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Novel attenuated poliovirus: PV-1 mono-cre-X

A polio virus technology, applied in the field of new attenuated polio virus, can solve the problem of attenuation

Inactive Publication Date: 2014-12-10
THE RES FOUND OF STATE UNIV OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, codon pair deoptimization (addition of underrepresented codon pairs relative to overrepresented codon pairs) in one or several ORFs of a virus can lead to large attenuation

Method used

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  • Novel attenuated poliovirus: PV-1 mono-cre-X
  • Novel attenuated poliovirus: PV-1 mono-cre-X
  • Novel attenuated poliovirus: PV-1 mono-cre-X

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Specific infectivity and attenuation of poliovirus and recombinant vaccine candidates.

[0074] Comparison of specific infectivity and attenuation of recombinant poliovirus vaccine candidates with PVM wild-type poliovirus. Viruses were grown on HeLa cells. Virus particle concentration was measured by optical density (1OD 260 =9.4x10 12 particles / ml). Plaque forming units / ml were determined by HeLa(R19) plaque assay and the ratio of viral particles to PFU was calculated. Relative specific infectivity was determined by normalizing particles / PFU to wild-type values. The virus was administered to CD155 transgenic mice (which express the PV receptor) by intracerebral injection, and the dose causing paralysis in 50% of the mice was determined (PLD 50 ).

[0075] Table 3 shows that engineering of the cre element in the genome of wt PV significantly reduced the neurovirulence and relative specific infectivity of the virus. Surprisingly, characterization of PV1-X showed t...

Embodiment 2

[0079] Viruses with back mutations are neuroneurally attenuated.

[0080] Neuroattenuation of polioviruses is usually caused by mutations at a small number of positions. For example, point mutations within the IRES of the Sabin poliovirus vaccine strain are determinants of the attenuated phenotype. Thus, such attenuated polioviruses frequently revert to the fully neurotoxic wild-type phenotype. After repeated passage in neuronal cells, PV1-mono-cre virus (Ld 50 >10 8 ) and PV1-mono-cre-X virus (Ld 50 >10 8 ) shows A 133 G mutation, which increases neuropathy in mice, although the mutated virus compared with wt poliovirus (LD 50 =10 1.9 ) is still attenuated. A 133 GPV1-mono-cre virus and A 133 LD of G PV1-mono-cre–X virus 50 10 respectively 4.5 and 10 5.6 .

[0081] The relative specific infectivity of PV1-mono-cre virus and PV1-mono-cre-X virus was 0.25 and 0.084, respectively. At the same time, A 133 G PV1-mono-cre virus and A 133 The relative specific infec...

Embodiment 3

[0084] Plasmid construction and DNA manipulation.

[0085] Neurovirulent poliovirus type 1 (Mahoney) is the strain used in the laboratory (Cello, 2002). The poliovirus cDNA sequence was that used for cDNA synthesis by Cello et al. (2002) (plasmid pT7PVM) (van der Werf, et al., 1986, Proc Natl Acad Sci USA 83:2330-4). "pT7PVMcre(2C ATP酶 ) mutant” is a full-length poliovirus cDNA clone, in which 2C ATP酶 The native cre element in the coding region was inactivated by introducing three mutations at nt4462 (G to A), 4465 (C to U) and 4472 (A to C) (Yin, et al., 2003, J. Virol. 77 : 5152–66; Paul, 2003, In: Semler BL, Wimmer E, eds. Molecular biology of picornaviruses. Washington (DC): ASM Press; 2002. pp. 227–46; Rieder, et al., 2000, J. Virol .74:10371–80). The dual-cre PV is a derivative of pT7PVM with two active cre elements; one at nt102 / 103 of the 5'-NTR on which a new NheI restriction site is created. The second cre element at 2C ATP酶 in the coding region ( figure 1 A)....

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Abstract

A novel and stable attenuated poliovirus is produced by engineering an indigenous replication element (cre), into the 5′ non-translated genomic region (with inactivation of the native ere element located in the coding region of 2C (mono-crePV), and replacing the nucleic acid sequence of all or part of the capsid coding region (PI) with a substitute PI coding region having reduced codon pair bias. The stably attenuated poliovirus is effective for vaccines and immunization.

Description

field of invention [0001] The present invention relates to novel attenuated polioviruses. Attenuated polioviruses exhibit excellent growth phenotypes in tissue culture while stably maintaining very low neurotoxicity. Therefore, attenuated viruses are preferably used in vaccines. [0002] Cross References to Related Applications [0003] This application claims priority to US Application No. 61 / 576,706, filed December 16, 2011, which is hereby incorporated by reference in its entirety. Background of the invention [0004] The poliovirus ("PV") genome contains a hairpin structure, called a cis-acting replication element (cre), which is required for genome replication. This cre element is located in the coding region of the 2CATPase protein (within the P2 domain). Specific mutations within this loop result in a replication-free phenotype. The PV genome with an inactive cre in P2 can be restored to activity by inserting a cre synthesized elsewhere into the PV genome, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04
CPCC12N2770/32321C12N7/00C12N2770/32334C12N2770/32361C12N2770/32662A61P31/14
Inventor E.维默J.塞洛Y.刘
Owner THE RES FOUND OF STATE UNIV OF NEW YORK
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