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Vaccine composition useful for HPV and hepatitis B infections and a method for preparing the same

A vaccine composition, hepatitis B technology, applied in chemical instruments and methods, biochemical equipment and methods, vaccines, etc., can solve problems such as cost hindering global delivery

Inactive Publication Date: 2011-06-29
BHARAT BIOTECH INTERNATIONAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although approximately 70% of cervical cancer cases are caused by these two types, and there is evidence of some degree of cross-protection against other closely related types, the cost of these current vaccines prevents sustained global delivery, especially is in a third world country with a high incidence of HPV infection

Method used

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  • Vaccine composition useful for HPV and hepatitis B infections and a method for preparing the same
  • Vaccine composition useful for HPV and hepatitis B infections and a method for preparing the same
  • Vaccine composition useful for HPV and hepatitis B infections and a method for preparing the same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0046] 1. Example 1: Cloning strategy

[0047] a) Strategy 1 : Chimeric fusion of multiple epitopes from early proteins to HbsAg:

[0048] This sequence is composed of the fusion of small hepatitis B antigens and series multi-epitopes derived from HPV16 and HPV18E7 proteins, and then the multi-epitopes are chimerically fused with the C-terminus and N-terminus of HbsAg protein. (HbsAg protein sequence:

[0049] MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPACPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLISLLVLLDY QGMLPVCPPLLGKSNTTTTSTGPCKTCTMPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWM

[0050]The following epitopes derived from E7 and / or E6 proteins were arranged in tandem to obtain: 5'EYMLDLQPETTEEDEIDGPAGQAEPDRAHYNIDEIDGVNHQHL 3', ie 43 amino acids (129 nucleotides). This major epitope is a combination of the following and selected from the list of T-cell and B-cell epitopes: HPV16E7 T-cell epitope: amino acids 20-29: TDLYCYEQLN; amino acids 45-...

example 2

[0118] 2. Example 2: Gene cloning and the construction of HbsAg-HPV antigen chimera:

[0119] a) Utilize N-terminal and C-terminal primers containing EcoR1 and BamH1 restriction sites respectively, amplify the hepatitis B gene encoding the full-length HbsAg protein by PCR to obtain a PCR fragment of about 700bp (see figure 1 ). The primers used for PCR amplification of HbsAg gene are:

[0120] HB N-terminal (HBNTERM):

[0121] 5'CACGAATTCACCATGGAGAACACAACATCAGG3'

[0122] HB C-terminus (HBCTERM):

[0123] 5'TCAGGATCCAATGTATGCCCAAAAGACAACAGG3'

[0124] The conditions for PCR amplification were denaturation at 94°C for 45 s, annealing at 65°C for 40 s and extension at 72°C for 1.5 min for 30 cycles, followed by a final extension at 72°C for 10 min. 10 ng of plasmid DNA obtained by cloning the HbsAg gene into pBluescript SKII+ was used as a template for the PCR reaction. Reactions consisted of 1x Taq DNA polymerase buffer, 0.25 mM dNTPs, 20 picomoles of each primer, and 1.5...

example 3

[0174] Example 3: Cloning and expression in Pichia pastoris:

[0175] The correctness of chimeric sequences and reading frames was verified by sequencing both DNA strands of all chimeric sequences, followed by yeast transformation. All the chimeric sequences mentioned in the previous sections were digested with EcoR1 and Not1, followed by gel purification, and combined with EcoR1 and Not1 digested pPIC3.5K vector (Invitrogen Corporation, Carlsbad, USA ( Carlsbad, USA))) and ligated overnight with T4 DNA ligase. Escherichia coli DH5a competent cells were transformed with the ligation mixture, and spread on LB agar plates containing 100 μg / mL ampicillin. Plasmid DNA for each of the constructs was isolated on a large scale from transformed clones, purified and digested with Bgl II to linearize the plasmids for yeast transformation.

[0176] Substantially linearized plasmid DNA encoding the chimeric sequences SEQ ID No. 1 to SEQ ID No. 15 was transformed into Pichia GS115 follow...

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Abstract

The invention describes a vaccine compositions comprising chimeric fusions of the HPV antigens with viral or bacterial proteins conferring enhanced immunogenicity useful for Hepatitis B virus as well as human papillomavirus (HPV) infections.

Description

technical field [0001] The present invention relates to compositions comprising human papillomavirus (HPV) antigens and polypeptides derived from said antigens, alone or in combination, as chimeric fusions with bacterial or viral antigens, useful for prophylactic and / or Therapeutic treatment of HPV and hepatitis B infections in mammals, especially humans. [0002] The present invention relates to a pharmaceutical formulation capable of eliciting a protective antibody response and a cytotoxic T cell response against human papillomavirus infection (HPV). Immunogenic formulations contain purified recombinant HPV antigens in a stable formulation. The present invention also discusses methods of eliciting protective antibody responses and cytotoxic T cell responses using monovalent, bivalent and multivalent chimeric vaccines. Immunogenic compositions are formulated suitable for in vivo administration to humans. Background technique [0003] Human papillomavirus (HPV) is a doubl...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61K39/285C07K14/025
CPCC07K2319/00A61K39/12C07K14/005C12N2730/10122C12N7/00A61K39/292C12N2730/10134A61K39/00C12N2710/20022A61K2039/55505A61K2039/70C12N2710/20034
Inventor 克里希纳·默斯·艾拉坎达斯瓦弥·苏玛斯
Owner BHARAT BIOTECH INTERNATIONAL
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