JP2002521494A - Gastrointestinal bacterial antibody factory - Google Patents

Abstract

  (57) [Summary] Neonates 30 days after birth are particularly susceptible to the pathogen. Because the immune system is not yet fully functioning. Even adults can become susceptible to pathogens if their immune system is compromised by disease or suddenly exposed to large amounts of GI pathogens. The present invention provides a method of immunizing newborns and adults against a pathogen by orally administering recombinant commensal bacteria expressing antibodies to the pathogen. These commensal bacteria may be administered with antibodies that are immunologically specific for the pathogen. The invention further provides compositions comprising recombinant commensal bacteria expressing antibodies to the pathogen, and methods of immunizing newborns and adults with the compositions.

Description

DETAILED DESCRIPTION OF THE INVENTION

This application claims priority to US Provisional Application No. 60/094697, filed Jul. 30, 1998, which is incorporated herein by reference. 35 U.S. S. C. In accordance with §202 (c), the United States Government has certain rights in the inventions described herein, in part, the United States Department of Agri
culture Grant No. 94-37204-0857; Hatch Nos. 3948 and 3822; and Nation
Funds from the al Institutes of Health Grant CA07175 and CA22484 were used.

FIELD OF THE INVENTION [0002] The present invention relates to the field of genetically engineered bacteria for therapeutic and prophylactic purposes. In particular, the present invention relates to recombinant commensal bacteria expressing antibodies to common gastrointestinal pathogens, and methods of using them in the treatment and prevention of disease.

BACKGROUND OF THE INVENTION Several publications in parentheses are referred to in this application in order to more fully describe the state of the art in the relevant fields of the invention. The disclosure of each of these publications is incorporated herein by reference.

[0004] For about thirty days after birth, humans and animals have no functional immunity to pathogens. As a result, when grown, infection by pathogenic organisms can be overcome by the action of a functional immune system, but infants are at increased risk of severe illness and death due to infection by pathogenic organisms. For example, in humans, cattle and pigs, rotavirus infection during the first few months of life causes the most serious life-threatening diarrheal infection. About 20% of newborn cattle and swine in the United States die from such infections. In chickens, it is common for all hatching chicks from Salmonella to get recurrent infections, a recurring event that causes the death of most of the population of chickens and the human food chain. Getting inside causes human illness.

[0005] Immunosuppression in adults caused by certain diseases, such as, for example, acquired immunodeficiency syndrome (AIDS) or by treatment with immunosuppressive drugs,
It usually increases the susceptibility of an individual to be able to provide immune defense to pathogenic organisms. Such organisms include, inter alia, pathogenic Escherichia coli, Cryptosporidium, Sal
monella, Vibrio cholera, Listeria monocytogenes and Helicobacter pylori
(Related to ulcer development). In addition, unusual pathogens or large amounts of pathogens (eg, Cryptosporidium syrup in drinking water or Sal in food and milk)
(Monella) exposed healthy adults would benefit from measures to reduce or prevent GI infection by the pathogen. In the United States, transmission of such large amounts of pathogens is known to occur in isolated systems (eg, patients in nursing homes and cruise ship passengers) or more reliably in more extreme situations such as war or famine refugees. Have been. In this regard, persons, such as military personnel who may be intentionally exposed to infectious organisms (ie, biological warfare), also benefit from measures to reduce or prevent such infections. Accordingly, there is a need to provide immune-mediated protection to newborns and / or immunosuppressed adults to treat or avoid serious and life-threatening diseases associated with infection by certain gastrointestinal (GI) pathogens. .

[0006] Three basic approaches have been developed in an attempt to protect newborn animals from GI pathogens. One approach is to use pathogenic organisms (eg, pathogenic Escherichi
immunization (vaccination) of a pregnant mother with an antigen associated with the E. coli strain. Its purpose is to stimulate the production of antibodies to pathogens in maternal colostrum given to the newborn on the day of delivery and on the second day. Antibodies consumed by some species of newborns are retained for a period of time (ie, absorbed and circulated in the blood),
Produces some immune defense against subsequent exposure to pathogens. In neonatal humans, colostrum antibodies are not retained and therefore this approach is not effective.

[0007] A second approach is to immunize a newborn against the pathogen with an antigen associated with the pathogenic organism. Oral administration of live xenogeneic and allogeneic rotavirus protected neonatal and adult mice from challenge with virulent murine rotavirus (Feng et al., 1994, J. Virology 68: 7766-7773; Burns et al.,
1995, Virology 207: 143-153). However, this approach has very limited success if the neonatal immune system is essentially nonfunctional. In human neonates, especially the immune system is marginally functional, and it is unlikely that humans will benefit from neonatal immunity.

A third approach is to administer an antibody or semi-purified antiserum to the pathogen of interest to a newborn animal as a bolus, usually by intranasal injection. If the antibody concentration is high enough on the GI mucosal surface, this procedure will provide the animal with a protective measure against the GI pathogen if the injections are repeated frequently enough to keep the antibody concentration high. However, since this approach requires repeated handling of the individual's newborn and multiple doses of the purified antibody or serum, which can be expensive, this approach is cost-impractical and time / human. Infeasible.

From the above, it appears that such attempted approaches are not a good solution to the problem of providing immune protection against infants or other immunosuppressed individuals. It is an object of the present invention to provide such a solution, namely an effective and feasible means for conferring immunity against GI and other pathogens to newborns and other immunosuppressed individuals.

SUMMARY OF THE INVENTION [0010] The present invention provides compositions and methods for supplementing or replacing an immune response to a selected pathogen in an individual in need of treatment. In general, the present invention provides
It is directed to newborn animals or humans, or to immunosuppressed or immunodeficient adults, or to healthy individuals who have been suddenly exposed to the GI pathogen bolus. The compositions of the invention include a commensal microorganism that has been genetically modified to express a recombinant antibody (rAB) to a selected pathogenic product. In a preferred embodiment of the present invention, the commensal microorganism is a GI tract colonizing bacterium or a GI tract-binding bacterium, such as Lactococcus, Bifidobacteria, Eubacteria, a non-pathogenic strain of Escherichi coli, E. coli F18 and E. coli Nissle 1917. Shares, or L. casei, L. pla
ntarum, L. paracasei, L. acidophilus, L. fermentum, L. zeae and L. gass
Various strains of Lactobacillus such as eri. In a preferred embodiment, the pathogenic organism is rotavirus, calicivirus, reovirus, coronavirus, enterovirus, adenovirus, Norwalk virus, enterotoxigenic Escherichia c.
oli, Campylobacter jejuni, Yersinia enterocolitica, Cryptosporidium spp.
, Giardia lamblia, Entamoeba histolytica, Helicobacter pylori, Isospora
GI pathogens such as belli, Clostridium spp. and Vibrio cholera (the last two cause GI disease by toxin secretion); and coxsackievirus, poliovirus, hepatitis A virus, Salmonella and Shigella spp., Listenia
Non-GI pathogens such as monocytogenes and L. strongyloides. In a preferred embodiment, antibodies expressed by commensal bacteria are adapted for expression in bacteria. Although any one bacterial cell produces only one type of recombinant antibody, multiple clones of the producing bacterium can be mixed to create a symbiotic population that simultaneously produces recombinant antibodies against multiple pathogens. This is a further advantage provided by the present invention. This is because, in general, vaccination of an animal simultaneously produces a single productive immune response to a single antigen. Symbiotic bacteria can express a mixed population of antibodies immunologically specific for one or more pathogens. In a particularly preferred embodiment, the commensal organism is one that produces rotavirus neutralizing antibodies. In another preferred embodiment, the composition of the present invention further contains lactose, maltodextrin and / or a desiccant.

The present invention is practiced by providing an antibody-expressing symbiotic organism to an animal or human in need of such treatment. The organism colonizes or becomes attached to the individual's GI tract and then continues to grow and proliferate, producing and secreting anti-pathogen antibodies. Thus, a steady-state immune state against the selected pathogen is formed in the individual's GI tract, whereby the individual is not infected by the pathogen, even if the individual is unable to mount its own immune response. Defended. In a preferred embodiment, the individual in need of this treatment is a newborn, an immunosuppressed or immunodeficient adult, or a healthy individual who has been rapidly exposed to one or more pathogens. In a preferred embodiment, a supplemental bolus of antibody immunologically specific for at least one antigen is administered with the antibody-expressing commensal. In another preferred embodiment, the antibody-expressing probiotic is transformed with 40 × 10 ⁹ colony forming units (cfu)
Orally twice a day for up to 8 weeks as a bolus containing
Antibody-expressing commensals are immunologically specific for at least one pathogen.
It may additionally contain up to mg / kg body weight / day of at least one antibody.
In another preferred embodiment, the antibody-expressing probiotic is administered to an adult three times daily for 10 days as a bolus containing up to 80 × 10 ⁹ colony forming units or until the external threat of the pathogen is eliminated. Further, the bolus may contain up to 25 mg / kg body weight / day of at least one antibody immunologically specific for at least one pathogen. Other features and advantages of the invention will be apparent from the description herein and the examples which follow.

DETAILED DESCRIPTION OF THE INVENTION The present invention should be carried out according to the following basic steps. (1) identifying the pathogen or pathogen mixture for which immune protection is desired in a neonate or other at-risk individual; (2) in a conventional system (eg, mouse, rabbit, sheep) against the pathogen. Obtaining a neutralizing antibody; (3) cloning the antibody gene in a form suitable for expression in the selected commensal bacterium; (4) transforming the bacterium with the cloned antibody gene so that the bacterium is Allowing the antibody to express and secrete; and then (5) providing the bacterium producing the recombinant antibody to an animal or human subject in whom immune protection is desired. The sections below describe each of these steps in more detail.

[0013] The present invention is applicable to the prevention or control of diseases caused by pathogens in which a gastrointestinal immune response is effective. Although the present invention is particularly applicable to GI pathogens, it can provide adequate protection (ie, the first line of defense) against ingested pathogens whose primary target is not the GI tract.

GI pathogens that are considered targets for the practice of the present invention include enteroviruses, adenoviruses, Norwalk-type viruses, enterotoxic Escherichia coli, Campylobacter
jejuni, Yersinia enterocolitica, Clostridium spp., Vibrio cholera, Cryp
tosporidium spp., Giardia lamblia, Entamoeba histolytica, Helicobacter p
ylori, and Isospora belli, but are not limited to these. Non-GI pathogens that enter the body through the GI tract, which are considered targets for the practice of the present invention, are Salmonel
la and Shigella spp., including, but not limited to, Listenia monocytogenes and strongyloideswo.

[0015] Neutralizing antibodies to one or more selected pathogens can be obtained using standard methods. Generate neutralizing antibodies to pathogen antigenic determinants, including intact pathogens, pathogen-specific surface antigens, epitopes of those surface antigens, or any other part of the organism that can elicit an immune response be able to. In a preferred embodiment, the species is immunized with a recombinant antibody from the particular species.

In a preferred embodiment of the invention, monoclonal antibodies immunologically specific for a particular antigen of the pathogen are obtained according to standard methodology. Some such antibodies are already known and are commercially available. For example, Example 1 describes a neutralizing monoclonal antibody against coronavirus serotype G3. In addition, hybridomas are available from the American Type Culture collection (ATCC Accession No. HB8178), and the hybridomas are characterized by a K found on enterotoxic strains of E. coli.
It secretes monoclonal antibodies against 99 cilia proteins. The advantage of using a monoclonal antibody is that it is directed to a single antigen or epitope and can be pressed as "neutralizing", i.e., binding to antigens on pathogens that are important for infection, replication, etc. That is what can be cleaned. Other monoclonal antibodies and fragments thereof of interest are those specific for the hepatitis B surface antigen (
Passafume et al., 1998, FEBS Let. 441: 407-412), specific to Puumala virus (Liang et al., 1997, Virology 235: 252-260), specific to HIV-1 (Burton) et al., 1994, Science 266: 1024-1027) and those specific to the human rhinitis virus (Condra et al., 1990, J. Biol. Chem. 265: 2292-2295).
), But not limited thereto.

A mixed population of monoclonal antibodies or a population of polyclonal antibodies may be used in the present invention. For example, instead of using a single hybridoma cell line,
Spleens may be removed from immunized mice and used to clone a mixed population of antibodies to the antigen used for immunization. The advantage of this approach is that the mixed antibody population can target a diverse array of epitopes on the pathogen and neutralize the pathogen at different sites in the physiologically changing conditions of the mammalian or avian GI tract.

Finally, recombinant antibodies suitable for use in the present invention may be synthetic variants.
One or more recombinant antibodies specific for a site on the selected pathogen may be isolated by starting from a library of recombinant antibodies. In this antibody library, three short domains in the variable region of the heavy chain (residues 31-33, 50-54, 95-98) and three in the variable region of the light chain determine high affinity binding to the antigen. The short domains (residues 32, 50, 91-96) were carefully mutated using partially degenerate primers to create a vastly diverse synthetic library (Pini et al. , 1998, J. Biol. Chem. 273: 2
1769-21776).

[0019] Methods for cloning antibodies and expressing them in bacteria are well known in the art. For example, as described in more detail in the Examples, kits are commercially available for cloning antibodies into phagemids for expression in E. coli.
In a preferred embodiment of the invention, selected antibodies are cloned using such kits and protocols set forth herein and tested in vitro for the ability to neutralize the target pathogen. The cloned antibody is then adapted for transformation and expression in the selected commensal bacteria. In another preferred embodiment, the gene encoding the recombinant antibody isolated in the first round of selection is subjected to random mutagenesis using degenerate primers for both heavy and light chain antigen binding domains, Isolates with increased binding affinity for neutralization sites on pathogens of P. et al. (Pini et al., 1998, J. Biol. Chem.
273: 21769-21776).

After isolation, the sequence of the antibody protein may be changed to an extension to increase binding affinity for neutralizing sites on the pathogen and / or stability in the GI tract. In a preferred embodiment, a residue in the linker sequence between the _VH and _VL domains is mutated to a cysteine residue, forming a disulfide bond between the rAb monomers to yield a dimeric rAb with increased binding affinity. In another preferred embodiment, the codon in the protease digestion site is changed to a structurally neutral amino acid to eliminate the digestion site. In this embodiment, if the site is within one of the known antigen recognition domains on the recombinant antibody, the mutation is combined with reselection to lose the protease site but retain high antigen binding affinity Identify mutants that have

An antibody that is immunologically specific for the selected antigen can also be administered in combination with a recombinant commensal bacterium that expresses the antibody. These antibodies may be isolated or may be administered while present in the antibody-expressing cells. An isolated antibody may be secreted by a recombinant commensal, or it may be a purified natural or recombinant antibody. Administration of an antibody in combination with a recombinant commensal bacterium that expresses the antibody has the advantage of rapidly establishing immune protection, while the commensal bacterium colonizes the gastrointestinal tract and begins to secrete the recombinant antibody.

Symbiotic bacteria suitable for the present invention include non-pathogenic bacteria, preferably physiologically beneficial bacteria that colonize or bind or settle in the gastrointestinal tract of the selected subject or human. . Preferably, these bacteria are species of the genus Lactobacillus. However, Lactococcus, Bifidobacteria, Eubacteria and non-pathogenic Escherichia coli strains (Gibson and Robertfroid, J. Nutrition 125: 1401-1)
412, 1995) may be used. A particularly interesting nonpathogenic Escherichia coli is E. coli strain F18 (Cohen et al., 1983, Inf.
ect. Immun. 40: 62-69; Myhal et al., 1982, Eur. J. Clin. Microbial.
86-192), E. coli Nissle 1917 strain (E. coli DSM 6601, Mutafluor ^R ) (Lodinov
a-adnikova and Sonnenborn, 1997, Biol. Neonate 1: 224-232, incorporated herein by reference).

The selection of symbiotic strains for use in the present invention may be determined by a combination of factors that include the normal site of infection of the pathogen and the normal site of colonization of antibody-producing bacteria. For example, one way to neutralize rotavirus infection, which typically infects the GI epithelium 20-30% later in the GI tract, is to secrete anti-rotavirus recombinant antibodies. And colonize the lower GI tract to secrete antibodies,
Providing a mixture of E. coli F18 or K12 with overlapping rotavirus infection sites and Lactobacillus braevis cells secreting anti-rotavirus recombinant antibodies that secrete antibodies by colonizing the very top of the GI tract. Is included. Without limiting the function of this embodiment to any one mode, using this approach, the ingested virus was bound to the antibody early in the GI tract and colonized at the normal site of infection. F18 E. coli cells further enhance antibody concentration.

Symbiotic bacterial strains suitable for delivering previously synthesized antibodies are those that do not secrete recombinant antibody, but rather sequester the antibody within the cell membrane or cell wall. Such strains include laboratory strains of E. coli derived from the original symbiotic strain E. coli K12,
Not limited to this. Advantageously, these E. coli laboratory strains, such as HB2151, are selected for their weakened cell walls. Without limiting the function of this embodiment to any one mode, those E. c expressing at least one recombinant antibody
The oli strain will lyse in the upper GI tract when exposed to normal bile salts. Upon lysis, these cells will release their contents of the recombinant antibody into the GI lumen, where it will mix with other rumen components. This embodiment provides an inexpensive method for delivering pure recombinant antibodies to the upper GI tract.

[0025] Lactobacilli are a large and diverse group of Gram-positive bacilli that are common components of the 25 normal endemic flora of humans and other animals.
Lactobacillus is considered "health-promoting," but is rarely pathogenic in any case and is a good candidate for use in the present invention. In fact,
Lactobacillus has been used as a vaccine delivery vehicle, ie, to express antigens in the GI tract of immunologically functional individuals for the purpose of eliciting an immune response (eg, Gram-Positive Bacteria as Vaccine Vehicles for Mucos
al Immunization, (G. Pozzi & JM Wells, eds.), Landes Bioscience, 1997
See Chapter 6 by Rush et al.).

Different strains and species of Lactobacillus can colonize or bind differently to the GI tract of individual animals. Two approaches can be used to select an appropriate strain to deliver the antibody to a particular animal species (including humans): (1) strains known to be capable of colonizing mucosal surfaces of animals. (Eg, Lactobacillus GG is known to colonize the human GI tract); and / or (2) has a limited interaction with the host GI tract,
Selection of strains found naturally in food that can nevertheless live in the GI tract for a considerable amount of time.

[0027] The fate of live Lactobacillus in the mammalian GI system depends on many factors. 1
Extreme bacterial cells mix easily with the undigested fiber of fed food, travel along the GI system, and secrete antibodies into the mucosal matrix that lines the inner surface of the GI epithelium. In this situation, there is little colony formation by Lactobacillus cells. Conversely, extreme cells find host sites in the mucosal matrix, remain there and replicate (colonize), and continue to secrete antibodies into the surrounding mucosal matrix. The factors that determine whether a cell can colonize, or merely settle in a site in the GI system, are not well understood. Some parameters that affect the ability of commensal bacteria to colonize include: pH of individual GI sites (Acidofels means eosinophilic, these strains adapt to low gastric and duodenal pH). The ability to compete for nutrition with bacterial strains; and other types of bacteria encountered on the mucosal surface of the GI epithelium. Transient or colony forming strains can exert their intended antibody secretion function, so that the appropriate site can be selected based on how long such secretion is required in the treated individual. Alternatively, a combination of different strains, some of which are transient strains and some of which are colonizing strains, can be used to obtain short and long term immune protection.

A number of Lactobacillus species have been shown to be applicable for transformation as well as expression / secretion of recombinant proteins. In general, Lactobacillus can be modified through two mechanisms: (1) by introducing an ectopic plasmid carrying the foreign gene; or (2) stably integrating the foreign gene into the genome. By that. One advantage over the latter approach is that potential losses of foreign genes that can result from plasmid shedding while the bacterial culture is growing are avoided. Thus, the potential low expression associated with stable integration of a foreign gene can be offset by more stable retention of that gene and the ability of the bacterium to express and secrete recombinant antibodies over time.

There are many examples in the art for cloning and expression vectors designed for use in Lactobacillus species. Besides, especially L.
casei, L. braevis, L. plantarum, L. paracasei, L. acidophilus, L. ferme
Successful introduction and expression of foreign genetic material in various Lactobacillus species, including ntum and L. zear, have been described in the art (Rush et al., supra.
1997; Rush et al., Microbiol. Biotechnol. 47: 537-542, 1997; Hols et al.
., Microbiology 143: 2733-2741, 1997). Example 3 below describes a vector design and protocol for transformation of L. gasseri that colonizes the murine upper GI tract. This protocol follows standard protocols for transformation of Lactobacillus by electroporation (eg, Auku
rst et al., Chapter 20 in Methods in Molecular Biology, Vol. 47, (JA
Nickoloff, ed.), Humana Press Inc., Totowa, NJ).

[0030] The stable integration of foreign genes into the Lactobacillus genome via the temperate phage mv4 has also been described (Dupont et al., J. Bact. 179: 586-595,
1995; Avuray et al., J. Bact. 179: 1837-1845, 1997). Originally, this bacteriophage was present in L. delbuckii subsp.bulgaricus, and L. plantarum,
It has been modified for use in integrating foreign genes into the genome of L. casei and L. lactis.

Vectors currently available in the art for the transformation of Lactobacillus were not designed for uptake by humans or animals. In particular, most vectors use antibiotic resistance as a selectable marker. Since it is not desirable to introduce antibiotic resistance into animal or human patients, currently available vectors should be modified to include alternative means of choice, such as a color indicator. Selection means that do not rely on antibiotic resistance are known in the art and can be used.

Once commensal bacteria expressing recombinant antibodies have been obtained, they are used to protect against invasion by the selected pathogen in newborns or other individuals in need of such treatment. The mode of administration is by oral or intranasal injection or by ingestion (alone or in the subject's food or meal). For use in newborns, bacteria are preferably introduced into the GI tract at birth and then periodically until the infant's immune system is functional. In this regard, La that is currently widely marketed
It should be noted that ctobacillus acidophilus is sold as a gel capsule containing a lyophilized mixture of a solid carrier such as bacterial cells and mannitol. If the gel capsule is consumed with the liquid, the lyophilized cells are rehydrated and become viable clonogenic bacteria. Thus, in one preferred embodiment, the Lactobacillus cells are added to the newborn milk substitute by spraying as a powdered lyophilized formulation. The rehydrated live Ralto Bacillus cells then settle and colonize sites throughout the upper and lower gastrointestinal tract and constitutively secrete recombinant antibodies to provide protection against the appropriate pathogen.

For other individuals, the bacteria should be administered as soon as immunosuppression or exposure to extreme GI pathogens occurs, or (if possible) before. For example,
For the defense of troops dispatched to the war zone, symbiotic strains of the present invention that settle in the GI tract can be fed continuously as gel capsules as long as the potential threat remains. After ingestion of such a capsule, the symbiotic cells are slowly diluted from the GI tract. For infrequent but recurring water pollution in metropolitan cities, for example by Cryptosporidium or Salmonella, rather than eliminating the infection extensively and slowly as if the pathogens were controlled by various means (if any), The commensal bacteria of the present invention are used to rapidly stop a wide range of disease states associated with such healthy public infections. In the case of Cryptosporidium, the treatment method of the present invention is particularly important, since chemotherapy is not currently available. In this context, the distribution and use of commensal cells secreting appropriate antibodies can provide immediate immune protection to the population, thereby rapidly reducing the number of infectious hosts and greatly reducing Significantly accelerate the disappearance of epidemics from cities.

For ease of administration and uniformity of dosage, the compositions of the present invention may be formulated in a single dosage form. Dosage form as used herein refers to physically discrete units of the composition suitable for the patient to be treated. A serving should contain a fixed amount of the active ingredient calculated to achieve the desired effect in relation to the selected pharmaceutical carrier. Suitable one
Methods for determining dosage forms are well known to those skilled in the art (Lodinova-adnikova and So
nnenborn, 1997, Biology of the Neonate 71: 224-232).

The composition comprising the commensal microorganism expressing the selected antibody is preferably administered orally, with or without supplemental antibodies. The bacteria and the antibodies that may be administered may be hydrated or lyophilized. There are several options for prescribing these boluses, tablets, capsules, solutions, syrups, elixirs,
Suspensions, lumps, gels and powders are included. The composition may include diluents, disintegrants, desiccants, coatings and colorings. Using an enteric coating,
Dissolution may be delayed until the tablet has passed through the intestine.

[0036] The composition of the present invention may further comprise a food product. A bolus of lyophilized and processed commensal bacteria, which is generally tasteless, may be dispersed in food to facilitate its consumption by adult livestock or adults. Freeze-dried bacteria have a shelf life of about one year in a refrigerator and about three to four months when stored without refrigeration. In the case of untreated commensal bacteria, the recombinant antibody-expressing bacteria are used to produce foods such as yogurt or kefil, or added to the finished food product to provide the desired protection when the food is consumed. It may be performed.

For both domestic and human newborns, the most common method for administering the compositions of the present invention is to use compositions containing lyophilized bacteria, lactose or maltodextrin as a carrier and / or filler. Orally in a milk replacer (for domestic animals) or a formula (for humans) to be fed to newborns and agitated multiple times daily. For adults, gel capsules or compressed tablets containing the same lyophilized bacteria and carrier / filler mixture used for neonates are:
It is considered to be the most effective way to consume commensal bacteria.

The examples in which lyophilized commensal bacteria are administered as described above are well known in the art and are commonly used by health food consumers in today's real world.
According to the manufacturer's recommendations, after a 10-day course of antibiotics, adults should consume 9-90 × 10 ⁹ colony forming units of commensal bacteria per day to re-colonize the human gastrointestinal tract. Is suggested. In commercial preparations of freeze-dried bacteria, there are about 10 ¹⁰ colony forming units per gram of dry fibrous powder. Therefore, as three gel capsules, powder or infant formula, which is stirred into food,
Alternatively, the daily dose can easily be consumed as a standard ingredient in food products such as yogurt.

In a preferred embodiment, the commensal microbial composition is administered to a newborn while the animal is at immunological risk. In ^general, administered twice daily bolus containing 3x10 ⁹ ~40x10 9 colony forming units. Depending on the relative binding affinity of the individual antibodies, add 0.1 mg to 25.0 mg of antibody to the neonatal bolls.
The concentration may be mg / kg body weight / day.

In another preferred embodiment, the commensal bacterial composition is administered three times daily as a boll containing 12 × 10 ^{9 to} 80 × 10 ⁹ colony forming units for at least 10 days. The actual duration of the administration will be largely determined by the duration of the external pathogen threat. The composition of the symbiotic substance to be administered to an adult will be the same as described above for the neonate, and the method of administration will include capsules, compressed tablets and dispersion in food. Lyophilized bacterial formulations up to 10 ¹⁰ colony forming units per gram may be prepared and then diluted with a filler / carrier such as maltodextrin for ease of handling.

In humans, the initial dose of commensal bacteria and antibodies is based on body weight,
The dose has been optimized in previous studies using research animals and using clinical studies in livestock. Based on the data from the experiments in mice shown in Example 4, doses of monoclonal antibodies in the range of 0.1-2.5 mg / kg body weight per day indicated that the desired GI infection of rotavirus or other pathogens For livestock or humans is estimated to be a protective dose. For recombinant antibodies with lower binding affinity, this estimated dose will be higher, for example 1.0-25 mg / kg body weight per day.

A suitable dose will be one that diminishes the signs of infection and / or protects against infection. Over time, the level of antibody in the stool sample can be monitored to determine the efficacy of the administration.

The following examples are provided to describe the invention in more detail. The examples should not be construed as limiting the invention. Unless otherwise specified, Sambrook et al., Molec
ular Cloning, Cold Spring Harbor Laboratory (1989) (Sambrook et al.) or Ausbel et al. (eds) Current Protocols in Molecular Biology, John Wiley
Use general cloning, biochemical and molecular biological methods as set forth in & Sons (1999) ("Ausbel et al.").

Example 1 Preparation of Anti-Rotavirus Antibodies for Expression and Secretion in E. coli In this example, we describe the cloning of a mouse anti-rotavirus monoclonal antibody in E. coli. This protocol is based on the commercial kit "Reco
mbinant Phage Antibody System ”(Pharmacia Biotech, Piscataway, New Jers
ey) and essentially according to the manufacturer's instructions.

A murine hybridoma cell line expressing the rotavirus serotype G3-neutralizing monoclonal antibody (# 159) was a kind gift of HB Greeberg, Stanford University. A culture of the # 159 cell line containing 〜5 × 10 ⁶ viable cells was prepared. QuickPrep® mRNA Purification Kit (Pharmacia, Piscat) according to manufacturer's instructions
Messenger RNA was isolated from the cultured cells using NJ away.

The mRNA was ethanol precipitated, resuspended in 20 μl of RNase-free water and heated to remove secondary structure. Next, first strand cDNA synthesis was performed according to the manufacturer's instructions. Using the first-chain antibody cDNA as a template for PCR amplification, appropriate amounts of antibody heavy and light chain DNA were obtained for cloning. Using primers that specifically hybridize to the appropriate DNAs encoding the heavy and light chains,
This was done with a standard PCR protocol (indicated by the manufacturer's instructions).
The amplified heavy and light chain PCR fragments were separated from the other components of the mixture by agarose gel electrophoresis, then the separated bands were excised and the fragments eluted from the agarose. The purified product was then quantified on a gel.

The antibody heavy and light chain DNAs were ligated into single chains using the linker DNA segments provided by the manufacturer. The resulting ligated DNA segment (referred to as ScFv DNA in the manufacturer's instructions) was amplified by PCR using primers containing either NotI or SfiI restriction sites. The final product was separated from the other components of the PCR mixture by centrifugation in a MicroSpin column, after which the ScFv DNA segment was quantified.

The ScFv DNA was digested with NotI and SfiI and ligated into the similarly digested phagemid vector pCANTAB 5E (attached to the kit). The phagemid displays the recombinant antibody domain. This is because the recombination domain is fused to an external filamentous gene 3 protein.

[0050] Competent E. coli TG1 cells were then transformed with the pCANTAB 5E vector containing the antibody ScFv insert. Two clones, clone 11 and clone 22, were selected for further study. The ７750 bp SfiI-NotI rAb insert (SEQ ID NO: 1; FIG. 1 and SEQ ID NO: 2; FIG. 2) of clone 11 and clone 22 were sequenced. TG1 cells contain a stop codon suppressor that keeps some of the recombinant antibodies bound to the phage. Recombinant phage were recovered from transformed E. coli according to the manufacturer's instructions.

[0050] Immunoblotting showed acidified recombinant antibody levels from E. coli clones 11 and 22 in the medium, and the rAb fraction was purified from the anti-E immunoaffinity column (FIG. 3). Cultures of HB2131 were induced with 1 mM IPTG for 4 hours. The cells were pelleted by centrifugation and a 1 ml aliquot of the medium was precipitated with 10% TCA on ice. Neutralize protein with Tris base, SD
The sample was dissolved in S sample buffer and electrophoresed on a 4-12% polyacrylamide gel. Transfer protein to PVDF membrane. Anti-E conjugated to horseradish peroxidase
Probed with tag antibody. Bands were colored using ABTS reagent. FIG. 3 shows that 1 ml of bacterial medium contains about 0.3 μg of the recombinant antibody. There are ２2 × 10 ⁹ bacteria in 1 ml of this culture, with 0.1 per cell per hour.
It is estimated that 2 pg of the recombinant antibody was produced.

Recombinant phage were tested for the expression of anti-rotavirus antibodies by immunological interaction of the native rotavirus or murine Mab with a specific epitope (if known). After confirming this, E. c
The oli may be reinfected. E. coli strain HB2151 (without stop codon repressor) was used for the production of soluble recombinant anti-rotavirus antibodies according to the manufacturer's instructions. The soluble rAb was purified on an anti-E tag immunoaffinity column according to the manufacturer's instructions (FIG. 4). 50 ml of bacterial medium of clone 11 cells induced by IPTG was applied to the column, the column was washed and then eluted with 0.1 M glycine (pH 3.0).

In connection with the 30 steps of panning and confirmation described above, we used RR in tissue culture.
MA-104 cells, host cells capable of replicating V2-1 rotavirus, were obtained. MA-104 cells are infected with the RRV2-1 rotavirus, causing lytic virus disruption in a tissue culture dish, and then a media supernatant containing virus particles is obtained. The virus-containing environment was subjected to both fluorocarbon extraction and sucrose gradient centrifugation to obtain a highly concentrated virus stock, which was then titrated and used for MA-104 monolayer culture in a standard plaque formation assay. Was used to determine the number of infectious virus particles in 1 ml. After one week of culture, the monolayers were stained with crystal violet and these plaques were counted by counting the resulting clear plaques. The number of clear plaques is equal to the number of infectious virus particles applied to the plate. Typically, virus stocks exceeded 1 × 10 ⁹ plaque forming units (pfu) per ml.

[0053] The titrated tissue culture supernatant is used for at least four purposes. First, in the initial isolation of clones of E. coli cells expressing recombinant antibodies to rotavirus, there may be slight differences between the clones with regard to the exact specificity and binding properties of the resulting recombinant antibodies. Virus particles are adsorbed to the bottom of a multi-well plate to identify E. coli clones that produce recombinant antibodies with the most desirable binding properties, and then are known to produce recombinant antibodies
Medium containing individual clones of E. coli cells is added. At this stage, the recombinant antibody remains fused to the end of the phage protein extended from the E. coli cell surface. Thus, by binding the recombinant antibody to the adsorbed rotavirus,
That is, by panning, a recombinant gene encoding an E. coli cell and a recombinant antibody can be recovered.

Second, in the step of selecting a soluble antibody, in order to select a soluble recombinant antibody having desirable properties with respect to the binding of rotavirus that is suspended but not adsorbed to the plastic surface, Use a more sophisticated method.
Here, detectably labeled (eg, biotinylated) rotavirus particles are added to a suspension of the individual recombinant antibodies. Where the antibody is secreted from Lactobacillus cells in the animal intestinal lumen, the conditions of the binding assay can be altered depending on the choice of recombinant antibody having particularly desirable binding characteristics. For example, the conditions may be rotavirus or ETEC E. coli at low pH typical of food leaving the stomach.
The ability to bind to the K99 antigen of E. coli may be used. In practice, panning can be performed in the presence of a dilution of the already sterilized GI lumen content to accurately reflect the conditions under which antigen-recombinant antibody binding occurs. The binding of the soluble recombinant antibody to the biotinylated rotavirus particles can be demonstrated by adding avidin-coated beads to bind to the biotinylated rotavirus and then pelleting. The resuspended beads are incubated with, for example, a fluorescein-labeled anti-mouse antibody. If the primary recombinant antibody binds to the rotavirus particles, the fluorescein-labeled secondary antibody binds and is monitored using a standard 96-well fluorescence monitor.

Third, the addition of a known number of plaque-forming units from the RRV stock to the MA-104 monolayer to form a known number of transparent plaques per tissue culture well, thereby neutralizing the ability of the recombinant antibody to neutralize Can be screened. Virus M
Prior to addition to the A-104 supernatant, dilutions of individual recombinant antibodies are preincubated with the titrated virus supernatant. A decrease in the number of clear plaques indicates the ability of the recombinant antibody to neutralize natural host cell binding and infection. In a first step of such a screen, test cells, such as MA-104 cells, are tested. These results are used to estimate the neutralizing effect of the antibody on normal GI epithelial cells of the host organism. In the case of the RRV2-1 strain of the virus, normal host organisms, ie, animals that develop a severe GI disorder when infected, include at least mice, humans and monkeys.

Fourth, in animal model systems, bacteria that secrete recombinant antibodies (the first laboratory symbiotic E. coli strains (HB2151, F-18, Nissle 1917) and then the Lactobacillus strain, eg, mouse Lactobacillus gass known to colonize in
eri) and to eat the mouse, then aliquots of titrated virus - ie aliquot of virus-containing supernatant (typically 10 7 ^pfu) given to newborn mice, antibody secretion bacteria in newborn mice Assess whether and to what extent diarrheal infections are suppressed or eliminated. Mice are scored for the presence of severe RRV infection by gently holding the abdomen and examining the anal area for the presence of yellow watery stools or the presence of yellow stools in the last 5 cm of the rectum.

Example 2 Modification of DNA Encoding Recombinant Anti-Rotavirus Antibodies for Expression and Secretion in Lactobacillus Using SvFc DNA encoding the anti-rotavirus described in Example 1 in Lactobacillus Produce a plasmid for transformation and expression. Lactobacillus expression vector pCMR102 (Rush et al., Microbial. Bi
otechnol. 47: 537-542, 1996) is used as the basic plasmid. This plasmid is a derivative of plasmid pZN17 and has a broad host range of Lactobacillus la
Includes a replicon of the ctis plasmid pSH71, E. coli and Lactobacillus l
It is a low copy number plasmid in both actis. In addition, the pCMR102 plasmid contains a synthetic derivative of protein A, EZZ, from Staphylococcus aureus. The pCMR102 plasmid has been shown to direct the production and export of heterologous proteins in several Lactobacillus species (Rush et al., 1996 supra).

MOMP V of pCMR102 by restriction digestion with EcoRI and PstI
The segment encoding D4 is excised from the plasmid and replaced with ScFv DNA encoding a rotavirus antibody that has been terminally modified to include the appropriate restriction sites. The new plasmid contains a DNA segment encoding an in-frame fusion protein of an anti-rotavirus antibody and an EZZ protein.

Once efficient expression and secretion of the recombinant antibody has been achieved using the gene encoded by the plasmid, the purified plasmid DNA is digested with appropriate restriction enzymes and intact, containing the 5 ′ and 3 ′ regulatory sequences. The coding sequence for the DNA cassette, the recombinant antibody, and the gene product (a gene product that allows for colorimetric or immunological screening of bacterial colonies that synthesize the encoded gene product) is removed. Under conditions that have already been optimized, the expression cassette is electroporated into a suitable strain of symbiotic host cell that grows logarithmically over time. The cells are then plated under non-selective conditions and a number of commensal bacterial colonies are screened for (1) a colony that synthesizes and secretes recombinant antibodies, and / or (2)
) Detecting colonies expressing the originally linked gene, allowing for colorimetric screening.

The steps of transferring the above DNA fragment into bacterial cells and then allowing the cells to grow to allow the integration of the gene expression cassette into a neutral or preferred site of the selected commensal bacterium are described in two parts below. Performed for reasons: (1) to ensure that bacteria do not have drug resistance genes in them when they are released and consumed as products by healthy individuals; and (2) on ectopic plasmids. To significantly reduce or eliminate the possibility of recombinant antibody gene efflux from the bacterial genome as compared to placing the recombinant antibody gene. DN into a preferred site in the Lactobacillus genome to allow sufficient cell growth without loss of the recombinant gene
For the introduction of a recombinant gene on the A fragment, methods described in the literature are available (eg, Raya et al., J. Bact. 174: 5584-5592, 1992; DuPont et al.).
., J. Bact. 177: 586-595, 1995). Such a method would be equally useful for other commensal bacteria.

Example 3 Transformation of Lactobacillus gasseri Lactobacillus species is suitable for transformation by electroporation. This fruit
In the example, we devised a process devised for the transformation of Lactobacillus gasseri.
Explain the protocol. Medium: 1) MRS broth: 10.0 g peptone, 8.0 g meat extract, 4.0 g yeast extract
, 20.0 g glucose, 1 ml monooleate (Tween 80), 2.0 g K
₂HPO₄5.0 g sodium acetate 3H₂O, 2.0g (NH₃)₃−Sito
Rate, 0.2g MgSO₄7H₂O, 0.05g MnSO₄4H₂O, distillation
Add water to make 1L. 2) MRS plate: solidify MRS broth on 1.5% agar. 3) MRSSM: 0.5M sucrose, 0.1M MgCl₂Containing MRS
. Electrophoresis solution: 1) SM: 326 g sucrose (952 mM), 0.71 g MgCl₂6H₂
O (3.5 mM) and distilled water are added to make 1 L. 2) DNA: Plasmid pGK12 was replaced with TE (10 mM Tris-HCl, 1 m
M EDTA, pH 7.5) to make 0.10-1.0 pg / μL. Communicating
The ligation mixture should be ethanol precipitated, washed and dissolved in TE before electrophoresis
. Method: 100 ml of MRS or 25 ml of L. gasseri preculture in logarithmic growth phase
Glycine supplemented MRS was inoculated. A to inoculate₆₀₀= 0.25. A₆₀₀ Cultures were cultured at 30 ° C. until = 0.6. Minimum speed (5 minutes at 1500g
The cells were harvested by centrifugation in (1), and the supernatant was decanted. Cells are treated with 100 ml SM (alternatively 100 ml MgCl 2₂Resuspend in
, Then washed and repelleted as described above. 100 ml of pellet
Resuspend in SM, or alternatively 30% PEG, then wash,
And resuspended. The cells were resuspended in 1 ml SM (alternatively 30% PEG). 10⁹~ 1
0¹⁰Aliquots containing 10 ml / ml were collected for microporation for electroporation.
Moved to the lab. DNA in 1/20 volume of the cell suspension was added just before electroporation.
Was. The mixture was ice cold electroporated with a 2 mm electrode gap.
Moved to bet. Different electrical pulses were given as shown in Table 1 below. Immediately after the discharge, 1 ml of MRSSM was added to the cuvette, and the resulting diluted cell suspension was obtained.
Transfer the solution to a microfuge tube and incubate at 30 ° C for 2 hours.
It was cubified. Undiluted and serial dilutions of the cell suspension are combined with the appropriate components
(Eg, antibiotics, color indicator) spread on MRS plate containing
. Plates were incubated for 3 days at 30 ° C. and colonies of transformed cells were removed.
Propagated.

The results of electroporation of L. gasseri are shown in Table 1.

Table 1: Electroporation of pGK12 plasmid into L. gasseri 100-5 strain a. 5.0 pg / ml chloramphenicol was used for selection. b. A cuvette with a 2 mm electrode gap was used for electroporation. c. The resistance setting was the only variable parameter, the other settings were: voltage 1.5 kV and capacitance 25 pF. d. 3 pg of pGK12 plasmid and 100 pl of 100-5 cells were used for all electroporations. e. No colonies were found on negative control plates consisting of 100-5 cells that were not mixed with pGK12 but were subjected to the same electroporation conditions as above.

Example 4 Use of Rotavirus Antibodies to Immunize Neonates in Mice Oral administration of rotavirus monoclonal antibody M159 prevents the onset of signs of diarrhea in mice. A 7-day-old mouse infant was given a stomach bolus using a feeding tube, followed by oral rotavirus administration. 50μg to 1μ
A gastric bolus containing less than g of M159 rotavirus antibody conferred complete protection against rotavirus (7.5 × 10 ⁶ pfu) given immediately thereafter (FIG. 5). Unprotected mouse infants showed 100% infection on day 3. 25 μg of M15
A stomach bolus of 9 antibodies was obtained by oral administration of rotavirus (7.5 hours later).
x10 ⁶ pfu), while control mouse infants received 1 day 3
It showed 00% infection (FIG. 6).

The present invention is not limited to the embodiments described and illustrated above, but can be modified and modified without departing from the scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1. 図 750 in pCANTAB 5E cloning / expression vector.
1 shows the DNA sequence analysis of the base pair SfiI-NotI insert (clone 11) (SEQ ID NO: 1). The open reading frame in the insert is M15
Contains the mouse variable heavy (V _H ) and variable light (V _L ) domains found in monoclonal antibodies secreted by 9 hybridomas. The open reading frame also contains a 13 amino acid E-tag domain that allows for immunodetection and immunoaffinity purification of the rAb molecule. The SfiI and NotI sites are r
The _VH domain, which defines the Ab insert region and exhibits 97-99% sequence identity to other mouse heavy chain genes, the linker region, and 9% to other mouse light chain genes.
All _VL domains showing 5-99% sequence identity as well as an E-tag domain of 13 amino acids are shown.

FIG. 2. ７750 in pCANTAB 5E cloning / expression vector.
1 shows the DNA sequence analysis of the base pair SfiI-NotI insert (clone 22) (SEQ ID NO: 2). Two codons in the linker region, as well as the encoded r
Identical to the sequence found in clone 11, except for two codons in the _VH region that can slightly alter Ab recognition and binding affinity. The SfiI and NotI sites define the rAB insert region, the _VH domain showing 97-99% sequence identity to other mouse heavy chain genes, the linker region, and 95-99 for other mouse light chain genes. _VL domains with 13% sequence identity and 13
All amino acid E-tag domains are shown.

FIG. 3 shows Western blot analysis of recombinant antibody levels in bacterial media taken from the induction media of clones 11 and 22. Also shown is a purified recombinant antibody after immunoaffinity chromatography on a column to which an anti-E tag antibody has been attached. The recombinant antibodies in the blot are visualized by probing the blot with an anti-E tag antibody conjugated to horseradish peroxidase.
Color development is performed using ABTS reagent. Lane 1 contains 1 μg of the recombinant antibody standard. Lane 2 contains 1 ml of TCA precipitated Clone 11 medium. Lane 3 contains 1 ml of TCA precipitated Clone 22 medium. Lane 4 contains a partial sample of purified clone 11 rAb eluted from the anti-E tag immunoaffinity column.

FIG. 4. Purification of recombinant antibodies from the culture medium of clone 11 using an anti-E tag immunoaffinity column. An aliquot of fraction 6 was analyzed by Western blot. It is shown in lane 4 of the immunoblot in FIG.

FIG. 5. After oral administration of rotavirus (10 ⁷ pfu), 0 μg, 2 μg
The percentage of neonatal mice with diarrhea after administration of a gastric bolus containing 5 μg, 8 μg and 1 μg of M159 rotavirus monoclonal antibody is shown.

FIG. 6. Percentage of neonatal mice with diarrhea 3 days after administration of a gastric bolus containing 25 μg of M159 rotavirus antibody at 0, 8, 16, and 25 hours after oral administration of rotavirus. Is shown. The control treatment consisted of a gastric bolus without M159 rotavirus antibody 25 hours after oral administration of rotavirus.

[Procedure amendment]

[Submission date] May 28, 2001 (2001.5.28)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 A61P 31/04 31/12 31/12 // A61K 39/395 A61K 39/395 N C12N 1 / 21 C12N 1/21 15/09 (C12N 1/21 ZNA C12R 1: 225) (C12N 1/21 C12R 1: 225) C12N 15/00 ZNAA (C12N 15/09 A C12R 1: 225) (81) Designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), E (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Jeffrey J. Letchworth United States 53711 Wadden Hill Drive 1022 Madison, Wisconsin 5372 (72) Invention Gerald Sea Muller United States 53705 Madison, Wisconsin Plymouth Circle 3926 (72) Inventor Adam Kay Savage United States 53703 Madison, Wisconsin North Blair Street 311 (72) Inventor Deborah Lou United States 53706 Wisconsin Dison, University Avenue 1400 F-term (reference) 4B024 AA01 BA50 BA51 CA04 DA05 DA06 EA03 GA14 4B065 AA26 AA30 AB01 AC01 AC20 BA03 CA25 CA44 4C084 AA13 CA53 MA52 NA14 ZA662 ZB082 ZB092 4C085 AA02 A08 AA13 BC BC59 MA52 NA14 ZA66 ZB33 ZB35

Claims (27)

[Claims] 1. A composition for supplementing or replacing an immune response against one or more selected pathogens in an individual in need of treatment, said composition comprising immunologically against at least one selected pathogen. A composition comprising a commensal microorganism genetically modified to express a recombinant antibody specific for the microorganism. 2. The composition of claim 1, further comprising a supplementary bolus of an antibody immunologically specific for one or more pathogens. 3. The composition of claim 2, wherein the antibody of the supplemental bolus is immunologically specific for the same pathogen that the antibody produced by the commensal microorganism is immunologically specific. 4. The antibody of the supplemental bolus is immunologically specific for a different pathogen from which the antibody produced by the commensal microorganism is immunologically specific.
3. The composition of claim 2. 5. The composition of claim 1, wherein the pathogen is a gastrointestinal pathogen. 6. The pathogen is rotavirus, calicivirus, reovirus,
Coronavirus, enterovirus, adenovirus, Norwalk virus, enterotoxigenic Escherichia coli, Campylobacter jejuni, Yersinia enterocolitic
a, Clostridium spp., Vibrio cholera Cryptosporidium spp., Giardia lambl
6. The composition of claim 5, wherein the composition is selected from the group consisting of ia, Entamoeba histolytica, Helicobacter pylori, and Isospora belli. 7. The composition of claim 1, wherein the pathogen is a gastrointestinal pathogen. 8. The pathogen may be a coxsackievirus, poliovirus, hepatitis A virus, Salmonella and Shigella spp., Listeria monocytogenes and S
The composition of claim 7, wherein the composition is selected from the group consisting of trongyloides. 9. The symbiotic organism is Lactobacillus, Bifidobacteria, Eubacteria
2. The composition of claim 1, wherein the composition is selected from the group consisting of: a non-pathogenic strain of Escherichia coli, E. coli F18 and E. coli Nissle 1917 strain. 10. The composition of claim 1, wherein the symbiotic organism is of the genus Lactobacillus. 11. The Lactobacillus species is L. casei, L. plantarum, L. para
11. The composition of claim 10, wherein the composition is selected from the group consisting of casei, L. acidophilus, L. fermentum, L. zeae, and L. gasseri. 12. The composition of claim 1, wherein said recombinant antibody has been adapted for expression in bacteria. 13. The composition of claim 1, wherein said recombinant antibody is polyclonal. 14. The composition of claim 1, wherein said recombinant antibody is monoclonal. 15. The composition of claim 1, wherein the recombinant antibodies comprise a mixed population of antibodies immunologically specific for one pathogen. 16. The composition of claim 1, wherein the recombinant antibody comprises a mixed population of antibodies immunologically specific for one or more pathogens. 17. The composition of claim 1, wherein said composition comprises a plurality of symbiotic organisms, wherein each organism produces at least one recombinant antibody. 18. The composition of claim 1, wherein the commensal organism expresses a rotavirus neutralizing monoclonal antibody. 19. The composition of claim 1, further comprising at least one compound selected from the group consisting of lactose, maltodextrin and a desiccant. 20. A method for supplementing or replacing an immune response against one or more selected pathogens in an individual in need of treatment, comprising a period effective to confer an immune response against the selected pathogen, and Genetically, in a manner such that the commensal microorganism secretes the recombinant antibody into the individual's GI tract in an amount that is immunologically specific for one or more selected pathogens. A method comprising administering a chemically modified symbiotic bacterium to an individual. 21. The method of claim 20, further comprising administering a supplemental bolus of antibody immunologically specific for one or more selected pathogens. 22. The individual in need of treatment is selected from the group consisting of neonatal animals or humans, immunosuppressed or immunodeficient adults, and healthy individuals suddenly exposed to large amounts of one or more pathogens. 20 methods. 23. The individual is a newborn and the symbiotic bacterium is 3 × 10⁹~ 40x10 ⁹ 22. The method of claim 20, which is administered orally twice daily as a bolus containing colony forming units.
Method. 24. The method of claim 23, wherein the bolus further comprises at least one antibody immunologically specific for at least one pathogen from 0.1 to 25.0 mg / kg body weight / day. . 25. The individual is an adult, and the symbiotic bacterium is 12 × 10⁹~ 80x10 ⁹ 21. The method of claim 20, which is administered orally three times a day as a bolus containing colony forming units.
Method. 26. The method of claim 25, wherein the bolus further comprises at least one antibody immunologically specific for at least one pathogen from 0.1 to 25.0 mg / kg body weight / day. . 27. The method of administering the composition of claim 1, wherein the composition is administered orally.