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To help rationally design an antibody for oral administration, we examined how different isotypes (IgG, IgA and sIgA) with the same variable sequence affect antibody stability across digestion. We compared the degradation of recombinant palivizumab (IgG1), and recombinant IgA and sIgA versions of palivizumab spiked in human milk to the degradation of naturally-occurring anti-respiratory syncytial virus (RSV) sIgA/IgA and IgG in human milk from four donors across gastric and intestinal phases of an in vitro model of infant digestion via a validated RSV F protein ELISA. Palivizumab IgG and IgA formats were less stable than the sIgA version after complete simulated gastrointestinal digestion: palivizumab IgG, IgA and sIgA decreased across complete simulated gastrointestinal digestion by 55%, 48% and 28%, respectively. Naturally-occurring RSV F protein-specific IgG was stable across digestion, whereas naturally-occurring sIgA/IgA was stable in the gastric phase but decreased 33% in the intestinal phase of simulated digestion.

Oral administration of engineered immunoglobulins has the potential to prevent enteric pathogen-induced diarrhea in infants. To prevent infection, these antibodies need to survive functionally intact in the proteolytic environment of the gastrointestinal tract. This research examined both ex vivo and in vivo the functional survival across infant digestion of palivizumab, a model FDA-approved recombinant antibody against respiratory syncytial virus (RSV) F protein. Palivizumab-fortified feed (formula or human milk), infant gastric, and intestinal samples were incubated to simulate in vivo digestion (ex vivo digestion). Palivizumab-fortified human milk was also fed to infants, followed by collection of gastric and intestinal samples (in vivo digestion). Palivizumab was purified from the samples of digestate using protein G spin columns followed by filtration through molecular weight cut-off membranes (30 kDa). Palivizumab functional survival across ex vivo and in vivo digestion was determined via an anti-idiotype ELISA and an RSV plaque reduction neutralization test. Palivizumab concentration and RSV neutralization capacity both decreased when incubated in intestinal samples (ex vivo study). The concentration and neutralization activity of orally-supplemented palivizumab also decreased across infant digestion (in vivo study). These results indicate that if recombinant IgGs were selected for oral supplementation to prevent enteric infections, appropriate dosing would need to account for degradation occurring in the digestive system. Other antibody formats, structural changes, or encapsulation could enhance survival in the infant gastrointestinal tract.

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.

Orally delivered antibodies may be useful for the prevention of enteric pathogen infection, but to be effective they need to survive intact across digestion through the gastrointestinal tract. As a test case, we fed a recombinant human antibody, palivizumab, spiked into human milk to four infants and collected gastric, intestinal and stool samples. We identified a tryptic peptide from palivizumab (LLIYDTSK) that differs from all endogenous human antibodies and used this for quantitation of the intact palivizumab. To account for dilution by digestive fluids, we co-fed a non-digestible, non-absorbable molecule-polyethylene glycol 28-quantified it in each sample and used this value to normalize the observed palivizumab concentration. The palivizumab peptide, a stable isotope-labeled synthetic peptide and polyethylene glycol 28 were quantified via a highly sensitive and selective parallel-reaction monitoring approach using nano-liquid chromatography/Orbitrap mass spectrometry. On average, the survival of intact palivizumab from the feed to the stomach, upper small intestine and stool were 88.4%, 30.0% and 5.2%, respectively. This approach allowed clear determination of the extent to which palivizumab was degraded within the infant digestive tract. This method can be applied with some modifications to study the digestion of any protein.

Hepcidin belongs to the antimicrobial peptide family but has weak activity with regards to bacterial killing. The regulatory function of hepcidin in humans serves to maintain an iron-restricted environment that limits the growth of pathogens; this study explored whether hepcidin affected bacterial iron homeostasis and oxidative stress using the model organism Escherichia coli. Using the Miller assay it was determined that under low iron availability exposure to sub-inhibitory doses of hepcidin (4-12µM) led to 2-fold and 4-fold increases in the expression of ftnA and bfd, respectively (P < 0.05), in both a wild type (WT) and Δfur (ferric uptake regulator) background. Quantitative real-time PCR analysis of oxyR and sodA, treated with 4 or 8 µM of hepcidin showed that expression of these genes was significantly (P < 0.05) increased, whereas expression of lexA was unchanged, indicating that hepcidin likely mediated oxidative stress but did not induce DNA damage. [ABSTRACT FROM AUTHOR]