Delayed antiviral plus immunomodulator treatmentstill reduces mortality in mice infected by highinoculum of influenza A/H5N1 virusBo-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*†‡�

*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 canexceed 80%. The high mortality and its poor response to theneuraminidase inhibitor oseltamivir have been attributed to un-controlled virus-induced cytokine storm. We challenged BALB/cmice with 1,000 LD50 of influenza A/Vietnam/1194/04. Survival,body weight, histopathology, inflammatory markers, viral loads, Tlymphocyte counts, and neutralizing antibody response were doc-umented in infected mice treated individually or in combinationwith zanamvir, celecoxib, gemfibrozil, and mesalazine. To imitatethe real-life scenario, treatment was initiated at 48 h after viralchallenge. 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 loadto a similar extent. Insignificant prolongation of survival wasobserved when individual agents were used alone. Significantlyhigher levels of CD4� and CD8� T lymphocytes and less pulmonaryinflammation were also found in the group receiving triple ther-apy. Zanamivir alone reduced viral load but not inflammation andmortality. The survival benefits of adding celecoxib and mesalazineto zanamivir could be caused by their synergistic effects in reducingcytokine dysfunction and preventing apoptosis. Combinations of aneuraminidase inhibitor with these immunomodulators should beconsidered in randomized controlled treatment trials of patientssuffering from H5N1 infection.

zanamivir � celecoxib � mesalazine

The mortality of patients suffering from pneumonia andmultiorgan involvement caused by influenza A/H5N1 virus

(H5N1) varies between 45% and 81% since earlier reports (1, 2).Subsequent use of oseltamivir has not reduced the mortalityassociated with this virus. The unsatisfactory outcome of itstreatment was attributed to either deficiencies in antiviral ad-ministration or the induction of a severe cytokine storm (3). Thepoor response to oseltamivir can also be the result of delayedinitiation of treatment because of the nonspecific initial mani-festations of H5N1 infection, high initial viral load, poor oralbioavailability of oseltamivir in the seriously ill, lack of i.v.preparations of oseltamivir, and the emergence of resistanceduring therapy (4, 5). Attempts to use antiinflammatory doses ofcorticosteroids to control excessive inflammation were associ-ated with severe side effects without any improvement in sur-vival (6). Moreover, cytokine and chemokine knockout mice orsteroid-treated wild-type mice did not have survival advantageover wild-type mice after viral challenge (7). This paradox canbe explained if both a high initial viral load and the commen-surate degree of excessive inflammation are important in thepathogenesis and outcome of H5N1 infection. Here, we testedthe hypothesis that the combination of a parenterally adminis-tered neuraminidase inhibitor, zanamivir, together with the

cyclooxygenase-2 (COX-2) inhibitor celecoxib and mesalazinecould be effective in reducing mortality. To imitate the clinicalsituation, we delayed combination therapy for 48 h after chal-lenging the BALB/c mice with an inoculum of 1,000 LD50 of ahighly virulent influenza A virus, human isolate A/Vietnam/1194/04. Our results demonstrate that combination therapyconsisting of an inhibitor of the viral neuraminidase (zanamvir)and two inhibitors of inflammation (celecoxib and mesalazine)greatly increased the survival rate of mice infected with a highlypathogenic strain of influenza A/H5N1 virus.

ResultsAll mice survived with early institution of i.p. zanamivir treat-ment (Fig. 1A). The survival rate of mice was decreased to 13.3%(2/15) if the treatment with zanamivir was delayed for 48 h,although the mean survival time was prolonged to 10.7 � 1.6days compared with 6.6 � 1.6 days in controls (Fig. 1B). Asexpected, all PBS-treated controls died, whereas all mice onimmunomodulators alone died with a trend toward increasedmean survival time to �8.5 days for mice given celecoxib ormesalazine and �9.5 days for those given both celecoxib andmesalazine, but only �7.5 days for those given gemfibrozil aloneor both celecoxib and gemfibrozil. Therefore, we did not selectgemfibrozil for further study. Single use of any of these immu-nomodulators did not confer survival benefit. However, whenzanamivir was combined with both immunomodulators, thesurvival rate increased to 53.3% (8/15) (P � 0.02) and the meansurvival time increased to 13.3 days (P � 0.0179) compared withzanamivir alone (survival rate 13.3% and survival time 8.4 days).The body weight of all infected mice steadily decreased to aminimum at day 11 and then increased again for those thatsurvived (Fig. 1C).

Significant decrease (�2.5 logs) of viral titers in tracheal-pulmonary lavage (TPL) by TCID50 or copies of viral RNAgenomes in lung tissues by real-time quantitative RT-PCR wasfound in groups treated by zanamivir with or without immuno-modulators at days 6 and 8 postchallenge (Fig. 2). Levels ofinflammatory markers IL-6, IFN-�, TNF-�, macrophage inflam-matory protein 1 (MIP-1), and leukotriene assayed by enzymeimmunoassays were significantly higher in TPL obtained from

Author contributions: B.-J.Z. and K.-Y.Y. designed research; B.-J.Z., K.-W.C., Y.-P.L., G.-Y.Z.,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.,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. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

�To whom correspondence may be addressed. E-mail: kyyuen@hkucc.hku.hk.

This article contains supporting information online at www.pnas.org/cgi/content/full/0711942105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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the mice treated with zanamivir alone and controls than thosetreated by triple therapy (P � 0.01 or 0.05) or uninfected normalmice (Fig. 3). However, IL-1 levels were only slightly lower inthose treated with zanamivir alone and controls (P � 0.05),whereas prostaglandin E2 (PGE2) levels were found to besignificantly higher in the samples collected at day 8 postchal-lenge from the group receiving triple therapy (Fig. 3). Asexpected, their serum cytokine and chemokine changes weresimilar to those of their TPL [supporting information (SI) Fig.S1]. Furthermore, levels of both CD4� and CD8� T lymphocyteswere significantly higher in the blood taken at days 6 and/or 8from the mice given triple therapy than those taken fromzanamivir-treated and PBS control mice (Fig. 4A).

The degree of lung damage as evidenced by increased albuminlevels in the TPL (Fig. 3) and higher elastase activity in the TPL(Fig. S1) was significantly lower in groups treated by thecombination therapy compared with the groups treated byzanamivir alone (P � 0.01) or PBS (P � 0.03). Histopathologicexamination further showed that the alveolar damage and in-terstitial inflammatory infiltration in mice treated by the com-bination were much less severe than those treated by zanamiviralone (Fig. 4B). Immunohistochemical staining with antiinflu-enza nucleoprotein mAb demonstrated strong expression of this

protein in the cytoplasm of pulmonary alveolar epithelial cells(Fig. 4C). Tissue damage was primarily confined to lung tissues.However, there was mild perivascular mononuclear cell infiltra-tion in the cerebral cortex from the mice treated with zanamiviralone but not in those from mice treated by both zanamivir andimmunomodulators, whereas focal dense mononuclear cell in-filtration in the cerebral cortex was observed in brain tissuestaken from the untreated mice (Fig. S2 A). Reactive lymphoidcells that appeared paler in staining were found in spleensobtained from zanamivir-treated and PBS control mice, in whichreactive lymphoid cells were present along with frequent apo-ptotic bodies with prominent nuclear fragmentation, but not inthose collected from mice treated with zanamivir and immuno-modulators (Fig. S2B). Nevertheless, no significant pathologicchanges or tissue damages could be detected in liver (Fig. S2C)and kidney (Fig. S2D) from all mice.

As shown in Fig. S3A, 12 surviving mice with undetectableviral load in lung tissues at day 21 after viral challenge also hada neutralizing antibody titer of 80. Western blot confirmed thatthe neutralizing antibody reacted specifically with baculovirus-expressed nucleoprotein and hemagglutinin of H5N1 (Fig. S3B).Interestingly, two surviving mice treated with triple therapy stillhad a detectable low viral load and a neutralizing antibody titerof 40. Compared with the zanamivir-treated group whoseTCID50 titer in the TPL was below our detectable limit, the tripletherapy group had a TCID50 titer of 5.1 � 102 � 4.9 � 102, whichwas still 2.5 log below the titer of 2.7 � 105 � 2.0 � 105 in thePBS control group (Fig. 2 A). This finding is not completelyunexpected because the immunomodulators may still have somedegree of immunosuppression that is not clinically apparent.Consistent with these findings, these two mice [zanamivir �celecoxib � mesalazine (2)], together with the surviving mousefrom the zanamivir-treated group, also had inflammatory infil-

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Fig. 1. Survival times, survival rates, and body weight for infected micetreated with zanamivir, celecoxib, mesalazine, and gemfibrozil. (A) Shown isthe survival rate and time of the mice (five mice per group) treated withzanamivir (Z), celecoxib (C), mesalazine (M), gemfibrozil (G), celecoxib/mesalazine (C�M), celecoxib/gemfibrozil (C�G), and PBS (control) at 4 hpostchallenge. (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 postchal-lenge were monitored for 21 days. (C) Body weights of the mice were moni-tored for 21 days (survival mice) or until death.

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Fig. 2. Detection of viral load in infected mice treated with zanamivir,celecoxib, and mesalazine. (A) Titers of released virus in TPL collected at theindicated days from mice treated with zanamivir alone (Z), zanamivir/celecoxib/mesalazine (Z�C�M), and PBS, which were started at 48 h postchal-lenge, as measured by TCID50. The detection limit (undetectable) is 1:20. (B)Viral RNA copies in lung tissue from the above mice were determined byreal-time RT-PCR and normalized by �-actin. The P values between groupsZ�C�M and Z or PBS are indicated.

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trate in their alveoli on histologic examination, whereas nosignificant inflammation was observed in the other survivingmice [zanamivir � celecoxib, zanamivir� mesalazine, and zana-mivir � celecoxib � mesalazine (6)], which was similar to thosefound in normal mice (Fig. 4B and Fig. S3C).

DiscussionThere is an urgent need to find an effective treatment strategyagainst H5N1 infection in humans because of the substantialmortality associated with this virus. Although oseltamivir ishighly effective in mouse models, humans treated with this drugstill exhibited high fatality, which can be attributed to delayedinitiation of therapy. Many antiviral treatment studies of mousemodels infected by H5N1 used an inoculum of �10 LD50 ofH5N1. Good treatment results were obtained if the antiviral wasstarted 4 h before, soon after, or within 36 h after inoculation (8,9). Only a few studies showed good results when the antiviraltreatment was started after 36 h. However, in those series, eithera low viral inoculum was used or a duck H5N1 virus adapted tomice was used instead of a human virus for inoculation (10–12).Thus the pathophysiologic status of the infected mice in thosestudies could be quite different from the real clinical situationwhen patients often do not enter the hospital until 2–4 days afterthe onset of symptoms, when the viral load in respiratorysecretions is already high. The high inoculum and delayedtherapy in the presently reported mouse model provided a morerealistic simulation for testing various forms of therapy. To avoidthe confounding effects of poor oral bioavailability of oseltami-

vir in sick mice and the known risk of emergence of oseltamivirresistance during therapy, i.p. zanamivir was used. However, asin the case of oseltamivir, 87% of the mice died when thezanamivir treatment was delayed for 48 h, although the survivaltime was insignificantly prolonged. Our animal model providedan ideal situation for testing combination therapy with immu-nomodulators that had no antiviral effects or any significanteffect on survival if used alone.

Our study showed that even if the viral replication had beensuppressed in the mice treated with antiviral, levels of cytokinesand chemokines were still similar to the untreated mice, whichwere significantly higher than those in the mice receiving com-bination therapy. This finding suggests that once the viralinfection has triggered the cytokine storm, even if viral replica-tion is suppressed by antiviral therapy, the proinflammatorycytokines and chemokines will continue to drive the immuno-pathologic progression. Previous studies showed that antiinflam-

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Fig. 3. Detection of proinflammatory cytokines, chemokines, prostaglan-dins, and albumin in TPL. Concentrations of IL-1, IL-6, IFN-�, IFN-�, MIP-1, PGE2,leukotrienes, and albumin in TPL collected from mice treated with zanamiviralone (Z), zanamivir/celecoxib/mesalazine (Z�C�M), untreated control (PBS),and uninfected (normal) mice at the indicated days were determined by ELISAand compared between different groups. Their P values are shown.

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Fig. 4. T lymphocyte counts in peripheral blood and histopathologic changesin lungs. (A) Numbers of CD3�/CD4� and CD3�/CD4� T lymphocytes in 10,000blood cells taken from the mice in the indicated days were counted by flowcytometry, and the P values between zanamivir alone (Z), zanamivir/celecoxib/mesalazine (Z�C�M), and PBS groups are shown. (B) Histopathologic changesin mouse lung tissues collected at the indicated days postinfection are shown.Representative histologic sections of the lung tissues from these mice anduninfected mice (Normal) were stained with H&E (original magnification:�100). Inflammatory infiltrate and alveolar damage are seen as thickening ofthe alveolar septum with obliteration of some alveolar spaces at this magni-fication. (C) Viral infection in lung of the mice was further demonstrated byimmunohistochemical staining. Positivity is indicated by brown staining in thecytoplasm. Representative histologic sections of the lung tissues taken fromthese mice and uninfected mice (Normal) at the indicated day were stainedwith an antiinfluenza NP mAb (Original magnification: �400.)

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matory dose of steroid was ineffective in mice in the absence ofantiviral treatment (7) and was associated with significant sideeffects in human infected by the H5N1 virus without improvingsurvival (5, 6). Therefore other immunomodulators have to beconsidered.

Because COX-2 knockout mice had significantly better survivalafter challenge with mouse-adapted influenza A H3N2 virus thanwild-type BALB/c mice (13), i.p. celecoxib was chosen in this study.Sulfasalazine and related compounds such as mesalazine and5-aminosalicylic acid are also chosen in this study because they arehighly active in alimentary tract epithelial cells and are commonlyused in the treatment of inflammatory bowel diseases. They havediverse effects on the immune system including inhibition oflipoxygenase and COX pathways, which decrease proinflammatorycytokines and eicosanoids, and therefore decrease the activation ofinflammatory cells such as macrophages and neutrophils. In addi-tion, sulfasalazine and 5-aminosalicylic acid inhibit NF-�B activa-tion and promote the synthesis of phosphatidic acid. Both actionsinhibit the potent stimulatory effects of ceramides on apoptosis (14,15). The combined actions of mesalazine (the effective moiety ofsulfasalazine) and celecoxib have synergistic effects in counteract-ing virus-induced cytokine dysregulation and apoptosis. Both drugsare relatively inexpensive, currently used in humans, not known tocause immunosuppression, and relatively free from adverse druginteractions or major side effects with short-term use.

The main target of action of the fibrates such as gemfibrozilis peroxisome proliferators-activated receptor � (PPAR�).PPARs are members of the nuclear receptor superfamily thataffects the lipid and glucose metabolism and inflammatoryresponses. PPAR� activation inhibits NF-�B, COX-2 activity,and production of proinflammatory cytokines such as IL-6 andTNF-� (16). Therefore, activation of the PPAR� by gemfibrozilis expected to damp down the excessive inflammatory response.Budd et al. (17) demonstrated that gemfibrozil improved survivalof mice infected by influenza A/H2N2 virus from 26% (controls)to 52% (treated). However, no improved survival was notedwhen the hypervirulent H5N1 virus was used in this study. Thisdiscrepancy could be related to the different pathophysiologybetween H2N2 and H5N1 viruses or the relatively weak agonisticactivities of gemfibrozil on PPAR�.

The association between higher levels of PGE2 and micesurvival is compatible with the known immunologic profiles ofhuman and experimental H5N1 infection. Among other cyto-kines and chemokines, severe H5N1 infections are associatedwith increased RANTES and MIP-1, and their synthesis isinhibited by PGE2. Our triple therapy also showed a reductionin MIP-1 levels without suppressing PGE2. PGE2 has antiin-f lammatory and antiapoptotic properties, both of which mayplay a beneficial role in preventing excessive tissue and cellulardamage. Previous reports showed that COX-2�/� knockout micehad a significantly lower mortality, lesser degree of inflamma-tory cell infiltrates in the lungs, and lower levels of proinflam-matory cytokines (TNF�, IL-1�, IFN-�, IL-6) in the TPL ascompared with wild-type and/or COX-1�/� knockout mice afterinfection by influenza A/H3N2 virus (13). But the PGE2 levelsin the TPL and the viral load in the lungs were significantlyhigher in COX-2�/� mice. Our findings of lower leukotrienes andhigher PGE2 levels in the TPL in mice treated by combinationtherapy is compatible with the above findings. Although me-salazine or celecoxib have not been shown to cause immuno-suppression, two surviving mice after triple therapy still had alow, but detectable, viral load and a low level of neutralizingantibody. This finding is not unexpected because the sameimmunologic factors causing tissue damage during the mountingof the immune response may also be critical for viral clearance(18). The combination of mesalazine and celecoxib may causemild subclinical immunosuppression. IL-1 was speculated to be

protective because infected IL-1 receptor knockout mice showedincreased morbidity, mortality, lung viral titer, and inflamma-tory infiltrate when infected with a low-lethality HK/486 virus(19). In this study, mice treated by triple therapy had improvedsurvival without significant suppression of IL-1 in TPL despitethe use of a hypervirulent virus.

H5N1-infected patients who succumbed often had persistentlyhigh levels of serum proinflammatory cytokines and chemokines(3, 5). Therefore, the pathogenesis was initially attributed tovirus-induced cytokine storm. However, studies with knockoutmice deficient in TNF-�, TNF receptor 1, TNF receptor 2, IL-6,chemokine (C-C motif) ligand 2, MIP-1�, and IL-1R (7) did notconfer better survival after viral challenge when antivirals werenot given. Moreover, recent studies showed that the levels ofserum proinflammatory cytokines and chemokines correlatedclosely with the viral load (5). Therefore, the pathogenesisshould involve the interplay between a rising viral load and theresulting proinf lammatory response. An effective therapyshould consist of combinations of an effective antiviral agent andimmunomodulatory agents to control viral load and cytokinestorm, respectively. This scenario is especially true if patientspresent late in the course of influenza, when local and systemicproinflammatory cascade are fully activated.

Postmortem examination of patients who succumbed to H5N1infection often showed severe lymphopenia and lymphoid atro-phy or necrosis in the spleen and other lymphoid tissues (1, 3).Our study also showed that both CD4� and CD8� T lymphocyteswere significantly decreased in antiviral-treated and untreatedmice during disease progression. However, unlike the use of thesteroid or other immunosuppressants, the use of celecoxib andmesalazine with zanamivir maintains significantly higher levelsof CD4� and CD8� T lymphocytes at days 6 and 8 postchallenge.Histopathologic examination also showed that reactive lymphoidcells with frequent apoptotic bodies were found in spleensobtained from zanamivir-treated and untreated mice, but wereinfrequent in spleens from mice treated with zanamivir andimmunomodulators. Thus, the antiapoptotic effects of celecoxibplus mesalazine may play a role in averting this type of damage.

Our results provide a sound theoretical and experimental basisfor further studies on the role of triple therapy. An antiviral andthe combined use of celecoxib and mesalazine may causesynergistic reduction in proinflammatory cytokines, chemo-kines, and leukotrienes via different pathways. These inhibitoryactivities, together with the antiapoptotic activities of the amino-salicylates, reduce cell death and tissue damage in the host (20).Apoptosis in pulmonary alveoli and lymphoid tissues leading tolymphopenia are prominent pathological features in patientsdying of H5N1 infection. The concomitant use of an effectiveantiviral is essential, not only to limit the extent of viral repli-cation (which drives the cytokine dysfunction) from naturalinfection, but also to counteract the possible increase in viralload after COX-2 inhibition. We suggest that H5N1 avianinfluenza could be treated with an effective antiviral like i.v.zanamivir, in conjunction with immunomodulating drugs likecelocoxib and mesalazine to control the symptoms associatedwith cytokine storm. Triple therapy offers some hope forsurviving the devastating consequences associated with a pan-demic influenza outbreak.

Materials and MethodsAnimal Model and Viral Challenge. BALB/c female mice, 5–7 weeks old, werekept in biosafety level-3 housing and given access to standard pellet feed andwater ad libitum. All experimental protocols followed the standard operatingprocedures of the approved biosafety level-3 animal facilities and were ap-proved by the Animal Ethics Committee (21). Aliquots of stocks of influenza Avirus strain A/Vietnam/1194/04 were grown in embryonated eggs. Virus-containing allantoic fluid was harvested and stored in aliquots at �70°C. TheLD50 was determined in mice after serial dilution of the stock. One thousand

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LD50 were used for viral challenge in all of the experiments. Infection wasestablished by intranasal inoculation of mice anesthetized by isoflurane.

Antiviral and Immunomodulatory Treatments. Antiviral and immunomodula-tors were administered by the i.p. route using 0.5-ml, 29-gauge ultrafineneedle insulin syringes. The administered dosage for each agent followedprotocols as described (18, 22–25). Control mice were given PBS i.p. on thesame days (Table 1). Survival, body weight, and general conditions weremonitored for 21 days or until death.

Experiments were conducted in duplicates or triplicates of five mice foreach group of treated or control mice. Six mice in each of groups (groups 8, 11,and 12 in Table 1) were killed on days 4, 6, and 8 postchallenge, respectively.Blood, TPL, lung, brain, kidney, liver, and spleen tissue samples were collectedfrom these mice, normal uninfected mice, and the surviving mice for his-topathologic, immunologic, and virologic assays.

Virologic Tests. Titers of released virus in TPL were determined by TCID50 asdescribed, whereas viral RNA in lung tissues was quantified by real-timeRT-PCR (26, 27). Briefly, total RNA in lysed lung tissues was extracted by usinga RNeasy Mini kit (Qiagen) and reverse-transcribed to cDNA by using appliedSuperScript II Reverse Transcriptase (Invitrogen). Viral nucleoprotein gene andinternal control �-actin gene were measured by the SYBR Green Mx3000Real-Time PCR System (Stratagene), using primers NP-forward, 5-GAC CAGGAG TGG AGG AAA CA-3; NP-reverse, 5-CGG CCA TAA TGG TCA CTC TT-3;�-actin-forward, 5- CGT ACC ACT GGC ATC GTG AT-5; and �-actin-reverse,5-GTG TTG GCG TAC AGG TCT TTG-3.

ELISA. Proinflammatory cytokines and chemokines IL-1, IL-6, IFN-�, TNF-� (BDBiosciences), PGE2, MIP-1� (R&D Systems), leukotriene (GE Healthcare), andlung injury indicator albumin (Bethyl Laboratories) in TPL and serum sampleswere tested by ELISA using the protocol as described (3) with modificationsaccording to the instructions of the kit suppliers.

Elastase Activity Assay. Elastase activity in TPL was measured by the additionof the elastase-specific chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma) at a final concentration of 1 mM. After 30 minat room temperature, the change in optical density at a wavelength of 405 nmwas measured.

Neutralization Assay. Neutralizing antibody levels in mice sera were deter-mined by neutralization assay by using the same virus strain for challenge inMDCK cells as described (3).

Western Blot. Influenza A viral proteins NP from H5N1 strain A/Indonesia/5/2005, HA1 from H5N1 strain A/Vietnam/1203/2004 (Immune Technology), andHA2 from strain A/Vietnam/1194/04, which was expressed in baculovirusvector (BD Bioscience), were separated in 12% SDS/PAGE gel and then elec-troblotted onto PVDF membrane. The membranes were incubated withmouse sera at 1/200 dilution, washed, and then incubated with HRP-conjugated anti-mouse IgG mAb at a dilution of 1/1,000 (Abcam). The blotswere detected by the ECL Western blotting detection system (AmershamBiosciences).

Flow Cytometry. Blood cells from the mice were stained with fluorescein-labeled mAbs specific for mouse CD3, CD4, and CD8 (BD Pharmingen) andfixed with 4% p-formaldehyde overnight. The fixed blood cells were analyzedby flow cytometry (FACSCaliber; BD) as described (21).

Histopathologic Analysis. The lung, brain, spleen, kidney, and liver tissues ofchallenged mice were immediately fixed in 10% buffered formalin and em-bedded in paraffin wax. Sections 4–6 �m in thickness were mounted on slides.Histopathologic changes were examined by H&E staining under a light mi-croscope as described (3).

Immunohistochemical Assay. Lung sections were stained as described (3) byusing an antiinfluenza nucleoprotein mAb (HB65; ATCC) at 1:5,000 dilution,goat anti-mouse IgG, H and L chain-specific biotin conjugate (Calbiochem)at 1:2,000 dilution, and streptavidin/peroxidase complex reagent (VectorLaboratories).

Statistical Analysis. Statistical analysis of survival time and rate were per-formed by the log rank Kaplan-Meier and �2 tests, respectively, whereas theothers were calculated by Student’s t test with Stata statistical software.Results were considered significant at P � 0.05. The Cox proportional hazardsmodel was used to estimate hazard ratios.

ACKNOWLEDGMENTS. This work is partly supported by the ProvidenceFoundation Limited in memory of the late Dr. Lui Hac Minh, the ResearchGrant Council, the Hong Kong Special Administrative Region ResearchFund for the Control of Infectious Diseases of the Health, Welfare, andFood Bureau, the Hong Kong University Special Research AchievementAward, Croucher Senior Medical Research Fellowship 2006 –2007, and TheShaw Foundation.

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Table 1. Treatment regimens containing zanamivir, celecoxib, mesalazine, and gemfibrozilused alone or in combination for infected mice

Group Treatment regimen No.

1 3 mg zanamivir in PBS i.p. once every 12 h � 8 days* 52 2 mg celecoxib in 10%DMSO/PBS i.p. once per day � 8 days* 53 1 mg mesalazine in ddH2O i.p. once per day � 8 days* 54 1 mg gemfibrozil in propylene glycol i.p. once per day � 8 days* 55 2 mg celecoxib � 1 mg mesalazine i.p. once per day � 8 days* 56 2 mg celecoxib � 1 mg gemfibrozil once per day � 8 days* 57 PBS i.p. once per day � 8 days* 58 3 mg zanamivir i.p. once every 12 h � 6 days† 33‡

9 3 mg zanamivir � 2 mg celecoxib i.p. � 6 days† 1010 3 mg zanamivir � 1 mg mesalazine i.p. � 6 days† 1011 3 mg zanamivir � 2 mg celecoxib � 1 mg mesalazine i.p. � 6 days† 33‡

12 PBS i.p. once per day � 6 days 33‡

BALB/c mice (female, ages 5–7 weeks) were intranasally challenged with 1,000 LD50 of H5N1 virus strainA/Vietnam/1194/04.*The treatments started 4 h postchallenge.†The treatments started 2 days postchallenge.‡ Experiments were conducted in triplicates of five mice for each group. Furthermore, six mice in each of thesegroups were killed on days 4, 6, and 8 postchallenge, while all survived mice were killed on day 21 postchallenge.Blood, TPL, lung, brain, kidney, liver, and spleen were collected from these mice.

Zheng et al. PNAS � June 10, 2008 � vol. 105 � no. 23 � 8095

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