SPECIAL FOCUS y Emerging Zoonotic & Mosquito-Borne Virus Diseases

Review
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Current and emerging strategies for the

diagnosis, prevention and treatment of
Lassa fever

Jessica N Hartnett1, Matthew L Boisen1,2, Darin Oottamasathien†,2, Abigail B

Jones2, Molly M Millett2, Diana S Nelson2, Ivana J Muncy2, Augustine Goba3,
Mambu Momoh3,4, Mohammed Fullah†,3, Chad E Mire5, Joan B Geisbert5, Thomas
W Geisbert5, Debra L Holton1, Julie A Rouelle1, Chandrika B Kannadka1, Ashley A
Reyna1, Lina M Moses1, S Humarr Khan†,3, Sahr M Gevao6,7, Donald S Grant3,6, James E
Robinson8, Christian Happi9, Kelly R Pitts2, Robert F Garry1,10, Luis M Branco*,10 & the
Viral Hemorrhagic Fever Consortium

Abstract Lassa fever (LF) is a potentially fatal disease that affects an estimated
300,000–500,000 people in endemic areas of west Africa each year. Though past studies have
identified fatality rates of 5–20% in patients suspected to have contracted Lassa virus (LASV),
new studies using more precise clinical diagnoses and modern diagnostic assays show
fatalities rates above 60% in acutely ill patients from endemic regions. Currently, there are no
approved vaccines or therapeutics, and only one Comformité Européenne (CE) marked rapid
immunodiagnostic for acute LASV infection. Therefore, preventing LASV transmission is the
primary goal in endemic regions. Development of rapid immunodiagnostics and research
into the efficacy of current treatment options continues toward saving lives in west Africa as
well as creating a line of defense against the nefarious use of LASV in bioterrorism settings.

Lassa fever (LF) is an often fatal disease that affects an estimated 300,000–500,000 people each year Keywords
in endemic areas of Sierra Leone, Guinea, Liberia, Nigeria and other west African countries [1–3] . Past • arenaviruses
studies have identified fatality rates of 5–20% in suspected Lassa fever patients [4] . Recent studies • bioterrorism • diagnostics
in Sierra Leone showed fatalities rates of greater than 60% in acutely ill patients presenting to a • epidemiology
healthcare facility while viremic [5,6] . Pleural and pericardial effusions, hemorrhage and spontane- • hemorrhagic fever • Lassa
ous abortion are common acute complications, while bilateral deafness is reportedly a common, fever • Lassa virus • public
irreversible side effect of LF in convalescent patients [7–11] . health • therapeutics
Lassa fever was first reported in Nigeria in 1969 after three missionary-nurses contracted the • vaccines
illness [12] . After the deaths of the first two nurses, a third nurse was evacuated from Nigeria and
recovered in the United States where the etiological agent Lassa virus (LASV) was identified [13–15] .
1
Department of Microbiology, Tulane University School of Medicine,1430 Tulane Avenue, SL-38, New Orleans, LA 70112, USA
2
Corgenix Medical Corporation, Broomfield, CO 80020, USA
3
Kenema Government Hospital, Kenema, Sierra Leone
4
Eastern Polytechnic College, Kenema, Sierra Leone
5
Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX 77555, USA
6
Ministry of Health and Sanitation, Freetown, Sierra Leone
7
University of Sierra Leone, Freetown, Sierra Leone
8
Department of Pediatric Infectious Diseases, Tulane University School of Medicine, New Orleans, LA 70112, USA
9
Redeemer’s University, Ogun State, Nigeria
10
Zalgen Labs, LLC, Germantown, MD 20876, USA
*Author for correspondence: lbranco@zalgenlabs.com
†
Deceased part of

10.2217/FVL.15.41 © 2015 2015 Future Medicine Ltd Future Virol. (2015) 10(5), 559–584 ISSN 1746-0794 559
Review Hartnett, Boisen & Oottamasathien et al.

LASV is a member of the family Arenaviridae. subunit in a fully assembled GPC complex has
Though it was discovered in man in 1969, LASV not been well [34–39] elucidated. The GP1 subunit
likely diverged from other members of the fam- of the GPC complex initiates viral infection by
ily Arenaviridae over 1000 years ago [16,17] . binding to a cell surface receptor. The known
Currently, LASV is comprised of four major lin- receptor for LASV is α-dystroglycan [40–42] .
eages. Lineages I, II and III circulate in Nigeria, Activation of the prefusion GPC complex by an
while a newer lineage IV is responsible for disease acidic pH shift is followed by structural reor-
in Sierra Leone and surrounding countries [18] . ganization of the GP2 subunit and formation
LASV belongs to the Old World arenavi- of a highly stable six-helix bundle structure with
rus subfamily, grouped with the well-studied membrane fusion properties. The arenaviral
lymphocytic choriomeningitis virus (LCMV). envelope glycoprotein is therefore classified as a
The counterpart subfamily is comprised of the class I virus fusion protein. Arenaviral GPCs are
New World arenaviruses. Because the viruses unique among class I envelope glycoproteins in
of the New and Old World complexes share that they retain their cleaved signal peptides in
common features – they have highly conserved the mature complex as an essential subunit for
genomic structures and gene sequence similar- viral infectivity [43–45] .
ity and function – studies with one arenavirus The genome is packaged into a spherical, pleo-
allow general predictions to be made about morphic lipid-enveloped virion ranging in size
characteristics of related counterparts [19] . Less from 50 to 300 nm in diameter [46] . Electron
virulent arenaviruses, such as Pichinde (PICV), microscopy of LASV particles reveals a sandy
Mobala (MOBV) and Mopeia (MOPV), can morphology due to the presence of electron-
provide good representations of replication and dense structures within the virion. These struc-
pathogenesis observed with more virulent are- tures are believed to be host ribosomes based on
naviruses that can only be studied in high con- the findings of abundant 28S and 18S ribosomal
tainment (BSL-4) laboratories, such as Lassa, RNA [47] , but there has not been a formal dem-
Lujo (LUJV) and Machupo (MACV). Junin onstration that arenaviruses incorporate intact
virus (JUNV) can be presently manipulated in and functional ribosomes.
BSL-3 laboratories if personnel are vaccinated LASV infection is characterized by a broad
with Candid#1, a live vaccine that has demon- range of symptoms including fever, malaise,
strated clinical efficacy against this virus, and is headache, sore throat, cough, myalgia, gas-
approved for human use in Argentina. troenteritis, abdominal or retrosternal pain,
Arenaviruses have a bisegmented, single- myocarditis, pleuritis and can progress to a
stranded RNA genome encoding four genes in late stage involving encephalopathy and hem-
an ambisense fashion. The L RNA encodes the orrhage [4,7,12,48,49] . Symptoms typically pre-
viral RDRP, also known as L polymerase, and sent 1–3 weeks after exposure to LASV, and
the Z matrix protein [20–22] . The S RNA encodes the differential presentation poses significant
the NP, possessing known 3’– 5’ dsRNA exonu- challenges to accurately diagnose LF. Major
clease function and immune evasion activity [23– symptoms of LF, such as conjunctival injection,
31] and the GPC precursor. The mature GPC is facial edema and frank bleeding can occur and
comprised of three independent subunits that generally correlate with a poor prognosis [50–52] .
arise from post-translational cleavage of the pre- Among positively diagnosed LF patients, those
cursor glycoprotein. Cellular signal peptidases under the age of 29 have a higher case–fatal-
cleave the SSP, a 58 amino acid stable signal ity rate without distinction between males and
polypeptide with membrane spanning domains. females [6] . However, pregnant women with LF,
The SSP associates with the GP1-GP2 precursor particularly in their third trimester, appear to
and allows trafficking of the complex from the have a higher case–fatality rate and a high rate
endoplasmic reticulum to the Golgi apparatus. of fetal abortion [5,6,53–55] .
In the Golgi cellular SKI-1/S1P proteases cleave Currently, there are no approved vaccines, or
the GP1-GP2 precursor into distinct GP1 and therapeutics and only one commercially avail-
GP2 glycoprotein subunits [19,20,32,33] (Figure 1) . able diagnostic for LASV infection [56] . The
The GPC complex is transported to the cell sur- antiviral therapeutic ribavirin is available as an
face and decorates the membrane with trimers of off-label treatment for LF and increases sur-
noncovalently associated GP1 and GP2 subunits vival if administered early in infection [4,6,57–59] .
and the associated SSP, although the ratio of each It has recognized side effects if improperly

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 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

GP1
GP2

SSP

NP
Small RNA
(encoding GP1,
GP1, SSP
and NP)

Ribosome (???)
Z, matrix

Large RNA
(encoding L
and Z)

Figure 1. Lassa virion structure. Arenaviral particles are enveloped, with a bilayer lipid membrane
containing anchored protein spikes corresponding to the assembled GPC complex (GP1, GP2,
SSP). Arenaviral particles are commonly round or pleiomorphic particles 50–300 nm in diameter
and contain several electron dense particles approximately 25 nm in size thought to be host cell
ribosomes, although presence of these cellular organelles have not been confirmed. The dense
particles gave rise to the use of the Latin “arena” which means “sandy” for the family name. The
genome of arenaviruses consists of two segments (Large, L and Small, S) of single-stranded
ambisense RNA. The L segment encodes the RDRP and Z protein genes. The S segment encodes
the GPC precursor and NP genes. Enveloped virions contain copies of each RNA segment, RDRP, NP,
and processed GPC molecules, in addition to Z matrix protein-derived scaffolding. The NP protein
protects the viral RNA from degradation, and it has crucial immunomodulatory functions in the viral
replication cycle. Presence of RDRP in virions permits de novo synthesis of RNA upon viral entry and
uncoating in the cytoplasm of infected cells. Without the RDRP the negative stranded genomic RNA
could not be transcribed to sense messenger RNA for de novo translation of arenaviral proteins, or
replicated for incorporation into new virions during viral biogenesis. The SSP is unique among signal
peptides in that it is incorporated in the processed glycoprotein complex and is crucial for arenaviral
virion infectivity.
GP1/2: Glycoprotein 1/2; L: L-polymerase; NP: Nucleoprotein; SSP: Stable signal peptide; Z: Zinc.
Drawing courtesy of Dr Robert F Garry.

administered and can be toxic [57,60–69] , par- cases, if necessary. Ribavirin is approved by the
ticularly to fetuses [53–54,57,70] . Ribavirin has not FDA for treatment of HCV infections and res-
been approved by the US FDA for the treatment piratory syncytial virus (RSV) infections in chil-
of LF. Controlled clinical efficacy studies with dren [71,72] . Therefore, because there is no safe or
Ribavirin treatment in LF patients have not been clinically proven way to treat LASV infection,
performed. The cost–benefit of conducting such preventing LASV transmission is the primary
studies and projected low financial returns of goal in endemic regions. The National Institutes
using approved Ribavirin as a LF therapy under- of Allergy and Infectious Diseases (NIAID) and
score the reticence by manufacturers to pursue the Centers for Disease Control (CDC) classify
FDA licensure of the drug. The drug is available LASV as a Category A pathogen and a BSL-4
in the USA for off-label use to treat imported LF agent. Fear of nefarious release of LASV as a

future science group www.futuremedicine.com 561

Review Hartnett, Boisen & Oottamasathien et al.

bioterrorism agent has resulted in significant Outreach groups in endemic regions attempt
interest in development of new and effective to combat these forms of transmission by edu-
LASV diagnostics, vaccines and therapeutics. cating communities on how to prevent rodents
from entering homes, how to safely store food
Prevention and water and the importance of cooking meat
LASV infection has a high prevalence in com- to the proper temperature [80] . Moreover, efforts
munities in west Africa according to serosurveys undertaken to create and implement rodent traps
based on LASV-specific IgG antibodies. Reports made from strictly local supplies may begin to
from the latter half of the twentieth century curb Lassa fever transmission chains. Education
identified that 7–20% of people in endemic campaigns also focus on mitigation of human-
areas have been exposed to the virus [1,73–74] . to-human transmission of LF. Although much
However, recent studies of apparently healthy remains to be learned about LF transmission –
blood donors from districts of Sierra Leone the role of fomites, sexual transmission and direct
previously thought to be non-endemic for LF contact with bodily fluids of infected individuals
showed even higher incidences of exposure [rf in the early stages of infection prior to develop-
garry et al., Unpublished observations] . These obser- ment of clinical manifestations – certain societal
vations may be indicative of the ease with which practices contribute to the spread of the disease
the virus is transmitted from reservoir-to-human between people. Notably, caring for infected
and human-to-human. individuals during acute manifestations of LF is
The natural reservoir for LASV is Mastomys likely a major source of human-to-human trans-
natalensis, which is referred to as the multi- mission, of which caretakers are mostly unaware.
mammate mouse or rat [75] . This ubiquitous Further, burial practices in endemic regions are
sub-Saharan rodent is found throughout LASV steeped in tradition and place great emphasis on
endemic regions and lives commonly near caring for the corpse such as washing and wrap-
human dwellings and crop fields [1,75–76] . A study ping of the deceased, which likely shed live virus
that examined the seroconversion of members of in the acute stages of LF (via bleeding, diarrhea,
two mining communities and two agricultural possibly other bodily fluids). These observations
communities showed that those of the mining are purely anecdotal and the rate of human-to-
communities had more exposure to LASV [1] . human transmission outside hospital settings has
The report suggested that dwellings of the min- not been established.
ing communities were constructed poorly, food Nosocomial infections have caused docu-
was not stored safely and homes in the communi- mented outbreaks in Sierra Leone and Nigeria [81–
ties were generally more ‘disheveled’ than those 85] . The reuse of syringes has been the source of
of the agricultural communities [1] . The inves- outbreaks in past settings [82,83,86] . An effort to
tigators surmised that rodent access to dwell- educate healthcare providers with the dangers of
ings was an important part of the transmission reusing needles – dangers to both themselves and
chain. However, the investigators were unable to to patients – has presumably slowed this route
demonstrate a statistical correlation between the of transmission in hospital settings. However,
percentage of humans in these communities with direct contact with blood or bodily secretions
antibodies against LASV and the level of LASV- continues to account for a large portion of
infected Mastomys. It is possible that human-to- nosocomial infections [82–84] . Proper barrier
human transmission contributed and continues practices while treating LF patients are key to
to contribute to LASV seroprevalence [1] . preventing transmission to healthcare workers.
Due to the lack of rodent-proof construc- Recommendations of disposable coveralls, filter
tion of most dwellings and inadequate food and masks and face shields provide adequate protec-
water storage, Mastomys are often found scav- tion from both ‘aerosolization’ of LASV droplets
enging inside of west African homes. Mastomys, and fluid splatters [82,86,87] . Moreover, patient iso-
which shed LASV in their excreta, can contam- lation and the use of an anteroom are important
inate surfaces and food and water supplies in for preventing viral transmission to health staff
homes [1,75,77] (Figure 2) . The excreta potentially and patients or visitors [86,87] . Another factor that
infect humans either via oral or respiratory routes can limit transmission in healthcare settings is
or through microabrasions in the skin [76,78] . corpse decontamination. As observed for other
Consumption of Mastomys as a food source has VHFs, such as Ebola, ritual burials that involve
also been implicated in LASV infection [79] . the touching and ‘cleansing’ of the corpse can

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 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

contribute greatly to transmission. The handling afflicted pregnant women (usually stillbirths)
of a corpse without proper protection can lead and treatment of infected individuals in adja-
to transmission from infected body fluids, as cent hospital wards by unsuspecting staff are all
outlined for non-nosocomial human-to-human recorded modes of nosocomial transmission of
transmission above. While practically more dif- LF [6,84–85] .
ficult in resource-limited settings, nosocomial In a recent study, an innovative modeling
transmission of LASV is preventable. approach was employed to analyze data from past
Nosocomial transmission of VHFs is com- LF outbreaks in Sierra Leone to estimate the rela-
monplace in Africa. The 2014 and still ongo- tive contributions of zoonotic and anthroponotic
ing Ebola virus disease (EVD) outbreak in west transmission in LF [88] . This study challenged the
Africa highlighted the ease with which EBOV perception that human-to-human transmission
can be transmitted in hospital settings, particu- of LF is low in outbreak settings. According to
larly from infected patients to medical staff. At the model, approximately 20% of patients with
the height of the outbreak in mid-2014 health- LF presenting to Kenema Government Hospital
care centers were quickly overwhelmed by large (KGH) likely acquired the disease from another
numbers of suspected EVD cases being triaged infected person [88] . Furthermore, the patterns
and admitted to treatment wards. Overcrowding of disease spread during outbreaks pointed to
and understaffing quickly led to conditions that a small number of people being responsible for
greatly facilitated the spread of EVD in hospi- such infections, in what the authors termed
tals. Fortunately, outbreaks of LF are generally ‘super-spreaders’. Super-spreaders are defined as
less severe; the rate of patient influx to health acutely ill individuals who can infect a dispro-
care facilities and duration of illness are histori- portionately number of susceptible subjects [89] .
cally lower. Despite this contrast, LASV trans- In the Lo Iacono et al. study data were analyzed
mission to wholly unprotected or poorly pro- from two nosocomial LF outbreaks: Jos, Nigeria
tected healthcare workers, improper handling in 1970 [90] and Zorzor, Liberia in 1972 [91] and
of bodies postmortem, delivery of fetuses in a nearly 2-year study at the KGH from 2010 to

A Photo credits: E
Lina Moses, PhD
Tulane

B C

Figure 2. Lassa fever rodent vector. Mastomys natalensis (natal mastomys, or multimammate “rat”)
is the main reservoir of Lassa virus, and are peridomestic and highly prolific breeders. As the name
indicates female mastomys have multiple mammary ducts (A) permitting the sustenance of large
litters of offspring in each breeding cycle. Grain harvested from fields is processed, dried, and
often stored in open spaces inside dwellings (B). Food stored in case homes inside containers often
shows evidence of rodent entry (C). Dwellings are usually built with readily available local lumber
or handmade bricks and are not rodent proof. Rats can burrow through weaknesses in dwellings,
often through outside walls (D) or under doors that offer substandard barrier against entry by small
animals (E). Lassa fever is acquired through contact with excreta or preparation of “rat” for food.

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Review Hartnett, Boisen & Oottamasathien et al.

2012 [6] . The datasets were disparate in terms of specialized suite for manipulation of samples
subject numbers and in the qualitative methods from suspected cases of LF. The building is
used to classify LF cases. Yet, Lo Iacono et al. equipped with redundant power sources, includ-
established a mathematical appropriateness in ing municipal power (which is extremely spo-
comparing data from the three independent sites radic), a solar power system and dual 100 KVa
nearly 40 years apart. The resulting fit can be diesel generators.
simplified as a ‘20/80 rule’, whereby 20% of ini- KGH currently maintains a year-round 25-bed
tial cases cause 80% of the subsequent human- ward for the care of patients with LF and evaluates
to-human transmission [89,92] . These findings approximately 600 suspected cases yearly (Figure 4).
suggest that addressing human-to-human trans- The ward is staffed with a full-time team of doc-
mission chains may be as important as educating tors, nurses and cleaners. The staff members, the
communities on how to prevent rodent-borne majority of whom have over a decade of experience
infections. with LASV infections, have extensive training and
Since its identification in 1969, imported experience in treating and caring for LF patients.
cases of LF have been identified and recorded The KGH team is nearing completion on a new
in infected individuals traveling from developed 48-bed Lassa Ward that will replace this historic,
nations to endemic regions. In some instances but timeworn facility (Figure 4) .
LF is suspected in travelers prior to returning to KGH has assembled two expert teams that
their countries of origin. In others, LF has been dispatch to communities on demand if LF cases
identified only after extensive medical investiga- are suspected, and for education and research
tions. Mortality rates of those diagnosed with LF purposes – the outreach and ecology teams.
appear to be higher in non-Africans, which is of The outreach team at KGH is a highly expe-
concern because LASV is the most commonly rienced group of community outreach workers
exported hemorrhagic fever. Cases of imported (Figure 5) . The team conducts LF case investi-
LF from west Africa have been documented in gations countrywide, which includes contact
the USA, Canada, Germany, UK, Netherlands, tracing and updating local medical and com-
Belgium, Switzerland, Japan and Israel, dated to munity leaders. The team also is heavily involved
1973 [48,93–117] . in community education for the prevention and
early detection of LF including implementation
Capacity building of rodent control interventions and sanitation
Controlling Lassa fever requires development marketing. They are well versed in survey meth-
of infrastructure to diagnose, and treat the ill- ods including sampling strategies, questionnaire
ness in west African countries. There are only administration and collection of blood and other
two institutions with continuously operated specimens in community settings. The KGH
facilities dedicated to treatment of viral hemor- ecology team has extensive knowledge on trap-
rhagic fevers: KGH in Sierra Leone and Irrua ping of small mammals for zoonotic disease
Specialist Teaching (ISTH) Hospital in Nigeria. research. They have expertise in field survey
Experiences at these two facilities can provide methods, live-capture trapping and sample pres-
guidance to other areas with endemic VHFs. ervation and storage. They also are experienced
rodent control and hygiene experts and provide
●●KGH Lassa Fever Ward & Lassa Fever a valuable resource for the development of LF
Laboratory, Sierra Leone p­revention programs.
KGH is a 350-bed facility situated in the heart Both teams are heavily integrated into Sierra
of the region with the world’s highest incidence Leone’s highly-structured health surveillance
of Lassa fever. Because of the importance of LF system, working in close partnership with dis-
as a public health threat, KGH has cultivated trict medical and surveillance officers. The
an advanced clinical and laboratory research outreach and ecology team’s knowledge of the
capacity. The KGH Lassa Laboratory is located communities in eastern Sierra Leone is extensive,
on the grounds of the hospital, but in a stand- and team members speak the majority of dia-
alone building constructed in 2005 (Figure 3) . lects indigenous to Sierra Leone including Krio,
The laboratory is comprised of an approximately Mende, Temne and Kono. During the 2014
5500 square-feet building divided into a general EVD outbreak, the KGH LF ward was con-
clinical laboratory for routine diagnostics that verted to an Ebola treatment center. Likewise,
services the entire hospital and a 700 square-feet the focus of the nearby LF laboratory shifted to

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 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

the diagnosis of suspected EVD patient blood

samples, with negligible follow-up testing for LF A
or other diseases (e.g., malaria, diarrheal agents,
etc.). Both the entire KGH facility and the LFL
remained open, functional, and led the diagno-
sis, treatment and containment efforts against
EVD in eastern Sierra Leone throughout 2014.
The emergence of EVD in the LASV endemic
regions of far west Africa will impact the diagno-
B
sis of EBOV and LASV infections in the future.
It is reasonable to expect that future outbreaks
or, at a minimum, that sporadic cases of EVD
will emerge with some periodicity. Therefore,
going forward every suspected VHF case should Figure 3. The Kenema Government Hospital
include testing for EBOV and LASV so that Lassa Laboratory. (A) The single story building
appropriate containment responses are imple- houses the Lassa laboratory and other clinical
mented and effective in preventing an outbreak. laboratories and offices. The solar panels provide
To this end, EBOV-specific RDTs, ELISA and essential power for refrigerators, freezers,
quantitative polymerase chain reaction(qPCR) and other critical instruments. (B) The Lassa
platforms have been developed by the VHFC laboratory is outfitted with modern ELISA plate
and implemented at the KGH LF laboratory that washers and readers, classical polymerase chain
will accurately, rapidly and differentially diag- reaction and quantitative polymerase chain
nose the two VHFs. Additionally, recent stud- reaction instruments, tabletop centrifuges,
ies at the KGH that tested serum samples from +4C, -20C, -80C refrigeration units, laminar flow
suspected LF patients that were diagnosed with hoods, incubators, dessicator, water purification
a non-LF febrile illness revealed a multitude of station, flow cytometry, digital imaging station,
other possible infectious agents that potentially Enzyme-Linked ImmunoSpot reader, mass
contribute to observed morbidity and mortal- spectroscopy, and numerous internet enabled
ity in the local human population [118] . These state-of-the-art computer stations.
studies paved the way toward the development Photos courtesy of Drs Robert F Garry (A) and
and eventual inclusion of additional testing for Erica Ollmann Saphire (B).
high-impact infectious diseases in the region.
and Control is the first of its kind in Nigeria
●●Irrua Specialist Teaching Hospital, Nigeria (Figure 6) .
As such, with its reputation as a Lassa
We have established a study site at the Specialist Fever Center for Excellence, hundreds of Lassa
Teaching Hospital in Irrua, a rural area in Nigeria and Fevers of Unknown Origin (FUO) patients
where LF is endemic with yearly outbreaks. ISTH are admitted to ISTH annually for diagnostics
has 140 physicians and about 16,000 annual and treatment. At ISTH, all laboratories imple-
patients. Based on a 2003–2004 study including ment important safety precautions, inactivation
31 febrile patients, we expect around 800–1600 protocols and waste management procedures.
patients positive for LF at ISTH annually and Lab workrooms are separated from the rest of
an overall exposure of the population at about their buildings by changing rooms and double
30% [119] . The Lassa Laboratory and Ward sets of sliding doors. The ISTH employs pri-
(Figure 6) employs two Senior Research Scientists marily LASV RT-PCR in the diagnosis of acute
(PhDs), four Medical Doctors with specialization LF [120] . With the aid of sequencing efforts, a
in Public Health, eight laboratory technicians, large collection of Nigerian LASV strains has
12 community health workers and 12 research been catalogued [16,18,121–125] and has potenti-
assistants. The laboratory is capable of LF sam- ated the development of new, clade-specific
ple preparation, PCR, gel electrophoresis and rapid diagnostics, such as RDT and confirma-
ELISA. A 24-bed ward specially designed for LF tory ELISA platforms. These platforms are under
patients was recently made operational at ISTH current development by the VHFC, toward clini-
(Figure 6) . Construction has been completed on cal validation, regulatory approval and eventual
a new research, conference and administration implementation of rapid and sensitive pan-LASV
center. The Institute for Lassa Fever Research diagnostics across the LF endemic region.

future science group www.futuremedicine.com 565

Review Hartnett, Boisen & Oottamasathien et al.

Diagnosis by reverse transcription PCR (RT-PCR) using

Early detection of a disease is key to treating oligonucleotide primers to highly conserved
it and preventing its spread. The most sensitive regions of the GPC gene [2,120,127] . Laboratories
diagnostic procedure for a virus infection is viral in endemic areas are often not equipped to per-
isolation in cell culture. In LF endemic regions in form PCR. As a result, doctors without this
west Africa, high containment (BSL-4) laborato- technology are left to diagnose patients based
ries with cell culture capabilities are non-exist- on clinical symptoms alone [7] . Often, though,
ent, and the capacity to safely handle high con- patients present with symptoms that are similar
tainment pathogens under lower biosafety levels to other common infections in LASV endemic
remains exceedingly rare [50,74,126] . The ‘gold areas such as malaria, typhoid, or influenza.
standard’ diagnostic procedure for LASV infec- Only during advanced stages of LASV infection
tion is viral RNA isolation from blood followed do patients experience the telltale symptoms of

A B

Figure 4. The Lassa Ward. The current Lassa Ward is a 25-bed facility with a full-time clinical
and nursing staff dedicated to the treatment of Lassa fever patients (A & B). This facility will be
replaced with a much-needed 48-bed facility (funded by Naval Facilities Engineering Command)
with construction by Fajaha International Construction expected to be completed by Q2 2015
(C & D). The new ward will contain offices, a pharmacy, medical records room as well as high and
low containment wards. The rooms were specifically designed to deal with infection control issues
associated with VHFs. (C) Frontal view. The white structure is the final resting place of the Sierra
Leonean national hero Dr S Humarr Khan, who selflessly risked his life to treat countless patients in
the first half of the 2014 Ebola virus disease outbreak. (D) Inner compound view showing the two-
sided segregated “high” (infectious) and “low” (non-infectious) containment sectors. Design of the
facility was developed with technical assistance from Jason Moses (Jason Moses Projects, NYC) and
the VHFC.
Photos courtesy of Jessica N Hartnett.

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 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

A B

C D

Figure 5. Outreach and surveillance activities of the LF program. (A) Community meetings with
the KGH LF OutreachTeam are done in evenings when subsistence farmers return from their fields.
In addition to case investigations, the team also traps and distributes rodent control products to
case households to prevent additional infections. (B) Mr Lansana Kanneh, outreach team supervisor,
performing informed consent and blood draws. (C) James Koninga (right) sets a Sherman live-
capture trap in a cornfield targeting Mastomys natalensis. Willie Robert (left) records position on
transect line and documents GPS location for spatial studies. (D) James Koninga (left) Kandeh Kargbo
(middle) and Lina Moses (right) performing a necropsy on a juvenile Mastomys natalensis. In addition
to biological specimens for virological testing, the team collects morphometric data for ecology-
focused research.
Photos courtesy of Dr Lina M Moses.

LF – mucosal bleeding, conjunctivitis, enceph- because of a lack of a consistent electrical power

alitis and seizures. These symptoms, unfortu- supply, easy contamination of samples and per-
nately, are indicative of a poor prognosis when sonnel technical expertise shortcomings. More
treatment with ribavirin is usually no longer than these environment- and resource-depend-
beneficial. ent limitations, a failure to amplify divergent
In the early 1980s, immunofluorescent anti- strains of LASV with a single primer set has
body assays (IFA) for screening patient samples been recorded, even from a geographically close
for the presence of viral antigens were imple- sample group [130,131] . Nevertheless, several
mented [126,128–129] . IFA made it possible for laboratories in endemic areas successfully make
laboratory diagnosis of LF, which provided use of RT-PCR as a diagnostic, including the
important advantages over clinical diagno- Irrua Specialist Teaching Hospital in Nigeria
sis alone. However, similar to viral isolation, and Kenema Government Hospital Lassa Fever
this method was not readily implementable in Laboratory in Sierra Leone [55,121] . Because LASV
field settings and fell out of favor when PCR and a host of other select agents remain a threat
and ELISA were developed for the detection of to first world countries by accidental exportation
LASV [2,120] . Utilizing RT-PCR for the diagnosis and nefarious weaponization, advanced diag-
of LF in endemic areas proved to be challenging nostic technologies such as microarray assays

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Review Hartnett, Boisen & Oottamasathien et al.

are also under development that could fill a with subsequent need to redesign oligonucleo-
niche market capable of s­upporting the use of tide primer sets [2,127,133] . The first ELISA for the
advanced instrumentation [132] . detection of LASV antigen and LASV-specific
The ELISA platform, while still requiring antibody was developed in the mid-1980s [7,134] ,
laboratory equipment, is much less prone to and in the ensuing two decades ELISA were used
contamination, can better accommodate incon- in some endemic areas. The definition of a posi-
sistent power supply, and results in higher sen- tive LASV diagnosis was broad, though – a four-
sitivity and less cross-reactivity and subjectivity fold rise in IgG antibody titer, or LASV-specific
than IFA [50,126] . Moreover, ELISA may be able IgM and/or IgG accompanying LF clinical symp-
to detect a broader range of viral strains because toms [50] – given that the dynamics of the host
the assay is based on polyclonal antibody-antigen humoral immune response remained relatively
interaction rather than the highly specific interac- obscure. Since then, ELISA diagnostics have been
tions of PCR primers with the viral genome [131] . optimized for sensitivity and strain-specificity,
The broader detection range is sustainable when and have provided more insight into the host
employing polyclonal antibodies for both capture immune response. Recent studies employing the
and detection of antigens, even if strain variants use of modern, recombinant protein based diag-
emerge as a result of point mutations or more nostics have helped redefine the emergence and
significant antigenic shifts. PCR is prone to loss diagnostic role of immunoglobulins following
of sensitivity if mutations develop within previ- infection with LASV (Figure 7) [6] .
ously conserved target segments of viral RNA, In Sierra Leone, the KGH LFL diagno-
particularly those affecting the three regions of ses LF cases for the county. The laboratory
oligonucleotides critical for PCR polymerase currently employs three different diagnostic
binding and initiation of primer-template exten- ELISA for the detection and monitoring of
sion. This phenomenon has been previously iden- LASV [5–6,50,55,126,137] . To determine if an indi-
tified in PCR detection of clinical LASV variants, vidual is experiencing an acute infection, LASV

A B

C D

Figure 6. The Irrua Specialist Teaching Hospital Lassa Laboratory. (A) View of the Institute for
Lassa Fever Research and Control from entry road. (B) Lassa Laboratory. (C) Lassa Ward (24 beds).
(D) Research, administration and conference center under construction
Photos courtesy of Dr Robert F Garry (2013).

568 Future Virol. (2015) 10(5) future science group

 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

RT-PCR
ReLASV Ag RDT
ReLASV Ag ELISA
Acute
lassa Convalescent
Prodrome fever lassa fever

IgG
Ribavirin (?)
effective (?)

IgM
Serum
(?)
LASV
Ag or
RNA

sGP1 (?) Reservoir Ag (?)

0 1 2 3 4 5 6 7 8 9 10 40+
Time (weeks) years

Figure 7. Natural history of Lassa fever. Lassa fever is preceded by a 1–3 week incubation period of
the Lassa virus (LASV) infection. As Lassa fever (LF) progresses viral titers and antigenemia increase
along with development of more specific LF signs. Development of humoral and cellular immune
responses neutralizes viremia leading to LF convalescence. Anti-LASV nucleoprotein has been found
to be a dominant titer in the immune response which can also include persistent anti-LASV IgM
titers. Some convalescent patients have sustained IgM titers months or years into convalescence
(dashed IgM line), suggesting that the classic IgM to IgG seroconversion may be permanently
disabled by LASV infection. Some patients seroconvert from classical IgM (dashed, decreasing IgM
line) to IgG responses and remain only IgG positive into convalescence. Dynamics of long lasting
humoral responses in LF have not been extensively investigated and are marked with (?) in the
graph to outline this gap in knowledge. The biological role of soluble LASV glycoprotein 1 subunit
of (sGP1) has not been well elucidated, but sGP1 could be a target for early detection of infection,
before NP-containing virions emerge from infected cells during viral biogenesis. Generation of sGP1
has been demonstrated with recombinant GPC constructs in vitro [33,135], and has been detected in
some patients [136]. The duration and role of sGP1 in vivo is poorly understood, but it may wane as
infection progresses and in convalescence if it functions as an early decoy to innate immunity, as
suggested for the sGP of Ebola virus (sGP1[?], fading box). Some patients shed viral antigens weeks to
months into convalescence. It is not known if LASV antigen in convalescent patients is indicative of
presence of low levels of virus in reservoirs, shed antigen from dead infected cells, or if it is infectious
(Reservoir Ag[?], fading box, and broken black antigen line). LASV antigen-specific IgG can persist for
years in convalescent patients [6], sometimes for decades [15]. The (?) symbols represent anecdotal
evidence for the observed phenomena, but little experimental evidence exists to fully support
these assertions. In suspected LF cases where the rise of nucleoprotein antigen early in infection or
its decline in treated acute patients has been possible to ascertain the ReLASV RDT and ELISA have
lagged PCR detection of LASV antigen by one to two days [RF Garry et al., Unpublished observations] [55].
LASV: Lassa virus; RT- PCR: Reverse transcription polymerase chain reaction.
Drawing courtesy of Drs. Robert F. Garry and Luis M. Branco.

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Review Hartnett, Boisen & Oottamasathien et al.

antigen-capture ELISA, which identifies the NP 30-40 μl of blood, serum, or plasma samples,
antigen, is performed for the detection of circu- obtained by pricking a fingertip with a safely
lating antigen in biologic samples. LASV IgM lancet. This method reduces risk of exposure to
and IgG antibody capture ELISA are performed the healthcare provider and reduces the amount
to determine if an individual’s immune system of blood collected from the potentially hemo-
is responding to the infection and if they have dynamically unstable patient. The LFI can be
had previous LASV infections. Recombinant interpreted visually for a dichotomous scoring
proteins coated on the ELISA plate are bound of positive or negative, can be scored on a scale
by LASV NP-, GPC- and Z-specific IgM or IgG from 0 to 5 indicating the relative viral load,
antibodies from patient samples [5,137] . Though or can be semiquantitated by an optical reader
these diagnostics require significantly less time which provides a numeric value (Figure 8) . The
and specialized equipment than PCR, IFA and time from addition of the biological sample to
virus isolation, they are still prohibitive in LASV reading of the result is between 10 and 15 min.
endemic areas that lack laboratory infrastructure. The minimally invasive nature of sample col-
A rapid, lateral flow immunoassay (LFI) that lection for analysis, the visual assessment, the
provides point-of-care (POC) diagnosis of LASV short time to result and the portability and sta-
infection from a single finger prick has been bility make the LFI an ideal diagnostic tool for
developed to detect LASV infections in resource- the endemic, resource-limited settings. Though
limited settings. Based on principles similar to proving to be less sensitive than RT-PCR, this
ELISA, the LFI captures LASV antigen from diagnostic is an adequate supplement to a clinical

A B Positive Normal
Invalid results
control control
Blood sample
[LASV NP] Gold particle- Test Control
conjugated Abs zone zone
Control
Control
Contro
Con zone
trol
tro l zone
zone
Flow Test
Tes
estzone
es
Test t zzone
o e
one

Serum Conjugate Wicking

separator
p pad
p pad
p

Positive results
High Low Neg.

Test Control
zone zone
positive valid
Control
Con
Co
Con rozone
ntro
tro
tr
Control l zone
zone
one
ne
Test
Testzone
Test zone
zone

Figure 8. Lassa fever lateral flow immunoassays. (A) Schematic diagram of Lassa fever lateral flow immunodiagnostic (adapted from
a Google image). After patient sample, containing NP protein in enveloped LASV virions, is applied to the Sample Pad the dipstick
is inserted into a test tube containing Sample Buffer. Absorption of the sample and sample buffer initiates a linear flow through the
reagent pads releasing the gold nanoparticle conjugate to interact with the antigen present in the sample. Nanoparticle immune
complexes are captured by the antigen specific Test Line antibody and the resulting signal strength is proportional to the amount of
LASV NP antigen in the sample. (B) Lassa fever lateral flow immunodiagnostic interpretation from package insert. The rapid diagnostic
test generates a visible Test Zone line if antigen is present, and no line if it is absent. The Control Zone line validates the test result.
A rapid diagnostic test is visually scored on a scale of 0 to 5, with the signal strength proportional to the relative amount of antigen
present in the sample.
LASV: Lassa virus; NP: Nucleoprotein.

570 Future Virol. (2015) 10(5) future science group

 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

diagnosis in field settings where no laboratories Similarly, oral ribavirin did not reduce mortality
are present. This test, ReLASV® Antigen Rapid enough to warrant its use as the preferred therapy
Test (Corgenix Medical Corporation, CO, USA), in acute patients, though it has long been consid-
is a first-generation, CE marked rapid diagnos- ered a postexposure prophylactic (PEP) treatment
tic platform. The ReLASV LFI was developed option (current opinions suggest oral ribavirin is
with reagents that detect Sierra Leonean strains ineffective as a PEP) [4,70,94,110,140–142]. Intravenous
of LASV (Clade IV). While still capable of pro- ribavirin treatment, on the other hand, signifi-
viding a broader range of detection due to strain cantly reduced mortality compared with patients
variation than PCR, second-generation immuno- who received no therapy for LASV infection,
assays that are inclusive of all circulating strains and reduced mortality nearly fivefold when
in west Africa have been development and are treated within the first 6 days of fever compared
commercially available (Pan LASV, ReLASV with those who received treatment after 7 days
Antigen ELISA and ReLASV IgG/IgM ELISA, since fever onset. Moreover, significant toxicity
Corgenix Medical Corporation). Not only will associated with administration of intravenous
Pan-LASV diagnostics be superior options in and oral ribavirin was not reported in the 1986
endemic zones to permit detection of divergent study [57] . These results were supported by stud-
strains transmitted from human-to-human or ies using NHP models, though were never again
via geographic expansion of the rodent vector, formally investigated in humans because of the
but they also augment the biodefense armamen- difficulty of conducting a clinical trial in endemic
tarium. Being a Category A agent, weaponization regions [60,70,94,110,140,143] . Therefore, intravenous
of LASV is a concern of homeland defense. These ribavirin is recommended for use in the early
diagnostics would be able to quickly detect the stages of acute LASV infection. It is also believed
nefarious release of nearly any strain of LASV, that fluid replacement and blood transfusions
allowing a swift response to a potential disaster. are beneficial, though they must be m­onitored
carefully [51] .
Treatment The pharmaceutical company SIGA
Ribavirin, a purine nucleoside-analogue drug, Technologies, Inc., has discovered and evaluated
has activity against a number of RNA viruses several compounds believed to possess antiviral
including hepatitis C virus and respiratory syn- properties against arenaviruses [144] . Their stud-
cytial virus. Its mechanism(s) of action have ies have demonstrated protection and tolerance
not been clearly determined, but is suggested to in animals that have received ST-193 following
include inhibition of viral RNA replication, inhi- a lethal challenge with LASV. One additional
bition of inosine monophosphate dehydrogenase compound has been identified, ST-161, with sub-
(IMPDH), endogenous guanosine 5 ‘cap binding nanomolar activity against LASV and submicro-
of mRNA, immunomodulation and mutagene- molar activity against other arenaviruses in vitro.
sis [57] . Ribavirin is the only therapeutic currently To date, SIGA Technologies has not reported the
used for the treatment of LASV infection. In a progression of these drugs to advanced preclinical
1986 study of the efficacy of various therapies studies in relevant animal models of LF.
including oral and intravenous ribavirin and Another potential alternative to the toxic
convalescent plasma transfusion from a LF sur- ribavirin for treatment of LF is the drug favi-
vivor, intravenous ribavirin was shown to be the piravir (T-705). This drug is a broadly active
most effective. In the study, convalescent plasma nucleotide analog, which is licensed in Japan
to treat LF was used alone in pregnant women and is currently undergoing Phase III clinical
or concurrently with ribavirin in nonpregnant studies in the USA for the treatment of influ-
patients [57] . Despite showing advantageous results enza infections. Favipiravir has been tested in a
in cynomolgus monkeys [60,138] , in these human hamster model of Pichinde (PICV) virus infec-
clinical trials convalescent plasma was shown to be tion (a New World arenavirus distantly related
ineffective [57] . Additionally, the plasma was pro- to LASV), with higher reported activity than
hibitively expensive to isolate and difficult to store ribavirin, reflected by a greater therapeutic
in endemic areas. Therefore, convalescent plasma index [145] . Studies on the efficacy of favipiravir
has since been disregarded as a viable treatment in animal models of LF have not been reported.
option for LF, although it is the current treat- Another promising approach to antiviral
ment of choice for Argentine hemorrhagic fever drug development is the use of monoclonal
caused by JUNV, a New World arenavirus [139] . antibody therapy. The reported ineffectiveness

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Review Hartnett, Boisen & Oottamasathien et al.

of convalescent human LF serum as a source animal models used for LASV challenge have
of protective antibodies might have precluded been NHP and guinea pigs. Both develop fatal
efforts to develop immunotherapeutics for the infections with LASV. As expected, the disease
post-exposure treatment of LASV infections. in NHP more closely resembles that of humans.
However, recent efforts to define and charac- In a limited study (three immunized NHP given
terize protective and pathogenic human B cell a single dose), γ-inactivated LASV failed to pro-
epitopes in LF by the Viral Hemorrhagic Fever tect rhesus macaques [156] . Another study found
Consortium (VHFC [223]) have led to the isola- that inactivated LASV protected another species
tion of the largest known panel of human mono- of NHP after a single injection [157] . In these
clonal antibodies (huMAbs) specific to LASV prior studies it was observed generally that pro-
glycoprotein and nucleoprotein antigens. To tection from LASV challenge did not correlate
date, greater than 125 huMAbs reactive with with the magnitude of the humoral immune
full length LASV GP1, GP2 and GPC have been response. Interestingly, antibodies against LASV
independently derived from Sierra Leonean LF structural proteins were generated in the study in
survivors. Selected LASV huMAbs with signifi- which γ-inactivated LASV failed to protect from
cant in vitro neutralizing activity have been tested lethal challenge [156] .
in vivo. Antibodies that protect a minimum of Given practical limitations of nonlive vaccines,
75% of guinea pigs are candidates for testing in other vaccine strategies to protect against LASV
NHP. Due to significantly larger amounts of each were explored. Live viral vaccines have tradition-
antibody needed for NHP studies, constructs of ally been thought to offer the most effective long-
interest have been stably transfected into serum- term protection against LASV, in part because
free medium-adapted mammalian NS0 (murine they deliver antigen endogenously and are effec-
myeloma background) cells for generation of stable tive at inducing antigen specific activation of
cell lines. The CHOLCelect system [146] gener- CD8 T cells. Subunit vaccines typically have
ates stable NS0 cell lines for large-scale produc- not provided as much durability, presumably
tion of recombinant molecules in approximately because exogenous antigens are typically taken
2.5 months, thus reducing timelines to in vivo up by antigen presenting cells via phagocytic or
testing. To date, eight huMAbs protected 100% endocytic processes and activate antigen-specific
of guinea pigs against lethal LASV challenge, and CD4 T cells. Fortunately, additional types of
six of the MAbs tested so far in NHP protected modern adjuvants (e.g., ADP-ribosylating pro-
75–100% of animals in lethal challenge studies, tein adjuvants and TLR agonists) and new deliv-
with each antibody administered independently ery strategies (e.g., mucosal and transcutaneous
and on the same day as infection with LASV [RF immunization) can be effective at activating both
Garry et al., Unpublished data] . Moreover, most CD4 and CD8 T cells to exogenous antigens via
huMAbs have conferred complete protection in subunit vaccines [164–167] . These adjuvants and
guinea pigs and NHP without visible signs of LF delivery systems have been shown to induce both
throughout the study, and with transient or unde- humoral and cellular responses against a number
tected viremia [RF Garry et al., Unpublished data] . of exogenous proteins, broadening the immune
These encouraging results are driving the devel- repertoire to include both neutralizing antibodies
opment of potent immunotherapeutic huMAb and cytotoxic T lymphocyte (CTL) responses.
cocktails for the treatment and prevention of While neutralizing antibodies have not histori-
LF. This approach parallels the more advanced cally been a dominant protective factor in pre-
preclinical and clinical development of ZMapp, venting LASV infections, it has been suggested
a cocktail of chimerized murine antibodies with that they are synergistic with CTL responses
protective properties against EBOV infections, against LASV and other arenaviruses [161,168,169] .
which is currently under evaluation as a leading Moreover, LASV neutralizing antibodies do not
candidate drug in the fight against the ongoing typically develop until late in convalescence and
EVD outbreak in west Africa [147–155] . it is unknown if pre-existing high-titer virus
neutralizing antibodies induced by vaccination
Vaccines would have a major impact on infectivity.
Development of an effective LASV vaccine is Recombinant vaccinia virus (rVV) express-
crucial both for bioterrorism preparedness and ing LASV GP protected both guinea pigs and
to improve public health in endemic areas. In macaques, while rVV vector expressing only
prior vaccine studies (Table 1) , the two major NP was protective in guinea pigs, but not

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 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

Table 1. Prior Lassa virus vaccine studies.

Vaccine platform Animal challenge Routes vaccine/# of Results Study (year) Ref.
model immunizations/
challenge
rVV/LASV NP Guinea pig ID/1X/IM Complete protection Clegg and Lloyd [158]
(1987)†
γ-inactivated LASV Rhesus macaque IM/1X 0% survival McCormick et al. (1992) [156]
γ-inactivated LASV Papio hamadryas IM/1X, 2X/IM Complete protection w/ Krasnianskiă et al. [157]
IM challenge (1993)
50% survival w/
respiratory challenge
rVV/LASV‡ w/ Rhesus and ID/1X/SC 90% survival w/ Fisher-Hoch et al. [159]
Cynomolgus GP1+2+NP (2000)
macaque
GP1and 2, or 88% survival w/ GPC
GP1,2, or NP 20% survival w/ NP
rVV/LASV NP 0% survival w/ GP1 or GP2
alone
Complete protection w/
GP1 + GP2 administered
at different sites
VRP§/LASV GPC or NP Guinea pig SC/3X/SC Complete protection with Pushko et al. (2001) [160]
either GP or NP
rVSV#/LASV GPC Cynomolgus IM/1X/IM Complete protection Geisbert et al. (2005) [161]
macaque
YFV17D¶/LASV GPC Guinea pig SC/1X/SC 80% survival Bredenbeek et al. [162]
(2006)
Mopeia-LASV live Guinea pig SC/1X/SC 30 days post- complete protection Carrion et al. (2007) [163]
attenuated immunization SC/1X/ w/ homologous or
SC 0 – 2 days post- heterologous LASV
immunization strain 60–100% survival
w/ homologous or
heterologous LASV strain
LASV VLP w/Z, GPC, Guinea pig SC/1X/IM 40% survival [Branco et al.
NP unpublished data]
VEEV TC-83††/LASV Mouse n/a Complete protection [Lukashevich et al.
GPC unpublished data]
†
Recombinant vaccinia virus.
‡
Recombinant vesicular stomatitis virus.
§
Similar results reported by [170].
¶
Yellow fever virus vaccine strain 17D.
#
Venezuelan equine encephalitis virus-like replicon particles.
††
Advanced alphavirus replicon (Venezuelan equine encephalitis virus-like particle vectors).
ID: Intradermal; IM: Intramuscular; n/a: Not available; SC: Subcutaneous.

NHP [158,159,170] . Further, rVV vectors express- GP), protected both mice and guinea pigs from
ing each glycoprotein subunit alone were tested Lassa fever challenge [172] . Remarkably, this vac-
in guinea pig challenge studies, but neither con- cine delivered on the same day as the LASV chal-
ferred protection. Conversely, co-administration lenge protected seven of nine guinea pigs. While
of rVV GP1 and GP2 vectors at different injection protection is paramount, an important limita-
sites conferred full protection. These results sug- tion of most live recombinant virus vaccines is
gest a strong interdependence of both subunits the potential for prior immunity to the vector
for protection [158,159,170,171] . A different vaccine either through natural exposure or prior immu-
platform, in which an attenuated reassortant virus nization [173,174] . Another critical issue is safety,
containing the L genome segment from Mopeia particularly in patient populations in Africa where
virus (a nonlethal arenavirus) and the S genome AIDS and other immunosuppressive illnesses are
segment from LASV (thusly expressing LASV common. Live vaccines based on Venezuelan

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Review Hartnett, Boisen & Oottamasathien et al.

equine encephalitis virus (VEEV) replicons, an seroprevalence of VSV is negligible, except in

alphavirus, expressing either individual LASV or enzootic regions of central America [179,180] . A
EBOV glycoproteins, or co-expression of both similar vaccine, based on attenuated recombinant
antigens (bivalent) conferred complete protec- VSV that expresses the EBOV or Marburg virus
tion in guinea pigs against lethal LASV and (MARV) glycoprotein similarly protected nonhu-
EBOV challenges. In these studies guinea pigs man primates against lethal challenge with both
were immunized with either dual-expression par- filoviruses [118,135–136,160–161,181–206] . The demon-
ticles or with a mixture of VEEV/LASV GPC and strated success of VSV-based vaccines in relevant
VEEV/EBOV GP particles [160] . Testing of this animal models of disease, high safety profile and
vaccine platform in nonhuman primates has not relative ease of manufacture at large scale has
been reported. In another study a recombinant recently resulted in the early phase clinical trial
yellow fever 17D (YFV 17D) vaccine was gener- evaluation of VSV-ZEBOV in humans for safety
ated that expressed the LASV GPC. Recombinant and its ability to induce an immune response in
YFV 17D/LASV-GPC virus was replication- healthy adults who are given two intramuscular
competent in cell culture, but replicated poorly doses. VSV-ZEBOV is a vaccine against the Zaire
in guinea pigs. Despite poor replication in vivo the strain of EBOV, the causative agent of the current
vaccine elicited specific antibodies against both west African EVD outbreak [207,208] .
LASV GPC and YFV 17D antigens and pro- Since subunit vaccines are easier to produce,
tected 80% of animals against LF after a single store and deliver than live vaccines, and may be
subcutaneous injection. The YFV 17D vaccine safer in areas where LASV is endemic, develop-
strain has an excellent safety record since being ment of nonlive platforms warrants serious consid-
first implemented in the 1950s, and is included eration for LASV and other biothreat agents. To
in vaccination regimens for persons traveling this end, LASV virus-like particles (VLPs) were
to endemic regions of west Africa. Despite its generated in human embryonic kidney cells via
safety record, the endemicity of yellow fever and transient transfection with ZP matrix, NP and
other flaviviruses (dengue, Japanese encepha- GPC expression constructs. These VLPs gener-
litis, West Nile virus) in west Africa make the ated robust antibody titers in mice [101] . Studies
development of a YFV-based vaccine impractical. performed in guinea pigs with a prime and one
Seroprevalence against flaviviruses in the region is boost strategy, 2 weeks apart, each with VLPs for-
significant [118,162] , and immunity against a YFV- mulated in RIBI adjuvant, followed by challenge
based vector may render the vaccine largely inef- with a lethal dose of LASV, protected 40% (2/5)
fective in conferring population-wide protection. of animals [RF Garry et al., Unpublished data] . This
A live, replication competent vaccine based nonoptimized vaccination strategy warrants fur-
on attenuated recombinant vesicular stomatitis ther investigation, including modified administra-
virus (VSV) expressing the LASV glycoprotein tion schedules and adjuvants. A nonlive vaccine
was developed by Geisbert et al. [161] , that resulted that protects greater than 80% of animals would
in the development of a commercially viable LF be a major step forward in the development of a
vaccine platform. A single intramuscular injec- robust nonlive LASV vaccine [209] .
tion with the VSVΔG/LVGPC vaccine elicited Recently, the use of advanced alphavirus
a fully protective immune response in a nonhu- RNA replicon technology to derive VLP vec-
man primate model of LF against a lethal LASV tors (VLPV) expressing LASV GPC as a vac-
challenge. The vaccine elicited strong humoral cine candidate against LF was reported ([210]
and cellular immune responses, without fever or with permission). In these studies a modified
other vaccination-associated symptoms, and vac- LASV GPC with cross-presenting capabili-
cine shedding was not detected in the primates. ties was cloned within the alphavirus replicon
Vaccinated animals did not show evidence of (VEEV TC-83) to produce VLPVs decorated
clinical disease despite transient LASV viremia, with the arenaviral envelope protein. Bivalent
unlike unvaccinated animals that displayed typi- VLPV expressing modified and wild-type LASV
cal signs of LF and succumbed to LASV chal- GPCs from distantly related LASV lineages in
lenge. The VSV-based vaccine platform offers the same virus completely protected mice against
many advantages over other live virus vectors: it fatal challenge with LASV. Further, these biva-
has been a highly effective vaccine platform in lent VLPV induced cross-reactive cell-mediated
several animal models of disease, can be adminis- immune responses against all LASV lineages
tered by the mucosal route [175–178], and worldwide currently circulating in west Africa.

574 Future Virol. (2015) 10(5) future science group

 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

Another potential LF vaccine approach is based bleeding. Further, electroporation-based vaccine

on peptide epitopes that directly activate CD8+ delivery is highly unlikely to be a viable method
T cells, the cytotoxic T cell (CTL) subset largely of vaccinating populations in endemic areas [214] .
responsible for controlling primary LASV infec- The ability of a vaccine to confer cross-protec-
tion. This approach has been met with mixed tion against heterologous strains within the same
results in small animal models of arenaviral infec- clade (LASV clades I–IV) or different groups
tions [168,210–211] . In addition to destroying infected (LASV, Old World and JUNV, New World), or
cells CTL release cytotoxic cytokines, such as within a family (EBOV and MARV), is a highly
IFN-γ and TNF-α that induce some degree of desirable characteristic. The arenaviridae are a
immunopathology. Vaccination of naive individu- highly diverse family of viruses, and achieving
als with peptide-based vaccines would most likely cross-protection between divergent Old World
be safe since CTL responses are HLA-restricted, strains alone (between lineage II, III, IV for exam-
and LASV-specific CD8 + T cells would not be ple, the currently circulating LASV clades in west
present in individuals previously not exposed to Africa) would greatly enhance the protective cov-
the virus. However, some of the systemic symp- erage, vaccination strategies and even commercial
toms of LASV infections are caused by disregu- appeal of a vaccine platform. To this end some
lated immune responses rather than the direct studies have investigated cross-protective potential
effects of the virus itself [212] . Therefore, significant of arenaviral vaccines. One approach included the
pathology could be observed in individuals vac- bicistronic expression of heterologous glycoprotein
cinated with peptides who were infected recently precursor genes (GPC) from JUNV and Machupo
with LASV asymptomatically, or in those with a (MACV) viruses in the same VEEV TC-83-based
previous infection that developed a robust CTL vector. To date, the efficacy of this vaccine plat-
response. Thus, this approach could have promis- form in conferring cross-protection between these
ing applications in vaccination campaigns in indi- two New World Clade B pathogenic arenaviruses
viduals from non-endemic regions. Though, in in animal models has not been reported. A 2007
the LF endemic regions of west Africa the human study by Carrion et al. employing the ML29
HLA gene variability is high, thereby requiring a reassortant MOPV/LASV vaccine candidate
large number of peptides to afford broad induction demonstrated complete cross-protection when
of protective CTL responses. Development and single-dose, 30-day immunized Strain 13 guinea
validation of a large number of peptides in a vac- pigs were challenged with a heterologous Nigerian
cine platform raises concerns about the feasibility strain of LASV (LASV-803213/NIG, lineage II,
of this approach. Ultimately, there is currently no GenBank accession number AF181854) [18] . This
licensed peptide-based vaccine so the concept is platform also protected 60–100% of guinea pigs
largely unproven in a human setting, which fur- when vaccine and challenge were administered on
ther complicates the feasibility of this type of vac- the same day or 2 days apart. These results are
cine platform in an already underfunded disease. highly significant since the GPC gene in the ML29
DNA-based immunizations have also been reassortant is derived from LASV Josiah (clade IV,
investigated as alternative LF vaccine platforms. Sierra Leone), and LASV-803213/NIG is a line-
This approach has not been extensively studied, age II strain, which is more divergent from clade
although complete protection of guinea pigs and IV than clade III Nigerian strains. The studies
macaques immunized with DNA constructs outlined above that employed advanced alphavi-
expressing the full length LASV GPC have been rus VLPV expressing bivalent, modified and wild-
reported [213] . DNA immunization studies tar- type LASV GPCs from distantly related LASV
geting common arenaviral CD8 + CTL epitopes lineages induced cross-reactive, cell-mediated
have produced mixed protection results in vivo. immune responses against all LASV lineages cur-
Epitope-specific DNA-based immunizations rently circulating in west Africa (clades I–IV) [215] .
share the potentially untoward consequences of Development of multivalent arenaviral vac-
peptide epitope-based strategies in populations cines have also been investigated, namely in the
with high prevalence of previous LASV expo- form of novel DNA vaccines that could generate
sure, as discussed above. Recent developments in protective immune responses in subjects against
electroporation technologies have significantly multiple viruses. In a recently published patent
enhanced the immunogenicity of DNA-based application, this approach was proposed for the
vaccine candidates, albeit with enhanced adverse simultaneous protection against LASV, LCMV,
side effects at the site of injection, such as pain and MACV, JUNV, Guanarito virus (GTOV),

future science group www.futuremedicine.com 575

Review Hartnett, Boisen & Oottamasathien et al.

White Water Arroyo virus (WWAV) and PICV. meet with a high regulatory and financial bur-
The proposed approach would entail the expres- den, in addition to poor medical infrastructure
sion of peptides derived from short sequences of and a population difficult to recruit due socio-
the GPC gene of each virus known to elicit strong economic constraints, including mistrust and
CTL responses [216] . Although this approach skepticism of western medical practices.
would meet with the same potential untoward Opportunity and promise toward development
effects of hyper CTL responses in vaccinated of an effective LASV vaccine may currently rest
individuals with preimmunity, it could also with the success of Candid#1, a live-attenuated
result in the discovery of potentially protective, vaccine against Argentine hemorrhagic fever
new arenaviral epitopes. Botten et al. proposed a (AHF), caused by JUNV. This vaccine was
similar approach by using a panel of HLA class jointly developed by Argentine and US scien-
I-restricted peptides from the same region of the tists, with support from the national Ministry
GPC gene of LASV, LCMV, JUNV, MACV, of Health and the US Army Medical Research
GTOV and WWAV, which were recognized by Institute of Infectious Diseases (USAMRIID),
CD8 + T cells in a cross-reactive manner follow- under supervision of the United Nations
ing LCMV infection or peptide immunization Development Program and Pan American Health
of HLA-restricted transgenic mice [211] . These Organization (PAHO). In 2006, after successful
studies demonstrated the feasibility of develop- clinical trials, the Candid#1 vaccine was regis-
ing a cell-mediated vaccine that could protect tered by the National Regulatory Authorities and
against multiple, antigenically distinct arenavi- is currently manufactured in Argentina. With a
rus species. Cross-protection against highly path- clinically proven efficacy of 95% against AHF,
ogenic New World arenaviruses, such as JUNV, Candid#1 was incorporated in the Argentinian
by vaccinating animals with non-pathogenic National Immunization Plan and has tremen-
counterparts, such as Tacaribe virus (TCRV), dously impacted the magnitude of epidemic
have also been reported [163] . Validation of cross- outbreaks [209,217–218] . In the USA Candid#1 has
protective vaccine candidates against related and an Investigational New Drug (IND) status and
unrelated arenaviruses will need to be performed is currently used to vaccinate military and labo-
in NHP models of arenaviral disease in order to ratory personnel with potential exposure to live
gain acceptance as viable platforms. Junin virus. Based on the availability and proven
To date, none of the LASV vaccine platforms clinical record of Candid#1, the US CDC and
that have demonstrated efficacy in NHP models NIH have permitted research with JUNV in
of the disease have progressed through human BSL-3 containment laboratories, provided that
safety clinical trials. Though impetus toward personnel are immunized with this vaccine [219] .
development of safe and protective vaccines Despite its use and proven clinical record, it is
against pathogenic arenaviruses for public health unlikely that Candid#1 will progress toward FDA
and biodefense exists, there is an absence of posi- licensure. Such decision would not be unique in
tive market-driving forces. Profitability in this vaccinology. Absence of regulatory approval for
market is low, therefore funding for clinical trials Candid#1 in the long run would parallel the
and product commercialization in the neglected VEEV vaccine platform VEEV TC-83.
tropical diseases space is limited. Moreover, a VEEV TC-83 was developed in 1961 and to
particularly involved and largely untested FDA date more than 8000 vaccinations have been
Animal Rule regulation of biodefense biologics administered to protect military and laboratory
has deterred investigators. Since its implementa- personnel [220–222] . In the five decades since its
tion in 2002, there are no FDA-approved vac- inception, there has not been an impetus to seek
cines using the Animal Rule. This rule is well- FDA approval for VEEV TC-83, despite its
meaning and provides sponsors with available extensive clinical history, effectiveness and lack
options to derive clinically relevant efficacy on of significant reported adverse events. Unlike
vaccine platforms and therapeutic compounds VEEV and JUNV, which cause sporadic out-
when performing such studies in humans would breaks in human populations, LASV is endemic
be unethical. Lassa fever is endemic through- in west Africa, with an annual incidence that is
out a large geographic area of west Africa and still not well defined. Recent projections esti-
vaccine safety and efficacy studies in human mate that 59 million people are at risk of pri-
beings are therefore possible. Sponsors of human mary LASV infection, with an incidence of as
vaccine studies in the endemic LF zone would many as 3 million cases annually, and as many as

576 Future Virol. (2015) 10(5) future science group

 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

67,000 deaths [51] . The actual number of people estimate if a previously undetected mutation
at risk of contracting LF may be as high as 200 will give rise to an extremely pathogenic and
million, since the reported endemic zone spans highly infectious strain of LF that could rival
from Guinea to Nigeria with defined LASV the current EVD outbreak. Development and
seroprevalence detected in additional adjacent deployment of rapid diagnostics and research
nations. This estimate was published in 2003, at into the efficacy of current treatment options
the conclusion of the Blood Diamonds Civil War should continue and will save lives in west
in Sierra Leone and Liberia, at a time when the Africa as well as create a line of defense against
diagnosis, monitoring and treatment of LF had LASV bioterrorism.
largely been interrupted for over a decade [51] . Biodefense-funding initiatives over the past
The modern diagnostics and surveillance devel- decade have spurred renewed research interest
oped and implemented at clinical sites such as in pathogenic arenaviruses. Fortuitously, the
the KGH and ISTH present new opportunities applied research initiatives have resulted in sig-
to redefine these estimates. It is clear, despite nificant public health benefits. Just a few of the
fluctuations in projected annual incidence of positive outcomes potentiated by the enhanced
LF in endemic countries, that a safe and effec- interest in LF research over the past decade are:
tive LASV vaccine would have a major positive readily available, validated and rapid diagnos-
health, social, economic and political impact tics for LF, countrywide education campaigns
in the endemic LF subregion. Development on prevention and identification of signs of the
of a LASV vaccine for implementation in the disease, international cooperation between sci-
national vaccination programs of endemic coun- entists and medical staff, significant support
tries will require unprecedented collaboration by Ministries of Health in endemic zone coun-
and financing by international scientists, foun- tries, laboratory and hospital capacity build-
dations and monetary funds. Unfortunately, and ing at centralized sites, training and education
despite its well-established impact on human initiatives, and positive economic, social and
populations in the endemic zone, LF remains political impacts. The next decade could pro-
a neglected tropical disease, with underfunded duce yet another leap forward in diagnosis,
research and clinical efforts toward successful management, control and treatment of LF:
prevention and eradication. improved rapid diagnostics will be validated
and implemented; a safe and well-tolerated
Future perspective vaccine platform capable of conferring broad
LASV has been the known cause of a fatal viral protection may be developed, although regula-
hemorrhagic fever for decades, and has been cir- tory approval will remain a challenge; novel
culating in west Africa for centuries. However, therapeutics will progress toward advanced test-
our knowledge of this disease is limited. To date, ing, and even into human clinical trials at sites
only one diagnostic tool has been developed that equipped to perform such studies in endemic
is appropriate for the environment where LF is regions; far reaching education campaigns will
endemic. While several chains of transmission reduce the time from infection to diagnosis as
have been identified and strategies to sever these afflicted individuals seek medical care sooner
chains exist, education and implementation are after the onset of symptoms. This enthusiasm
still somewhat limited in the endemic regions. And may be tempered by the possible emergence
though a therapeutic for LF exists, not enough is of new and more virulent and infectious viral
known about its effects on LASV or the patient. strains and the spread of endemicity across the
The recent and still ongoing EVD out- risk belt of LF across west Africa. The growing
break in the LF endemic zone of west Africa economic impact of the African continent on
makes a very strong case for this cautionary the world economy with the greatly enhanced
tale. Despite decades of well-funded research, expansion of trade and travel between west
vaccine and therapeutic development, monitor- Africa and the rest of the globe will also likely
ing, design and implementation of predictive potentiate an uptick in the number of imported
models of viral hemorrhagic fever outbreaks, LF cases. The true impact of LF in future
the current EVD was unanticipated. Much years is a matter of speculation, however tools
like EBOV, LASV is an RNA virus that can to detect, prevent and treat LF should remain
undergo a very high rate of mutation within a high priority in future global public health and
short period of time. It is nearly impossible to biodefense initiatives.

future science group www.futuremedicine.com 577

Review Hartnett, Boisen & Oottamasathien et al.

Executive summary
Lassa virus infection is difficult to prevent without proper education
●● It is suspected that many people become infected from the rodent reservoir, Mastomys, contaminating their food and
water supplies and from people eating the rodent.
●● Outreach teams in endemic areas educate communities on how to prevent transmission from rodents.
●● Nosocomial infections can cause outbreaks in hospitals where barrier nursing techniques and safe handling of corpses are
disregarded.
●● Human-to-human transmission is likely an underestimated chain of transmission.
Diagnostics are evolving from platforms that can only be used in laboratory settings to field-deployable assays
●● Virus isolation and polymerase chain reaction (PCR) are currently the ‘gold standards’ for Lassa virus (LASV) infection but
are difficult to implement in endemic areas because they require equipment that is not sustainable in these settings, are
time consuming and error prone.
●● In endemic regions ELISA and lateral flow immunodiagnostics (LFI) are becoming more popular because they take less time
and less equipment and can detect a broader range of strain variation.
●● ELISA requires less equipment, will not be severely affected by a short power outage, utilizes reagents that are more shelf-
stable and takes less time.
●● LFI can be utilized in field settings with no equipment, takes 10–15 min to obtain a valid result and requires only a drop of
blood from a finger prick.
●● Continued development of next-generation LFI platform with greater sensitivity and the ability to detect different strains
of LASV (Pan-LASV).
The only treatment shown to be clinically effective against LASV is ribavirin
●● Intravenous ribavirin has shown effectiveness against LASV infection if administered early enough in the disease course.
●● ST-193 (SIGA Technologies, Inc., OR, USA) is a promising therapeutic candidate as demonstrated in animal models.
●● Additional small molecule oasis: entry inhibitors (e.g., ST-161) have been evaluated in vitro.
●● Favipiravir (T-705) is a broadly active nucleotide analog licensed in Japan.
●● Currently undergoing Phase III clinical studies in the USA for the treatment of influenza infections.
●● LASV huMAbs are highly protective in guinea pig and nonhuman primate models of LF.
Vaccine platforms have been developed and investigated but have not reached human clinical trials
●● Killed whole virus and recombinant virus vaccines have proven to be somewhat effective but face significant regulatory
hurdles.
●● Subunit vaccines using modern adjuvants can activate innate adaptive immune responses.
●● Candid#1 is a vaccine against Junin virus, a New World Arenavirus.
●● It is used in military personnel and in Argentine vaccination regimens.
●● It is safe and effective but will likely not undergo US FDA regulatory approval.
●● Studies investigating cross-protection by Candid#1 against Old World Arenaviruses have not been reported, but it is
unlikely this vaccine would protect against LASV infections based on dissimilarities between the two viral classes.
●● The absence of human efficacy studies, the FDA Two-Animal Rule and a severe lack of funding impede further vaccine
development.

578 Future Virol. (2015) 10(5) future science group

 Current & emerging strategies for the diagnosis, prevention & treatment of Lassa fever Review

Executive summary (cont.)

Future perspectives
●● EBOV outbreak demonstrates the necessity for advancement of therapeutics, vaccines and diagnostics for viral
hemorrhagic fevers.
●● Fear of bioterrorism will urge funding agencies to support efforts towards preparedness.

In memoriam Financial & competing interests disclosure

Tragically, two co-authors, who contributed greatly to pub- This work was performed as partial fulfilment of PhD dis-
lic health and VHF research efforts in Sierra Leone, con- sertation requirements for JN Hartnett. ML Boisen, AB
tracted EVD and lost their battle with the disease before Jones, MM Millett, DS Nelson, IJ Muncy, and KR Pitts
this manuscript could be published: Mohamed Fullah and are employed by Corgenix Medical Corporation, Inc. Robert
Sheik Humarr Khan. A third author who assisted in the F Garry and Luis M Branco are co-founders of Zalgen Labs,
development of the ReLASV® diagnostics also lost her life LLC.
all too soon, due to unrelated circumstances: Darin No writing assistance was utilized in the production of
Oottamasathien. We wish to honor their memory. this manuscript.

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