WO2000033882A1 - A vi-repa conjugate vaccine for immunization against salmonella typhi - Google Patents

Abstract

This invention relates to conjugates of the Vi polysaccharide of S. typhi with the carrier Pseudomonas aeruginosa recombinant exoprotein A (rEPA), and compositions thereof, and to methods of using af these conjugates and/or compositions thereof for eliciting an immunigenic response in mammals, including responses whith 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 against S. typhi and are useful to prevent and/or treat illnesses caused by S, typhi.

Description

A V I-REPA CONJUGATE VACCINE FOR IMMUNIZATION AGAINST SALMONELLA TYPHI

FIELD OF THE INVENTION

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.

BACKGROUND

Typhoid fever remains a common, serious and now increasingly difficult disease to treat especially in developing countries. 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 ciprofioxacin. Limitations of the three licensed vaccines, i.e., attenuated S. typhi Ty21a, killed whole cell vaccines, and Vi polysaccharide vaccine, explain the failure to achieve routine immunization in countries that endure high endemic rates of typhoid fever.

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 Ty21 a 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 >5 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 l-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-rEPA^ or adipic acid dihydrazide (ADH, Vi- rEPAπ) as linkers, were compared sequentially in adults, 5-14 year-olds and then 2-4 year olds in Vietnam. [Manuscript in preparation].

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the invention to provide methods of using conjugates of the capsular polysaccharide of Salmonella typhi (Ni) 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-rEPAπ conjugates of this invention induce a strong initial IgG antibody response in humans. In this respect, they have a significant advantage over the conjugates.

DETAILED DESCRIPTION OF THE INVENTION

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-rEPAn) 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 > 80% of the immunized population, of a > 8-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-rEP An vaccine containing 25 μg ofS. typhi Ni polysaccharide, of inducing in the serum of the human a level of anti-Ni 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-rEPAn vaccine composition containing 25 μg 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-rEPAn vaccine composition containing 25 μg 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.

Definitions

Vi is a linear homopolymer of α(l- 4)-D-GalpA, which is N- acetylated at C-2 and O-acetylated at C-3.

As used herein, the terms "immunoreact" and "immunoreactivity" 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 "antibody" 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, Fab', F(ab') 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 l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, l-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 "unit dose" 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, IgG], 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 "prophylactic" or "therapeutic" 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.

EXAMPLE 1 MATERIALS AND METHODS

Clinical protocol. The study was approved by the Ministry of Health of Vietnam, the Institutional Review Board of the National Institute of Child Health and Development, NIH and FDA. Informed consent was obtained from adults or from parents or guardians of vaccinees under 18 years old. The site was Cao Lanh District, Dong Thap Province in the Mekong Delta region of Vietnam. The vaccines were stored at 4°C and injected intramuscularly into the deltoid muscle in 0.5 mL aliquots, containing 25 μg of Vi alone or as a conjugate. Twenty two teachers or administrative personnel of the Bon Sang Nursery, My-Tho Town, Cao Lanh District, received an injection of Vi-rEPA_π (BB IND 6990). 157 5 to 14 year-olds, recruited from the elementary, middle and high school of Cao Lanh District, received 1 injection of 0.5 mL of either Vi-rEPAi (BB IND 4334), Vi-rEPAn or Vi (Lot Kl 140, manufactured by Pasteur-Merieux Serums et Vaccins and distributed by Connaught Laboratories, U.S. License 384).

A group of 203 2 to 4 year-olds, recruited from the Bon Sang Nursery, were randomized to receive either 1 or 2 injections of Vi-rEPAi or of Vi-rEPAn spaced 6 weeks apart. The groups are uneven because some individuals refused the second injection. The teachers and the parents were instructed to examine the children at 6, 24 and 48 hours following the injection. Children who were absent on the ensuing 2 days were visited at home by the District Health medical staff. None of the recipients had erythema >1 cm at the injection site and none of the recipients had fever for 2 days following the injection.

Blood samples were taken before, and at 6 and 26 weeks after injection of the adults and 5-14 year-olds. An additional blood sample was taken from the 2-4 year-olds 10 weeks after the first injection.

Reagents: Dialysis tubing: Spectra/por, 45mm, mwco=3500, 32mm, mwco=8000, from Spectrum, Houston, Tx; Biodesign #D102, 15.5, mwco=8000, Carmel, NY; YM10 membrane, 62 mm, mwco=10,000 from Amicon, Beverly, MA; Filters: 150 mL unit, C.A., 0.45μm, from Nalgene, Rochester, NY, 25 mm, 0.45 μm, Uniflow, from Schleicher & Schuell, Keen, NH; Chemicals: 2-[N-morpholino]- ethanesulfonic acid [MES], acid form, sodium form, [MES buffer (pH5.6), titrated with MES-Na and MES-H]; hydroxylamine, resorcinol, adipic acid dihydrazide [ADH], l-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC], thimerosal, from Sigma Chemical Co, St. Louis, Mo; Tris, GIBCO, NY; Sephacryl S-1000, Sephadex G-50, from Pharmacia, Piscataway, NJ; ammonium sulfate, Mallinckcrodt, Paris, KY; Limulus amebocyte lysate (LAL), lot 12-56-648, from Associates of Cape Cod Ind. Woods Hole, MA; U.S. standard endotoxin (RSE), lot EC-5, from Bureau of Biologies (CBER), FDA, Bethesda, MD; Goat anti-exotoxin, lot GAE-02A from List Biological Lab., Inc., Campbell, CA; 2,4,6-trinitrobenzene sulfuric acid, Pierce Chemicals, Rockford, II; Coomassie blue reagent, standard bovine serum albumin (BSA) solution (2 mg/mL), from Pierce Chemicals, II.

Assays. EDC, protein, hydrazide were measured as described [31]. Vi content of conjugates was measured by determination of the O acetyl with Vi as a standard. Sterility, pyrogenicity, and general safety was assayed according to the Code of Federal Regulations (CFR) 610.126.

Vi (Lot 112A). Vi (3.2 μmol O-acetyl/mg, 1.2% nucleic acid, <0.01% protein) was obtained from Pasteur-Merieux, Serums et Vaccins, Lyon, France. This Vi (985 mg) was extracted with cold phenol 10 times. The water phase was dialyzed 4 times against 6 L of pyrogen-free water (PFW), 4°C and freeze-dried. The final yield of Vi was -50%. The endotoxin content, determined by Limulus Amebocyte Lysate (LAL), was 25-50 EU/μg. rEPA. Recombinant exoprotein A (rEPA) is a genetically manipulated non-toxic, fully antigenic derivative of Pseudomonas aeruginosa exotoxin A (ETA) secreted by the recombinant strain of Escherichia coli BL21(1DE3) carrying plasmid pVC45D. Fermentation of E. coli BL21(IDE3) and purification of rEPA was performed as described [10]. Fractions containing rEPA were pooled, dialyzed against pyrogen-free saline (PFS), 50 mM sodium phosphate (PBS), pH 7.2, sterile filtered and ultracentrifuged at 100,000 x g for 5 hours at 4°C. The pellet was discarded and the supernatant (25 mL) was sterile-filtered. The endotoxin content of rEPA was <1 EU/mg. rEPA showed no toxicity in mice at 500 times the lethal dose of ETA.

Vi-rEPAi (SPDP). Vi-rEPAi (lot 6141 1) was prepared using N- succinimidyl-3-(2-pyridyl dithio) propionate (SPDP) as a linker [51, 54]. Cystamine (360 mg), dissolved in 20 mL of PFS, was mixed with 120 mg of Vi (lot 112A) and the pH brought to 5.0 with 0.1M NaOH in an autotitrator. EDC was added to a final concentration of 0.1M and the pH maintained at 5.0 for 3 hours by addition of 0.1M HCl. The reaction mixture was dialyzed against PFW at 5°C and freeze-dried. The SH concentration was 1.3% w/w.

SPDP, 14 mg/1.6 mL ethanol, was added to 7 mL of rEPA (10 mg/mL) with stirring for 2 hours at room temperature and overnight at 4°C. The reaction mixture was passed through a Bio-Gel P-6DG in PBS, 1 mM EDTA, pH 7.2, the void volume concentrated, sterile-filtered, and stored at 4°C. The SPDP/rEPA was 10.6 mol/mol. A single line of precipitation was formed between rEPA and the rEPA- SPDP derivative (not shown).

Dithiothreitol (DTT) (37.2 mg) was added to 3 mL of Vi-cystamine (10 mg/mL in PFS, 10 mM sodium phosphate, pH 7.2 (PBS)) for 2 hours at room temperature. The reaction mixture was passed through Bio-Gel P6DG in PFS. Void volume fractions were sterile-filtered and added to 4.0 mL of rEPA-SPDP (31.5 mg). The reaction mixture was stirred at room temperature for 2 hours and passed through a column of S-1000 Sephacryl in PBS, pH 7.2 at 4°C. Fractions were monitored for protein, O-acetyl, and by immunodiffusion. A pool of conjugate-containing fractions (71 μg/mL protein and 75 μg/mL Vi) was denoted as Vi-rEPAi Rabbit anti-ETA and burro anti-Vi reacted with an identical precipitation line with Vi-rEPAi and did not enter 10% PAGE in 1% SDS (not shown).

Vi-rEPA„ (ADH). 0.5M MES buffer, pH 5.6 (4.6 mL), was added to 24.6 mL of rEPA (300 mg): the resultant pH was 5.7. With stirring, ADH (1.05 g) was added followed by EDC (60.8 mg) and maintained for 1 hour at room temperature. The pH was stable at 5.6. The reaction mixture was dialyzed against PBS at 4°C, centrifuged at 14,500 x g for 30 minutes at 4°C, and the supernatant passed through a 5 x 87 cm column of Sephadex G-50 in 0.2 M NaCl, 0.25 mM phosphate, pH 7.0. The void volume fractions were concentrated over a YM-10 membrane at N₂ pressure of 150 kPa and sterile-filtered. The ratio of hydrazide/protein of rEPA-AH was 0.023 (w/w) or 8.7 (mol/mol). SDS-PAGE (8% acrylamide) showed a similar pattern of rEPA-AH compared to rEPA (not shown). rEPA-AH and rEPA formed a line of identity with goat anti-ETA (not shown).

Vi, 100 mg of Vi (10 mg/mL PFS) was mixed with 2.4 mL of 0.5 M MES buffer pH 5.6 at room temperature. With mixing, 63 mg of EDC was added and after 2 minutes, 100 mg of rEPA-AH (10.1 mg/mL) was added dropwise. The reaction mixture was brought to 33.3 mL with 11 mL of PFS so that the concentration of Vi and rEPA was 3 mg/mL and 10 mM for EDC. The pH rose gradually from 5.5 to 5.7 within 3 hours of reaction, then the pH was brought up to 7.0 with several drops of 1M sodium phosphate buffer, pH 7.0. The mixture was stored at 4°C overnight, centrifuged for 30 minutes at 14,500 x g, 10°C and passed through a 2.5 x 90 cm Sephacryl S-1000 column in phosphate buffered saline, pH 7.0, (PBS, pyrogen-free saline containing 5mM sodium phosphate pH 7.0, and 0.01% thimerosal). Fractions #23 through 50 were pooled and the bulk of Vi-rEPA contained 200.3 μg Vi/ml and 171.1 μg rEPA/ml. The bulk was 4-times diluted with the PBS and the resultant Vi- rEPAn was bottled. The bottled conjugate vaccine contained 48 μg Vi/ml and 43 μg rEPA ml. Immunogenicity in mice and in guinea pigs: Vi or the Vi conjugates were diluted to 25 μg/mL in saline and 0.1 mL injected subcutaneously three times 2 weeks apart in 6 week-old female general purpose mice (10 mice/group). Controls were injected once with saline or three times with Vi alone. Mice were exsanguinated 7 days after each injection and assayed for total Vi antibody by ELISA using a pooled murine hyperimmune serum, containing 500 μg anti-Vi/mL, as a reference.

Three vaccines, containing 5 μg of saccharide, were injected into groups of 4 6-week-old Duncan-Hartley guinea pigs and serum anti-Vi assayed by ELISA as described (performed by Pasteur Merieux).

Double immunodiffusion. Vi, rEPA, rEPA-AH, and the two conjugates were reacted with burro anti-Vi (B260) and goat anti-EPA and sera from mice after their second injection of conjugates were assayed by double immunodiffusion against 100 μg Vi/mL as described [31].

ELISA. Total anti-Vi was assayed in murine and in guinea pig sera as described [31]. IgG was extracted from 500 mL of plasma from an adult vaccinated with Vi polysaccharide typhoid vaccine. The anti-Vi content of this human IgG was assayed by RIA by Pasteur Merieux Serum et Vaccins, Lyon, France. Sera were assayed for IgG, IgM and IgA anti-Vi by ELISA [31]. Goat anti-human IgG (Jackson Immuno Research Laboratories, Inc) and IgM (Sigma Chemical Company) conjugated to alkaline phosphatase were used as secondary antibodies. Serum from a typhoid carrier with high titer of IgM anti-Vi IgM was assigned a value 100 EU and used as a reference for this Ig. The levels of anti-Vi were calculated as a percent of the standard and expressed as the geometric mean and the 25-75 centiles for IgG and for IgM anti- Vi. Confirming previous results, the correlation coefficient was r=0.964 between the level of IgG anti-Vi assayed by RIA and ELISA and 0.084 for IgM. Antibody levels are expressed as the geometric mean and the 25th and 75th centiles.

Data analysis. Comparisons of geometric means were performed by paired and unpaired t tests when appropriate. EXAMPLE 2 COMPOSITION OF THE VACCINES Because the immunogenicity is related to M_r of Vi, we used the highest molecular weight Vi available for our conjugates. Vi passes through Sephacryl S-1000 and the CL-2B Sepharose starting from the void volume. SDS-PAGE of the fractions showed a Coomassie Blue-staining band that did not move through the gel: there were no bands in the gel. Double immunodiffusion showed a stained circle around the edge of the antigen wells and two precipitation lines that did not cross, one positive with anti-Vi and the other with anti-ETA sera. Accordingly, we cannot yet distinguish bound (conjugated) from unbound Vi. The specifics for the sterility and general safety tests in Code of Federal Regulations 610.11 should be met.

Vi-rEPAn did not give a positive reaction with the sensitivity of the assay at 2.6 x 10^*5 M carbodiimide. 10% SDS PAGE of Vi-rEPA, or of Vi-rEPAn showed one band at the top of the gel (did not enter the gel). No bands corresponding to the rEPA-AH or rEPA were detected. In HPLC profiles of Vi(lot 126 A), Vi-rEPAi and of Vi-rEPAn, and rEPA on TSK-G6000, Vi eluted as a single broad peak at 19.96 minutes and rEPA-AH eluted at 24.27 minutes. Both Vi-rEPA] and Vi-rEPAn showed one peak at 16.67 minutes with A₂₈o.

Table 1. Vi polysaccharides from Pasteur Merieux, Serum et Vaccins, Lyon, France.

Lot 65332 (Vi-rEPAn) Lot 51706 (Vi-rEPA;,)

Pasteur Merieux VU 12A Vil04A

O-acetyl 3.19 μmoles/mg 2.97 μmoles/mg

M_r 60.3% <Kd 0.25 60.5% <Kd 0.25

Nucleic acid 1.2% 1.4%

Protein <0.1% <0.1%

Pyrogen Passes 0.01 μg/kg Passes 0.01 μg/kg

Both lots of Vi pass the r ≥quirements of the World Hea th Organization for Vi typhoid polysaccharide vaccine. EXAMPLE 3 IMMUNOGENICITY IN MICE

After one injection, mice immunized with either conjugate had higher levels of anti-Vi than Vi alone (13,4, 112.5 vs 5.78 p=0.01). In contrast to Vi, both conjugates elicited a booster response after the second injection (79.5 vs 12.5, 109 vs 13.4, p=0.01): Vi-rEPAn elicited higher levels of anti-Vi than Vi-rEPAi (109 vs 79.5, p=0.05). See Table 2. Sera from the mice after the second injection of either conjugate precipitated with Vi in double immunodiffusion (not shown).

Table 2.

Geometric mean serum anti-Vi (μg/mL) elicited in mice by subcantaneous injection of Vi, Vi-rEPAi and Vi-rEPAn

*Reported in Reference 31 as Vi-rEPA . Mice were injected with 2.5 μg Vi alone or as a conjugate as described in MATERIALS AND METHODS.

Numbers for Vi Lot 104A and for related conjugate Vi-rEPAi are reported in Ref. 54 (see general purpose mice experiment). Results are in μg anti-Vi/mL (measured by RIA).

Vi-rEPAn and related Lot of Vi 112A (used for making this conjugate) were made and tested in animals years after Vi-rEPAi was made and tested in mice, guinea pigs and adults. Immunogenicity of Vi-rEPAn was tested by ELISA, not RIA, but because results are expressed in μg anti-Vi-mL using the same standard serum, the numbers in Table 2 are comparable. EXAMPLE 4

IMMUNOGENICITY IN GUINEA PIGS As reported, Vi did not elicit anti-Vi in guinea pigs after two injections [31]. Neither conjugate induced anti-Vi after the first injection and both conjugates elicited anti-Vi after the second injection, but only Vi-rEPAn elicited a statistically significant rise of the GM anti-Vi level after the third injection. See Table 3.

Table 3

Geometric mean serum IgG anti-Vi (ELISA units) elicited in guinea pigs (n=4) by injection of Vi, Vi-rEPAi and Vi-rEPAn

*Reported in Reference 31 as Vi-rEPA₈. Mice were immunized with Vi vaccines as described in MATERIALS AND METHODS. Geometric mean serum anti-Vi is expressed as percent of a reference serum.

Vi-rEPAi is reported in Ref. 54. Results from the guinea pig experiment with Vi-rEPAi and Vi Lot 104 cannot be compared with Vi-rEPAn, because they are expressed in arbitrary ELISA units referring to different standard sera.

EXAMPLE 5

CLINICAL REACTIONS

None of the volunteers had fever following either the first or second injection. Local reactions were confined to mild pain in a small fraction of the vaccinees at any age.

Anti-Vi in adults (Table 4). In the present study, only Vi-rEPAn was evaluated in adults. All volunteers had significantly higher pre-existing anti-Vi than those of the 5-14 year-olds (9.62 vs 0.44, 0.42, 0.61, p=0.0001). Six weeks after injection, all vaccinees responded with >4 rise in anti-Vi of the 3 Ig classes: 48-fold rise of IgG (465 vs 9.62, p=0.0001), 5-fold rise of IgM (19.0 vs 4.76, p=0.0001) and a 43-fold rise of IgA (8.85 vs 0.20, p=0.0001). The IgG anti-Vi fell to 1 19 (3.9-fold decline) at 26 weeks, but this level was 12.4-fold higher than the pre-vaccination levels (119 vs 9.62, p=0.0001). Similarly, at the 26 weeks interval, IgM and IgA anti- Vi declined but were significantly higher than the pre-immune values (p<0.01).

Table 4

Serum anti-Vi (μg/mL) in adults (n=22) elicited by one injection of Vi-rEPA_π

Pre-injection 6 weeks 26 weeks

IgG: Geometric mean 9.62 465 119 25-75 centiles 5.0-20.8 293-894 52.8- 277

465, 119 vs 9.62, p=0.0001; 465 vs 119, p=0.0001

IgM: Geometric mean 4.76 19.0 9.34 25-75 centiles 2.68-7.48 6.27-36.2 4.78-18.2

19.0, 9.34 vs 4.76, p<0.0.01; 19.0 vs 9.34, NS

IgA: Geometric mean 0.20 8.85 4.99 25-75 centiles 0.10-0.30 1.92-18.2 1.22-10.7

8.85, 4.99 vs 0.20, p=0.0001; 8.85 vs 4.99, p<0.0001 NS = Not statistically significant Vi-rEP A, was evaluated in adults in a previous study using 15ug polysaccharide per injection, as reported in reference 54.

Anti-Vi in 5-14 year-olds (Table 5). On a random basis, the 5-14 year-olds were injected once with Vi or 1 of the 2 conjugates. All 4 groups had similar levels of pre-injection anti-Vi that were significantly lower than those of adults (vide supra).

Table 5

Serum anti-Vi of 5 to 14 year-olds injected with Vi, Vi-rEPA, or Vi-rEPAn

Geometric mean ELISA Units (25-75th centiles) Vi Vi-rEPAi Vi-rEPA_π

gG: Pre- 0.44 (0.28-0.59) 0.42 (0.24-0.53) 0.67 (0.24-1.81)

6 wks 18.9 (7.84-44.1) 22.8 (7.86-58.9) 169.0 (80.8-290)

26 wks 13.4 (6.01-29.4) 10.8 (3.64-28.8) 30.0 (14.1-45.5)

30.0 vs 13.4, 10.8; p<0.001

IgM: Pre- 6.47 (4.02-9.90) 6.75 (4.16-10.2) 5.79 (3.33-8.25)

6 wks 25.2 (17.4-40.3) 48.0 (21.0-81.1) 92.1 (51.5-154)

26 wks 12.3 (6.64-21.2) 26.2 (13.0-49.0) 31.3 (17.9-56.7)

31.3 vs 26.2 NS; 31.3 vs 12.3 p=0.0001; 31.3, 26.2 vs 12.3 p=0.0002

gA: Pre- 0.05 (0.03-0.07) 0.03 (0.02-0.04) 0.05 (0.02-0.10)

6 wks 2.64 (0.81-7.59) 1.99 (0.73-5.13) 16.5 (9.19-43.5)

26 wks 2.04 (0.81-6.72) 0.99 (0.35-2.77) 4.99 (3.34-28.9)

4.99 vs 2.04, NS; 4.99 vs 0.99, p=0.02

All three vaccines elicited significant rises of anti-Vi of the 3 isotypes at 6 and at 26 weeks over the pre-immune levels. Vi-rEPAn elicited higher levels of anti-Vi at all intervals than Vi-rEP Ai and Vi. IgG anti-Vi. At 6 weeks, all responded with >4-fold rises of anti-Vi: 43 -fold for Vi, 54-fold for Vi-rEPA! and 252-fold for Vi-rEP A_π. Vi-rEP A_n elicited higher levels of anti-Vi than Vi-rEPA, or Vi (169 vs 22.8, 18.9 p=0.0001). Twenty six weeks later, the IgG anti-Vi of all groups declined but remained >4 fold higher than the pre-immune levels: Vi-rEPAn > Vi > Vi-rEP Aj (30.0 vs 13.4,10.8, p=0.0001). Of interest, is that similar levels of IgG anti-Vi were elicited by Vi-rEPA, and Vi at both 6 and 26 weeks following vaccination.

IgM anti-Vi. Pre-immune levels of the three groups were similar. At the six weeks interval, all the vaccines elicited significant rises of anti-Vi (25.2 vs 6.47, 48.0 vs 6.75, 92.1 vs 5.79; p=0.0001, for Vi, Vi-rEP Ai, for Vi-rEP A_π, respectively). Vi-rEPA, induced higher anti-Vi than Vi alone at both post vaccination intervals (p<0.0002). At 26 weeks, the GM IgM anti-Vi of the three groups were higher than the pre-immune levels: the levels in the recipients of the conjugates were higher than that of Vi (31.3,26.2 vs 12.2; p<0.01).

IgA anti-Vi. The pre-immune levels of the three groups were similar and almost at the level of detection. Vi-rEPA,, elicited the highest IgA anti-Vi of the 3 vaccines (16.5 vs 1.99, 2.64; p<0.002). The levels of the 3 groups declined at 26 weeks but this order of IgA anti-Vi was retained at 26 weeks (4.99 for Vi-rEP A„ vs Vi- rEP Ai, 2.04; NS; 4.99 vs 0.99, p=0.02). The 26 week level elicited by Vi-rEPAn (4.99) was higher than the 6 week level in the groups receiving Vi (2.04) and Vi- rEPA, (1.99).

One vs 2 injections of Vi conjugates in 2 to 4 year-olds (Table 6) Vi was not administered to the 2-4 year-olds. On a random basis, 2-4 year-olds were administered 1 or 2 injections of Vi-rEP Ai or Vi-rEPA,, 6 weeks apart: blood was taken before each injection and 4 and 26 weeks after the second injection. The pre- immune levels of the 4 groups were similar and slightly lower than those of the 5-14 year-olds. Six weeks after the first injection, all responded with >4 fold rise of anti-Vi of each Ig class and there was no significant difference for each conjugate between the groups destined to receive 1 or 2 injections. Table 6. Serum anti-Vi of 2-4 year-olds injected 1 or 2 times 6 weeks apart with Vi-rEP Ai or Vi-rEP A_π (~50/group)

IgM: Pre- 4.72 (2.67-7.90) 5.00 (3.06-7.48) 3.61 (2.50-4.80) 3.93 (2.84-5.18) 6 wk 37.7 (24.1-55.2) 41.8 (26.0-62.7) 47.5 (27.8-81.5) 39.8 (22.9-57.5)

M 10 wk 35.7 (20.3-65.2) 82.5 (51.2-155) 34.8 (20.1-58.6) 31.8 (19.3-48.6) 26 wk 19.5 (12.2-29.4) 36.2 (21.8-62.1) 20.1 (13.1-32.3) 19.5 (12.8-30.6)

IgA: Pre- 0.02 (0.01-0.02) 0.02 (0.01-0.02) 0.02 (0.01-0.02) 0.02 (0.01-0.02) 6 wk 1.76 (1.30-2.54) 1.32 (0.71-3.34) 6.23 (2.79-18.1) 5.68 (2.22-12.9) 10 wk 1.48 (1.03-2.68) 2.00 (0.74-3.69) 4.21 (1.86-9.90) 4.99 (2.24-11.8) 26 wk 0.70 (0.50-1.12) 0.85 (0.50-2.02) 3.00 (1.37-8.49) 2.62 (1.09-7.29)

3.00, 2.62 vs 0.70. 0.85 p<0.02; 6.23 vs 5.68, 4.21 vs 4.99, 3.00 vs 2.62, NS

On a random basis, 2-4 year-olds were injected 1 or 2 times 6 weeks apart with Vi-rEPA_! or Vi-rEP A_π. Blood was drawn before each injection and 4 and 20 weeks after the 2nd injection. NS = not statistically significant.

IgG anti-Vi: Vi-rEPAπ elicited higher levels of anti-Vi than Vi-rEPA, (77.2,69.9 vs 30.2, 28.9, p=0.0001). The levels of anti-Vi in the 2-4 year-olds receiving Vi-rEPAπ were higher than those administered Vi alone in the 5-14 year- olds (77.2,69.9 vs 18.9; p=0.0001). Four weeks after the second injection, both conjugates elicited a rise in anti-Vi (2.87-fold for Vi-rEP Ai and 1.36-fold for Vi- rEPAπ: levels elicited by 2 injections of Vi-rEPAπ were only slightly higher than those by Vi-rEP Ai (95.4 vs 83.0, NS). The second injection of both conjugates elicited higher levels of anti-Vi than one injection of the Vi or Vi-rEPA, in the 5-14 year-olds (95.4, 83.0 vs 18.9, 22.8; p=0.0001). At 26 weeks, IgG anti-Vi of the recipients of 2 injections of Vi-rEP A„ were the highest (30.6 vs 20.4, 12.8, 5.50), similar to that of the 5-14 year-olds receiving the same conjugate but higher than the recipients of Vi alone in that age group (30.6 vs 13.4, p=0.0001). Serum IgG anti-Vi in the recipients of 2 injections of Vi-rEP A„ at 26 weeks were slightly different for the 3 (20.7, n=19) and 4 year-olds (31.4, n=12) compared to the 2 year-olds (20.7, n=6) but these differences were not statistically significant.

IgM anti-Vi. IgM anti-Vi levels in all groups were similar and slightly lower than the 5-14 year-olds. One injection of either conjugate elicited about an 8- fold increase: there were no significant differences between the two conjugates. Unexpectedly, reinjection of Vi-rEP Aj (82.5 vs 41.8; p=0.0003), but not Vi-rEP A„ (31.8 vs 39.8; NS), elicited a booster response. At 26 weeks, the group that received Vi-rEPA, had the highest level; the levels were similar for the others (36.2 vs 19,5, 20.1, 19.5; NS)

IgA anti-Vi. Both conjugates elicited a significant rise of IgA anti-Vi: Vi-rEPAn higher than Vi-rEP Ai (6.23, 5.68 vs 1.87, 1.36; p=0.02). Reinjection of Vi- rEP Ai only elicited a slight rise of IgA anti-Vi (2.00 vs 1.32, NS). The levels of all groups declined at 26 weeks although all were significantly higher than those of the pre-immune sera: the recipients of 1 or 2 injections of Vi-rEP A„ were similar and higher than those of the groups that received Vi-rEPA, (3.00, 2.62 vs 0.70, 0.85; p<0.02).

Anti-rEPA. Anti Vi-rEPA elicited higher levels of IgM anti-Vi than Vi alone in 5-14 year olds (data not shown). DISCUSSION

As shown for other polysaccharides, such as Haemophilus influenzae type b, the immunogenicity of Vi is improved by covalently binding it to a protein. Previously, we reported the enhanced immunogenicity of conjugates compared to Vi, similar to Vi-rEPA,, in adults [54].

Since reinjection of Vi and other polysaccharide-protein conjugates in older children or in adults does not elicit a booster response, only 1 injection of Vi conjugates and Vi were compared in 5 to 14 year-olds. Unexpectedly, at 6 and at 26 weeks after vaccination, IgG anti-Vi levels elicited by Vi-rEPA, and by Vi were similar at 6 (22.8 vs 18.9) and at 26 weeks (10.8 vs 13.4). Vi-rEP Ai, however, elicited higher levels of IgM anti-Vi than Vi at 6 (48.0 vs 25.2) and at 26 weeks (26.2 vs 12.3). Vi-rEPAπ elicited higher levels of IgG, IgM and IgA anti-Vi at all intervals in the 5 to 14 year-olds than Vi-rEPA, and Vi.

In the 2-4 year-olds at 26 weeks, IgG anti-Vi levels elicited by Vi- rEP An were higher than those elicited by Vi-rEPA, after 1 (20.4 vs 5.50, p=0.01) and 2 injections (30.6 vs 20.4), but these latter differences were not statistically significant. Two injections of Vi-rEP A„, elicited higher levels than Vi in the 5-14 year-olds and, therefore, it can be predicted that this Vi conjugate will elicit greater than 70% efficacy when injected 2 times in individuals >2 years of age.

There is evidence that a critical (protective) level of serum IgG anti-Vi is sufficient to confer immunity to typhoid fever. In passive immunization experiments with serum taken mice and humans injected wih cellular vaccines, IgG anti-Vi accounted for the protection against challenge with S. typhi. By analogy to H. influenzae type b and other capsulated pathogens it is antibodies of the IgG isotype, not IgM or IgA, that exude onto the epithelial surface and account for most of the serum anti-Vi in the intestine. We suggest that measurement of serum IgG anti-Vi will be essential and sufficient to standardize Vi conjugate vaccines.

We are yet unable to demonstrate by physico-chemical or immunologic methods whether there is some free Vi (unbound to protein) in our new conjugate. Vi is molecularly polydisperse material that cannot be 100% effectively separated from Vi conjugates on the available gel filtration media or polyacrylamide gels. Similarly, double immunodiffusion with antibodies to the Vi and to the carrier protein (rEPA) does not yield a precipitin line of identity. For the present, our only method for identifying that the Vi and protein are covalently bound is to demonstrate the increased immunogenicity of our conjugate in mice and in guinea pigs compared to mixtures of Vi and the adipic hydrazide-derivatized protein. [See, e.g., ref 31].

SUMMARY

A Vi conjugate, prepared by treatment of an adipic hydrazide derivative of rEPA and Vi with EDC (Vi-rEP A„), is safe and more immunogenic in mice, guinea pigs and in young children and adults than a similar construct made with SPDP. Vi- rEPAπ elicited a booster response in 2-4 year-olds that results in levels of IgG anti-Vi significantly higher than those achieved by Vi alone in 5 to 14 year-olds. This new Vi conjugate is safe and can be expected to confer a high degree and long-lived immunity against typhoid fever in children as well as in adults.

One injection of Vi-rEP A„ into adults (n=22) elicited 465 ELISA U/mL (48-fold geometric mean rise) of IgG anti-Vi at 6 weeks that fell to 119 after 26 weeks: similar patterns were observed for IgM and IgA anti-Vi. In 5-14 year-olds (~50/group), one injection of Vi elicited a 43-fold rise of IgG anti-Vi, Vi-rEP Ai a 54- fold rise (54 vs 43, NS) and Vi-rEP A„ a 252-fold rise (252 vs 54,43 p=0.0001). At 26 weeks, Vi-EPA„ elicited 30.0 units of IgG anti-Vi that was higher than that induced by Vi-rEPA, and Vi (10.8 vs 13.4, NS); all were higher than pre-immune levels (p=0.0001). Vi-rEP A„ elicited the highest IgM and IgA anti-Vi at 6 weeks and at 26 weeks.

One or two injections of Vi-rEPA, and Vi-rEPAπ were evaluated in the 2-4 year-olds (-50 group). After 6 weeks there was a 406-fold rise of IgG anti-Vi in recipients of Vi-rEP A„ and 94-fold rise in recipients of Vi-rEPA, (p=0.0001). Four weeks after a second injection, recipients of Vi-rEPA, and Vi-rEP A„ had a rise of IgG anti-Vi: 83.0 from 28.9 and 95.4 from 69.9, respectively. At 26 weeks, the IgG anti-Vi levels of all vaccinees were higher than the pre-immune levels (p=0.0001). IgG anti- Vi levels elicited by 2 injections were higher than those with only 1 injection (30.6 vs 20.4 for Vi-rEP A„ and 12.8 vs 5.50 for Vi-rEP Ai): IgG anti-Vi levels elicited by 2 injections of Vi-rEP A„ were higher than those elicited by Vi in the 5-14 year-olds (30.6 vs 13.4, p=0.01). In all three age groups, Vi-rEP A„ was more immunogenic than Vi-rEPA,. Similar values were obtained for IgM and IgA anti-Vi. One injection of Vi-rEP A„ should confer a higher degree of immunity to typhoid fever than Vi in individuals >5 years: 2 injections should confer comparable immunity in 2 to 4 year- olds to that in individuals >5 years of age.

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62. U. S. Patent 5,204.098, Polysaccharide Protein Conjugates, Szu et al.

Claims

CLAIMS We claim:

1. A method of inducing, in a human, serum antibodies which protect against infection with S. typhi, comprising administering to said human, a composition comprising a molecular conjugate of the S. typhi Vi polysaccharide covalently bound through a carboxylic acid dihydrazide linker to Pseudomonas aeruginosa recombinant exoprotein A in a pharmaceutically acceptable carrier.

2. The method of claim 1 wherein the S. typhi Vi polysaccharide is covalently bound to the rEPA by means of an adipic acid dihydrazide linker.

3. The method of claim 1 or 2 wherein said conjugate molecule is administered at a dose of about 3 micrograms to about 50 micrograms of S. typhi Vi polysaccharide.

4. The method of claim 3 wherein said conjugate molecule is administered at a dose of about 25 micrograms of Vi polysaccharide.

5. The method of claim 1 or 2 wherein the antibodies protect the human against infection by S. typhi.

6. A composition comprising antibodies which are immunoreactive with S. typhi Vi polysaccharide, said antibodies being obtained from a human after administration to said human of a composition comprising a conjugate molecule comprising the S. typhi Vi polysaccharide covalently bound through a carboxylic acid dihydrazide linker to Pseudomonas aeruginosa recombinant exoprotein A.

7. The composition of claim 6, further comprising antibodies which are immunoreactive with ETA.

8. The composition of claim 6, wherein the composition is chosen from the group consisting of plasma, serum, and gamma globulin fraction.

9. The composition of claim 7, wherein the composition is chosen from the group consisting of plasma, serum, and gamma globulin fraction.

10. An antibody which is immunoreactive with S. typhi Vi polysaccharide which is obtained from a human, after administration to said human of a composition comprising a conjugate molecule comprising the S. typhi Vi polysaccharide covalently bound through a carboxylic acid dihydrazide linker to Pseudomonas aeruginosa recombinant exoprotein A.

11. A method of passively immunizing a mammal against S. typhi , comprising administering to said mammal an immunologically sufficient amount of a composition according to any one of claims 6-9.

12. A method for vaccinating a human against S. typhi infection, comprising administering to the human an immunizing amount of a composition comprising a molecular conjugate of S. typhi Vi polysaccharide covalently bound through a carboxylic acid dihydrazide linker to Pseudomonas aeruginosa recombinant exoprotein A in a pharmaceutically acceptable carrier.

13. The method of claim 12 wherein the S. typhi Vi polysaccharide is covalently bound to the Pseudomonas aeruginosa recombinant exoprotein A by means of an adipic acid dihydrazide linker.

14. A vaccine composition comprising an immunologically effective amount of a molecular conjugate of S. typhi Vi polysaccharide covalently bound through a carboxylic acid dihydrazide linker to Pseudomonas aeruginosa recombinant exoprotein A, in a pharmaceutically acceptable carrier.

15. The vaccine composition of claim 14 wherein the S. typhi Vi polysaccharide is covalently bound to the Pseudomonas aeruginosa recombinant exoprotein A by means of an adipic acid dihydrazide linker.