Compositions of influenza viral proteins and methods of use thereof

An influenza and protein technology, applied in the direction of viral peptides, viral antigen components, chemical instruments and methods, etc., can solve the problems of unsatisfactory symptoms, expensive, mutation protection, etc., to prevent or treat influenza infection, The effect of avoiding serious illness and death

Inactive Publication Date: 2007-12-12
VAXINNATE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such strategies can be costly to supply and maintain demand, and therefore supply is limited; can lead to variant protection and uns...

Method used

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  • Compositions of influenza viral proteins and methods of use thereof
  • Compositions of influenza viral proteins and methods of use thereof
  • Compositions of influenza viral proteins and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1: Flagellin-M2e fusion protein

[0159]M2e is conserved among multiple A subtypes (also referred to herein as "strains") of influenza. M2e is at least a portion of the M2 protein, specifically the 24 amino-terminal amino acids of the M2 protein (also referred to herein as the "external domain"). The M2 external domain is a considerably smaller amino acid sequence (24 amino acids) compared to HA (about 566 amino acids) and neuraminidase (about 469 amino acids). The M2e sequences of exemplary avian influenza A isolates differ from those of human isolates, but are highly conserved among avian isolates (see Table 1 above). Four tandem copies of M2e fused to the carboxyl terminus of flagellin STF2 (full length or deleted STF2 hinge region) were produced. STF2 without a hinge region is referred to herein as "STF2Δ".

[0160] Construction of fusion protein:

[0161] The synthetic 4XM2e sequence (four contiguous 24 amino acid sequences) fused to the carboxyl termin...

Embodiment 2

[0240] Example 2: Expression and purification of flagellin (STF2 and STF2Δ) fusion protein constructs encoding influenza A M2 external domain sequences

[0241] Consistent M2e sequences among influenza A strains from several human and avian sources are described in Table 1. To facilitate cloning of M2e sequences, two vector cassettes pMT / STF2 and pMT / STF2Δ were generated, each containing a multiple cloning site (MCS) (see Figures 17A and 17B). To generate pMT / STF2, the 1.5 kilobase pair gene encoding the full-length S. typhimurium type 2 fljb or STF2 flagellin was fused to the pMTBIP / V5-His vector (Invitrogen Corporation, Caspian, CA, USA) The Ig-binding protein (BIP) secretion signal for expression in Drosophila. A BiP sequence was incorporated at the 5' end of the construct as a secretion signal for expression in Drosophila. The chemically synthesized 4xM2e gene representing the H1, H2 and H3 consensus sequence SLLTEVETPIRNEWGSRSNDSSDP (SEQ ID NO. 47, Table 1) was cloned i...

Embodiment 3

[0247] Embodiment 3: Construction and expression of flagellin-hemagglutinin (HA) construct

[0248] The gene encoding HA was isolated from genomic DNA of the in-house laboratory strain PR8 (an attenuated derivative of A / Puerto Rico / 8 / 34) (SEQ ID NO: 68, coding sequence ID NO: 67). The gene was fused to the STF2Δ cassette, which had been previously constructed in pPICZΔ to generate STF2Δ.HAPR8 (SEQ ID NO: 63, CODING SEQUENCE ID NO: 62) (see Figure 18). The purified recombinant protein was tested for immunogenicity and efficacy in BALB / c mice. The H5N1 gene encoding the A / Vietnam / 1203 / 04 strain was custom-synthesized and fused to the STF2Δ cassette to generate STF2Δ.HAH5 (SEQ ID 61, CODE ID 60). Both human and avian HA constructs were transformed into P. pastoris strains GS115 and X-33 (Invitrogen Corporation, Casper, CA, USA). Selected clones were screened for expression by layering on SDS-PAGE gels and by Coomassie blue staining, and Western blot analysis using anti-HA and a...

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Abstract

Compositions, fusion proteins and polypeptides comprise at least one pathogen-associated molecular pattern and at least a portion of at least one integral membrane protein of an influenza viral antigen. The compositions, fusion proteins and polypeptides are used to stimulate an immune response in a subject.

Description

[0001] related application [0002] This application claims U.S. Provisional Application Nos. 60 / 638,254 (filed December 21, 2004); 60 / 638,350 (filed December 21, 2004); 60 / 645,067 (filed January 2005 19); Nos. 60 / 653,207 (filed 15 February 2005); 60 / 666,878 (filed 31 March 2005); 60 / 682,077 (filed 18 May 2005 ); and priority of No. 60 / 741,202 (filed November 30, 2005). The entire teachings of all of the above applications are incorporated herein by reference. Background technique [0003] Influenza is a contagious disease usually caused by RNA viruses. Three types of influenza viruses are known: influenza A, B and C. The natural host of influenza A is waterfowl. Influenza A viruses can infect humans, birds, farm animals (eg, pigs, horses), and aquatic animals (eg, seals). Influenza B viruses are usually only found in humans. Influenza infection is generally characterized by fever, myalgia, headache, cough and muscle pain. In the elderly and infirm, infection with influ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/11A61K39/12A61K39/39
Inventor 汤玛斯・J・普威尔弗利立安・纳卡尔朗秋・尚威廉・F・麦当劳杜安・D・黑威特
Owner VAXINNATE
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