVAC private website.The applications were approved by the regional ethical committee in May 2010 and by the Medical Product Agency in July 2010. An amendment of the protocol was approved in November, 2010. Standard operational procedures, case report forms, laboratory analytical plan and other documents were approved by the sponsor prior to the clinical trial.Methods for determination of pertussis specific IgG producing plasma and memory B cells, IFN and IL.2 producing T cells and lymphocyte proliferation were developed, optimised and validated. The value of these exploratory assays will be evaluated during the study. The laboratory analytical plan contained in total 38 standard operation procedures (SOPs) and 42 other documents. The major SOPs were included in a laboratory handbook, which has been shared with the project consortium.A start meeting for the clinical trial all staff members at the clinic and the laboratory was held on 10 August 2010 at the clinical trial site Karolinska Trial Alliance, Karolinska University Hospital. The first volunteer was recruited on 11 August 2010 and the last visit of the last volunteer was on 15 June 2011.In total 107 volunteers were recruited of which 48 healthy Swedish males aged 19 - 31 years meeting the inclusion and exclusion criteria were vaccinated once with drops administered by tuberculin syringes, one syringe containing 0.1 mL for each nostril. The volunteers were recruited in a step-wise fashion with 16 individuals in each of three dose groups. 12 individuals in each group were vaccinated once intranasally with a low dose, a medium dose and a high dose, respectively. Four individuals in each group got placebo (the diluent alone).The administration of vaccine or diluent alone was performed in a double-blind fashion with administration of coded vials with exception for the two first study subjects in each dosage group, who received active substance (unblinded).The volunteers visited the clinic for providing written informed consent and screening two to six weeks before vaccination. On the day of vaccination the volunteers stayed at the study centre for six hours after administration of the vaccine. Thereafter the volunteers visited the clinic at day 4, 7, 11, 14 and 28 and at 5-6 months after vaccination for physical examination, sample collection and questions concerning general and local adverse events and questions about unsolicited adverse events.The following safety parameters were assessed:- general safety, i.e. general well-being of the volunteers and any symptoms felt by the volunteers with onset within one month after vaccine administration;- vital signs: blood pressure, heart rate, respiratory rate, oral temperature;- abnormalities in blood cell count;- specific side effects: local symptoms from the respiratory tract: sneezing, swollen nose, cough, bleeding from the nose, pain or other symptoms from the ear, symptoms from the eyes (redness, secretion).The safety data were reported to the sponsor and to the independent data monitoring committee (IDMC) between the dosage groups for decision before dose escalation. A final evaluation meeting was held prior to the unblinding of the study results.One serious adverse event was reported during the clinical trial. It was judged by the IDMC, the sponsor and everyone involved in the study as not vaccine related.Any differences in adverse events between placebo and the different dosage groups were not detectable. Among the 48 volunteers (36 vaccinated with an active vaccine and 12 with placebo) the most frequent adverse events were rhinorrhea, sneezing, nasal congestion, headache and tiredness. Many of the volunteers had common cold during the follow up time, which might explain at least a part of the symptoms.Bacterial culture of nasopharyngeal aspirate was performed at five time points during four weeks after vaccination. The vaccine strain was detected between day 4 and 28 after vaccination and the ability of the vaccine strain to colonise the nasopharyngeal mucosa was dose-related. The objective of the immunogenicity analysis was to assess the B and T cell immune responses to five different B. pertussis antigens: pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae 2/3 and whole Bordetella pertussis cell lysate before and after vaccination with BPZE1.Immune responses were determined by:- serum IgG and IgA and saliva IgA by ELISA;- IgG producing plasma and B cells by ELISpot assay;- IFN and IL-2 producing T cells by ELISpot assay;- T lymphocyte proliferating CD4+ and CD8+ T cells by flow cytometry-based assay;- cytokine production in cell culture supernatants by Luminex-based assay.Except for serum IgG antibodies, the immunological assays were regarded as exploratory assays, which value will be evaluated during the study.More than 3500 blood samples, 480 saliva samples and 750 nasopharyngeal aspirates were processed during the clinical trial and approximately 60 000 individual test results were produced and monitored according to good clinical laboratory practice.The effect of the vaccination was measured by the proportion of subjects with a positive immune response and median titres (serum IgG) in the different dosage groups. The serum antibodies appeared 14 days after vaccination and remained at high levels after 5-6 months follow up. Plasma cells producing pertussis specific IgG were detected already on day 7 after vaccination, while other pertussis specific B and T cell responses were demonstrated from day 14. The pertussis specific immune responses were strongly correlated to the colonisation of the nasopharyngeal mucosa. The safety data and immune responses have been summarised in a statistical report and will be described in detail in a clinical study report (available by the end of April 2012) and in scientific papers.Potential impact:General information was provided to all partners during the project's final meeting on possible dissemination / commercial exploitation strategies developed by Inserm / IT for further development of the BPZE1 vaccine platform. IT is exploring various scenarios and opportunities in order to maximise the dissemination / exploitation chances of the generated foreground, notably via licensing procurement to major pharmas or targeted biotech companies. Once an agreement will be concluded with an industrial partner, IT and Inserm engage to come back to the relevant CHILD-INNOVAC partners in order to discuss potential opportunities of new project(s) to boost the translation of the generated foreground into innovative applications for health where applicable (via for instance new FP7 bids opportunities) and depending on the business opportunity choosen. During the CHILD-INNOVAC PROJECT, we have developed four main dissemination activities: - Adapted communication tools: The CHILD-INNOVAC public website, presentation brochure and poster. The CHILD-INNOVAC public website (see http://www.child-innovac.org/ online) was presenting the consortium, the context of the project, its objectives and monitored the progress of the project. In addition, sections about the documentation, as well as for news and announcements, were proposed on the site. A brochure and a poster presenting the project were also issued to support the dissemination activities to a wider public. - Workshops and conference presentations: When appropriate, the participants presented the project and related results to scientific and clinical conferences and workshops in Europe and outside Europe (such as the European Congress of Clinical Microbiology and Infectious Disease). Attendance to these meetings gave the partners the opportunity to present the work of the consortium and develop interest of other experts but also develop contact with industrial partners. - Publications in peer-reviewed and / or open access journals: Scientific results of the project were disseminated via publications in peer-reviewed and open access journals and in abstracts of national and international conferences. The CHILD-INNOVAC project led to the publication of 19 articles in renowned in peer-reviewed journals and several are still under preparation.- Outreach the general public: In addition to our public website, several press release have been published in newspapers, an interviewed was made for a national French TV channel and a video is available on you tube to present the CHILD-INNOVAC project. WP2: Preclinical studies on B. pertussis BPZE1The work performed under WP2 has a great impact in several ways:1) The safety profile, including in severely immunocompromised mice, has allowed BPZE1 to be downgraded from biosafety level 2 (the international biosafety level for Bordetella pertussis) to biosafety level 1 in several European countries. This has been particularly essential for BPZE1 to undergo GMP production, toxicology studies and the first-in-man phase I clinical safety trial in healthy adult human volunteers. Without the safety information gathered in this WP, these product and clinical developments would have been very difficult if not impossible. Several papers have been published, and further papers are in preparation: - Skerry C. M., Cassidy J. P., English K., Feunou-Feunou P., Locht C., Mahon B.; 2009. A live attenuated Bordetella pertussis candidate vaccine does not cause disseminating infection on gamma interferon receptor knockout mice. Clin. Vaccine Immunol. 16, 1344-1351. - Kavanagh H., Noone C., Cahill E., English K., Locht C., Mahon B.; 2010. Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen-induced immunity and prevents allergic pulmonary pathology in a murine model. Clin. Exp. Allergy 40, 933-941. The work has also been presented at numerous national and international scientific meetings, both in Europe and elsewhere. The work has also led to a patent application: - Vaccine for prophylaxis or treatment of an allergen-driven airway pathology. H. Kavanagh, B. Mahon, C. Locht. Submission number 1000052155; Application number EP09395371.8; date of receipt April 28th, 2009. 2) The longevity of BPZE1-induced protection may also have a strong impact on public health in the long run, especially in the developed countries, where booster vaccinations with current vaccines are often difficult to implement. In contrast, nasal vaccination at birth may be the most effective way to ensure a large vaccination coverage. Although BPZE1 is designed as a priming vaccine in the neonatal period, so that the current vaccine schedules can be the viewed as boosters, the observed longevity of protection, if confirmed in humans, may prolong protection also in non-boosted children. In addition, the observation that BPZE1 induces rapid protection is also of great impact, as it implies that neonates vaccinated soon after birth will benefit very quickly. Finally, BPZE1 also appears to protect against other Bordetella species, such as B. parapertussis and B. bronchiseptica, both respiratory infections for which non vaccine is currently available.Again, several papers have been published, and others are still in preparation- Feunou Feunou P., Bertout J., Locht C.; 2010. T- and B-cell-mediated protection induced by novel, live attenuated pertussis vaccine in mice. Cross protection against parapertussis. PLoS One 5, e10178- Feunou Feunou P., Kammoun H., Debrie A. S., Mielcarek N., Locht C.; 2010. Long-term immunity against pertussis induced by a single nasal administration of live attenuated B. pertussis BPZE1. Vaccine 28, 7047-7053. - Skerry C. M., Mahon B. P.; 2010. A live, attenuated Bordetella pertussis vaccine provides long-term protection against virulent challenge in a murine model. Clin. Vaccine Immunol. 18, 187-193. - Mielcarek N., Debrie A. S., Mahieux S., Locht C.; 2010. Dose response of attenuated Bordetella pertussis BPZE1-induced protection in mice. Clin. Vaccine Immunol. 17, 317-324.3) The findings of the effect of BPZE1 on human dendritic cell maturation is also of importance as possible explanations for in vivo effects of BPZE1 in humans. This has also lead to a publication - Fedele G., Bianco M., Debrie A. S., Locht C., Ausiello C. M.; 2011. Attenuated Bordetella pertussis vaccine candidate BPZE1 promotes human dendritic cell CCL21-induced migration and drives Th1/Th17 responses. J. Immunol. 186, 5388-5396. 4) Finally, the genetic stability, i.e. non-reversion to virulence, has also a major impact on the clinical development of BPZE1. Reversion to virulence would have lead to a no-go for clinical development. This also has lead to a publication, and an additional publication is in preparation. - Feunou P. F., Ismaili J., Debrie A. S., Huot L., Hot D., Raze D., Lemoine Y., Locht C.; 2008. Genetic stability of the live attenuated Bordetella pertussis vaccine candidate BPZE1. Vaccine 26, 5722-5727. The CHILD-INNOVAC project, including the pre-clinical studies, has been the theme of a video that can be seen at http://www.youtube.com/watch?v=mIpeBiWR4Qs and http://www.comed-project.org/index.php?id=17.WP3: Immune responses to B. pertussis antigens in childrenThe technique to detect antigen-specific memory B cell responses was described earlier by colleagues of partner 10 RIVM, but during the CHILD-INNOVAC project, this technique underwent further optimisation and this resulted in a less time-consuming experiment. For the T cell memory assay, we can now present a technique that amplifies the antigen-specific response during a five-day culture, allowing the detection of immune responses that are too small to be detected in a short term culture. A longer stimulation period, as proposed in our assay, seems to overcome the problem with antigens for which low frequencies of responding T cells circulate, and might therefore be useful for other pathogens as well. It might be especially useful in studies evaluating vaccine effectiveness months or even years after vaccination, in which case the assays that are currently used to evaluate vaccine effectiveness after e.g. one month, will not be sufficiently sensitive to detect a response. Each of the partners of WP3 is writing manuscripts describing immune responses induced by the vaccine schedules used in their country. Together, this will lead to a better overall view on vaccine or infection induced immunity, both with respect to duration and characteristics (type and magnitude). WP4: Construction and evaluation of recombinant B. pertussis / RSVBecause of the delays in obtaining recombinants that were immunogenic, publications from this WP are still in preparation. So far, the work has been presented in abstract form and as posters at meetings. The work is in preparation for publication and will be the subject of press releases and public dissemination activities at that time. However, we anticipate at least three publications arising from the current body of work and further publications arising in the future as a result of the novel recombinant available beyond the duration of the CHILD-INNOVAC grant. In terms of the broad implications, this WP adds substantially to the evidence that BPZE1 is not only safe but might also have beneficial non-specific protective effects against a wide variety of respiratory pathogens by accelerating mucosal maturation. This new direction of research could have wider implications beyond the proposal to study RSV-specific immunity induced by a recombinant BPZE1 expressing RSV antigens. However, the very recent availability a novel BPZE1 recombinant expressing RSV antigen now opens the possibility of producing a combined B. pertussis and respiratory syncytial virus vaccine. A single live attenuated vaccine capable of protecting against common multiple respiratory pathogens of childhood would be a truly important and novel breakthrough. Although this ultimate goal eluded us during the timeframe of the grant, this ultimate outcome of the CHILD-INNOVAC project appears within our grasp.WP5: GMP production of vaccine lot to be used in phase I clinical trials on B. pertussis BPZE1The results of this first-in-man clinical trial are very encouraging, which encourages further product and clinical development. There is a need for further development of the formulation and the method for application and optimisation of the dose to improve the vaccine take as determined by the rate of colonisation and the development of immune responses.The results will be described in detail in a clinical study report (available by the end of April 2012) and will be published in scientific papers in peer reviewed journals:- Thorstensson R., Trollfors B., et al.: Nasal vaccination with a genetically modified strain of Bordetella pertussis, BPZE1, is safe and immunogenic. - Jahnmatz M. et al.: Optimisation of plasma and B cell ELISpot assay for detection of IgG production.- Jahnmatz M. et al.: Induction of pertussis specific IgG producing plasma and B cells after nasal vaccination with a genetically modified strain of B. pertussis.- Jahnmatz M.: PhD thesis at Karolinska Institutet, Stockholm, Sweden- Wehlin L., Kostic L. et al.: Induction of pertussis specific T cell responses after nasal vaccination with a genetically modified strain of B. pertussis.Partners involved:Partner 1: Institut National de la Santé et de la Recherche Médicale (INSERM, coordinator), FrancePartner 2: Inserm-Transfert (IT), FrancePartner 3: Université Libre de Bruxelles (ULB), BelgiumPartner 4: Istituto Superiore di Sanità (ISS), ItalyPartner 5: National University of Ireland, Maynooth (NUIM), IrelandPartner 6: Imperial College London, United KingdomPartner 7: Swedish Institute for Infectious Disease Control (SMI), SwedenPartner 10: National Institute for Public Health and Environment (RIVM), the NetherlandsPartner 11: Innogenetics N.V. (INNX), BelgiumPartner 12: Ministerie Van Volkgezondheid, Welzijn en Sport (VWS-NVI), the Netherlands.Project coordinator's contact: Project coordinator: Camille Locht - INSERM E-mail: Camille.Locht@pasteur-lille.fr Website of the project: http://www.child-innovac.orgDuration of the project: 48 months (1 March 2008 - 29 February 2012) Related documents Final Report - CHILD-INNOVAC (Nasal vaccination against respiratory infections in young children)
