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. 2005 Mar;79(5):2910-9.
doi: 10.1128/JVI.79.5.2910-2919.2005.

Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection

Takeshi Ichinohe  1 , Izumi Watanabe, Satoshi Ito, Hideki Fujii, Masami Moriyama, Shin-Ichi Tamura, Hidehiro Takahashi, Hirofumi Sawa, Joe Chiba, Takeshi Kurata, Tetsutaro Sata, Hideki Hasegawa
Affiliations

Synthetic double-stranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection

Takeshi Ichinohe et al. J Virol. 2005 Mar.

Abstract

The mucosal adjuvant effect of synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I:C)] against influenza virus was examined under intranasal coadministration with inactivated hemagglutinin (HA) vaccine in BALB/c mice and was shown to have a protective effect against both nasal-restricted infection and lethal lung infection. Intranasal administration of vaccine from PR8 (H1N1) with poly(I:C) induced a high anti-HA immunoglobulin A (IgA) response in the nasal wash and IgG antibody response in the serum, while vaccination without poly(I:C) induced little response. Intracerebral injection confirmed the safety of poly(I:C). In addition, we demonstrated that administration of poly(I:C) with either A/Beijing (H1N1) or A/Yamagata (H1N1) vaccine conferred complete protection against PR8 challenge in this mouse nasal infection model, suggesting that poly(I:C) possessed cross-protection ability against variant viruses. To investigate the mechanism of the protective effect of poly(I:C), mRNA levels of Toll-like receptors and cytokines were examined in the nasal-associated lymphoid tissue after vaccination or virus challenge. Intranasal administration of HA vaccine with poly(I:C) up-regulated expression of Toll-like receptor 3 and alpha/beta interferons as well as Th1- and Th2-related cytokines. We propose that poly(I:C) is a new effective intranasal adjuvant for influenza virus vaccine.

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Figures

FIG. 1.
FIG. 1.
Anti-PR8 HA-specific antibody titer and PR8 virus titer. Anti-PR8 HA-specific IgA and IgG responses in BALB/c mice that received primary intranasal immunization with 0.1 to 10 μg of poly(I:C) as an adjuvant. Secondary immunization was performed 4 weeks after primary immunization with or without the adjuvant. The nasal wash and serum samples were collected 2 weeks after the second immunization. The antibody titers of five mice from each group were measured by ELISA. The same groups of mice were infected intranasally with 1,000 PFU of PR8 influenza virus at 2 weeks after the second immunization. Nasal wash fluids were collected 3 days after virus challenge. The virus titers were measured by plaque assay. De, heat-denatured poly(I:C). Each column represents the mean ± SD. The virus titers were statistically compared to those of nonimmunized mice.
FIG. 2.
FIG. 2.
(A) Virus titers in the lung wash fluids from the mice that received primary intranasal immunization with 1 to 10 μg of poly(I:C) as an adjuvant. Secondary immunization was performed 4 weeks after primary immunization with or without the adjuvant. The mice were infected intranasally with 1,000 PFU (40 LD50) of PR8 influenza virus 2 weeks after the second immunization. The survival rate of each experimental group is shown on the right side of the bar graph. Each column represents the mean ± SD. N.D., not determined. (B) Body weight change after virus challenge. Each point represents the relative ratio for initial body weight (mean) of 5 mice for each day after the challenge. (C) Histopathological finding of a lung immunized intranasally with A/PR8 vaccine with poly(I:C) followed by 1,000 PFU (40 LD50) of A/PR8 virus at 8 days after the challenge (×40; H&E). (D) Histopathological finding of a lung immunized intranasally with B/Aichi vaccine with poly(I:C) followed by 1,000 PFU (40 LD50) of A/PR8 virus at 8 days after the challenge (×40; H&E).
FIG. 3.
FIG. 3.
(A) Cross-protective antibody responses against PR8 HA in the mice immunized intranasally with A/PR8 (H1N1), A/Beijing (H1N1), A/Yamagata (H1N1), A/Guizhou (H3N2), B/Ibaraki, B/Yamagata, and B/Aichi vaccine with poly(I:C) as an adjuvant. Secondary immunization was performed 4 weeks after primary immunization without the adjuvant. The same groups of mice were infected with 1,000 PFU in 2 μl of PR8 influenza virus 2 weeks after the second immunization. The nasal wash fluid was collected 3 days after virus challenge. The virus titer was measured by plaque assay. Each column represents the mean ± SD. The serum collected at 2 weeks after the booster was analyzed for the presence of neutralizing antibodies against homologous or heterologous influenza virus. Inhibition of the virus was assessed by the additional reduction in infectivity beyond the background of naive mice. Samples were run in duplicate, and data are presented per group, where the ability to inhibit 50% of infection at the indicated dilution is shown. The dash indicates lack of reduction of infectivity. (B) The survival curve of the mice immunized with poly(I:C) and the various vaccines after the lethal A/PR8 (H1N1) challenge. Mice were immunized with 3 μg of A/PR8 (H1N1), A/Yamagata (H1N1), A/Guizhou (H3N2), and B/Ibaraki vaccine with 10 μg of poly(I:C) as an adjuvant. Secondary immunization was performed 4 weeks after primary immunization with the same amount of vaccine with the adjuvant. The survival rates of the mice until 10 days after the virus challenge are presented in a line graph.
FIG. 4.
FIG. 4.
In vitro responses of A/PR8 (H1N1) influenza virus-specific T cells derived from mice vaccinated with A/PR8 (H1N1), A/Yamagata (H1N1), A/Guizhou (H3N2), and B/Ibaraki viruses. The mice were intranasally administered 1 μg of each vaccine with 10 μg of poly(I:C) and then boosted with the same dose of the reagents at 3 weeks after priming. Spleens were isolated at 1 week after the boost and stimulated with T-cell-depleted splenocytes that had been pulsed with the indicated concentration of A/PR8 vaccine. These cells were cultured for 4 days and [3H]thymidine was added 8 h prior to the harvest. (B) Production of IFN-γ in the culture supernatant of the cells prepared in the same manner as the cells shown in Fig. 4A. The results are represented as a means of two independent experiments.
FIG. 5.
FIG. 5.
Expression of TLR3 and TLR4 mRNAs in the NALTs. Total RNAs were extracted from the NALTs of mice infected with 1,000 PFU of A/PR8 (A) and intranasally immunized with the HA vaccine with poly(I:C) (B) and the HA vaccine alone (C). To determine the mRNA expression levels of TLR3 or TLR4 in the NALTs, real-time quantitative RT-PCR was performed (n = 3). *, P < 0.05 versus the pretreated group (0 h). ND, not determined.
FIG. 6.
FIG. 6.
Expression of cytokine mRNAs in the NALTs. Total RNA was extracted from the NALTs of mice intranasally treated with 1 μg of PR8 vaccine with or without poly(I:C). The mRNA levels of IFN-α (A), IFN-β (B), IFN-γ (C), IL-4 (D), IL-6 (E), and IL-12 p40 (F) in the NALTs were determined with real time RT-PCR (n = 3). *, P < 0.05 versus the pretreated group (0 h).
FIG. 7.
FIG. 7.
(A) Body weight of mice with intranasal administration of 10 μg of poly(I:C) or 10 μg of CTB* daily for 9 days. Each point represents the mean relative ratio to initial body weight (mean ± SD [%]) of 5 mice in each day. (B to D) Histopathological findings of the nasal cavities of the mice intranasally administered 10 μg of poly(I:C) (B), 10 μg of CTB* (C), and PBS (D) daily for 9 days (×100; H&E). Black arrows indicate the mucus exudation with inflammatory cells.
FIG. 8.
FIG. 8.
(A) Body weight of mice after intracerebral injection of various doses [poly(I:C), 0.25, 2.5, and 25 μg; CTB*, 2.5, 10, and 25 μg] of poly(I:C) and CTB*. Each point represents the relative ratio to initial body weight (mean ± SD [%]) of 5 mice in each day. Histopathological findings of the brains injected with 10 μg of poly(I:C) (B), 10 μg of CTB* (C), and PBS (D) at day 4 after injection (×100; H&E).
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