Lactobacillus based oro-mucosal therapies against rotavirus

Abstract: Rotavirus is the most important agent for severe infantile diarrhea, responsible for over 2 million diarrhea episodes and 600,000 deaths annually, mainly in the developing countries. Introduction of successful vaccine programs could help reduce the burden of rotavirus disease. However, at a deplorable rate of 70 children dying every hour there is an urgent need of finding alternative treatments. Passive immunization with orally delivered protective antibodies provides immediate protection against rotavirus. Over the last few decades the role of probiotics especially from Lactobacillus spp in managing rotavirus diarrhea has also been increasingly recognized. The objective of this work has been to optimize oral delivery of passive immunity against rotavirus using antibodies directly or through genetically engineered lactobacilli that express antibody fragments. Oral combination treatment of different lactobacilli with bovine anti-rotavirus antibodies was evaluated in mouse pups challenged with rotavirus (paper I). Lactobacillus rhamnosus GG, a well known probiotic was found to synergize with antibodies and helped in early recovery from diarrhea in mice while saving up to 90% of antibodies. These components do not require special storage conditions and could be used to complement existing therapies. Members of the family Camilidae express unconventional IgG antibodies composed of only heavy chains. The variable part of these antibodies (VHH) is composed of a single polypeptide and has remarkable thermo- and acid-stability allowing its use in the gastrointestinal tract. A phage display library of llama VHH fragments against rotavirus was constructed. VHH fragments were selected by stringent panning and were expressed in yeast. Purified VHH proteins were tested in mouse pups challenged with rotavirus. A VHH fragment with high neutralizing activity in vitro and ability to reduce diarrhea in vivo was identified (paper II). Genetically engineered lactobacilli that can sustain continuous in situ production of antibodies are suitable candidates for delivery of passive immunity against rotavirus. The VHH fragment selected in paper II was expressed in Lactobacillus paracasei both in cell surface anchored and in secreted forms. VHH fragments produced by lactobacilli conferred significant reduction of infection in cell culture. Oral delivery of lactobacilli expressing cell surface anchored VHH fragments alleviated diarrhea symptoms and reduced viral load in the intestine. Genetically engineered lactobacilli expressing functional VHH fragments may thus form the basis of a novel form of therapy against rotavirus (paper III). Targeting a single epitope has limitations in terms of cross-reactivity to the circulating serotypes of virus and typically requires high amounts of antibodies for neutralization. Therefore, two VHH fragments (VHH1 and VHH3) that recognize unique epitopes on rotavirus and in combination synergistically reduce infection were expressed in L. paracasei as cell surface anchored dimers. The VHH3-VHH1 dimer expressing lactobacilli had stronger affinity for rotavirus than lactobacilli expressing monomers. These lactobacilli reduced diarrhea symptoms when administered to mice challenged with rotavirus. Thus VHH fragments can be used to build modular designs that can be expressed by lactobacilli (paper IV).