Lactobacillus mediated targeting of gastrointestinal pathogens

The mucosal membranes of the gastrointestinal, genitourinary and respiratory tract are the main targets for infection in the human body and are repeatedly challenged by pathogens. Passive immunization using delivery of antibodies at the mucosal membrane may thus provide a new first line of defense against these pathogens.

The objective of this work was to genetically modify Lactobacillus, a natural inhabitant of the gastrointestinal tract, for in situ delivery of passive immunity against gastrointestinal infections.

With the aim of identifying colonizing strains of Lactobacillus that could be used as vehicle for delivery of passive immunity in the gastrointestinal tract, a collection of ninety- three Lactobacillus strains, derived from human fecal samples, was screened for markers for survival and persistence in the gastrointestinal tract (paper I). Five strains showed promising results and will be taken forward for testing of survivability in human gut and confirm their safety in human volunteers.

A novel expression system for production and delivery of therapeutic molecules in Lactobacillus was constructed based on the framework of the apf gene from L. crispatus. This expression system was tested for delivery of antibody fragments both in a secreted form and cell wall anchored on the surface of the Lactobacillus. The expression cassettes were stably integrated on the chromosome using the integrase gene from the bacteriophage A2 to create “food grade” strains of Lactobacillus, devoid of antibiotic markers, for delivery of antibody fragments (paper II).

The feasibility of delivering passive immunity against bacterial toxins in the gastrointestinal tract was tested with L. paracasei BL23 engineered to express a single chain antibody binding the anthrax protective antigen. The engineered Lactobacillus was able to provide protection in mice orally challenged with the anthrax edema toxin, validating the concept of in situ toxin neutralization in the gastrointestinal tract (paper III). Members of the family of Camilidae, to which llamas belong, express a subset of their IgG antibodies as heavy chain only antibodies. Their antigen binding domain is encoded in the single variable domain (VHH) that can be produced as a single polypeptide. The VHH has higher acid and proteolytic stability compared to conventional IgG making them ideally suitable for therapeutic use in the gastrointestinal tract. Llamas were immunized with the toxins A and B, the two causative elements of C. difficile associated diarrhea. A range of VHH neutralizing the dominant virulence factor, toxin B, were isolated and cloned for expression in L. paracasei. When expressed either in a secreted or cell wall anchored form, the Lactobacillus produced VHH were able to provide protection against the cytotoxic effects of toxin B. Prophylactic treatment with a combination of two strains of engineered L. paracasei expressing two toxin B neutralizing VHH could delay and provide partial protection against the effect of a toxin B producing strain of C. difficile in an in vivo hamster model (paper IV).

In summary this work has shown the potential of using Lactobacillus for the delivery of passive immunity against gastrointestinal infections. Lactobacillus strains colonizing the gastrointestinal tract for delivery of antibody fragments could represent a potential new approach to management of the C. difficile associated diarrhea that could be used both prophylactically or for the prevention of recurrent infections.

List of scientific papers

I. Screening and evaluation of human intestinal lactobacilli for the development of novel gastrointestinal probiotics. Kõll P, Mändar R, Smidt I, Hütt P, Truusalu K, Mikelsaar RH, Shchepetova J, Krogh-Andersen K, Marcotte H, Hammarström L, Mikelsaar M. Current Microbiology. 2010 Dec;61(6):560-566.
https://doi.org/10.1007/s00284-010-9653-y

II. Integrative expression system for delivery of antibody fragments by lactobacilli. Martín MC, Pant N, Ladero V, Günaydin G, Andersen KK, Alvarez B, Martínez N, Alvarez MA, Hammarström L, Marcotte H. Applied and Environmental Microbiology. 2011 Mar;77(6):2174-2179.
https://doi.org/10.1128/AEM.02690-10

III. In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli. Andersen KK, Marcotte H, Álvarez B, Boyaka PN, Hammarström L. BMC Biotechnology. 2011 Dec 20;11:126.
https://doi.org/10.1186/1472-6750-11-126

IV. Neutralization of Clostridium difficile toxin B mediated by engineered lactobacilli producing single domain antibodies. Andersen KK, Strokape NM, Hultberg A, Kai Truusalu, Imbi Smidt, Raik-Hiio Mikelsaar, Mikelsaar M, Verrips T, Hammarström L, Marcotte H. [Submitted]