Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s004300100061
O RI GI N AL IN V ES T IG A T IO N
Abstract It is not known whether the small 11-kDa Z (P=0.001). These data provide evidence for appearance
protein of Lassa virus is immunogenic during human of antibodies to Z protein and NP following Lassa vi-
Lassa virus infection. To obtain evidence for the exis- rus infection. A recombinant blot for detection of both
tence of an antibody response and to test the suitability antibody speci®cities seems to be speci®c but less sen-
of these antibodies for serosurveys, sera from Lassa sitive than IIF.
fever endemic regions (Guinea and Nigeria, n=75) were
tested for co-reactivity to Z protein and nucleoprotein Keywords Lassa virus á Antibody response á
(NP). Sera from a non-endemic region (Uganda, n=50) Immuno¯uorescence á Western blot
served as a speci®city control. Z protein and NP were
expressed in Escherichia coli, anity-puri®ed, and used
as antigen in Western blot. Indirect immuno¯uores- Introduction
cence (IIF) with Lassa virus-infected cells was per-
formed for comparison. Due to high unspeci®c Lassa virus belongs to the family Arenaviridae and is
reactivity of the African sera, Western blot testing was endemic in West African countries. Transmission of the
performed with a 1:1,000 serum dilution. Under these virus from its rodent reservoir to humans causes Lassa
conditions, none of the control sera but 12% of the sera fever which is associated with a wide spectrum of clinical
from endemic regions co-reacted with both Z protein manifestations including organ failure, encephalopathy
and NP. Reactivity to Z protein was signi®cantly and hemorrhage. Up to 20% of susceptible persons se-
associated with NP reactivity (P<10±6). NP and Z roconvert to Lassa antibodies per year in selected vil-
protein-speci®c antibodies were co-detected in 33% of lages in Sierra Leone, often without experiencing severe
the IIF-positive sera and in 5% of the IIF-negative sera illness [8]. Serosurveys reveal a prevalence of Lassa an-
tibodies of up to 50% in some areas of West Africa [7, 8,
10, 13]. Lassa virus expresses only four proteins, two of
which ± nucleoprotein (NP) and glycoprotein ± have
S. GuÈnther (&) á O. KuÈhle á D. Rehder á P. Emmerich
J. ter Meulen á H. Schmitz
been shown to induce antibodies following infection
Bernhard-Nocht-Institut fuÈr Tropenmedizin, [2, 6, 7, 10]. An NP-speci®c T cell response has recently
Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany been demonstrated in persons from Lassa fever endemic
E-mail: guenther@bni.uni-hamburg.de regions with serological evidence of previous Lassa virus
Tel.: +49-40-42818421 infection [11]. Whether the newly identi®ed 11-kDa Z
Fax: +49-40-42818 378
protein [3] elicits an immune response upon Lassa virus
G. N. Odaibo á D. O. Olaleye infection is not known. There are also no corresponding
Department of Virology, College of Medicine,
University of Ibadan, Ibadan, Nigeria data for other arenaviruses. According to studies with
lymphocytic choriomeningitis virus, the Z protein is a
J. ter Meulen á H. Schmitz virion component and physically interacts with NP [12].
Projet de Recherche sur les FieÁvres HeÂmorragiques en GuineÂe,
Conakry, Republic of Guinea This interaction may facilitate the simultaneous recog-
nition of both proteins by the immune system. However,
J. ter Meulen
EPICENTRE (Groupe EuropeeÂne d'Expertise en the small size of the Z protein and, thus, the potential
Epidemiologie), Paris, France lack of appropriate B and T cell epitopes may interfere
Present address: J. ter Meulen
with its immune recognition. In a ®rst attempt to char-
Institute of Virology, Philipps University, acterize the immune response to Z protein, we studied
35037 Marburg, Germany whether antibodies to Z protein co-occur with antibodies
226
of antigen on the blot, the dilution of serum and sec- protein or NP were con®rmed in independent experi-
ondary antibody, the composition of the binding buer, ments with a dierent lot of strips, demonstrating the
the incubation time, and the type of TMB were opti- reproducibility of the method.
mized. Stringent binding conditions with a high dilution Applying the optimized assay conditions, none of the
of serum (1:1,000) and conjugate (1:5,000), and a high 50 sera from Uganda, where Lassa fever is not observed,
concentration of two detergents (0.5% Tween 20 and reacted with both Z protein and NP. Five sera reacted
1% NP-40) in the binding buer were found to suppress either with Z protein or with NP, but most of them
unspeci®c reactivity. It is noteworthy that a panel of sera weakly (Fig. 2A, bottom). In contrast, of the 75 sera
from German blood donors did not show unspeci®c collected in high endemic areas for Lassa fever, Guinea
reactivity with Z protein when tested at a dilution of and Nigeria, 11 (15%) reacted with Z protein and 17
1:200. Although both proteins were anity-puri®ed to (23%) reacted with NP (Fig. 2A, top). Reactivity with Z
near homogeneity (Fig. 1), some sera reacted with a protein was found in 9 of the 17 (53%) sera reacting with
protein of lower electrophoretic mobility than NP NP, but in only 2 of the 58 (3.4%) sera that did not react
(Fig. 2A). Binding to this protein was independent of with NP. Thus, the occurrence of antibodies to Z protein
the reactivity to Z protein or NP, and was therefore was statistically signi®cantly associated with the pres-
most likely caused by antibodies to a contaminating ence of antibodies to NP (P<10±6, v2-test). Taken to-
bacterial protein. Additional bands below that of NP gether, the results with the sera from the non-endemic
appeared only in sera with strong reactivity to NP region demonstrate a high speci®city of the assay, in
(Fig. 2A) and, therefore, probably resulted from a minor particular if reactivity to both proteins is considered.
fraction of co-puri®ed NP cleavage products. A corre- Therefore, the frequent co-reactivity of sera from Lassa
sponding faint band was seen in Coomassie-stained gel fever endemic regions with Z protein and NP strongly
loaded with the NP eluate (Fig. 1B, lane puri®ed NP). suggests that the assay detects NP and Z protein-speci®c
However, neither of the additional bands aected in- antibodies resulting from Lassa virus infection rather
terpretation of the blot. Positive reactions with either Z than unrelated cross-reactive antibodies.
To evaluate the suitability of the recombinant blot recombinant test compared with IIF and an NP dot blot
for serosurveys, it was compared with an IIF test using assay published previously [10]. The double-staining IIF
Lassa virus-infected cells, which is the current standard technique allows the identi®cation of even very faint
method for detecting Lassa virus-speci®c antibodies. Lassa virus-speci®c signals in the face of a high back-
The IIF test was improved by the following modi®ca- ground staining. Of advantage in terms of the speci®city
tion. In parallel with the human sera, cells were incu- of the Western blot assay was the possibility of
bated with a Lassa virus NP-speci®c mAb. While the co-detection of antibodies to two dierent proteins. While
mAb was detected with a rhodamine-labeled secondary (unspeci®c) reactivity to either Z protein or NP was
antibody to mouse IgG (Fig. 2B, left), human anti- observed in some sera from the non-endemic region,
bodies were detected with a FITC-labeled antibody to none of these sera reacted with both proteins. Therefore,
human IgG (Fig. 2B, right). Only if both antibodies despite its lower sensitivity compared with IIF and NP
recognized identical structures within the cells, was the dot blot [10], the recombinant NP/Z Western blot may
test interpreted as positive (Fig. 2B, compare left and be applicable if very high speci®city rather than sensi-
right panels). This double staining increases the speci- tivity is required. It is noteworthy that three sera were
®city of the IIF test, since unspeci®c signals can be anti-NP and anti-Z protein positive in the recombinant
disregarded, as well as the sensitivity, since faint signals blot but negative in IIF (Fig. 2C). Each of the two blots
that may normally escape detection within a high and IIF seem to detect dierent antibody speci®cities,
background can be speci®cally searched for in infected probably depending on the dierent degree of denatur-
cells (Fig. 2B, bottom right). Lassa virus-speci®c anti- ation of the proteins in the assays. Therefore, the blot
bodies were detected by this IIF test in 24% of the sera techniques may be used in serosurveys in addition to IIF
from Guinea and Nigeria. One third of the IIF-positive to con®rm as well as to extend the IIF results. On the
sera was also positive for both Z protein and NP- other hand, it is doubtful whether a Western blot tech-
speci®c antibodies, while 95% of the IIF-negative sera nique using highly denatured proteins, allowing mainly
were negative for one or both of these antibody spec- detection of antibodies directed to linear epitopes, can
i®cities (Fig. 2C). Although the results of both tests approach the sensitivity of the IIF test at all. Our data
showed a signi®cant correlation (P=0.001, v2-test), may suggest that eorts in the development of sensitive
which corroborates the conclusion that the recombi- recombinant assays for Lassa virus should focus on the
nant blot detects NP and Z-protein-speci®c antibodies ELISA format. The simple puri®cation procedure of the
resulting from Lassa virus infection, the blot seems to Z protein from the cytoplasm may facilitate the appli-
be less sensitive than IIF. cation of native Z protein in such assays.
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