Patent Application
20210401960
The following is related to the field of polysaccharide conjugate immunogenic compositions such as vaccines. More particularly, to the field of polysaccharide conjugate compositions. The combined conjugate vaccine compositions of this present invention is for prophylaxis against infections caused by Salmonella and Non-typhoidal Salmonella infections that cause typhoid fever.
In humans, the infection caused by Salmonella enterica depends primarily on the infecting serovar. The majority of invasive infections causing typhoid fever is from the serovar known as Salmonella enterica serotype Typhi (S. typhi); S. enterica serotypes Paratyphi A, B, and C.
Salmonella enterica is a motile aerobic or facultatively anaerobic, gram-negative bacillus, nonspore-forming bacteria known to cause clinical infections. Salmonella enterica encompasses more than 2500 sero-variants which are known to cause human clinical infections. Serovar S. typhi, a human-restricted pathogen, causes typhoid fever, which is characterized by fever, abdominal discomfort and frontal headaches. These sero-variants include both typhoidal serovars and non-typhoidal serovars. For instance, serovars such as Salmonella enterica serovar Typhimurium (S. Typhimurium) cause self-limiting gastroenteritis, while Salmonella enterica serovar Typhi (S. typhi) result in typhoid fever.
These typhoidal serovariants were known to cause clinical infection in human but the non-typhoidal serovars (NTS) can cause invasive clinical disease in infants, immunosenescent elderly and immune-compromised people. Hence together these serovariants are responsible for notable drift on the morbidity and mortality rate across the world.
The Sub-Saharan populations especially children seem to be more affected with both the typhoid and invasive NTS (iNTS) disease, wherein school age children are more affected with typhoid while infants and toddlers were attacked and affected by iNTS. Nontyphoidal Salmonella can be invasive and cause paratyphoid fever, which requires immediate treatment with antibiotics. In Saharan Africa, iNTS infections are mainly caused by serovars such as S. enteritidis and S. typhimurium which results in fatality rates of 12-28%.
Some regions exhibit only typhoid disease whereas others only iNTS, and yet others have both types of infections, indicating variability in geographic prevalence. The iNTS strains which are found in sub-Saharan Africa are fundamentally distinct from the conventional gastroenteritis NTS strains which are found in the USA and Europe.
Analyses of S. typhimurium and S. enteritidis revealed novel multi-locus sequence types and patterns of genetic degradation analogous to those present in typhoid and paratyphoid serovars. Pathotypic analyses of a prototype sub-Saharan African S. typhimurium blood isolate found reduced inflammation and enhanced intracellular survival in cultured cells, and the absence of diarrhea in a non-human primate model—phenotypes generally associated with S. typhi.
Despite ongoing research efforts, the reservoir for iNTS infection has not yet been identified, hampering the ability to implement traditional environmental interventions for interruption of transmission, Crump et al., “A Perspective on Invasive Salmonella Disease in Africa” Clin. Infect. Dis. 61(Suppl. 4), S235-S240 (available on-line Oct. 7, 2015). Although several types of typhoid vaccines are available, no human NTS vaccines are currently in use.
U.S. Pat. No. 5,738,855 (Shousun Chen Szu et al.) teaches the method of making a modified saccharide and immunogenic conjugate. It can be a modified plant, fruit or synthetic oligosaccharide or polysaccharide which has been structurally altered so as to render the modified saccharide antigenically similar to the Vi of Salmonella typhi. The modified saccharide may be conjugated to a carrier to form a conjugate that is immunogenic against S. typhi. Antibodies produced in response to the immunogenic conjugate are protective against typhoid fever.
U.S. Pat. No. 3,856,935 (R Germanier) teaches about the oral typhoid vaccine and the method of preparing a vaccine thereof using Salmonella typhi strain ty 2 which is subjected to ultraviolet light. The mutants so produced are screened for selection of strains defective in the enzyme uridine diphosphogalactose-4-epimerase. A live vaccine is prepared from the selected and carefully isolated strains in the usual manner. The strains are also identified by their sharply reduced galactokinase and galactose-1-phosphate uridylyl transferase activity, as compared to the parent strain.
China Patent No. 1404873A teaches that the typhoid Vi polysaccharide is covalently conjugated on the carrier protein to form conjugative vaccine that can be used to generate active immunity in humans and other mammals, and for preventing typhoid infection. After the typhoid Vi polysaccharide is conjugated with protein, the recognition mode of organism to typhoid Vi antigen is changed into T cell depended antigen that can be used for immunity of all people and possesses obvious effect for boosting immunity.
U.S. Pat. No. 9,011,871 (Myron M. et al.) teaches about a multivalent Salmonella enterica serovar conjugate vaccines comprising conjugates of S. typhimurium, S. enteritidis, S. choleraesuis, S. typhi, S. paratyphi A and S. paratyphi B, wherein the conjugates comprise a hapten antigen and a carrier antigen, wherein at least one of the hapten antigens or carrier antigens is characteristic of the Salmonella enterica serovar. The disclosure also provides Salmonella enterica serovar reagent strains to produce the multivalent conjugate vaccines and attenuated Salmonella enterica serovars for use as vaccines.
U.S. Pat. No. 9,050,283 (Myron M. et al.) is drawn to attenuated Salmonella serovar strains S. typhimurium and S. enteritidis, conjugate vaccines derived from these attenuated strains of S. typhimurium and S. enteritidis, comprising an O polysaccharide covalently linked to a flagellin protein, and methods for inducing an immune response in a subject.
WO 2015/029056 A1 (Ella et al.) teaches stable conjugate vaccine formulations for protections against Salmonella typhii, and methods of conjugation between Vi-polysaccharide of S. typhii to tetanus toxoid as the carrier protein, responsible for producing improved T-dependent immune response against Typhoid fever caused by Salmonella typhi. The methods disclosed in embodiments of the invention and the resulting formulations are capable of inducing immunity against typhoid fever including in children below 2 years of age, through only a single injection to comprise a complete vaccination schedule.
A conjugate vaccine against typhoid Vi (Typbar-TCV®) was distributed in 2013. That vaccine has been given to large numbers of infants, toddlers, children and adults in India and also other countries. Thus, there is already a substantial track record of safety and immunogenicity for Typbar-TCV®.
Although different forms of typhoid vaccines are in use, no human NTS vaccines are available. Thus, there is a need in the field for immunogenic compositions that provide protection against Salmonella infections, which cause typhoid, and Non-typhoidal Salmonella infections (NTS), which cause paratyphoid fever.
An aspect relates to an immunogenic composition comprising two or more of antigen of typhoidal serovars and/or non-typhoidal serovars of Salmonella, wherein each antigen is conjugated to one or more carrier molecules.
Another aspect is to provide a method for preventing or treating a Salmonella infection comprising administering to a patient an effective amount of the immunogenic composition comprising antigens of two or more typhoidal serovars and/or non-typhoidal serovars of Salmonella.
Yet another aspect is to provide a method for manufacturing immunogenic composition comprising antigens of two or more typhoidal serovars and/or non-typhoidal serovars of Salmonella.
Embodiments of the invention are directed to polysaccharide conjugate immunogenic compositions, and combined immunogenic compositions of polysaccharide conjugate immunogenic compositions. Compositions of embodiments of the invention provide prophylaxis against infections caused by Salmonella and Non-typhoidal Salmonella infections that cause typhoid fever. Types of Salmonella and Non-typhoidal Salmonella infections that can be treated or prevented include but are not limited to infections of Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi A, B and/or C, and combinations thereof. Immunogenic compositions may also be generated for the prophylaxis or treatment of Meningococcal infections.
The main aspect of embodiments of the invention is to provide an immunogenic composition comprising two or more of:
In some embodiment, there is provided the above described immunogenic composition, wherein the one or more carrier molecules comprises one or more of tetanus toxin, tetanus toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertusis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, rEPA, Hemophilus influenzae protein D, Flagellin Fli C, Horseshoe crab Haemocyanin, and fragments, derivatives, and modifications thereof.
In some embodiment, the immunogenic composition comprises a pharmaceutically acceptable buffer such as PBS and Tween 80.
In some embodiment, the immunogenic composition comprises a stabilizer such as 2-phenoxy ethanol.
In some embodiment, the immunogenic composition comprises an adjuvant.
In some embodiment, each of the antigenic conjugate is present in the immunogenic composition at a dose range of about 5 μg/dose to about 30 μg/dose.
In some embodiment, the immunogenic composition comprises two antigenic components selected form the following:
In some embodiment, the immunogenic composition comprises three antigenic components selected form the following:
In another aspect, there is provided a method for preventing or treating a Salmonella infection comprising administering to a patient an effective amount of the immunogenic composition according to embodiments of the invention. In some embodiments, there is provided a method for preventing or treating a Salmonella infection comprising parenteral administration to a patient an effective amount of the immunogenic composition according to embodiments of the present invention. In some other embodiment. In some embodiment, there is provided a method for preventing or treating a Salmonella infection comprising administering to a patient an effective amount of the immunogenic composition according to embodiments of the present invention, wherein administration results in an eight-fold rise in antibody titer.
In yet another aspect, there is provided a method for the manufacture of the immunogenic composition, comprising:
In some embodiment, the conjugation method for the manufacture of the immunogenic composition is performed by:
One embodiment of the invention is directed to combination conjugate immunogenic compositions comprises one or more of: an antigen of Salmonella enteritidis; an antigen of Salmonella typhimurium; an antigen of Salmonella typhi; an antigen of Salmonella paratyphi A; and an antigen of a non-typhoidal Salmonella microorganism, with each antigen conjugated to a carrier protein or peptide. The antigen for each may comprise an isolated immunogenic portion of the microorganism such as a polysaccharide, and/or capsular polysaccharide, and/or a portion of or a whole attenuated microorganism, In an embodiment, the carrier proteins or peptides of the immunogenic composition comprises one or more of tetanus toxin, tetanus toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertusis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, rEPA, Hemophilus influenzae protein D, Flagellin FliC, Horseshoe crab Haemocyanin, and fragments, derivatives, and modifications thereof, or another conventional protein conjugate. In an embodiment each conjugate of the immunogenic composition is formulated at a dose range of from about 5 μg/dose to about 30 μg/dose. Also, In an embodiment, the composition contains a pharmaceutically acceptable stabilizer and/or a pharmaceutically acceptable buffer. Stabilizers include, for example, 2-phenoxy ethanol, and buffers include, but are not limited to PBS buffers with or without a non-ionic detergent, such as, for example Tween 80 and at a pH of about 6.5 to about 7.5.
In an embodiment, the immunogenic composition comprises two or more of: an antigen of Salmonella enteritidis conjugated to a first carrier protein; an antigen of Salmonella typhimurium conjugated to a second carrier protein or peptide; an antigen of Salmonella typhi conjugated to a third carrier protein or peptide; an antigen of Salmonella paratyphi A conjugated to a fourth carrier protein or peptide, and an antigen of a non-typhoidal Salmonella microorganism conjugated to a fifth carrier protein or peptide, the immunogenic composition comprises three or more of an antigen of Salmonella enteritidis conjugated to a first carrier protein; an antigen of Salmonella typhimurium conjugated to a second carrier protein or peptide; an antigen of Salmonella typhi conjugated to a third carrier protein or peptide, an antigen of Salmonella paratyphi A conjugated to a fourth carrier protein or peptide, and an antigen of a non-typhoidal Salmonella microorganism conjugated to a fifth carrier protein or peptide. Each carrier molecule may be the same or different. Especially combinations comprise an antigen from each of Salmonella enteritidis, Salmonella typhimurium, and Salmonella typhi, or an antigen from each of Salmonella enteritidis and Salmonella typhimurium, or an antigen from each of Salmonella typhi Vi and Salmonella paratyphi A.
Another embodiment of the invention comprises one or more antigens of a Meningococcal microorganism, such as a polysaccharide or glycoconjugate that may be combined with any other immunogenic compositions of this disclosure.
Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of embodiments of the invention.
Some of the embodiments will be described in detail, with references to the following Figures, wherein like designations denote like members, wherein:
Typhoid fever, also known simply as typhoid, is a bacterial infection attributed to Salmonella. These bacteria colonize the intestines and blood of affected individuals causing significant morbidity and mortality. In 2000, typhoid fever caused an estimated 21.7 million illnesses and approximately 217,000 deaths, most often in children and young adults between 5 and 19 years old. The species and subspecies of Salmonella generally responsible include Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, and the multiple serotypes or serovars of each. The two main types of the subspecies enterica are ST1 and ST2, based on MLST subtyping scheme. Non-typhoidal serovars (NTS) of Salmonella cause invasive clinical disease especially in infants, the elderly and immune-compromised individuals. Although a vaccine against typhoid fever exist, there are currently no vaccines against iNTS and many of the different serovars. The typhoid vaccines which are globally available are generally impractical for administration to the most vulnerable such as infants and the elderly.
Immunogenic composition comprised of typhoidal serovars and/or non-typhoidal serovars of Salmonella, and methods for the manufacture and use of these immunogenic compositions, have been surprisingly discovered that are effective for the prevention and/or treatment of Salmonella infections and, in particular, for prevention and/or treatment of infections caused by S. enteritidis, S. typhimurium, S. typhi, S. partyphi A, B and C, NTS and iNTS. The immunogenic composition comprises one or more of: an antigen of Salmonella enteritidis; an antigen of Salmonella typhimurium; an antigen of Salmonella typhi; an antigen of Salmonella paratyphi A; and an antigen of a non-typhoidal Salmonella microorganism, with each antigen conjugated to a carrier protein or peptide. The antigen for each may comprise an isolated immunogenic portion of the microorganism such as an immunogenic polysaccharide, and/or capsular polysaccharide, and/or a portion of or a whole attenuated microorganism. In an embodiment, the carrier proteins or peptides of the immunogenic composition comprises one or more of tetanus toxin, tetanus toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, rEPA, Hemophilus influenzae protein D, Flagellin Fli C, Horseshoe crab Hemocyanin, and fragments, derivatives, and modifications thereof, or another conventional protein conjugate. In an embodiment, each antigen is conjugated to a carrier protein or peptide, although multiple antigenic components may be conjugated to the same or multiple carrier molecules. Conjugation of polysaccharides to protein carriers typically improves immunogenicity. In an embodiment the conjugation involves coupling via a conventional conjugation procedure, such as, for example, cyanylating agent such as, for example, 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP), 1-cyanobenzotriazole (1-CBT), 2-cyanopyridazine-3 (2H)-One (2-CPO), 1-cyanoimidazole (1-CI), 1-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT), carbodiimide mediated modification of the polysaccharide with adipic acid dihydrazide (ADH) treatment of the carrier, modification at the polysaccharide terminal 2-keto-3-deoxyoctonate (KDO) carbonyl with an aminooxy thiol linker that forms an oxime bond with the KDO ketone that is further reduced with sodium cyanoborohydride, antigen derivatized with the amine reactive reagent succinimidyl 4-maleimidylbutyrate (GMBS) to introduce a maleimide moiety that is then linked to the reactive sulfhydryl of the derivatized COPS molecule via formation of a thio ether bond, or a modification thereof.
In some embodiment, the immunogenic composition comprises two antigenic components selected form the following:
In some embodiment, the immunogenic composition comprises three antigenic components selected form the following:
In an embodiment, the composition contains one or more of a pharmaceutically acceptable stabilizer (e.g., 2-phenoxy ethanol), buffer, protecting agent such as a preservative (e.g., Thiomersal), amino acid, salt, bulking agent, antioxidant, and/or dispersant. Stabilizers and protecting agents are used to help the composition maintain effectiveness during manufacture and/or storage and, in particular, where transportation conditions may be an issue. Instability can cause loss of antigenicity and decreased effectiveness. Factors affecting stability include temperature, pH, hydrolysis and protein or polysaccharide aggregation, for example. Stabilizing agents include, for example, pharmaceutically acceptable sugars, gelatins, amino acids, alcohols, oils, and salts. Stabilizers include, for example, 2-phenoxy ethanol, and buffers include, but are not limited to, PBS (phosphate buffered saline) buffers with or without a non-ionic detergent, which is based on a polyoxyethylene or a glycoside. Non-ionic detergents include, for example, Tween (e.g., Tween 20; Tween 80), Triton (e.g., TX-100), and the Brij series of detergents. In an embodiment, the pH of the composition is from about 5.0 to about 8.5, from about 6.0 to about 8.0, or from about 6.5 to about 7.5. In an embodiment each conjugate of the immunogenic composition is formulated at a dose range of from about 1 μg/dose to about 100 μg/dose, from about 2.5 μg/dose to about 50 μg/dose, or from about 5 μg/dose to about 30 μg/dose, although individual patients and situations may warrant more of less per dose.
In an embodiment, immunogenic compositions of embodiments of the the invention may include adjuvants. An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants may be added to a vaccine to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Adjuvants include, but are not limited to analgesic adjuvants, inorganic compounds such as alum, aluminium hydroxide, aluminium phosphate, calcium phosphate hydroxide, mineral oil, paraffin oil, bacterial products such as killed bacteria (e.g., Bordetella pertussis, Mycobacterium bovis, bacterial toxoids), cytokines (e.g., IL-1, IL-2, IL-12), Freund's complete adjuvant, Freund's incomplete adjuvant, and combinations thereof.
In yet another aspect, there is provided a method for the manufacture of the immunogenic composition, comprising:
In some embodiment, the conjugation method for the manufacture of the immunogenic composition is performed by:
One embodiment of the invention comprises S. typhi Vi conjugated by carbodiimide mediated modification of S. typhi Vi polysaccharide with adipic acid dihydrazide (ADH) that introduces reactive hydrazide groups that are then used to link to tetanus toxoid (TT) via a second carbodiimide step.
Another embodiment of the invention comprises S. paratyphi conjugated via by either 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry to link S. enteritidis core-0 polysaccharide (COPS) to the homologous serovar FliC flagellin protein subunits that had been derivatized with ADH using carbodiimide or by modification at the polysaccharide terminal 2-keto-3-deoxyoctonate (KDO) carbonyl with an aminooxy thiol linker. The aminooxy forms an oxime bond with the KDO ketone that is further reduced with sodium cyanoborohydride.
Another embodiment of the invention comprises S. typhimurium FliC protein conjugate derivatized with the amine reactive reagent succinimidyl 4-maleimidylbutyrate (GMBS) to introduce a maleimide moiety that is then linked to the reactive sulfhydryl of the derivatized COPS molecule via formation of a thio ether bond.
Another embodiment of the invention comprises a S. typhimurium conjugate component generated by modification at the polysaccharide terminal 2-keto-3-deoxyoctonate (KDO) carbonyl with an aminooxy thiol linker. The aminooxy forms an oxime bond with the KDO ketone that is further reduced with sodium cyanoborohydride. S. typhimurium FliC protein is derivatized with the amine reactive reagent succinimidyl 4-maleimidylbutyrate (GMBS) to introduce a maleimide moiety that is then linked to the reactive sulfhydryl of the derivatized COPS molecule via formation of a thioether bond. There is no conjugate vaccine for human use that utilizes the specific method of conjugation employed for Salmonella typhimurium glycoconjugate as presented in this disclosure.
Another embodiment of the invention comprises a S. enteritidis conjugate generated by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry to link S. enteritidis core-0 polysaccharide (COPS) to the homologous serovar FliC flagellin protein subunits that had been derivatized with ADH using carbodiimide.
In another aspect, there is provided a method for preventing or treating a Salmonella infection comprising administering to a patient an effective amount of the immunogenic composition according to embodiments of the present invention. In some embodiment, there is provided a method for preventing or treating a Salmonella infection comprising parenteral administration to a patient an effective amount of the immunogenic composition according to embodiments of the present invention. In some embodiments, there is provided a method for preventing or treating a Salmonella infection comprising administering to a patient an effective amount of the immunogenic composition according to embodiments of the present invention, wherein administration results in an eight-fold rise in antibody titer.
Another embodiment of the invention is directed to administering a conjugate of the disclosure to a patient. Administration may be for prophylaxis or treatment, and may comprise oral administration, nasal administration, injection such as administration via intravenous, intramuscular, or intraperitoneal, or a combination thereof. In an embodiment administration comprises a single dose, but may comprises multiple doses such as two, three, four or more doses.
The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of embodiments of the invention.
The combined glycol-conjugate vaccine compositions of the present disclosure are provided in Table 1.
Salmonella paratyphi A conjugate vaccine (Single dose) 5-30 μg/dose, PBS buffer pH: 6.5-7.5
Salmonella paratyphi A conjugate vaccine (multi dose) 5-30 μg/dose, 2-Phenoxy ethanol,
typhimurium conjugate (Single dose) 5-30 μg each/dose, PBS buffer pH: 6.5-7.5
typhimurium conjugate (multidose) 5-30 μg each/dose, PBS buffer with tween 80, 2-Phenoxy
A. Preparation of S. Enteritidis COPS:FliC Conjugate:
Conjugation of S. Enteritidis COPS to S. Enteritidis FliC is accomplished with cyanylation chemistry to generate a lattice conjugate that is cross-linked at multiple random COPS hydroxyls to multiple random primary amines and hydrazides on the protein. For this, the protein is first derivatized at protein carboxyls with adipic acid dihydrazide using carbodiimide. Separately, COPS is activated with CDAP to introduce a reactive cyanate group at polysaccharide hydroxyls. The derivatized protein and the activated polysaccharide are then mixed together as the protein and polysaccharide are linked to each other at multiple points; this forms a heterogeneous lattice that varies both in molecular weight and distribution of linkage points. The
Protein activation: S. Enteritidis FliC (stored in 0.9% saline) is concentrated to >6.0 mg/ml and then brought to 0.1% Tween 20, at which point it is confirmed to be monomeric form by HPLC-SEC. If found to be polymeric, it is then subjected to monomerization by lowering to pH 2 for 30 mins at 5±3° C., then raising to pH 7 with NaOH. The FliC is then derivatized with 0.5M adipic acid dihydrazide (ADH) using 0.5M ADH in 100 mM MES pH 6.5 by making the solution 5 mg/mL EDC for 17±1 hours at 5±3° C. The FliC-ADH is then diafiltered against 40-diavolumes of borate-buffer pH 9.2 using 10 kDa TFF. Protein ADH labeling and removal of free ADH are confirmed using the TNBS assay prior to proceeding to conjugation.
Polysaccharide activation: S. Enteritidis COPS is brought to 10 mg/ml in WFI at 5±3° C. CDAP (100 mg/ml in acetonitrile) is then added at a ratio of 0.5 mg:mg to the dissolved COPS, and incubated for 30 seconds at 5±3° C., at which point 2.5 M DMAP is added to bring the pH to 9.5. After incubation for 6 minutes at 5±3° C., the activated COPS is used immediately for conjugation by adding FliC-ADH.
Conjugation process: Freshly activated COPS-CN is added to FliC-ADH at a 1:1 mg:mg ratio and incubated with continuous stirring for 17±1 hour at 5±3° C. The conjugation reaction is quenched by bringing to 0.2M Glycine-HCl and stirring for 24 hrs at room temperature.
Purification of DS from unreacted components: The crude conjugate is centrifuged at 4° C., 4000±200 rpm for 15 minutes and the insoluble material pellet is discarded. The cleared supernatant is fractionated in 10 mM PBS using a Superdex 200 PG columns attached to a K-Prime chromatography system. Fractions containing high molecular weight conjugate are analyzed for protein and carbohydrate. Selected qualified fractions are pooled and then concentrated and diafiltered with 10 kDa TFF against 15 diavolumes of 10 mM PBS pH 7.1. The final concentrated COPS: FliC conjugate bulk is sterile filtered through 0.22 μm under sterile conditions and stored at 2-8° C.
Capping of unreacted active groups: Activation of the COPS with CDAP, and FliC with ADH introduces cyanate esters and hydrazide groups, respectively, to these molecules that react with each other during conjugation to form an amide bond. Although this reaction is efficient and robust with chemical linkages consistently formed, a portion of these activated groups remain unreacted during conjugation. In order to quench the residual COPS cyanate esters, an excess of glycine is added to the mixture which reacts with the remaining unreacted cyanate groups and caps them by forming a covalent link to glycine. This capping approach is specific for the cyanate esters, and hence no other changes are expected to occur upon glycine addition. A comparable approach is not readily implementable for quenching unreacted hydrazides, as the reactive group is a primary amine and a quenching reagent would react broadly with primary amines present on the protein amino acid side chains (e.g., lysine) as well, generating neo-epitopes. We have found that during conjugation, many of the hydrazides are consumed by the reaction, some remain uncapped (see report in Appendix VII-9).However, the calculated residual level of <2 μg of ADH per dose is less than the calculated permitted daily exposure for a single administration of 120 μg (see Gadd Toxicology evaluation, Appendix VII-10) does not explicitly cap unreacted hydrazides. This vaccine is licensed and prequalified by WHO and has been administered to hundreds of thousands of adults and children in whom it has been found to be safe and efficacious.
B. Preparation of S. Typhimurium COPS:FliC Conjugate:
S. Typhimurium COPS is variably 0-acetylated at both the abequose and rhamnose monosaccharides in the OPS repeating unit. In order to preserve the OPS 0-acetyls, S. Typhimurium COPS:FliC conjugates are thus synthesized with a method that is conducted at neutral to slightly acidic pH levels. This is accomplished by derivatizing both COPS and FliC with linkers that react with each other to form the conjugate. The KDO carbonyl is modified with an aminooxy-thiol linker that upon reduction with sodium cyanoborohydride generates a sulfhydryl group that is linked to the OPS reducing-end terminus with an oxime bond. The protein is derivatized with GMBS that places thiol-reactive maleimides at protein amino acids with primary amine containing side chains (e.g., lysine). The conjugate is rapidly formed upon mixing of the activated COPS and FliC, generating a “sun-type” conjugate that is linked at multiple FliC sites to the reducing-end of different COPS molecules. The
Polysaccharide activation: S. Typhimurium COPS is brought to 10 mg/ml in WFI and derivatized at the polysaccharide reducing end 2-keto-3-deoxyoctonate (KDO) carbonyl with 1:5 mg:mg ratio of an aminooxy thiol linker (O-3-Mercapto-propyl-hydroxyl-amine). In this reaction, the linker aminooxy group forms an oxime bond with the KDO carbonyl. The reaction is incubated at pH 5.3 with gentle stirring for 18±1 hrs at room temperature, at which point the oxime bond is reduced by bringing the mixture to 10 mM sodium cyanoborohydride and incubating for an additional 2.5 hours at room temperature. This mixture is centrifuged at 4000 rpm at 4° C. and supernatant is collected and diafiltered 20-fold against 10 mM PBS 5 mM EDTA 0.1% Tween 20 (pH 6.8) using a 10 kDa TFF membrane to remove unreacted linker prior to use in the conjugation reaction. COPS labeling with the aminooxy thiol linker is confirmed prior to use for conjugation by assessing the thiol:polysaccharide ratio with the resorcinol polysaccharide assay to measure total COPS and the DTNB assay to measure free thiol groups.
Protein activation: S. Typhimurium FliC (stored in 0.9% saline) is concentrated to >6.0 mg/ml, confirmed for monomeric form by HPLC-SEC, and then brought to 0.1% Tween 20. If found polymeric then it is subjected to monomerization by lowering to pH 2 for 30 mins at 5±3° C., then rising to pH 7 with NaOH. FliC monomers are brought to 100 mM PBS pH 7.4 and then labeled with the amine reactive reagent succinimidyl 4-maleimidylbutyrate (GMBS) to introduce a maleimide moiety at protein lysines. For this, GMBS is prepared in DMSO, and then added to FliC at a 30:1 molar ratio for 1 hour at room temperature with gentle stirring. The labeled protein is then immediately purified from unreacted linker and solvents with 10 kDa TFF and 10 diavolumes of 10 mM PBS+5 mM EDTA pH 6.8 and used for conjugation to maximize reactivity of the maleimide group that undergoes slow hydrolysis in aqueous solution.
Conjugation Process: The purified COPS-SH is added to FliC-GMBS at a ratio of 2.8:1 mg:mg and incubated at pH 7.4 at room temperature for 12±01 hrs under gentle stirring. The reaction is quenched by addition of 50 mM 2-mercaptothanol and incubation for lhr at RT under stirring. Thiol-labeled COPS and maleimide-labeled FliC are mixed at a ratio of 2.8:1 COPS-thiol:FliC-maleimide and incubated for 12-18 hours at 2-8° C. with gentle mixing.
Purification of DS from unreacted components: The crude conjugate is centrifuged at 4° C. at 4000±200 rpm for 15 mins and the insoluble material pellet is discarded. The cleared supernatant is fractionated in 10 mM PBS using a Superdex 200G columns attached to a K-Prime chromatography system. Fractions containing high molecular weight conjugate are analyzed and selected for pooling. Selected qualified fractions are pooled, and then concentrated and diafiltered by 10 kDa TFF against 15 diavolumes of 10 mM PBS pH 7.1. The final concentrated COPS:FliC conjugate bulk is sterile filtered through 0.22 μm under sterile conditions and stored at 2-8° C.
Capping of unreacted active groups: The aminooxy-thiol linker used for COPS derivatization is a hetero-bifunctional linker with unique groups (aminooxy and sulfhydryl) at either end. When the aminooxy group reacts with the COPS (Carbonyl on KDO) this caps the aminooxy functionality, and the unreacted linker is removed from the labeled COPS. Thus, in the absence of free linker, the aminooxy groups are considered to be capped by COPS. Linkage of the sulfhydryl present at the end of the aminooxy-thiol labeled COPS obligate capping of the active group. Any remaining uncapped thiols are thus inherently associated with unlinked free OPS. The FliC protein is decorated with maleimide groups following modification with GMBS. Residual unreacted GMBS linker is removed by TFF filtration. Maleimides are unstable at neutral pH, and rapidly hydrolyze; hence the labeled/activated polysaccharide is rapidly added following FliC derivatization in order to maximize conjugation efficiency. In order to cap any possible unreacted maleimides that may remain during conjugation, a molar excess of β-mercaptoethanol (βME) is added whereby the βME sulfhydryls react with the remaining active maleimide groups, functionally extinguishing their activity.
C. Preparation of S. Typhi ViPs-TT Conjugate (Typbar-TCV™)
Typbar-TCV™ is comprised by the purified Vi capsule polysaccharide of S. Typhi linked to tetanus toxoid. Synthesis of the conjugate is accomplished by first derivatizing Vi with ADH at the polysaccharide carboxyl groups with carbodiimide chemistry, followed by then linking the ADH derivatized Vi to tetanus toxoid carboxyls with same carbodiimide approach. The
Polysaccharide activation: S. Typhi Vi PS bulk (stored at −20° C.) is thawed at room temperature. Hydrolysis of the bulk to partially de-O-acetylate Vi is performed by addition of 0.45 M sodium carbonate and bicarbonate buffer under gentle stirring for 15 minutes at 2-8° C., after which the pH is adjusted to 7 using 50% glacial acetic acid and the material is then buffer exchanged at RT against 0.1M MES/50 mM NaCl pH 6(MES buffer) with 30 kDa TFF. The partially de-O-acetylated bulk is then treated with ADH and EDC for 4 hours at 2-8° C. to label the carboxylic acid groups with ADH. The activated polysaccharide is then buffer exchanged and concentrated first against PBS, and then MES buffer using 300 kDa TFF.
Conjugation process: The Tetanus Toxoid bulk is buffer exchanged and concentrated against MES buffer pH 5.8 with 30 kDa TFF. The ADH-derivatized Vi and Tetanus Toxoid bulks are then mixed at a 1:1 mg: mg ratio, in the presence of EDC at 2-8° C., and mixed with gentle stirring until viscosity of the conjugate bulk reaches a specified viscosity limit (cP), whereupon the reaction is quenched by adjusting pH to 7.4 slowly by adding 20 mM EDTA pH 8.5 under stirring for 10-15 minutes.
Purification of DS from unreacted components: The quenched Vi:TT is purified by 1,000 kDa TFF with 10 diavolumes of 10 mM MES, 50 mM NaCl, pH 6.0 and then 10 diavolumes of 10 mM PBS pH 7.1. This process is sufficient to remove free Vi and protein to levels below 20% and 1% respectively. The final purified conjugate bulk is sterile filtered through 0.22 μm filter under sterile conditions and stored at 2-8° C. pending QC approval.
Capping of unreacted active groups: Vi is modified in a limiting fashion with ADH, and following addition of an excess of TT protein, the hydrazide groups on the activated Vi are effectively capped. It was found that the remaining level of uncapped hydrazide in Typbar-TCV™ is indeed very low, presumably due to factors including: i.) derivatization with ADH is under sub-saturating conditions (i.e., few hydrazides per molecule) and ii.) an excess of TT is added to the conjugation reaction, thus the molar ratio of protein carboxyls available for conjugation is in excess to the polysaccharide hydrazides.
Studies were performed to evaluate the potential local and systemic toxicity and the persistence or reversibility of any effects during a 14-day treatment free period of bivalent conjugate vaccine (containing Salmonella enteritidis and S. typhimurium vaccines comprised of Core-O polysaccharide [COPS] conjugated for phase 1 flagellin [MC]), and trivalent conjugate vaccine (containing S. Enteritidis and S. Typhimurium COPS:FliC conjugate vaccines and S. typhi Vi conjugate vaccine [Typbar-TCV™]) when administered by intramuscular injection to male and female New Zealand White rabbits.
All animals survived to scheduled termination. There were no test article-related adverse changes in mortality, clinical observations, physical exams, injection site scores, body weights, food consumption, organ weights, organ to body weight ratios, body temperature, ophthalmology or urinalysis.
Following administration of the vaccine boosts, test article groups exhibited changes in clinical pathology and histopathology indicative of an acute inflammatory response.
Pathological changes at Day 86/87 in the test article groups included inflammatory lesions at the injection sites, consisting microscopically of heterophilic and/or mixed cell inflammation of the dermis, subcutis and/or muscle and myocyte necrosis or degeneration. Inflammatory cells were present in the medullary sinuses of the draining iliac lymph nodes as an indirect effect of the inflammation observed at the injection sites. The reactions were more prevalent and/or severe on the left side as a result of the Day 85 dose. Overall the trivalent multiple dose vaccine group (Group 4), was mildly more affected in severity than bivalent vaccine dose groups. Unlike bivalent groups, Group 4 females were observed to have inflammation at injection sites in both the right (last injection Day 57) and left side (last injection Day 85).
Additional indicators of an acute inflammatory reaction in the test article dosed groups included increased neutrophil and monocyte counts on Day 86/87 and higher fibrinogen and C-Reactive Protein concentrations on Days 3 and 86/87 following dosing on Days 1 and 85.
These changes were not present at the recovery sacrifice on Day 99/100 indicating the resolution of the inflammation during the 14-day post-dose period.
Both bivalent and trivalent formulations were subjected for toxicity studies whereas only trivalent vaccine alone was taken forward for immunogenicity studies. Both bivalent and Trivalent formulations were designed with the same buffer constituents and same strengths of individual antigens. For evaluating immunogenicity studies, only trivalent formulation was chosen the reason being that that the trivalent formulation covers the components of bivalent formulation. The results indicated that the trivalent formulation has given high fold rise of antibodies by which it is expected that the bivalent as a separate vaccine should also express the same level of immunogenicity for the respective diseases.
Immunogenicity studies of the Trivalent Salmonella Conjugate Vaccine Drug Product:
The trivalent Salmonella conjugate vaccine drug product in multi-dose vials and the bivalent Salmonella conjugate vaccine drug product in both multi-dose and mono-dose vials were maintained in controlled storage at 2-8° C. with stability being monitored only for the Vi conjugate component because qualified assays were not available during 21 months for monitoring the S. enteritidis and S. typhimurium components.
Embodiments of the invention describea methodology to monitor the components of the trivalent and bivalent conjugate vaccines. This was accomplished by measuring total COPS by resorcinol (which is not affected by the Vi capsular polysaccharide). The S. typhimurium COPS is then measured by an inhibition ELISA using a potent monoclonal antibody specific for the immunodominant antigen 4 of Group B Salmonella; since serovar S. typhimurium is a member of Group B Salmonella, it reacts with this monoclonal antibody. The S. typhimurium COPS value is then subtracted from the total resorcinol COPS value to provide a quantification of the S. enteritidis COPS. This test was effective in measuring both total COPS and free (unconjugated) COPS.
The resorcinol subtraction method has proved to be a reliable method. Two studies of rabbit immunogenicity were performed in which animals were vaccinated with a single dose of trivalent Salmonella conjugate vaccine to assess the immunogenicity of the vaccine components 7 months and 8 months after 20 rabbits had been vaccinated in the rabbit toxicology study with the same trivalent Salmonella conjugate vaccine. The doses of trivalent conjugate vaccine came from the remaining vials of vaccine that were stored at 2-8° C. under GLP (Good Laboratory Practices) conditions at CRO that performed the rabbit toxicology study. In total, results from three separate rabbit immunogenicity studies spread over 8 months were obtained that provided data for monitoring the immunogenicity of the trivalent salmonella conjugate vaccine. The design and features of the rabbit immunogenicity studies are described below in Table 2.
Rabbit Toxicology Study—Analysis of the antibody titers from Group 4 of the rabbit toxicology study performed from the 20 rabbits that received the trivalent Salmonella conjugate vaccine, documenting the immunogenicity of the trivalent Salmonella conjugate vaccine when it was 14 months from time of release. In particular, the results show the immunogenicity four weeks after administration of the first dose of the trivalent conjugate (assessed by comparing the day 1 and day 29 antibody titers), thereby providing evidence of the immune response following a single dose of the trivalent Salmonella conjugate vaccine.
Rabbit Immunogenicity Study #1—An immunogenicity study in which 10 rabbits were given a single dose of trivalent conjugate vaccine and blood was drawn on day 1 (the day of immunization) and four weeks later (day 29). Paired sera were available from nine rabbits.
Rabbit Immunogenicity Study #2—A second smaller immunogenicity study was carried out 6 weeks after animals in Rabbit Immunogenicity Study #1 were vaccinated. Five rabbits, all female, were immunized with a single dose from the last final remaining vial of trivalent Salmonella conjugate vaccine.
Methods—the 10 rabbits that were utilized in Rabbit Immunogenicity Study #1 (yielding paired sera from nine animals) and the five rabbits utilized in Rabbit Immunogenicity Study #2 (yielding paired sera from all five rabbits) were all Special Pathogen Free (SPF) juvenile animals of the same variety (New Zealand White), age (10-16 weeks at time of dosing) and weight (>2.2 kg), and obtained from the same vendor (Charles River Laboratories), as the rabbits that were used in the Rabbit Toxicology Study. All rabbits were acclimated for at least 7 days prior to dosing. The serum specimens were collected on the identical time points, day 1 and day 29, as in the larger (20-animal) Rabbit Toxicology Study. Sera from all three studies were tested by the identical serological assay (IgG ELISA), using the identical source of S. enteritidis COPS, S. typhimurium COPS and S. typhi Vi antigens. Thus, methods and animals were standardized across all three immunogenicity studies to minimize sources of variability.
S. Enteritidis COPS IgG (EU/ml)
S. Enteritidis COPS IgG (EU/ml)
S. Enteritidis COPS IgG (EU/ml)
S. Typhimurium COPS IgG (EU/ml)
S. Typhimurium COPS IgG (EU/ml)
S. Typhimurium COPS IgG (EU/ml)
S. Typhi Vi IgG (EU/ml)
S. Typhi Vi IgG (EU/ml)
S. Typhi Vi IgG (EU/ml)
Results—Table 2 summarizes the serological results from the three sets of rabbits immunized with a single dose of trivalent Salmonella conjugate vaccine (Toxicology Study; N=20 rabbits), Immunogenicity Study #1; (N=9 rabbits) [with paired serum specimens]) and (N=5 rabbits). Serological data from these three time points provide an opportunity to establish whether or not the three different conjugate vaccines within the trivalent Salmonella conjugate vaccine remain comparably immunogenic over several months or whether there is evidence of an important fall in titers to one or another component conjugate.
Within each individual study, the fold-rise in titer of each rabbit to each of the three vaccine antigens (S. enteritidis COPS, S. typhimurium COPS and S. typhi Vi) is the key measurement.
The four key parameters to be compared across the studies should be:
i) Percent of rabbits demonstrating a >4-fold rise in titer among the total rabbits immunized.
ii) Geometric Mean Fold-Rise in titer
iii) Minimum fold-rise in titer
iv) Maximum fold-rise in titer
Conclusions from Analysis of the Serological Data:
The Rabbit Toxicology Study results with 20 rabbits (10 female, 10 male) and the Immunogenicity Study #1 with 9 rabbits (5 female, 4 male) provide a reasonable N (No. of animals) per study for comparison. Comparing the Geometric Mean Fold-Rise in titer and the minimum and maximum rise in titer for each of the three antibodies (anti-enteritidis COPS, anti-typhimurium COPS and anti-Vi PS) from the Rabbit Toxicology Study and Immunogenicity Study #1, each individual conjugate in the trivalent conjugate vaccine is seen to have retained its ability to stimulate serum antibodies, despite a difference of 7 months in the age of the drug product when used in the two studies.
In comparing serological results from Immunogenicity Study #2 with Immunogenicity Study #1 and with results of the Rabbit Toxicology Study, all three conjugates elicited at least a four-fold rise in titer to each vaccine antigen in each rabbit. Indeed, all but one rabbit exhibited a >8-fold rise in titer to each vaccine antigen. Thus, the Salmonella trivalent conjugate vaccine remained immunogenic. The Geometric Mean Fold Rise was somewhat lower for S. typhimurium and Vi antibodies in the small rabbit Immunogenicity Study #2. However, the minimum fold rise in titer and the maximum fold rise in titer were similar to those recorded in the Rabbit Toxicology Study and in Immunogenicity Study #1. This suggests that the difference in Geometric Mean Fold-Rise is due to the small numbers, particularly in Rabbit Immunogenicity Study #2.
The fold-rise in titer for each group of animals for each antibody assay was compared by Wilcoxon Rank Sum to determine if the fold rises in the 20 rabbits in the Toxicology Test were significantly different (p<0.05) for any of the three antibodies from the fold-rises of the nine rabbits in Immunogenicity Test #1. There were no significant differences. The same statistical test (Wilcoxon Rank Sum) was conducted to compare fold rises between day 1 and day 29 for each antibody comparing the nine rabbits in Immunogenicity Study #1 with the 5 rabbits in Immunogenicity Study #2. No significant differences were observed.
Controlled experiments were conducted as described in Table 3.
Eighty (80) rabbits (40/sex) were divided into four (4) groups of twenty (20) animals (10/sex/group). See Table 2 for group description, test/control article administration, and necropsy days. The procedures followed are listed in Table 4.
aBivalent vaccine contains S. enteritidis and S. typhimurium COPS:FliC conjugate vaccines.
bTrivalent vaccine contains S. enteritidis and S. typhimurium COPS:FliC conjugate vaccines and S. typhi Vi conjugate vaccine [Tybar-TCV ™].
The mean C reactive protein (CRP concentrations increased markedly and statistically significantly in vaccine-dosed groups on Days 3 and 86/87, relative to dosing on Day 1 or 85, respectively, indicating acute inflammation. The higher values on Day 86/87 correlated with the aforementioned microscopic observation of inflammation primarily at the injection site at terminal necropsy. With the exception of one control male, the CRP values were well below 32 μg/mL in all control and vaccine-dosed animals by Day 99/100, indicating the reversibility of this change. CRP values were statistically significantly different compared to control animals (see Table 5).
The immunogenic compositions according to embodiments of the invention were subjected to various stability studies. Real-time stability testing was carried for evaluating the stability of vaccine compositions according to embodiments of the invention at recommended storage conditions of 2-8° C.
S. Typhimurium
S. Enteritidis
S. Typhimurium
S. Enteritidis
Dose size: 0.5 mL
Analysis Results: Shown in Table 8
S. Typhimurium and S. Typhi Vi Conjugates when stored at 5° C. ± 3° C.
S. Typhimurium
S. Enteritidis
S. Typhi
S. Typhimurium
S. Enteritidis
S. Typhi
Thus, compositions according to embodiments of the invention were found to be stable at recommended storage conditions of 2-8° C.
Although the invention has been illustrated and described in greater detail with reference to the preferred exemplary embodiments, the invention is not limited to the examples disclosed, and further variations can be inferred by a person skilled in the art, without departing from the scope of protection of the invention.
For the sake of clarity, it is to be understood that the use of “a” or “an” throughout this application does not exclude a plurality, and “comprising” does not exclude other steps or elements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201841017672 | Nov 2018 | IN | national |
This application claims priority to PCT Application No. PCT/IN2019/050825, having a filing date of Nov. 7, 2019, which is based on IN 201841017672, having a filing date of Nov. 10, 2018, the entire contents both of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2019/050825 | 11/7/2019 | WO | 00 |
