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. 2016 Mar 1;11(3):e0150213.
doi: 10.1371/journal.pone.0150213. eCollection 2016.

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus

Akira Higuchi  1 , Hiroko Toriniwa  2 , Tomoyoshi Komiya  2 , Tetsuo Nakayama  3
Affiliations

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus

Akira Higuchi et al. PLoS One. .

Abstract

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

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Conflict of interest statement

Competing Interests: On behalf of all co-authors, the following competing interests are declared: TN had research funding from Daiichi Sankyo. AH is an employee of Kitasato-Daiichi Sankyo Vaccine, Division of Vaccine Production. HT and TK are staff members of Kitasato-Daiichi Sankyo Vaccine, Division of Vaccine Development. Daiichi Sankyo provided JEV vaccine strain. There are no patents, products in development or marketed products to declare. This did not alter the adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Construction strategy of recombinant cDNA.
The PrM-E region was amplified from the Beijin strain of Japanese encephalitis virus and cloned into the P/M junction of pMVAIK, using Nco I and Not I restriction enzyme sites.
Fig 2
Fig 2. Virus growth in Vero cells at different temperatures of 33, 35, 37, and 39°C.
Virus infectivity was assayed as TCID50 in Vero cells and each bar represents the mean ± 1.0 SD.
Fig 3
Fig 3. Expression of JEV E and measles N proteins.
Monoclonal antibodies against the JEV E and measles N proteins were used, and visualized by anti-mouse antibodies conjugated with FITC or rhodamine.
Fig 4
Fig 4. Protein expression of JEV of culture fluid and purified measles virus particles.
Vero cells were infected with a recombinant virus, and cell lysates, culture supernatants, and cell culture fluids were then immunoprecipitated using a polyclonal antibody against JEV. They were subjected to Western blotting (4A). Recombinant virus particles were purified through sucrose discontinuous density gradients. Fractions 1, 2, and 3 were collected and subjected the Western blotting. Polyclonal antibodies against measles virus (left panel) and monoclonal antibodies against the JEV E protein were used (4B).
Fig 5
Fig 5. Antibody response against measles virus.
Three cotton rats were immunized with a recombinant virus and reimmunized eight weeks after the first immunization. Serum samples were obtained after 1, 3, 5, 8, and 12 weeks. Measles antibodies were examined using a PA antibody kit.
Fig 6
Fig 6. Antibody responses of EIA and NT antibodies against JEV.
Three cotton rats were immunized with a recombinant MVAIK/JEVprM-E virus and reimmunized eight weeks after the first immunization. Serum samples were obtained after 1, 3, 5, 8, and 12 weeks. EIA and NT antibodies against JEV were measured.
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