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Method for inactivating zika virus and related methods

a zika virus and inactivation technology, applied in the field of methods for inactivating a zika virus, can solve the problems of less desirable two-plasmid system for the development of a zika vaccine, and achieve the effect of high sensitivity and efficient inactivation

Inactive Publication Date: 2021-12-30
TAKEDA VACCINES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present disclosure is based, at least in part, on the surprising finding that Zika virus can efficiently be inactivated with a low concentration of formaldehyde which is applied to the virus for a relatively short time at room temperature. Additionally, an assay was developed which allows to determine with a high sensitivity whether infectious virions are still present after inactivation. Finally, a method was developed which allows to detect low levels of residual formaldehyde in the final drug product or pharmaceutical composition.

Problems solved by technology

While Zika virus poses a substantial public health threat, no FDA-approved vaccine or treatment currently exists, and the only preventative measures for controlling Zika virus involve managing mosquito populations.
However, a two plasmid system for the development of a Zika vaccine is less desirable.

Method used

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  • Method for inactivating zika virus and related methods
  • Method for inactivating zika virus and related methods
  • Method for inactivating zika virus and related methods

Examples

Experimental program
Comparison scheme
Effect test

example 1

ka Virus Strain Generation

[0345]This example describes the production of Zika virus (ZIKAV) strains with a known research history.

Materials and Methods

Vero Cell Maintenance

[0346]One vial of WHO Vero 10-87 cells was rapidly thawed in a water bath and directly inoculated into 19 mL pre-warmed DMEM (Dulbecco's modified minimal essential medium) containing penicillin-streptomycin, L-glutamine 40 mM, and 10% FBS in a T-75 cm2 flask at 36° C.+ / 2° C., at 5% CO2. Cells were allowed to grow to confluency and subcultured using TryplE. This flask was expanded to two T-185 cm2 flasks, grown to confluency and subcultured to 31 xT-185 cm2 flasks and grown until the cells reached 100% confluency. Cells were harvested by trypsinization, centrifuged at 800×g for 10 minutes, and resuspended in DMEM containing 10% FBS and 10% DMSO at a concentration of 1.9×107 cells / mL. One vial of the Vero cells was rapidly thawed and resuscitated as described above into a T-75 cm2 flask. These were subcultured twice...

example 2

al Immunogenicity and Efficacy of a Purified Inactivated Zika Virus Vaccine (PIZV) Derived from the P6b and P6e Strains

[0367]The following example describes the preclinical immunogenicity and efficacy in CD1 and AG129 mice of an inactivated Zika virus vaccine (PIZV) derived from the P6b and P6e strains. As described in Example 1, six clones were generated from the epidemically relevant PRVABC59 strain, and two (P6b and P6e) were chosen for further preclinical immunogenicity and efficacy studies.

Materials and Methods

Purification, Inactivation and Formulation of a Zika Virus Vaccine

[0368]A lot of inactivated ZIKAV vaccine, suitable for use in preclinical immunogenicity and efficacy studies, was generated and characterized. Virus was amplified from the P6b and P6e strains by infecting flasks of confluent Vero cells at a MOI of 0.01. Virus was adsorbed for 1 hour at 36° C.±2° C. / 5% CO2. Following adsorption, 20 mL of cDMEM-0%-FBS was added to each flask, and incubated at 36° C.±2° C. / 5%...

example 3

al Assessment of the Phenotype of the P6a and P6e Strains

Materials and Methods

[0395]AG129 mice (lacking interferon α / β and γ receptors) are susceptible to ZIKV infection and disease, including severe pathologies in the brain. 14-week-old AG129 mice were intraperitoneally infected with 104 and 103 pfu of the ZIKV passage 6 clones a and e.

[0396]Mice were weighed and monitored daily (up to 28 days) for clinical signs of illness (weight loss, ruffled fur, hunched posture, lethargy, limb weakness, partial / full paralysis). Additionally, analysis of viremia was performed by plaque titration of serum samples collected three days post-challenge as described in Example 1.

Results

[0397]Infection with preMVS P6e resulted in 100% mortality (median survival time=12.5 days), while infection with preMVS P6a resulted in only 33% mortality (median survival time=undetermined) (FIG. 22). In agreement with this, preMVS P6e infected mice showed greater weight loss as compared to PRVABC59 P6a infected mice...

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Abstract

The present disclosure relates to methods for inactivating a Zika virus which can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation and to a method for determining the residual formaldehyde content in a pharmaceutical composition comprising an inactivated virus.

Description

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH[0001]This invention was made with government support under Contract No. HHSO100201600015C with the Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority. This invention was created in the performance of a Cooperative Research and Development Agreement with the Centers for Disease Control and Prevention, an Agency of the Department of Health and Human Services. The Government of the United States has certain rights in the invention.FIELD OF THE INVENTION[0002]The present disclosure relates to methods for inactivating a Zika virus which can be used in vaccines and immunogenic compositions. The present disclosure also relates to a method for determining the completeness of inactivation of an arbovirus preparation and to a method for determining the residual formaldehyde content in a pharmaceutical composition comprising an inactivat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N7/00A61K39/12A61P31/14C12Q1/22
CPCC12N7/00A61K39/12A61K2039/5252C12Q1/22A61P31/14C12N2770/24121C12N2770/24134C12N2770/24163C07K16/1081C07K2317/76Y02A50/30A61K2039/545C12N2770/24151G01N30/74
Inventor LIVENGOOD, JILL AGIEBLER, HOLLIDEAN, HANSISATOU, TATSUKIRAO, RAMANMARKS, JACKIELYONS, MARKSHINTANI, ASAEGIFFORD, JAMESOGASAWARA, NAOMISAKI, MASAFUMIADACHI, SATOSHI
Owner TAKEDA VACCINES INC
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