Lactobacilli expressing antibody fragments against pathogens

Adherence to mucosal surfaces has been shown to be of great importance in the pathogenesis of various infections in the gastro-intestinal tract. Live recombinant bacteria can be used to deliver active or passive immunity at the mucosal surface, which is the site of entry for the majority of the pathogens. GRAS (Generally Regarded As safe) organisms with probiotic properties, such as Lactobacillus, are suitable candidates as vectors for delivery of foreign antigens or antibody fragments especially in the human gastrointestinal tract. The aims of these investigations have been to evaluate the potential of lactobacilli expressing antibody fragments against various types of pathogens in order to block adherence of these pathogens to their target sites.

Vectors, with a promoter inducible with mannitol, encoding a scFv antibody fragment, which recognizes the streptococcal antigen I/II (SA I/II) adhesion molecule of S. mutans (main etiological agent of dental caries), were constructed and expressed in lactobacilli. The expression and functionality of the single chain (scFv) antibody fragments, secreted into the supernatant or expressed on the surface of the bacteria, was verified in vitro. Oral administration of scFv-expressing bacteria in desalivated rats resulted in decreased number of S. mutans bacteria in the oral cavity and lower development of dental caries (Paper I).

In order to evaluate if continuous expression of antibodies in situ in the oral cavity might be therapeutically superior to intermittent production, we compared the anti-cariogenic effect of lactobacilli expressing the scFv against the SA I/II adhesin under the control of an inducible or constitutive promoter. Both lactobacilli expressed equal amounts of scFv on the surface and agglutinated S. mutans bacteria expressing SA I/II to a similar level. In a rat caries model, transformed lactobacilli could be detected in the oral cavity throughout the duration of the study. Transformants containing the constitutive promoter were slightly more protective than those containing the inducible promoter suggesting that in situ expression might increase protection (Paper II).

Selected llama IgG subclass antibodies are composed of an elongated heavy chain but no light chain. The variable domain of llama heavy chain antibodies (VHHs) which are formed by a single polypeptide are markedly stable and can be easily engineered more easily, while retaining the correct functional conformation. These properties make them suitable for therapy against gastrointestinal infections. We first analysed the possibility of expressing functional model VHH fragments under the control of an inducible promoter. Two VHH fragments, VHH2 (directed against the capsid of the phage) and VHH5 (directed against the receptor-binding protein of the phage tail) against a lactic acid bacteriophage, p2, were respectively expressed as an anchored and secreted product. Both VHH fragments were efficiently expressed and shown to inhibit phage infection of lactococci in a neutralization assay. This may be of future use for prevention of lysis of starters and destruction of cheese batches caused by bacteriophages (Paper III).

Subsequently, a VHH directed against rotavirus (VHHI) was expressed constitutively by lactobacilli, both in secreted and in cell surface anchored forms. Expression of VHH fragments in lactobacilli conferred significant reduction of infection in cell cultures in a neutralization assay. When administered orally, lactobacilli producing surface expressed VHH markedly shortened disease duration, severity and viral load in a mouse model of rotavirus-induced diarrhea both when given fresh or in a freeze-dried form. Such "lactobodies" may form the basis of a novel form of therapy against rotavirus infections and other diarrheal diseases worldwide (Paper IV).

Our experiments suggest that lactobacilli are potent expressers of functional antibody fragments against various pathogens, thus making them future candidates as delivery vehicles of passive immunity.

List of scientific papers

I. Kruger C, Hu Y, Pan Q, Marcotte H, Hultberg A, Delwar D, van Dalen PJ, Pouwels PH, Leer RJ, Kelly CG, van Dollenweerd C, Ma JK, Hammarstrom L (2002). In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotechnol. 20(7): 702-6.
https://pubmed.ncbi.nlm.nih.gov/12089555

II. Kruger C, Hultberg A, van Dollenweerd C, Marcotte H, Hammarstrom L (2005). Passive immunization by lactobacilli expressing single-chain antibodies against Streptococcus mutans. Mol Biotechnol. 31(3): 221-31.
https://pubmed.ncbi.nlm.nih.gov/16230772

III. Hultberg A, Tremblay DM, de Haard H, Verrips T, Moineau S, Hammarström L, Marcotte H (2006). III. Lactobacilli expressing llama VHH fragments for prevention of phage infection in lactococci. [Manuscript]

IV. Pant N, Hultberg A, Zhao Y, Svensson L, Pan-Hammarstrom Q, Johansen K, Pouwels PH, Ruggeri FM, Hermans P, Frenken L, Boren T, Marcotte H, Hammarstrom L (2006). Lactobacilli Expressing Variable Domain of Llama Heavy-Chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-Induced Diarrhea. J Infect Dis. 194(11): 1580-8.
https://pubmed.ncbi.nlm.nih.gov/17083044