Author(s): Neha Pant 1 , Harold Marcotte 1 , Pim Hermans 2 , Sandra Bezemer 2 , Leon Frenken 3 , Kari Johansen 4 , Lennart Hammarström [[dagger]] 5
antibodies; diarrhea; lactobacilli; passive immunization; rotavirus
Gastroenteritis due to rotavirus infection is an important health problem in developing countries, causing over 125 million episodes of diarrhea annually and 527,000 deaths among children less than 5 years of age  . Given the high contagiousness and small infectious dose of rotavirus, infections can often increase to epidemic proportions  . Efficient vaccines are currently available but are not suitable in an epidemic situation. Passive immunity is, at present, the only available intervention that provides immediate protection and may thus represent the prophylaxis or therapy of choice for outbreaks in developing countries or selected groups of children such as hospitalized children or immunocompromised patients.
Rotavirus-neutralizing mucosal antibodies, either host-derived secretory IgA or orally delivered antibodies from heterologous sources, have previously been shown to mitigate severe diarrhea in animal models and in naturally infected children [3,4] . Thus, optimizing delivery of specific antibodies to the intestinal lumen may be of considerable importance in achieving protection against rotavirus. Llamas and other camelids contain a unique class of antibodies in their serum that lack light chains, the heavy-chain only antibodies. The variable region of these antibodies, VHH, is the smallest naturally occurring antigen-binding protein domain described  . Llama VHH fragments are of major interest in biotechnology as they are markedly acid resistant and could therefore be advantageous for use against infectious agents in the GI tract. They can also be easily produced in yeast and bacteria while retaining a correct functional conformation  . As their projecting complementarity determining region loops may be able to access deep recesses of protein, some VHH domains have demonstrated improved penetration against cryptic (immuno-evasive) target antigens such as trypanosome surface glycoproteins  and/or the active site of enzyme [8,9] . Such VHH domains are well suited to target cryptic sites in the virus capsid that are poorly accessible to conventional antibodies. The llama single-domain antibodies are also the perfect modular building units for bivalent and bispecific proteins that can be simply engineered through fusion of VHH domain encoding genes [10,11] . For many applications, it is advantageous to engineer monovalent antibody fragments into multivalent formats to increase functional affinity (termed avidity) or to produce bispecific antibody fragments that can simultaneously bind to different antigens. However, while some bivalent or bispecific VHH show up to 500-times higher potency than monovalent molecules  others show low  or no increased activity  .
Lactobacilli are generally regarded as safe (GRAS) microorganisms that constitute an important component of the normal mucosal microbiota. Many strains of lactobacilli show probiotic properties and have been successfully used for intervention against rotavirus diarrhea  . In a proof of concept study, we previously showed that oral administration of lactobacilli producing monospecific VHH antibody fragments against rotavirus reduced the morbidity of diarrhea in a mouse model  . Modified lactobacilli producing antibodies could be administered and distributed in the form of a food product, thus allowing both...