Title of Invention	POLYSACCHARIDE CONJUGATION WITH DETOXIFIED E. COLI HEAT LABILE ENTEROTOXIN (LT) USED AS VACCINE
Abstract	A detoxified recombinant E. coli heat-labile enterotoxin mutant LTS61K is employed as a carrier protein to conjugate polysaccharide. The LTS61K contains a mutated mature sub-unit A (LTA) that includes lysine at amino acid position 61 and a wild-type mature sub-unit B (LTB). Various types of bacterial capsular polysaccharide antigens were chemically conjugated with the LTS61K protein by a reductive amination reaction. The conjugated polysaccharide-LTS61K products were physically chemically and biochemically identified as soluble form. Rabbits were immunized intramuscularly to determine the immunogenicity of conjugated vaccines by ELISA to detect anti- polysaccharide antigen IgG titers and serum bactericidal assay thereby determining the functional activity of the antibodies. Study results show that conjugated polysaccharide-LTS61K vaccines induce higher polysaccharide-specific IgG titers and greater bactericidal activity in sera than that of polysaccharide alone or polysaccharide mixed with LTS61K. The presence of anti-LTS61K serum IgG antibody alleviates travel diarrhea caused by E. coli

Title of Invention

POLYSACCHARIDE CONJUGATION WITH DETOXIFIED E. COLI HEAT LABILE ENTEROTOXIN (LT) USED AS VACCINE

Abstract

A detoxified recombinant E. coli heat-labile enterotoxin mutant LTS61K is employed as a carrier protein to conjugate polysaccharide. The LTS61K contains a mutated mature sub-unit A (LTA) that includes lysine at amino acid position 61 and a wild-type mature sub-unit B (LTB). Various types of bacterial capsular polysaccharide antigens were chemically conjugated with the LTS61K protein by a reductive amination reaction. The conjugated polysaccharide-LTS61K products were physically chemically and biochemically identified as soluble form. Rabbits were immunized intramuscularly to determine the immunogenicity of conjugated vaccines by ELISA to detect anti- polysaccharide antigen IgG titers and serum bactericidal assay thereby determining the functional activity of the antibodies. Study results show that conjugated polysaccharide-LTS61K vaccines induce higher polysaccharide-specific IgG titers and greater bactericidal activity in sera than that of polysaccharide alone or polysaccharide mixed with LTS61K. The presence of anti-LTS61K serum IgG antibody alleviates travel diarrhea caused by E. coli

Full Text	FORM 2 THE PATENTS ACT 1970 (39 of 1970) & The Patents Rules 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. POLYSACCHARIDE CONJUGATION WITH DETOXIFIED E. COLI HEAT LABILE ENTEROTOXIN (LT) USED AS VACCINE 2. 1. (A) DCB-USA LLC (B) UNITED STATES OF AMERICA (C) 1007 NORTH ORANGE STREET 9TH FLOOR NEW CASTLE COUNTY WILMINGTON DELAWARE 19801 UNITED STATES OF AMERICA The following specification particularly describes the invention and the manner in which it is to be performed. Cross-Reference to Related Applications: [0001] Priority is claimed to U.S. Provisional Patent Application Serial No. 61/330 650 filed on May 3 2010 the disclosure of which is incorporated herein by reference in its entirety. Background of the Invention: [0002] Polysaccharide vaccines when prepared without carrier protein lack immune memory responses. Currently known conjugated vaccines include bacterial capsular polysaccharides such as Haemophilus influenzae type b Neisseria meningitidis group C and Streptococcus pnemoniae serotypes 1 3 4 5 6B 7F 9V 14 19F 23F. These vaccines are covalently conjugated to a carrier protein such as tetanus toxoid diphtheria toxoid CRM197 a mutant nontoxic diphtheria toxin or Neisseria meningitidis outer membrane protein. These polysaccharide conjugate vaccines could induce T-cell dependent response especially in infants below the age of two years; and they could prime long term immunologic memory produce high affinity antibody and could lower the rate of nasopharyngeal colonization and transmission. However the majority of the currently marketed bacterial polysaccharide conjugate vaccines applied tetanus toxoid (TT) or diphtheria toxoid (DT) as a carrier protein. TT and DT these two toxoid proteins are regular vaccines for infants/children; high frequency vaccination of TT and DT within a short time could have impact on the immunogenicity and safety. (Reduced response to multiple vaccines sharing common protein epitopes that are administered simultaneously to infants. Infect. Immun. 1998; 66(5):2093-8; Immunogenicity and safety of a combination pneumococcal-meningococcal vaccine in infants: a randomized controlled trial. JAMA 2005 ;293( 14): 1751-8). Therefore this invention provides a new type of the carrier protein LTS61K for its use on the conjugate vaccine. Summary of the Invention: [0003] This invention includes polysaccharide conjugation with a detoxified E. coli heat labile enterotoxin (LT) useful as vaccine to protect or immunize against the effects of infectious bacteria such as Haemophilus influenzae and Streptococcus pneumoniae and alleviate travel diarrhea caused by enterotoxigenic E. coli. [0004] One aspect of this invention relates to covalently conjugated polysaccharide-LTS61K vaccines isolated in purified form. These conjugated products have unexpectedly superior immunogenic and bactericidal properties in mammals. Another aspect relates to a method of administering an effective amount of conjugated polysaccharide-LTS61K vaccine to a mammal in need of protection from Haemophilus influenzae type b (Hib). The vaccines of the present invention stimulate T-helper cell response exhibit strong booster response upon re-exposure and have high antibody titers. [0005] Yet another aspect of the invention relates to the method of producing conjugated polysaccharide-LTS61K by reductive amination and isolating purified conjugated product. In accordance with the present invention it was discovered that the preferred method of conjugating polysaccharide and LTS61K for making the vaccine of the present invention is periodate oxidation of native PS followed by reductive amination. [0006] The LTS61K employed in the conjugates of the present invention is described in PCT Application Number PCT/US2007/075801 filed August 13 2007; in US Applications Numbers US 1 1/779419 filed July 18 2007 US 12/120 953 filed May 15 2008 and US 12/729 649 filed March 23 2010; and in Taiwan Patent Application No. 95139707 filed October 27 2006 and issued January 2009. The subject matter described in each of these applications is hereby incorporated by reference. Brief Description of the Figures: [0007 ] Figure 1 is a generalized depiction of a LT carrier protein. [0008] Figure 2 is the structural representation of Hib PRP saccharides. [0009] Figure 3 shows several conjugation methods. [0010] Figure 4 shows a reductive amination method according to the present invention. [0011] Figure 5 depicts PRP and LTS61K conjugation by reductive amination. [0012] Figure 6 shows the HPLC-SEC-RI elution profile of oxidized-PRP after NaI04 treatment. [0013] Figure 7 is an NMR spectrum of oxidized-PRP. This confirms the formation of aldehyde groups on PRP after periodate oxidation. [0014] Figure 8 shows a Far-UV CD spectra that confirms no secondary structure difference between LTS61K and PRP-LTS61K conjugated samples; and also a Fluorescence spectra which confirms no ? max tertiary structure difference between LTS61K and PRP-LTS61K conjugated samples. [0015] Figure 9 shows amino acid analyses that confirm the successful conjugation of PRP to LTS61K and the formation of a covalent bond. [0016] Figure 10 shows SDS-PAGE Western Blotting analysis to confirm the covalent conjugation between PRP and LTS61K. [0017] Figure 11 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE. [0018] Figure 12 shows IEF Western Blotting to confirm the covalent conjugation between PRP and LTS61K. [0019] Figure 13 confirms the purity of purified conjugate vaccine via HPLC-SEC-UV-MALLS-RI. [0020] Figure 14 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE to confirm the purity of purified conjugate vaccine. [00211 Figure 15 summarizes a rat immunogenicity study of Hib P P-LTS61 conjugates of the present invention. [0022] Figure 16 summarizes a rabbit immunogenicity study of Hib PRP-LTS61 K conjugates of the present invention. [0023] Figure 17 illustrates results of a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titer assay and anti-PRP lgG Ab titers. [0024] Figure 18 illustrates additional information about the rabbit immunogenicity study. [0025] Figure 19 illustrates results of a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titers (BA) and anti-PRP lgG Ab titers (OD). [0026] Figure 20 shows results of additional rabbit immunogenicity ELISA of anti-PRP and anti-LTS61K antibodies response studies. [0027] Figure 21 shows the results of additional rabbit serum bactericidal assay and anti-PRP IgG Ab. [0028] Figure 22 shows results of additional rabbit serum bactericidal assay and anti-PRP IgG Ab after a fourth immunization. [0029] Figure 23 shows the immunogenicity in mouse on pneumococcal PS serotype 14-LTS61K conjugates. Detailed Description of the Invention: [0030] We have now discovered that conjugated polysaccharide-LTS61K vaccines made in accordance with the present invention surprisingly induce higher polysaccharide-specific IgG antibody titers and greater bactericidal activity in sera than that of polysaccharide or polysaccharide mixed with LTS61K. [0031] A list of abbreviations employed herein is as follows: CFU: colony forming unit ELISA: enzyme linked immunosorbent assay Hib: Haemophilus influenzae type b IEF: isoelectric focusing LT: heat labile enterotoxin MALLS: multiple angle laser light scattering OD: optical density PNPS: pneumococcal polysaccharide PRP: polyribosyl ribitol phosphate PS: polysaccharide SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC_HPLC: size exclusion high pressure liquid chromatography RI: reflective index [0032] The new carrier protein employed in the conjugates of the present invention is detoxified recombinant E. coli heat-labile enterotoxin mutant LT more specifically LTS61K. In the LT mutant the A and B sub-units form a typical AB5 holotoxin structure. The detoxified LT mutant (LTS61K) contains a mutated mature sub-unit A (LTA) that includes K at amino acid position 61 and a wild-type mature sub-unit B (LTB). LTS61K renders the product significantly less toxic than wild-type LT. The carrier protein is depicted in Figure 1 of the drawings. [00331 The invention of the LTS61K which was selected as one of the starting materials for the present invention is based on the unexpected discovery that an LT containing a mutated LTA exhibits reduced toxicity compared to its wild type counterpart while retaining immunogenicity. This mutated LTA has an amino acid substitution at the position corresponding to position 61 of a wild-type LTA whose amino acid sequence SEQ ID NO: 5 is shown in the cited patent applications incorporated by reference herein. Accordingly LTS61K features an isolated polypeptide including a mutated LTA that contains an amino acid residue other than S T and F at the position corresponding to position 61 of SEQ ID NO: 5. The substituting amino acid residue can be D E H I K L N P Q R Y or W. It can be a naturally occurring amino acid or a non-naturally occurring amino acid e.g. a D-amino acid or a ß-amino acid. In one example the LTA has the amino acid sequence of SEQ ID NO:2 4 8 or 10. An LT containing this mutated LTA exhibits reduced toxicity i.e. [0034] In accordance with the present invention Haemophilus influenzae type B was cultured and its capsular polysaccharide antigen polyribosylribitol phosphate (PRP) was purified. The PRP is linear has a negative charge and is hydrophilic. The Hib PRP saccharide with molecular weight 345 and formula 10C 18H 1 10 IP is depicted as in Figure 2 of the drawings. [0035] PRP is conjugated onto LTS61K by a chemical reductive amination reaction employing appropriate molar ratios of polysaccharide PRP to protein (LTS61K) which produce conjugate vaccines. Ranges of molar ratios of PRP:LTS61K are shown in Table 1 below. Preferably the range of molar ratios of PRP:LTS61K is between about 3 : 1 and about 60:1 and mole /mole I047PRP is between about 0.1 and about 0.4. Table 1. Different ratios of PRP/LTS61K conjugation test [0036] Further in accordance with the present invention a variety of different types of the bacterial capsular polysaccharides antigens from Haemophilus influenzae type b and Streptococcus pneumoniae are chemically conjugated with the LTS61K protein by reductive amination reactions. A variety of different molar ratios of NaI04:PRP as shown in Table 1 above were employed to oxidize polysaccharides to produce short lengths of polysaccharides with different repeating units. Following the proper molar ratio of the polysaccharide reacted with LTS61K the successfully conjugated and purified polysaccharide-LTS61K conjugate products were obtained. The products were in soluble form and were physically and chemically evaluated by SEC-HPLC orcinol SDS-PAGE western blotting IEF GM1 -binding activity circular dichroism and fluorescent assays to identify the successful chemical conjugation of the polysaccharide antigen and LTS61K carrier protein. Example of the procedure of making polysaccharide conjugated with LTS61K: [0037] A variety of potential conjugation processes are illustrated in Figure 3 of the drawings. A generalized flow diagram of the reductive amination process selected in accordance with the present invention is found at Glyconjugate J. 1989.6:489 and is reproduced as Figure 4 of the drawings. [0038] In accordance with the present invention a polysaccharide is cleaved by periodate oxidation to smaller fragments to produce aldehyde end groups. Thereafter conjugation of the oxidized polysaccharide to LTS61K protein is carried out by reductive amination. More specifically an example of the process is as follows: [0039] A. Polysaccharide activation (periodate oxidation): Native polyribosylribitol phosphate (PRP) which is purified capsular polysaccharide of Haemophilus influenzae type b in an amount of 5 mg/mL is mixed with periodate (I04") 0.3 or 0.6 or 1.2 mg/mL (molar ratio (m/m) of I04" to PRP = 0.1 0.2 0.4). This mixture was allowed to stand at 4°C in the dark for 24 hours. Then glycerol was added to terminate the reaction. The resulting oxidized PRP underwent dialysis against ddH20 using 3.5 K membrane to remove impurities. The resulting product was sterile filtered with 0.22 um filter. The periodate oxidation procedure provided fragmentation of the polysaccharide PRP into different chain lengths. The range of average number of repeating units of PRP was about 40 to 10. [0040] B. Polysaccharide-LTS61K conjugation: Preparation of PRP and LTS61K conjugate mixture of oxidized PRP obtained from above purified LTS61K (which was disclosed in the previous obtained/filed patents) and sodium cyanoborohydride (NaBH3CN) in 10 mg/mL 3 mg/mL and 10 mg/mL respectively the reaction was earned for 2 weeks at 4°C in the dark. The reaction was terminated by adding sodium borohydride NaBH4 to quench unreacted aldehydes on PRP the preparation was then dialyzed against ddH20 with 10 K membrane. [0041] C. Characterization of the polysaccharide conjugated LTS61K products: Following the proper molar ratio of the polysaccharide reacted with LTS61K the successfully conjugated and purified polysaccharide-LTS61K conjugate products were obtained. The products with soluble form were physically chemically and biologically evaluated by SEC-HPLC Orcinol SDS-PAGE Western blotting IEF GM1 -binding activity circular dichroism and fluorescent assays to confirm the successful chemical conjugation of the polysaccharide antigen and LTS61K earner protein ratio of polysaccharide to LTS61K protein and its immunogenicity. Figure 5 of the drawings summarizes reaction conditions and assays employed in accordance with specific exemplification of the present invention. [0042] Reference is now made to the Figures of drawings which pertain to confirmation of structure and purity of the conjugates and vaccine. [0043] Figure 6 shows the HPLC-SEC-RI elution profile of oxidized-PRP after NaI04 treatment. This figure illustrates the process of the invention and results are readily reproducible. [0044] Figure 7 is an NMR spectrum of oxidized-PRP. This confirms the formation of aldehyde groups on PRP after periodate oxidation. [0045] Figure 8 shows a Far-UV CD spectra that confirms no secondary structure difference between LTS61K and PRP-LTS61K conjugated samples; and also a fluorescence spectra which confirms no ? max tertiary structure difference between LTS61K and PRP-LTS61K conjugated samples. [0046] Figure 9 shows amino acid analyses that confirm the successful conjugation of PRP to LTS61K and the formation of a covalent bond. [0047] Figure 10 shows SDS PAGE Western Blotting analysis to confirm the covalent conjugation between PRP and LTS61K. [0048] Figure 1 1 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE. [0049] Figure 12 shows IEF Western Blotting to confirm the covalent conjugation between PRP and LTS61K. [0050] Figure 13 confirms the purity of purified conjugate vaccine via HPLC-SEC-UV-MALLS-RI. [0051] Figure 14 shows purified PRP-LTS61 conjugates analyzed by IEF PAGE to confirm the purity of purified conjugate vaccine. [0052] Table 2 below is the result of a GM1 binding assay that confirms that LTS61K protein retained its binding activity following conjugation. Table 2 Mammalian pre-clinical studies showing efficacy of the polysaccharide-LTS61K conjugates: [0053] New Zealand White rabbits were immunized intramuscularly three times at two week intervals using the intended human dose of the polysaccharide polysaccharide mixed with LTS61K or polysaccharide-LTS6K conjugate to evaluate the potency of the conjugate vaccines. All vaccine preparations contained no A1P04 or other related adjuvant. The immune responses of all vaccines were determined by ELISA to detect anti-polysaccharide antigen specific IgG titers and serum bactericidal assay to detect the functional activity of the antibodies. The study results indicated that only successfully conjugated polysaccharide-LTS61 K vaccines could induce higher poly sac charide-specific IgG antibody titers and greater bactericidal activity in sera than that of polysaccharide alone or polysaccharide mixed with LTS61K only. The animal immunogenicity studies suggest that detoxified E. coli heat-labile enterotoxin holotoxin including LTS61K protein is useful as carrier protein of polysaccharide to significantly stimulate a specific immune response of polysaccharide antigens. [0054] Reference is now made to additional Figures of drawings that pertain to results of initial mammalian studies. [0055] Figure 15 summarizes a rat immunogenicity study of Hib PRP-LTS61K conjugates of the present invention. [0056] Figure 16 summarizes a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention. [0057] Figure 17 illustrates results of a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titer assay and anti-PRP IgG Ab titers. Only the PRP-LTS61K conjugates were able to induce anti-PRP IgG Ab titers. The extra added LTS61K into the PRP-LTS61K conjugates did not enhance the anti-PRP IgG Abs. [0058] Figure 18 illustrates additional information about the rabbit immunogenicity study. [0059] Figure 19 illustrates results of a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titers (BA) and anti-PRP lgG Ab titers (OD). [0060] Additional studies were conducted employing the procedures and observing the results set forth below: [0061] New Zealand White rabbits were immunized intramuscularly three times and four times at two week intervals using the intended human dose of the polysaccharide polysaccharide mixed with LTS61K and polysaccharide-LTS61K conjugate to evaluate the • potency of the conjugate vaccines. All preparations contained no A1P04 or other related adjuvant. The immune responses of all vaccines were determined by ELISA to detect anti-polysaccharide antigen specific IgG titers and serum bactericidal assay to show the functional activity of the antipolysaccharide antibodies. The responses of sera anti-LTS61K IgG titers were also determined by ELISA and in vitro Caco-2 cell cAMP induction and Y-l adrenal cell toxicity rounding up study were used to determine the sera anti-LTS61K Ab neutralizing ability with the wild type LT. In vivo rabbit ileal loop challenge studies were conducted to examine the function of the anti-LTS61K antibodies. The animal immunogenicity studies suggest that detoxified E. coli heat-labile enterotoxin holotoxin including LTS61K protein is useful as earner protein of polysaccharide to significantly stimulate a specific response of polysaccharide antigens. LTS61K itself can be used as an immunogen and effectively produces antibodies against wild type LT. [0062] A. Rabbit immunogenicity studies. Rabbit immunization intramuscularly using 10 ug dose of PRP with test articles including: PRP PRP conjugated with LTS61K and PRP mixed with LTS61K; two and three boosters were given at biweekly intervals. Blood samples were collected at 14 days after each immunization and collected serum samples were stored at -80°C until use. [0063] B. Anti-polysaccharide and anti-LTS61K antibodies determined by ELISA. PRP-BSA conjugate was coated on the 96- well plate in the concentration of PRP at 100 ng/well for anti-PRP antibodies determination and coated with LTS61K at 250 ng/well for the anti-LTS61K antibodies determination. The coated plates were incubated for 16 hours at 4°C and following incubated with 5% skim milk as blocking buffer for one hour at 37°C . Animal sera were tested starting from a dilution of 1 :50. Specific antibodies were measured using a horseradish peroxidate-conjugated goat anti-rabbit IgG incubated for 1 hour at 37°C and revealed the antibodies by adding TMB (tetramethyl benzidine) peroxidase substrate and after 10 minutes the reaction was terminated by the addition of 12% H2S04 and the absorbance was read at OD (optical density) 650-450 nm (reference wavelength 650 mn). The ELISA titers were expressed as the reciprocal of the last dilution which gave OD 450 =0.5. [0064] C. Serum bactericidal assay. Antibody dependent complement mediated bactericidal activity was conducted for the serum diluted titers that kill more than 50% of the Haemophilus influenzae type b (Hib) colonies. Serum samples were pretreated in 56°C for 30 minutes to inactivate the complement of rabbit serum. Two fold serial dilutions of the serum samples with volume of 10 uL in 96-well U-bottom plates were prepared. Following added 20 uL of the diluted Hib culture which was prepared in 1000 CFU/20 uL. The mixtures were further incubated at 37°C with 5% C02 for 15 minutes and added 50 uL of the 1 : 1 hank""s buffer diluted baby rabbit complement into each well and incubated the mixture at 37°C with 5% C02 for 60 minutes. Plated 5 uL of the baby rabbit complement added mixture in the chocolate agar plates and incubated the plates at 37°C with 5% C02 for 16 hours. Counted the Hib colony forming units and determined the serum dilution titers that killed more than 50% Hib colonies. All the serum samples are compared with the reciprocal number of dilution titers in graphics. [0065] D. Induction of cAMP study in Caco-2 cells by the neutralized wild type LT with rabbit anti-LTS61K serum antibody was performed using a cyclic AMP (cAMP) EIA kit (Enzo Life Science). Ten nanograms of wild type LT were incubated with 150 uL anti-LTS61K rabbit serum (1 : 100 dilution) at room temperature. After one hour incubation the mixture was added to the Caco-2 cell plate (5xl04cell/well) and Caco-2 cells were further incubated at 37°C with 5% C02 for 2 hours. Following cells were washed with PBS and lysed with 0.1M HCL (200 uL per well) and neutralized with 0.1 M NaOH. The cell lysis products were collected by centrifugation at 660 xG for 10 mins at room temperature. The resultant suspensions were assayed for intracellular cAMP levels by utilizing the commercial EIA kit. [0066] E. Neutralization study of wild type LT with serious dilutions of rabbit anti-LTS61K serum was examined on Y-l mouse adrenal tumor cells. For the Y-l cell assay serial two fold dilutions of rabbit sera 5 OX 100X tol02 400X were premixed with wild type LT 10" 5ug (which is the EC 50 that the toxin concentration of the wild type LT to produce greater than 50% cell rounding up on Y-l adrenal cell) following the mixture were added to Y-l adrenal cell 5 x 104 cells/well. The cells were observed for morphological changes (cell rounding up) after 24 hours incubation. [0067] F. Rabbit ileal loop fluid accumulation study: the neutralization of the rabbit anti-LTS61K serum antibody against the wild type LT was conducted in the rabbit ileal loop assay. Rabbit ileal loops with 5.5 cm segments were made on the rabbits after being immunized with PRP or PRP conjugated with LTS61K or PRP mixed with LTS61K. Wild type LT in the concentration ranges from 0.01 to 1 ug were administrated to each study animal; the amount of fluid accumulated in each segment was measured after 18 hours. Results of the immunogenicity effect of polysaccharide conjugated with LTS61K: mammalian pre-clinical studies showing efficacy of the polysaccharide-LTS61K conjugates [0068] The study results are described below with reference to Figures 20 through 23 of the drawings and Tables 3 through 5. [0069] Antibodies to PRP and to LTS6 IK were determined by ELISA. Results of immunogenicity in rabbits were shown in Figure 20; only rabbits given PRP -LTS6 IK conjugates three injections elicit high anti-PRP IgG antibody titers and were 1000-fold greater than PRP or PRP mixed with LTS61K. The major immunoglobulin induced by PRP-LTS61K conjugates vaccine was IgG. The presence of high titer of anti-LTS61K IgG antibody did not interfere with the immune response of anti-PRP IgG Ab. [0070] In Figure 20 which shows the rabbit immunogenicity ELISA of anti-PRP and anti-LTS61K antibodies response results all animals received a total of three intramuscular doses each of 10 ug conjugated PRP at 2 week intervals. Sera were collected and assayed at one week post dose 1 (1 WPD1) one week post dose 2 (1 WPD2) and two weeks post dose 3 (2WPD3) as depicted in Figure 20. [0071 ] The protective potential of the anti-PRP antibodies induced by the conjugates were evaluated by testing the bactericidal activity of the rabbit sera. The bactericidal titers were determined against Haemophilus influenzae type b (Hib) Eagen strain data were presented as reciprocal serum dilution that killed more than 50 % of the Hib colonies. Figure 21 shows that rabbits after three times intramuscular immunization at two week intervals in PRP 10 ug per dose the anti-PRP IgG Abs and bactericidal titers were similar in all of the three batches of in-house prepared PRP-LTS61K conjugates with the conjugated PRP average repeating units in 40 or 10 and the PRP-LTS61K conjugates doses 10 ug/48 ug to 10 ug/30 ug per dose. Meanwhile PRP and PRP mixed with LTS61K immunized rabbit sera were unable to kill the Hib bacterial which reciprocal titers [00721 Figure 21 shows rabbit serum bactericidal assay and anti-PRP IgG antibodies responses after rabbits received three intramuscular doses each of 10 ug conjugated PRP at 2 week intervals. Tested sera were collected at day 43 two weeks post dose 3. [0073] A re-boosted study was conducted on rabbits after they received a primary -three immunization of the PRP-LTS61K conjugates. The results are summarized in Figure 22 which demonstrated bactericidal titers and anti-PRP IgG antibodies titers gradually decreased with time although 6WPD3 (6 weeks post dose 3) rabbit sera bactericidal titers were lower than the rabbit sera 2WPD3 the rabbit sera anti-PRP IgG antibodies and bactericidal titers could be effectively enhanced after a following dose of PRP-LTS61K conjugates. However these phenomena could not be observed on the PRP or PRP mixed with LTS61K immunized rabbits. [0074 j Regarding Figure 22 rabbits were immunized with different ratios of PRP-LTS61K conjugates. The sera were obtained at different times and ELISA anti-PRP IgG Abs and bactericidal assays were performed. The results demonstrate correlations between the serum anti-PRP IgG antibodies and the serum bactericidal activities and that a 4 immunization on rabbits with PRP-LTS61K conjugates effectively re-boosts the anti-PRP Abs. [0075] Table 3 shows that wild type LT toxin at 10 ng was unable to stimulate an increase of intracellular releasing of the cAMP levels in Caco-2 cells only when rabbit sera with high anti-LTS61K antibodies; those were PRP-LTS61K conjugates and PRP mixed with LTS61K immunized rabbit sera. In contrast rabbit sera obtained from PRP immunized rabbits did not prevent wild type LT from increasing cAMP levels in Caco-2 cells. Table 3 : Induction of cAMP study in Caco-2 cells by the neutralized wild type LT with rabbit anti-LTS61K serum antibody. [0076] A neutralization study of wild type LT with serial dilutions of rabbit anti-LTS61K serum was conducted on Y-1 mouse adrenal tumor cells. The results in Table 4 demonstrate that only those rabbits"" sera present with the anti-LTS61K antibodies effectively neutralize the toxicity of the wild type LT which occurred on PRP- LT61K conjugates and PRP mixed with LTS61K immunized rabbit sera but not in sera of rabbits immunized with PRP. Table 4 Neutralization Study [0077] The data in Table 5 shows that at the concentration of 0.01 ug per 5.5 cm segment ileal loop of wild type LT fluid accumulation was induced in the rabbit with no anti-LTS61K antibodies; however in the rabbit with anti-LTS61K antibodies there was no fluid accumulation. These results confirm those previously obtained in vitro Caco-2 cAMP induction and Y-l adrenal cell neutralization studies that serum anti- LTS61K antibodies are able to neutralize the wild type LT. Severe hemorrhagic lesions on ileum mucosa were observed on the rabbit without anti-LTS61K Ab administered with wild type LT in 0.5 and 1.0 ug per segment. Table 5 : Rabbit ileal loop fluid accumulation study. Fluid accumulation is measured in each 5.5 cm segment of rabbit ileal loop. Severe hemorrhagic lesions on ileum mucosa were observed on the rabbit without anti-LTS61K Ab administered wild type LT in 0.5 and 1.0 ug per segment. [0078] The results of an animal immunogenicity study as an example of pneumococcal polysaccharide serotype 14 covalent conjugated with LTS61K is shown in Figure 23. Antibody titers to PNPS serotype 14 were determined by ELISA. Samples were PNPS 14-LTS61K conjugates with PS average repeating units of 94 or 40 PNPS 14 alone PNPS 14 mixed with LTS61K and PBS. Only the conjugated products were able to induce high anti-PNPS 14 IgG antibodies. The presence of the chemical adjuvant aluminum hydroxide during the immunization program did not benefit or significantly enhance the anti-PNPS 14 antibodies. 10079] Figure 23 shows the immunogenicity in mice on pneumococcal PS serotype 14-LTS61K conjugates. Only animals given PNPS 14-LTS61K conjugates elicit high anti-PS IgG antibody titers and were 500-fold greater than PNPS 14 or PNPS 14 mixed with LTS61K. [0080] Many modifications and alterations of the present invention will become apparent to those skilled in the art without departing from the spirit and scope of the present invention which is defined by the claims. We Claim:- What is claimed is: 1. A conjugate of a polysaccharide and a detoxified E. coli heat labile enterotoxin (LT) useful as vaccine. 2. A conjugate of claim 1 which is to protect or immunize against the effects of an infectious bacteria. 3. A conjugate of claim 1 where the infectious bacteria is Haemophilus influenzae or Streptococcus pnemoniae. 4. A conjugate of claim 1 where (LT) comprises a mutant protein LT61. 5. A conjugate of claim 1 where (LT) comprises the mutant protein LTS 6 IK. 6. A conjugate of claim 1 where the polysaccharide is polyribosylribitol phosphate. 7. A vaccine for administration to mammals which comprises a purified conjugate of claim 1. 8. A vaccine for administration to mammals which comprises a purified conjugate of claim 4. 9. A vaccine for administration to mammals which comprises a purified conjugate of claim 5. 10. A method of protecting or immunizing mammals from the effects of infectious bacteria which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1. 1 1. A method of protecting or immunizing mammals from the effects of infectious bacteria which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 5. 12. A method of conjugating a polyribosylribitol phosphate and a mutant protein antigen (LT) of an infectious bacteria which comprises periodate oxidation of a polysaccharide followed by reductive amination of the resulting polysaccharide and an (LT) species and isolating the resulting conjugate. 13. A method of claim 12 wherein the mutant protein antigen (LT) species comprises LTS61K. 14. A covalent conjugate of a bacterial capsular polysaccharide antigen of Haemophilus influenzae type b or Streptococcus pneumoniae and a mutant protein LTS61K. 15. A method of producing a covalent conjugate of a polysaccharide and LTS61K which comprises subjecting a bacterial capsular polysaccharide antigen of Haemophilus influenzae type b or Streptococcus pneumoniae to reductive amination and then isolating the resulting covalent conjugate. 16. A method of claim 14 wherein the range of molar ratios of PRP:LT is between about 3 : 1 and about 60: 1 and mole/mole I04/T>RP is between about 0.1 and about 0.4. 17. A method of protecting or immunizing mammals from the effects of travel diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1. 18. A method of protecting or immunizing mammals from the effects of diarrhea from enterotoxigenic E. coli which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1. 19. A method of protecting or immunizing mammals from the effects of diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 4. 20. A method of protecting or immunizing mammals from the effects of diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 5. Dated this 23rd day of October 2012

Full Text

FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. POLYSACCHARIDE CONJUGATION WITH DETOXIFIED E. COLI HEAT LABILE ENTEROTOXIN (LT) USED AS VACCINE

2.

1. (A) DCB-USA LLC
(B) UNITED STATES OF AMERICA
(C) 1007 NORTH ORANGE STREET 9TH FLOOR NEW CASTLE COUNTY
WILMINGTON DELAWARE 19801 UNITED STATES OF AMERICA

The following specification particularly describes the invention and the manner in which it is to be performed.

Cross-Reference to Related Applications:

[0001] Priority is claimed to U.S. Provisional Patent

Application Serial No. 61/330 650 filed on May 3 2010 the disclosure of which is incorporated herein by reference in its entirety.

Background of the Invention:

[0002] Polysaccharide vaccines when prepared without carrier protein lack immune memory responses. Currently known conjugated vaccines include bacterial capsular polysaccharides such as Haemophilus influenzae type b Neisseria meningitidis group C and Streptococcus pnemoniae serotypes 1 3 4 5 6B 7F 9V 14 19F 23F. These vaccines are covalently conjugated to a carrier protein such as tetanus toxoid diphtheria toxoid CRM197 a mutant nontoxic diphtheria toxin or Neisseria meningitidis outer membrane protein. These

polysaccharide conjugate vaccines could induce T-cell dependent response especially in infants below the age of two years; and they could prime long term immunologic memory produce high affinity antibody and could lower the rate of nasopharyngeal colonization and transmission.

However the majority of the currently marketed bacterial

polysaccharide conjugate vaccines applied tetanus toxoid (TT) or diphtheria toxoid (DT) as a carrier protein. TT and DT these two toxoid proteins are regular vaccines for infants/children; high frequency vaccination of TT and DT within a short time could have impact on the immunogenicity and safety. (Reduced response to multiple vaccines

sharing common protein epitopes that are administered simultaneously to infants. Infect. Immun. 1998; 66(5):2093-8; Immunogenicity and safety of a combination pneumococcal-meningococcal vaccine in infants: a randomized controlled trial. JAMA 2005 ;293( 14): 1751-8). Therefore this invention provides a new type of the carrier protein LTS61K for its use on the conjugate vaccine.

Summary of the Invention:

[0003] This invention includes polysaccharide conjugation with a detoxified E. coli heat labile enterotoxin (LT) useful as vaccine to protect or immunize against the effects of infectious bacteria such as Haemophilus influenzae and Streptococcus pneumoniae and alleviate travel diarrhea caused by enterotoxigenic E. coli.

[0004] One aspect of this invention relates to covalently conjugated polysaccharide-LTS61K vaccines isolated in purified form. These conjugated products have unexpectedly superior immunogenic and bactericidal properties in mammals. Another aspect relates to a method of administering an effective amount of conjugated polysaccharide-LTS61K vaccine to a mammal in need of protection from Haemophilus influenzae type b (Hib). The vaccines of the present invention stimulate T-helper cell response exhibit strong booster response upon re-exposure and have high antibody titers.

[0005] Yet another aspect of the invention relates to the method of producing conjugated polysaccharide-LTS61K by reductive amination and isolating purified conjugated product. In accordance with the present invention it was discovered that the preferred method of conjugating polysaccharide and LTS61K for making the vaccine of the

present invention is periodate oxidation of native PS followed by reductive amination.

[0006] The LTS61K employed in the conjugates of the present invention is described in PCT Application Number

PCT/US2007/075801 filed August 13 2007; in US Applications

Numbers US 1 1/779419 filed July 18 2007 US 12/120 953 filed May 15 2008 and US 12/729 649 filed March 23 2010; and in Taiwan Patent Application No. 95139707 filed October 27 2006 and issued January 2009. The subject matter described in each of these applications is hereby incorporated by reference.

Brief Description of the Figures:

[0007 ] Figure 1 is a generalized depiction of a LT carrier protein.

[0008] Figure 2 is the structural representation of Hib PRP saccharides.

[0009] Figure 3 shows several conjugation methods.

[0010] Figure 4 shows a reductive amination method according to the present invention.

[0011] Figure 5 depicts PRP and LTS61K conjugation by reductive amination.

[0012] Figure 6 shows the HPLC-SEC-RI elution profile of oxidized-PRP after NaI04 treatment.

[0013] Figure 7 is an NMR spectrum of oxidized-PRP. This confirms the formation of aldehyde groups on PRP after periodate oxidation.

[0014] Figure 8 shows a Far-UV CD spectra that confirms no secondary structure difference between LTS61K and PRP-LTS61K conjugated samples; and also a Fluorescence spectra which confirms no ? max tertiary structure difference between LTS61K and PRP-LTS61K conjugated samples.

[0015] Figure 9 shows amino acid analyses that confirm the successful conjugation of PRP to LTS61K and the formation of a covalent bond.

[0016] Figure 10 shows SDS-PAGE Western Blotting analysis to confirm the covalent conjugation between PRP and LTS61K.

[0017] Figure 11 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE.

[0018] Figure 12 shows IEF Western Blotting to confirm the covalent conjugation between PRP and LTS61K.

[0019] Figure 13 confirms the purity of purified conjugate vaccine via HPLC-SEC-UV-MALLS-RI.

[0020] Figure 14 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE to confirm the purity of purified conjugate vaccine.

[00211 Figure 15 summarizes a rat immunogenicity study of Hib P P-LTS61 conjugates of the present invention.

[0022] Figure 16 summarizes a rabbit immunogenicity study of Hib PRP-LTS61 K conjugates of the present invention.

[0023] Figure 17 illustrates results of a rabbit

immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titer assay and anti-PRP lgG Ab titers.

[0024] Figure 18 illustrates additional information about the rabbit immunogenicity study.

[0025] Figure 19 illustrates results of a rabbit

immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titers (BA) and anti-PRP lgG Ab titers (OD).

[0026] Figure 20 shows results of additional rabbit

immunogenicity ELISA of anti-PRP and anti-LTS61K antibodies response studies.

[0027] Figure 21 shows the results of additional rabbit serum bactericidal assay and anti-PRP IgG Ab.

[0028] Figure 22 shows results of additional rabbit serum bactericidal assay and anti-PRP IgG Ab after a fourth immunization.

[0029] Figure 23 shows the immunogenicity in mouse on pneumococcal PS serotype 14-LTS61K conjugates.

Detailed Description of the Invention:

[0030] We have now discovered that conjugated

polysaccharide-LTS61K vaccines made in accordance with the present invention surprisingly induce higher polysaccharide-specific IgG antibody titers and greater bactericidal activity in sera than that of polysaccharide or polysaccharide mixed with LTS61K.

[0031] A list of abbreviations employed herein is as follows:

CFU: colony forming unit

ELISA: enzyme linked immunosorbent assay

Hib: Haemophilus influenzae type b

IEF: isoelectric focusing

LT: heat labile enterotoxin

MALLS: multiple angle laser light scattering

OD: optical density

PNPS: pneumococcal polysaccharide

PRP: polyribosyl ribitol phosphate

PS: polysaccharide

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC_HPLC: size exclusion high pressure liquid chromatography

RI: reflective index

[0032] The new carrier protein employed in the conjugates of the present invention is detoxified recombinant E. coli heat-labile enterotoxin mutant LT more specifically LTS61K. In the LT mutant the A and B sub-units form a typical AB5 holotoxin structure. The detoxified LT mutant (LTS61K) contains a mutated mature sub-unit A (LTA) that includes K at amino acid position 61 and a wild-type mature sub-unit B (LTB). LTS61K renders the product significantly less toxic than wild-type LT. The carrier protein is depicted in Figure 1 of the drawings.

[00331 The invention of the LTS61K which was selected as one of the starting materials for the present invention is based on the unexpected discovery that an LT containing a mutated LTA exhibits reduced toxicity compared to its wild type counterpart while retaining immunogenicity. This mutated LTA has an amino acid substitution at the position corresponding to position 61 of a wild-type LTA whose amino acid sequence SEQ ID NO: 5 is shown in the cited patent applications incorporated by reference herein. Accordingly LTS61K features an isolated polypeptide including a mutated LTA that contains an amino acid residue other than S T and F at the position corresponding to position 61 of SEQ ID NO: 5. The substituting amino acid residue can be D E H I K L N P Q R Y or W. It can be a naturally occurring amino acid or a non-naturally occurring amino acid e.g. a D-amino acid or a ß-amino acid. In one example the LTA has the amino acid sequence of SEQ ID NO:2 4 8 or 10. An LT containing this mutated LTA exhibits reduced toxicity i.e.

[0034] In accordance with the present invention

Haemophilus influenzae type B was cultured and its capsular

polysaccharide antigen polyribosylribitol phosphate (PRP) was purified. The PRP is linear has a negative charge and is hydrophilic. The Hib PRP saccharide with molecular weight 345 and formula 10C 18H 1 10 IP is depicted as in Figure 2 of the drawings.

[0035] PRP is conjugated onto LTS61K by a chemical reductive amination reaction employing appropriate molar ratios of polysaccharide PRP to protein (LTS61K) which produce conjugate vaccines. Ranges of molar ratios of PRP:LTS61K are shown in Table 1 below. Preferably the range of molar ratios of PRP:LTS61K is between about 3 : 1 and about 60:1 and mole /mole I047PRP is between about 0.1 and about 0.4.

Table 1. Different ratios of PRP/LTS61K conjugation test

[0036] Further in accordance with the present invention a variety of different types of the bacterial capsular polysaccharides antigens from Haemophilus influenzae type b and Streptococcus pneumoniae are chemically conjugated with the LTS61K protein by reductive amination reactions. A variety of different molar ratios of NaI04:PRP as shown in Table 1 above were employed to oxidize polysaccharides to produce short lengths of polysaccharides with different repeating units. Following the proper molar ratio of the polysaccharide reacted with LTS61K the successfully conjugated and purified polysaccharide-LTS61K conjugate products were obtained. The products were in soluble form and were physically and chemically evaluated by SEC-HPLC orcinol SDS-PAGE western blotting IEF GM1 -binding activity circular dichroism and fluorescent assays to identify the successful chemical conjugation of the polysaccharide antigen and LTS61K carrier protein.

Example of the procedure of making polysaccharide conjugated with LTS61K:

[0037] A variety of potential conjugation processes are illustrated in Figure 3 of the drawings. A generalized flow diagram of the reductive amination process selected in accordance with the present invention is found at Glyconjugate J. 1989.6:489 and is reproduced as Figure 4 of the drawings.

[0038] In accordance with the present invention a

polysaccharide is cleaved by periodate oxidation to smaller fragments to produce aldehyde end groups. Thereafter conjugation of the oxidized polysaccharide to LTS61K protein is carried out by reductive amination. More specifically an example of the process is as follows:

[0039] A. Polysaccharide activation (periodate oxidation): Native polyribosylribitol phosphate (PRP) which is purified capsular polysaccharide of Haemophilus influenzae type b in an amount of 5 mg/mL is mixed with periodate (I04") 0.3 or 0.6 or 1.2 mg/mL (molar ratio (m/m) of I04" to PRP = 0.1 0.2 0.4). This mixture was allowed to stand at 4°C in the dark for 24 hours. Then glycerol was added to terminate the reaction. The resulting oxidized PRP underwent dialysis against ddH20 using 3.5 K membrane to remove impurities. The resulting product was sterile filtered with 0.22 um filter. The periodate oxidation procedure provided fragmentation of the polysaccharide PRP into different chain lengths. The range of average number of repeating units of PRP was about 40 to 10.

[0040] B. Polysaccharide-LTS61K conjugation: Preparation of PRP and LTS61K conjugate mixture of oxidized PRP obtained from above purified LTS61K (which was disclosed in the previous

obtained/filed patents) and sodium cyanoborohydride (NaBH3CN) in 10 mg/mL 3 mg/mL and 10 mg/mL respectively the reaction was earned for 2 weeks at 4°C in the dark. The reaction was terminated by adding sodium borohydride NaBH4 to quench unreacted aldehydes on PRP the preparation was then dialyzed against ddH20 with 10 K membrane.

[0041] C. Characterization of the polysaccharide conjugated LTS61K products: Following the proper molar ratio of the

polysaccharide reacted with LTS61K the successfully conjugated and purified polysaccharide-LTS61K conjugate products were obtained. The products with soluble form were physically chemically and biologically evaluated by SEC-HPLC Orcinol SDS-PAGE Western blotting IEF GM1 -binding activity circular dichroism and fluorescent assays to confirm the successful chemical conjugation of the polysaccharide antigen and LTS61K earner protein ratio of polysaccharide to LTS61K protein and its immunogenicity. Figure 5 of the drawings summarizes reaction conditions and assays employed in accordance with specific exemplification of the present invention.

[0042] Reference is now made to the Figures of drawings which pertain to confirmation of structure and purity of the conjugates and vaccine.

[0043] Figure 6 shows the HPLC-SEC-RI elution profile of oxidized-PRP after NaI04 treatment. This figure illustrates the process of the invention and results are readily reproducible.

[0044] Figure 7 is an NMR spectrum of oxidized-PRP. This confirms the formation of aldehyde groups on PRP after periodate oxidation.

[0045] Figure 8 shows a Far-UV CD spectra that confirms no secondary structure difference between LTS61K and PRP-LTS61K conjugated samples; and also a fluorescence spectra which confirms no ? max tertiary structure difference between LTS61K and PRP-LTS61K conjugated samples.

[0046] Figure 9 shows amino acid analyses that confirm the successful conjugation of PRP to LTS61K and the formation of a covalent bond.

[0047] Figure 10 shows SDS PAGE Western Blotting analysis to confirm the covalent conjugation between PRP and LTS61K.

[0048] Figure 1 1 shows purified PRP-LTS61K conjugates analyzed by IEF PAGE.

[0049] Figure 12 shows IEF Western Blotting to confirm the covalent conjugation between PRP and LTS61K.

[0050] Figure 13 confirms the purity of purified conjugate vaccine via HPLC-SEC-UV-MALLS-RI.

[0051] Figure 14 shows purified PRP-LTS61 conjugates analyzed by IEF PAGE to confirm the purity of purified conjugate vaccine.

[0052] Table 2 below is the result of a GM1 binding assay that confirms that LTS61K protein retained its binding activity following conjugation.

Table 2

Mammalian pre-clinical studies showing efficacy of the polysaccharide-LTS61K conjugates:

[0053] New Zealand White rabbits were immunized intramuscularly three times at two week intervals using the intended human dose of the polysaccharide polysaccharide mixed with LTS61K or polysaccharide-LTS6K conjugate to evaluate the potency of the conjugate vaccines. All vaccine preparations contained no A1P04 or other related adjuvant. The immune responses of all vaccines were determined by ELISA to detect anti-polysaccharide antigen specific IgG titers and serum bactericidal assay to detect the functional activity of the antibodies. The study results indicated that only successfully conjugated polysaccharide-LTS61 K vaccines could induce higher poly sac charide-specific IgG antibody titers and greater bactericidal activity in sera than that of polysaccharide alone or polysaccharide mixed with LTS61K only. The animal immunogenicity studies suggest that detoxified E. coli heat-labile enterotoxin holotoxin including LTS61K protein is useful as carrier protein of polysaccharide to significantly stimulate a specific immune response of polysaccharide antigens.

[0054] Reference is now made to additional Figures of drawings that pertain to results of initial mammalian studies.

[0055] Figure 15 summarizes a rat immunogenicity study of Hib PRP-LTS61K conjugates of the present invention.

[0056] Figure 16 summarizes a rabbit immunogenicity study of Hib PRP-LTS61K conjugates of the present invention.

[0057] Figure 17 illustrates results of a rabbit

immunogenicity study of Hib PRP-LTS61K conjugates of the present invention results of the rabbit serum bactericidal titer assay and anti-PRP IgG Ab titers. Only the PRP-LTS61K conjugates were able to induce anti-PRP IgG Ab titers. The extra added LTS61K into the PRP-LTS61K conjugates did not enhance the anti-PRP IgG Abs.

[0058] Figure 18 illustrates additional information about the rabbit immunogenicity study.

[0059] Figure 19 illustrates results of a rabbit

immunogenicity study of Hib PRP-LTS61K conjugates of the present

invention results of the rabbit serum bactericidal titers (BA) and anti-PRP lgG Ab titers (OD).

[0060] Additional studies were conducted employing the procedures and observing the results set forth below:

[0061] New Zealand White rabbits were immunized intramuscularly three times and four times at two week intervals using the intended human dose of the polysaccharide polysaccharide mixed with LTS61K and polysaccharide-LTS61K conjugate to evaluate the • potency of the conjugate vaccines. All preparations contained no A1P04 or other related adjuvant. The immune responses of all vaccines were determined by ELISA to detect anti-polysaccharide antigen specific IgG titers and serum bactericidal assay to show the functional activity of the antipolysaccharide antibodies. The responses of sera anti-LTS61K IgG titers were also determined by ELISA and in vitro Caco-2 cell cAMP induction and Y-l adrenal cell toxicity rounding up study were used to determine the sera anti-LTS61K Ab neutralizing ability with the wild type LT. In vivo rabbit ileal loop challenge studies were conducted to examine the function of the anti-LTS61K antibodies. The animal immunogenicity studies suggest that detoxified E. coli heat-labile enterotoxin holotoxin including LTS61K protein is useful as earner protein of polysaccharide to significantly stimulate a specific response of polysaccharide antigens. LTS61K itself can be used as an immunogen and effectively produces antibodies against wild type LT.

[0062] A. Rabbit immunogenicity studies. Rabbit

immunization intramuscularly using 10 ug dose of PRP with test articles including: PRP PRP conjugated with LTS61K and PRP mixed with LTS61K; two and three boosters were given at biweekly intervals. Blood samples were collected at 14 days after each immunization and collected serum samples were stored at

-80°C until use.

[0063] B. Anti-polysaccharide and anti-LTS61K antibodies determined by ELISA. PRP-BSA conjugate was coated on the 96- well plate in the concentration of PRP at 100 ng/well for anti-PRP antibodies determination and coated with LTS61K at 250 ng/well for the anti-LTS61K antibodies determination. The coated plates were incubated for 16 hours at 4°C and following incubated with 5% skim milk as blocking buffer for one hour at 37°C . Animal sera were tested starting from a dilution of 1 :50. Specific antibodies were measured using a horseradish peroxidate-conjugated goat anti-rabbit IgG incubated for 1 hour at 37°C and revealed the antibodies by adding TMB (tetramethyl benzidine) peroxidase substrate and after 10 minutes the reaction was terminated by the addition of 12% H2S04 and the absorbance was read at OD (optical density) 650-450 nm (reference wavelength 650 mn). The ELISA titers were expressed as the reciprocal of the last dilution which gave OD 450 =0.5.

[0064] C. Serum bactericidal assay. Antibody dependent complement mediated bactericidal activity was conducted for the serum diluted titers that kill more than 50% of the Haemophilus influenzae type b (Hib) colonies. Serum samples were pretreated in 56°C for 30 minutes to inactivate the complement of rabbit serum. Two fold serial dilutions of the serum samples with volume of 10 uL in 96-well U-bottom plates were prepared. Following added 20 uL of the diluted Hib culture which was prepared in 1000 CFU/20 uL. The mixtures were further incubated at 37°C with 5% C02 for 15 minutes and added 50 uL of the 1 : 1 hank""s buffer diluted baby rabbit complement into each well and incubated the mixture at 37°C with 5% C02 for 60 minutes. Plated 5 uL of the baby rabbit complement added mixture in the chocolate agar plates and incubated the plates at 37°C with 5% C02 for 16 hours. Counted the Hib colony forming units and determined the serum dilution titers that killed more than 50% Hib colonies. All the serum samples are compared with the reciprocal number of dilution titers in graphics.

[0065] D. Induction of cAMP study in Caco-2 cells by the neutralized wild type LT with rabbit anti-LTS61K serum antibody was performed using a cyclic AMP (cAMP) EIA kit (Enzo Life Science). Ten nanograms of wild type LT were incubated with 150 uL anti-LTS61K rabbit serum (1 : 100 dilution) at room temperature. After one hour incubation the mixture was added to the Caco-2 cell plate

(5xl04cell/well) and Caco-2 cells were further incubated at 37°C with

5% C02 for 2 hours. Following cells were washed with PBS and lysed with 0.1M HCL (200 uL per well) and neutralized with 0.1 M NaOH. The cell lysis products were collected by centrifugation at 660 xG for 10 mins at room temperature. The resultant suspensions were assayed for intracellular cAMP levels by utilizing the commercial EIA kit.

[0066] E. Neutralization study of wild type LT with serious dilutions of rabbit anti-LTS61K serum was examined on Y-l mouse adrenal tumor cells. For the Y-l cell assay serial two fold dilutions of rabbit sera 5 OX 100X tol02 400X were premixed with wild type LT 10" 5ug (which is the EC 50 that the toxin concentration of the wild type LT to produce greater than 50% cell rounding up on Y-l adrenal cell) following the mixture were added to Y-l adrenal cell 5 x 104 cells/well.

The cells were observed for morphological changes (cell rounding up) after 24 hours incubation.

[0067] F. Rabbit ileal loop fluid accumulation study: the neutralization of the rabbit anti-LTS61K serum antibody against the wild type LT was conducted in the rabbit ileal loop assay. Rabbit ileal loops with 5.5 cm segments were made on the rabbits after being immunized with PRP or PRP conjugated with LTS61K or PRP mixed with LTS61K. Wild type LT in the concentration ranges from 0.01 to 1 ug were administrated to each study animal; the amount of fluid accumulated in each segment was measured after 18 hours.

Results of the immunogenicity effect of polysaccharide conjugated with LTS61K: mammalian pre-clinical studies showing efficacy of the polysaccharide-LTS61K conjugates

[0068] The study results are described below with reference to Figures 20 through 23 of the drawings and Tables 3 through 5.

[0069] Antibodies to PRP and to LTS6 IK were determined by ELISA. Results of immunogenicity in rabbits were shown in Figure 20; only rabbits given PRP -LTS6 IK conjugates three injections elicit high anti-PRP IgG antibody titers and were 1000-fold greater than PRP or PRP mixed with LTS61K. The major immunoglobulin induced by PRP-LTS61K conjugates vaccine was IgG. The presence of high titer of anti-LTS61K IgG antibody did not interfere with the immune response of anti-PRP IgG Ab.

[0070] In Figure 20 which shows the rabbit immunogenicity ELISA of anti-PRP and anti-LTS61K antibodies response results all animals received a total of three intramuscular doses each of 10 ug conjugated PRP at 2 week intervals. Sera were collected and assayed at one week post dose 1 (1 WPD1) one week post dose 2 (1 WPD2) and two weeks post dose 3 (2WPD3) as depicted in Figure 20.

[0071 ] The protective potential of the anti-PRP antibodies induced by the conjugates were evaluated by testing the bactericidal activity of the rabbit sera. The bactericidal titers were determined against Haemophilus influenzae type b (Hib) Eagen strain data were presented as reciprocal serum dilution that killed more than 50 % of the Hib colonies. Figure 21 shows that rabbits after three times intramuscular immunization at two week intervals in PRP 10 ug per dose the anti-PRP IgG Abs and bactericidal titers were similar in all of the three batches of in-house prepared PRP-LTS61K conjugates with the conjugated PRP average repeating units in 40 or 10 and the PRP-LTS61K conjugates doses 10 ug/48 ug to 10 ug/30 ug per dose. Meanwhile PRP and PRP mixed with LTS61K immunized rabbit sera were unable to kill the Hib bacterial which reciprocal titers

[00721 Figure 21 shows rabbit serum bactericidal assay and anti-PRP IgG antibodies responses after rabbits received three intramuscular doses each of 10 ug conjugated PRP at 2 week intervals. Tested sera were collected at day 43 two weeks post dose 3.

[0073] A re-boosted study was conducted on rabbits after they received a primary -three immunization of the PRP-LTS61K

conjugates. The results are summarized in Figure 22 which

demonstrated bactericidal titers and anti-PRP IgG antibodies titers

gradually decreased with time although 6WPD3 (6 weeks post dose 3) rabbit sera bactericidal titers were lower than the rabbit sera 2WPD3 the rabbit sera anti-PRP IgG antibodies and bactericidal titers could be effectively enhanced after a following dose of PRP-LTS61K conjugates. However these phenomena could not be observed on the PRP or PRP mixed with LTS61K immunized rabbits.

[0074 j Regarding Figure 22 rabbits were immunized with different ratios of PRP-LTS61K conjugates. The sera were obtained at different times and ELISA anti-PRP IgG Abs and bactericidal assays were performed. The results demonstrate correlations between the serum anti-PRP IgG antibodies and the serum bactericidal activities and that a 4 immunization on rabbits with PRP-LTS61K conjugates effectively re-boosts the anti-PRP Abs.

[0075] Table 3 shows that wild type LT toxin at 10 ng was unable to stimulate an increase of intracellular releasing of the cAMP levels in Caco-2 cells only when rabbit sera with high anti-LTS61K antibodies; those were PRP-LTS61K conjugates and PRP mixed with LTS61K immunized rabbit sera. In contrast rabbit sera obtained from PRP immunized rabbits did not prevent wild type LT from increasing cAMP levels in Caco-2 cells.

Table 3 : Induction of cAMP study in Caco-2 cells by the neutralized wild type LT with rabbit anti-LTS61K serum antibody.

[0076] A neutralization study of wild type LT with serial dilutions of rabbit anti-LTS61K serum was conducted on Y-1 mouse adrenal tumor cells. The results in Table 4 demonstrate that only those rabbits"" sera present with the anti-LTS61K antibodies effectively neutralize the toxicity of the wild type LT which occurred on PRP- LT61K conjugates and PRP mixed with LTS61K immunized rabbit sera but not in sera of rabbits immunized with PRP.

Table 4 Neutralization Study

[0077] The data in Table 5 shows that at the concentration of 0.01 ug per 5.5 cm segment ileal loop of wild type LT fluid accumulation was induced in the rabbit with no anti-LTS61K antibodies; however in the rabbit with anti-LTS61K antibodies there was no fluid accumulation. These results confirm those previously obtained in vitro Caco-2 cAMP induction and Y-l adrenal cell neutralization studies that serum anti- LTS61K antibodies are able to neutralize the wild type LT. Severe hemorrhagic lesions on ileum mucosa were observed on the rabbit without anti-LTS61K Ab administered with wild type LT in 0.5 and 1.0 ug per segment.

Table 5 : Rabbit ileal loop fluid accumulation study.

Fluid accumulation is measured in each 5.5 cm segment of rabbit ileal loop. Severe hemorrhagic lesions on ileum mucosa were observed on the rabbit without anti-LTS61K Ab administered wild type LT in 0.5 and 1.0 ug per segment.

[0078] The results of an animal immunogenicity study as an example of pneumococcal polysaccharide serotype 14 covalent

conjugated with LTS61K is shown in Figure 23. Antibody titers to PNPS serotype 14 were determined by ELISA. Samples were PNPS 14-LTS61K conjugates with PS average repeating units of 94 or 40 PNPS 14 alone PNPS 14 mixed with LTS61K and PBS. Only the conjugated products were able to induce high anti-PNPS 14 IgG antibodies. The presence of the chemical adjuvant aluminum hydroxide during the immunization program did not benefit or significantly enhance the anti-PNPS 14 antibodies.

10079] Figure 23 shows the immunogenicity in mice on pneumococcal PS serotype 14-LTS61K conjugates. Only animals given PNPS 14-LTS61K conjugates elicit high anti-PS IgG antibody titers and were 500-fold greater than PNPS 14 or PNPS 14 mixed with LTS61K.

[0080] Many modifications and alterations of the present invention will become apparent to those skilled in the art without departing from the spirit and scope of the present invention which is defined by the claims.
We Claim:-

What is claimed is:

1. A conjugate of a polysaccharide and a detoxified E. coli heat labile enterotoxin (LT) useful as vaccine.

2. A conjugate of claim 1 which is to protect or immunize against the effects of an infectious bacteria.

3. A conjugate of claim 1 where the infectious bacteria is Haemophilus influenzae or Streptococcus pnemoniae.

4. A conjugate of claim 1 where (LT) comprises a mutant protein LT61.

5. A conjugate of claim 1 where (LT) comprises the mutant protein LTS 6 IK.

6. A conjugate of claim 1 where the polysaccharide is polyribosylribitol phosphate.

7. A vaccine for administration to mammals which comprises a purified conjugate of claim 1.

8. A vaccine for administration to mammals which comprises a purified conjugate of claim 4.

9. A vaccine for administration to mammals which comprises a purified conjugate of claim 5.

10. A method of protecting or immunizing mammals from the effects of infectious bacteria which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1.

1 1. A method of protecting or immunizing mammals from the effects of infectious bacteria which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 5.

12. A method of conjugating a polyribosylribitol phosphate and a mutant protein antigen (LT) of an infectious bacteria which comprises periodate oxidation of a polysaccharide followed by reductive amination of the resulting polysaccharide and an (LT) species and isolating the resulting conjugate.

13. A method of claim 12 wherein the mutant protein antigen (LT) species comprises LTS61K.

14. A covalent conjugate of a bacterial capsular polysaccharide antigen of Haemophilus influenzae type b or Streptococcus pneumoniae and a mutant protein LTS61K.

15. A method of producing a covalent conjugate of a

polysaccharide and LTS61K which comprises subjecting a bacterial capsular polysaccharide antigen of Haemophilus influenzae type b or Streptococcus pneumoniae to reductive amination and then isolating the resulting covalent conjugate.

16. A method of claim 14 wherein the range of molar ratios of PRP:LT is between about 3 : 1 and about 60: 1 and mole/mole I04/T>RP is between about 0.1 and about 0.4.

17. A method of protecting or immunizing mammals from the effects of travel diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1.

18. A method of protecting or immunizing mammals from the effects of diarrhea from enterotoxigenic E. coli which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 1.

19. A method of protecting or immunizing mammals from the effects of diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 4.

20. A method of protecting or immunizing mammals from the effects of diarrhea which comprises administering an effective dose of a vaccine comprising a purified conjugate of claim 5.

Dated this 23rd day of October 2012

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Patent Number

279735

Indian Patent Application Number

2449/MUMNP/2012

PG Journal Number

05/2017

Publication Date

03-Feb-2017

Grant Date

30-Jan-2017

Date of Filing

24-Oct-2012

Name of Patentee

DCB-USA LLC

Applicant Address

1007 NORTH ORANGE STREET 9TH FLOOR NEW CASTLE COUNTY WILMINGTON DELAWARE 19801 UNITED STATES OF AMERICA

Inventors:

#	Inventor's Name	Inventor's Address
1	HSU YU-SHEN	NO. 101 LANE 169 KANGNING ST. XIZHI DIST. NEW TAIPEI CITY 22180 TAIWAN R.O.C.
2	KOU I-LING	NO. 101 LANE 169 KANGNING ST. XIZHI DIST. NEW TAIPEI CITY 22180 TAIWAN R.O.C.
3	HUNG KUO-CHAN	NO. 101 LANE 169 KANGNING ST. XIZHI DIST. NEW TAIPEI CITY 22180 TAIWAN R.O.C.
4	LU YUAN-HSIN	NO. 101 LANE 169 KANGNING ST. XIZHI DIST. NEW TAIPEI CITY 22180 TAIWAN R.O.C.
5	YUAN TA-TUNG	NO. 101 LANE 169 KANGNING ST. XIZHI DIST. NEW TAIPEI CITY 22180 TAIWAN R.O.C.

PCT International Classification Number

A61K39/385,A61K39/02

PCT International Application Number

PCT/IB2011/001429

PCT International Filing date

2011-04-29

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	61/330,650 (US)	2010-05-03	U.S.A.