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. 2008 Jun 10;105(23):8091-6.
doi: 10.1073/pnas.0711942105. Epub 2008 Jun 3.

Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus

Bo-Jian Zheng  1 , Kwok-Wah Chan, Yong-Ping Lin, Guang-Yu Zhao, Chris Chan, Hao-Jie Zhang, Hong-Lin Chen, Samson S Y Wong, Susanna K P Lau, Patrick C Y Woo, Kwok-Hung Chan, Dong-Yan Jin, Kwok-Yung Yuen
Affiliations

Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus

Bo-Jian Zheng et al. Proc Natl Acad Sci U S A. .

Abstract

The mortality of human infection by influenza A/H5N1 virus can exceed 80%. The high mortality and its poor response to the neuraminidase inhibitor oseltamivir have been attributed to uncontrolled virus-induced cytokine storm. We challenged BALB/c mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, body weight, histopathology, inflammatory markers, viral loads, T lymphocyte counts, and neutralizing antibody response were documented in infected mice treated individually or in combination with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate the real-life scenario, treatment was initiated at 48 h after viral challenge. There were significant improvements in survival rate (P = 0.02), survival time (P < 0.02), and inflammatory markers (P < 0.01) in the group treated with a triple combination of zanamivir, celecoxib, and mesalazine when compared with zanamivir alone. Zanamivir with or without immunomodulators reduced viral load to a similar extent. Insignificant prolongation of survival was observed when individual agents were used alone. Significantly higher levels of CD4+ and CD8+ T lymphocytes and less pulmonary inflammation were also found in the group receiving triple therapy. Zanamivir alone reduced viral load but not inflammation and mortality. The survival benefits of adding celecoxib and mesalazine to zanamivir could be caused by their synergistic effects in reducing cytokine dysfunction and preventing apoptosis. Combinations of a neuraminidase inhibitor with these immunomodulators should be considered in randomized controlled treatment trials of patients suffering from H5N1 infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Survival times, survival rates, and body weight for infected mice treated with zanamivir, celecoxib, mesalazine, and gemfibrozil. (A) Shown is the survival rate and time of the mice (five mice per group) treated with zanamivir (Z), celecoxib (C), mesalazine (M), gemfibrozil (G), celecoxib/mesalazine (C+M), celecoxib/gemfibrozil (C+G), and PBS (control) at 4 h postchallenge. (B) Survival time and rate of the mice (10–15 mice per group) treated with zanamivir (Z), zanamivir/celecoxib (Z+C), zanamivir/mesalazine (Z+M), zanamivir/celecoxib/mesalazine (Z+C+M), and PBS at 48 h postchallenge were monitored for 21 days. (C) Body weights of the mice were monitored for 21 days (survival mice) or until death.
Fig. 2.
Fig. 2.
Detection of viral load in infected mice treated with zanamivir, celecoxib, and mesalazine. (A) Titers of released virus in TPL collected at the indicated days from mice treated with zanamivir alone (Z), zanamivir/celecoxib/mesalazine (Z+C+M), and PBS, which were started at 48 h postchallenge, as measured by TCID50. The detection limit (undetectable) is 1:20. (B) Viral RNA copies in lung tissue from the above mice were determined by real-time RT-PCR and normalized by β-actin. The P values between groups Z+C+M and Z or PBS are indicated.
Fig. 3.
Fig. 3.
Detection of proinflammatory cytokines, chemokines, prostaglandins, and albumin in TPL. Concentrations of IL-1, IL-6, IFN-γ, IFN-α, MIP-1, PGE2, leukotrienes, and albumin in TPL collected from mice treated with zanamivir alone (Z), zanamivir/celecoxib/mesalazine (Z+C+M), untreated control (PBS), and uninfected (normal) mice at the indicated days were determined by ELISA and compared between different groups. Their P values are shown.
Fig. 4.
Fig. 4.
T lymphocyte counts in peripheral blood and histopathologic changes in lungs. (A) Numbers of CD3+/CD4+ and CD3+/CD4+ T lymphocytes in 10,000 blood cells taken from the mice in the indicated days were counted by flow cytometry, and the P values between zanamivir alone (Z), zanamivir/celecoxib/mesalazine (Z+C+M), and PBS groups are shown. (B) Histopathologic changes in mouse lung tissues collected at the indicated days postinfection are shown. Representative histologic sections of the lung tissues from these mice and uninfected mice (Normal) were stained with H&E (original magnification: ×100). Inflammatory infiltrate and alveolar damage are seen as thickening of the alveolar septum with obliteration of some alveolar spaces at this magnification. (C) Viral infection in lung of the mice was further demonstrated by immunohistochemical staining. Positivity is indicated by brown staining in the cytoplasm. Representative histologic sections of the lung tissues taken from these mice and uninfected mice (Normal) at the indicated day were stained with an antiinfluenza NP mAb (Original magnification: ×400.)
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