Live attenuated zika virus with 3'utr deletion, vaccine containing and use thereof

A technology for Zika virus deletion, which is applied in the field of live attenuated Zika virus with 3'UTR deletion, vaccines containing the virus and its application, and can solve the problems of reducing safety and the like

Pending Publication Date: 2019-10-25
BOARD OF RGT THE UNIV OF TEXAS SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Live-attenuated vaccines usually provide rapid and durable immunity, but sometimes with reduced safety; whereas inactivated and subunit vaccines provide enhanced safety at the expense of reduced immunogenicity and often require multiple doses and regular enhancer

Method used

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  • Live attenuated zika virus with 3'utr deletion, vaccine containing and use thereof
  • Live attenuated zika virus with 3'utr deletion, vaccine containing and use thereof
  • Live attenuated zika virus with 3'utr deletion, vaccine containing and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1: Generation of Live Attenuated ZIKV Strains with 3'UTR Deletion

[0115] Materials and methods:

[0116] Viruses: The ZIKV Cambodian strain FSS13025 (GenBank No. KU955593.1) was generated from the infectious cDNA clone pFLZIKV as previously described (ref. 10). All cell lines tested negative for mycoplasma.

[0117] Plasmid construction. For all plasmid constructions, standard molecular biology procedures were performed. Standard overlap PCR was performed to amplify the DNA fragment between the unique restriction enzyme sites EcoRI and ClaI using the corresponding primer pair. DNA fragments containing 3'UTR deletion mutations were introduced individually into pFLZIKV and pZIKV Rep (replicon cDNA plasmid, ref. 11) via EcoRI and ClaI. All constructs were confirmed by DNA sequencing. Primer sequences are available upon request. All restriction enzymes were purchased from New England BioLabs (Ipswitch, MA).

[0118] result:

[0119] We chose to pursue ...

Embodiment 2

[0120] Example 2: Analysis of replication and IFN-β inhibition of ZIKV 3'UTR deletion mutants

[0121] Materials and methods:

[0122] cells and antibodies. Vero cells were purchased from the American Type Culture Collection (ATCC, Bethesda, MD) and maintained at 37 °C, 5% CO 2 High glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA) (Invitrogen, Carlsbad, CA). The following antibodies were used in this study: mouse monoclonal antibody (mAb) 4G2 cross-reactive with flavivirus E protein (ATCC); ZIKV-specific HMAF (Hyperimmune Ascites), Emerging Viruses at The University of Texas Medical Branch and Arbovirus World Reference Center (World Reference Center of Emerging Viruses and Arboviruses) (WRCEVA); horseradish peroxidase-labeled anti-mouse IgG (H+L) antibody (KPL, Gaithersburg, MD) and Alexa Fluor 488 Conjugated goat anti-mouse IgG (The...

Embodiment 3

[0133] Embodiment 3: the stability of mutant virus

[0134] Materials and methods:

[0135] Stability, RNA extraction, and RT-PCR of 3'UTR mutants. To check the stability of the 3'UTR mutants, we passaged them on Vero cells for 5 rounds (5 days per round). Simply put, the 1.5×10 6 Vero cells were seeded into T-25 flasks. Virus from RNA transfection (defined as PO) was used to infect Vero cells. On day 5 post-infection, culture broth (100 μl) was transferred to a new T-25 flask containing Vero cells in 5 ml medium. After five rounds of this passage (P5), viral RNA was extracted from P5 cultures using the QIAamp Viral RNA Kit (Qiagen). Viral RNA was amplified by RT-PCR using the SuperScript III One-Step RT-PCR Kit (Invitrogen). Complete genome-length sequencing of P5 viruses. Three independent passages were performed for each mutant virus.

[0136] Immunostaining colony assay. Wild-type and P5 mutant viruses were analyzed on Vero cells using an immunostaining colony a...

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Abstract

The present invention discloses a live attenuated strain of Zika virus (ZIKV) having a deletion in the 3' untranslated region (3'UTR) of the viral genome, which may affect viral RNA synthesis and sensitivity to type I interferon inhibition, but may not affect viral RNA translation. The present invention also discloses the use of these live attenuated ZIKV strains in the preparation of ZIKV vaccines and for providing immunoprotection against ZIKV infection and congenital ZIKV syndrome, particularly in pregnant females.

Description

[0001] Statement Regarding Federally Sponsored Research or Development [0002] This invention was supported by NIH Grant No. AI120942. [0003] related application [0004] This PCT application claims priority to U.S. Provisional Application No. 62 / 458,839, filed February 14, 2017, the contents of which are hereby incorporated by reference in their entirety. technical field [0005] The present invention generally relates to the development of live attenuated strains of Zika virus (ZIKV) and vaccine compositions comprising such strains. Strains and vaccines comprising said strains can be used in humans and animals to treat or provide immune protection against ZIKV, which can cause congenital ZIKV syndrome and Guillan-Barré syndrome (Guillan-Barré syndrome). ). Specifically disclosed are methods of preventing congenital ZIKV syndrome, including microcephaly. Background technique [0006] Mosquito vector ZIKV has recently posed a global threat to public health. The most ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/00
CPCA61K39/12A61K2039/53A61K2039/55A61K2039/57C12N2770/24121C12N2770/24134C12N2770/24162A61P31/14Y02A50/30A61K2039/555
Inventor P-Y·史X·谢C·单
Owner BOARD OF RGT THE UNIV OF TEXAS SYST
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