Invented by Annaliesa Sybil Anderson, Kathrin Ute Jansen, Mark Edward Ruppen, Justin Keith Moran, Susan Kay Hoiseth, Wyeth LLC
Traditionally, lipidated antigens have been used in vaccine development for Neisseria meningitidis. Lipidation is a process where lipids are attached to proteins, enhancing their immunogenicity and stability. However, recent research has shown that non-lipidated variants of Orf2086 antigens can also induce a strong immune response against Neisseria meningitidis.
One of the main advantages of non-lipidated variants is their potential for improved safety. Lipidated antigens can cause adverse reactions in some individuals, leading to concerns about their use in vaccines. Non-lipidated variants eliminate this risk, making them a safer alternative for vaccine development.
Another advantage of non-lipidated variants is their potential for increased efficacy. Research has shown that non-lipidated Orf2086 antigens can induce a robust immune response, including the production of antibodies and activation of T cells. This immune response is crucial for preventing and controlling Neisseria meningitidis infections.
The market for non-lipidated variants of Orf2086 antigens is expected to grow due to the increasing demand for safer and more effective vaccines against Neisseria meningitidis. The global burden of meningococcal disease remains high, particularly in low-income countries with limited access to vaccines. Therefore, there is a need for affordable and accessible vaccines that can provide protection against this deadly bacterium.
Several pharmaceutical companies and research institutions are actively involved in the development of non-lipidated Orf2086 antigens. These organizations are conducting preclinical and clinical trials to evaluate the safety and efficacy of these antigens in vaccine formulations. If successful, these trials could pave the way for the commercialization of non-lipidated Orf2086 antigen-based vaccines.
The market for non-lipidated variants of Orf2086 antigens is also driven by technological advancements in vaccine production. Novel vaccine platforms, such as recombinant DNA technology and protein subunit vaccines, enable the production of non-lipidated antigens in large quantities. This scalability is crucial for meeting the global demand for Neisseria meningitidis vaccines.
Furthermore, the market for non-lipidated variants of Orf2086 antigens is supported by government initiatives and public health organizations. These entities recognize the importance of preventing meningococcal disease and are actively promoting the development and distribution of effective vaccines. Their support provides a favorable environment for the growth of this market.
In conclusion, the market for non-lipidated variants of Neisseria Meningitidis Orf2086 antigens is expanding rapidly. The demand for safer and more effective vaccines against Neisseria meningitidis is driving the development of non-lipidated antigens. With ongoing research and clinical trials, these antigens have the potential to revolutionize the prevention and control of meningococcal disease worldwide.
The Wyeth LLC invention works as follows
The present invention is a composition containing an ORF2086 polypeptide that has not been pyruvylated or lipidated, and a method of preparing it. The compositions described in an exemplary embodiment are immunogenic. “The present invention also relates to compositions which elicit an immune response against an ORF2086 polypeptide of serogroup B Neisseria Meningitidis in a mammal, and related methods.
Background for Non-lipidated Variants of Neisseria Meningitidis Orf2086 Antigens
rLP2086″ is a recombinant lipoprotein of 28 kDa that induces cross reactive bacterial antibodies to a number Neisseria Meningitidis (MnB), or more specifically, serogroup (MnB), strains. Using deduced sequence homology of amino acids, two subfamilies, A and B, were identified. These two subfamilies have been used to formulate the MnB rLP2086 samples. Each sample contains 20, 60,120, and 200?g/mL in 10 mM Histidine, 150 mM sodium chloride, and 0.5mg/mL of aluminum, with different levels of Polysorbate 80. Native LP2086 can be classified as a lipoprotein. Fletcher et al. Fletcher et al. vol. 72(4):2088-2100 (2004) showed that mice were more immune to rLP2086 containing an amino terminal lipid than versions without it. Preclinical and Clinical studies have shown that combining these two lipidated protein can provide coverage for the entire fHBP-family. Meningococcal septicemia is a deadly disease that can kill young children and adults in just a few hours, despite antibiotics being available. “There is still a need for serogroup B Meningococcal immunegenic compositions.
To meet these needs and others for a meningococcal vaccination, additional compositions were evaluated to provide coverage when using non-lipidated polypeptides from N. meningitidis. In a first aspect, the invention provides an immunegenic composition containing a non-lipidated ORF2086 polypeptide, wherein ORF2086 is a variant of B44, B02, B03, B22, B24, B09, A05, A04, A12 or A22. The ORF2086 is in some embodiments a variant of B44, B22, B09, A05, A12 or A22.
Another aspect” of the invention is an immunogenic composition containing a non-lipidated ORF2086 polypeptide (P2086 subfamily B). In some embodiments the P2086 Subfamily B Polypeptide can be a B44 variant, B02, B03, B22, B24 or B09 variant. In certain embodiments, an ORF2086 polypeptide P2086 Subfamily B variant is also included in the immunogenic composition. The P2086 subfamily A polypeptide can be an A05, A04, A12 or A22 variant in some embodiments.
In some embodiments the immunogenic composition comprises a further adjuvant. In some embodiments the adjuvant can be an aluminum adjuvant or a saponin. It could also be a CpG sequence, or a combination of these. In some embodiments the aluminum adjuvant can be AlPO4, Al(OH), Al2(SO4)3, alum, or Al(OH). In certain embodiments, the concentration of aluminium in the immunogenic formulation is between 0.125?g/ml to 0.5?g/ml. In certain embodiments, the concentration of aluminium in the immunogenic formulation is 0.25?g/ml. In a preferred embodiment the concentration of aluminium in the immunogenic formulation is between 0.125mg/ml to 0.5mg/ml. In some preferred embodiments, the concentration of aluminium in the immunogenic formulation is 0.25 mg/ml.
In some embodiments, an immunogenic composition can be used to induce an immune response against Neisseria Meningitidis following administration of multiple doses to a subject. In some embodiments the immunogenic reaction is conferred upon the subject after two doses. In some embodiments the immunogenic reaction is conferred upon the subject after three doses have been administered.
Another aspect” of the invention is a composition that confers increased immunogenicity to a non-lipidated P2086-antigen. The composition includes a saponin, and at least one P2086-antigen. In certain embodiments, saponin is present in an immunogenic composition at a concentration between 1 g/ml to 250 g/ml. In certain embodiments, saponin is present in an immunogenic composition at a concentration between 10 g/ml to 100 g/ml. In certain embodiments, saponin is present in the immunogenic formulation at a concentration of 10?g/ml. In some embodiments the concentration of saponin in the immunogenic formulation is 100?g/ml. The saponin in some embodiments is QS-21, ISCOMATRIX.
In some embodiments the composition also contains aluminum. In some embodiments the aluminum can be found as AlPO4, Al(OH), Al2(SO4)3 or alum. In certain embodiments, the composition contains between 0.125 and 0.5 mg/ml of aluminum. In certain embodiments, the composition contains 0.25 g/ml of aluminum. In a preferred embodiment the concentration of aluminium in the composition ranges between 0.125 mg/ml to 0.5 mg/ml. In some preferred embodiments, the aluminum concentration in the composition can be as low as 0.25 mg/ml.
In some embodiments, an immunogenic composition can be used to induce an immune response against Neisseria Meningitidis following administration of multiple doses to a subject. In some embodiments the immunogenic reaction is conferred upon the subject after two doses. In some embodiments the immunogenic reaction is conferred upon the subject after three doses have been administered.
In some embodiments the non-lipidated P2086 is a P2086 subfamily B polypeptide. In some embodiments the P2086 Subfamily B Polypeptide can be a B44 variant, or it could be a B02 polypeptide, B03 polypeptide, B22 polypeptide, B24, B09, etc. In some embodiments the non-lipidated P2086 antibody is a P2086 subfamily A polypeptide. P2086 Subfamily-A polypeptides can be A05, A04, A12 or A22 in some embodiments.
In some embodiments the composition contains at least two P2086 non-lipidated antigens. The two P2086 non-lipidated antigens include at least one P2086 Subfamily-A polypeptide, and at least a P2086 Subfamily-B polypeptide. In some embodiments the non lipidated P2086 subfamily A polypeptide has an A05 variant, and the non lipidated P2086 subfamily B polypeptide has a B44 variation. In some embodiments the non-lipidated P2086 subfamily A polypeptide can be an A05 and the non lipidated P2086 subfamily B polypeptide can be a B22. In some embodiments the non-lipidated P2086 subfamily A polypeptide can be an A05 and the non lipidated P2086 subfamily B polypeptide can be a B09.
Another aspect” of the invention is a method of conferring immunity on a subject to a Neisseria Meningitidis bacterium, the method comprising the step of administering an immunogenic composition containing a non-lipidated P2086 subfamily B polypeptide. In certain embodiments, the P2086 Subfamily B is a B44 variant, or it can be a B02 polypeptide, B03 polypeptide, B22 polypeptide, B24, B09, etc. In certain embodiments, an immunogenic composition also contains a polypeptide from the P2086 subfamily A. In some embodiments the P2086 subfamily A polypeptide can be an A05, A04, A12 or A22 variant.
In some embodiments the immunogenic composition comprises an adjuvant. In some embodiments the adjuvant can be an aluminum adjuvant or a saponin. It could also be a CpG sequence, or a combination of these. In some embodiments the aluminum adjuvant can be AlPO4, Al(OH), Al2(SO4)3, alum, or Al(OH). In certain embodiments, aluminum concentration in the immunogenic formulation is between 0.125?g/ml to 0.5?g/ml. In certain embodiments, aluminum concentration in the immunogenic formulation is 0.25?g/ml. In a preferred embodiment the concentration of aluminium in the immunogenic formulation is between 0.125mg/ml to 0.5mg/ml. In some embodiments the concentration of aluminium in the immunogenic formulation is 0.25 mg/ml.
In some embodiments the concentration of saponin in the immunogenic formulation is between 1 g/ml to 250 g/ml. In certain embodiments, the concentration of saponin in the immunogenic formulation is between 100?g/ml – 10?g/ml. In certain embodiments, saponin is present in the immunogenic formulation at a concentration of 10?g/ml. In some embodiments the concentration of saponin in the immunogenic formulation is 100?g/ml. The saponin in some embodiments is QS-21, ISCOMATRIX.
In some embodiments, an immunogenic composition may be administered to a subject over multiple doses according to a schedule. In some embodiments the immunogenic composition may be administered in two doses to the subject over a schedule. In some embodiments the immunogenic composition may be administered to the subject over a period of time in three doses.
The invention also provides a method for producing a non lipidated P2086 variation, which includes the following steps: (a) cloning the ORF2086 nucleic acids into an expression vector in order to create an ORF2086 vector; (b), transforming bacteria using the OFR2086 vector; (c), causing expression of the P2086 from the ORF2086 vector; and (d), isolating expressed P2086 protein. In some embodiments the bacteria is E. coli. In some embodiments expression is induced through IPTG addition.
In some embodiments, a codon that encodes the N-terminal cys of the P2086 variation is deleted. In some embodiments the codon that encodes the N-terminal cys of the P2086 is mutated into an Ala Gly or Val code. In some embodiments the P2086 is a variant A05, B01 or B44. In some embodiments the P2086 is a B09 version.
In some embodiments, a mutation is made to the N terminal tail to add Ser residues and extend the Gly/Ser stem immediately downstream of the Cys N-terminal. In some embodiments the total number Gly and Ser residues within the Gly/Ser stem is at minimum 7, at most 8, at least 9 or at least 10.
In some embodiments, point mutagenesis is used to optimize the codons in the P2086 variant’s N-terminal. In some embodiments the codons in the ORF2086’s N-terminal are optimized through point mutagenesis. The codon that encodes the fifth amino acids of the ORF2086 is identical to the nucleotides of SEQ NO: 8, and the code for the thirteenth amino acids of the ORF2086 is identical to the nucleotides of SEQ NO: 8. In some embodiments the N-terminal of the non-lipidated ORF2086 is optimized so that the 5?45 amino acids are identical to the 1-45 of SEQID NO: 8. In some embodiments the N-terminal of the non-lipidated ORF2086 is optimized so that the 5?45 nucleic acids are 100% identical to the 1-45 nucleic acids in SEQ ID NO: 8. 42 nucleic acid are 100% identical with nucleic acid 4-45 of SEQID NO: 8. In certain embodiments, the N terminus of the non-lipidated ORF2086 is optimized so that the 5? 39 nucleic acid are 100% identical with nucleic acid 4-42 in SEQ ID No: 8. In certain embodiments, in comparison to the amino acids 1-15 from SEQ ID No: 18, the N-terminal of the variant P2086 that is not lipidated contains at least one substitution. In some embodiments the N-terminal of the non-lipidated P2086 version contains two amino acid substitutes in comparison to amino acids 1-15 from SEQ ID No: 18. In some embodiments the N-terminal of the non-lipidated P2086 version contains at least one substitution of amino acids compared to the amino acids 2-15 in SEQ ID No: 18. In some embodiments the N-terminal of the non-lipidated P2086 version comprises at least one amino acid substitution compared to the amino acids 2-15 in SEQ ID No: 18. In certain embodiments, the substitutions of amino acids are conservative amino acid replacements.
In one embodiment, this invention relates stable formulations of Neisseria Meningitis ORF2086 subfamily B Antigens within immunogenic compositions.
The present invention also relates methods for preserving the conformation and potency of Neisseria Meningitis rLP2086 Antigens.
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