Recombinant antigen multiagent diagnostic assays for Lassa and
Transcript of Recombinant antigen multiagent diagnostic assays for Lassa and
Recombinant antigen multiagent diagnostic assays for Lassa and
other arenavirusesPresented by Robert F. Garry
for the Arenavirus Diagnostic Consortium
USAMRIID
CIRIT-Guinee
Kenema GovernmentHospital
Sierra Leone
Instituto Nacional de Enfermedades Virales Humanas-Argentina
Develop modernDevelop modern IgMIgM--,,IgGIgG-- and antigenand antigen--capture capture assays for diagnosis of assays for diagnosis of infections by Old and infections by Old and New WorldNew World arenavirusesarenaviruses..
GOALS
ArenaviridaeArenaviridae• Name derived from “arenosus” (Latin “sandy”)
describing appearance by EM• Enveloped virus, round or pleomorphic, 50-300
nm in diameter
•• Name derived from Name derived from ““arenosusarenosus”” (Latin (Latin ““sandysandy””) ) describing appearance by EMdescribing appearance by EM
•• Enveloped virus, round orEnveloped virus, round or pleomorphicpleomorphic, 50, 50--300 300 nm in diameternm in diameter
• Single-stranded linear genome with 2 RNA segments: small (~3.4 kb) and large (~7.3 kb)
• 2 genes/ambisense segment, encoding 5 proteins.
•• SingleSingle--stranded linear stranded linear genome with 2 RNA genome with 2 RNA segments: small (~3.4 kb) segments: small (~3.4 kb) and large (~7.3 kb) and large (~7.3 kb)
•• 2 genes/2 genes/ambisense ambisense segment, encoding 5 segment, encoding 5 proteinsproteins. F. A. Murphy
LASSA
IPPY
MOPEIA
MOBALA
JUNIN
TAMIAMI
LCM
MACHUPO
GUANARITO
TACARIBEAMAPARI
FLEXAL PICHINDE
LATINO
PARANASABIA
ArenavirusArenavirus Phylogeny Phylogeny Old and New World Old and New World ArenavirusesArenaviruses
Adapted fromBowen et al., 2000
Lassa FeverLassa Fever• Hemorrhagic fever caused by Lassa virus, an
Old World Arenavirus.• First report of cases: Lassa, Nigeria in 1969.• Endemic in areas of West Africa
– No symptoms in 80% of people infected– Remaining 20% have multisystemdisease– Most common complication is deafness (1/3 of cases)
• Occasional nosocomial or epidemic outbreaks in which case fatality rates can reach 50%
• Ribovirin can reduce mortality if used early in infection
Photo: D. Bausch
Lassa EpidemiologyLassa Epidemiology
• Endemic in areas of West Africa, including Nigeria, Liberia, Sierra Leone, and Guinea
• Estimated 100,000-300,000 infections/year, with 5000 deaths
• Rodent reservoir– Rodent-to-human transmission (the “multimammate rat”,
Mastomys species-complex)– Rodent excreta contaminating food– Direct contact (consumption or bite)– Aerosol
• Secondary human-to-human transmission with the potential for nosocomial outbreaks with high case-fatality
• Endemic in areas of West Africa, including Nigeria, Liberia, Sierra Leone, and Guinea
• Estimated 100,000-300,000 infections/year, with 5000 deaths
• Rodent reservoir– Rodent-to-human transmission (the “multimammate rat”,
Mastomys species-complex)– Rodent excreta contaminating food– Direct contact (consumption or bite)– Aerosol
• Secondary human-to-human transmission with the potential for nosocomial outbreaks with high case-fatality
• “Multimammate rat”• Peri-domestic• Inhabit fields and cleared forest• Prolific breeders (~8-12 pups/litter)• Infected at birth and become chronic asymptomatic carriers of Lassa virus• Shed virus in the urine and feces• Numerous sub-species
MastomysMastomys Species ComplexSpecies Complex
Photos: J. Fair & D. Bausch
Lassa Virus Rodent ReservoirLassa Virus Rodent Reservoir
Distribution of LassaDistribution of LassaAntibodies in Humans: Antibodies in Humans:
SeroSero--EpidemiologicEpidemiologicStudiesStudies
MastomysdistributionMastomys
distribution
00
60
1 0
0
024
0
023
3516
IFAIFA
ELISAELISA
Anecdotal reports/CFA Anecdotal
reports/CFA
Slide courtesy of D. Bausch
Lassa Diagnostic DifficultiesLassa Diagnostic Difficulties• Presenting syndrome very non-specific
All age groupsIncubation period: 10 days (3 - 21 days)Common symptoms: fever, headache, weakness, retrosternal pain, abdominal pain, vomitingRare symptoms: facial and neck swelling, bleeding, (maculopapular rash)
• Broad differential diagnosis (malaria, typhoid fever, yellow fever, influenza, measles, shigellosis, leptospirosis, meningococcemia, Rickettsial infections, bacterial sepsis, other viral hemorrhagic fevers)
• Even an experienced clinician cannot easily distinguish Lassa fever from other common febrile illnesses
• No widely available diagnostics available
Lassa: Clinical Presentation at Admission
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10
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100
Fever
Malaise
Headac
heChes
t pain
Arthral
gia
Lumber pain
Myalgia
Vomiting
Vertigo
CoughSore
throat
Rales/r
honchi
Abdominal pain
Tinnintus
Conjunctiva
l injec
tion
Pharyngitis
Diarrh
eaBlee
ding
Hearin
g loss
Gait an
omaly
Disorie
ntation
Edema
Dysuria
Tachyc
ardia
Jaundice
Hypoten
sion
Adenopath
y
Mouth ulcers
Splenogem
agly
Hepato
megaly
Low pulse pres
sure
Icteru
sArrh
ythmiaRas
hPete
chia
Ascite
sBrad
ycard
ia
Sign or symptom
Perc
enta
ge
Lassa fever (n=21)
Non-Lassa febrile illness (n=268)
Lassa Fever: Clinical Presentation at Admission
FROM:Bausch et al., Vector Borne and Zoonotic Diseases, 2001
Lassa cannot be distinguished from other febrile illnesses by clinical presentation
Laboratory Diagnosis of LassaLaboratory Diagnosis of Lassa• Enzyme-Linked Immunosorbent Assay (ELISA)
– Mainstay of diagnosis (simple equipment)– Detection of viral specific antigen and IgM antibodies
for acute disease– Detection of IgG antibodies for past exposure (ie.
surveillance)• Polymerase Chain Reaction (PCR)
– Sensitive tool, but risk of false positives (PCR contamination) necessitates confirmation-another test
– Important research tool (requires complex equipment)• Immunofluorescent Antibody test (IFA)
– Less reliable than ELISA• Virus Isolation
– Requires maximum safety laboratory (BSL-4)– Primarily a research tool Slide courtesy of D. Bausch
Numerous problems are associated with the current Lassa ELISA
• Lack of sensitivity.• Viral antigens must be produced at BSL-4.• Not available to reference laboratories in
endemic areas.• Expensive to produce (BSL-4 time,
personnel, space, etc.)
PROJECT MANAGEMENT
Luis BrancoDarryl SampeyAlex Matchiner
Russell WilsonPeter Kulakosky F. Jon Geske
USAMRIID
CIRIT-Guinee
Augustin Goba, Allen Seissay
Kenema Government Hospital
Sierra Leone
PROTEIN EXPRESSION/MONOCLONALPRODUCTION
ASSAY PRODUCTION/SCALE-UP
SAMPLE COLLECTION-ASSAY FIELD TESTING - Dan Bausch/Joseph Fair
VALIDATION (PCR/virus)BSL-4 ASSAYS ABSL-4
OVERALL PROJECT MANAGEMENT Robert Garry, Dan Bausch, Joseph Fair, Allyson Haislip
Instituto Nacional de Enfermedades ViralesHumanas - Argentina
Delia Enria
ASSAY DEVELOPMENT
Mary GuttieriJoseph FairPhil FerroKat Cashman
Mamadou CoulibalyCorina Monagin
EXTERNAL ADVISORY GROUP
Clarence J. Peters - University of Texas at Galveston
Tom Geisbert - USAMRIID
Connie Schmaljohn - USAMRIID
Thomas Voss - Tulane University Health Sciences Center
Cristain Apetrei - Tulane National Primate Research Center
Andrew Lackner - Tulane National Primate Research Center
May Chu - World Health Organization
DELIVERABLESMILESTONE 1MILESTONE 1••Prototype Lassa Virus antigenPrototype Lassa Virus antigen--capture ELISA using monoclonal capture ELISA using monoclonal antibodiesantibodies••Prototype Lassa Virus Prototype Lassa Virus IgMIgM--capture ELISA using recombinant capture ELISA using recombinant GP1, GP2 and NPGP1, GP2 and NP••Prototype Lassa Virus Prototype Lassa Virus IgGIgG--capture ELISA using recombinant capture ELISA using recombinant Lassa GP1, GP2 and NPLassa GP1, GP2 and NP
MILESTONE 2MILESTONE 2••Commercial grade Lassa virus antigenCommercial grade Lassa virus antigen--, , IgMIgM--, and , and IgGIgG--capture capture ELISAsELISAs using monoclonal antibodies and recombinant proteinsusing monoclonal antibodies and recombinant proteins
MILESTONE 3MILESTONE 3••Commercial grade Commercial grade arenavirusarenavirus antigenantigen--, , IgMIgM--, and , and IgGIgG--capture capture ELISAsELISAs using monoclonal antibodies and recombinant Lassa, using monoclonal antibodies and recombinant Lassa, JuninJunin, etc, virus proteins, etc, virus proteins
Arenavirus protein expression• Bacterial and mammalian cell lines stably
expressing the Lassa virus glycoprotein precursor (GPC), glycoprotein 1 (GP1), glycoprotein 2 (GP2), and NP will be engineered.
• Cell lines producing murine monoclonal antibodies (mMAbs) to each antigen will be selected and scaled-up.
• Similar strategies will be used to produce Juninvirus antigens and monoclonal antibodies. Other arenavirus proteins and MAbs will be produced as needed for multiagent antigen-, IgM- and IgG-capture ELISAs.
Cloning Strategy : Bacterial• Secreted forms of each protein (lacking the
signal sequence and transmembrane anchors) were inserted into the pMALp2x expression system and transformed into the Rosetta strain of E.coli.
• pMALp2x contains a periplasm export sequence. The periplasm contains “helper” proteins which aid in the proper folding of proteins containing cysteine linkages.
• The Rosetta strain of E. coli allows for expression from multiple “rare-for-E.coli”codons.