key: cord-1052791-h5qf2qld authors: Carlander, David; Kollberg, Hans; Wejåker, Per-Erik; Larsson, Anders title: Peroral immunotheraphy with yolk antibodies for the prevention and treatment of enteric infections date: 2000 journal: Immunol Res DOI: 10.1385/ir:21:1:1 sha: 83c206ed163d6024781f1b508c3b536721d69d63 doc_id: 1052791 cord_uid: h5qf2qld Oral administration of specific antibodies is an attractive approach to establish protective immunity against gastrointestinal pathogens in humans and animals. The increasing number of antibiotic-resistant bacteria emphasize the need to find alternatives to antibiotics. Immunotherapy can also be used against pathogens that are difficult to treat with traditional antibiotics. Laying hens are very good producers of specific antibodies. After immunization, the specific antibodies are transported to the egg yolk from which the antibodies then can be purified. A laying hen produces more than 20 g of yolk antibodies (IgY) per year. These antibodies also have biochemical properties that make them attractive for peroral immunotherapy: They neither activate mammalian complement nor interact with mammalian Fc receptors that could mediate inflammatory response in the gastrointestinal tract. Eggs are also normal dietary components and thus there is practically no risk of toxic side effects of IgY. Yolk antibodies have been shown in several studies to prevent bacterial and viral infections. There is an increasing prevalence of antibiotic-resistant bacteria, which makes traditional antibiotics less effective. It is thus important to find alternatives to the use of antibiotics. Oral immunotherapy with specific antibodies is a treatment strategy that has been actively pursued in laboratory studies and early-stage clinical studies for the last decade. Several sources of antibodies have been used in these studies, including human IgG, monoclonal antibodies, bovine colostrum, and chicken egg yolk antibodies. Polyclonal antibodies recognize more epitopes on the bacterial surface, which makes it difficult for bacteria to avoid the antibodies by mutations. They are also cheaper to produce than monoclonal antibodies. Egg yolk is a very interesting source of specific antibodies for oral immunotherapy. Hens, which have been immunized with microbes, respond by producing specific antibodies against the microbe. These antibodies are actively transported to the egg yolk in large quantities from the blood of laying hens. A laying hen produces more yolk antibodies than a rabbit can produce during the same time period, and the animal care costs are lower for the chicken compared to the rabbit. Egg yolk antibodies will thus be an inexpensive way of producing large amounts of specific antibodies. Chicken antibodies offer many advantages to the traditional mammalian antibodies when used for the detection of mammalian antigen. Owing to the evolutionary difference chicken IgY will react with more epitopes on a mammalian antigen, which will give an amplification of the signal (1). Chicken antibodies can also be used to avoid interference in immunological assays caused by the human complement system, rheumatoid factors, human anti-mouse IgG antibodies (HAMA), or human and bacterial Fc receptors (1) . The antibodies can be purified in large amounts from egg yolk, making laying hens highly efficient producers of polyclonal antibodies. Several studies show that bacterial and viral infections can be prevented with egg yolk immunoglobulin (IgY) in a dose-dependent manner. Peroral administrations of specific antibodies from egg yolk have been used successfully to treat bacterial and viral infections in animals (2) (3) (4) (5) . The low-molecular-weight serum antibody found in birds is IgY, whereas mammals have IgG. IgY is the main serum immunoglobulin in chicken. Chicken IgY (6), or chicken IgG as it was called earlier, is the functional equivalent of mammalian IgG in birds, but it differs in many functional aspects to mammalian IgG (1) . Chicken IgY consists of two light chains and two heavy chains and has a molecular weight of approx 180,000 Da. The heavy chain (upsilon, υ) has a weight of 66,000 Da and the light chain 22,000 Da. IgY is transported from the mother to the embryo via the egg yolk, and the egg yolk thus contains high concentrations of chicken IgY. Other immunoglobulin classes are present only in negligible amounts in the egg yolk. The IgY concentration, in egg yolk is higher than the serum concentration and a single egg yolk (approx 15 mL) contains more than 100 mg IgY. The use of chicken egg yolk as a source for antibody production represents a reduction in animal use as chickens produce larger amounts of antibodies than laboratory rodents. It also makes it possible to eliminate the collection of blood, which is painful for the animal. The European Centre for the Validation of Alternative Methods (ECVAM) thus recommends that yolk antibodies should be used instead of mammalian antibodies for animal welfare reasons (7) . When an antibody reacts with the antigen, an immune complex is formed. Antigen-antibody complexes containing mammalian antibodies will activate the human complement system (1). Activated complement components are potent inflammatory mediators. Theoretically, immune complexes containing mammalian antibodies may interact with Fc and complement receptors in the mammalian gastrointestinal tract, causing cell activation. Immune complexes containing IgY do not activate the mammalian complement system and do not interact with mammalian Fc and complement receptors. In summary, yolk IgY seems to be well suited for peroral immunotherapy. The transit time for orally administered antibodies in the gastrointestinal tract is between 12 and 36 h in infants and children (8, 9) . Orally administered antibodies are subjected to denaturation by the acidic pH of the stomach and degradation by proteases. In neonates, the gastric fluid has a pH close to neutral but the pH rapidly decreases to below 3.0 during the first days. Studies have shown that part of the antibodies remain intact in pepsin and trypsin digests but there is a considerable cleavage of the antibodies into Fab, Fab' 2 and Fc fragments. However, Fab' 2 , and Fab fragments still have the capability to bind to the antigen and exhibit neutralizing activity. Despite the degradation in the gastrointestinal tract, active antibodies can still be detected in stool samples. The percentage active antibody in the stool varies between very low levels to up to 50% of the orally given dose in different studies. These differences are probably due to differences in the pH of the stomach, enzyme activity, presence of antigen, and the gastrointestinal passage time. The methods used to detect the antibodies in stool may also influence the results. A large portion of the antibodies is digested to Fab' 2 or Fab fragments. These fragments still bind to the antigen, but many immunological tests give different results with Fab fragments than with whole antibodies. Antibodies are absorbed from the intestine in young piglets and calves. Low levels of IgY can be detected in the circulation, of calves or piglets that are treated orally with yolk antibodies during the first 24 to 48 h post natum (10) . After this time period there will be no absorption of active antibodies from the gastrointestinal tract. No absorption of intact antibodies has been shown in humans. Blum et al. (11) and Eibl et al. (12) showed that there was no increase in serum immunoglobulin levels after oral administration of human immunoglobulins to infants. Also, Losonsky et al. (9) could not detect any high-molecularweight radioactivity in blood after oral administration of 125 I-labeled IgG. Thus, no systemic effects can be expected after oral administration of yolk antibodies to humans. Consumption of eggs is accepted in most cultures. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY. The mean egg consumption is 0.5-1 egg/d in Sweden. This amount of IgY is similar to or higher than the amounts used in several of the immunotherapy studies. However, allergic reactions to egg-derived immunoglobulins and to residual egg proteins may occur (13) . It is important to consider egg allergy before starting immunotherapy with yolk antibodies in humans. Oral administration of spray dried yolk antibodies specific against Salmonella typhimurium or S. dublin has been shown to prevent Salmonella infections in calves. The antibodies were administered three times a day for 7-10 d after inoculation with S. typhimurium or S. dublin. All calves in the control group died, whereas only diarrhea and fever were observed in the group treated with the highest antibody titer. Thus, the antibodies could protect against fatal salmonellosis (14) . Yolk preparations from chickens immunized with purified outer membrane proteins, lipopolysaccharide, flagella, and fimbriae of Salmonella have also been tested in mouse models of experimental salmonellosis. The mice treated with specific antibodies had a significantly higher survival rate than mice fed normal egg yolk antibodies (14, 15) . Yolk immunoglobulins have also been used to protect neonatal piglets against experimental enterotoxigenic Escherichia coli infections. Oral treatment with antibody preparations against the fimbrial adhesins K88, K99, and 987P considerably reduced the severity of diarrhea and reduced the mortality (16) . Yolk immunoglobulins have also been shown to prevent diarrhea in rabbits challenged with E. coli (17) . A mouth rinse containing egg yolk antibodies to Streptococcus mutans has been used to reduce the establishment of these bacteria in dental plaque of humans. The antibodies inhibited S. mutans adherence to saliva-coated hydroxyapatite discs in vitro and decreased the percentage of S. mutans per total streptococci in vivo (4) . We have also shown that gargling once a day with a yolk preparation specific to Pseudomonas aeruginosa prevents the colonization of these bacteria in the lungs of patients with cystic fibrosis (18) . One of these patients has now been treated for more than 4 yr without any chronic colonization with P. aeruginosa. Egg yolk and colostrum powders containing specific antibodies against bovine coronavirus (BCV) antigen were evaluated in a challenge model with a standardized dose of virulent BCV strain. Daily treatment with these antibody preparations started 6 h until 7 d post-challenge. Control calves, which received no antibody, had severe diarrhea and all died within 6 d after infection. In contrast, calves fed milk containing egg yolk or colostrum with high antibody titers all survived and had positive weight gain. The study showed that specific antibody preparations protected against BCV-induced diarrhea in neonatal calves and that the egg yolk used provided a higher degree of protection compared to colostrum powder on a titer basis (19) . A field trial with oral administration of chicken IgY specific for bovine rotavirus (BRV) resulted in a significantly increased mean body weight (p < 0.05) and a decrease in number of calves shedding high titer of BRV (G6) in stool compared to control calves (p < 0.01) (20) . Oral administered yolk immunoglobulins have also been shown to protect suckling mice against experimental bovine rotavirus-induced diarrhea (21) . Oral treatment with bovine antirotavirus colostrum resulted in a reduction of rotavirusassociated diarrhea in infants (22) . It also reduced the infection rate in children (23) . Cryptosporidium is a zoonotic pathogen that infects the respiratory or gastrointestinal tracts of a large number of hosts including fish, birds, reptiles, and mammals. Cryptosporidium infections are usually associated with immunosuppressed hosts, but the infection may also occur in individuals with a normal immune system. The use of yolk preparations with high anti-Crypto-sporidium activities caused a significant parasite reduction in a neonatal mouse model (24) . Treatment with bovine anti-Crypto-sporidium colostrum immunoglobulin has also been shown to induce a 100-fold reduction of oocyte excretion in healthy human volunteers (25) . Laying hens are a cost-efficient method for the production of large quantities of specific antibodies. Yolk antibodies do not activate the mammalian complement system nor interact with mammalian Fc receptors that could mediate inflammatory response in the gastrointestinal tract. Eggs are also normal dietary components and thus there is practically no risk of toxic side effects of IgY. Oral immunotherapy with yolk antibodies is thus an attrac-tive approach for treatment of gastrointestinal infections in humans and animals that are difficult to treat with traditional antibiotics. The increasing number of antibiotic resistant bacteria emphasize the need to find alternative to antibiotics. Chicken IgG: Utilizing the evolutionary advantage Prevention of murine rotavirus infection with chicken egg yolk immunoglobulins Oral passive immunization against dental caries in rats by use of hen egg yolk antibodies specific for cell-associated glucosyltransferase of Streptococcus mutans Passive immunization against dental plaque formation in humans: effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans Passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk Chicken immunoglobulins: biological halflives and normal adult serum concentrations of IgM and IgY The production of avian (egg yolk) antibodies: IgY Use of bovine milk concentrate containing antibody to rotavirus to treat rotavirus gastroenteritis in infants Oral administration of human serum immunoglobulin in immunodeficient patients with viral gastroenteritis. A pharmacokinetic and functional analysis Detection of passage and absorption of chicken egg yolk immunoglobulins in the gastrointestinal tract of pigs by use of enzyme-linked immunosorbent assay and fluorescent antibody testing Survival of oral human immune serum globulin in the gastrointestinal tract of low birth weight infants Prevention of necrotizing enterocolitis in lowbirth-weight infants by IgA-IgG feeding Allergenicity of orally administered immunoglobulin preparations in food-allergic children Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonellaspecific antibodies Passive immunisation against experimental salmonellosis in mice by orally administered hen egg-yolk antibodies specific for 14-kDa fimbriae of Salmonella enteritidis Passive protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets Oral ingestion of egg yolk immunoglobulin from hens immunized with an enterotoxigenic Escherichia coli strain prevents diarrhea in rabbits challenged with the same strain Proceedings from the 2nd international symposium on egg nutrition and newly emerging ovo-biotechnologies Passive protection of neonatal calves against bovine coronavirus-induced diarrhea by administration of egg yolk or colostrum antibody powder Field evaluation of chicken egg yolk immunoglobulins specific for bovine rotavirus in neonatal calves Passive protection against bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg yolk Gastroenteritis in suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a proteinbound poly-saccharide preparation (PSK) Passive immunisation of children with bovine colostrum containing antibodies to human rotavirus Hyperimmune hens as a novel source of anti-Crypto sporidium antibodies suitable for passive immune transfer Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum