Final Report Summary - LACTOBODY (Production and delivery of antibody fragments against gastrointestinal pathogens by lactobacilli)
Executive Summary:
Enteric infections such as rotavirus and C. difficile induced diarrhea, remain a major cause of morbidity and mortality globally, accounting for an estimated 2 millions deaths each year. Effective preventive and therapeutic interventions are not yet available for many etiological agents of diarrheal diseases. Furthermore, even where vaccines are available, the lag time needed to induce an immune response can be critical in epidemic situations.
The aim of this project was to develop an effective treatment against rotavirus and Clostridium difficile based on lactobacilli producing VHH and scFv antibody fragments. As a proof of principle, a lead VHH fragment (called ARP1) against rotavirus was tested in a community-based, double-blind placebo controlled interventional trial in south India. A large batch of ARP1 was produced in yeast and prophylactically administered to children. In parallel, we have generated, selected and expressed new scFv and VHH fragments against the gastrointestinal pathogens rotavirus and C. difficile in lactobacilli. The modified bacteria were tested for their protective capacity in vitro and animal models.
The genes encoding the ARP1 fragment against rotavirus was subsequently cloned using pharmaceutical grade and biologically contained expression systems in a probiotic Lactobacillus strain. The modified bacteria were tested in healthy human volunteers to ensure safety and to measure their immunogenicity.
Our approach, which falls into the priority "Innovative approaches and interventions" and the work program "Development and production of new generation antibodies" represents a novel system for the induction of passive immunity that can be rapidly applied to populations at risk (for example through the drinking water, rehydrating solution or as a food supplement). The results obtained in this project could be applied to therapy against a vast number of human/animal pathogens in the gastrointestinal tract.
Project Context and Objectives:
The LACTOBODY project is a four year project that falls into the topic “Development and production of new generation antibodies” of the FP7 Health work program. The main objective of this topic was the development of new, efficient and safe preventive strategies and/or therapies by combining high specificity and effector functions with stable production, preclinical studies and good manufacturing practice (GMP). The LACTOBODY project is based on an idea that may revolutionize passive immunisation: engineering bacteria “generally regarded as safe” (GRAS) to produce antibody fragments directly at the mucosal site. The consortium was composed of seven groups in Europe and one in India (see list of participants in Annex). The project started in February 2008 and the present summary submitted in June 2012 covers the work performed during the whole project.
Enteric infections remain a major cause of morbidity and mortality globally, accounting for an estimated 2 millions deaths each year. In developing countries, over 125 million episodes of rotavirus diarrhea are observed annually in children under the age of 5 years and close to 500 000 succumb to the infection (Black et al. 2003). Clostridium difficile is responsible of 15-25% of cases of antibiotic-associated diarrhea and has been implicated in large outbreaks in hospital settings (Kuijper et al 2006). Effective preventive and therapeutic interventions are not yet available for many etiological agents of diarrheal diseases. Furthermore, even where vaccines are available, the lag time needed to induce an immune response can be critical in epidemic situations. Passive administration of antibodies may thus represent the therapy of choice for both epidemic and endemic gastrointestinal infections, particularly in children and those suffering from malnutrition or immunodeficiency. Oral administration of antibodies of human origin, or derived from bovine colostrum or chicken egg yolk, has previously been successfully used in children with acute diarrhea (for review see Weiner et al. 1999). The production of and purification of antibodies from colostrum and egg yolk are, however, expensive and time consuming procedures.
Other sources of antibodies that have been used for passive immunization include engineered antibody fragments such as single-chain antibodies (scFv) or llama antibody fragments (VHH). Single-chain variable fragments (scFv) are genetically engineered antibody fragments that consist of the variable heavy chain (VH) and light chain (VL) of an immunoglobulin joined together by a flexible peptide linker. These fragments show similar binding specificities as the original antibodies, a low degree of immunogenicity and are more easily manipulated than the bivalent parent antibody. Antibody fragments can neutralize toxin, block adhesion and attachment of bacteria or viruses to cell surfaces and inhibit their infectivity. Functional immunoglobulins containing a heavy chain only have been found in camels and llamas (Hamers-Casterman et al. 1993). The variable domain of llama heavy chain antibodies (VHH) consists of a single immunoglobulin domain and constitutes the smallest naturally occurring antigen-binding molecule known to date, thus making it an alternative to conventional antibodies or antibody fragments. The VHH exhibit several advantages over scFv’s as they are smaller, markedly more acid- and heat-resistant, and, as they are formed by a single polypeptide, easier to express in a recombinant form with an intact spatial structure (Frenken et al. 2000). These properties make them suitable for therapy against gastrointestinal infections.
An attractive idea is to use lactobacilli as vectors for in situ delivery of antibody fragments in the gastrointestinal tract. In situ production of antibody fragments locally in the intestine not only circumvents the need for large-scale manufacturing and purification but also avoids the practical problem of degradation of orally administered antibodies in the stomach. Bacteria represent one of the cheapest antibody production systems available to date and therapeutic application of the lactobacilli producing antibody fragments would both be easy (requiring minimum handling and storage) and cost-effective. If increased avidity or multispecificity is the key to efficient neutralisation of pathogens, it is possible to improve the system by concomitant expression of antibody fragments of varying specificities or by using a mixture of modified clones. Due to their low cost of production and long shelf life when lyophilized, engineered lactobacilli may thus have a major health impact in both developed and developing countries.
The LACTOBODY project aimed at developing effective approaches towards rotavirus and Clostridium difficile based on lactobacilli producing VHH and scFv antibody fragments. These modified lactobacilli producing antibodies (named lactobodies) (See Figure 1 and 2 in annex), taken with food such as water, milk derived products, or rehydratation solutions would colonize the gastrointestinal tract allowing production of protective antibodies fragments in situ.
The main objectives included:
- Develop a safe and efficient prophylactic and therapeutic approach against C. difficile and rotavirus based on antibody fragments and lactobacilli
- Standardisation of the production and processing of such antibody fragments and modified lactobacilli according to food and/or clinical GMP requirements
- Demonstrate the efficacy and safety of the product in animal models and/or human trials
The project involved the generation of scFv and VHH antibody fragment against rotavirus and C. difficile toxins and evaluation of their activity in vitro and in vivo, the selection of lactobacilli with probiotic activity that can be use as vectors for delivery of antibody fragments, the expression of antibody fragments in lactobacilli using both plasmid and food grade expression systems, and the activity of the modified lactobacilli in rodent models of rotavirus and C. difficile infection. In order to show the feasibility of this approach in humans, one lead VHH fragment, directed against rotavirus, was produced in a large amount in yeast according to food GMP standards and tested as a food supplement in a prophylactic study in India. Finally, biologically contained modified lactobacilli expressing VHH fragment against rotavirus are currently being tested in human volunteers.
Project Results:
GENERATION OF ANTIBODY FRAGMENTS
The first objective was the generation of antibody fragments that can be cloned in lactobacilli. Both previously and newly generated VHH against rotavirus were used in the project.
Rotavirus
A number of anti-rhesus rotavirus (RRV) specific VHH fragments (ARP1, ARP3) from llama were previously generated (van der Vaart et al. 2006). In order to select for antibody fragments with a broader range of cross reactivity, additional VHH fragments against rotavirus were selected from new libraries derived from llamas immunised with human rotavirus strains. Different biopanning procedures were used to select phage displaying cloned VHHs that bind to human rotavirus.
C. difficile toxins
Two llamas were immunised with a mixture of inactivated toxin A and B of C. difficile. Phage display libraries were constructed using a phagemid vector and selection was performed by panning on native toxin A and B. Using this method, a wide variety of VHHs against toxin B were selected, but only a limited variation was found in the toxin A recognizing VHHs.
In addition, two scFv, one against toxin A (6cdtA) and one against toxin B (10cdtB), were derived from well characterized monoclonal antibodies.
ACTIVITY OF ANTIBODY FRAGMENTS IN VITRO AND IN ANIMAL MODELS
The VHH antibody fragments were first produced E. coli and/or yeast in order to test their neutralising activity before expression in lactobacilli.
Anti-rotavirus
E. coli and yeast produced VHH were tested for their capacity to prevent the infection of MA104 (monkey kidney) cell monolayers by various rotavirus strains (of different serotype and genotypes). The VHH fragments were later tested in the mouse pup model of rotavirus infection.
The two VHH fragments, ARP1 and ARP3, originating from a VHH phage library against RRV appear to be non competitive in binding to rotavirus in ELISA and may be binding to exclusive epitopes on the virus. The combination of ARP1 and ARP3 was superior to either of the VHH fragment by themselves at reducing infection by RRV in cell cultures and in our mouse pup model of rotavirus infection, suggesting that ARP1 and ARP3 may be interacting synergistically (Pant et al 2011). ARP1 and ARP3 fragments were also shown to bind and neutralise a wide range of rotavirus strains of different G and P types from clinical samples (Aladin et al. 2012).
VHH from llamas immunised with human rotavirus were purified and tested for neutralization. One VHH family was shown to neutralise rotavirus with the same breadth and efficacy as ARP1 and ARP3. The fragments were also tested in the mouse pup model and were shown as efficient as ARP1 and ARP3 in reducing rotavirus induced diarrhea
Anti-C. difficile
A toxin killing in vitro assay based on the cell line MA104 (rhesus monkey kidney epithelial cells) was set up for screening the neutralising capacities of the developed VHH. VHH fragments shown to neutralize the toxins in vitro were subsequently tested for their ability to protect hamsters from challenge with C. difficile spores.
E. coli periplasmic extracts of anti-toxin A and anti-toxin B clones were tested in the in vitro assay. The unique anti-toxin A VHH’s and anti-toxin B VHH’s were purified and re-tested. Five VHH anti-toxin B were shown to neutralise toxin B in vitro while the anti-toxin A were non neutralisers. It was thus decided to continue only with the VHH anti-toxin B. For subsequent characterisation of binding epitopes, toxin B was cloned as 4 sub domains in the expression vector pET28 and fused to a C-terminal VSV- and His-tag. The VHH anti-toxin B have been shown to recognise the C-terminal receptor binding domain.
The three best non-competing anti-toxin B fragments were produced in yeast and purified. A toxin A deleted C. difficile 630 strain (A-B+) generated by inactivation of the toxin A gene was obtained to test the efficacy of the VHH against toxin B (Kuehne et al 2010). We showed that the fragments can neutralise the toxin B produced by the toxin A deleted 630 strain (A+B-) in vitro. The hamster model was subsequently optimised using the toxin A deleted C. difficile 630 strain. However, a mix of the three VHH fragments did not appear to be protective in the hamster model under the conditions tested.
ACTIVITY OF LACTOBACILLI EXPRESSING ANTIBODY FRAGMENTS IN VITRO AND IN ANIMAL MODELS
ROTAVIRUS
The model strain L. paracasei has been transformed to express ARP1 and ARP3 as monovalent and bivalent proteins (homo- and hetero-dimers). The transgenic lactobacilli were tested in our infant mouse model of rotavirus diarrhea both in a prophylactic and therapeutic setting. Daily administration of lactobacilli expressing the ARP3-ARP1 heterodimer reduced signs of diarrhea in mice in both treatment modalities. ARP3-ARP1 expressing lactobacilli were superior at reducing the rate of diarrhea as compared to lactobacilli expressing monomeric ARP, lactobacilli expressing homodimers or a mixture of monovalent ARP1 and ARP3 expressing lactobacilli (Pant et al. 2011).
We also used these two well characterized ARPs to develop various double expression cassettes for co-expression of two antibody fragments in secreted or cell wall anchored forms. Three different double expression cassettes were constructed where the two ARP fragments can be both secreted in the medium, one secreted and the other anchored on the cell surface, or both covalently anchored on the cell surface. Expression of ARPs and display of surface anchored ones were evaluated by Western blot and flow cytometry respectively. Their binding efficiencies were analyzed by flow cytometry and ELISA. The expression and binding activity of ARP1 and ARP3 fragment was shown to be similar between lactobacilli co-expressing the two ARPs and lactobacilli producing only one fragment. The main advantage of the lactobacilli co-expressing ARPs will be the targeting of different epitopes on rotavirus in order to reduce the risk of escape mutants.
C. difficile
The scFv fragment against toxin A and the one against toxin B were expressed on the surface of lactobacilli. After showing that the lactobacilli displaying the scFv on their surface were neutralising in vitro, the lactobacilli were tested in the hamster model. A mix of lactobacilli (L. paracasei) expressing scFv antibody fragments against both C. difficile toxin A and toxin B or control non expressing L. paracasei were given by intra-gastrical gavage before and after challenge with C. difficile spores. No difference in mortality was noted between the group administered a control strain of L. paracasei and the group treated with L. paracasei producing scFv antibodies against tox A and B.
Furthermore, VHH against toxin B secreted in the supernatant by Lactobacillus or anchored on the bacteria surface were shown to be protective in vitro. Lactobacilli producing VHH against toxin B have also been tested in the hamster model of C. difficile infection. A mix of two Lactobacillus producing surface anchored VHH was shown to delay the symptoms of the disease but it did not confer full protection.
SELECTION OF LACTOBACILLI FOR ANTIBODY EXPRESSION
Some Lactobacillus strains have been shown to display probiotic and antagonistic activities against infection caused by rotavirus and C. difficile. We investigated candidate lactobacilli strains both for their probiotic effect and as potential carriers of vectors encoding suitable antibodies. We wanted to express antibody fragments in Lactobacillus strains that are naturally protective against C. difficile and rotavirus infection, in order to obtain optimum activity.
Lactobacillus strains were screened for their capacity to inhibit C. difficile in vitro as well as to reduce C. difficile infection in the hamster model. Commercial probiotics and/or Lactobacillus strains previously isolated from the human gastrointestinal tract were tested for their ability to reduce severity and duration of rotavirus infection in the mouse pup model. The best candidate strains were tested for their transformability by electroporation. The Lactobacillus strain with the best probiotic properties has been selected and has subsequently been used for expression of VHH antibody fragments against rotavirus.
DEVELOPMENT OF PHARMACEUTICAL GRADE EXPRESSION SYSTEM
As previously mentioned, one of the VHH fragments (ARP1) against rotavirus has been shown to be broadly cross reactive and to reduce the frequency and severity of rotavirus induced diarrhea in the mouse pup model. It was decided to further develop lactobacilli producing ARP1 as a potential medical product for prevention and therapy against rotavirus.
The use of genetically engineered lactobacilli for medical purposes must guarantee their stability, safety and containment within the host. In order to increase the stability, the expression cassette fused to the gene encoding ARP1 was integrated on the chromosome of Lactobacillus.
As a proof of concept, the ARP1 fragment encoding gene has initially been integrated on the chromosome of the model strain L. paracasei and shown to be well expressed and functional (Martín et al. 2011). In the mouse model of rotavirus infection, the chromosomally integrated Lactobacillus construct was as good as the plasmid version to reduce diahrrea. This work represented an important step forward in the development of live lactobacilli for delivery of antibody fragments.
The system was further developed for integration of the expression cassette mediating surface display of ARP1 in the selected Lactobacillus strains. In order to meet the requirement of the authorities, additional modifications have been performed on the expression cassette before chromosomal integration including the E-tag. N-terminal sequencing of purified secreted fragments has clarified that the signal peptide has been cut at the exact position immediately before the ARP1 sequence. Since the nucleotide sequence of E-tag was removed, two anti-VHH antibodies were used for detecting ARP1 in different applications such as Western blot, ELISA and flow cytometry.
The optimized cassette was subsequently cloned in the integrative vector pEM76 and integrated on the chromosome of the selected thyA deleted Lactobacillus using site specific integration generating Lactobacillus thyA deleted EM270. In this strain, the thymidine A (thyA) gene was knocked out for biological containment purposes. The thyA gene disruption was shown to prevent the growth of Lactobacillus in culture medium not containing thymine or thymidine. Lactobacillus thyA deleted EM270 showed expression of ARP1 and binding to RRV similar to the same strain transformed with the equivalent plasmid-based expression system.
SAFETY TRIAL WITH MODIFIED LACTOBACILLI
The modified lactobacilli (Lactobacillus thyA deleted EM270) and corresponding non modified lactobacilli have been produced using clinical GMP (Good Manufacturing Practice) standard for the clinical trial. The phase I clinical trial is currently being performed using the service of the Karolinska Trial Alliance at the Karolinska University Hospital Huddinge. Volunteers receive lyophilised thyA deleted lactobacilli producing ARP1 daily for a period of 7 days. During treatment and the following 10 days, the volunteers will be assessed daily for the presence of potential adverse effects by direct questioning and, when necessary, additional testing. Individuals will be monitored for abdominal pain, headache, appetite, nausea, flatulence and vomiting. Blood samples will be analyzed for hematological parameters and blood chemistry. The detection of the modified lactobacilli and the expression of ARP1 in feces of the volunteers by real-time PCR will subsequently be performed in our laboratory. TaqMan Real-time PCR has been developed for that purpose. Antibodies against Lactobacillus and the ARP1 will be measured in serum and fecal samples to assess if any antibody response has been developed against the modified lactobacilli.
CLINICAL TRIAL WITH THE LEAD FRAGMENT ARP1 IN INDIA
The lead ARP1 fragment, cross reactive against various human rotavirus strains, was tested in an intervention study carried out in an urban slum area in Vellore, South India. ARP1 was produced at a large scale in fermenters under Good Manufacturing practice and lyophilised. Children between the ages of 6 and 12 months were recruited for participation in the study. They were randomized to receive supplementation with ARP or placebo, and were followed for a period of one year, with weekly home visits and monitoring of all episodes of diarrhoea. In addition, every two weeks, surveillance stool samples were collected and tested for rotavirus to identify asymptomatic infections by PCR. All episodes of diarrhoea were investigated intensively for bacterial, viral and parasitic agents of diarrhoea using both conventional and molecular techniques and all cases were treated appropriately at the study clinic or referral hospital as required. Monthly anthropometric measurements were carried out to estimate rates of growth and to identify growth faltering. Blood samples were taken at 4, 8 and 12 months for estimation of anti-rotavirus IgA and IgG antibodies. Studies on intestinal absorption and permeability were carried out at recruitment, 6 and 12 months by the lactulose:mannitol test (measured by HPLC detection of sugars). The clinical trial started in February 2011 and was completed in June 2012. The analysis of the samples will not be completed before September 2012.
Potential Impact:
MAIN RESULTS OBTAINED DURING THE PROJECT
During the project, new functional antibody fragments against C. difficile and rotavirus as well as modified lactobacilli producing these antibodies have been generated and characterised. Furthermore, after completion of the clinical trials we will demonstrate if the use of ARP1 is protective in children and if lactobacilli producing antibody fragments are biologically contained and safe in human volunteers.
IMPACT ON GLOBAL HEALTH
Despite numerous scientific advances regarding the pathogenesis, diagnosis and treatment of infectious enteritis, diarrhoeal diseases remain major threats to health worldwide. Successful implementation of oral rehydration therapy has resulted in a fall in mortality, but effective prophylactic measures are urgently needed. Furthermore, even when vaccines are available, the lag time needed to induce an immune response can be particularly critical in epidemic situations and emergence of new serotypes.
The results obtained during the LACTOBODY project could thus result in the development of a new form of a Functional Food (prophylactic) or a Medicine (therapeutic) against infections in the gastrointestinal tract of humans. The local delivery of antibody fragments in the gastro-intestinal tract by lactobacilli may represent an efficient and cheap alternative to existing therapies both in Europe and developing countries. Passive administration of yeast or Lactobacillus produced VHH may represent the intervention of choice for prevention or containment of both epidemic and endemic gastrointestinal infections, particularly in young children, those suffering from malnutrition or immunodeficiency and ageing people. Similarly, it may be well-placed as an intervention against nosocomial infections.
IMPACT AT EUROPEAN LEVEL
LACTOBODY may provide the industrial partners with the knowledge, the technology and the insights to develop products to successfully enter the enormous, but currently intangible, market of the middle to lower socio-economic classes in developing countries. Furthermore, current sales of functional foods in Europe are estimated at 30 billion Euros, and novel functional foods that can reduce infectious diarrhoea have vast market opportunities in Europe as well. They are applicable to susceptible persons especially children in day care centres, hospital patients or the elderly, all groups who are susceptible to rotavirus, C. difficile and other infectious intestinal diseases. The successful implementation of the project is likely to have a high impact on Europe’s technological capability to develop effective food-based preventive and therapeutic products.
DISSEMINATION OF RESULTS
The coordinator has created a public access website which includes a description of the consortium and the project. The objective is to inform the public about the concept of using genetically modified lactobacilli for prophylaxis and therapy and its potential impact. Additional material include a list of relevant publications. The webmail addresses are www.lactobody.com www.lactobody.se and www.lactobody.eu.
During the project, seven papers have been published in peer-reviewed journal. Three manuscripts are in preparation, one on selection of VHH against C. difficile toxins and two on expression of antibodies against rotavirus. Three additional manuscripts are expected next year including selection of VHH against human rotavirus, clinical trial in India with ARP1 and clinical trial with genetically modified lactobacilli. In addition, communications have been presented at international meetings and two patents have been filed.
Paper
Marcotte H et al. 2008. Engineered lactobody-producing lactobacilli: a novel form of therapy against rotavirus infection. Future Virology 3:327-341.
Kõll P et al. 2010. Screening and Evaluation of Human Intestinal Lactobacilli for the Development of Novel Gastrointestinal Probiotics. Current Microbiology 61:560-566
Martín MC et al. 2011. Integrative expression system for delivery of antibody fragments by lactobacilli. Applied and Environmental Microbiology 6:2174-2179
Hütt P et al. 2011. Safety and persistence of orally administered human Lactobacillus sp. strains in healthy adults. Beneficial Microbes 2:79-90.
Pant N et al. 2011. Lactobacilli expressing non-competing bispecific llama derived anti-rotavirus proteins effectively combat rotavirus induced diarrhea. Future Microbiology 6:583-93.
Mikelsaar M. 2011. Human microbial ecology: lactobacilli, probiotics, selective decontamination. Anaerobe. 17:463-7.
Aladin F. et al. 2012. In vitro neutralisation of rotavirus infection by two broadly Specific recombinant monovalent llama-derived antibody fragments. PLOS ONE 7(3): e32949
Thesis
Neha Pant. Lactobacillus based oro-mucosal therapies against rotavirus. Department of Laboratory medicine, Karolinska Institutet, May 30 2008
Pirje Hütt. Functional properties, persistence, safety and efficacy of potential probiotic lactobacilli. Department of Microbiology, University of Tartu; supervisor professor Marika Mikelsaar, Tartu, June 18, 2012.
Communication
Neha Pant. The Lactobody Project: an EU funded project on antibody technology. Animal Cell Technology Industrial Platform (ACTIP), Cambridge, UK, November 17-18, 2008.
Truusalu K., I. Smidt, P. Hütt, S. Kõljalg, R.H. Mikelsaar, P. Naaber, E. Sepp, J. Stsepetova, M. Mikelsaar. In vitro and in vivo comparison of Clostridium difficile PCR ribotype 027 and non 027 strains, 19th ECCMID meeting, Helsinki, 2009, Poster no 1259
Lennart Hammarström. Lactobodies: Lactobacilli Expressing Variable Domain of Llama Heavy-chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-induced Diarrhea. Single domain antibodies come of age (SDA) 2010 - October 14-15, 2010 - Ghent
Marika Mikelsaar. Human microbial ecology: environmental, nutritional and medical factors.
SOMED XXXIII CONGRESS, Greece, Sept, 2011.
Jelena Štšepetova, Epp Sepp, Helgi Kolk, Krista Lõivukene, Epp Songisepp, Marika Mikelsaar. Diversity and metabolic impact of intestinal Lactobacillus sp. in healthy adults and the elderly. SOMED XXXIII CONGRESS, Greece, Sept, 2011.
P. Naaber, J. Stsepetova, I. Smidt, E. Shkut, M. Rätsep, S. Kõljalg, K. Lõivukene, R. Mändar, I.H. Löhr, O.B. Natås, E.Sepp. Intestinal lactoflora in patients with antibiotic associated diarrhea (ADD). SOMED XXXIII CONGRESS, Greece, Sept, 2011.
Mª Cruz Martín, Noelia Martínez and Miguel A. Alvarez. Genetically modified lactobacilli as mucosal delivery vectors. Second International Congress on Pharmacology of Vaccines (VacciPharma 2012), scheduled for June 16th to 20th, 2012 at Cayo Santa María, Cuba
Miguel A. Alvarez has participated as a Scientific Advisor of WONDERFOOD, a Science Dissemination Project included in the European Project 2WAYS. One of the activities has been to present the LACTOBODY Project to high school students from different European countries.
EXPLOITATION OF RESULTS
The exploitable deliverables include affordable novel food- or pharmaceutical-based prophylactic and therapeutic approaches for sustainable reduction of the risk of infectious diarrhoea in both developed and developing countries.
During the project, the industrial partners of LACTOBODY ensured that the knowledge gathered in the different workpackages was protected. When appropriate, the results were patented and licenced according to the terms of the Consortium Agreement.
A plan was established for the development of prophylactic or therapeutic products against rotavirus and C. difficile.
REFERENCES
Aladin F., Einerhand AWC, Bouma J, Bezemer S, Hermans P, Wolvers D, Bellamy K, Frenken LGJ, Gray J, Iturriza-Gómara M. In vitro neutralisation of rotavirus infection by two broadly Specific recombinant monovalent llama-derived antibody fragments. PLOS ONE 7(3): e32949
Black RE, Morris SS, and Bryce J. 2003. Where and why are 10 million children dying every year? Lancet 361:2226-2234.
Frenken LG, van der Linden RH, Hermans PW, Bos JW, Ruuls RC, de Geus B, and Verrips CT. 2000. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J Biotechnol 78:11-21.
Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, and Hamers R. 1993. Naturally occurring antibodies devoid of light chains. Nature 363:446-448.
Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. The role of toxin A and toxin B in Clostridium difficile infection. Nature. 2010 Oct 7;467(7316):711-3. Epub 2010 Sep 15.
Kuijper EJ, Coignard B, Tull P; and the ESCMID Study Group for Clostridium difficile (ESGCD)*; EU Member States and the European Centre for Disease Prevention and Control (ECDC). 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12 Suppl 6:2-18.
Martín MC et al. 2011. Integrative expression system for delivery of antibody fragments by lactobacilli. Applied and Environmental Microbiology 6:2174-2179
Pant N et al. 2011. Lactobacilli expressing non-competing bispecific llama derived anti-rotavirus proteins effectively combat rotavirus induced diarrhea. Future Microbiology 6:583-93.
van der Vaart JM, Pant N, Wolvers D, Bezemer S, Hermans PW, Bellamy K, Sarker SA, van der Logt CPE, Svensson L, Verrips CT, Hammarström L, and van Klinken BJW. 2006. Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments.Vaccine 24:4130-4137.
Weiner C, Pan Q, Hurtig M, Boren T, Bostwick E, and Hammarström L. 1999. Passive immunity against human pathogens using bovine antibodies. Clin Exp Immunol 116:193-205.
List of Websites:
http://lactobody.eu/site/
Enteric infections such as rotavirus and C. difficile induced diarrhea, remain a major cause of morbidity and mortality globally, accounting for an estimated 2 millions deaths each year. Effective preventive and therapeutic interventions are not yet available for many etiological agents of diarrheal diseases. Furthermore, even where vaccines are available, the lag time needed to induce an immune response can be critical in epidemic situations.
The aim of this project was to develop an effective treatment against rotavirus and Clostridium difficile based on lactobacilli producing VHH and scFv antibody fragments. As a proof of principle, a lead VHH fragment (called ARP1) against rotavirus was tested in a community-based, double-blind placebo controlled interventional trial in south India. A large batch of ARP1 was produced in yeast and prophylactically administered to children. In parallel, we have generated, selected and expressed new scFv and VHH fragments against the gastrointestinal pathogens rotavirus and C. difficile in lactobacilli. The modified bacteria were tested for their protective capacity in vitro and animal models.
The genes encoding the ARP1 fragment against rotavirus was subsequently cloned using pharmaceutical grade and biologically contained expression systems in a probiotic Lactobacillus strain. The modified bacteria were tested in healthy human volunteers to ensure safety and to measure their immunogenicity.
Our approach, which falls into the priority "Innovative approaches and interventions" and the work program "Development and production of new generation antibodies" represents a novel system for the induction of passive immunity that can be rapidly applied to populations at risk (for example through the drinking water, rehydrating solution or as a food supplement). The results obtained in this project could be applied to therapy against a vast number of human/animal pathogens in the gastrointestinal tract.
Project Context and Objectives:
The LACTOBODY project is a four year project that falls into the topic “Development and production of new generation antibodies” of the FP7 Health work program. The main objective of this topic was the development of new, efficient and safe preventive strategies and/or therapies by combining high specificity and effector functions with stable production, preclinical studies and good manufacturing practice (GMP). The LACTOBODY project is based on an idea that may revolutionize passive immunisation: engineering bacteria “generally regarded as safe” (GRAS) to produce antibody fragments directly at the mucosal site. The consortium was composed of seven groups in Europe and one in India (see list of participants in Annex). The project started in February 2008 and the present summary submitted in June 2012 covers the work performed during the whole project.
Enteric infections remain a major cause of morbidity and mortality globally, accounting for an estimated 2 millions deaths each year. In developing countries, over 125 million episodes of rotavirus diarrhea are observed annually in children under the age of 5 years and close to 500 000 succumb to the infection (Black et al. 2003). Clostridium difficile is responsible of 15-25% of cases of antibiotic-associated diarrhea and has been implicated in large outbreaks in hospital settings (Kuijper et al 2006). Effective preventive and therapeutic interventions are not yet available for many etiological agents of diarrheal diseases. Furthermore, even where vaccines are available, the lag time needed to induce an immune response can be critical in epidemic situations. Passive administration of antibodies may thus represent the therapy of choice for both epidemic and endemic gastrointestinal infections, particularly in children and those suffering from malnutrition or immunodeficiency. Oral administration of antibodies of human origin, or derived from bovine colostrum or chicken egg yolk, has previously been successfully used in children with acute diarrhea (for review see Weiner et al. 1999). The production of and purification of antibodies from colostrum and egg yolk are, however, expensive and time consuming procedures.
Other sources of antibodies that have been used for passive immunization include engineered antibody fragments such as single-chain antibodies (scFv) or llama antibody fragments (VHH). Single-chain variable fragments (scFv) are genetically engineered antibody fragments that consist of the variable heavy chain (VH) and light chain (VL) of an immunoglobulin joined together by a flexible peptide linker. These fragments show similar binding specificities as the original antibodies, a low degree of immunogenicity and are more easily manipulated than the bivalent parent antibody. Antibody fragments can neutralize toxin, block adhesion and attachment of bacteria or viruses to cell surfaces and inhibit their infectivity. Functional immunoglobulins containing a heavy chain only have been found in camels and llamas (Hamers-Casterman et al. 1993). The variable domain of llama heavy chain antibodies (VHH) consists of a single immunoglobulin domain and constitutes the smallest naturally occurring antigen-binding molecule known to date, thus making it an alternative to conventional antibodies or antibody fragments. The VHH exhibit several advantages over scFv’s as they are smaller, markedly more acid- and heat-resistant, and, as they are formed by a single polypeptide, easier to express in a recombinant form with an intact spatial structure (Frenken et al. 2000). These properties make them suitable for therapy against gastrointestinal infections.
An attractive idea is to use lactobacilli as vectors for in situ delivery of antibody fragments in the gastrointestinal tract. In situ production of antibody fragments locally in the intestine not only circumvents the need for large-scale manufacturing and purification but also avoids the practical problem of degradation of orally administered antibodies in the stomach. Bacteria represent one of the cheapest antibody production systems available to date and therapeutic application of the lactobacilli producing antibody fragments would both be easy (requiring minimum handling and storage) and cost-effective. If increased avidity or multispecificity is the key to efficient neutralisation of pathogens, it is possible to improve the system by concomitant expression of antibody fragments of varying specificities or by using a mixture of modified clones. Due to their low cost of production and long shelf life when lyophilized, engineered lactobacilli may thus have a major health impact in both developed and developing countries.
The LACTOBODY project aimed at developing effective approaches towards rotavirus and Clostridium difficile based on lactobacilli producing VHH and scFv antibody fragments. These modified lactobacilli producing antibodies (named lactobodies) (See Figure 1 and 2 in annex), taken with food such as water, milk derived products, or rehydratation solutions would colonize the gastrointestinal tract allowing production of protective antibodies fragments in situ.
The main objectives included:
- Develop a safe and efficient prophylactic and therapeutic approach against C. difficile and rotavirus based on antibody fragments and lactobacilli
- Standardisation of the production and processing of such antibody fragments and modified lactobacilli according to food and/or clinical GMP requirements
- Demonstrate the efficacy and safety of the product in animal models and/or human trials
The project involved the generation of scFv and VHH antibody fragment against rotavirus and C. difficile toxins and evaluation of their activity in vitro and in vivo, the selection of lactobacilli with probiotic activity that can be use as vectors for delivery of antibody fragments, the expression of antibody fragments in lactobacilli using both plasmid and food grade expression systems, and the activity of the modified lactobacilli in rodent models of rotavirus and C. difficile infection. In order to show the feasibility of this approach in humans, one lead VHH fragment, directed against rotavirus, was produced in a large amount in yeast according to food GMP standards and tested as a food supplement in a prophylactic study in India. Finally, biologically contained modified lactobacilli expressing VHH fragment against rotavirus are currently being tested in human volunteers.
Project Results:
GENERATION OF ANTIBODY FRAGMENTS
The first objective was the generation of antibody fragments that can be cloned in lactobacilli. Both previously and newly generated VHH against rotavirus were used in the project.
Rotavirus
A number of anti-rhesus rotavirus (RRV) specific VHH fragments (ARP1, ARP3) from llama were previously generated (van der Vaart et al. 2006). In order to select for antibody fragments with a broader range of cross reactivity, additional VHH fragments against rotavirus were selected from new libraries derived from llamas immunised with human rotavirus strains. Different biopanning procedures were used to select phage displaying cloned VHHs that bind to human rotavirus.
C. difficile toxins
Two llamas were immunised with a mixture of inactivated toxin A and B of C. difficile. Phage display libraries were constructed using a phagemid vector and selection was performed by panning on native toxin A and B. Using this method, a wide variety of VHHs against toxin B were selected, but only a limited variation was found in the toxin A recognizing VHHs.
In addition, two scFv, one against toxin A (6cdtA) and one against toxin B (10cdtB), were derived from well characterized monoclonal antibodies.
ACTIVITY OF ANTIBODY FRAGMENTS IN VITRO AND IN ANIMAL MODELS
The VHH antibody fragments were first produced E. coli and/or yeast in order to test their neutralising activity before expression in lactobacilli.
Anti-rotavirus
E. coli and yeast produced VHH were tested for their capacity to prevent the infection of MA104 (monkey kidney) cell monolayers by various rotavirus strains (of different serotype and genotypes). The VHH fragments were later tested in the mouse pup model of rotavirus infection.
The two VHH fragments, ARP1 and ARP3, originating from a VHH phage library against RRV appear to be non competitive in binding to rotavirus in ELISA and may be binding to exclusive epitopes on the virus. The combination of ARP1 and ARP3 was superior to either of the VHH fragment by themselves at reducing infection by RRV in cell cultures and in our mouse pup model of rotavirus infection, suggesting that ARP1 and ARP3 may be interacting synergistically (Pant et al 2011). ARP1 and ARP3 fragments were also shown to bind and neutralise a wide range of rotavirus strains of different G and P types from clinical samples (Aladin et al. 2012).
VHH from llamas immunised with human rotavirus were purified and tested for neutralization. One VHH family was shown to neutralise rotavirus with the same breadth and efficacy as ARP1 and ARP3. The fragments were also tested in the mouse pup model and were shown as efficient as ARP1 and ARP3 in reducing rotavirus induced diarrhea
Anti-C. difficile
A toxin killing in vitro assay based on the cell line MA104 (rhesus monkey kidney epithelial cells) was set up for screening the neutralising capacities of the developed VHH. VHH fragments shown to neutralize the toxins in vitro were subsequently tested for their ability to protect hamsters from challenge with C. difficile spores.
E. coli periplasmic extracts of anti-toxin A and anti-toxin B clones were tested in the in vitro assay. The unique anti-toxin A VHH’s and anti-toxin B VHH’s were purified and re-tested. Five VHH anti-toxin B were shown to neutralise toxin B in vitro while the anti-toxin A were non neutralisers. It was thus decided to continue only with the VHH anti-toxin B. For subsequent characterisation of binding epitopes, toxin B was cloned as 4 sub domains in the expression vector pET28 and fused to a C-terminal VSV- and His-tag. The VHH anti-toxin B have been shown to recognise the C-terminal receptor binding domain.
The three best non-competing anti-toxin B fragments were produced in yeast and purified. A toxin A deleted C. difficile 630 strain (A-B+) generated by inactivation of the toxin A gene was obtained to test the efficacy of the VHH against toxin B (Kuehne et al 2010). We showed that the fragments can neutralise the toxin B produced by the toxin A deleted 630 strain (A+B-) in vitro. The hamster model was subsequently optimised using the toxin A deleted C. difficile 630 strain. However, a mix of the three VHH fragments did not appear to be protective in the hamster model under the conditions tested.
ACTIVITY OF LACTOBACILLI EXPRESSING ANTIBODY FRAGMENTS IN VITRO AND IN ANIMAL MODELS
ROTAVIRUS
The model strain L. paracasei has been transformed to express ARP1 and ARP3 as monovalent and bivalent proteins (homo- and hetero-dimers). The transgenic lactobacilli were tested in our infant mouse model of rotavirus diarrhea both in a prophylactic and therapeutic setting. Daily administration of lactobacilli expressing the ARP3-ARP1 heterodimer reduced signs of diarrhea in mice in both treatment modalities. ARP3-ARP1 expressing lactobacilli were superior at reducing the rate of diarrhea as compared to lactobacilli expressing monomeric ARP, lactobacilli expressing homodimers or a mixture of monovalent ARP1 and ARP3 expressing lactobacilli (Pant et al. 2011).
We also used these two well characterized ARPs to develop various double expression cassettes for co-expression of two antibody fragments in secreted or cell wall anchored forms. Three different double expression cassettes were constructed where the two ARP fragments can be both secreted in the medium, one secreted and the other anchored on the cell surface, or both covalently anchored on the cell surface. Expression of ARPs and display of surface anchored ones were evaluated by Western blot and flow cytometry respectively. Their binding efficiencies were analyzed by flow cytometry and ELISA. The expression and binding activity of ARP1 and ARP3 fragment was shown to be similar between lactobacilli co-expressing the two ARPs and lactobacilli producing only one fragment. The main advantage of the lactobacilli co-expressing ARPs will be the targeting of different epitopes on rotavirus in order to reduce the risk of escape mutants.
C. difficile
The scFv fragment against toxin A and the one against toxin B were expressed on the surface of lactobacilli. After showing that the lactobacilli displaying the scFv on their surface were neutralising in vitro, the lactobacilli were tested in the hamster model. A mix of lactobacilli (L. paracasei) expressing scFv antibody fragments against both C. difficile toxin A and toxin B or control non expressing L. paracasei were given by intra-gastrical gavage before and after challenge with C. difficile spores. No difference in mortality was noted between the group administered a control strain of L. paracasei and the group treated with L. paracasei producing scFv antibodies against tox A and B.
Furthermore, VHH against toxin B secreted in the supernatant by Lactobacillus or anchored on the bacteria surface were shown to be protective in vitro. Lactobacilli producing VHH against toxin B have also been tested in the hamster model of C. difficile infection. A mix of two Lactobacillus producing surface anchored VHH was shown to delay the symptoms of the disease but it did not confer full protection.
SELECTION OF LACTOBACILLI FOR ANTIBODY EXPRESSION
Some Lactobacillus strains have been shown to display probiotic and antagonistic activities against infection caused by rotavirus and C. difficile. We investigated candidate lactobacilli strains both for their probiotic effect and as potential carriers of vectors encoding suitable antibodies. We wanted to express antibody fragments in Lactobacillus strains that are naturally protective against C. difficile and rotavirus infection, in order to obtain optimum activity.
Lactobacillus strains were screened for their capacity to inhibit C. difficile in vitro as well as to reduce C. difficile infection in the hamster model. Commercial probiotics and/or Lactobacillus strains previously isolated from the human gastrointestinal tract were tested for their ability to reduce severity and duration of rotavirus infection in the mouse pup model. The best candidate strains were tested for their transformability by electroporation. The Lactobacillus strain with the best probiotic properties has been selected and has subsequently been used for expression of VHH antibody fragments against rotavirus.
DEVELOPMENT OF PHARMACEUTICAL GRADE EXPRESSION SYSTEM
As previously mentioned, one of the VHH fragments (ARP1) against rotavirus has been shown to be broadly cross reactive and to reduce the frequency and severity of rotavirus induced diarrhea in the mouse pup model. It was decided to further develop lactobacilli producing ARP1 as a potential medical product for prevention and therapy against rotavirus.
The use of genetically engineered lactobacilli for medical purposes must guarantee their stability, safety and containment within the host. In order to increase the stability, the expression cassette fused to the gene encoding ARP1 was integrated on the chromosome of Lactobacillus.
As a proof of concept, the ARP1 fragment encoding gene has initially been integrated on the chromosome of the model strain L. paracasei and shown to be well expressed and functional (Martín et al. 2011). In the mouse model of rotavirus infection, the chromosomally integrated Lactobacillus construct was as good as the plasmid version to reduce diahrrea. This work represented an important step forward in the development of live lactobacilli for delivery of antibody fragments.
The system was further developed for integration of the expression cassette mediating surface display of ARP1 in the selected Lactobacillus strains. In order to meet the requirement of the authorities, additional modifications have been performed on the expression cassette before chromosomal integration including the E-tag. N-terminal sequencing of purified secreted fragments has clarified that the signal peptide has been cut at the exact position immediately before the ARP1 sequence. Since the nucleotide sequence of E-tag was removed, two anti-VHH antibodies were used for detecting ARP1 in different applications such as Western blot, ELISA and flow cytometry.
The optimized cassette was subsequently cloned in the integrative vector pEM76 and integrated on the chromosome of the selected thyA deleted Lactobacillus using site specific integration generating Lactobacillus thyA deleted EM270. In this strain, the thymidine A (thyA) gene was knocked out for biological containment purposes. The thyA gene disruption was shown to prevent the growth of Lactobacillus in culture medium not containing thymine or thymidine. Lactobacillus thyA deleted EM270 showed expression of ARP1 and binding to RRV similar to the same strain transformed with the equivalent plasmid-based expression system.
SAFETY TRIAL WITH MODIFIED LACTOBACILLI
The modified lactobacilli (Lactobacillus thyA deleted EM270) and corresponding non modified lactobacilli have been produced using clinical GMP (Good Manufacturing Practice) standard for the clinical trial. The phase I clinical trial is currently being performed using the service of the Karolinska Trial Alliance at the Karolinska University Hospital Huddinge. Volunteers receive lyophilised thyA deleted lactobacilli producing ARP1 daily for a period of 7 days. During treatment and the following 10 days, the volunteers will be assessed daily for the presence of potential adverse effects by direct questioning and, when necessary, additional testing. Individuals will be monitored for abdominal pain, headache, appetite, nausea, flatulence and vomiting. Blood samples will be analyzed for hematological parameters and blood chemistry. The detection of the modified lactobacilli and the expression of ARP1 in feces of the volunteers by real-time PCR will subsequently be performed in our laboratory. TaqMan Real-time PCR has been developed for that purpose. Antibodies against Lactobacillus and the ARP1 will be measured in serum and fecal samples to assess if any antibody response has been developed against the modified lactobacilli.
CLINICAL TRIAL WITH THE LEAD FRAGMENT ARP1 IN INDIA
The lead ARP1 fragment, cross reactive against various human rotavirus strains, was tested in an intervention study carried out in an urban slum area in Vellore, South India. ARP1 was produced at a large scale in fermenters under Good Manufacturing practice and lyophilised. Children between the ages of 6 and 12 months were recruited for participation in the study. They were randomized to receive supplementation with ARP or placebo, and were followed for a period of one year, with weekly home visits and monitoring of all episodes of diarrhoea. In addition, every two weeks, surveillance stool samples were collected and tested for rotavirus to identify asymptomatic infections by PCR. All episodes of diarrhoea were investigated intensively for bacterial, viral and parasitic agents of diarrhoea using both conventional and molecular techniques and all cases were treated appropriately at the study clinic or referral hospital as required. Monthly anthropometric measurements were carried out to estimate rates of growth and to identify growth faltering. Blood samples were taken at 4, 8 and 12 months for estimation of anti-rotavirus IgA and IgG antibodies. Studies on intestinal absorption and permeability were carried out at recruitment, 6 and 12 months by the lactulose:mannitol test (measured by HPLC detection of sugars). The clinical trial started in February 2011 and was completed in June 2012. The analysis of the samples will not be completed before September 2012.
Potential Impact:
MAIN RESULTS OBTAINED DURING THE PROJECT
During the project, new functional antibody fragments against C. difficile and rotavirus as well as modified lactobacilli producing these antibodies have been generated and characterised. Furthermore, after completion of the clinical trials we will demonstrate if the use of ARP1 is protective in children and if lactobacilli producing antibody fragments are biologically contained and safe in human volunteers.
IMPACT ON GLOBAL HEALTH
Despite numerous scientific advances regarding the pathogenesis, diagnosis and treatment of infectious enteritis, diarrhoeal diseases remain major threats to health worldwide. Successful implementation of oral rehydration therapy has resulted in a fall in mortality, but effective prophylactic measures are urgently needed. Furthermore, even when vaccines are available, the lag time needed to induce an immune response can be particularly critical in epidemic situations and emergence of new serotypes.
The results obtained during the LACTOBODY project could thus result in the development of a new form of a Functional Food (prophylactic) or a Medicine (therapeutic) against infections in the gastrointestinal tract of humans. The local delivery of antibody fragments in the gastro-intestinal tract by lactobacilli may represent an efficient and cheap alternative to existing therapies both in Europe and developing countries. Passive administration of yeast or Lactobacillus produced VHH may represent the intervention of choice for prevention or containment of both epidemic and endemic gastrointestinal infections, particularly in young children, those suffering from malnutrition or immunodeficiency and ageing people. Similarly, it may be well-placed as an intervention against nosocomial infections.
IMPACT AT EUROPEAN LEVEL
LACTOBODY may provide the industrial partners with the knowledge, the technology and the insights to develop products to successfully enter the enormous, but currently intangible, market of the middle to lower socio-economic classes in developing countries. Furthermore, current sales of functional foods in Europe are estimated at 30 billion Euros, and novel functional foods that can reduce infectious diarrhoea have vast market opportunities in Europe as well. They are applicable to susceptible persons especially children in day care centres, hospital patients or the elderly, all groups who are susceptible to rotavirus, C. difficile and other infectious intestinal diseases. The successful implementation of the project is likely to have a high impact on Europe’s technological capability to develop effective food-based preventive and therapeutic products.
DISSEMINATION OF RESULTS
The coordinator has created a public access website which includes a description of the consortium and the project. The objective is to inform the public about the concept of using genetically modified lactobacilli for prophylaxis and therapy and its potential impact. Additional material include a list of relevant publications. The webmail addresses are www.lactobody.com www.lactobody.se and www.lactobody.eu.
During the project, seven papers have been published in peer-reviewed journal. Three manuscripts are in preparation, one on selection of VHH against C. difficile toxins and two on expression of antibodies against rotavirus. Three additional manuscripts are expected next year including selection of VHH against human rotavirus, clinical trial in India with ARP1 and clinical trial with genetically modified lactobacilli. In addition, communications have been presented at international meetings and two patents have been filed.
Paper
Marcotte H et al. 2008. Engineered lactobody-producing lactobacilli: a novel form of therapy against rotavirus infection. Future Virology 3:327-341.
Kõll P et al. 2010. Screening and Evaluation of Human Intestinal Lactobacilli for the Development of Novel Gastrointestinal Probiotics. Current Microbiology 61:560-566
Martín MC et al. 2011. Integrative expression system for delivery of antibody fragments by lactobacilli. Applied and Environmental Microbiology 6:2174-2179
Hütt P et al. 2011. Safety and persistence of orally administered human Lactobacillus sp. strains in healthy adults. Beneficial Microbes 2:79-90.
Pant N et al. 2011. Lactobacilli expressing non-competing bispecific llama derived anti-rotavirus proteins effectively combat rotavirus induced diarrhea. Future Microbiology 6:583-93.
Mikelsaar M. 2011. Human microbial ecology: lactobacilli, probiotics, selective decontamination. Anaerobe. 17:463-7.
Aladin F. et al. 2012. In vitro neutralisation of rotavirus infection by two broadly Specific recombinant monovalent llama-derived antibody fragments. PLOS ONE 7(3): e32949
Thesis
Neha Pant. Lactobacillus based oro-mucosal therapies against rotavirus. Department of Laboratory medicine, Karolinska Institutet, May 30 2008
Pirje Hütt. Functional properties, persistence, safety and efficacy of potential probiotic lactobacilli. Department of Microbiology, University of Tartu; supervisor professor Marika Mikelsaar, Tartu, June 18, 2012.
Communication
Neha Pant. The Lactobody Project: an EU funded project on antibody technology. Animal Cell Technology Industrial Platform (ACTIP), Cambridge, UK, November 17-18, 2008.
Truusalu K., I. Smidt, P. Hütt, S. Kõljalg, R.H. Mikelsaar, P. Naaber, E. Sepp, J. Stsepetova, M. Mikelsaar. In vitro and in vivo comparison of Clostridium difficile PCR ribotype 027 and non 027 strains, 19th ECCMID meeting, Helsinki, 2009, Poster no 1259
Lennart Hammarström. Lactobodies: Lactobacilli Expressing Variable Domain of Llama Heavy-chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-induced Diarrhea. Single domain antibodies come of age (SDA) 2010 - October 14-15, 2010 - Ghent
Marika Mikelsaar. Human microbial ecology: environmental, nutritional and medical factors.
SOMED XXXIII CONGRESS, Greece, Sept, 2011.
Jelena Štšepetova, Epp Sepp, Helgi Kolk, Krista Lõivukene, Epp Songisepp, Marika Mikelsaar. Diversity and metabolic impact of intestinal Lactobacillus sp. in healthy adults and the elderly. SOMED XXXIII CONGRESS, Greece, Sept, 2011.
P. Naaber, J. Stsepetova, I. Smidt, E. Shkut, M. Rätsep, S. Kõljalg, K. Lõivukene, R. Mändar, I.H. Löhr, O.B. Natås, E.Sepp. Intestinal lactoflora in patients with antibiotic associated diarrhea (ADD). SOMED XXXIII CONGRESS, Greece, Sept, 2011.
Mª Cruz Martín, Noelia Martínez and Miguel A. Alvarez. Genetically modified lactobacilli as mucosal delivery vectors. Second International Congress on Pharmacology of Vaccines (VacciPharma 2012), scheduled for June 16th to 20th, 2012 at Cayo Santa María, Cuba
Miguel A. Alvarez has participated as a Scientific Advisor of WONDERFOOD, a Science Dissemination Project included in the European Project 2WAYS. One of the activities has been to present the LACTOBODY Project to high school students from different European countries.
EXPLOITATION OF RESULTS
The exploitable deliverables include affordable novel food- or pharmaceutical-based prophylactic and therapeutic approaches for sustainable reduction of the risk of infectious diarrhoea in both developed and developing countries.
During the project, the industrial partners of LACTOBODY ensured that the knowledge gathered in the different workpackages was protected. When appropriate, the results were patented and licenced according to the terms of the Consortium Agreement.
A plan was established for the development of prophylactic or therapeutic products against rotavirus and C. difficile.
REFERENCES
Aladin F., Einerhand AWC, Bouma J, Bezemer S, Hermans P, Wolvers D, Bellamy K, Frenken LGJ, Gray J, Iturriza-Gómara M. In vitro neutralisation of rotavirus infection by two broadly Specific recombinant monovalent llama-derived antibody fragments. PLOS ONE 7(3): e32949
Black RE, Morris SS, and Bryce J. 2003. Where and why are 10 million children dying every year? Lancet 361:2226-2234.
Frenken LG, van der Linden RH, Hermans PW, Bos JW, Ruuls RC, de Geus B, and Verrips CT. 2000. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J Biotechnol 78:11-21.
Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, and Hamers R. 1993. Naturally occurring antibodies devoid of light chains. Nature 363:446-448.
Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. The role of toxin A and toxin B in Clostridium difficile infection. Nature. 2010 Oct 7;467(7316):711-3. Epub 2010 Sep 15.
Kuijper EJ, Coignard B, Tull P; and the ESCMID Study Group for Clostridium difficile (ESGCD)*; EU Member States and the European Centre for Disease Prevention and Control (ECDC). 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12 Suppl 6:2-18.
Martín MC et al. 2011. Integrative expression system for delivery of antibody fragments by lactobacilli. Applied and Environmental Microbiology 6:2174-2179
Pant N et al. 2011. Lactobacilli expressing non-competing bispecific llama derived anti-rotavirus proteins effectively combat rotavirus induced diarrhea. Future Microbiology 6:583-93.
van der Vaart JM, Pant N, Wolvers D, Bezemer S, Hermans PW, Bellamy K, Sarker SA, van der Logt CPE, Svensson L, Verrips CT, Hammarström L, and van Klinken BJW. 2006. Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments.Vaccine 24:4130-4137.
Weiner C, Pan Q, Hurtig M, Boren T, Bostwick E, and Hammarström L. 1999. Passive immunity against human pathogens using bovine antibodies. Clin Exp Immunol 116:193-205.
List of Websites:
http://lactobody.eu/site/
