This invention relates to methods of using conjugates of the capsular polysaccharide of Salmonella typhi, Vi, bound to the carrier Pseudomonas aeruginosa recombinant exoprotein A (rEPA) with a carboxylic acid dihydrazide linker, preferably an adipic acid dihydrazide (ADH) linker, and compositions of these conjugates, for eliciting serum antibody responses in humans, including responses which provide protection against, or reduce the severity of, S. typhi bacterial infections. The conjugates, and compositions thereof, are useful as vaccines to induce serum antibodies which are useful to prevent and/or treat illnesses caused by S. typhi. 
In developing countries, typhoid fever is common, serious, and increasingly difficult to treat because of resistance of the bacillus to antibiotics. [24, 63-66]. For example, more than 80% of Salmonella typhi from the Mekong Delta region of Vietnam are resistant to chloramphenicol and to ampicillin and even more expensive antibiotics such as ciprofloxacin. Typhoid fever has been thought of as a disease of mostly older children and young adults. In children less than 5 years of age, typhoid fever was often unrecognized due to atypical clinical symptoms, difficulties in drawing blood and less-than-optimal culture media. [66-69]. Similar to recent findings in other parts of Southeast Asia [70-72], a preliminary survey in 3 communes of the Dong Thap province of Vietnam showed that the annual attack rate of typhoid fever was highest among children less than 15 years of age: it was 413/100,000 in this age group and 358/100,000 for 2 to 4 year-olds. [73].
Unfortunately, it is unlikely that safe drinking water and foodstuffs will be available in many developing countries, especially in rural areas, in the near future. [24, 66, 74]. Control of typhoid fever by routine vaccination, especially in countries that endure high endemic rates of typhoid fever, has not been adopted because of the limitations of the three licensed vaccines (parenteral inactivated cellular vaccines, oral attenuated S. typhi Ty21a, and parenteral Vi polysaccharide). These vaccines confer only approximately 70% immunity to older children and adults but do not protect young children. [24, 1, 30, 75, 76].
Orally administered attenuated S. typhi Ty21a requires at least 3 doses, has a low rate of efficacy in areas with a high rate of typhoid fever and in travelers from developed countries and is not immunogenic in young children. Neither the protective antigens nor the vaccine-induced host immune responses have been identified which hinders improvement of the Ty21a vaccine.
Although effective in areas with high rates of typhoid fever, killed whole cell parenteral vaccines elicit a high rate of adverse reactions and have not been shown to be effective in young children. In 1952, Landy concluded that the protective antigen of cellular vaccines is the capsular polysaccharide (Vi) of S. typhi. 
In two randomized, double-blinded, vaccine-controlled clinical trials, one injection of Vi induced about 70% efficacy in xe2x89xa75 year-olds in the Kathmandu Valley of Nepal and in the Eastern Transvaal region of the Republic of South Africa: these regions had a high rate of endemic typhoid (0.4 to 1% per year) [1]. Recently, similar results were obtained by the Lanzhou Institute of Biologic Products in the People""s Republic of China [manuscript in preparation]. Vi is easily standardized. The World Health Organization has published requirements for Vi polysaccharide typhoid vaccine and this product is licensed in about 50 countries including the United States [59,60]. But Vi induces only short-lived antibody responses in children two to five years of age and does not elicit protective levels in children less than two years old: in adults, reinjection restores the level of vaccine-induced anti-Vi but does not elicit a booster response. These age-related and T-independent immunologic properties are similar to most other polysaccharide vaccines.
We proposed that it is the vaccine-induced serum IgG anti-Vi that confers immunity. Accordingly, the level of serum IgG anti-Vi should predict the efficacy of Vi vaccine. In order to improve its immunogenicity, Vi was conjugated to proteins using SPDP [51, 52, 54, 62]. The protein carriers for the SPDP linked conjugates included cholera toxin (CT), tetanus toxoid (TT), the B subunit of the heat-labile cholera-like enterotoxin (LT-B) of Escherichia coli and the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa (i.e., the nontoxic recombinant form of exotoxin from Pseudomonas aeruginosa (ETA) cloned into and secreted by E. coli). [Id.]. Recently, we employed another synthesis that treated rEPA with adipic acid dihydrazide (ADH) and bound the hydrazide derivative of rEPA (rEPA-AH) to Vi with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [31]. The safety and immunogenicity of the Vi-rEPA conjugates prepared either with N-succinimidyl-3-(2-pyridyl dithio) propionate (SPDP, Vi-rEPAI) or adipic acid dihydrazide (ADH, Vi-rEPAII) as linkers, were compared sequentially in adults, 5-14 year-olds and then 2-4 year olds in Vietnam. The data set forth in Example 5 herein demonstrate that the resultant conjugate (Vi-rEPA) both enhanced the immunogenicity of and conferred T-cell dependent properties to Vi. Vi-rEPA elicited a booster response in 2 to 4 year-olds with IgG anti-Vi levels approximately 3 times higher than those elicited by Vi in 5 to 14 year-olds. None of the vaccinees had a temperature  greater than 38.5xc2x0 C. or swelling  greater than 2.5 cm following injection. On the basis of these results, we initiated a double-blinded placebo-controlled randomized trial to determine the efficacy of Vi-rEPA in 2 to 5 year-old Vietnamese children, an age group for which there is yet no effective typhoid vaccine. The results of that efficacy trial are set forth in Example 6 herein.
It is an object of the invention to provide methods of using conjugates of the capsular polysaccharide of Salmonella typhi (Vi) bound to the carrier rEPA (as carrier protein) with a carboxylic acid dihydrazide linker, preferably an adipic acid dihydrazide (ADH) linker, and/or compositions thereof, for eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity of, bacterial infections. More particularly, it is an object of the invention to provide methods of using such conjugates, and/or compositions thereof, to induce serum antibodies against the capsular polysaccharide of S. typhi, called Vi. The conjugates, and compositions thereof, are useful as vaccines to induce serum antibodies which are useful to prevent typhoid fever.
It is also an object of the invention to provide antibodies which immunoreact with the Vi polysaccharide of S. typhi and/or the rEPA carrier, that are induced by these conjugates and/or compositions thereof. Such antibodies may be isolated, or may be provided in the form of serum containing these antibodies.
It is also an object of the invention to provide a method for the treatment or prevention of S. typhi infection in a mammal, by administration of compositions containing the antibodies of the invention, or serum containing the antibodies of the invention.
The invention also provides methods and kits for identifying, detecting, and/or diagnosing S. typhi infection or colonization using the antibodies which immunoreact with the Vi polysaccharide of S. typhi. The invention also relates to methods and kits for identifying, detecting and/or diagnosing the presence of P. aeruginosa and/or P. aeruginosa exotoxin A (ETA).
The Vi-rEPAII conjugates of this invention induce a strong initial IgG antibody response in humans. In this respect, they have a significant advantage over the Vi-rEPAI conjugates.
The invention provides methods of using conjugates of an S. typhi Vi polysaccharide which is covalently bound to the carrier rEPA with a dicarboxylic acid dihydrazide linker, preferably an adipic acid dihydrazide linker, and compositions thereof. The present invention also encompasses methods of using mixtures such S. typhi-rEPA conjugates and/or compositions thereof as part of a composition containing other immunogens, to form a multivalent vaccine for broad coverage against various pathogens. The S. typhi-rEPA conjugates, and/or compositions thereof, may also be administered concurrently with other vaccines, such as the DTP vaccine.
The invention also provides methods of using such S. typhi-rEPA conjugates, and/or compositions thereof, to induce in mammals, in particular, humans, the production of antibodies which immunoreact with the Vi polysaccharide of S. typhi. In the preferred embodiment, antibodies which immunoreact with ETA of P. aeruginosa are also produced. The antibodies which immunoreact with Vi of S. typhi may be useful for the identification, detection, and/or diagnosis of S. typhi colonization and/or infection. Antibodies against S. typhi may be useful to prevent and/or treat illnesses caused by S. typhi. Antibodies which immunoreact with ETA may be useful to prevent or treat illnesses caused by P. aeruginosa. 
Pharmaceutical compositions of this invention are capable, upon injection into a human, of inducing serum antibodies against S. typhi. In general, the exemplified Vi-rEPA conjugate vaccine of this invention using ADH as the linker (i.e., Vi-rEPAII) is capable of inducing serum IgG antibody levels which are statistically significantly higher than those induced by Vi alone or by Vi conjugated to rEPA using SPDP as the linker (i.e., Vi-rEPAI). The induction by the immunogen, in xe2x89xa780% of the immunized population, of a xe2x89xa78-fold increase in anti-Vi IgG at four to six weeks after a proscribed course of vaccination with the immunogen has been completed, is usually required for an effective vaccine against typhoid fever.
Preferably, the method of the invention is capable, upon injection into an adult human of an amount of Vi-rEPAII vaccine containing 25 xcexcg of S. typhi Vi polysaccharide, of inducing in the serum of the human a level of anti-Vi IgG antibody which, when measured six weeks after the injection, is at least about 48-fold higher than the anti-Vi IgG levels prior to injection.
Also preferably, the method of the invention is capable, upon injection into a five- to fourteen-year-old human of an amount of Vi-rEPAII vaccine composition containing 25 xcexcg of S. typhi Vi polysaccharide, of inducing in the serum of the human a level of anti-Vi IgG antibody which, when measured six weeks after the injection, is at least about 252-fold higher than the anti-Vi IgG levels prior to injection.
Also preferably, the method of the invention is capable, upon injection into a two- to four-year-old human of an amount of Vi-rEPAII vaccine composition containing 25 xcexcg of S. typhi Vi polysaccharide, of inducing in the serum of the human a level of anti-Vi IgG antibody which, when measured six weeks after the injection, is at least about 400-fold higher than the anti-Vi IgG levels prior to injection.
The Vi-rEPA vaccines of this invention are intended for active immunization for prevention of S. typhi infection, and for preparation of immune antibodies. The vaccines of this invention are designed to confer specific immunity against infection with S. typhi, and to induce antibodies specific to S. typhi Vi and ETA. The S. typhi conjugate vaccine is composed of non-toxic bacterial components, suitable for infants, children of all ages, and adults.
The methods of using the Vi-rEPA conjugates of this invention, and/or compositions thereof will be useful in increasing resistance to, preventing, ameliorating, and/or treating S. typhi infection in humans.
This invention also provides compositions, including but not limited to, mammalian serum, plasma, and immunoglobulin fractions, which contain antibodies which are immunoreactive with S. typhi Vi, and which preferably also contain antibodies which are immunoreactive with ETA. These antibodies and antibody compositions may be useful to prevent, treat, or ameliorate infection and disease caused by the microorganism. The invention also provides such antibodies in isolated form.
High titer anti-Vi sera, or antibodies isolated therefrom, may be used for therapeutic treatment for patients with S. typhi infection. Antibodies elicited by the Vi-rEPA conjugates of this invention may be used for the treatment of established S. typhi infections, and may also be useful in providing passive protection to an individual exposed to S. typhi. 
The present invention also provides diagnostic tests and/or kits for S. typhi infection and/or colonization, using the conjugates and/or antibodies of the present invention, or compositions thereof.
The invention is intended to be included in the routine immunization schedule of infants and children, and in individuals at risk for S. typhi infection. It is also planned to be used for intervention in epidemics caused by S. typhi. Additionally, it is may be used as a component of a multivalent vaccine for S. typhi and other pathogens, useful for example for the routine immunization of infants.
Vi is a linear homopolymer of xcex1(1xe2x86x924)-D-GalpA, which is N-acetylated at C-2 and O-acetylated at C-3.
As used herein, the terms xe2x80x9cimmunoreactxe2x80x9d and xe2x80x9cimmunoreactivityxe2x80x9d refer to specific binding between an antigen or antigenic determinant-containing molecule and a molecule having an antibody combining site, such as a whole antibody molecule or a portion thereof.
As used herein, the term xe2x80x9cantibodyxe2x80x9d refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art as Fab, Fabxe2x80x2, F(abxe2x80x2) and F(v), as well as chimeric antibody molecules.
Polymeric Carriers
Carriers are chosen to increase the immunogenicity of the polysaccharide and/or to raise antibodies against the carrier which are medically beneficial. Carriers that fulfill these criteria are well known in the art. A polymeric carrier can be a natural or a synthetic material containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, or carboxyl groups. Carrier can be water soluble or insoluble. The present invention concerns methods of using Vi conjugates with rEPA as a carrier.
Methods for Attaching Vi to rEPA
Methods for binding a polysaccharide to a protein, with or without a linking molecule, are well known in the art. See for example reference [8b], where 3 different methods for conjugating Shigella O-SP to tetanus toxoid are exemplified. See also, reference [31], which describes methods for conjugating S. typhi Vi and adipic hydrazide-derivatized protein.
In the present invention, attachment of the S. typhi Vi polysaccharide to a protein carrier is preferably accomplished by first coupling a dicarboxylic acid dihydrazide linker to rEPA, by treatment with a peptide coupling agent, preferably a water-soluble carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the like, to produce a hydrazide-functionalized carrier protein. Adipic acid dihydrazide is a preferred linker, but conjugates employing other linkers, such as the dihydrazides of succinic, suberic, and sebacic acids, are contemplated to be within the scope of the invention. The S. typhi polysaccharide, Vi, is then coupled to the hydrazide-functionalized carrier protein, again preferably with a water-soluble carbodiimide.
Regardless of the precise method used to prepare the conjugate, after the coupling reactions have been carried out the unbound materials are removed by routine physicochemical methods, such as for example gel filtration or ion exchange column chromatography, depending on the materials to be separated. The final conjugate consists of the polysaccharide and the carrier bound through a dihydrazide linker.
Dosage for Vaccination
The present inoculum contains an effective, immunogenic amount of a Vi-rEPA polysaccharide-carrier conjugate. The effective amount of polysaccharide-carrier conjugate per unit dose sufficient to induce an immune response to S. typhi depends, among other things, on the species of mammal inoculated, the body weight of the mammal, and the chosen inoculation regimen, as is well known in the art. Inocula typically contain polysaccharide-carrier conjugates with concentrations of polysaccharide from about 1 micrograms to about 500 micrograms per inoculation (dose), preferably about 3 micrograms to about 50 micrograms per dose, and most preferably about 5 micrograms to 25 micrograms per dose.
The term xe2x80x9cunit dosexe2x80x9d as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (polysaccharide) calculated to produce the desired immunogenic effect in association with the required diluent.
Inocula are typically prepared in physiologically and/or pharmaceutically tolerable (acceptable) carriers, and are preferably prepared as solutions in physiologically and/or pharmaceutically acceptable diluents such as water, saline, phosphate-buffered saline, or the like, to form an aqueous pharmaceutical composition. Adjuvants, such as aluminum hydroxide, may also be included in the compositions.
The route of inoculation may be intramuscular, subcutaneous or the like, which results in eliciting antibodies protective against S. typhi. In order to increase the antibody level, a second or booster dose may be administered approximately 4 to 6 weeks after the initial injection. Subsequent doses may be administered as indicated herein, or as desired by the practitioner.
Antibodies
An antibody of the present invention in one embodiment is characterized as comprising antibody molecules that immunoreact with S. typhi Vi.
An antibody of the present invention is typically produced by immunizing a mammal with an immunogen or vaccine containing an S. typhi Vi-rEPA polysaccharide-protein carrier conjugate to induce, in the mammal, antibody molecules having immunospecificity for the immunizing polysaccharide. Antibody molecules having immunospecificity for the protein carrier will also be produced. The antibody molecules may be collected from the mammal and, optionally, isolated and purified by methods known in the art.
Human or humanized monoclonal antibodies are preferred, including those made by phage display technology, by hybridomas, or by mice with human immune systems. The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies may be produced by methods known in the art. Portions of immunoglobulin molecules, such as Fabs, may also be produced by methods known in the art.
The antibody of the present invention may be contained in blood plasma, serum, hybridoma supernatants and the like. Alternatively, the antibodies of the present invention are isolated to the extent desired by well known techniques such as, for example, ion chromatography or affinity chromatography. The antibodies may be purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, IgG1, IgG2, IgG3, IgG4 and the like. Antibodies of the IgG class are preferred for purposes of passive protection. The antibodies of the present invention have a number of diagnostic and therapeutic uses. The antibodies can be used as an in vitro diagnostic agents to test for the presence of S. typhi in biological samples or in meat and meat products, in standard immunoassay protocols. Such assays include, but are not limited to, agglutination assays, radioimmunoassays, enzyme linked immunosorbent assays, fluorescence assays, Western blots and the like. In one such assay, for example, the biological sample is contacted with first antibodies of the present invention, and a labeled second antibody is used to detect the presence of S. typhi to which the first antibodies have bound.
Such assays may be, for example, of direct format (where the labeled first antibody is reactive with the antigen), an indirect format (where a labeled second antibody is reactive with the first antibody), a competitive format (such as the addition of a labeled antigen), or a sandwich format (where both labeled and unlabelled antibody are utilized), as well as other formats described in the art.
The antibodies of the present invention are also useful in prevention and treatment of infections and diseases caused by S. typhi. 
In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending upon such factors as the mammal""s age, weight, height, sex, general medical condition, previous medical history and the like.
In general, it is desirable to provide the recipient with a dosage of antibodies which is in the range of from about 1 mg/kg to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered. The antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, or lessen or attenuate the severity, extent or duration of the infection by S. typhi. Antibodies which immunoreact with ETA are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of an infection by ETA producing organisms, such as P. aeruginosa. 
The administration of the agents of the invention may be for either xe2x80x9cprophylacticxe2x80x9d or xe2x80x9ctherapeuticxe2x80x9d purpose. When provided prophylactically, the agents are provided in advance of any symptom. The prophylactic administration of the agent serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the agent is provided at (or shortly after) the onset of a symptom of infection. The agent of the present invention may, thus, be provided prior to the anticipated exposure to S. typhi (or other Shiga toxin producing bacteria), so as to attenuate the anticipated severity, duration or extent of an infection and disease symptoms, after exposure or suspected exposure to these bacteria, or after the actual initiation of an infection.
For all therapeutic, prophylactic and diagnostic uses, the polysaccharide-carrier conjugates of this invention, as well as antibodies and other necessary reagents and appropriate devices and accessories may be provided in kit form so as to be readily available and easily used.
The following examples are exemplary of the present processes and incorporate suitable process parameters for use herein. These parameters may be varied, however, and the following should not be deemed limiting.