Definitions

• This invention relates generally to vaccines against Haemophilus influenzae.
• it relates to a conjugate vaccine in which a synthetic oligosaccharide corresponding to a fragment of the polysaccharide capsule of H. influenzae type b has been coupled to an H. influenzae adhesin protein.
• the vaccine may be used against both invasive and non-invasive H. influenzae infec ⁇ tion of humans, particularly very young infants, and other mammals.
• H. influenzae (Hi) are divided into two groups, those strains that possess a polysaccharide capsule and those that do not.
• the encapsulated strains are typed by a serological reaction of a capsule with reference antisera. Types a-f have been identified.
• the non-encapsulated strains, which fail to react with any of the reference antisera, are known as non-typeable.
• Hi are a significant health problem worldwide.
• the type b strain (Hib) is the most virulent of the Hi strains, causing meningitis, acute epiglottitis, and other life- threatening infections in children five years old and younger.
• Non-typeable Hi also cause various diseases, including pneumonia, bacteremia, meningitis, postpartum sepsis, bronchitis, sinusitis, conjunctivitis, and otitis media.
• the non-typeable Hi cause about 20-40% of all otitis media in children and young adults.
• Current therapy for chronic or repeated occurrences of otitis media generally involves antibiotic administration. Children may experience multiple infections because infection does not confer a lasting immunity.
• the polysaccharide is obtained from natural sources. Although purified, the polysaccharide fragments are of various lengths and, therefore, not as well characterized as desirable. This creates problems with respect to reproducibility and variable potency. Also, since naturally occurring polysaccharide must be isolated from a pathogen, safety concerns must be addressed with respect to both manufacture and use of the vaccine.
• conjugate vaccines have been shown to be safe and more immunogenic than the conventional polysaccharide vaccines in children, particularly infants.
• the data suggest that the conjugate vaccines are functioning as T-cell dependent antigens.
• a T-cell- dependent response provides for a better overall immune response in a patient.
• One of the conjugate vaccines has been approved in the United States for children 15-18 months of age.
• Two of the conjugate vaccines have been licensed in the United States for infants as young as two months old.
• the vaccines currently available to the medical practitioner have several major limitations. First, they do not protect against other Hi infections besides Hib. The polysaccharide is not found in non-typable U____ influenzae; therefore, antibodies to it are non-protective against these strains. Second, they raise problems with respect to reproducibility, potency, and safety.
• Hib capsule consists of a linear homopolymer of alternating molecules of ribose and ribitol joined by a phosphodiester linkage represented by the following formula:
• the polymer is known as polyribosylribitol phosphate and abbreviated PRP.
• the PRP obtained from natural sources is crude degraded polysaccharide. It varies in molecular weight between 200 KD and 200,000 KD. A few groups have been able to synthesize small PRP oligosaccharides.
• European Patent Office Publication 0 320 942 dated June 21, 1989, incorporated herein by reference discloses the synthesis of synthetic PRP oligosaccharides of 2-20 units and their covalent attachment to immunogenic proteins, specifically tetanus or diptheria toxins or toxoids.
• the oligosaccharides are linked to the proteins through a spacer. A phosphite triester synthetic procedure was used for the oligomerization.
• the oligosaccharides are prepared using the phosphotriester synthetic procedure for oligomerization. Both of these involved solution-type synthetic techniques for the preparation of the PRP oligosaccharides.
• Synthetic PRP is chemically well-defined and characterized. It would be of superior quality and less prone to produce side effects in humans. Its use would also obviate problems relating to reproducibility, potency, and safety associated with PRP obtained from natural sources.
• synthetic PRP can be conjugated through a single point, which creates less undesired epitopes.
• At least one group has conjugated an Hib outer membrane protein to PRP fragments.
• P2 protein 2
• protein b/c protein b/c because they occur as a doublet.
• the molecular weight depends upon the strain from which they are obtained. They are cross-reactive, have very similar amino acid compositions, and have the same amino and carboxy terminal sequences.
• the proteins are coupled to PRP fragments by reductive amination. The PRP fragments are obtained from naturally occurring PRP using standard techniques.
• the application states that the carrier proteins themselves may confer immunity.
• the outer membrane proteins may vary among Hib types or serotypes within a particular type. Granoff, et al., in S.H. Sell and P.F. Wright (ed.), Haemophilus Influenzae: Epidemiology. Immunology, and Prevention, (New York: Elsevier Biomedical (1982)). Therefore, a vaccine based upon a particular outer membrane protein may not be effective against the broader spectrum of pathogenic H. influenzae bacteria and may not even be effective against all strains of Hib.
• Hi proteins and peptides alone as vaccine candidates.
• PCT Publica ⁇ tion No. WO 90/02557 published March 22, 1990, incorporated herein by reference.
• This application discloses two antigenically related Hi outer membrane proteins with a molecular weight of about 16 KD. It further discloses related fusion proteins and peptide frag ⁇ ments of the outer membrane proteins, methods of purifying the proteins, and methods of making them by genetic engineering. All of these are claimed to be useful as immunogens in vaccines. Such vaccines will also have the drawbacks mentioned immediately above.
• the present invention overcomes the limitations of the existing technology and meets that need. It provides a novel synthetic PRP conjugated to newly isolated and purified H. influenzae adhesin proteins.
• adhesin proteins are believed to be highly conserved among strains of a particular type of bacteria. This is because they are the protein molecules that mediate attachment by bonding bacteria to host cells, the initial step in the infection process. Thus, the adhesins would be expected to be present in all strains (both encapsulated and unencapsulated) of Haemophilus. Therefore, the present vaccine would be effective against a broad array of types and strains of Hi.
• vaccines based upon adhesin proteins should be more ef ⁇ fective than those based upon other outer membrane proteins, even for those bacterial strains from which the outer membrane proteins are derived. Antibodies to the adhesin protein would prevent adherence of the bacteria to the tissue of the host animal.
• Adherence is the initial step in Hi infection. Stopping the infection at this point would be the best approach possible.
• the novel PRP of the invention also has advantages over the existing technology. It is better defined and characterized, and it is of superior quality when compared to PRP obtained from natural sources. Also, it has been more efficiently produced than the synthetic PRP described above.
• Another object of the invention is to provide a vaccine for protecting a mammal against H. influenzae.
• Yet another object of the invention is to provide a method of inducing an immune response to H. influenzae in a mammal.
• a further object of the invention to provide purified H. influenzae adhesin proteins.
• a still further object of the invention is to provide a purified polypeptide capable of eliciting an antigenic response to H. influenzae in an animal host.
• Yet another object of the invention is to provide methods for producing purified H. influenzae adhesin proteins.
• a further object of the invention is to provide DNA coding for the adhesin proteins and derived polypeptides, vectors containing the DNA, microorganisms transformed by such DNA and vectors, and methods for preparing such materials.
• a still further object of the invention is to provide a composition of matter consisting essentially of synthetic PRP oligosaccharides having the same number of monomeric units and a method of preparing the synthetic PRP.
• Another object of the invention is to provide compounds useful as intermediates in the preparation of synthetic PRP and methods of preparing such compounds.
• the present invention provides an immunogenic oligosaccharide-protein conjugate useful in a vaccine for protecting a mammal against H. influenzae.
• the conjugate is made up of a PRP fragment, preferably a synthetic oligosaccharide, coupled to an H. influenzae adhesin protein.
• the oligosaccharides contain from 2- 30 ribosylribitol phosphate monomers, and from 1-30 of such oligosaccharides are attached to the protein.
• the oligosaccharide is bound to a polypeptide that is an active site of the adhesin protein.
• the conjugate is represented by the following formula:
• m is 1-30, n is 2-30, R is (CH2)pCH2NH or (CH2CH 0)pCH2CH 2 NHCSNH where p is an integer from 1-3, and X is an H. influenzae adhesin protein or a fragment thereof containing an active site of the protein.
• the vaccine comprises an immunologically effective amount of the conjugate in a pharmaceutically acceptable carrier.
• the vaccine also contains an adjuvant.
• the administration of the vaccine or conjugate to a human or other mammal induces a T-cell dependent protective immune response.
• the invention further comprises an isolated and a purified H. influenzae adhesin protein and modified proteins and polypeptides derived from the adhesin protein, provided such derived proteins and polypeptides are immunologically cross-reactive with the adhesin protein.
• such derivatives are one or more epitopes of the adhesin protein.
• the epitope is also a receptor binding site.
• the proteins and polypeptides may also be used in vaccines without being conjugated to the synthetic PRP.
• the adhesin protein is a minor H. influenzae outer membrane protein with a molecular weight of about 41,000 da1tons. In another preferred embodiment, the adhesin protein is an H. influenzae outer membrane protein with a molecular weight of about 47,000 daltons.
• the adhesin protein is purified from H. influenzae bacteria.
• Hi membranes are solubilized.
• the solubilized material contains the adhesin protein. This material is separated from the insoluble material and contacted with receptors for the adhesin protein for period of time sufficient for the protein molecules to bind to the receptors.
• the receptors are attached to an insoluble solid support. As a result, the protein is separated from the solubilized material. The protein molecules are then removed from the receptors thereby being recovered in purified form.
• adhesin proteins and related polypeptides of the invention are preferably recombinant proteins and polypeptides that have been produced through genetic engineering techniques. They are produced by an appropriate host cell that has been transformed by DNA that codes for such proteins or polypeptides.
• Adhesin protein receptors or antibodies to the adhesin are used to screen a geno ic library containing H. influenzae DNA.
• the library is made of clones which contain different sequences of the DNA which have been operably and recoverably inserted into a vector, with each of the vectors containing only one sequence of the DNA.
• the monoclonal antibodies or receptors identify the clones that produce the adhesin.
• the clone is then isolated.
• the exogenous DNA sequences are recovered from the clone.
• the invention further comprises isolated or substantially purified DNA derived from this DNA, for example, by single or multiple mutations.
• DNA hybridizes with the DNA obtained from the genomic library under conditions of high stringency.
• the invention further comprises a synthetic PRP oligosaccharide represented by the following formula:
• n is an integer from 2 to 30 and R 1 is (CH2)pCHO or (CH2CH2 ⁇ )pCH2CH2NH 2 where p is an integer from 1 to 3.
• the invention provides a compound useful as an intermediate in the preparation of synthetic PRP of the invention. It is represented by the formula:
• n is an integer from 2 to 30, Bn is benzyl, and R 2 is (CH2) p CH(OR 3 )2 or (CH 2 CH2 ⁇ )pCH 2 CH2R 4 where p is an integer from 1 to 3, R 3 is an alkyl group 1-4 carbons in length, and R 4 is a group that can be converted into an amino group.
• the monomer for chain initiation is a compound represented by the following formula:
• Bn is benzyl and R 2 is (CH2)pCH(OR 3 ) 2 or (CH2CH2 ⁇ ) p CH2CH2R 4 where p is an integer from 1 to 3, R 3 is an alkyl group 1-4 carbons in length, and R 4 is a group that can be converted into an amino group.
• the phosphonate groups of the support-bound oligomer are then oxidized to form phosphate groups.
• the resulting compound is then removed from the solid support and recovered.
• R 2 is (CH2CH 2 ⁇ ) p CH 2 CH2R 4 .
• R 2 is (CH2)pCH(OR 3 ) 2
• the hydrogenated compound is further subjected to selective acid hydrolysis.
• the preferred conjugate of the invention is then prepared by coupling the synthetic PRP with the Hi adhesin protein by reductive amination where R 1 is (CH2)pCHO or, where R 1 is (CH2CH2 ⁇ ) p CH2CH2NH2, by preparing the cor ⁇ responding isothiocynate and then coupling the isothiocynate with the protein.
• R 1 is (CH2)pCHO or, where R 1 is (CH2CH2 ⁇ ) p CH2CH2NH2
• Figure 1 shows an analysis of outer membrane preparations by SDS-polyacrylamide gel electrophoresis.
• Samples included the following (lanes) : 1, total outer membrane protein preparation from Haemophilus influenzae type b stained with Coomassie blue; 2, autoradiography of 3 5 S -labeled total outer membrane proteins; 3, autoradiography of 35 S-labeled adhesin protein eluted from immobilized receptor asialo-GMi; 4, autoradiography of material eluted from immobilized globoside, a nonsense glycolipid.
• Arrow indicates the adhesin migrating between PI and P2 with a molecular weight of about 41 kD.
• Figure 2 shows the neutralization of Haemophilus adhesin to the glycolipid receptor asialo-GMi- [ 35 S]methionine-labeled membranes from Haemophilus influenza type b were incubated with serial dilutions of mouse sera and then allowed to bind to receptor (0.5 microgram/well) .
• the mouse sera used was obtained from 5 mice, designated M-0 through M5, which had been immunized with Haemophilus membranes.
• the sera from an unchallenged mouse (NMS) was used as a negative control.
• Figure 3 shows inhibition of Haemophilus membrane binding to asialo-GMi with selected monoclonal antibodies.
• [ 35 S]methionine-labeled membranes from Haemophilus were incubated with supernatants of hybridoma cultures and then allowed to bind to receptor (0.5 microgram/well) .
• a negative receptor control of Gb indicates the specificity of the receptor-ligand interaction.
• Mouse sera (M-2) (1:500 dilution) used in Figure 2 shows strong, positive inhibition.
• Media shows no inhibition of binding by membranes to asialo-GMi.
• Two classes of positively inhibiting hybridomas were found.
• Hib 10 shows total inhibition of binding.
• Hib 30 and Hib 43 show partial (about 35%) inhibition. Most hybridoma cultures, such as Hib 2, showed no inhibition. All hybridoma cultures tested for binding reacted positively with membranes in an ELISA. Error bars are included to demonstrate the variability between duplicate wells.
• Figure 4 shows the identification and characterization of the 47 kDa Haemophilus adhesin.
• the monclonal antibody which partially inhibited membrane binding, Hib 43 was reacted on Western blot to identify the molecular weight of the protein it recognizes.
• Whole cells were run after no proteinase K treatment or either treatment with proteinase K prior to lysis in sample buffer or treatment with proteinase K after lysis in sample buffer (reading from left to right) .
• Non-treatment identifies the 47 kDa protein; treatment of whole cells by proteinase K prior to lysis indicates the sensitivity of the protein to this protease in its native location; and treatment after lysis by proteinase K demonstrates the general sensitivity to this protease after disruption from that native location.
• the Escherichia coli XL-1 transformed with pMClOl, expresses the 47 kDa Haemophilus protein, which reacts with Hib 43.
• the 47 kDa protein was also sensitive to proteinase K treatment of XL-1/pMClOl whole cells.
• Figure 5 shows a restriction map of the region in Haemophilus influenza type b that encodes the 47 kDa adhesin.
• a 10.5 kbp Eco Rl fragment that produces the 47 kDa protein which reacts with Hib 43 monclonal antibody was cloned from an Haemophilus lambda ZAPII genebank.
• the helper phage R408 was used to induce a plasmid containing this insert in the vector pSK(-) .
• Figure 6 shows the glycolipid binding phenotype of Escherichia coli that express the Hib 47 kDa protein.
• XL-1/pMClOl binds with high affinity to these receptors, similar to Haemophilus.
• Figures 7A and 7B show the nucleotide sequence of hin47 (SEQ ID NO:l) and the deduced Hin47 amino acid sequence (SEQ ID NO:2).
• the nucleotide sequence is numbered above each line and the deduced Hin47 amino acid sequence is shown below the line.
• the open reading frame for the hin47 gene is between nucleotide 115 and 1503.
• the end of the putative leader sequence and beginning of the putative mature polypeptide is indicated at nucleotide base 189.
• the predicted molecular weight of the mature polypeptide is 46399 and it has a pi of 5.86.
• Figure 8 shows the Haemophilus influenza type B, strain 9795 Hin47 amino acid sequence compared with the sequences of 5 non-typable strains as designated. Identical amino acids are indicated by ⁇ . Amino acid differences that are conservative with respect to charge are noted by lower case letters. Amino acids that differ with respect to charge are noted in upper case letters.
• the invention comprises an immunogenic oligosaccharide-H. influenzae adhesin protein conjugate useful as a vaccine against H. influenzae. purified H. influenzae adhesin proteins and related proteins and polypeptides, DNA coding for the proteins and polypeptides, host cells containing the DNA and producing the proteins and polypeptides, synthetic PRP oligosaccharides and intermediates useful for their synthesis, and methods of making and using these materials.
• Immunogenic Conjugate useful as a vaccine against H. influenzae. purified H. influenzae adhesin proteins and related proteins and polypeptides, DNA coding for the proteins and polypeptides, host cells containing the DNA and producing the proteins and polypeptides, synthetic PRP oligosaccharides and intermediates useful for their synthesis, and methods of making and using these materials.
• the conjugate comprises a polyribosylribitol phosphate fragment chemically coupled to a purified H. influenzae adhesin protein.
• the PRP fragment is a synthetic oligosaccharide. From 1 to about 30 and preferably from about 5 to 20 of the natural fragments or synthetic oligosaccharides are attached to the protein.
• the fragments are attached to the protein by known techniques for covalently attaching polysaccharides to proteins or polypeptides, applied to the teachings contained herein.
• the preferred techniques here are reductive amination or isothiocyanate coupling.
• the purified adhesin protein is a minor Hi outer membrane protein with a molecular weight of about 41,000 daltons, distinct from PI or P2.
• the purified adhesin protein is an Hi outer membrane protein with a molecular weight of about 47,000 daltons, distinct from Pl- P6.
• the protein may be replaced by a polypeptide that is an active site of the adhesin protein.
• active site means an epitope (antigenic determinant) or an H. influenzae receptor binding site, which may or may not also be an epitope.
• receptor is a macromolecule that binds to an Hi adhesin protein. The macromolecule is preferably a glycosphingolipid. Without intending to limit the scope of the invention, it is believed that the binding site is an epitope.
• the PRP fragment When the PRP fragment is obtained from natural sources, it is of varying lengths, but preferably about 8 to 120 monomers in length. Such fragments are obtained by known techniques, such as those described in the above- referenced European Patent Office Publication No. 0 338 265.
• Synthetic PRP is a linear homopolymer of alternating molecules of ribose and ribitol joined by a phosphodiester linkage and represented by the formula:
• n is 2 to 30 and preferably 5-20.
• Such synthetic PRP's include those known in the art as well as the novel ones of the invention.
• the previously mentioned European Patent Office Publications 0 320 942 and 0 276 516 disclose synthetic PRP's that could be used in the conjugate of the invention.
• the synthetic PRP is a compound represented by the formula:
• n is an integer from 2 to 30 and R 1 is (CH 2 ) p CHO or (CH2CH2 ⁇ )pCH2CH 2 NH2 where p is an integer from 1 to 3.
• n is 5-20 and p is 1.
• the synthetic PRP will be associated with a counter ion.
• the ion is sodium (Na + ) .
• the synthetic oligosaccharides usually contain a chemical spacer or linker by which they are attached to the protein.
• a spacer may be any chemical linkage that serves to connect the PRP and the protein and that has limited or no adverse effect to the animal host when the conjugate is administered.
• Such spacers may include those known in the art as well as the novel spacers of the inven ⁇ tion.
• Known spacers include those disclosed in the previ ⁇ ously mentioned European Patent Office Publications 0 320 942 and 0 276 516 as well as those disclosed in U.S. Patent 4,830,852 issued May 16, 1989 to Marburg, et al., the latter of which is incorporated herein by reference.
• the chemical spacer is a moiety represented by the formula:
• R is (CH 2 ) p CH 2 NH or (CH 2 CH2 ⁇ )pCH2CH 2 NHCSNH and p is an integer from 1-3, preferably 1.
• the conjugate is represented by the following formula:
• m is 1-30, n is 2-30, R is (CH 2 ) p CH2NH or (CH2CH2 ⁇ )pCH 2 CH 2 NHCSNH where p is integer from 1-3, and X is an H. influenzae adhesin protein or a fragment thereof containing an active site of the protein.
• m is 5-20, n is 5-20, and p is 1.
• the symbol X in the above- referenced formula may also represent certain derived or modified proteins or polypeptides discussed below.
• the conjugate will be associated with a counter ion.
• the ion is Na + .
• the invention further comprises an isolated H. influenzae adhesin protein.
• isolated means that the protein is significantly free of other proteins. That is, a composition comprising the isolated protein is between 70% and 94% pure by weight.
• the protein is purified.
• purified and related terms means that the protein is at least 95% pure by weight, preferably at least 98% pure by weight, and most preferably at least 99% pure by weight.
• the protein binds to a receptor selected from the group consisting of fucosylasialo-GMl, asialo-GMl, and asialo-GM2, all of which contain the structure N-acetylgalactosamine(beta 1- 4)galactose(beta 1-4)glucose-(beta l-l)ceramide abbreviated GalNAc(betal-4)Gal(betal-4)Glc(betal-l)Cer.
• the protein also binds to another receptor, phosphatidylethanolamine.
• the protein is a minor outer membrane protein with a molecular weight of about 41 KD as determined by SDS PAGE. It is distinguishable from the various major outer membrane proteins that have been identified for Hi. In particular, the protein appears as a fainter band between the bands on a polyacrylamide gel for the outer membrane proteins known as PI and P2. See Figure 1.
• Hi adhesin protein is prepared preferably from natural sources as follows.
• Hi bacterial membranes are obtained by standard techniques and solubilized, using a solubilizing compound, such as a detergent.
• a solubilizing compound such as a detergent
• the membranes are mixed with the detergent, and the mixture is sonicated.
• the most preferred solubilizing agent is a solution containing about 1.0% to about 1.5% and preferably about 1.3% octyl- glucopyranoside.
• the adhesin protein is in the solubilized material.
• the remaining insoluble material from the membrane is separated, preferably by centrifuging.
• the supernatant is contacted with receptors that bind the protein and are attached to an insoluble solid support or matrix, such as a microtiter well or a gel, for a period of time and under conditions sufficient for the protein to bind to the receptors, thus separating the protein from the other material.
• the preferred receptors for the adhesin protein are fucosylasialo-GMl, asialo-GMl, asialo-GM2, and phosphatidylethanolamine. These receptors can be prepared in accordance with the procedures disclosed in Krivan, et al., Proc. Natl. Acad. Sci. USA. 85:6157-6161 (1988) , incorporated herein by reference.
• the most preferred receptor, asialo-GMl is also commercially available. All of these receptors, except phosphatidylethanolamine, contain the carbohydrate sequence GalNAc(betal-4)Gal(betal-4)Glc, which, accordingly, may also be used as a receptor for the purification of the adhesin protein. This sequence can be prepared using standard carbohydrate synthesis techniques.
• the adhesin protein is then eluted using the appropriate agent.
• This may be free receptor in solution, SDS elution buffer, or a chaotropic agent, such as KSCN, NaCl, or quanidine hydrochloride.
• the eluted protein is then tested against the receptor to confirm that the protein does bind to it.
• the purity of the isolated protein is analyzed by SDS-PAGE. Preferably, it will be about 99% pure after affinity purification with the most preferred receptor.
• the receptor is immobilized onto a hydrophobic gel support, such as octyl- agarose.
• This matrix is prepared by adsorbing the receptors to the hydrophobic gel in the presence of salt as described by Hirabayashi, et al. for other glycolipids. Hirabayashi, et al., J. Biochem.. 94:327-330 (1983), incorporated herein by reference.
• Photoactivatable heterobifunctional crosslinking agents have also been used to prepare glycolipid affinity matrices. Lingwood, C. , J. Lipid Res.. 25:1010-1012 (1984), incorporated herein by reference.
• the receptor-active lipid is covalently crosslinked to the gel support.
• the column is then preferably washed extensively with an appropriate buffer solution, such as TMS-buffer saline, before the protein is eluted.
• a more preferred method is to purify the adhesin by affinity chromatography using an anti-adhesin monoclonal or polyclonal antibody prepared by standard techniques.
• the antibodies are covalently linked to agarose gels activated by cyanogen bromide or succinamide esters (Affi-Gel, BioRad Inc.) or by other methods known by those skilled in the art.
• the sonic extract is loaded on the top of the gel as described above.
• the adhesin proteins comprise an H. influenzae outer membrane protein with a molecular weight of about 47,000 daltons.
• Figures 7A and 7B show the protein amino acid sequence as well as the designated nucleotide sequence of the open reading frame (ORF) encoding a 49kDa protein.
• the 49kDa protein comprises 463 amino acids (amino acids 1-463 in Figure 7A and 7B) , includes a putative signal sequence of approximately 2.5 kDa and 25 amino acids, thereby resulting in a mature protein of approximately 47 kDa and 438 amino acids (amino acids 26 through 463 on Figure 7A and B) , herein designated Hin47.
• This protein is distinguishable from the known Hi proteins P1-P6 on the basis of molecular weight and the fact that those proteins are integral membrane proteins, while this protein is an outer membrane protein.
• This protein also binds to the previous mentioned receptors as well as to sulfatide, (S ⁇ 3 ⁇ -galactose(beta 1- l)ceramide) and it is soluble in 1% Sarkosyl (N- lauroylsarcosine) .
• This protein is preferably prepared in purified form as follows. Hi membranes are extracted with a solution that removes membrane associated proteins, which produces an extract containing the adhesin protein along with other membrane associated proteins.
• this solution is a nonionic detergent, such as Sarkosyl or octylglucopyranoside.
• the insoluble material is separated from the extract, preferably by centrifugation. This produces a supernatant that contains the adhesin protein.
• the supernatant is then brought into contact with a monoclonal antibody which recognizes the adhesin protein.
• the antibody is bound to an insoluble solid support. The contact is for a period of time and under standard reaction conditions sufficient for the adhesin protein to bind to the monoclonal antibody.
• the solid support is a material used in a chromatographic column.
• the adhesin protein is then removed from the antibody, thereby permitting the recovery of protein in purified form.
• the nonionic detergent solution is removed from the supernatant before the supernatant is subjected to the affinity chromatography. Such removal is preferably accomplished by dialyzing the supernatant to produce a dialysate that is substantially free of the detergent.
• mice are immunized with Hi membranes.
• Hybridomas are prepared by fusing spleen cells from the mice with myeloma cells.
• the fusion products are screened for those producing antibodies that bind to the Hi membranes.
• the positive clones are then screened to identify those whose binding with the Hi membranes is inhibited by an Hi adhesin receptor.
• the positive hybridomas clones are isolated, and the monoclonal antibodies are recovered from those clones.
• the outer membrane proteins could be separated on a gel.
• the 47 kDa band could be cut out and injected into the mice.
• the hybridomas could be prepared and screened as described above.
• the adhesin proteins of the invention are preferably produced through genetic engineering techniques. In this case, they are produced by an appropriate host cell that has been transformed by DNA that codes for the proteins.
• the host cell is a bacterium, and most preferably the bacterium is E. coli. B. subtilis. or Salmonella.
• the DNA of the invention is an isolated or substantially purified DNA sequence (i.e., polydeoxyribonucleotide molecule) encoding a protein or polypeptide that binds to the previously mentioned receptors.
• the DNA of the invention includes an open reading frame (ORF) sequence (nucleotides 115 through 1503 in Figures 7A and B) , designated hin47. encoding an approximate 49 kDa and 463 amino acid protein, designated Hin47, as shown in Figures 7A and B.
• the DNA comprises that part of the ORF that does not code for the signal sequence (nucleotides 191 through 1503 in Figures 7A and B) .
• the term “isolated” and variations thereof means that the DNA is in isolation from DNA encoding other proteins or polypeptides normally accompanying the Hi adhesin proteins.
• the DNA of the invention includes DNA encoding the protein or polypeptide when that DNA has been cloned into a microbial vector, such as a plasmid, or into a viral vector that may be harbored by a bacteriophage, provided that such clones are isolated from clones that contain DNA encoding other proteins or polypeptides normally accompanying this one.
• the term “substantially pure” and variants thereof means that the DNA is substantially free of DNA and RNA that does not encode the proteins or polypeptides of the invention. That is, there will be no more than about 1% by weight of other DNA and RNA and preferably no more than about 0.2% by weight of other DNA and RNA in any sample that contains the DNA of the invention.
• the DNA is obtained by using either the receptors or monoclonal antibodies to the adhesins to screen an appropriate genomic library that contains H. influenzae DNA.
• a library comprises colonies of a single type of microorganism, generally bacteria like E. coli K12 (XL-1) , into which pieces of the foreign DNA have been inserted, generally by being incorporated into a plasmid, cosmid, or phage vector compatible with the microorganism.
• the library comprises clones of vectors into which different sequences of the DNA have been operably and recoverably inserted, each of the vectors containing only one sequence of the DNA.
• the vectors may be plasmids, cosmids, phagemids, or phage genomes.
• segments of DNA will have been inserted into the vectors in a manner that they will be expressed under appropriate conditions (i.e., in proper orientation and correct reading frame and with appropriate expression sequences, including an RNA polymerase binding sequence and a ribosomal binding sequence.)
• the microorganisms will be ones that do not express the adhesin protein, such as E. coli HB101.
• Clones from the library are brought into contact with the receptors or antibodies to identify those clones that bind.
• the clones are isolated and the exogenous DNA sequence is recovered from one of the clones.
• the sequence is preferably evaluated to determine if it encodes the protein.
• the genomic library comprises bacteria, such as E. coli infected by phage, preferably bacteriophage lambda. Plaques produced by the phage infected bacteria are screened by monoclonal antibodies to identify those plaques containing bacteria that produce the adhesin protein. The screening involves contacting the plaques with the monoclonal antibody to determine if binding has occurred, using standard techniques. Preferably, immunoassays are used.
• the positive clones are then isolated by purifying the positive plaques and inducing plasmid formation in the bacteria in the purified plaque with a helper phage according to standard techniques.
• colonies containing DNA that encodes an Hi adhesin protein could be detected using DYNA Beads according to Olsvick et al., 29th ICAAC, Houston, Tex. 1989, incorporated herein by reference.
• the previously described receptors would be crosslinked to tosylated DYNA Beads M280, and these receptor-containing beads would then be used to adsorb to colonies expressing the adhesin protein. Colonies not expressing the adhesin would be removed by washing, and this process would be repeated to obtain an appropriate enrichment.
• Putative adhesin expressing colonies would then be plated and confirmed by metabolically labeling each colony with 35S-methionine and testing the ability of the colony to bind to the receptor as previously described.
• the DNA from several adherring clones would be compared to identify shared sequences, and these shared sequences would be further subcloned and characterized.
• the receptors could be nonspecifically immobilized to a suitable support, such as silica or Sealite resin. This material would then be used to adsorb to colonies expressing the adhesin protein as described in the preceding paragraph.
• the gene for a specific adhesin would be localized and identified by constructing non-adherent mutants of a specific pathogen. This would be accomplished by creating mutants using a transposable element such as TnPhoA as described in Manoil et al., Proc. Natl. Acad. Sci. USA. 82:81129-81133 (1985), incorporated herein by reference. Alkaline phosphatase positive mutants would indicate mutations within exported proteins. Since the adhesin for each pathogen is located on the outer membrane surface and therefore exported, this set of mutants would contain a much reduced subset of mutants. They would then be screened for a loss in binding activity.
• a transposable element such as TnPhoA as described in Manoil et al., Proc. Natl. Acad. Sci. USA. 82:81129-81133 (1985), incorporated herein by reference. Alkaline phosphatase positive mutants would indicate mutations within exported proteins. Since the adh
• a DNA sequence for an Hi adhesin protein can be modified by known techniques in view of the teachings disclosed herein.
• different codons can be substituted that code for the same amino acid as the original codon.
• the substitute codons may code for a different amino acid that will not affect the immunogenicity of the protein or which may improve its immunogenicity.
• oligonucleotide directed, site specific mutagenesis or other techniques to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis," Science. 229:1193-1210 (1985), which is incorporated herein by reference, can be employed. Since such modified DNA can be obtained by the application of known techniques to the teachings contained herein, such DNA is within the scope of the claimed invention.
• the DNA sequence (or fragments thereof) of the invention can be used to obtain other DNA sequences that hybridize with it under conditions of moderate to high stringency (including the derived sequences discussed in the preceding paragraph) , using general techniques known in the art. That is, the hybridizing sequences are at least 90% homologous and preferably at least 95% homologous to hin47. Accordingly, the DNA of the invention includes such DNA.
• the DNA of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform a microorganism for the expression and production of the adhesins of the invention.
• Such techniques include those disclosed in U.S. Patent Nos.
• the DNA of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host cell.
• the companion DNA would depend upon the nature of the host cell, the manner of the introduction of the DNA into the host cell, and whether episomal maintenance or integration is desired.
• the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
• an expression vector such as a plasmid
• the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector.
• the vector is then introduced into the host through standard techniques.
• the vector will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
• selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
• the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
• the preferred expression vector for use in the invention is the plasmid pMClOl.
• the preferred host cell is E. coli.
• the transformed host cells express the proteins or polypeptides of the invention. Such cells are cultured by known techniques, and the proteins or polypeptides are recovered by known techniques. Depending upon the host and expression system used, the recombinant proteins and polypeptides of the invention may be part of a fusion protein produced by the transformed host cells. Such proteins are recovered by known techniques, and the undesired part may be removed by known techniques. Alternatively, the fusion protein itself may be more immunogenic than the recombinant protein or polypeptide alone and, therefore, may itself be used in a vaccine.
• adhesins can be further purified by the application of standard protein purification techniques, modified and applied in accordance with the discoveries and teachings described herein.
• standard protein purification techniques include electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (including ion exchange chromatography, affinity chromatography, immunoadsorbent affinity chromatography, reverse-phase high performance liquid chromatography, and gel permeation high performance liquid chromatography) , isoelectric focusing, and variations and combinations thereof.
• One or more of these techniques are employed sequentially in a procedure designed to separate molecules according to their physical or chemical characteristics. These characteristics include the hydrophobicity, charge, binding capability, and molecular weight of the protein.
• the various fractions of materials obtained after each technique are tested for their ability to react with the adhesin receptors. Those fractions showing such activity are then subjected to the next technique in the sequential procedure, and the new fractions are tested again. The process is repeated until only one fraction reactive with the receptors remains and that fraction produces only a single band when subjected to polyacrylamide gel electrophoresis.
• the adhesins are purified by receptor affinity chromatography or antibody affinity chromatography.
• the adhesins of the invention may be modified by known protein modification techniques. Such modifications include breaking the protein into fragments that contain at least one active site or the addition, substitution, or deletion of one or more amino acids to the protein or a fragment thereof.
• derived proteins or polypeptides are immunologically cross-reactive with the Hi adhesin proteins, thus being capable of eliciting an antigenic response to H. influenzae in an animal host.
• H. influenzae receptor selected from the group consisting of fucosylasialo-GMl, asialo-GMl, and asialo- GM2.
• polypeptide also includes shorter chains of amino acids that are often referred to as peptides.
• modifications may enhance the immunogenicity of the protein or have no effect on such activity.
• the modification techniques include those disclosed in U.S. Patent No. 4,526,716, issued July 2, 1985 to Stevens, incorporated herein by reference.
• the proteins of the invention may contain one or more amino acid sequences that are not necessary to their immunogenicity. It may be the case, for example, that only the amino acid sequences of a particular epitope of the antigen will be necessary for immunogenic activity. Unwanted sequences can be removed by techniques well-known in the art. For example, unwanted amino acid sequences can be removed via limited proteolytic digestion using enzymes such as trypsin, papain, or related proteolytic enzymes.
• the protein binds to several related receptors having a consensus sequence, the protein should have a well conserved region that acts as the receptor binding site. This site is the particularly preferred polypeptide of the invention.
• polypeptides corresponding to various immunogenic epitopes and/or the receptor binding site of the protein may be chemically synthesized by methods well-known in the art, given the teachings contained herein. These include the methods disclosed in U.S. Patent No. 4,290,944, issued September 22, 1981 to Goldberg, incorporated herein by reference.
• Modified proteins or polypeptides can be prepared that are substantially homologous to the Hi adhesin protein or to the polypeptides discussed above through the use of known techniques and routine experimentation in view of the teachings contained herein.
• substantially homologous means immunologically cross- reactive.
• Such a protein or polypeptide may be identified by the fact that it will bind to antibodies that were made to the adhesin protein of the invention, which antibodies can be prepared by standard techniques. Some of such modified proteins or polypeptides may have enhanced immunogenicity compared to the one from which they are derived.
• the invention includes a class of derived proteins and polypeptides, including synthetically derived peptides or fragments of the adhesin protein, having common elements of origin, structure, and mechanism of action, such as immunogenic effect or being able to bind to the previously mentioned receptors, that are within the scope of the present invention because they can be prepared by persons skilled in the art without undue experimentation, once given the teachings of the present invention. Moreover, since persons skilled in the art can make modifications to or derivatives of epitopes or the receptor binding site on the proteins or polypeptides of the invention, once such epitopes or site are identified, such modifications or derivatives are within the scope of the invention. Such derived proteins and polypeptides are preferably pure as that term was previously defined herein.
• the Hi adhesin protein of the invention (as well as the related proteins and polypeptides derived therefrom) has utility not only in the conjugate vaccine but as an immunogen in its own right. Thus, it can be used in a vaccine for animals, including mammals, rodents, primates, and humans. The preferred use is a vaccine for humans, preferably children, and most preferably young infants.
• Such a vaccine can be prepared by techniques known to those skilled in the art and would comprise, for example, the antigen, a pharmaceutically acceptable car ⁇ rier, an appropriate adjuvant, and other materials traditionally found in vaccines.
• An immunologically effec ⁇ tive amount of the antigen to be used in the vaccine is determined by means known in the art in view of the teach ⁇ ings herein.
• the invention further comprises novel synthetic PRP represented by the formula:
• n is an integer from 2 to 30, preferably 5-20
• R 1 is (CH2) p CHO or (CH2CH2 ⁇ ) p CH2CH2NH2 where p is an integer from 1 to 3, preferably 1.
• the PRP of the invention is prepared by a combina ⁇ tion of solid phase synthesis and the highly efficient H- phosphonate method for the construction of the phosphodiester linkage. It also involves the use of gels with higher levels of functionalization, which are better suited for commercial scale operations.
• the general approach is to prepare a protected oligomeric ribosylribitol phosphate derivative by the following steps. First, the monomer for chain initiation is coupled to a solid phase. The monomer is represented by the formula:
• Bn is benzyl and MMTr is monomethoxytrityl. See Compound 7, Table 1.
• the preferred solid phase is a Merrifield-type amino resin.
• the chain initiation monomer (Compound 7) is coupled to the solid phase by known techniques, such as reaction with succinic anhydride, followed by coupling of the obtained succinate of Compound 7 to amino groups of the solid phase.
• the loading is determined by colorimetric quantification of the trityl cation released on acid treatment.
• the coupled compound is then detritylated, such as by treatment with trifluoroacetic acid in dichloromethane. Chain elongation is accomplished by coupling the detritylated chain initiation monomer with a compound represented by the formula:
• Bn is benzyl and MMTr is monomethoxytrityl. See Compound 8, Table 1.
• the compound will be associated with a counter ion.
• the ion is an organic cation, such as triethyl ammonium.
• the coupling is accomplished by using a condensing reagent, such as pivaloyl chloride.
• the resulting compound is then detritylated.
• the chain elongation-detritylation steps are repeated a sufficient number of times to prepare an oligosaccharide of the desired length.
• n represents the desired number of PRP monomers in the oligosaccharide
• the chain elongation-detritylation cycles are repeated n-2 times after the coupling of the chain initiation monomer and the first chain elongation monomer.
• the chain is terminated by coupling it with a chain termination monomer represented by the following formula: 0 — R
• Bn is benzyl and R 2 is (CH2)pCH(OR 3 ) 2 or (CH2CH2 ⁇ )pCH2CH2R where p is 1-3, R 3 is an alkyl group 1-4 carbons in length, and R 4 is a group that can be converted into an amino group. See Compounds 10 and 12, Table 2. (The compound will be associated with a counter ion. Preferably, the ion is an organic cation, such as triethyl ammonium.) Preferably, p is 1, and R 3 is methyl or ethyl. Preferably, R 4 is N 3 , trifluoroacetyl, benzyloxycarbonyl, or fluorenylmethoxycarbonyl.
• the phosphonate groups of the solid-bound oligomer are then oxidized to form phosphate groups. Preferably, this is accomplished by treatment with iodine in aqueous pyridin .
• the resulting compound is then removed from this solid support, preferably through cleavage by methanolysis,
• the recovered compound is represented by the formula: - 48 -
• n is an integer from 2 to 30, preferably 5 -2 0
• Bn is benzyl
• R 2 is defined as above.
• the resulting compound is then deprotecte d by hydrogenation with palladium on charcoal.
• R 2 is (C H 2)p CH(OR ) 2
• the hydrogenated compound is further su b jected to selective acid hydrolysis, such as by treatment with aqueous trifluoroacetic acid.
• the resulting PRP ol i go ers are purified by standard techniques, preferably by ion-exchange chromatography, HPL C or gel filtration. See Compounds 14 and 1 6 , Table 3 .
• Table l shows the synthesis of the chain initiation monomer.
• C ompound 7 and the chain elongation monomer.
• the readily available methyl 2, 3 - isopropylidene-beta-D-ri b ofuranoside (Compound l ) ( Leonard ⁇ - al., J. Het. C hem. 3:485 (1966), incorporate d herein by' reference ) is used as starting material. Allyiation of Compound 1 with allyl bromide/sodium hydroxide in N,N- dimethylformamide gives the expected 5-O-allyl Compound 2 as an oil that can be distilled.
• This compound is subjected to a sequence of reactions comprising hydrolysis with aqueous formic acid, sodium borohydride reduction, tritylation with triphenylmethylchloride/pyridine, benzylation with benzyl chloride/sodium hydroxide in N,N- dimethylformamide, and hydrolysis with aqueous acetic acid.
• the resulting Compound 3 is purified by silica gel chromatography.
• Glycosylation can be accomplished by several methods.
• A Compound 6 is treated with trimethylsilyl chloride to give the cor ⁇ responding glycosyl chloride, which, when treated with Compound 3 in the presence of molecular sieves, gives a ribitol glycoside.
• B Compound 6 is transesterified in the presence of Compound 3. The resulting ribitol orthoester is then rearranged in situ to give the ribitol glycoside.
• the ribitol glycoside is then subjected to debenzoylation with sodium methoxide in methanol and benzylation with benzyl chloride/sodium hydroxide in N,N- dimethylformamide.
• the resulting 5-0-allyl-2,3,4-tri-0- benzyl-1-O-(3-0-allyl-2,5-di-O-benzyl-beta-D- ribofuranosyl)-D-ribitol is deallylated by treatment with, successively, tris-(triphenylphosphine)rhodium(I)chloride and aqueous acetic acid and monomethoxytritylated with monomethoxytrityl chloride.
• the resulting chain initiation monomer (Compound 7) is purified by chromatography.
• Table 2 shows the synthesis of the monomers for chain termination.
• Compound 6 is reacted with trimethylsilyl chloride to give the corresponding chloride, which is reacted with the appropriate alcohols in the pres ⁇ ence of molecular sieves to give beta-glycosides of the alcohols.
• the alcohols are 2-(2- azidoethoxy)ethanol, 2-[2- benzyloxycarbonylamido)ethoxy]ethanol, or 2,2- diethoxyethanol.
• the beta-glycosides are subjected to the reaction sequence debenzoylation, benzylation, and deallylation, as in the preparation of Compound 7, which gives Compounds 9 or 11.
• Table 3 shows the specific PRP oligomers obtained after solid phase synthesis employing Compounds 7, 8, and 10 or 12.
• Compounds 13 and 15 are the protected oligomers after removal from the solid support, and Compounds 14 and 16 are the final oligomers after deprotection.
• the preferred use of the novel PRP is in the preparation of the novel immunogenic conjugates.
• the oligomer is coupled to one of the proteins or polypeptides of the invention by standard techniques applied to the teachings contained herein.
• the preferred technique is reductive amination using sodium borohydride as described in Roy, et al., J. Carbohydr. Chem. 6:161-165 (1987) and Lee, et al., Carbohvdr. Res.. 77:149-156 (1979), both of which are incorporated by reference.
• the PRP is converted into the isothiocynate by treatment with an activated thiocarbonic acid derivative, such as thiophosgene, and then coupled to the protein at a pH of 9-10 in accordance with the procedures described in Kallin, et al., Glycoconiugate J.. 3:311-319 (1986) and Zopf, et al., Methods Enzvmol.. 50:171-175 (1978) , both of which are incorporated herein by reference.
• the ratio of protein/carbohydrate is determined by a combination of Lowry protein determination and ribose determination.
• the ratio is primarily a function of the ratio of carbohydrate to protein in the initial reaction mixture and the type of spacer used. As shown in Example 3, the use of a spacer terminating in an amino group (Compound 16) results in a greater number of oligosaccharides being coupled to the protein than the use of a spacer terminating in an aldehyde group (Compound 14) . Table 4 shows the formulas of the final conjugates.
• the adhesin-oligosaccharide conjugates may be used in vaccines against both invasive and non-invasive strains of H. influenzae.
• the conjugate vaccines should have greatest utility against H. influenzae type b.
• the vaccines comprise an immunologically effective amount of the immunogen in a pharmaceutically acceptable carrier.
• the combined immunogen and carrier may be an aqueous solution, emulsion, or suspension.
• An im ⁇ munologically effective amount is determinable by means known in the art without undue experimentation, given the teachings contained herein. In general, the quantity of immunogen will be between 0.1 and 100 micrograms per dose.
• the carriers are known to those skilled in the art and include stabilizers, diluents, and buffers. Suitable stabilizers include carbohydrates, such as sorbitol, lactose, manitol, starch, sucrose, dextran, and glucose and proteins, such as albumin or casein.
• Suitable diluents include saline, Hanks Balanced Salts, and Ringers solution.
• Suitable buffers include an alkali metal phosphate, an alkali metal carbonate, or an alkaline earth metal carbonate.
• the vaccine may also contain one or more adjuvants to improve immunogenicity.
• Suitable adjuvents include aluminum hydroxide, aluminum phosphate, or aluminum oxide or a composition that consists of a mineral oil, such as Marcol 52, or a vegetable oil and one or more emulsifying agents.
• the vaccine may also contain other immunogens.
• Such a cocktail vaccine has the advantage that immunity against several pathogens can be obtained by a single administration.
• Examples of other immunogens are those used in the known DPT vaccines.
• the vaccines of the invention are prepared by techniques known to those skilled in the art, given the teachings contained herein. Generally, the immunogens are mixed with the carrier to form a solution, suspension, or emulsion. One or more of the additives discussed above may be in the carrier or may be added subsequently.
• the vac ⁇ cine preparations may be dessicated, for example, by freeze drying for storage purposes. If so, they may be subsequently reconstituted into liquid vaccines by the ad ⁇ dition of an appropriate liquid carrier.
• the vaccines are administered to humans or other mammals, including rodents and primates. Preferably, they are administered to human children, most preferably children younger than 18 months of age. They can be administered in one or more doses.
• the vaccines may be administered by known routes of administration for this type of vaccine.
• the preferred routes are intramuscular or subcutaneous injection.
• the invention also comprises a method for inducing an immune response to Hi in a mammal in order to protect the mammal against infection by invasive or non-invasive Hi.
• the method comprises administering an immunologically effective amount of the immunogens of the invention to the host and, preferably, administering the vaccines of the invention to the host.
• the conjugates, protein/polypeptides, and oligomers of the invention are also useful as reagents for scientific research on the properties of pathogenicity, virulence, and infectivity of Hi, as well as host defense mechanisms.
• the DNA of the invention can be used in an oligonucleotide probe to identify the DNA of other microorganisms that might encode an adhesin for such organism.
• the protein of the invention could be used to make a monoclonal antibody that could be used to further purify compositions containing the protein by affinity chromatography.
• the protein could also be used in standard immunoassays to screen for the presence of antibodies to H. influenza in a sample.
• a composition in accordance with the present invention useful as an investigational reagent contains an amount of conjugate, protein/polypeptide, or oligomer effective to provide the information or analysis sought.
• the determination of the amount necessary to accomplish a particular research goal depends upon the specific type of investigation involved and is readily within the routine skill of one engaged in such research, once given the teachings contained herein.
• Methyl 5-0-allyl-2,3-O-isopropylidene-beta-D- ribofuranoside (Compound 2) A solution of methyl 2,3-O-isopropylidene-beta-D- ribofuranoside (Compound 1, 50.0 g) , N,N-dimethyl formamide (250 ml), and powdered sodium hydroxide (55.0 g) was stirred while allyl bromide (50.0 ml) was added dropwise. After 2h, the excess allyl bromide was destroyed by addi ⁇ tion of methanol (50 ml) . After being stirred for another hour, the mixture was partitioned between water and toluene. The organic phase was washed with water, dried with magnesium sulfate, and concentrated. Barium carbonate (250 mg) was added and the oil was distilled at 90-95°C, 0.75 mm Hg. The yield of Compound 2 was approximately 90%.
• Methyl 5-0-allyl-2,3-O-isopropylidene-beta-D- ribofuranoside (Compound 2, 1.5 g) in aqueous formic acid (25 ml) was heated on an oil bath at 100°C for 10 hrs and was then concentrated and coevaporated twice with water.
• the obtained syrupy material consisting mainly of 5-0- allyl-D-ribose and residual formic acid, was dissolved in water (25 ml), and the pH was adjusted to 7 with aqueous ammonia.
• Sodium borohydride (0.5 g) was added, and the mixture was stirred for 3h, then adjusted to pH 7 with acetic acid, and concentrated.
• the combined organic extracts, containing mainly methyl 5-O-benzyl-beta-D- ribofuranoside were concentrated. Dry pyridine (50 ml) was added, the mixture was concentrated, then dry pyridine (150 ml) was added again. The mixture was cooled in ice while benzoyl chloride (34 ml) was added dropwise. The mixture was further stirred at room temperature overnight, then water (2 ml) was added to destroy excess benzoyl chloride. The mixture was then partitioned between water (1000 ml) and dichloromethane (500 ml) . The organic layer was washed with 2 M aqueous sulfuric acid, then with 1 M aqueous sodium hydrogen carbonate.
• a solution of hydrogen bromide in dichloromethane was prepared by mixing dichloromethane (150 ml) , methanol (3.0 ml), and acetyl bromide (6.0 ml). Then methyl 2,3-di- O-benzoyl-5-O-benzyl-beta-D-ribofuranoside (Compound 5, 4.62 g) was added, and the mixture was stirred at room temperature for 30 min.
• the residue containing mainly 3-0-benzoyl-5-0-benzyl-l,2-O-methoxybenzylidene- alpha-D-ribofuranose, was dissolved in methanol (50 ml) , and a solution of sodium methoxide in methanol (0.5 M, 20 ml) was added. After 2h at room temperature, the mixture was neutralized by addition of C ⁇ 2(s), then concentrated and co-concentrated once with N,N- dimethylformamide. The residue was dissolved in N,N- dimethylformamide (50 ml) and stirred at room temperature while powdered sodium hydroxide (3.0 g) was added, followed by allyl bromide (3.0 ml).
• the mixture was concentrated and taken up in acetic acid-water (30ml, 9:1 by volume) and the mixture was heated to 80°C for 1 hour, concentrated and the residue was partitioned between diethyl ether and water, dried, and concentrated.
• the residue containing mainly 2,3,4-tri-0-benzyl-l-0-(2,5- di-O-benzyl-beta-D-ribofuranosyl)-D-ribitol, was taken up in dry pyridine (50 ml) , and monomethoxytrityl chloride (3.5 g) was added. The mixture was stirred overnight, then methanol was added to destroy the excess chloride.
• the succinate obtained above (10 equivalents over the resin amino group content) was dissolved in dichloromethane (5 ml/g) and mixed with a solution of dicyclohexylcarbodiimide (5 equivalents over the resin amino group content) in a small volume of dichloromethane. The mixture was stirred for 15 min. at room temperature, then concentrated. The residue was dissolved in N,N- dimethylformamide (5 ml/g) and the solution was filtered, then added to Merrifield-type aminomethyl resin (pre-washed with N,N-dimethylformamide) . After 6 h, the resin was washed with N,N-dimethylformamide, then with pyridine.
• the resin was treated with 9:1 pyridine-acetic anhydride for 2 hr., washed with pyridine, then washed with dichloromethane.
• the degree of functionalization was determined by treating a dried and weighed amount of resin with 0.5% trifluoroacetic acid in 1,2-dichloroethane, and estimating the trityl cation content in the supernatant by spectrophotometry (495 nm) .
• a typical value was 0.5 mmol/ g-
• the solid-phase synthetic operations were carried out in a semi-automated apparatus, consisting of a reaction vessel with a glass filter bottom, agitation device (small scale batches were agitated by pressing dry nitrogen through the bottom filter) , liquid outlet (bottom) , and liquid inlet (top) . Liquid was removed from the vessel through the bottom filter by suction, and added at the top by pressing with nitrogen from other vessels through teflon tubing.
• the resin was treated with a 0.5% solution of trifluoroacetic acid in dichloromethane until no more trityl cation was released (as determined spectrophotometrically) , then the resin was washed with dichloromethane, followed by 4:1 dichloromethane-pyridine.
• the resin was treated with a freshly prepared 1% solution of iodine in 98% aqueous pyridine for 30 min. , then washed with, successively, pyridine and dichloromethane.
• the resin was treated with sodium methoxide 1:1 dioxane-methanol (0.05M) for 16 hours at room temperature, acetic acid was added, and the mixture was then filtered and the filtrate was concentrated.
• the residue according to NMR analysis, contained compound 13 (if 10 was used for chain termination) or 15 (if 12 was used for chain termination) , together with impurities.
• the material that was removed from the resin as described above was dissolved in 1:2:2 ethylacctate- ethanol-water(0.1 ml/mg material) containing acetic acid (0.3%), and 10% Pd/C (0.5-2 mg/mg material) was added.
• the mixture was hydrogenated at 60 °C and atmospheric pressure overnight, then filtered, adjusted to pH 7, and concentrated.
• the residue was partitioned between diethyl ether and water. The aqueous layer was separated and concentrated. The residue was taken up in 50% aqueous trifluoroacetic acid at 0°C.
• the mixture was neutralized at 0°C with ammonia to pH 1 , then the mixture was concentrated to a volume of approximately 10 mg/ml, and applied to a column of Fractogel TSK HW-50, packed and eluted with lOmM ammonium bicarbonate buffer, pH6.2. The appropriate fractions were collected, concentrated, and redissolved in water (0.1 ml/mg material). This solution was slowly passed through a column of Dowex-50 x 8 (Na form, packed and eluted with water) . The appropriate fractions were collected and lyophilized.
• NMR spectroscopy in D2O solution showed, inter alia, signals from the anomeric protons in the region 4.9-5.1 ppm and signals from the spacer unit (aldehyde proton, dihydrate form) at 5.1- 5.2 ppm.
• the amount of successful coupling cycles was verified by integration over the anomeric signals and the spacer signals, respectively.
• the material that was removed from the resin was treated essentially as described above for conversion of Compound 13 to 14, except that the trifluoroacetic acid treatment was omitted.
• NMR spectroscopy in D2O solution of the lyophilized product showed, inter alia, signals from the anomeric protons in the region 4.9-5.1 ppm and signals from the spacer unit (CH2N triplet) at 3.2 ppm.
• the amount of successful coupling cycles (that is, the value of n in the formula for Compound 16) was verified by integration over the anomeric signals and the spacer signals, respectively.
• the purification of 14 and 16 could also be effected by preparative HPLC on Nucleosil C-18, using 0.1 M aqueous triethylammonium acetate (pH5.3) with 2.5% acetonitrite as eluant.
• Bacteria were grown 24 h in defined media and labeled metabolically with 35 S-methionine. Cells were harvested and washed by centrifugation three times in saline and suspended in approximately 20 ml of 10 mM Hepes buffer, pH 7.4, and chilled on ice. The bacterial suspen ⁇ sion was then sonicated on ice 6 times for 30 seconds each at a setting of 4 on a Bronson Sonicator. The sonic extract was centrifuged at 10,000 x g for 10 min. at 4° C, and the resulting outer membrane protein (OMP) pellet was stored until use in Hepes buffer containing protease inhibitors (PIC I & PIC II) .
• PIC I & PIC II protease inhibitors
• OMPs were next centrifuged at 100,000 x g for 30 min. at 4° C and the resulting pellet was suspended in 4 ml of 10 mM Hepes, pH 8.0, containing 1.3% octyl- glucopyranoside (Sigma) , sonicated 5 min. , and incubated at room temperature for 30 min.
• the resulting solubilized OMPs were centrifuged again at 100,000 x g for 30 min. at 4° C, and the supernatant containing partially purified adhesin was decanted and saved.
• the adhesin was purified by a receptor-affinity solid phase procedure as follows. The supernatant was diluted 1/10 in 50 mM Tris-HCl, pH 7.8, containing 150 mM NaCL and 1% bovine serum albumin (BSA) and incubated in receptor-coated microtiter wells (0.8 microgram ⁇ of gangliotetraosylceramide/well) which had been previously blocked with BSA. Control wells lacking receptor were also used. After a 2 h incubation at room temperature, wells were washed 4 times with cold saline. The receptor-bound adhesin was eluted by incubating the wells for 30 min.
• BSA bovine serum albumin
• the adhesin can be purified by using an affinity chromatography column where the lipid receptor is immobilized onto an appropriate gel solid support.
• the sonic extract is loaded on the top of the gel and the column is washed to remove unbound material.
• the adhesin is then eluted with SDS elution buffer or a chaotropic agent, such as NaCl or KSCN, and dialyzed and analyzed by SDS-PAGE and autoradiography.
• FIG. 1 shows the sample analysis in the following lanes: 1, total outer membrane protein preparation from Haemophilus influenzae type b stained with Coomassie blue; 2, autoradiography of 35 S-labeled total outer membrane proteins; 3, autoradiography of 35 S-labeled adhesin protein eluted from immobilized receptor asialo- GM ⁇ ; 4, autoradiography of material eluted from immobilized globoside, a nonsense glycolipid.
• Arrow indicates the adhesin migrating between PI and P2 with a molecular weight of about 41 kD.
• mice were injected IP with 10 micrograms of partially purified adhesin protein (Hib OMPs) in complete Freunds adjuvant (1:1). After one month, the mice were boosted with a second IP injection (10 micrograms of protein) using incomplete Freunds adjuvant followed by a third injection 10 days later.
• Hib OMPs partially purified adhesin protein
• Antiserum was then tested for neutralizing activity against 35 S-labeled Hib adhesin in a receptor binding assay.
• antiserum and normal mouse serum at various dilutions were incubated with 35 S-labeled Hib adhesin protein for one hour at room temperature and then added to microtiter wells coated with asialo-GMl or globoside as a negative control. After incubation of the microtiter plates for 2 hours at room temperature, the microtiter wells were washed, cut from the plates and radioactivity was quantified using a Beta-scintillation counter. The results are shown in Figure 2. The results show that the adhesin is immunogenic and that antibodies to the adhesin effectively neutralize the adhesin's receptor binding activity.
• Membrane proteins were prepared as follows. Haemophilus influenzae type b (ATCC 9795) were grown to stationary phase, pelleted, resuspended in saline buffer, and sonically disrupted. This material was then centrifuged at 12,000 x g for 15 min, and the supernatant was centrifuged at 100,000 x g for 1 h. The resultant pellet contained Haemophilus membranes, which were resuspended in saline and tested for adhesin activity as described in Krivan, et al. Proc. Natl. Acad. Sci. USA. 85:6157-6161 (1988), incorporated herein by reference.
• membranes were prepared from [ 35 S] methionine metabolically-labeled cells (1 micro-Ci/ml of media) . Glycolipids were resuspended in chloroform:methanol (1:1, vol:vol) and serially diluted into 96-well microtiter plates. These plates were allowed to dry, washed 5 times with Tris/BSA (25 mMTris, pH7.5, 1% bovine serum albumin) , then 2 X 10 6 CPM of labeled membranes were added to each well and incubated at room temperature for 2 h. The plates were then washed with Tris/BSA 5 times, and the individual wells cut out and counted on a scintillation counter to determine the amount of CPM bound to each well. This showed that Hi membranes bound similar to Hi whole cells.
• mice were immunized with membranes from Haemophilus influenzae type b (ATCC 9795) , and their sera was tested for the development of antibody that inhibited membranes from binding to receptor ( Figure 2) .
• Spleens from these mice were used to isolate splenocytes for fusion with SP2/o-AG14 (ATCC CRL 8287) mouse myeloma cells according to Harlow, et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (1988) , incorporated herein by reference.
• ELISA ELISA was performed as follows. Membranes containing 1 microgram of protein were used to coat 96-well microtiter plates. The coated wells were washed with PBS (phosphate buffered saline, 10 mM sodium phosphate, pH 7.5, 167 mM sodium chloride), then incubated with 100 microliters of hybridoma culture supernatant. The wells were washed, incubated with 100 microliters of secondary goat anti-mouse antibody conjugated with horseradish peroxidase for 1 h, then bound antibody was detected colorimetrically (Biorad) .
• PBS phosphate buffered saline, 10 mM sodium phosphate, pH 7.5, 167 mM sodium chloride
• Cells were resuspended in PBS and iodinated with lodogen (Pierce) according to the manufacturer's recommendation. Cells were then solubilized in radioimmune precipitation buffer (RIPA buffer, 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxcholate, 0.1% SDS, ImM PMSF), and then incubated with Gammabind beads (Pharmacia) overnight at 4°C. The beads were then pelleted by centrifugation (2000 x g, 5 min), washed 5 times with PBS containing 0.05% Tween-20, and resuspended in SDS-PAGE sample buffer.
• RIPA buffer 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxcholate, 0.1% SDS, ImM PMSF
• This ligation was then packaged into phage particles and used to tranfect the Escherichia coli host strain, XL-1 (according to Statagene protocol) to obtain phage plaques which express Haemophilus proteins. These plaques were used in an immunoblot screen with Hib 43 using a Stratagene Picoblue detection kit. Positive reacting plaques were purified and used to induce the production of a plasmid through the use of the helper phage R408 (according to Stratagene protocol) . These plasmids carried the Haemophilus insert DNA which encoded the Hin47 immunoreactive protein. The restriction map for one of these plasmids, designated pMClOl, is shown in Figure 5.
• All plasmids which expressed the Hin47 protein contained the 10.5 kbp DNA from Hi.
• the location of the gene encoding this protein was determined by deletion analysis of pMClOl. Deletion analysis was performed by generation of subclones of pMClOl containing various restriction fragments in the vector pSK(-) (Stratagene) . These subclones are represented on Figure 5 with an indication of whether each expresses a Hib 43 immunoreactive protein.
• the deletion analysis suggested that the Hin47 was encoded by a gene which was bounded by an approximate 2.4 kbase Pst 1 to BamHl fragment.
• Hin47 This could result in a mature protein of approximately 47 kDa and 438 amino acids as indicated by prior Western blot analysis.
• This ORF was designated hin47.
• the expression of the Hin47 protein was similar irrespective of the orientation of the gene with respect to the beta- galactosidase promoter contained in pSK(-) , indicating this protein is expressed in __ coli under its own promoter.
• the Hin47 adhesin is a novel protein.
• a series of major integral membrane proteins has been characterized by several investigators (Gonzales et al., Infect. Immun.. 55:2993-3000 (1987), incorporated herein by reference). These include PI, which is approximately 43 kDa, and P6, which is approximately 18 kDa.
• the Hin47 adhesin was analyzed to insure that it was not any of these previously characterized proteins. Using an E. coli clone that expressed PI or P6, neither clone reacted with Hib 43, demonstrating that this antibody does not recognize either of these proteins.
• the PI protein is similar in size to the Hin47 adhesin, we demonstrated by heat modification that the Hin47 adhesin was not PI.
• the E. coli which expressed PI was separated by SDS-PAGE after treatment at room temperature or 100°C. PI has previously been shown to be heat modifiable (Gonzales et al.). After treatment at 100°C, the protein migrates at about 43 kDa, while after treatment at room temperature, PI migrates at about 32 kDa.
• the Hin47 protein was shown not to be heat modifiable. A comparison of the sequence of the 2.4 kbase Pst 1 to BamHl fragment of pMC102 confirmed that hin47 has no homology with the gene that encodes PI.
• Hin47 adhesin Purification of the Hin47 adhesin.
• the Hin47 protein was purified to homogeneity using the monoclonal antibody Hib 43 as an immunoabsorbent according to Krivan et al., Inf. and Immun.. 55:1873-1877 (1987). Briefly, antibody was coupled to cyanogen activated sepharose 4CL beads (Pharmacia) according to the manufacturer's recommendation. A 4 ml column containing about 8 mg of coupled antibody was used.
• the Hin47 protein was produced by XL-1/pMClOl grown to stationary phase in a 4 L culture in Luria Broth. The cells were pelleted by centrifugation (12,000 x g, 15 min), resuspended in PBS, and sonicated.
• the sonicate was pelleted by centrifugation (12,000 x g. 15 min) and the supernatant pelleted by centrifugation (1000,000 x g, 1 h) .
• the resultant membrane pellet was resuspended in 0.5% octylglucopyranoside (Sigma Chemical) and pelleted by centrifugation (100,000 x g, 1 h) .
• the supernatant was exhaustively dialyzed against 50 mM Tris, pH 8.5 and applied to a DEAE-sepharose column (Sigma Chemical) .
• a fraction containing Hin47 was eluted from the column using 125 mM NaCl, 50 mM Tris, pH 8.5.
• This fraction was dialyzed against PBS, then applied to the antibody column. The column was then washed with PBS, and bound protein was eluted with 100 mM glycine, pH 2.8 and immediately neutralized. This material was dialyzed against PBS and analyzed by separation on SDS-PAGE. The gel was stained by silver (Biorad) . The Hin47 protein appeared as a single species, indicating purification to homogeneity.
• the retained material was lyophilized and purified by gel filtration on Bio-Gel P4. The appropriate fractions were collected and lyophilized.
• the degree of functionalization (as haptens/protein molecule) was estimated by a combination of Lowry protein determination and orcinol ribose determination. Generally, a value of 10-20 haptens/protein molecule was obtained.
• DNA from each strain was used for amplification with a primer 5' and a primer 3' to the structural hin47 gene. See Hinf3 and Hinf4 on Figures 7A and 7B.
• the amplification was performed using an Amplitaq DNA amplification kit (Perkin Elmer Cetus, Norwalk, Conn.) by the methods provided by the manufacturer.
• Each amplified gene was cloned into the PCR cloning vector PCR II (Invitrogen, San Diego, CA) according to the manufacturer's methods and subsequently subjected to DNA sequence analysis. The sequence analysis was performed using the dideoxy double stranded sequencing method of Sanger et al., Science.
• a candidate subunit vaccine must be strongly im ⁇ munogenic and have the ability to generate a protective immune response.
• Several animal experiments were performed to assess these properties for Hin47.
• the protective activity of anti-Hin47 antibodies in an infant rat model of ________ influenza-mediated bacteremia was used as one measure of the ability of Hin47 to act as a protective antigen.
• the methodology was that Moxon et al., J. Infectious Diseases, 129:154-162 (1974) and Loeb, Infection and Immunity. 55:2612-2618 (1987), both of which are incorporated by reference.
• Rabbit anti-Hin47 antiserum was generated by immunizing a rabbit with 3 doses of 100 ug of purified Hin47 in the presence of complete Freund's adjuvant on day 1 and in the presence of incomplete Freund's adjuvant on day 28 and 42.
• IL_ influenzae PI, P2, and P6 proteins and polyribitol phosphate
• Groups of five 5-day old infant rats were injected subcutaneously with either rabbit anti- Hin47 antibody, prebleed serum, or saline. After 24 hours, the infant rats were challenged with 200 colony forming units (CFU) of the virulent ⁇ [____ influenzae Minn A strain (Munson and Grass, Infection and Immunity. 56:2235-2242 (1988) , incorporated herein by reference) by intraperitoneal injection.
• CFU colony forming units
• the results are expressed as the average CFU/ 0.1 ml of blood for each group of animals, the number of animals with bacteremia in each group, and the percentage of bacteremia compared to the saline control. Animals receiving Hin47-specific antibodies were significantly reduced in bacteremia, and 3 of 5 animals had no detectable organisms in their blood.
• the second animal disease model used to assess the efficacy of Hin47 as a vaccine candidate was the chincilla otitus media model.
• the methodology was that Bakaletz et al.. Infection and Immunity. 57:3226-3229 (1989), incorporated herein by reference.
• Chincilla were immunized with three doses (50 ug per dose) of Hin47 protein in the presence of complete Freund's adjuvant on day 1, and in the presence of incomplete Freund's adjunvant on day 28 and 42.
• Two control animals were injected with saline.
• the chincillas were challenged with 2,000 CFU of a non- typable H. influenzae strain designated strain 12.
• Ear infection was monitored by otoscopic examination and tympanometry on day 1, 2, and 6 post-infection. Fluid was collected through epitympanic bulla by injecting 0.2 ml of saline into the middle ear cavity and then aspirating the fluid. The fluid was plated on chocolate agar plates and incubated at 37'C overnight. Positive control protection animals were either an animal that had recovered from an ear infection or animals that were immunized with heat killed strain 12 whole cells. The results are summarized in Table 9. Two of four Hin47-immunized animals were negative by typanogram analysis and had significantly reduced bacteremia at day 2. These data suggest that Hin47 has protective value in the active protection chincilla model for human otitus media.
• Table 8 Protective activity of anti-Hin47 antibodies in the infant rat model.
• MOLECULE TYPE DNA (genomic)
• HYPOTHETICAL NO
• ANTI-SENSE NO
• GCC AAA GCC TTT AAT GTA AGC GCG CAA CAA GGT GCA TTT GTA AGT GAA 1029 Ala Lys Ala Phe Asn Val Ser Ala Gin Gin Gly Ala Phe Val Ser Glu 290 295 300 305
• GAT GAT GGT AGC CAA CTT TCC TCA AAA ACT GAG TTG CCT GCA TTA GAT 1269 Asp Asp Gly Ser Gin Leu Ser Ser Lys Thr Glu Leu Pro Ala Leu Asp 370 375 380 385
• MOLECULE TYPE protein

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