Meeting report: 4th ISIRV antiviral group conference: Novel antiviral therapies for influenza and

18 Pages·2016·0.34 MB·English

AntiviralResearch129(2016)21e38
ContentslistsavailableatScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Meeting report
Meeting report: 4th ISIRV antiviral group conference: Novel antiviral
therapies for influenza and other respiratory viruses
Jennifer L. McKimm-Breschkin a,*, Alicia M. Fry b
aCSIRO,343RoyalParade,Parkville,Australia
bInfluenzaDivision,CentersforDiseaseControlandPrevention,Atlanta,GA,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: TheInternationalSocietyforInfluenzaandotherRespiratoryVirusDiseases(isirv)heldits4thAntiviral
Received21January2016 GroupConferenceattheUniversityofTexason2e4June,2015.Withemergingresistancetothedrugs
Accepted22January2016 currentlylicensedfortreatmentandprophylaxisofinfluenzaviruses,primarilytheneuraminidasein-
Availableonline9February2016 hibitoroseltamivirphosphate(Tamiflu)andtheM2inhibitorsamantadineandrimantadine,andthelack
of effective interventions againstotherrespiratory viruses, the 3-dayprogrammefocused on the dis-
Keywords: coveryanddevelopmentofinhibitorsofseveralvirustargetsandkeyhostcellfactorsinvolvedinvirus
Respiratoryvirus replication or mediating the inflammatory response. Virus targets included the influenza haemag-
Isirv-AVG
glutinin,neuraminidaseandM2proteins,andboththerespiratorysyncytialvirusandinfluenzapoly-
Antiviral
Influenza merases and nucleoproteins. Therapies for rhinoviruses and MERS and SARS coronaviruses were also
discussed. With the emerging development of monoclonal antibodies as therapeutics, the potential
implications of antibody-dependent enhancement of disease were also addressed. Topics covered all
aspects from structural and molecular biology to preclinical and clinical studies. The importance of
suitableclinicaltrialendpointsandregulatoryissueswerealsodiscussedfromtheperspectivesofboth
industry and government. This meeting summary provides an overview, not only for the conference
participants,butalsoforthoseinterestedinthecurrentstatusofantiviralsforrespiratoryviruses.
©2016ElsevierB.V.Allrightsreserved.
Contents
1. Background ................................................................ ....................................................... 23
2. Keynotelectures.............................................................. ..................................................... 23
2.1. Publichealthimpactofantiviraltherapyforrespiratorydiseases ................................... ...............................23
2.2. Clinicaldevelopmentofantiviralsforrespiratorydiseases ....................................... ..................................23
3. Inhibitorsofviruspolymerases,nucleoproteinsandaccessoryproteins .................................... ............................... 24
3.1. Influenza.....................................................................................................................24
3.1.1. Keynotelecture:structure,mechanismanddrugtargetingofinfluenzapolymerase ....................... ....................24
3.1.2. SafetyandefficacyofJNJ-63623872(VX-787) .............................................................................24
3.1.3. Thenucleoproteinofinfluenzavirus,atargetfornewantivirals ................................ ............................24
3.1.4. Identificationandcharacterizationofinfluenzavariantsresistanttoaviralendonucleaseinhibitor,L-742,001 .....................25
3.1.5. Structure-baseddevelopmentofanewclassofinfluenzaendonucleaseinhibitors ....................... .....................25
3.1.6. Clinicalandanti-influenzaviruseffectsoffavipiravir,anovelanti-RNAvirus,anti-influenzaagent................ ..............25
3.2. Respiratorysyncytialvirus ...................................................... ...............................................25
3.2.1. DiscoveryanddevelopmentofALS-8176,anucleosideanalogueinhibitoroftheRSVRNApolymerase...........................25
3.2.2. RSVpolymeraseandnucleoproteininhibitors:mechanismofactionandresistance....................... ....................25
3.3. InfluenzaNS1 ............................................................ ....................................................26
3.3.1. NovelbroadspectrumantiviralagainstinfluenzablocksdsRNAbindingtoNS1Aproteinandrestoresantiviralresponses .... .....26
* Correspondingauthor.CSIRO343RoyalPde,Parkville,3052Australia.
E-mail addresses: [email protected] (J.L. McKimm-Breschkin), [email protected]
(A.M.Fry).
http://dx.doi.org/10.1016/j.antiviral.2016.01.012
0166-3542/©2016ElsevierB.V.Allrightsreserved.
22 J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38
4. NewinhibitorsofinfluenzaNAandM2activities....................................................................................... 26
4.1. Neuraminidase ........................................................... ....................................................26
4.1.1. Theinfluenzaneuraminidase-oldtarget,newapproaches .................................. ..............................26
4.1.2. NewinhibitorsofinfluenzaAneuraminidases ........................................ ...................................26
4.1.3. DelayedoseltamivirandT-705combinationtherapyprotectsmiceagainstlethalinfluenzaA(H5N1)infection.....................26
4.1.4. Influenzaviralloadandperamivirkineticsafterasingleadministrationinchildren....................... ....................26
4.2. InfluenzaM2............................................................. ....................................................27
4.2.1. IsM2agoodtargettocombatdrugresistanceintheinfluenzaAviruses? ...................................................27
5. Inhibitorsofvirionattachment/fusionproteins......................................................................................... 27
5.1. Influenza.....................................................................................................................27
5.1.1. TheinfluenzaHAasanantiviraltarget ............................................ ......................................27
5.1.2. Blockinginfluenzavirusbystabilizingthepre-fusionconformationofHA ....................................................27
5.1.3. Prophylacticandtherapeuticprotectionagainstinfluenzabyacomputationallyengineeredprotein..............................27
5.1.4. Novelfamilyofpeptideswithpotentantiviralactivityagainstinfluenzaviruses ........................ ......................27
5.2. Respiratorysyncytialvirus ...................................................... ...............................................28
5.2.1. DiscoveryandproofofconceptofGS-5806inRSVdisease .................................. ..............................28
5.2.2. RSVantivirals:fusioninhibitorsandbeyond..............................................................................28
6. Inhibitorsofseasonalandemergingthreats................................................. .......................................... 28
6.1. Antiviralstrategiesforpreventionandtreatmentofrhinovirusinfections ............................... ............................28
6.2. SevereillnessassociatedwithEV-D68infectionintheUnitedStatesandapproachestomanagement .................. ................28
6.3. Newtargetsandapproachesforcoronavirusantiviralinhibitors ....................................................................29
6.4. Functionaldipeptidylpeptidase(DPP4)inminksupportsentryandreplicationofMiddleEasternRespiratorySyndromeCoronavirus:American
mink(Neovisionvision),anovelinvivomodelofMERS-CoVinfection ................................. .............................29
6.5. PhaseIIImulti-centerclinicaltrialofnitazoxanideinadultpatientswithuncomplicatedinfluenzaAandBandotherinfluenza-likeillness:results
on1876subjectsfromtheUnitedStates,Canada,AustraliaandNewZealand.............................. ..........................29
7. Monoclonalantibodiesastherapeutics.................................................... ............................................ 29
7.1. Monoclonalantibodiesastherapeuticsagainstrespiratoryviruses ..................................................................29
7.2. Newantibody-basedstrategiesagainstviralrespiratorydiseases.................................... ...............................29
7.3. VIS410monoclonalantibodydemonstratespotentefficacyagainstneuraminidaseinhibitors-susceptibleanderesistantinfluenzaA(H7N9)viruses
andprotectsmicefromARDS ...................................................................................................30
8. Antibody-dependentenhancement(ADE)ofdisease:implicationsfortherapeuticmonoclonalantibodydevelopment .......................... 30
8.1. InfluenceofantibodiesandTcellsondenguediseaseoutcome .................................... ................................30
8.2. Influenzavaccine-inducedanti-HA2antibodiespromotevirusentryandenhancelungpathologyafterinfluenzaAinfection...............30
8.3. Doesantibody-dependentenhancementofdiseaseoccurininfluenzainfections?............................ ........................30
8.4. VIS410,abroadlyneutralizingantibodytoinfluenzaA:characterisationandpotentialforADE .........................................31
9. Hostcelltargets:factorsinvolvedinvirusreplicationormediatingtheinflammatoryresponse......................... ..................... 31
9.1. Usinggeneticapproachestodiscoverhostevirusinteractions.......................................................................31
9.2. Sphingosine-1-phosphatereceptormodulationofcytokineamplification ................................ ............................31
9.3. HostcellfactorsininfluenzaAvirusuncoating ............................................ ......................................31
9.4. UpdateonDAS181.......................................................... ..................................................31
9.5. Anovelclassofhost-directedantiviralswithbroadspectrumactivityagainstrespiratoryviruses..................... ..................32
9.6. Potentanti-influenzaandanti-inflammatoryactivityofVerdinexor,aselectiveinhibitorofnuclearexport(SINE)acrossabroadpanelofinfluenza
strains,includingavianinfluenzaAH7N9 .............................................. .........................................32
9.7. Targetingsirtuins,novelviralrestrictionfactors,tolimitacquiredresistance..........................................................32
9.8. Repurposingofsignaltransductioninhibitorstofighttheflueanupdate................................ ...........................32
9.9. Eritoran(E5564),aTLR4antagonisteffectiveagainstinfluenzainduceddisease............................. .........................32
10. Diagnosticsandresistance ......................................................... ................................................. 33
10.1. Detectionofdrugresistanceininfluenza:currentstatusandfuturedirections............................. .........................33
10.2. CharacterisationofalargeclusterofinfluenzaA(H1N1)pdm09viruscross-resistanttooseltamivirandperamivirduringthe2013e2014influenza
season ............................................................... ......................................................33
10.3. SixyearsofmonitoringemergentoseltamivirresistanceinpatientswithinfluenzaAvirusinfectionsintheInfluenzaResistanceInformation
Study(IRIS) ............................................................ ....................................................33
10.4. Thechanginglandscapeofinfluenzadiagnosticsandtheeffectonclinicalmanagement ........................ .....................33
11. Regulatoryissuesandclinicaltrialendpoints........................................................................................... 33
11.1. Regulatoryperspectivesonantiviraldrugdevelopmentforinfluenzaandendpointconsiderations.................... .................33
11.2. Humanrespiratoryviruschallengemodels:aworthwhilechallenge................................................................34
11.3. Thehumanviruschallengemodel-acceleratingdrugandvaccinedevelopment ............................ .........................34
11.4. Designandconductofadrugdevelopmentprogramforsevere/complicatedinfluenza:lessonsfromtheIVzanamivirexperience..... ....34
11.5. Challengesindesigninginformativeclinicaltrialsinpatientshospitalizedwithinfluenza:theperamivirexperience........... ..........34
11.6. Antibody-basedtherapyforinfluenzaB................................................ .........................................34
11.7. Supportingadvanceddevelopmentofnovelinfluenzaantiviraltherapeutics .............................. ..........................34
11.8. Multi-centerevaluationofoutpatientendpointsforRSVandotherrespiratoryvirusantivirals.........................................35
12. Conclusions ................................................................ ....................................................... 35
Acknowledgements ............................................................ ....................................................35
References................................................................. ........................................................35
J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38 23
1. Background administeredintravenously, as well as a long-acting derivative of
zanamivir, laninamivir (Inavir), have been approved in some
The International Society for Influenza and other Respiratory countries. The US CDC recommends the use of NAIs for patients
Virus Diseases (isirv) is an independent, international scientific withsuspectedorconfirmedinfluenzawhoarehospitalisedorat
professional society promoting the prevention, detection, treat- highriskforcomplicationsduetoinfluenza,includingpatientsless
ment, and control of influenza and other respiratory virus dis- than 2 and more than 65 years of age, those with underlying
eases. The isirv Antiviral Group (isirv-AVG) was established medical conditions or compromised immunity, and pregnant
following the merger in April 2011 of the former Neuraminidase women(CDC,2015b).Cliniciansareadvisedtouseclinicaljudge-
InhibitorSusceptibilityNetwork(NISN)withisirv.TheGroupaims ment to guide NAI treatment of outpatients who are not at high
topromoteunderstandingofthemeansofprevention,treatment risk. One study showed that antivirals are under-prescribed for
and control of influenza and other respiratory virus diseases, high-riskoutpatients,withonly19%receivingaprescriptionwhen
including the emergence of antiviral resistance. To communicate presenting at less than 48 h post symptom onset (Havers et al.,
preclinical and clinical developments of potential novel/new an- 2014). Furthermore, only 7.5% of all laboratory-confirmed influ-
tivirals three meetings have been held annually in different re- enzaoutpatientsweregivenanantiviralprescription, versus 30%
gionsoftheworld. whowereprescribedantibiotics.Withtherecentemergenceofthe
The 4th isirv Antiviral Group Conference was held at the novel influenza A(H5N2) circulating in poultry in the USA, CDC
UniversityofTexason2e4June,2015.Withemergingresistance recommends antiviral treatmentas soon as possibleforhospital-
to the drugs currently licensed for treatment and prophylaxis isedpatientswithnovelinfluenzaAvirusesassociatedwithsevere
of influenza (primarily the neuraminidase inhibitor (NAI) osel- infection.
tamivir phosphate (Tamiflu) and the M2 inhibitors amantadine Whileinfluenzavaccinesarestillrecommendedasthemeansof
and rimantadine) and the lack of effective interventions against primaryprotection(CDC,2015a),in2014anewhumanH3N2strain
other respiratory viruses, the 3-day programme focused on the emergedwhichwasantigenicallydistinctfromtheH3N2vaccine
discoveryand developmentof inhibitors of several virus targets component. Interim vaccine effectiveness against H3N2 virus in-
and key host cell factors involved in virus replication or in fectionswasestimatedatonly23%inJanuary2015(Flanneryetal.,
mediatingtheinflammatoryresponse.Virustargetsincludedthe 2015). This new H3N2 virus resulted in the highest numbers of
influenza haemagglutinin, neuraminidase and M2 proteins, and H3N2hospitalizationsintheUSAsincerecordsbeganmorethan10
both the respiratory syncytial virus (RSV) and influenza poly- years ago. Thus the CDC focused communications on reminding
merases and nucleoproteins. Therapies for rhinoviruses and clinicians about recommendations for NAI use, regardless of
Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV). vaccinationstatus.
andsevereacuterespiratorysyndromecoronaviruses(SARS-CoV) Recently there has been controversy over the efficacy of the
were also discussed. With the emerging development of mono- NAIs.TheCochranegroupcarriedoutmeta-analysisofclinicaldata
clonal antibodies as therapeutics, the potential implications of from 20 oseltamivir and 26 zanamivir trials (all enrolled out-
antibody-dependent enhancement of disease were also patients with uncomplicated influenza), concluding that oselta-
addressed. Topics covered all aspects from structural and mo- mivir provided a 17 h benefit for adults and a 29 h benefit for
lecularbiologytopreclinicalandclinicalstudies.Theimportance children, and zanamivir, a 0.6 day benefit among all enrolled
ofsuitableclinicaltrialendpointsandregulatoryissueswerealso participants, regardless of influenza infection (intention to treat
discussed from the perspectives of both industry and govern- (ITT)population)(Jeffersonetal.,2014).Theyfailedtofindasig-
ment.Thismeetingsummaryprovidesanoverview,notonlyfor nificant decrease in the number of hospitalizations after treat-
the conference participants, but also for those interested in the ment,andconcludedthat,withtheincreasedriskofnauseafrom
currentstatus of antivirals for respiratoryviruses. oseltamivir(4%)andpossiblepsychosis,theharmfromtreatment
outweighed the benefits. However, the PRIDE study (Muthuri
2. Keynotelectures et al., 2014), using meta-analysis of global hospitalizations of
more than 29,000 individual patients in 78 observational studies
2.1. Publichealthimpactofantiviraltherapyforrespiratory from 2009 to 2010, showed that NAI treatment led to a 19%
diseases decreaseinmortalityinadults,and,withearlytreatment,toa50%
reduction in mortality. No significant benefit was observed in
NancyCox,CentersforDiseaseControlandPrevention,Atlanta, children.
USA. More recently, the Multi Party Group for Advice on Science
PriortolicensingoftheNAIsin1999,theaminoadamantanes (MUGAS) repeated a meta-analysis of randomised, placebo-
were the only drugs used for the treatment and prevention of controlled clinical trials of oseltamivir enrolling outpatient
influenza. These target the virus M2 ion channel protein, adultsusingindividuallevelpatientdata.Theydemonstrateda1
involved in virus uncoating in the endosome (see 9.3 below). day decrease in the time to alleviation of symptoms, a 44%
However, due to central nervous system complications in the reduction in antibiotic prescriptions and a 60% decrease in hos-
elderly and lack of efficacy against influenza B, they were not pitalizations in the ITT influenza-infected population (Dobson
widely employed. Additionally, since 2000 many viruses have et al.,2015).
acquired mutations in the M2 gene conferring resistance, New antivirals are needed, but they face higher benchmarks
including the current human A(H3N2) and A(H1N1)pdm09 vi- to prove efficacy, including demonstration of reduction of
ruses, and avian influenza A(H5N1) and A(H7N9) viruses which severe complications due to influenza, and a recent mistrust of
have caused sporadic human infections. Resistance initially studies funded by industry. This will lead to higher costs of
emergedinChina,possiblyrelatedtothereadyavailabilityofthe clinical trials and ultimately of the drugs. The dilemma is who
aminoadamantanes in over the counter medications, and use in will pay?
poultryfeed.
TheNAIsTamiflu(prodrugoseltamivirphosphate),takenorally 2.2. Clinicaldevelopmentofantiviralsforrespiratorydiseases
as a capsule, and Relenza (zanamivir), an inhaled powder, were
approved in 1999e2000. More recently, peramivir (Rapivab), FredHayden,UniversityofVirginia,USA.
24 J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38
Recent clinical trials were disappointing for both the inhaled further development of the polymerase inhibitors. Resistance
long-actingderivativeofzanamivir,laninamivir,inuncomplicated studiesinvitrohaveshownthatafter10passagesofA/Aichi/2/68
influenzaandintravenousperamivirinhospitalisedpatients,both (H3N2) virus in the presence of an endonuclease inhibitor there
failing to meet the primary clinical endpoint of alleviation of wasnochangeintheIC50.
symptomsornormalizationofvital signs.Additionally,DAS181,a
sialidase fusion protein, which acts by removing potential sialic 3.1.2. SafetyandefficacyofJNJ-63623872(VX-787)
acid receptors for influenza (Belser et al., 2007), showed some LorantLeopold,JanssenPharma,Titusville,USA.
decreaseinvirusloadinuncomplicatedinfluenza,butnosignifi- JNJ-63623872(VX-787)isanon-nucleosideinhibitortargeting
cant clinical benefit (Moss et al., 2012). Combination therapy, PB2(Byrnetal.,2015;Clarketal.,2014).Itinhibitsproductionof
mostlyofNAIswithotherdrugsagainstdifferenttargets(Hayden, viral mRNA, preventing death of infected cells, unlike the NAIs
2013), are currently under evaluation, to try to increase potency whichstillallowcelldeath.ItwaseffectiveagainstallinfluenzaA
andreducethelikelihoodofoseltamivirresistance.DAS181isalso strainstestedincellculture,includingthosewhichareresistantto
being tested against parainfluenza (PIV) viruses, which also use NAIs,andwasalsoeffectiveinreducingmortalityinbothH1N1and
sialicacidreceptors(Jonesetal.,2013). H5N1 lethal mouse models. At 3 mg/kg it resulted in up to 50%
Forrhinoviruses,pleconaril,acapsidbindinginhibitor,resulted decrease in mortality due to A/Puerto Rico/8/34 (PR8, H1N1)
insomedecreaseintotalsymptomseverity,butitwasnotbroadly infectioninmice.With10mg/kgitcouldbedeliveredupto120h
effectiveagainstthemultipledifferentstrainsofthevirus.Anewer post-infectionandstillleadtoan80%decreaseinmortality.Syn-
derivative, vapendivir, is being tested in adults with rhinovirus ergywasdemonstratedincombinationwithoseltamivir,zanamivir
infections and asthma (ClinicalTrials.gov, 2015). Many other in- orfavipiravirinvitro.While0.3mg/kgalonewasnotprotectivein
hibitorswerediscussedinmoredetailinthesubsequentprogram. mice, there was a synergistic effect with oseltamivir leading to
100%survival.
3. Inhibitorsofviruspolymerases,nucleoproteinsand In a human challenge study different doses were tested,
accessoryproteins including100or400mgoncedailyfor5days,oraloadingdoseof
900or1200mgonthefirstdayfollowedby600mgoncedailyfor4
3.1. Influenza days.Theprimaryendpointwastheareaunderthecurveforvirus
shedding,determinedbyvirusinfectivityincellcultureandqRT-
3.1.1. Keynotelecture:structure,mechanismanddrugtargetingof PCR. A statistically significant decrease in virus shedding was
influenzapolymerase seeninthe1200/600cohort,althoughthedurationofvirusshed-
StephenCusack,EMBL,Grenoble,France. dingwasnotaffected.Therewasalsoadose-dependentdecreasein
InfluenzaRNA-dependentRNApolymeraseisagooddrugtarget symptom scores and duration of symptoms. Resistance screening
sinceitsactivesiteishighlyconserved.Thepolymerasecomplex, revealedamutationM431IinthePB2geneinfourof72patients,
composedofthreeproteins,PA,PB1andPB2,isessentialfortran- which resulted in a 57-fold reduction in sensitivity. However,
scription,togeneratecappedviralmRNAs,replication,togenerate mutantviruseshadreducedfitness,estimatedtobe8%ofwildtype.
the full length vRNA genome for incorporation into new virions, Cusack commented that resistance was seen within 2 passages
andantigenomecRNA,providingthetemplateforfulllengthvRNA. invitro.Mutationofphenylalanine323,involvedintightbindingof
PB2bindshostcellpre-mRNAassociatedwiththecellPolII,then theinhibitortoinfluenzaA,toserineleadstoresistance.Lackofthis
short50 cappedRNAfragmentsarecleavedoffbythePAendonu- phenylalanineininfluenzaBPB2accountsinpartfornaturallypoor
clease, in a process known as cap snatching. Transcription elon- bindingandlackofactivityagainstinfluenzaB.Aphase2btrialis
gation is then initiated by PB1 using the capped primer. Early evaluating the dose range and once versus twice daily dosing in
attemptsatcrystallizationofsolublefragmentshadlimitedsuccess. healthypatients.
However more recently, expression of the complete trimeric
polymerases, using self-cleavable polyproteins, of the novel bat 3.1.3. Thenucleoproteinofinfluenzavirus,atargetfornew
influenzavirusH17N10andinfluenzaBenabledsolvingtheX-ray antivirals
crystalstructuresincomplexwiththeRNApromoter,comprising AnnySlama-Schwok,INRA,JouyenJosas,France.
16e18nucleotidesfromthe30and50endsofvRNA,tobetween2.7 Influenzaribonucleoprotein(RNP)complexesarecomposedof
and 3.4 Å resolution (Pflug et al., 2014; Reich et al., 2014). The thethreesubunitsofthepolymeraseassociatedwiththeviralRNA
structuressuggestthatthecap-bindingdomainofPB2rotatesby genomecoveredbymultiplecopiesofthenucleoprotein(NP).The
70(cid:2),firstallowingtheendonucleasetocleavethehostpre-mRNA, NPisahighlyconservedproteinandthuspotentiallyagoodtarget
toanalternativeconfigurationinwhichthecappedoligomercan forabroadly-reactiveantiviral.Therecentstructuresofthepoly-
enterthepolymeraseactivesiteandprimetranscription.Allthree merasecomplexandcryo-electronmicroscopystudiesoftheRNP
subunitsarethusinvolvedinRNAbindingandtranscription.When have provided the basis for in silico screening of the Sigma cata-
the influenza B polymerasewas complexed with 50 vRNAonly, it logue for inhibitors of NP-polymerase and NP-RNA interactions.
yieldedadifferentstructuretothatofthecomplexwithboththe30 Naproxen,aknownanti-inflammatoryinhibitingcellularcylcoox-
and 50vRNAs, which may represent an alternative conformation ygenase 2 (COX-2), was identified as a competitor of NPeRNA
activeincapsnatching. interaction, and protected the NP C-terminus against proteolysis.
TheLaCrossebunyaviruswhichhasonly3RNAsegmentshasa NaproxenreducedinfectionofbothH3N2andH1N1virusesincells
single L polymerase protein. This is similar in total size to the withanEC50of50mM,andat2e8mg/kgperdaydecreasedvirus
influenzacomplex,andstructuralanalysisshowedittohavesimilar titersinmice(Lejaletal.,2013).Twoderivativesweresynthesised
overallarchitectureanddomainstructureastheinfluenzaenzyme, tobemorespecificforviralRNP,naproxenAandCO(Tarusetal.,
despitetheirdivergence(Gerlachetal.,2015). 2015). Naproxen CO competes with RNA binding to NP and de-
Structure-guided drug design has been used to develop in- stabilizes NP-RNA oligomers while stabilizing monomeric NP. It
hibitors against the cap snatching PB2 subunit and the PA endo- was more potent with an IC50 of 2e3 mM, more soluble and less
nuclease (Kowalinski et al., 2012; Pautus et al., 2013). The PA toxic than naproxen, but no longer inhibited COX-2 in human
inhibitorchelatesthetwocriticalmanganeseionsintheactivesite A549cells.
oftheenzyme.Savira(anESRFspinoff)andRocheareundertaking Work on anti-inflammatory compounds targeting the NADPH
J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38 25
oxidase(NOX)areaimedatadualantiviralandanti-inflammatory 3.2. Respiratorysyncytialvirus
approach.
3.2.1. DiscoveryanddevelopmentofALS-8176,anucleoside
analogueinhibitoroftheRSVRNApolymerase
3.1.4. Identificationandcharacterizationofinfluenzavariants
JulianSymons,AliosBiopharmaInc,SanFrancisco,USA.
resistanttoaviralendonucleaseinhibitor,L-742,001
The RSV L protein is a viral RNA-dependent RNA polymerase
GyanendraKumar,StJudeChildren'sResearchHospital,Mem-
thatcontainsmultipleenzymeactivitiesrequiredforRSVreplica-
phis,USA.
tion.Usingacellbasedscreeningassay,ALS-8112,anovelcytidine-
L-742,001isadiketoacidinhibitoroftheinfluenzaviruspoly-
based analogue, and its orally available prodrug, ALS-8176, were
merase PA subunit (Stevaert et al., 2015). Stevaert et al. (2013) identified as potent and specific inhibitors of the replication of
previously described mutations which confer reduced sensitivity
laboratoryandclinicalstrainsofRSVAandB(Devaletal.,2015),
(Stevaertet al., 2013). Kumarcarried out random mutagenesis as
with EC50s of approximately 100 nM and a Ki of 90 nM for the
natural mutants did notemerge after 10 passages in cell culture. enzyme. Both a 20F and the 40ClCH2 groups contributed to
They generated a plasmid library, then used reverse genetics to
the selectivity of ALS-8112. In vitro, the inhibitor caused chain
generaterecombinantPR8viruses.Thesewerethenpassagedinthe
termination ofviralRNA synthesis, but did notinhibit cellularor
inhibitor for 3e4 passages, and then potentially resistant viruses
unrelated virus RNA polymerases. It also inhibited related para-
wereselected.MutationsincludedI79L,E119D,T20A(alsoseenby
myxoviruses, including human metapneumovirus and para-
Stevaert et al., 2013), and F105S. Mutations marginally reduced influenzavirus3.HighlevelsoftheALS-8112-triphosphateformed
polymeraseactivity,butvirusesdidnotappeartolosefitness,with
in A549 and primaryepithelial cells. Oral administration to non-
similar growth properties in vitro and similar weight loss, virus
human primates resulted in high levels of the NTP in the lung,
titersandMLD50inmice. withahalf-lifeof29h.
In human challenge studies in adult volunteers (DeVincenzo
3.1.5. Structure-baseddevelopmentofanewclassofinfluenza et al., 2015), ALS-8176 was administered either with a high
loadingdoseof750mgfollowedbyamaintenancedoseof150or
endonucleaseinhibitors
500mg,orasaconstantdoseof375mg.Intheplacebogroup,by
JosephBaumann,RutgersUniversity,USA.
CrystalsofanN-terminaldomainoftheendonuclease(PA)ofan quantitativeRT-PCRthepeakRSVRNAtiterwasaround104log10at
3.5dayspostchallenge.Inthe750/500mgand750/150mggroups
A(H1N1)pdm09viruswereusedforfragmentscreeningtoidentify
there was an immediate decrease in the RSV RNA levels, and a
new chemical entities binding to the enzyme active site. A 3-
decrease in symptom scores and mucus weight. In the 375 mg
hydroxy-2-pyridonescaffoldwasshowntobindat3sites,identi-
group there was an initial increase in virus RNA followed by a
fyinganewmodeofchelationoftheactivesitemetalions(Bauman
etal.,2013).AthirdMnþþion,notpreviouslyidentified,wasalso decrease,thusindicatingtheimportanceofthehighloadingdose.
ALS-8176 was well tolerated and no resistant virus was isolated,
detected. SAR studies led to improved binding, reducing the IC50
from16 mM to11 nM in a fluorescence based enzyme assay, and although in vitro after 35 passages mutations were found in the
11mMinaplaquereductionassay(PRA)againstPR8(H1N1).The activesiteoftheLprotein.Itiscurrentlyundergoingevaluationin
þþ þþ hospitalisedinfants.
IC50sinenzymeassayvarieddependingonwhetherMg orMn
was in the reaction. The most active compounds had an IC50 of
0.2mMinthePRA. 3.2.2. RSVpolymeraseandnucleoproteininhibitors:mechanismof
actionandresistance
QinYu,AstraZenecaR&DBoston,Waltham,USA.
3.1.6. Clinicalandanti-influenzaviruseffectsoffavipiravir,anovel AZ-27,apolymeraseinhibitor(Tiong-Yipetal.,2014),wasactive
anti-RNAvirus,anti-influenzaagent against both RSV A (average EC50 ¼ 24 ± 9 nM) and B (average
CarolEpstein,MedivectorInc,Boston,USA. EC50¼1.0±0.28mM)subtypesinmanycelllines,andwaseffective
Favipiravir,T-705,isaselectiveinhibitoroftheRNA-dependent when added up to 24 h post infection. There was no detectable
RNApolymeraseofinfluenzavirus,andisalsoreportedtoinhibita cytotoxicity at 100 mM. AZ-27 inhibits an early stage in mRNA
broad range of other RNAviruses (Furuta et al., 2013; Oestereich transcription,aswellasgenomereplication,byinhibitinginitiation
et al., 2014). Additionally, since it does not target the NA, it is ofRNAsynthesisfromthepromoter(Notonetal.,2015).Resistant
effectiveagainstoseltamivir-resistantviruses(Tarbetetal.,2014). viruseswereisolatedafter7passagesinculture;adominantmu-
Over1500patientshavebeenstudiedinclinicaltrials,evaluating tation,Y1631H,intheputativecappingenzymedomainofLprotein,
symptom improvement and emergence of resistance. In the first resultedinagreaterthan5000-foldincreaseinEC50againstAZ-27.
phase2trialof550patientsover5seasons,nosignificantbenefit TheRSVnucleoprotein(N)isessentialforvirusreplicationand
wasobserved.Thesecondphase2trialevaluatedtwice(BID)versus assemblyas partof the viral RNP complex. Previously, RSV604, a
three times (TID) daily administration with different dosing: benzodiazepine, has been reported to inhibit both RSV RNA syn-
1200 mg TID for 1 day, then 600 mg TID for 4 days; a 2400 mg thesis and the infectivity of released virus, with an EC50 of
loadingdoseandtwo600mgdosesonday1,followedby600mg approximately 1 mM (Challa et al., 2015). Its potency is cell line
TIDfor4days;or1800BIDonday1and800mgBIDondays2e5. dependent.Yuandcolleaguesreportedherethattheyhaveiden-
ThehigherloadingdosewasneededtomaintaintheCminlevelsof tified a novel thienodiazepine as an N inhibitor with improved
20 mg/ml. The BID and TID groups both showed a more rapid potency and broad spectrum activity against both RSV A and B
decreaseinvirustiterscomparedtotheplacebogroups.Interest- strains, although it was also cell type dependent. The compound
ingly, the BID group showeda statisticallysignificant decrease in demonstrateddirectbindingtorecombinantNfragmentsbysur-
the time to resolution of symptoms, but the TID did not. Among face plasmon resonance (SPR) with a Kd of 1.93 mM. An X-ray
individual symptoms, a statistically significant decrease was coecrystalstructureshowedthebindingsiteoverlappedtheNeP
demonstratedfor6symptoms,butnotforfever.Noresistancehas interactionsite.MutationsatresiduesN80andN28causedreduced
beendetectedinmorethan700clinicalsamplestestedsofar,with inhibition,beingresistantto2x,butnotto10x,theEC50.Theydid
morethan1500samplestotestfromaPhase3multi-countrytrial not,however,affectinhibitorbindingtotheNproteinasassessed
thatrecentlyconcludedenrolment. bySPR,suggestingthatthemechanismof‘resistance’maynotbe
26 J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38
duetoreducedinhibitoreNbinding.Thedualmechanismofaction In addition to the catalytic site, in group 1 NAs amino acids
fortheseN inhibitorshighlights thepotential forthis classofin- 149e152formaloop,whicheitherprojectsintotheactivesitein
hibitorindeliveringefficacyandsuppressingresistance. theclosedconformation,orremainsoutsidetheactivesiteinthe
open conformation, creating the 150 cavity (Adabala et al., 2013;
3.3. InfluenzaNS1 Amaro et al., 2011; Wang et al., 2011). Pinto and colleagues have
created a carbocyclic analogue of zanamivir, byreplacing the hy-
3.3.1. Novelbroadspectrumantiviralagainstinfluenzablocks drophilicglycerolsidechainwiththehydrophobicpentyloxygroup
dsRNAbindingtoNS1Aproteinandrestoresantiviralresponses ofoseltamivir(Kerryetal.,2013;Mohanetal.,2014).Thisinhibitor
Ji-YoungMin,InstitutPasteur,Korea. targetsonlythecatalyticsite,andshowednMpotencyintheNAI
The influenza A virus non-structural 1 (NS1A) protein is a assay (Ki ¼ 0.46 nM; Ki (zanamivir) ¼ 0.16 nM) and an EC50 of
knownantagonistofthehostantiviralresponse.Asmallmolecule, 80nMincellculture;activitywasonly10-foldloweragainstthe
namedBOC,inhibitedhumanseasonalH1N1,H3N2,andBviruses H275Yoseltamivirresistantmutant(100e1000foldresistant).This
andavianH5andH7subtypeviruses.ThedsRNAbindingpocketof appearstobeduetoincreasedflexibilityofthependantpentyloxy
theNS1Aproteinwasshowntobetheputativetargetbasedonan group and the ability to pivot about a strong hydrogen-bonding
in vitro dsRNA binding assay. Influenza A virus-infected cells network. However, its properties suggest that it would still only
showedincreasedNF-kBnucleartranslocationandelevatedlevels besuitablefortopicalapplication.
of various cytokines related to interferon-stimulated genes. BOC Pintoalso described a series of triazole-containing carbocyles,
administered to mice infected with an avian influenza virus relatedtooseltamivir,whichboundinboththecatalyticsiteand
reducedlungvirustitersintheearlystageofinfection.Resultsthus 150cavity(Adabalaetal.,2013;Amaroetal.,2011;Mohanetal.,
suggestBOCmayabrogatetheantagonisticresponsesoftheNS1A 2010; Wang et al., 2011). Inhibition of virus replication in vitro
protein. withtriazolederivativescontainingeitheranaminoorguanidino
modificationindicatedthattheguanidiniumcompoundhadhigher
4. NewinhibitorsofinfluenzaNAandM2activities efficacyagainstanN2subtype,ataconcentrationof20mM,anddid
not inhibit replication of an N1 subtype virus even at a concen-
4.1. Neuraminidase trationof100mM.Healsobrieflydescribedtheserendipitousdis-
coveryofapotentspirolactamNAinhibitor(Mohanetal.,2014).
4.1.1. Theinfluenzaneuraminidase-oldtarget,newapproaches
JennyMcKimm-Breschkin,CSIRO,Parkville,Australia. 4.1.3. DelayedoseltamivirandT-705combinationtherapyprotects
WhilethecurrentNAIsarebasedonthetransitionstateanalogue miceagainstlethalinfluenzaA(H5N1)infection
DANA,anewseriesofcompounds,basedonthenaturalsubstrate Bindumadhav Marathe, St Jude Chidren's Research Hospital,
sialic acid, 2,3, difluoro sialic acids (DFSA), have recently been Memphis,USA.
described(Kimetal.,2013).DFSAsrequirethecatalyticactivityof Due to the emergence of resistance to oseltamivir, several
theNAtobind,hencearetermedmechanism-basedinhibitors.They groups are investigating combinations of therapeutics directed
formacovalentlinktoatyrosineintheenzymeactivesite.Unlike against different virus targets. Oseltamivir (20 mg/kg), T-705
theNAIs,theyarenotcompetitiveinhibitorsandtheirefficacyde- (50mg/kg)oracombinationofthetwowasadministeredtomice
pends on a slowturnoverof the covalentlylinked inhibitor. Sub- at48,72,96or120hpost-infection.Allmicewereprotectedat48
stitution on the sugar ring, with either 4-amino or 4-guanidino and72h,butat96honlythecombinationgave100%protection.
groups,affectedaffinityandratesofbindinganddissociationinan After120hthesingledrugshada25%survivalrate,comparedto
IC50kineticsassay(Barrettetal.,2011).Evenmorestrikingwasthe 80%withthecombination.Therewasa2log10decreaseinmouse
effectoftheorientationofthe3fluorogroup,withtheequatorial lungvirustitersforthemonotherapy,anda4log10decreaseforthe
beingmorepotentthantheaxial.DFSAshavebroadefficacy,with combination therapy. Analysis of lung tissue by immunohisto-
IC50sinenzymeassaysandinplaquereductionassaysinthelownM chemistry and histomorphometry revealed that combination
tomMrange,andcanprotectmicefromalethalinfluenzainfection. therapyrestrictedvirusspreadandreducedtheextentoflesionsby
Theyarealsoeffectiveagainstvirusesresistanttooseltamivirand 30% compared with monotherapy. Combination therapy also
zanamivir.TheoreticallyresistancetoDFSAsshouldbemoredifficult reduced the pro-inflammatorycytokine response, which reduced
toselect,sincemutationswouldcompromisetheNAfunction.No diseaseseverityandfacilitatedrecovery.
resistant viruses were selected after 12 passages of an A(H1N1)
pdm09andaninfluenzaBvirusinvitro,withvirusreducedtoalmost 4.1.4. Influenzaviralloadandperamivirkineticsafterasingle
undetectablelevels.Afurther3passagesinalowerdrugconcen- administrationinchildren
tration,however,ledtoisolationofasmallplaquevariantfromthe MasaktokiSato,FukushimaMedicalUniversity,Japan.
A(H1N1)pdm09 virus, which was highly resistant in the plaque Whileperamivir(Rapivab)administeredasasingledose(i.v.)has
reduction assay. The mutant virus had barely detectable enzyme beenlicensedinJapanfortreatmentofadults,childrenandinfants,
activity,butreplicatedtocomparabletitersinvitroasthewildtype it has only been approved in the USA for acute uncomplicated
virus. Preliminary results suggest a novel mutation/s in the NA, influenzainpatients18yearsandolder.Thisstudyinvestigatedthe
whichremainstobeconfirmed.TheseDFSAsarealsoactiveagainst pharmacokineticsofperamivirinchildren(Satoetal.,2015).While
humanPIV3(Dirretal.,2015;Streltsovetal.,2015).SincetheNAsof themeanserumperamivirconcentrationamong28childrenat0.5h
human H3N2 (Mohret al., 2015) and H1N1 (Hooper et al., 2015) aftera10mg/kgdosewas88.9mM,after1,2,3,4and5daysitwas
viruses candemonstratereceptor bindingas well asthe catalytic reducedto83.2,25.1,17.1,6.2and4.6nM,respectively.Themean
function,witharecentfocusonantiviralstargetingtheHAweneed concentrationintheURTat0.5hafteradministrationwas5.1mM
tounderstandthissecondaryroleoftheNA,whichmaycompensate and48.3nMat1day,butthedrugwasundetectableafterday2.
forinhibitionoftheHAreceptorbindingfunction.Wemayneedto WhileviralRNAloadinbothH3N2andH1N1infectionsdecreased
targetboththeHAandNA. fromday0today3,itincreasedin9/12patientsafterday3.The
decrease in viral RNA load was much less for influenza B than
4.1.2. NewinhibitorsofinfluenzaAneuraminidases influenza A. No resistant virus was detected. Hence a single per-
MarioPinto,NSERC,Ottawa,Canada. amivir dose appears to be inadequate virologically, and
J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38 27
readministrationshouldbeconsideredinseverecases. A second antibody, MAb#14, derived from an H7N9 virus-
infectedpatientbindsnearthereceptorbindingsite.Itsitsabove
4.2. InfluenzaM2 the190helix,interactingpartlywiththe130and150loopsinthe
headoftheHA.HoweveritisspecificonlyfortheH7HA,sincethis
4.2.1. IsM2agoodtargettocombatdrugresistanceintheinfluenza regionisnotconserved.
Aviruses?
JunWang,UniversityofArizona,Tucson,USA. 5.1.2. Blockinginfluenzavirusbystabilizingthepre-fusion
Oncetheinfluenzavirusbindstosialicacidreceptors,itistaken conformationofHA
upintotheendosome,wherethelowpHactivatestheformationof MeganShaw,MountSinaiHospital,NewYork,USA.
the M2 ion channel, causing an influxof protons, into the virion High throughput screening used a recombinant influenza A/
interior.ThisresultsindissociationoftheM1matrixproteinfrom WSN/33viruswiththeluciferasegenereplacingtheHAgeneand
thevirusRNP,whichisthenreleasedintothecell.TheM2proteinis cellsexpressingtheHA.Luminescencewasonlydetectedifwhole
highly conserved, and can be inhibited by the adamantanes. viruswasproduced.Fromalmost106compounds,thehitratewas
However resistance arises rapidly, due to three major mutations, 0.5%,(4582),with744compoundsshowingadoseresponseeffect
L26F, V27A or S31N, S31N being the most common. The S31N (Whiteetal.,2015).Thetopfivewerescreenedagainstwildtype
mutanthasamorecontractedchannelcomparedwiththatofthe viruses,withthreeshowinginfluenzaAspecificinhibition,butnot
wild type (WT). It is hypothesised that the pore undergoes a broadlyreactive.Theothertwowerebroadlyreactive,M4reacting
þ
conformationalchangefromopen-closedtoclosed-openastheH againstasubsetofvirusesandM53showingreactivityagainstboth
goesfromoutsidetoinside.Guidedbymoleculardynamicssimu- RNA and DNA viruses, suggesting a host cell target. The three
lations, NMR and X-ray crystallography studies, the group devel- influenza-specific hits were S57, which had broader reactivity,
oped amantadine derivatives which are effective against the interactingwiththeC-terminusoftheNP,S119,reactingwiththe
resistantmutants(Wangetal.,2015,2013).Onecompoundwhich N-terminus of the NP protein, and S20, reacting with the HA of
contains a charged ammonium group, M2WJ332, sits inside the group1viruses,butnotH3N2orinfluenzaBviruses.Whentested
channel,andintheplaquereductionassayhasanEC50of153nM against wild type virus, S20 had an IC50 of 80 nM and caused a
againsttheS31Nmutantincomparisonto328nMforamantadine maximum reduction in virus titers of >3 log10 with low toxicity
against the WT M2. Four passages in drug concentrations (CC5040mM).TimeofadditionassaysanduseofanHA/NApseu-
increasingfrom300to600nMdidnotselectaresistantmutant. dotypeHIVparticleshowedthatS20inhibitedentry.Resistantvi-
However,greaterincreaseindrugconcentrations(330,376nM,1.6 ruses were selected after 4 passages in vitro, all with mutations
and35.9mM)selectedresistantmutantswithanL26Imutation,and affecting the HA, primarily around the “B” loop structure con-
by passage 6 a reversion of the N31S occurred, which decreased nectingthelargeandsmallhelicesoftheHA2subunit(Whiteetal.,
binding. However, the limitation remains that new compounds 2015).Inaredcellfusionassay,thepHoffusion(50%fusionatpH5)
need to bind both the wild type and mutant M2s, and the L26I wasloweredbelowpH5inthepresenceofS20,indicatingstabi-
mutation is found naturally in avian viruses isolated from wild lisation of the prefusion form by S20. S20 also protected the HA
birds.Additionally,theBM2ofinfluenzaBvirusesisnotinhibited fromconversiontothetrypsinsensitiveformunderlowpHcon-
byaminoadamantanes. ditions.Incontrast,thepHoffusionofaM59Imutantwasunaf-
fected by S20. In silico docking studies predicted that S20 binds
5. Inhibitorsofvirionattachment/fusionproteins boththeHA1andHA2inthestalkregion,correspondingtothearea
whereresistancemutationsoccurred.
5.1. Influenza
5.1.3. Prophylacticandtherapeuticprotectionagainstinfluenzaby
TheinfluenzavirusHAproteinisahomotrimer,inwhicheach acomputationallyengineeredprotein
monomeriscomposedoftwodisulfide-linkedsubunits,HA1and MerikaTreants,UniversityofWashington,Seattle,USA.
HA2.TheHAbindstosialicacidreceptorsonthecellsurface,andthe A computationally designed and engineered small protein
virusisthentakenupintoendosomes,wherethelowpHleadstoa HB36.6 (90 amino acids) protected mice against weight loss and
conformationalchangeintheHAtrimertoitsfusogenicform.HA1 deathwhenadministeredeitherasasingleprophylacticdose48h
formstheheadregionoftheprotein,whichisprimarilyinvolvedin priortoviruschallenge,orfrom24hpost-infectionwhenadmin-
receptorbinding, whereas HA2,referredtoasthe stem region, is istered daily at 3 mg/kg. A synergistic response providing full
involvedinpromotingvirusehostmembranefusion.Variousgroups protectionwasseenwithsuboptimaldosesofoseltamivir(5mg/kg)
havetargetedtheHAtotrytoeitherstabilizetheprefusionformor andHB36.6.Todeterminewhetheranimmuneresponsedeveloped
blockthereceptorbindingsite.Antibodiesdirectedagainstthehead totheproteinmicewereimmunisedwithmultipledosesandwere
regionappeartobemorepotent,whereasthosedirectedtothestalk then challenged with virus. No immune response to HB36.6 was
regionappeartobemorebroadlyreactive. seenasmicewerefullyprotected.IL-6,IL-10,IFN-gandIL-12p7all
ofwhichincreasedincontrolmice,werealllowerintheHB36.6-
5.1.1. TheinfluenzaHAasanantiviraltarget treated mice, correlating with lower virus titers and more rapid
GeorgeGao,ChineseAcademyofSciences,Bejing,China. recovery. HB36.6 also reduced clinical signs in influenza-infected
Gao described the discoveryofan anti-stem monoclonalanti- ferrets. After intravenous administration HB36.6 could not be
body, CT149, which was isolated from convalescent plasma from detectedinblood,hencepharmacokineticstudiesareusinginhaled
patientsinfectedwiththeA(H1N1)pdm09virusin2009(Wuetal., administration.Labelledcompoundappearstocoattherespiratory
2015).CT149neutralisedallgroup2H3N2virusestestedandtwo tract.
H7N9viruses,butsurprisinglyonlysomegroup1influenzaviruses,
including1/6H1,twoH5andtwoH9N2,inhibitinglowpH-induced 5.1.4. Novelfamilyofpeptideswithpotentantiviralactivityagainst
HA-mediated membrane fusion. Both the heavy and light chains influenzaviruses
are involved in binding, and the antibodycross-links two mono- SeemaJasim,TheRoslinInstitute&Royal(Dick)SchoolofVet-
mersofthetrimer.ThisantibodyprotectsagainstH3N2infection erinaryStudies,UniversityofEdinburgh,UK.
bothinvitroandinmice. Approaches to novel antiviral compounds include the
28 J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38
developmentofsyntheticpeptidesthatdisrupttheentryofvirusinto severe disease and faster virus clearance correlates with faster
cells.Twopeptides,“FluPep”(afamilyofshort,hydrophobicpeptides clinical improvement. Detection of virus mRNA by RT-PCR corre-
related to suppressors of cytokine signalling-1, SOCS, proteins) lateswithvirusRNAandismoresensitivethanvirusculture,with
and “Entry Blocker” (derived from the signal sequence of RNA detected often to day 13, although generally cultures are
fibroblast growth factor-4) were shown to have antiviral activity negativeafterday6(IgAinthesamplemayinhibitvirusgrowth).
againstapanelofvirusesinvitroandinvivo(Nicoletal.,2012).In Lung pathology shows high virus staining, and low numbers of
plaquereductionassaystheFluPepwasmoreeffectiveagainstH1 immunecells.Unlikeinfluenzawhereviruspeaksatdays2e3and
thanH3orH4,althoughPR8hadlowersensitivitycomparedtothe symptomsatdays3e4,forRSVvirusloadandsymptomspeakat
A(H1N1)pdm09 virus. IC50s ranged from the low nM to ~100 nM thesametime.Childrenhavehighervirustitersandshedvirusfor
(Nicoletal.,2012).InPR8recombinantswithdifferentHAs,sensi- longercomparedtoadults.Palivizumab/Motavizumabmonoclonal
tivitywasdependentontheHA.TheEntryBlockerdidnotshowthe anti-fusion antibodies, which are effective prophylactically
samestrainspecificity.Theyactedupstreamofthenuclearimportof (AAPCID,2014)showednoclinicalefficacyfortreatmentofchildren
theRNPs.Thepeptidescausedaggregationoffluorescentlylabelled hospitalisedwithRSV(Ramiloetal.,2014).
virus particles on the surface of host cells, and appeared to slow A different approach to those already discussed is the use of
internalisation.Givenprophylacticallyinmicetherewasadecreasein RNAi, forexample, ALN-RSV01 targeting the N gene (DeVincenzo
virustiter,andsomeprotectionagainstweightloss. et al., 2010). A trial using a nasal spray of ALN-RSV01, adminis-
tered daily for 2 days before and 3 days after RSV inoculation,
5.2. Respiratorysyncytialvirus showedantiviralactivityagainstRSVinfection,withtheproportion
ofculture-definedRSVinfections71.4and44.2%inplaceboandin
5.2.1. DiscoveryandproofofconceptofGS-5806inRSVdisease ALN-RSV01recipients,respectively.
SethTobackandMikePerron,Gilead,FosterCity,USA.
GS-5806 is an orally available RSV inhibitor that targets the 6. Inhibitorsofseasonalandemergingthreats
fusion protein, blocking virus envelope fusion with the host cell
membrane (DeVincenzo et al., 2014; Mackman et al., 2015). It is 6.1. Antiviralstrategiesforpreventionandtreatmentofrhinovirus
highly effective against both RSV A and B with a mean EC50 of infections
0.43nMagainst75clinicalisolates,(RSVA0.51nMandB0.35nM),
and low toxicity (SI > 23,000-fold). GS-5086 does not block RSV RonaldTurner,UniversityofVirginia,USA.
attachment to cells, but interferes with virus entry, and cellecell There are currently no antiviral agents licensed for the treat-
fusion.Inacell-basedfusioninhibitionassaytheEC50was~2nM. mentofrhinovirusinfections.Thecommoncold,generallyaself-
Resistantviruseswereisolatedafter9passagesinvitro,withmu- limiting disease, was an original target, but no broadly effective
tationsintheFprotein,includingL138ForF140L/N517IinRSVA, inhibitorhasbeendemonstrated;additionally,itwouldneedtobe
conferringmorethan2000-foldresistance,andF488LorF488Sin cheap with no side effects. Interferon, protease inhibitors, and
RSVB.Thesewerecross-resistanttoanotherRSVfusioninhibitor capsidbindinginhibitorsallshowedefficacyinlaboratorymodels,
VP-14637 (Douglas et al., 2005; McKimm-Breschkin, 2000), also butnotinpatients(Hayden,2013).Althoughrhinovirus(RV)isan
undergoingclinicaldevelopmentasaninhaledproduct(MDT-637). importantcauseofexacerbationsofCOPDandasthma,hencebeing
IncottonratsasingleIPdoseof0.3,3or30mg/kgGS-5806was alternativetargets,thereisnogoodclinicalmodelforevaluationof
administered1hpriortointranasalchallengewithRSV.A1.8log10 therapies.Prophylaxismaybemorefeasible.Inafamilystudythere
differencewasseeninvirusRNAloadinnasalwashandlunglavage was a significant decrease in asthma scores with pleconaril pro-
samples on day 4, in the 30 mg/kg group. In a small trial with 4 phylaxis. However, approaches to therapy have been further
calvesinfectedwith105pfuofnebulisedbovineRSV,andtreated complicatedbythediscoveryofanewspeciesofrhinovirus,RV-C,
1hpriortoinfectionandtwicedailyfor7dayswith2mg/kg,there in 2006, in addition to the known RV-A and RV-B viruses
wasanon-significant3-folddecreaseinvirustiter,butasignificant (Palmenberg and Gern, 2015). As with RV-B they are inherently
3-folddecreaseinthesymptomscore. resistanttocapsidbindersandhaveadifferentcellularreceptorto
Adoubleblindplacebo-controlledtrialofGS-5806wascarried both RV-A and RV-B (CDHR3 instead of ICAM-1). Host-directed
outinhealthysubjectsexperimentallyinfectedwith104pfuofthe therapies, such as TLR antagonists and probiotics, may be prom-
RSV Memphis-37b strain. Nasal wash samples from day 2 were ising, and recent studies have reported some beneficial effect of
testedforRSVbyRT-PCR,andwhenpositiveoratday5,whichever probiotics (Lactobacillusspp.) ontheoccurrenceofcommoncold
wassooner,subjectswererandomisedinto7cohorts,with~20in illnessesinchildren(BallengeeandTurner,2014).
each,andtreatedasfollows:Cohorts1e4(day150mg;days2e5
25mgdaily),Cohort5(day150mg;days2e325mgdaily),Cohort 6.2. SevereillnessassociatedwithEV-D68infectionintheUnited
6(day1100mg),Cohort7(day110mg;days2e55mgdaily).All Statesandapproachestomanagement
patientsintreatmentgroups1e4showedasignificantdecreasein
theareaunderthecurve(AUC)forlog10virusload,(250.7vs757.7 SueGerber,CentersforDiseaseControlandPrevention,Atlanta,
log10pfuequivalents),lowermucusweight(during5daysafterfirst USA.
dose6.9vs15.1g,p¼0.03),andlowersymptomscores.Cohorts Enterovirus EV-D68 was first identified in 1962. Unlike other
5e7 had comparable results, although Cohort 7 did not show a enteroviruses, which can cause febrile rashes and neurological
decrease in mucus weight. Resistance frequency was 5%, with syndromes,EV-D68iscommonlyassociatedwithrespiratoryillness
mutations the same as selected in vitro, but the viruses showed andmayplayaroleinasthmaexacerbation.Inthesummerandfall
decreasedfitness.Plansaretotestahighersingledose,>100mg,to of2014therewasalargeincreaseinsevererespiratoryillnessinthe
trytoavoidresistance. USA and the CDC and state health laboratories identified 1,529
people PCR positive for EV-D68 (Midgley et al., 2015); however
5.2.2. RSVantivirals:fusioninhibitorsandbeyond rhinovirus,Coxsackievirusandechoviruseswillalsobedetectedby
JohnDeVincenzo,UniversityofTennessee,Memphis,USA. therhinovirus/enterovirusmultiplexPCR.UsingVP1sequencingto
Johngaveanoverviewofclinicalobservationsandcorrelatesof identifyEV-D68,46%ofspecimenswerepositiveforEV-D68.Several
protection.Ahighervirusloadinthenosecorrespondswithmore cases of flaccid myelitis have been temporally related to EV-D68
J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38 29
infectionbutacausalroleofEV-D68hasnotbeenestablished.Anew includingagainstinfluenza,PIVandRSV.Nitazoxanideappearsto
real-timeRT-PCRtestisavailablefromtheCDC(Zhugeetal.,2015). blockthematurationoftheinfluenzavirusHA(Rossignol,2014).In
Threestrainshavebeenidentifiedwith92%belongingtoonestrain, a phase 2b/3 trial in 2010e11, comparing300 mge600 mg twice
7.4%tothesecond,andasingle caseofthethirdstrain.Thevirus dailyin624patients,onlytreatmentwith600mgtwicedailyfor5
grows in MK cells and human lung A549 cells and binds to a2,6 dayswasassociatedwithareductioninthedurationofsymptoms,
linked sialic acids. It is sensitive to pleconaril and other capsid and a 1 log10 decrease in virus titer, in recipients with acute un-
bindinginhibitorsinvitro,andarandomiseddoubleblindplacebo complicated influenza (Haffizulla et al., 2014). Nitazoxanide has
controlledtrialsuggestedashortertimetocultureandPCRnegative also shown synergistic effects in vitro with NAIs (Belardo et al.,
status with oral pleconaril. DAS181 which removes sialic acid re- 2015).InacurrentPhaseIIItrial,600mgofnitazoxanideisbeing
ceptorsisalsobeingevaluated(Abzugetal.,2015). comparedtonitazoxanideplusoseltamivir,oseltamivir,orplacebo.
6.3. Newtargetsandapproachesforcoronavirusantiviral 7. Monoclonalantibodiesastherapeutics
inhibitors
7.1. Monoclonalantibodiesastherapeuticsagainstrespiratory
MarkDenison,VanderbiltUniversity,Nashville,USA. viruses
Coronaviruses (CoVs) are endemic respiratory pathogens,
causing mainly colds, bronchiolitis and pneumonia. Additionally, WayneMarasco,HarvardMedicalSchool,Boston,USA.
zoonotic transmissions have occurred, with the SARS-CoV and In studies of respiratory viruses, this group has used non-
morerecentlyMERS-CoV.AlargenumberofendogenousbatCoVs immune human antibody phage display libraries as a source of
have been identified. They have the largest genome of any RNA humanmonoclonalantibodies(mAbs).Asacomplementarystrat-
virus, 26e32 kB, with uptothree proteases, and a proof reading egy,theyhaveusedsinglememoryBcellcloningtoisolatebroadly
exonuclease(nsp14-ExoN),endonucleaseandhelicase-ATPase.The neutralizingantibodies.ToisolateantibodiesagainsttheSARS-CoV
SARSExoN-minusvirusappearstoacquiremorelethalmutations andMERS-CoVbloodsampleswereobtainedfromChinaandAra-
with5-fluorouracilmutagentreatmentthanthewildtype(Smith bia.Binderswereidentifiedusingpseudotypedviruses. Aninitial
et al., 2013). It is also attenuated in mice. Hence high replication SARS-CoV mAb, 80R, which neutralised all 2002e3 isolates, in-
fidelityiscriticalforCoVs.Treatmentinvitrowithribonucleoside terfereswiththeglycoproteinspikeS,bindingtoitsACE2receptor
analoguesEV524andEV1081(Gilead)causeda5log10reductionin site.However,virusesisolatedfrompatientsinasmalloutbreakin
virus titer compared to the control. A combination of fidelity in- 2004 were all resistant to neutralization due to a single Asp/Gly
hibitors and nucleoside analogues may provide broad activity mutation.AnewmAbwasmadetocovertheseescapemutants(Sui
againstmultipleCoVs. etal.,2008).However,recentworkhasshownthateventwomAbs
incombinationdo notpreventtheemergenceofescapemutants
6.4. Functionaldipeptidylpeptidase(DPP4)inminksupportsentry (Suietal.,2014).
andreplicationofMiddleEasternRespiratorySyndrome MERS-CoVemerged in the Arabian Peninsulain 2012,but ap-
Coronavirus:Americanmink(Neovisionvision),anovelinvivo pearstohavebeenendemicformorethan20years.Manycamels
modelofMERS-CoVinfection are infected, including elite racing camels, although the primary
hostmaybebats.ToJune2015,therehadbeen1150caseswith434
ThomasVoss,SRIInternational,Harrisonburg,Virginia,USA. deaths.UsinganScFvphagelibrary,7mAbs,encompassingthree
Dipeptidylpeptidase4(DPP4/CD26)isthereceptorforMERS- different epitopes of the S1 spike, were identified, all inhibiting
CoV. It is a multifunctional protein and is a target for diabetic binding to the DPPD4 receptor in an SPR assay. Escape mutants
therapy,andapoptosis.Acelllinederivedfromminklungepithe- identified changes in five amino acids critical for neutralization
liumissusceptibletoinfectionbyMERS-CoV.Althoughferretcells escape.Lossofbindingtooneepitopedidnothaveamajorimpact
express DPP4, based on antibody reactivity, ferrets cannot be on binding of Abs directed to other epitopes, but mutants had
infected. Mink challenged with MERS-CoV showed no signs of reduced fitness, resulting in decreased virus amplification and
illnessorvirusinanytissue.Differentchallengeroutesweretried, reduced inflammation in the lungs of infected rhesus monkeys
intranasal,oral,intratrachealandintraocular,andchallengedoses (Tangetal.,2014).
from 106 -109, and different challenge strains, including one Screening for antibodies against influenza A(H5N1) identified
adaptedtominklungcells.Asanalternativeanimalmodel,while 10 mAbs which bound a common epitope on the HA stem, pre-
normal mice are not susceptible to MERS-CoV, expression of the venting the post-attachment fusion process (Sui et al., 2009). All
human DPP4 (hDPP4) overcomes the lack of susceptibility. The appeared to use the IGHV1-69 variable heavy chain region, with
transgenichDPP4micedevelopsevereandlethalrespiratorydis- highconservationofCDR-2.Theseantibodiesprotectedmiceand
ease upon inoculationwith MERS-CoV andarepotentiallya suit- decreasedthevirusload.Theantibodiescross-reactedwithother
able animal model for efficacy testing of therapeutic and subgroup1influenzaHAs,butnotwithsubgroup2.Whilegener-
prophylacticcountermeasures(vanDoremalenandMunster,2015). ationofescapemutantswasdifficult,thoseselectedhaddecreased
fitness;mutationswereoutsidetheepitope,causingdestabilization
6.5. PhaseIIImulti-centerclinicaltrialofnitazoxanideinadult of the trimer. Another mAb, I14, was broadlyactive against both
patientswithuncomplicatedinfluenzaAandBandotherinfluenza- groups 1 and 2, stabilizing the pre-fusion form of the HA, as
likeillness:resultson1876subjectsfromtheUnitedStates,Canada, assessedbyblockingtrypsincleavageoftheHA0toHA1andHA2.
AustraliaandNewZealand Unexpectedly,despitebeingselectedagainstagroup1H5N1strain,
itsefficacyinvitroandinvivoishigheragainstgroup2viruses.
Jean-FrancoisRossignol,RomarkLaboratories,Florida,USA.
Nitazoxanide has been approved for treatment of diarrhoea 7.2. Newantibody-basedstrategiesagainstviralrespiratory
associatedwithGiardiaandCryptosporidiuminfections.Ithasbeen diseases
usedinmorethan75millionpatientsintheUSAandLatinAmerica
alone, hence has a known safety profile. It is an orally available, QingZhu,Medimmune,Gaithersburg,USA.
smallMWdrug,withwhatappearstobebroadantiviralactivity, The humanised mAb Synagis (palivizumab), which is still the
30 J.L.McKimm-Breschkin,A.M.Fry/AntiviralResearch129(2016)21e38
onlymAbagainstaviraldiseaseapprovedforclinicalusebytheUS consequent binding of antibody-coated virus to the Fc receptors
FDA,hasbeenwidelyusedforpreventionofRSVinfectionsinne- (FcR)whichfacilitatesvirusentry,leadingtoenhancedinfection.In
onatesandimmunocompromisedindividuals.RespiGam®isahu- the case of secondary infections and of maternal antibody in
man polyclonal antibody preparation also used for prevention of newborns, there may be some cross-reactive antibodies, but
RSV,butthereissignificantbatchvariation.AnewmAb,MEDI8897, insufficienttoneutralizevirus.AmodelofDHF/DSS-likediseasehas
whichhas100-foldhigherpotencyandneutralizesbothRSVAandB been developed in AG129 mice lacking the type I IFN receptor
strains,hasanEC90inthecottonratmodelof6.5mg/ml,comparedto (IFNAR)andtheIFN-gR(IFNAR(cid:3)/(cid:3);IFN-gR(cid:3)/(cid:3)).Thismousemodel
57mg/mlforpalivizumab.Thehalflifewas~40daysinmonkeysand reproduceskeypathophysiologicalfeaturesofDHF/DSS,including
~80daysinaPhase1atrialinadulthumans.Theaimistoincrease similarcellularandtissuetropism,lethalvascularleakage,cytokine
thehalflifetoenableasingleintramuscularinjectiontobeeffective storm, low platelet count, elevated hematocrit, and hemorrhage
forthewholeseason,thusprovidingvaccine-likeimmunity. (Tangetal.,2015).Inordertotestthehypothesis,micewerepre-
AnothermAb,MEDI8852,neutralizesallinfluenzaAstrainsby treated with different levels of antibody IP, followed by DENV
bindingahighlyconservedepitopeintheHAstalk,withamedian challenge:0.5mgofantibodywastoolowtohaveanyeffect;sub-
IC50of0.76mg/ml.Ithasmultipleeffectsonvirus-inducedmem- neutralizing doses of 2e15 mg led to severe disease and death;
branefusion,HAmaturationandcellecellspread.Itisclaimedtobe whileafullyneutralizingdoseof500mgprotectedallmice.Virus
superior to oseltamivir when given late in infection in mice and load was significantly higher at 48 h in the liver of ADE mice
ferrets. Given 3 days post-infection in mice there was 100% pro- comparedwithnon-ADEmice.Theimportanceof theinteraction
tection, versus 40% protection by oseltamivir. A combination of between the Fc in the DENV-Ab complex and Fcg receptor was
MEDI8852 and oseltamivir at a suboptimal dose demonstrated a confirmedusinganantibodywithamutantFcregionthatcannot
synergistic effect. The aim is to generate a bispecific antibody bind Fcg receptors, or Fcg receptor-blocking antibody or the Fab
againstbothinfluenzaAandB. fragmentofanti-DENVAb.Theseinterventionscoulddecreasethe
enhancedvirustiterintheliversofADEmicetonon-ADElevels.In
7.3. VIS410monoclonalantibodydemonstratespotentefficacy anothermodelusingaVEErepliconandtheDENVE2protein(virus
againstneuraminidaseinhibitors-susceptibleanderesistant repliconparticles), AG129 immunised mice had a higher survival
influenzaA(H7N9)virusesandprotectsmicefromARDS rateandlowervirustiterswhenchallengedwithDENV(Zellweger
etal., 2013). However, passive transferof 50e1500ml of immune
Tatiana Baranovich, St Jude Children's Research Hospital, seraresultedinADEinrecipientmice.
Memphis,USA. TodeterminetheroleofTcells,CD4orCD8depletionwasused
VIS410isabroadlyneutralizinghumanIgG1anti-HAantibody, toshowthatwhereasCD4-depletedmicehadlowvirustiters,CD8
whichreactswitharegionoftheHAstalkconservedingroup1and depleted mice had high virus titers. Thus CD8 T cells conferred
2influenzaviruses(Tharakaramanetal.,2015).Itappearstohave greater protection. When mice were primed with UV-irradiated
loweraffinity toH7 HA, henceits efficacy was tested against the denguevirus,CD8TcellsprotectedagainstADE.Thusintermsof
oseltamivir-susceptibleA/Anhui/1/2013or-resistantA/Shanghai/1/ vaccine development, both cellular and humoral immune re-
2013 (R292K-NA) H7N9 influenza viruses in mice. VIS410 was sponsesneedtobeinduced,andtheFceFcRinteractioncontrolled.
administeredviaintraperitonealinjectionofeitherasingle50mg/
kgdose12hbefore(prophylaxis),ora2,10,or50mg/kgdose24h 8.2. Influenzavaccine-inducedanti-HA2antibodiespromotevirus
after (treatment) H7N9 virus inoculation. Weight loss, morbidity entryandenhancelungpathologyafterinfluenzaAinfection
andviruslungtitersweremonitored:Prophylacticadministration
oftheantibodyresultedin100%protectionofthemice;asingleIP SurenderKhurana,FDA,Bethesda,USA.
administrationof50mg/kg24hpostinfectionalsoprovided100% Duringthe2009influenzaA(H1N1)pandemic,severalreports
survival,whereasthe10mg/kgdoseprotected70%oftheAnhui- suggested priorvaccinationwith the 2008e2009 seasonal inacti-
infected and 90% of the Shanghai-infected mice. All doses of vated influenza vaccine increased severity of clinical disease
VIS410testedprotectedmicefromH7N9-inducedacuterespiratory following infection with the A(H1N1)pdm09 virus (Skowronski
distress syndrome (ARDS). There was a dose response in the etal.,2010). The mechanismofvaccine-associated enhancedres-
numbers of cells positive for NP. Mice developed an anti-HA im- piratory disease (VAERD) is not well understood. Using a swine
muneresponse,sufficienttoprovideprotectionagainsta25MLD50 model,Khuranaetal.(2013)showedthatvaccinationofpigswith
challengewiththehomologousvirus. anH1N2virusandchallengewithaA(H1N1)pdm09virusresulted
in the pigs having enhanced pneumonia and disease. Using re-
8. Antibody-dependentenhancement(ADE)ofdisease: combinantexpressionofHA1andHA2andSPRbindingassays,they
implicationsfortherapeuticmonoclonalantibody showedthattheH1N2serareactedwiththeA(H1N1)pdm09virus,
development butthatantibodiesboundonlytotheHA2oftheH1N1virus,and
competed with other stem-targeting antibodies. The H1N2 sera
8.1. InfluenceofantibodiesandTcellsondenguediseaseoutcome also enhanced A(H1N1)pdm09 infection of MDCK cells and pro-
motedvirusfusionactivity.OnlyadsorptionoftheserawithHA2
SujanShresta,LaJollaInstituteforAllergyandImmunology,La hadanyeffectinreducingtheenhancementeffect.Livevaccination
Jolla,USA. of pigs caused no ADE; howeveradministration of the same, but
ThefourtypesofDenguevirus(DENV)causeupto100million UV-inactivated, vaccine resulted in ADE. The findings of these
cases, with 2.1 million severe cases and up to 21,000 deaths per studies have generated concerns that clinical trials evaluating
year. Infectionsrangefromasymptomatic toa self-limited febrile universal vaccines and monoclonal antibody therapeutics should
illness,denguefever(DF),tolifethreateningdenguehemorrhagic monitorforantibody-associatedenhanceddisease.
fever(DHF)anddengueshocksyndrome(DSS).Therolesofanti-
bodies and T cells in mediating protection versus pathogenesis 8.3. Doesantibody-dependentenhancementofdiseaseoccurin
remains unclear. Antibody-dependent enhancement (ADE) was influenzainfections?
identifiedmorethan40yearsago,andishypothesisedtobedueto
the presence of subneutralizing levels of antibody and the Man-WahTan,Genentech,SanFrancisco,USA.