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

An HPV16L1 and codon technology, which is applied in the field of HPV16LI with optimized codons for the SALMONELLA vaccine strain against human papillomavirus type 16, can solve problems such as difficulty in obtaining vaccines

Inactive Publication Date: 2008-01-30
INDIAN IMMUNOLOGICALS LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 was flanked at 5' by Ncol restriction sites and at 3' by Hindlll restriction sites. This L1S Ncol-Hindlll fragment was inserted to replace the original LINcoI-HindlH fragment in plasmid pFS14nsdHPV16-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 4 989{Acya Acrp, (4)), x4990 (AcyaAcrp-cdt, (4)) and AaroA (SL7207(16)), a gift from Irene Corthesy-Theulaz, Lausanne, CH.

[0050] HPV16 LI and VLP Analysis

[0051] Expression of LI in Salmonella lysates was analyzed by Western blot using anti-HPV16 LI mAb, CAMVIR-1 (Anawa), as previously described 15 (31 ). Data were normalized to the level in bacteria as measured by the OD600 of the c...

Embodiment 2

[0064] Plasmid construction and bacterial strains used

[0065] In the plasmid pFS14nsd HPV16-L1S [Baud, 2004 #1439], 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):

[0066] 5'-GGG CCGCGG TGGTCAT GAACAATAA-3', and a 28-mer primer containing an Xbal restriction site (underlined):

[0067] 5'-GGG TCTAGA AGCTGTCAAACATGAGAAT-3'. Another SacII-XbaI large fragment containing the entire pFS14nsdHPV16-L1S plasmid sequence but without the ampicillin resistance gene was generated by inverse PCR with Expand High Fidelity PCR (Roche Molecular Biochemicals), using the following primers: 28-mer primer 5'-GGG containing a ...

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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 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 novel nucleic acid sequence (HPV16 L1S) encoding the antigenic HPV 16L1 protein as provided in SEQ ID NO: 1, wherein said sequence has at least one modified codon to optimize the recombinant plasmid during transformation in prokaryotic microorganisms The stability of the vector to enhance 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 attributabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/025A61K39/12C12N15/63
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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