Delayed Antiviral Plus Immunomodulator Treatment Still Reduces Mortality in Mice Infected by High Inoculum of Influenza A/H5N1 Virus
Total Page：16
File Type：pdf, Size：1020Kb
Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus Bo-Jian Zheng*†‡, 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¶, and Kwok-Yung Yuen*†‡ʈ *State Key Laboratory of Emerging Infectious Diseases, †Department of Microbiology, ‡Research Centre of Infection and Immunology, and Departments of §Pathology and ¶Biochemistry, University of Hong Kong, Pok Fu Lam, Hong Kong Edited by Tak Wah Mak, University of Toronto, Toronto, ON, Canada, and approved April 7, 2008 (received for review December 20, 2007) The mortality of human infection by influenza A/H5N1 virus can cyclooxygenase-2 (COX-2) inhibitor celecoxib and mesalazine exceed 80%. The high mortality and its poor response to the could be effective in reducing mortality. To imitate the clinical neuraminidase inhibitor oseltamivir have been attributed to un- situation, we delayed combination therapy for 48 h after chal- controlled virus-induced cytokine storm. We challenged BALB/c lenging the BALB/c mice with an inoculum of 1,000 LD50 of a mice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival, highly virulent influenza A virus, human isolate A/Vietnam/ body weight, histopathology, inflammatory markers, viral loads, T 1194/04. Our results demonstrate that combination therapy lymphocyte counts, and neutralizing antibody response were doc- consisting of an inhibitor of the viral neuraminidase (zanamvir) umented in infected mice treated individually or in combination and two inhibitors of inflammation (celecoxib and mesalazine) with zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitate greatly increased the survival rate of mice infected with a highly the real-life scenario, treatment was initiated at 48 h after viral pathogenic strain of influenza A/H5N1 virus. challenge. There were significant improvements in survival rate survival time (P < 0.02), and inflammatory markers (P < Results ,(0.02 ؍ P) MICROBIOLOGY 0.01) in the group treated with a triple combination of zanamivir, All mice survived with early institution of i.p. zanamivir treat- celecoxib, and mesalazine when compared with zanamivir alone. ment (Fig. 1A). The survival rate of mice was decreased to 13.3% Zanamivir with or without immunomodulators reduced viral load (2/15) if the treatment with zanamivir was delayed for 48 h, to a similar extent. Insignificant prolongation of survival was although the mean survival time was prolonged to 10.7 Ϯ 1.6 observed when individual agents were used alone. Significantly Ϯ ؉ ؉ days compared with 6.6 1.6 days in controls (Fig. 1B). As higher levels of CD4 and CD8 T lymphocytes and less pulmonary expected, all PBS-treated controls died, whereas all mice on inflammation were also found in the group receiving triple ther- immunomodulators alone died with a trend toward increased apy. Zanamivir alone reduced viral load but not inflammation and mean survival time to Ϸ8.5 days for mice given celecoxib or mortality. The survival benefits of adding celecoxib and mesalazine mesalazine and Ϸ9.5 days for those given both celecoxib and to zanamivir could be caused by their synergistic effects in reducing mesalazine, but only Ϸ7.5 days for those given gemfibrozil alone cytokine dysfunction and preventing apoptosis. Combinations of a or both celecoxib and gemfibrozil. Therefore, we did not select neuraminidase inhibitor with these immunomodulators should be gemfibrozil for further study. Single use of any of these immu- considered in randomized controlled treatment trials of patients nomodulators did not confer survival benefit. However, when suffering from H5N1 infection. zanamivir was combined with both immunomodulators, the survival rate increased to 53.3% (8/15) (P ϭ 0.02) and the mean zanamivir ͉ celecoxib ͉ mesalazine survival time increased to 13.3 days (P ϭ 0.0179) compared with zanamivir alone (survival rate 13.3% and survival time 8.4 days). he mortality of patients suffering from pneumonia and The body weight of all infected mice steadily decreased to a Tmultiorgan involvement caused by influenza A/H5N1 virus minimum at day 11 and then increased again for those that (H5N1) varies between 45% and 81% since earlier reports (1, 2). survived (Fig. 1C). Subsequent use of oseltamivir has not reduced the mortality Significant decrease (Ͼ2.5 logs) of viral titers in tracheal- associated with this virus. The unsatisfactory outcome of its pulmonary lavage (TPL) by TCID50 or copies of viral RNA treatment was attributed to either deficiencies in antiviral ad- genomes in lung tissues by real-time quantitative RT-PCR was ministration or the induction of a severe cytokine storm (3). The found in groups treated by zanamivir with or without immuno- poor response to oseltamivir can also be the result of delayed modulators at days 6 and 8 postchallenge (Fig. 2). Levels of initiation of treatment because of the nonspecific initial mani- inflammatory markers IL-6, IFN-␥, TNF-␣, macrophage inflam- festations of H5N1 infection, high initial viral load, poor oral matory protein 1 (MIP-1), and leukotriene assayed by enzyme bioavailability of oseltamivir in the seriously ill, lack of i.v. immunoassays were significantly higher in TPL obtained from preparations of oseltamivir, and the emergence of resistance during therapy (4, 5). Attempts to use antiinflammatory doses of corticosteroids to control excessive inflammation were associ- Author contributions: B.-J.Z. and K.-Y.Y. designed research; B.-J.Z., K.-W.C., Y.-P.L., G.-Y.Z., ated with severe side effects without any improvement in sur- C.C., H.-J.Z., and H.-L.C. performed research; K.-W.C., S.S.Y.W., S.K.P.L., P.C.Y.W., K.-H.C., vival (6). Moreover, cytokine and chemokine knockout mice or D.-Y.J., and K.-Y.Y. analyzed data; and B.-J.Z., S.S.Y.W., S.K.P.L., P.C.Y.W., and K.-Y.Y. wrote steroid-treated wild-type mice did not have survival advantage the paper. over wild-type mice after viral challenge (7). This paradox can The authors declare no conflict of interest. be explained if both a high initial viral load and the commen- This article is a PNAS Direct Submission. surate degree of excessive inflammation are important in the ʈTo whom correspondence may be addressed. E-mail: [email protected]. pathogenesis and outcome of H5N1 infection. Here, we tested This article contains supporting information online at www.pnas.org/cgi/content/full/ the hypothesis that the combination of a parenterally adminis- 0711942105/DCSupplemental. tered neuraminidase inhibitor, zanamivir, together with the © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711942105 PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 ͉ 8091–8096 Downloaded by guest on September 26, 2021 Z Z C A 100% M 1000000 Z+C+M G <0.05 80% C+M 100000 <0.01 PBS C+G 60% PBS 10000 40% 1000 20% 100 Undetectable Survival rates 0% Viral titres +SD 10 1 3 5 7 9 11 13 15 17 19 1 Days post-challenge 468 Z Days post-challenge 100% Z+C Z+M Z Z+C+M B 80% 1000000 <0.01 Z+C+M PBS <0.01 PBS 60% 100000 40% -actin 10000 20% >0.05 Survival rates 1000 0% 135791113151719 100 Days post-challenge 10 Viral copies/100 copies/100 Viral Z 1 Z+C 468 24 Z+M Z+C+M Days post-challenge 20 PBS Fig. 2. Detection of viral load in infected mice treated with zanamivir, 16 celecoxib, and mesalazine. (A) Titers of released virus in TPL collected at the indicated days from mice treated with zanamivir alone (Z), zanamivir/ 12 celecoxib/mesalazine (ZϩCϩM), and PBS, which were started at 48 h postchal- lenge, as measured by TCID50. The detection limit (undetectable) is 1:20. (B) Weight (g ±SD) 8 Viral RNA copies in lung tissue from the above mice were determined by 1 3 5 7 9 1113151719 real-time RT-PCR and normalized by -actin. The P values between groups Days post-challenge ZϩCϩM and Z or PBS are indicated. Fig. 1. Survival times, survival rates, and body weight for infected mice treated with zanamivir, celecoxib, mesalazine, and gemfibrozil. (A) Shown is protein in the cytoplasm of pulmonary alveolar epithelial cells the survival rate and time of the mice (five mice per group) treated with (Fig. 4C). Tissue damage was primarily confined to lung tissues. zanamivir (Z), celecoxib (C), mesalazine (M), gemfibrozil (G), celecoxib/ However, there was mild perivascular mononuclear cell infiltra- mesalazine (CϩM), celecoxib/gemfibrozil (CϩG), and PBS (control) at 4 h postchallenge. (B) Survival time and rate of the mice (10–15 mice per group) tion in the cerebral cortex from the mice treated with zanamivir treated with zanamivir (Z), zanamivir/celecoxib (ZϩC), zanamivir/mesalazine alone but not in those from mice treated by both zanamivir and (ZϩM), zanamivir/celecoxib/mesalazine (ZϩCϩM), and PBS at 48 h postchal- immunomodulators, whereas focal dense mononuclear cell in- lenge were monitored for 21 days. (C) Body weights of the mice were moni- filtration in the cerebral cortex was observed in brain tissues tored for 21 days (survival mice) or until death. taken from the untreated mice (Fig. S2A). Reactive lymphoid cells that appeared paler in staining were found in spleens obtained from zanamivir-treated and PBS control mice, in which the mice treated with zanamivir alone and controls than those Ͻ reactive lymphoid cells were present along with frequent apo- treated by triple therapy (P 0.01 or 0.05) or uninfected normal ptotic bodies with prominent nuclear fragmentation, but not in mice (Fig.