Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Periodic Report Summary 5 - STOPENTERICS (Vaccination against Shigella and ETEC: novel antigens, novel approaches)

Project Context and Objectives:
STOPENTERICS aims at developing novel vaccine candidates against Shigella and ETEC for children of the developing world. This consortium gathers a unique combination of laboratories, platforms, vaccinology centers from academia and industry in the North and in endemic areas. STOPENTERICS proposes novel solutions by imposing a paradigm switch (i) to break the dogma of serotype-specificity by seeking to induce cross-protective immunity. It exploits the potential of genomics, transcriptomics and proteomics to identify surface and virulence proteins conserved throughout Shigella or ETEC isolates. For ETEC, the development of a safe, immunogenic ST toxoid is also a major goal. For Shigella, the vaccine potential of bacterial outer membrane blebs (GMMA) is exploited; (ii) another approach is to improve the immunogenicity of Shigella glycoconjugates by using synthetic oligosaccharides mimicking the lipopolysaccharide (LPS) O-antigens (O-Ags).
The ultimate aim is to optimize chances for best coverage by combining - possibly in a single formulation - cross-protective protein antigens and the most prevalent serotype-defining synthetic oligosaccharides. STOPENTERICS has developed two platforms, one dedicated to the identification and production of novel protein antigens, and one dedicated to the evaluation of the protective capacities of candidate antigens in animal models. However, the predictive value of these models shows limitations, due to the human-specificity of both pathogens, thus a strong incentive to also push the new vaccine candidates through clinical evaluation as soon as possible.
A key issue is also to define the optimal route of immunization. STOPENTERICS aims at parenterally-administered formulations. A significant effort is therefore invested in optimizing the delivery route and the associated adjuvant in order to obtain maximum titers of specific serum IgG combined with significant titers of mucosal sIgA. Particular attention will also be paid to the elicitation of strong T cell responses and B/T cell memory.
Finally, two phase 1 clinical trials that are proposed as proofs–of-concept are both on a successful trajectory. (i) A GMMA-based vaccine (NGVH / now GSK) - that emerged from STOPENTERICS-based research - was produced under GMP conditions in 2013. Immunization of volunteers have been completed at the Paris Cochin-Pasteur Vaccine Center in 2014 and full results of this trial have been released at the STOPENTERICS annual meeting in Oxford in September 2015.
(ii) A glycoconjugate (IP) incorporating a synthetic oligosaccharide mimicking the Shigella flexneri 2a O-Ag (called SF2a-TT15) that will be tested at the Tel Aviv Sourasky Medical Center in 2016, thanks to a one-year extension allowed by EU. GMP lots of the synthetic polysaccharides are well advanced (Sanofi-Chimie) and they have been transferred for conjugation (Intravacc).
Hence a key objective of the program, mainly “to carry out at least one phase 1 clinical trial of a vaccine candidate” will be fulfilled beyond expectation.
Beyond the teams’ efforts to complete the individual work packages, the members of the SAB who have closely supervised STOPENTERICS all along, have consistently expressed pertinent comment, several of which addressing transversal issues with an aim to increase synergies. In summary:
– To attempt to further increase the level of exchanges between the representatives of the different work packages. A strong effort was made to organize “cross-WPs”, the website being used to increase the flux of exchanges thanks to securing sufficient funding to WP7.
– To early define the profile that one wishes to give to the vaccine candidates, beyond R&D and phase 1 stages. The exercise made for the GEMMA vaccine served as blue print.
– To increase the international visibility of STOPENTERICS, and prepare future funding for the “post-STOPENTERICS” era. STOPENTERICS was thus (re)presented in relevant international conventions such as VED (Thailand, UK), EU-sponsored meeting (Brussel), etc… STOPENTERICS has established solid interactions with the Bill & Melinda Gates Foundation, via PATH: common meetings and symposia, visit of the coordinator to the PATH representatives in Washington, successful application to a PATH-funded project on improvement of antibody production and gut delivery following vaccination by subunit vaccines. Last but not least, the last annual meeting held in September 2015 comprised a one-day symposium – entitled “What’s next ? Towards STOPENTERICS 2” - that gathered most of the international stakeholders and funding agencies, in addition to SAB members. This symposium was an opportunity to present STOPENTERICS achievements in front of an international and highly relevant audience, and to reflect on future funding of the actions launched by STOPENTERICS. Opportunities for applications to H2020, EDCTP and the Bill and Melinda Gates Foundation (among other possible options) were discussed in the final wrap-up round session.
In summary, STOPENTERICS is a lively and productive forum that crystallizes EU efforts and position in the field of diarrheal vaccine development, but the “post-STOPENTERICS” era becomes an issue of major importance, particularly in regards to the follow up on the phase 1 clinical trials.

Project Results:
WP1, “Getting ready for clinical trials” is led by Dani Cohen (TAU) and Armelle Phalipon (IP).

GMMA vaccine (Shigella sonnei 1790GAHB) trial is completed, and its results were released at the last annual meeting in Oxford in September 2015. Immunization phase of the phase 1 clinical trial was performed in Paris with no major vaccine-related side-effect. All relevant biological markers of human tolerance and immunogenicity are available. Significant immunogenicity opens the way to further steps, including a multivalent formulation. D1.5 can thus be considered fully delivered.

SF2a-TT15 conjugate incorporating the synthetic pentadecaccharide mimicking S. flexneri serotype 2a-specific determinants is in GMP production. Due to a significant delay in the delivery of the (pre-) GMP pentadecasaccharide by Sanofi-Chimie, due to unexpected issues of product degradation at the last stage of lyophilisation, a SF2a-TT15 GMP lot could not be produced in time to be delivered to Intravacc for conjugation to its carrier protein, quality control of the conjugate and final delivery of the GMP lot before the official end of STOPENTERICS on Oct. 30, 2015. Following intensive action by Sanofi-Chimie this technical issue was solved last year. In parallel, Institut Pasteur obtained a one-year extension for STOPENTERICS from EU. Following rescheduling, delivery of the SF2a-TT15 GMP lot is expected before April 2016, hence permitting initiation of the phase 1 trial in Tel Aviv before summer 2016, well before the official new date of completion of WP1 (i.e. Oct. 30, 2016). Meanwhile, all pre-clinical components have been or are successfullyconducted, including preparation of the clinical trial itself. In summary: D1.1, D1.2 (due M42) not delivered but postponed. According to this new schedule, D1.3 and D1.4 respectively due M52 and M60 will also be postponed, now with the guarantee of no further obstacle. There is still pending discussion about the necessity to on-btain a further six-month extension beyond October 2016 in order to safely include the full post-immunization surveillance period.

WP2, “Immunomonitoring and correlates of protection” is led by Dani Cohen and Ann Mari Svennerholm (UGO). It aims to provide the tools necessary to optimally evaluate the protective capacity of the vaccine candidates. Thus a strong emphasis on novel correlates of protection and markers for immunomonitoring. This WP engages several groups that have high experience in immunomonitoring of vaccine trials and immunological analysis of natural enteric infections. The relevant milestones were met, efforts continue with strong emphasis on B-cell memory with the aim to provide better biomarkers of immune protection. In summary, D2.1-3 were successfully delivered on time. D2.4 was partially delivered as the SF2a-TT15 clinical trial has not yet been conducted. D2.5 was very partially delivered as the yield of novel potentially cross-protective proteins from ETEC and Shigella that were not shared with commensal E. coli, and appeared biochemically “manageable”, remained very limited.

WP3, “Discovery and evaluation of novel Shigella cross-protective antigens” is led by Christoph Tang (UO). This program integrates transcriptomics and proteomics with a strong accent on in vivo expression. Both Shigella and Shigella-derived GMMAs are used for identification of cross protective proteins. This WP underwent a major delay as its leader moved to University of Oxford in Oct. 2011. Difficulties regarding RNA extraction from bacteria grown in vivo have been painfully solved and conditions for protein extraction from in vivo samples were established prior to (immuno)proteomics analysis. Hence D3.5 due M36 could not be met. The last year has been marked by considerable technical progress. It has been possible to establish proper conditions of bacterial RNA extraction from samples grown in vivo in conditions of infection. From these experiments, a small set of genes that are strongly induced in conditions of in vivo growth has been identified and the relevant proteins confirmed. A couple of these proteins have been selected and their role in virulence is currently studied, as well as their location at the bacterial surface, their possible purification and expected immunogenicity. In summary, D3.1-3 have been delivered. D3.4,5 are still pending in spite of slid progress.

WP4, “ETEC antigen discovery” is led by Halvor Sommerfelt (UiB). Bioinformatics work is pursued to identify ETEC-specific genes preferentially encoding putative surface exposed proteins. Encouraging results have been obtained, although like Shigella, bearing on a limited number of candidate proteins, one of which being already patented by another group. These antigens will be tested in the murine model of passive protection against ETEC. In consequence D4.4 can be considered delivered if one sticks to the strict definition of D4.4:”Description of a database pf ETEC antigens that potentially induce anti-colonizing immunity”. If, however, one requests final functional proof of concept, it has not yet been fully provided.
Cross-reactivity to uroguanylin is indeed a real concern for an ST vaccine, but careful choice of ST peptide variant and conjugation configuration may prevent such cross-reaction. Further progress on mutagenesis of the ST gene has provided mutants of low enterotoxicity, while maintaining immunogenicity with the help of a carrier. The major challenge set by STOPENTERICS to safely and efficiently protect against ST toxin may be in view but not solved yet. We still need complete assessment of the abilities of the conjugates to elicit neutralizing antibodies in mice, and their cross-reacting potential. Thisis in view for March 2016. In consequence D4.3 is not yet fully delivered.

WP5, “Synthetic oligosaccharides as immunogenic mimics of Shigella O-Ag” is led by Laurence Mulard (IP). This WP aims at synthesizing and optimizing the length of oligosaccharides mimicking the serotype-specific determinants of the 5 most prevalent Shigella serotypes. A library of oligosaccharides of different lengths is synthesized. Then, a battery of mouse monoclonal antibodies (mAbs) specific for each serotype is used to select the best recognized oligosaccharides. The major serotypes that were planned for synthesis have been achieved, particularly SF2A and 3a, but also SF1b and 6. Characterization is well advanced, and the issue of possible serotype dominance has even been addressed. In brief, D5.1-3 are completed. D.4,5 are on their wayand will be completed by the now set date of October 2016 that will mark the end of STOPENTERICS.

WP6, “Research and development, from antigens to vaccine formulations and preclinical testing” is led by Lina Bernardini (Uniroma). It is aimed at standardizing methods of protein production and at implementing relevant animal models to screen the protective potential of the candidate antigens. The Guinea pig model of Shigella colitis now operates in routine, but could not be adapted to ETEC. Erik Nygren at UGOT could develop an alternative model of passive immunization in the infant mouse.
Due to limitation in identification and validation of new cross-reactive protein antigens in Shigella and ETEC, D6.2 and D6.4 are delayed. On the other hand, D6.1 is completed and D6.3 is well on its way and will surely be completed by October 2016.

WP7, “Training in vaccinology” is led by Armelle Phalipon (IP) in collaboration with Jean-Pierre Kraehenbühl (HSeT). The STOPENTERICS website is regularly updated ( It is a very efficient tool as back up of vaccine courses, particularly through its e-learning portal. It has been enriched by the set up of a forum through which the STOPENTERICS participants can directly exchange. It also presents the administrative elements that regulate STOPENTERICS operation. This WP has been completed. A two-week course on clinical trials and immunomonitoring was successfully conducted at ICDDR,B, Bangladesh. Thus, in addition to the annual five-week vaccinology course (D7.4) due for M48, the ICDDR,B course (D7.5) was also completed on time (expected M48).

WP8, “Project management and coordination” is led by Sophie Ablott (IP) and by Thomas Wiest (IP) from 01/10/2014. They have jointly supervised amendments, ensured that the website contains all relevant administrative, financial and legal documents and organized the second, third and forth annual meetings. Efficient communication was maintained throughout the first three years period at the scientific and administrative levels. Five meetings were organised during the first 48 months. A report of each meeting was written, and all material presented and the minutes were made available to the partners under a password-protected portal.

Potential Impact:
Expected final results, their potential impact and use.
The major scientific and medical ambitions of STOPENTERICS were summed up in the introduction of this summary. After two years, most of the essential elements of this consortium have been put in place and most of the deliverables and milestones were met. If not, the reasons were of scientific nature and of technical nature in two cases. The issues will be dealt with within the end of the program. The focus has been strongly maintained towards the set objectives of a switch in paradigm in vaccination against Shigella and ETEC.

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