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

Arenavirus RNAs are ambisenseSt

raus

s and

Stra

uss,

VIR

USE

S A

ND

HU

MA

N D

ISEA

SE (2

001)

Tulane University

R. Garry R. Garry ‘‘0606

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

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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

0

10

20

30

40

50

60

70

80

90

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

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Mouth ulcers

Splenogem

agly

Hepato

megaly

Low pulse pres

sure

Icteru

sArrh

ythmiaRas

hPete

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Ascite

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Sign or symptom

Perc

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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.