Abstract
Oral administration of enteric pathogen-specific immunoglobulins may be an ideal approach for preventing infectious diarrhea in infants and children. For oral administration to be effective, antibodies must survive functionally intact within the highly proteolytic digestive tract. As an initial step toward assessing the viability of this approach, we examined the survival of palivizumab, a recombinant monoclonal antibody (IgG1κ), across infant digestion and its ability to neutralize respiratory syncytial virus (RSV). Human milk and infant digestive samples contain substances known to interfere with the RSV neutralization assay (our selected functional test for antibody survival through digestion), therefore, antibody extraction from the matrix was required prior to performing the assay. The efficacy of various approaches for palivizumab purification from human milk, infant's gastric and intestinal digestates, including casein precipitation, salting out, molecular weight cut-off, and affinity chromatography (protein A and G) were compared. Affinity chromatography using protein G with high-salt elution followed by 30-kDa molecular weight cut-off centrifugal filtration was the most effective technique for purifying palivizumab from human milk and infant digestates with a high yield and reduced background interference for the viral neutralization assay. This work is broadly applicable to the optimal isolation of antibodies from human milk and infant digesta for viral neutralization assays, enables the examination of how digestion affects the viral neutralization capacity of antibodies within milk and digestive samples, and paves the way for assessment of the viability of oral administration of recombinant antibodies as a therapeutic approach to prevent enteric pathogen-induced infectious diarrhea in infants.
Highlights
Enteric pathogen-induced infectious diarrhea is one of the leading causes of death in children in developing countries [1]
Knowledge of the extent to which palivizumab maintains its respiratory syncytial virus (RSV)-neutralizing function across the infant digestive system requires the isolation of the immunoglobulin, as the complex matrices of milk, and infant’s gastric and intestinal digestates have a variety of components, including proteases, protease inhibitors, immunoglobulins (SIgA, IgG, and IgM), β-casein, lactoferrin and lactoperoxidase, milk fat, cells and bacteria that can interfere with the RSV neutralization assay [4,5,6,7]
Substances present in milk and digestive samples are capable of interfering with the RSV neutralization assay and prevent accurate measurement of the neutralizing capacity specific to added palivizumab and its survival across digestion
Summary
Enteric pathogen-induced infectious diarrhea is one of the leading causes of death in children in developing countries [1]. One potential approach to preventing enteric pathogen-induced diarrhea in infants is oral administration of recombinant, pathogen-specific immunoglobulins. Such enteric pathogen-specific antibodies would have to survive functionally intact across the infant digestive. As a proxy for enteric pathogen-specific recombinant antibodies, the functional survival of orally delivered palivizumab, the recombinant monoclonal antibody (IgG1κ) against respiratory syncytial virus (RSV), was examined. This antibody has been approved by the FDA to provide passive immunity against infection by RSV in infants via intramuscular injection. Knowledge of the extent to which palivizumab maintains its RSV-neutralizing function across the infant digestive system requires the isolation of the immunoglobulin, as the complex matrices of milk, and infant’s gastric and intestinal digestates have a variety of components, including proteases, protease inhibitors, immunoglobulins (SIgA, IgG, and IgM), β-casein, lactoferrin and lactoperoxidase, milk fat, cells and bacteria that can interfere with the RSV neutralization assay [4,5,6,7]
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