Journal of Virological Methods, 39 (1992) 139-147 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00

139

VIRMET 01377

Antibodies to Puumala virus in humans determined by neutralization test Jan Hiirling”,

Pike Lundkvista3b,

John W. Huggi&

and Bo Niklassonb,d

“Department of Virology, National Bacteriological Laboratory, Stockholm (Sweden), bDepartment of Virology, Karolinska Institute, c/o National Bacteriological Laboratory, Stockholm (Sweden), ‘Department of Virology, USAMRIID, Fort Detrick. Frederick, MD (USA) and dNational Defense Research Establishment, Uned (Sweden)

(Accepted 24 March 1992)

Summary An assay for detection of neutralizing antibodies to Puumala virus using 96well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 14 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected l&20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NTELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests. Hantavirus; Puumala virus; Neutralization

Correspondence to:

Stockholm, Sweden.

test; ELISA

J. Hiirling, Department of Virology, National Bacteriological Laboratory, S-105 21

140

Introduction Viruses causing hemorrhagic fever with renal syndrome (HFRS) are widely distributed throughout the world (Lee et al., 1978). A milder form of HFRS, occurring in Scandinavia and Finland, is called Nephropathia epidemica (NE) (Lahdevirta et al., 1971). Puumala virus (PUU), the etiologic agent of NE, is a member of the Hantavirus genus in the Bunyaviridae family (Schmaljohn et al., 1985; Brummer-Korvenkontio et al., 1980). This virus was first isolated in Sweden from bank voles, the rodent reservoir (Niklasson et al., 1984). PUU virus is antigenically related to the Hantaan virus (HTN) (Svedmyr et al., 1979), the etiological agent of the more severe form of HFRS called Korean haemorrhagic fever (KHF), which is endemic in parts of Asia (Lee et al., 1978). Neutralization tests for PUU virus have been hampered by the inability of the virus to produce cytopathic effects in cell monolayers. This problem was overcome by visualizing infected cells as plaques with immunoperoxidase staining (Tanashita et al., 1984; Niklasson et al., 1991). However, this technique is both time consuming and requires large amounts of reagents, limiting the number of specimens that can be tested. We, therefore, adapted a neutralization assay, originally designed for screening of antiviral substances, for measurement of neutralizing antibodies to PUU virus. The new assay was evaluated as a sero-epidemiological tool and for diagnosis of patients. In addition, the test was used for screening hybridoma clones for neutralizing activity in the production of monoclonal antibodies.

Materials and Methods Reagents used in the neutralization

assay and FRNT

The Puumala virus (strain 83-223L) passaged in Vero E6 cells (CRL 1586; ATCC, Rockville, MD) was used in this study. PUU virus-specific antisera were produced in rabbits (Chinchilla breed), as described elsewhere (Niklasson et al., 1991). Neutralization

assay - NT ELBA

Endpoint titers of neutralization in sera were determined as follows. Sera were diluted serially four-fold in Eagle’s complete medium (Gibco, Paisley, UK) supplemented with 2% fetal calf serum, 60 pg/ml penicillin and 100 pg/ml streptomycin and mixed with an equal volume of medium containing 4&60 focus-forming units of virus per 100 ~1. The mixture was incubated for 1 h at 37°C and subsequently inoculated (200 ~1) into 96-well plates (Nunc, Denmark) containing confluent Vero E6 cell monolayers. After incubation for 16 h at 37°C in a humidified 5% CO:! atmosphere, the plates were emptied and 200 ~1 of new medium was added to each well. The virus dilution and incubation times

141

were determined by box titrations. After incubation for 12 days at 37°C in a humidified 5% CO2 atmosphere, the plates were washed twice in washing buffer (0.9% NaCl with 0.05% Tween 20) and fixed with 100 pi/well of 10% formaldehyde, 1% CaC12 for 15 min at 20°C. After five washes in washing buffer, 100 $/well of rabbit anti-PUU virus serum diluted 1:300 in dilution buffer (PBS with 5% fetal calf serum and 0.1% Tween 20), was added for 1 h at 37°C. The plates were washed as above and peroxidase-conjugated goat antibodies to rabbit IgG (Bio-Rad, Richmond, CA), 1:lOOO in dilution buffer, were added for 1 h at 37°C. Incubation times before fixation and dilution of rabbit anti-PUU virus serum were determined by titrations. Viral antigen was visualized by adding 2,2’-Azinobis(3-ethylbenzothiazoline6-sulfonic acid), diammonium salt, (ABTS-substrate) (Kirkegaard and Perry Laboratories, Gaithersburg, MD), after five washes, as above. The absorbances were read after 30 min in a spectrophotometer at 405 nm. The lowest serum dilution tested was 1:40 with the exception of the eight sera tested in parallel with FRNT, starting at 1:20. Supernatants from the bank vole-mouse heterohybridomas were added undiluted to the plate. A negative human serum was used as a standard in all tests. Each dilution of all serum specimens was compared to the corresponding dilution of the negative standard. Specimens were tested in duplicate wells and the serum dilutions giving a mean reduction of absorbance of more than 80% of the negative serum at the corresponding dilution, were considered as being neutralizing. A positive control serum (MI), with a neutralizing titer of 640 was included in all tests. Only plates where this titer was achieved were accepted. Focus-reduction

neutralization

test, FRNT

The FRNT was carried out, as described earlier (Niklasson et al., 1991). Briefly, sera were diluted serially four-fold and mixed with an equal volume containing 40-60 focus-forming units (FFU) of PUU virus per 100 ~1. The mixture was incubated for 1 h at 37°C and subsequently inoculated (200 ~1) into wells of six-well tissue culture plates containing confluent Vero E6 cell monolayers. The wells were overlaid with a mixture of agarose and basal Eagle’s medium. The tissue culture plates were incubated at 37°C in a humidified 5% COz atmosphere for 12 days. The agarose was removed from the wells and the cells were fixed by methanol for 5 min at 20°C. Rabbit antiPUU virus serum, followed by goat antibodies to rabbit IgG, labelled with peroxidase, were added to indicate virus-infected cells. 3, 3’ 5, fr’-tetramethylbenzidine (TMB) solution was used as substrate and foci were enumerated. An 80% reduction of the number of foci was used as the criterion for virus neutralization titers.

142

Human serum specimens

Sera collected from apparently healthy populations (n = 187) in two counties in the endemic area of northern Sweden (Norsjo and Sorsele) had been tested previously by immunofluorescence tests (IFT) and by an IgG-ELISA (Niklasson et al., 1990). Sequential sera were collected from a total of 19 NE-patients. Paired sera were available from 12 patients from whom the acute serum was collected day 1-13 post-onset and the convalescent serum 2-4 weeks later. Seven patients were bled three times; acute sera day 1-13, convalescent sera 2-4 weeks following the acute serum specimen and a third specimen 2 years after the onset of disease. In addition, single sera drawn 10-20 years after the illness were available from nine patients. All 45 sequential sera, as well as the nine 10-20 year follow-up sera, had been examined by IFT, IgG ELISA and IgM ELISA as a part of a previous study (Niklasson et al., 1990). The sera were not heatinactivated. To determine PUU virus neutralization by specific IgM antibodies, IgG antibodies were absorbed from NE-patient sera using RF-absorbent (Behring AG, Marburg, Germany). Eight sera collected day l-5 and found to have high IgM ELISA A’s, and one convalescent serum, with no detectable IgM, were diluted 1:5 in PBS and incubated with an equal volume of RF-absorbent (or PBS as control) for 16 h at 4°C. Bound IgG were removed by centrifugation for 5 min at 2000 x g. Absence of PUU virus specific IgG was confirmed by IgG ELISA. Eight convalescent sera (4 NE patients and 4 KHF patients) were tested in parallel to compare the neutralization assay in the present study with focus reduction neutralization test (FEINT). Bank vole-mouse heterohybridomas

As part of another study, bank vole-mouse heterohybridomas were produced by fusing spleen cells from a PUU virus-infected bank vole with the mouse myeloma cell line SP2fO (Lundkvist et al., 1991). Cell su~rnatants from 31 IgG ELISA-positive heterohybridomas were screened for neutralizing activity. Results

The virus dilution found to be optimal corresponded to 40-60 FFU, and was used in both the NT-ELISA and in the FRNT. Two different strategies for fixation of cells were used; methanol as in the FRNT, or 10% formaldehyde, 1% Ca&. It was found that the methanol fixation increased the background substantially, without increasing the specific signals. Hence, fo~aldehyde

143

ABSORBANCE 1.400 1.200 -

1.000 -

g

600 -

W

&

,!

:I

I I I I I I

:' :'

400 -

200 0

i I I

.?;

600 -

z

.,, ’

1:40

..

.

1:160

:’ / ,..

..

..’

’ / 1:640

I

I

1:2660

1:10240

I

SERUM DILUTIONS El0 Neg.aera MI _~ --*-_..* ._..

6OYbreduction

Fig. 1. The mean values of 30 negative sera (Neg. sera) as compared to the serum used as negative control (BO) and the serum used as positive control (MI) achieved in 10 separate tests. The vertical lines represent the maximum and minimum A values. The positive and negative sera show the variation between different tests and the 30 negative sera show variation between specimens. The dotted line represents the border for virus neutralization (80% reduction of the absorbance of the negative control serum).

fixation was used in all tests. The borderline of 80% reduction of absorbance was defined by running 30 antibody negative sera and these were compared with a positive and a negative control serum. (Fig 1.) Seroepidemiology In the previous study, 36/l 87 (19%) of sera from normal populations contained PUU virus-specific antibodies, measurable by both IFT and IgG ELISA (Niklasson et al., 1990). All 36 sera positive by IFT and IgG ELISA had neutralizing titers of 240. One of the 151 sera, negative by IFT and IgG ELISA, had a neutralizing titer of 40. The correlation coefficient (r) of the endpoint neutralizing titers of the 37 sera and their corresponding IgG ELISA A, was found to be 0.66. The correlation is depicted in Fig. 2. Patient sera The levels of IgM in NE-patients reach a peak 2 weeks after onset of disease, and are low or negative after 334 months. At the onset of disease, most patients have low, but detectable, levels of IgG. The IgG titers rise during the first two

144

ABSORBANCE 1.400

1.200

t

160

40

640

2!560

10240

NEUTFlALlZATlONTITER NT ELISA Fig. 2. Comparison of NT ELISA and IgG ELISA in 37 sera. The sera were selected from a set of 187 sera collected from a normal population on the basis of being NT ELISA positive. NT ELISA titers were compared with IgG ELISA A at a serum dilution of 1:400. r = 0.66, slope = 205.

years and are high even IO-20 years after disease (Niklasson

et al., 1990).

TABLE 1 Neutralisation

titres in 7 patients

1 SERUM SAMPLE 1

1 SERUM SAMPLE2

/ SERUMSAMPLE

PATIENT 1

160

(DAY 6)

160

(DAY 23)

640 QYEARS)

PATIENT 2

160

(DAY13)

160

(DAY 29)

640 (2YEARS)

1

/ PATIENT 3

j

160

(DAY 5)

1

640

(DAY 36)

I ~~

[ PATIENT 4

[

160

(DAY 6)

1

640

(DAY 33)

/

640 (2YEARS)

1

/ PATIENT 5

)

40

(DAY 1)

1

160

(DAY 32)

1

640 (2YEARS)

/

2560 (2 YEARS)

PATIENT 6

160

(DAY 4)

160

(DAY 20)

640 (2YEARS)

1

PATIEM

160

(DAY 11)

640

(DAY 36)

640 (2YEARS)

1

7

Acute-convalescent and 2-year convalescent sera from seven Swedish NE patients. The NT ELISA titres are expressed as reciprocal dilution giving A reduction of 80%. Given in parentheses are the timespost-onset of disease when the sera were drawn.

145

The neutralizing antibody level at different time intervals after onset of disease are shown in Fig. 3. The neutralizing titers of the seven patients followed for 2 years and bled three times are also shown in Table 1. Neutralizing antibodies were detected even in the early sera drawn l-4 days after onset of disease with titers ranging from 40 to 640. Only 10 out of the 19 patients showed a significant NT titer rise between the acute and convalescent specimens. However, a significant titer rise occurred in all seven patients followed over 2 years when comparing the acute and 2-year serum specimens (Table 1). As can be seen in both Table 1 and Fig. 3 there was a continuous increase in neutralizing titers in patient sera over time. This is further supported by the separate set of sera collected l&20 years post-infection, revealing very high neutralizing titers (Fig. 3). In all the early sera (n = S), identical NT-titers (160 (n = 5), 640 (n = 3)) were found before and after absorption of IgG, indicating presence of neutralizing IgM antibodies. The convalescent control serum with a NT-titer of 640 became negative after IgG absorption. The eight sera tested in parallel to compare the previously documented FRNT and the NT-ELISA had identical titers in the two tests (160, 640, 2560, 2560,