Codon-optimized HPV16LI for salmonella vaccine strains against human papillomavirus type 16

An HPV16L1 encoding technology, applied in the field of HPV16L1 with optimized codons for SALMONELLA vaccine strains against human papillomavirus type 16, can solve the problem of difficulty in obtaining vaccines and the like

Inactive Publication Date: 2013-05-08
INDIAN IMMUNOLOGICALS LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these expensive vaccines require multiple intramuscular doses to be effective, and since most cervical cancers occur in developing countries, they appear to be inaccessible to those who need them most

Method used

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  • Codon-optimized HPV16LI for salmonella vaccine strains against human papillomavirus type 16
  • Codon-optimized HPV16LI for salmonella vaccine strains against human papillomavirus type 16
  • Codon-optimized HPV16LI for salmonella vaccine strains against human papillomavirus type 16

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Plasmid construction and bacterial strains used

[0049] The L1S gene was synthesized by Microsynth, Buchs, Switzerland. The open reading frame is flanked at the 5' by Nco I restriction sites and at the 3' by Hind III restriction sites. This L1S Nco I-Hind III fragment was inserted to replace the original L1 Nco I-Hind III fragment in plasmid pFS14nsd HPV16-L1 (31). The resulting plasmid, pFS14nsd HPV16-L1S, was introduced into attenuated Salmonella enterica serovar Typhimurium strain PhoP by electroporation (37) c , (CS022(27)) and PhoP″(CS015(26)), both gifts from John Mekalanos, Boston, USA, x 4989 {Acya Acrp, (4)), x 4990 (Acya Acrp-cdd, (4)) and AaroA (SL7207(16)), a kind gift from Irene Corthesy-Theulaz, Lausanne, CH.

[0050] HPV16L1 and VLP analysis

[0051] Expression of L1 in Salmonella lysates was analyzed by Western blot using the anti-HPV16 L1 mAb, CAMVIR-1 (Anawa), as previously described (15, 31 ). Data were normalized to the level in bacteria as me...

Embodiment 2

[0063] Plasmid construction and bacterial strains used

[0064] In plasmid pFS14nsd HPV16-L1S, the ampicillin resistance coding sequence was replaced with the kanamycin resistance coding sequence as follows. The SacII-XbaI fragment containing the kanamycin coding sequence and promoter was generated by PCR using pET-9a (Novagen) plasmid DNA as a template. The primer used was a 25-mer primer located 54 nucleotides upstream from the first ATG of kanamycin and containing a SacII restriction site (underlined): 5'-GGG CCGCGG TGGTCATGAACAATAA-3', and a 28-mer primer containing an XbaI restriction site (underlined): 5'-GGG TCTAGA AGCTGTCAAACATGAGAAT-3'. Another large SacII-XbaI fragment containing the entire pFS14nsd HPV16-L1S plasmid sequence but without the ampicillin resistance gene was generated by inverse PCR using Expand High Fidelity PCR (Roche Molecular Biochemicals), using the following primers: 28-mer primer 5'-GGG located 92 nucleotides upstream from the ATG of ampicil...

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Abstract

The present invention relates to a novel nucleic acid sequence (HPV16 L1S) encoding antigenic HPV16 L1 protein as provided in SEQ ID NO: 1, wherein the said sequence has atleast one modified codon for optimum stability of recombinant plasmid vector when transformed in the prokaryotic micro-organism for improved immunogenicity of the resulting prokaryotic micro-organism. The invention further relates to constructing recombinant vectors pFS14nsdHPV16L1 and pFS14nsdHPV16 kan L1S harboring SEQ ID NO: 1, wherein the former carries Ampicillin and the latter, Kanamycin as a selection marker. The invention also relates to an attenuated strain of a prokaryotic micro-organism transformed with nucleic acid encoding HPV16 (Human Papillomavirus) major capsid protein and expressing the corresponding protein. In addition the invention discloses a process of producing a vaccine based on prokaryotic micro-organism for the treatment of papillomavirus infection and associated risk of cancer.

Description

technical field [0001] The present invention relates to a new nucleic acid sequence (HPV16 L1S) encoding the antigenic HPV16L1 protein as provided in SEQ ID NO: 1, wherein said sequence has at least one modified codon to optimize the recombinant plasmid vector when transformed in prokaryotic microorganisms stability to improve the immunogenicity of the resulting prokaryotic microorganisms. The invention also relates to an attenuated strain of a prokaryotic microorganism transformed with a nucleic acid encoding the major capsid protein of HPV16 (human papillomavirus) and expressing the corresponding protein. In addition, the present invention discloses a method of producing a prokaryotic microorganism-based vaccine for the treatment of papillomavirus infection and the associated risk of canceration. Background technique [0002] Cervical cancer is the second leading cause of cancer death in women worldwide, and virtually all of these tumors are attributable to infection with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/025A61K39/12C12N15/63A61P35/00
CPCA61K2039/523C12N2710/20023A61K39/12C12N2710/20034A61K2039/5258C12N2710/20022C12N7/00C07K14/005A61K2039/542A61K2039/543A61P31/12A61P35/00Y02A50/30
Inventor 丹尼斯·纳尔戴里
Owner INDIAN IMMUNOLOGICALS LIMITED
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