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. 2013 Sep;87(18):10324-33.
doi: 10.1128/JVI.00480-13. Epub 2013 Jul 17.

An inactivated cell culture Japanese encephalitis vaccine (JE-ADVAX) formulated with delta inulin adjuvant provides robust heterologous protection against West Nile encephalitis via cross-protective memory B cells and neutralizing antibody

Nikolai Petrovsky  1 , Maximilian Larena, Venkatraman Siddharthan, Natalie A Prow, Roy A Hall, Mario Lobigs, John Morrey
Affiliations

An inactivated cell culture Japanese encephalitis vaccine (JE-ADVAX) formulated with delta inulin adjuvant provides robust heterologous protection against West Nile encephalitis via cross-protective memory B cells and neutralizing antibody

Nikolai Petrovsky et al. J Virol. 2013 Sep.

Abstract

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m(-/-)) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m(-/-) mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8(+) T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.

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Figures

Fig 1
Fig 1
JE-ADVAX protection against heterologous WNV challenge. Groups of B6 mice (n = 20) were immunized with 2 doses of ccJE (0.5 μg) with or without Advax adjuvant or West Nile Innovator vaccine 3 weeks apart (A, C) or with a single dose of ccJE (5 μg) with or without Advax-1 or -2 (B, D). A negative-control group (n = 20) was immunized with saline. At 6 weeks postimmunization, mice were challenged with WNV (105 PFU s.c.). Mice were monitored for morbidity and mortality and for body weight loss for 21 days. Asterisks denote significant differences relative to the saline group determined by the log-rank test (**, P < 0.01; ***, P < 0.001).
Fig 2
Fig 2
Viral burden in vaccinated mice challenged with WNV. Groups (n = 5) of B6 mice were immunized twice with ccJE (0.5 μg) with or without Advax adjuvant or with West Nile Innovator vaccine 3 weeks apart or with a single dose of ccJE (5 μg) with or without Advax adjuvant. A negative-control group was immunized with saline. Six weeks after completion of the vaccination schedule, mice were challenged with WNV (105 PFU s.c.). (A) At 3 days postchallenge, mice were bled and the WNV content in plasma was determined. (B) Animals were euthanized at 6 days postchallenge to determine the virus content in brain. Each symbol represents an individual mouse, and horizontal lines indicate geometric mean titers. The lower detection limit of the assay is indicated by the dashed line. Asterisks represent significant differences relative to saline-treated negative-control mice determined by the Wilcoxon-Mann-Whitney rank-sum test (*, P < 0.05; **, P < 0.01). TCID50, 50% tissue culture infective dose.
Fig 3
Fig 3
JE-ADVAX immunization reduces histological lesions in midbrain of mice challenged with WNV. Representative hematoxylin-eosin-stained photomicrographs obtained from the midbrain of groups of three mice that were necropsied 6 days after WNV challenge are shown. (A) Severe lymphocyte and moderate neutrophil accumulation (arrows) around multiple small vessels were seen in the midbrain of placebo (saline)-treated mice. (B, D) Mice that received a single dose of ccJE (B) or ccJE plus Advax-1 (D) showed moderate lymphocyte and neutrophil (arrow) accumulation around the small vessels. (C, E) Mice that received two doses of ccJE alone (C) or ccJE plus Advax-1 (E) showed no accumulation of lymphocytes around small vessels. Photomicrographs in panels a to f are higher-magnification images of the images in panels A to F, respectively. Bars = 100 μm.
Fig 4
Fig 4
JE-ADVAX provides cross-protection against WNV in mice lacking CD8+ T cells. Groups of 8-week-old β2m−/− mice were immunized with two doses of JE-ADVAX (0.5 μg) delivered 2 weeks apart or sham vaccinated with saline. Six weeks after completion of the vaccination schedule, mice were inoculated s.c. with 103 PFU of WNV. Morbidity and mortality were recorded daily, and surviving mice were monitored for 21 days. The data shown were constructed from two independent experiments. Asterisks denote statistical significance (***, P < 0.001).
Fig 5
Fig 5
JE-ADVAX induces B cells able to mediate cross-protection against related flaviviruses. Eight-week-old B6 donor mice were immunized with two doses of JE-ADVAX (0.5 μg) delivered 2 weeks apart. Purified B and CD4+ T cells were isolated from spleens at 3 weeks postimmunization and adoptively transferred into 6-week-old B6 recipient mice. Naive B6 mice that did not receive any cells served as a control group. (A) One day after splenocyte transfer, all mice were challenged s.c. with 104 PFU of WNV and mortality was monitored in each group. (B) Purified B cells from JE-ADVAX-immunized or naive donor mice were adoptively transferred into 4-week-old B6 recipient mice, which were then challenged 1 day later s.c. with 105 PFU of MVEV and monitored twice daily for mortality. Asterisks denote statistical significance (*, P < 0.05). Combined statistical analysis of the survival data for groups of immune B-cell recipients challenged with either WNV or MVEV relative to those for the control groups showed that the difference was highly significant (P < 0.001, two-tailed Fisher's exact test).
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