_Archives
Vi rology
Arch Virol (1992) 126:93-105
© Springer-Verlag 1992 Printed in Austria
Bank vole monoclonal antibodies against Puumala virus envelope glycoproteins: identification of epitopes involved in neutralization ~. Lundkvist 1'2 and B. Niklasson 2'3 t,2 Departments of Virology, National Bacteriological Laboratory and Karolinska Institute, Stockholm, Sweden 3National Defense Research Establishment, Sundbyberg, Sweden
Accepted February 11, 1992
Smnmary. Bank vole (Clethrionomysglareolus) monoclonal antibodies (MAbs) against the two envelope glycoproteins (G 1 and G 2) of the Puumala (PUU) virus were generated and characterized. Analyses of the MAbs' antigen and epitope specificities showed non-overlapping reactivities of one anti-G 1 and two anti-G 2 MAbs. A significant neutralizing activity was shown by the antiG 1 and one of the anti-G2 MAbs, suggesting the existence of at least one neutralizing domain on each of the two glycoproteins. The two neutralizing MAbs reacted with eight PUU-related (serotype 3) virus strains, but did not react with Hantaan, Seoul, or Prospect Hill viruses in an immunofluorescence assay, indicating reactivity with epitopes unique for PUU virus. The nonneutralizing anti-G 2 MAb also reacted with Seoul virus, revealing the presence of a conserved G 2-epitope common for PUU and Seoul viruses, not involved in neutralization. Competitive binding of the MAbs and sera from nephropathia epidemica patients indicated that the defined neutralizing and non-neutralizing epitopes of the glycoproteins were immunodominant also in humans. In another experiment, magnetic beads coated with two MAbs were bound with the virus glycoproteins and used for selectiveenrichment of cells secreting anti-glycoprotein antibodies.
Introduction Hemorrhagic fever with renal syndrome (HFRS) refers to a number of human diseases with similar clinical symptoms characterized by fever and renal failure with or without hemorrhagic manifestations [1]. The etiologic agents of HFRS are Hantaan, Seoul, and Puumala viruses of Hantavirus, a separate genus of the family Bunyaviridae [2, 3]. The natural reservoirs ofhantaviruses are several species of persistently infected rodents [4]. Transmission to humans is believed to occur via aerosol [4].
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~. Lundkvist and B. Niklasson
It has been shown that hantaviruses isolated from different hosts may be differentiated into at least five serotypes based on the patterns of neutralization, immunofluorescence titrations and immunoprecipitation assays [2, 3, 5, 6]. The representatives of each serotype are Hantaan, Seoul, Puumala ~ U U ) , Prospect Hill, and Leakey viruses, respectively [1, 3]. Nephropathia epidemica (NE), a European form of HFRS, was first described in Sweden in 1934 [7]. The disease is caused by P U U virus or closely related virus strains and occurs in Scandinavia, Finland, western Russia, and central Europe [6, 8, 9-1. P U U virus consists of 4 structural proteins including a large protein (L), 2 glycoproteins (G 1 and G 2) and a nucleocapsid protein (N), with approximate molecular weights of 200, 68, 57, and 54 kDa, respectively [6, 103. Production of mouse monoclonal antibodies (MAbs) specific for N of P U U virus was first reported by Ruo etal. [11]. They described the successful use of Mongolian gerbils to increase the titers of the virus used for immunization. We have recently described the establishment of 8 stable bank vole-mouse heterohybridomas secreting N-specific MAbs against P U U virus and the use of these reagents for comparison of different hantavirus strains [12]. In the present study we selectively generated and characterized bank vole MAbs specific for the P U U virus glycoproteins, in order to gain a better understanding of the different viral proteins involved in infection and neutralization. The MAbs were used in an immunofluorescence assay for antigenic comparison of eight PUU-related virus strains isolated in different parts of Europe. We also examined the MAbs' serological cross-reactivities with virus strains representing 3 of the other recognized antigenic groups of hantaviruses. In addition, two MAbs were used for selective enrichment of cells producing antibodies to P U U virus glycoproteins. Materials and methods Virus strains
The history of the hantavirus strains used in this study is summarized in Table 1. All virus strains were propagated in Vero E 6 cells (CRL 1586; ATCC, Rockville, MD), cultivated in Eagle's MEM supplemented with 2% fetal calf serum (FCS), 2 mM L-glutamine, 60 lag/ ml penicillin and 100 lag/ml streptomycin. Generation of bank vole MAbs against the PUU virus glycoproteins
The generation and characterization of bank vole-mouse heterohybridomas producing MAbs against the nucleocapsid protein (N) of PUU virus has earlier been described in detail [12]. Briefly, colonized bank voles were injected with a suspension of PUU virusinfected bank vole lung tissue. Two months after the injection, spleen cells from one serumpositive animal were fused with the mouse myeloma cell line SP 2/0. Heterohybridomas were screened for secretion of IgG antibodies against PUU virus by ELISA and positive results were confirmed by immunofluorescence assay (IFA). All seeded wells containing ELISA-positive heterohybridomas (107/480) were transferred to and expanded in small tissue culture flasks. One to 10 × 10 6 cells from each flask,
Neutralizing MAbs against Puumala virus
95
Table 1. History of virus strains Virus strain
Species
Geographic loc.
Reference/source
Puumala Sotkamo
Finland
24, 2
83-223 L 83-L 20
Clethrionomys glareolus (lung) C. glareolus (lung) C. glareolus (lung)
Sweden Sweden
CG-13891 CG-1820 K-27 H-45
C. glareolus (lung) C. glareolus (lung) human (blood) human (blood)
Belgium U.S.S.R. Bashkiria U.S.S.R. Bashkiria U.S.S.R. Bashkiria
P-360
human (lung, fatal)
U.S.S.R. Saratov
Hantaan (76-118)
Apodemus agrarius (lung) Rattus norvegicus (lung) Microtus pennsylvanieus (lung)
Korea
25 trapped 1983, H/illnfis 26 27 6 E.A. Tkachenko, Moscow E.A. Tkachenko, Moscow 28
Korea
29
U.S.A. Maryland
30
Seoul (urban rat) 80-39 Prospect Hill
still ELISA-positive after propagation (n = 51), were cryopreserved in RPMI-1640 (Gibco, Paisley, Scotland) with 20% FCS and 10% dimethylsulphoxide. In order to selectively establish clones producing MAbs against the P U U virus glycoproteins, 30 supernatants from the cryopreserved heterohybridomas were screened by radio-immuno precipitation assay (RIPA) [6, 12] and by neutralization test as described below. The supernatants were also assayed for disparate reactivities against four different PUU-related virus strains by IFA. Eight heterohybridomas were selected for cloning, and monoclonality was assured by single-cell cloning three times.
Neutralization test Virus neutralization activity of the MAbs was examined in neutralization tests as described elsewhere [13]. In short, hybridoma culture supernatants were serially diluted 2-fold and mixed with equal volumes of approximately 50 focus forming units of P U U virus (strain 83-223 L) for 1 h, before incubation on Vero E6 cells. An irrelevant mouse IgG 1 MAb was used as a negative control. After adsorption for 1 h, the cells were overlaid with 0.5% agarose and incubated for 14 days. The agar layer was removed and the cells fixed with methanol. Viral antigen was visualized by addition of rabbit-anti PUU virus serum followed by peroxidase-conjugated goat antibodies to rabbit IgG and substrate. An 80% reduction of foci was used as the criterion for neutralization.
E L I S A additivity test The epitope specificities of the MAbs were investigated by antibody additivity immunoassays as described previously [12, 14]. Briefly, a saturating quantity of one MAb was incubated in P U U virus antigen coated microtiter wells. Unbound antibodies were removed by washing prior to addition of a second MAb. The amount of bound antibodies was determined by 7-chain specific alkaline phosphatase- or peroxidase-conjugated goat antibodies to mouse
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It. Lundkvist and B. Niklasson
IgG, followed by substrate. An additivity index (AI) was calculated in the following manner: AI = 100 ([(2 A1 + 2) / (A1 + Az) - 1] where A1, A2, and A1 + 2 are the absorbances reached with the first antibody alone, the second antibody alone, and the two antibodies together. Those antibody pairs giving an AI of more than 50% were considered to recognize different epitopes. Pairs with an index between 25 and 50% were considered to recognize partially overlapping epitopes. Antibody pairs with an index of < 25% were considered to recognize identical or entirely overlapping epitopes.
Competitive ELISA A comparison of the abilities of NE patient sera to compete with biotinylated anti-glycoprotein MAbs in binding to the antigen was performed by ELISA. The anti-G 2 MAbs 4G2 and 5B7, affinity purified on protein A-Sepharose columns and diluted to 10 gg MAb/ ml in 0.05 M carbonate buffer pH 9.6, were coated to microtiter plates over night at 4 °C. Ascitic fluid containing the anti-Hantaan-G 1 mouse MAb 8B6 ([8]; kindly provided by Dr. Connie Schmaljohn), cross reactive with PUU virus, was diluted 1 : 50 and coated in the same way. Unsaturated protein binding sites were blocked with 3% bovine serum albumin in PBS. Detergent treated PUU virus antigen, prepared as described below for cell separation, diluted 1 : 20, was added and plates were incubated for 1 h. Unlabelled sera diluted 1:40 from 15 serologically confirmed NE-patients [15]; 5 acute phase sera (drawn 1-8 days post-infection), 5 late convalescent sera (drawn 2 years post-infection) and 5 sera drawn > 10 years post-infection, were incubated in duplicate for I h. Serum from a noninfected human was used as an internal negative-standard in all tests. Each anti-glycoprotein MAb (0.1 gg/ml), conjugated to biotin as described elsewhere [16], was incubated for 1 h followed by incubation for 1 h with Extravidin-alkaline phosphatase or streptavidin-peroxidase (Sigma, St. Louis, MO), diluted 1 : 5,000 and 1 : 1,000, respectively. Washing was performed after each step and the plates were developed by addition of p-nitrophenylphosphate (Sigma) or 3, Y, 5, 5'-tetradimethyl benzidine (ICN Biochemicals, Cleveland, OH) according to the manufacturer's instructions.
Antigen-spec~'c selection of cells by antigen-coated magnetic beads Confluent Vero E 6 cells in roller-bottles were infected with PUU virus (strain 82-223 L). Fourteen days post infection, the cells were harvested by centrifugation and washed once in PBS. The cell-pellets were dissolved in 1.5 ml/roller-bottle of RIPA-buffer (0.01 M TrisHC1 pH7.8, 2% Triton X-100, 0.15M NaC1, 0.6M KC1, 5raM EDTA, 1% aprotinin (Sigma), and 1 mM PMSF). Remaining cell-debris was removed by centrifugation. The virus antigen was stored at - 20 °C until used. 100 ~tg of each affinity purified anti-G t and -G 2 MAbs 5A2 and 5B7 were incubated with 108 magnetic beads coupled with goat anti-mouse IgG according to the manufacturer's instructions (Dynal, Osto, Norway). Non-saturated binding sites were blocked with normal mouse IgG (Sigma) for 2 h. The beads were then incubated with virus antigen preparations diluted 1 : 2 in PBS over night followed by 5 washes in PBS with 1% FCS. The specificity of the antigen coupled beads was investigated by using the anti-G 2 MAb producing hybridoma, 4G2, in cell separation experiments. Three hybridoma clones secreting MAbs against N of PUU virus and a non-secreting human-mouse heterohybridoma [17] were used as negative controls. The cell-separations were performed as described elsewhere [18] with minor modifications. Briefly, cells were washed twice with cold RPMI1640 (Gibco) containing 10% FCS before incubation with the beads. Rosetting was performed for 2 h at 4 °C in the same medium during gentle agitation of the suspension in an end-over-end rotator. The rosette cells were separated from non-rosetted cells by placing the tube in a magnetic device (MPC-1, Dynal) for 60 s. The magnet bound cells were washed
Neutralizing MAbs against Puumala virus
97
5 times with 10 ml cold medium by resuspension followed by further magnetic separation. The percentage of magnet-separable rosettes was determined by microscopic cell counts. Results
Generation and characterization of bank vole MAbs RIPA revealed that 4 supernatants from 30 cryopreserved polyclonal heterohybridomas contained detectable antibodies against G 1 of the P U U virus (strain 83-223 L). Two of these were shown to have neutralizing activity. Of the remaining 26 supernatants, 22 precipitated a band correlating to either N or G 2 in RIPA, not distinguishable due to insufficient gel-separation. Adequate separation was later achieved by use of gradient gels. The supernatants were further tested by IFA against the PUU-related virus strains 82-223 L, 83-L20, CG1820, and K-27. Most of the supernatants reacted with all the tested strains, however, 4 gave different IFA-patterns; negative to K-27 and/or negative to CG-1820. Eight polyclonal heterohybridomas were selected for further singlecell clonations: 4 precipitating G t, 2 IFA-negative against K-27 and CG-1820 and 2 IFA-positive against all tested virus strains. Six of the 8 heterohybridomas could successfully be recloned with maintained stable antibody production. One MAb, designated 5A2, precipitated G 1 in RIPA (Fig. 1). The MAbs 4G2 and 5B7 both precipitated G2, although 4G2 also showed a weak reaction with G 1. MAbs 4E5, 5A3, and 5El showed specific reactivity with N. The heterohybridomas have to date remained stable antibody secretors for over 9 months in continuous cell culture.
Immunofluorescence patterns of viral antigens in acetone fixed cells The IFA-patterns in P U U virus-infected, acetone-fixed Vero E 6 cells, showed that different viral components could be distinguished by the MAbs. All anti-
Fig. 1. [3SS]methionine-labelledvirus (83-223 L) antigen was immunoprecipitated with: 1 human negative control serum; 2 NE patient serum, 3-7 bank vole MAbs 4E5, 4G2, 5A2, 5A3, and 5B7, respectively. 8 Polyclonal supernatant of heterohybridoma 5B7, containing antibodies against N and G 2, prior to recloning. The immunoprecipitates were separated by gradient gels ranging from 6 to 15% acrylamide and 0.08 to 0.30% N, N'-methylenebisacrylamide
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~. Lundkvist and B. Niklasson
N MAbs showed a similar speckled pattern of small to large granules or inclusions in the cytoplasm (Fig. 2 A). The anti-G 1 MAb 5A2 showed a staining pattern of mainly large, distinct dots (Fig. 2 B). The two anti-G 2 MAbs (4G2 and 5B7) showed similar smooth diffuse staining patterns (Fig. 2 C and D).
Virus neutralization activity of the MAbs The eight anti-N MAbs described previously [12] together with the six MAbs described in the present study were tested for neutralizing activity against PUU virus (strain 83-223 L). Only 4G2 and 5A2 showed neutralizing activity when tested as unconcentrated supernatants (approx. 10 gg MAb/ml) with reciprocal titers of 16 and 256, respectively. Serum from the splenectomized bank vole used for fusion showed a neutralizing titer of 5120.
Fig. 2. IFA staining pattern obtained by bank vole MAbs in PUU virus infected, acetone fixed, Vero E 6 cells. The fluorescent signal was enhanced for photography by using biotinlabelled rabbit immunoglobulins to mouse IgG followed by FITC-labelled Extravidin. A The anti-N MAb 5Et reacted with antigen found in the cytoplasm of infected cells. B The anti-G 1 MAb 5A2 gave a staining with mainly large, distinct dots. The anti-G 2 MAbs C 4G2 and D 5B7 showed a more diffuse staining pattern
Neutralizing MAbs against Puumala virus
99
Epitope mapping Additivity tests indicated that the anti-G 1 and the two anti-G 2 MAbs recognized unique epitopes (AI > 50%). The anti-N MAb 5El reacted with a unique N-epitope, whereas MAbs 4E5 and 5A3 in pair showed an additivity index of < 25%, indicating reactivities with identical or entirely overlapping epitopes. MAb 5El was further compared to the previously described MAb 3H9 [t2], which according to IFA-reactivity also recognized an epitope suggested to be specific for PUU-related virus strains. When paired, these two MAbs gave an AI of 67%, indicating reactivities with two different epitopes.
Competitive binding assay of MAbs and sera from NE-patients The assay was used to determine whether serum antibodies from NE-patients recognized epitopes similar to or different from those recognized by the bank vole anti-glycoprotein MAbs. The results demonstrated that antibodies in 5 acute phase sera competed with the 2 neutralizing MAbs 4G2 and 5A2 at less than 5% at a dilution of 1 : 40. All sera, however, were reactive with P U U virus in an IFA (Table 2). In contrast, 5 late convalescent sera and 5 sera drawn > 10 years after disease showed a mean competition of 64% at the same dilution. This competition can be interpreted as either reaction with similar, or spatially close, epitopes on the glycoproteins. Similar results were obtained for the nonneutralizing MAb 5B7. Acute phase sera showed a mean competition of 7% and convalescent sera a mean competition of 30%.
Antigenic comparbson of eleven hantavirus strains The MAbs were serially diluted 2-fold and assayed by IFA against eleven virus strains in the genus Hantavirus. No significant variation (i> 4-fold) in titers against the different virus strains were detected (data not shown). For this reason only the IFA-reactivities at the lowest dilution (1:2) are presented (Table 3). When the IFA-reactivities of the 8 anti-N MAbs previously described [12] were included, the variation in reactivity allowed a classification of the MAbs into six groups. The neutralizing anti-G 1 and -G2 MAbs (5A2 and 4G2) showed an exclusive specificity for PUU-related virus strains. The nonneutralizing anti-G 2 MAb (5B7) showed cross-reactivity with Seoul virus. Five different reactivity patterns were observed among the anti-N MAbs; i.e., those that only recognized the eight PUU-related strains; all PUU-related strains and Prospect Hill virus; all PUU-related strains and Hantaan virus; or all of the eleven virus strains. Three anti-N MAbs were reactive with all virus strains except two of the PUU-related virus strains; K-27 and CG-1820, isolated in western Russia.
Specific selection of anti-glycoprotein antibody producing hybridomas The antigen specificity of the magnetic beads, indirectly coated with the viral glycoproteins, and their applicability for selection of specific antibody secreting
100
~. Lundkvist and B. Niklasson Table 2. Summary of competitive ELISA
NE sera a
IFA titer b
Clone 5A2 (G 1)
4G2 (G2)
5B7 (G2)
Acute phase (1-8 days) 1
8
0°
0
6
2 3 4 5
32 16 128 1024
4 4 0 0
0 3 4 0
0 6 16 7
256 512 1024 1024 512
54 58 62 69 76
48 73 86 75 77
17 34 60 47 38
128 32 128 128 128
54 51 62 73 56
92 81 34 26 67
46 16 14 2 28
2 years 6 7 8 9 10 > 10 years 11 12 13 14 15
a PUU virus-positive patient sera, previously confirmed by ELISA and IFA b Reciprocal IFA titers to strain 83-223 L. Data previously published [15] ° Percentage inhibition by unlabelled polyclonal serum (competitor). Each figure represents the mean of duplicate of two replicate assay determinations
cells, were investigated by cell separation trials. One million cells o f the antiG 2 M A b secreting clone 4G2 were incubated with 3 x 106 beads for two hours. The anti-N M A b secreting clones 4C3 [12], 4E5 a n d 5E1 and the non-secreting h u m a n - m o u s e h y b r i d o m a SPAM-8 were used in parallel as negative controls. 10% (11% a n d 9%, respectively in two independent experiments) o f the 4G2 cells were still attached to the magnetic beads after 5 washes. In the negative controls, no rosetted cells could be detected after washing. The separated 4G2cells were grown in cell culture flasks together with the attached beads without any visible influence on the cell growth. Specific M A b secretion f r o m the separated h y b r i d o m a cells was confirmed by ELISA. Discussion
This paper describes, for the first time, the generation o f m o n o c l o n a l antibodies specific for the envelope glycoproteins of P U U virus. In a previous study, we reported the establishment o f eight b a n k vole M A b s directed to the nucleocapsid
+ a + + + + + + + . .
.
.
-
+ + + + + + + +
.
. + .
+ + + + + + + + . .
.
.
+ + + + + + + + .
5El
. . +
+ + + + + + + +
3G5 b
+
+ + + + + + + +
5F4 b
+ + + + + + + + + (+) (-)
4E5
+ + + + + + + + (+) -
5B5 b
o n l y a f e w infected cells stained weakly; - negative
+ + + + + + + + . .
3H9 b
5B7
5A2
4G2
anti-N
anti-G 1 anti-G2
Monoclonal antibody
a + Positive; ( + ) w e a k l y positive; ( - ) b D a t a previously p u b l i s h e d [ 1 2 ]
Puumala Sotkamo 83-223 L 83-L20 CG-13891 CG-1820 K-27 H-45 P-360 H a n t a a n 76-118 Seoul Prospect Hill
Virus strain
Table 3. M A b reactivity w i t h h a n t a v i r u s strains in I F A
+ + + + (-) + + + (+) +
1C12 b
+ + + + (-) + + + + +
3 E l 1b
+ + + + (-) + + + + +
5A3
+
(+) (+)
+ + + + + + + +
2E12 b
+ + + + +
(+)
+ + + + +
4C3 b
gD
o'o
s.
Z
102
A. Lundkvist and B. Niklasson
protein of this virus [12]. We speculated that our ELISA-screening system, which is based on virus antigen bound via polyclonal rabbit antibodies to PUU virus, was more sensitive for detection of anti-N than anti-glycoprotein antibodies. This problem was circumvented in the present study by additional screening by RIPA and NT. When the established MAbs were compared in the ELISA used for screening, we indeed found the test to be substantially less sensitive for detection of antibodies against the glycoproteins; the absorbances obtained with saturating amounts of the anti-glycoprotein MAbs were only 24 times the background signal. This should be compared to the absorbances of more than 20 times the background signal obtained with all anti-N MAbs. Therefore RIPA and NT, although cumbersome and laborious, seem to be preferable screening assays for selective production of MAbs specific for different PUU virus antigens. Two MAbs (5A2 and 4G2) showed a neutralizing activity against PUU virus. MAb 4G2 precipitated, in addition to G 2, a weak G 1-band in RIPA, which made the definition of a neutralizing G2-epitope by this MAb questionable. The neutralizing activity of MAb 4G2 may hypothetically be due to a weak cross-reactivity with G 1. However, when examined by ELISA, no reactivity with G 1 could be detected, indicating that the reactivity with G 1 is extremely weak and therefore not likely to be responsible for neutralization (data not shown). The smooth diffuse IFA-staining pattern obtained with the 2 anti-G 2 MAbs, was consistent with similar observations previously described for Hantaan virus and other hemorrhagic fever viruses [11, 19--21]. The anti-G 1 MAb 5A2, in contrast, showed an IFA-pattern of mainly large, distinct dots. The relevance of the disparate IFA-patterns obtained for the two glycoproteins remains to be elucidated. Altogether 14 anti-PUU virus MAbs have been produced, 3 against the glycoproteins and 11 against the nucleocapsid. None of the MAbs specific for the nucleocapsid showed any neutralizing activity. These results agree with investigations performed for Hantaan and Seoul viruses [11, 19, 22, 23], where only MAbs against epitopes on the envelope proteins were able to block the infectivity of these viruses. It was also reported that both glycoproteins of these two viruses contained neutralizing epitopes. The IFA against 11 hantavirus strains suggested that the 2 defined neutralizing epitopes are unique for PUU-related virus strains, since these epitopes were expressed on all 8 PUU-related virus strains tested, but not on any of the virus strains representing 3 of the other recognized serotypes. The IFA, in combination with additivity assays, also indicated the existence of two different N-epitopes specific for PUU-related virus strains, as defined by MAbs 5El and 3H9. Two virus strains isolated in western Russia, K-27 and CG-1820, reacted differently as compared to the other PUU-related virus strains. The differing nature of strain CG-1820 was also seen by cross-neutralization tests [13]. The results suggest that the large group of PUU-related hantaviruses now isolated
Neutralizing MAbs against Puumala virus
103
may be more complex than previously believed when convalescent sera were used in IFA or immunoprecipitation. Competitive binding assays indicated that the neutralizing and the nonneutralizing glycoprotein epitopes, defined by the MAbs, are immunogenic also in man. Since all tested convalescent sera inhibited all 3 anti-glycoprotein MAbs, it is suggested that these epitopes are immunodominant in humans. Although the sensitivity of the assays for the 3 MAbs may not be identical, it is interesting to note the differences in inhibition of the two neutralizing MAbs 4G2 and 5A2 as compared to the non-neutralizing MAb 5B7. All 10 convalescent sera inhibited MAb 5A2 at more than 50% and 7 of these sera also inhibited MAb 4G2 at more than 50%. Five convalescent sera inhibited MAb 5B7 at less than 30% and only 1 serum at more than 50%. These differences might depend on higher levels of antibodies directed to neutralizing epitopes in late convalescent sera. The results suggest that the defined neutralizing epitopes may represent common immunodominant neutralization sites, shared by humans and bank voles, which may be of biological importance in the induction of protective immunity. The results are of special interest for further investigations of neutralizing domains. The cell-separation experiments were performed to investigate the suitability of these reagents for a "one-step" affinity purification of crude virus antigen preparations, combined with specific separation of cells secreting anti-glycoprotein antibodies. Large amounts of P U U viral antigens are difficult to obtain in vitro, which makes purification with common methods, e.g., uttracentrifugation or affinity chromatography difficult. The described procedure is timesaving and excludes any influence on the antigen due to rough elution environments. The separation of cells secreting anti-G 2 antibodies, and the absence of binding to irrelevant cells, showed that the method is highly specific and efficient. Our intention is to adapt the method for separation of human B-cells, in order to establish human MAbs against viral antigens. Preliminary results indicate that specific isolation of Epstein-Barr virus-immortalized human Bcells can be achieved. We believe that this technique will be most useful, since isolation of specific antibody secreting cells is one of the major obstacles in production of human monoclonal antibodies.
Acknowledgements We thank Marona Engvall and Angelica Fatouros for excellent technical support. This project was supported by the U.S. Army Medical Research and Development Command DAMD 17-89-Z-9010.
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28. 29. 30.
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virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. J Gen Virol 70:1125-1132 Ruo SL, Mitchell SW, Kiley MP, Roumillat LF, Fisher-Hoch SP, McCormick JB (1991) Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies. J Gen Virol 72:549-555 Arikawa J, Schmaljohn AL, Dalrymple JM, Schmaljohn CS (1989) Characterization of Hantaan virus envelope glycoprotein antigenic determinants defined by monoclonal antibodies. J Gen Virol 70:615-624 Dantas JR Jr, Okuno Y, Asada H, Tamura M, Takahashi M, Tanashita O, Takahashi Y, Kurata T, Yamanishi K (1986) Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies. Virology 151:379-384 Brummer-Korvenkontio M, Henttonen H, Vaheri A (1982) Hemorrhagic fever with renal syndrome in Finland: ecology and virology of nephropathia epidemica. Scand J Infect Dis [Suppl] 36:88-91 Niklasson B, LeDuc J (1984) Isolation of the nephropathia epidemica agent in Sweden. Lancet i: 1012-1013 Van der Groen G, Beelaert G, Hoofd G, Leirs H, Verhagen R, Yamanishi H, Tkachenko EA, Ivanov AP (1987) Partial characterization of a hantavirus isolated from Clethrionomys glareolus captured in Belgium. Acta Virol 31: 180-184 Tkachenko EA, Bashkirtsev VN, Van der Groen G, Dzagurova TK, Ivanov AP, Ryltseva EV (1984) Isolation in Vero E 6 cells of hantavirus from Clethrionomys glareolus captured in the Bashkiria area of the USSR. Ann Soc Belg Med Trop 65: 121135 Lee HW, Lee PW, Johnson KM (1978) Isolation of the etiologic agent of Korean hemorrhagic fever. J Infect Dis 137:298-308 Lee HW, Back LJ, Johnson KM (1982) Isolation of Hantaan virus, the etiologic agent of Korean hemorrhagic fever, from wild urban rats. J Infect Dis 146:638-644 Lee PW, Amyx HL, Gajdusek DC, Yanagihara RT, Goldgaber D, Gibbs CJ Jr (1982) New haemorrhagic fever with renal syndrome-related virus in indigenous wild rodents in the United Stated. Lancet ii: 1405
Authors' address: A. Lundkvist, Department of Virology, National Bacteriological Laboratory, S-10521 Stockholm, Sweden. Received October 28, 1991
Vi rology
Arch Virol (1992) 126:93-105
© Springer-Verlag 1992 Printed in Austria
Bank vole monoclonal antibodies against Puumala virus envelope glycoproteins: identification of epitopes involved in neutralization ~. Lundkvist 1'2 and B. Niklasson 2'3 t,2 Departments of Virology, National Bacteriological Laboratory and Karolinska Institute, Stockholm, Sweden 3National Defense Research Establishment, Sundbyberg, Sweden
Accepted February 11, 1992
Smnmary. Bank vole (Clethrionomysglareolus) monoclonal antibodies (MAbs) against the two envelope glycoproteins (G 1 and G 2) of the Puumala (PUU) virus were generated and characterized. Analyses of the MAbs' antigen and epitope specificities showed non-overlapping reactivities of one anti-G 1 and two anti-G 2 MAbs. A significant neutralizing activity was shown by the antiG 1 and one of the anti-G2 MAbs, suggesting the existence of at least one neutralizing domain on each of the two glycoproteins. The two neutralizing MAbs reacted with eight PUU-related (serotype 3) virus strains, but did not react with Hantaan, Seoul, or Prospect Hill viruses in an immunofluorescence assay, indicating reactivity with epitopes unique for PUU virus. The nonneutralizing anti-G 2 MAb also reacted with Seoul virus, revealing the presence of a conserved G 2-epitope common for PUU and Seoul viruses, not involved in neutralization. Competitive binding of the MAbs and sera from nephropathia epidemica patients indicated that the defined neutralizing and non-neutralizing epitopes of the glycoproteins were immunodominant also in humans. In another experiment, magnetic beads coated with two MAbs were bound with the virus glycoproteins and used for selectiveenrichment of cells secreting anti-glycoprotein antibodies.
Introduction Hemorrhagic fever with renal syndrome (HFRS) refers to a number of human diseases with similar clinical symptoms characterized by fever and renal failure with or without hemorrhagic manifestations [1]. The etiologic agents of HFRS are Hantaan, Seoul, and Puumala viruses of Hantavirus, a separate genus of the family Bunyaviridae [2, 3]. The natural reservoirs ofhantaviruses are several species of persistently infected rodents [4]. Transmission to humans is believed to occur via aerosol [4].
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~. Lundkvist and B. Niklasson
It has been shown that hantaviruses isolated from different hosts may be differentiated into at least five serotypes based on the patterns of neutralization, immunofluorescence titrations and immunoprecipitation assays [2, 3, 5, 6]. The representatives of each serotype are Hantaan, Seoul, Puumala ~ U U ) , Prospect Hill, and Leakey viruses, respectively [1, 3]. Nephropathia epidemica (NE), a European form of HFRS, was first described in Sweden in 1934 [7]. The disease is caused by P U U virus or closely related virus strains and occurs in Scandinavia, Finland, western Russia, and central Europe [6, 8, 9-1. P U U virus consists of 4 structural proteins including a large protein (L), 2 glycoproteins (G 1 and G 2) and a nucleocapsid protein (N), with approximate molecular weights of 200, 68, 57, and 54 kDa, respectively [6, 103. Production of mouse monoclonal antibodies (MAbs) specific for N of P U U virus was first reported by Ruo etal. [11]. They described the successful use of Mongolian gerbils to increase the titers of the virus used for immunization. We have recently described the establishment of 8 stable bank vole-mouse heterohybridomas secreting N-specific MAbs against P U U virus and the use of these reagents for comparison of different hantavirus strains [12]. In the present study we selectively generated and characterized bank vole MAbs specific for the P U U virus glycoproteins, in order to gain a better understanding of the different viral proteins involved in infection and neutralization. The MAbs were used in an immunofluorescence assay for antigenic comparison of eight PUU-related virus strains isolated in different parts of Europe. We also examined the MAbs' serological cross-reactivities with virus strains representing 3 of the other recognized antigenic groups of hantaviruses. In addition, two MAbs were used for selective enrichment of cells producing antibodies to P U U virus glycoproteins. Materials and methods Virus strains
The history of the hantavirus strains used in this study is summarized in Table 1. All virus strains were propagated in Vero E 6 cells (CRL 1586; ATCC, Rockville, MD), cultivated in Eagle's MEM supplemented with 2% fetal calf serum (FCS), 2 mM L-glutamine, 60 lag/ ml penicillin and 100 lag/ml streptomycin. Generation of bank vole MAbs against the PUU virus glycoproteins
The generation and characterization of bank vole-mouse heterohybridomas producing MAbs against the nucleocapsid protein (N) of PUU virus has earlier been described in detail [12]. Briefly, colonized bank voles were injected with a suspension of PUU virusinfected bank vole lung tissue. Two months after the injection, spleen cells from one serumpositive animal were fused with the mouse myeloma cell line SP 2/0. Heterohybridomas were screened for secretion of IgG antibodies against PUU virus by ELISA and positive results were confirmed by immunofluorescence assay (IFA). All seeded wells containing ELISA-positive heterohybridomas (107/480) were transferred to and expanded in small tissue culture flasks. One to 10 × 10 6 cells from each flask,
Neutralizing MAbs against Puumala virus
95
Table 1. History of virus strains Virus strain
Species
Geographic loc.
Reference/source
Puumala Sotkamo
Finland
24, 2
83-223 L 83-L 20
Clethrionomys glareolus (lung) C. glareolus (lung) C. glareolus (lung)
Sweden Sweden
CG-13891 CG-1820 K-27 H-45
C. glareolus (lung) C. glareolus (lung) human (blood) human (blood)
Belgium U.S.S.R. Bashkiria U.S.S.R. Bashkiria U.S.S.R. Bashkiria
P-360
human (lung, fatal)
U.S.S.R. Saratov
Hantaan (76-118)
Apodemus agrarius (lung) Rattus norvegicus (lung) Microtus pennsylvanieus (lung)
Korea
25 trapped 1983, H/illnfis 26 27 6 E.A. Tkachenko, Moscow E.A. Tkachenko, Moscow 28
Korea
29
U.S.A. Maryland
30
Seoul (urban rat) 80-39 Prospect Hill
still ELISA-positive after propagation (n = 51), were cryopreserved in RPMI-1640 (Gibco, Paisley, Scotland) with 20% FCS and 10% dimethylsulphoxide. In order to selectively establish clones producing MAbs against the P U U virus glycoproteins, 30 supernatants from the cryopreserved heterohybridomas were screened by radio-immuno precipitation assay (RIPA) [6, 12] and by neutralization test as described below. The supernatants were also assayed for disparate reactivities against four different PUU-related virus strains by IFA. Eight heterohybridomas were selected for cloning, and monoclonality was assured by single-cell cloning three times.
Neutralization test Virus neutralization activity of the MAbs was examined in neutralization tests as described elsewhere [13]. In short, hybridoma culture supernatants were serially diluted 2-fold and mixed with equal volumes of approximately 50 focus forming units of P U U virus (strain 83-223 L) for 1 h, before incubation on Vero E6 cells. An irrelevant mouse IgG 1 MAb was used as a negative control. After adsorption for 1 h, the cells were overlaid with 0.5% agarose and incubated for 14 days. The agar layer was removed and the cells fixed with methanol. Viral antigen was visualized by addition of rabbit-anti PUU virus serum followed by peroxidase-conjugated goat antibodies to rabbit IgG and substrate. An 80% reduction of foci was used as the criterion for neutralization.
E L I S A additivity test The epitope specificities of the MAbs were investigated by antibody additivity immunoassays as described previously [12, 14]. Briefly, a saturating quantity of one MAb was incubated in P U U virus antigen coated microtiter wells. Unbound antibodies were removed by washing prior to addition of a second MAb. The amount of bound antibodies was determined by 7-chain specific alkaline phosphatase- or peroxidase-conjugated goat antibodies to mouse
96
It. Lundkvist and B. Niklasson
IgG, followed by substrate. An additivity index (AI) was calculated in the following manner: AI = 100 ([(2 A1 + 2) / (A1 + Az) - 1] where A1, A2, and A1 + 2 are the absorbances reached with the first antibody alone, the second antibody alone, and the two antibodies together. Those antibody pairs giving an AI of more than 50% were considered to recognize different epitopes. Pairs with an index between 25 and 50% were considered to recognize partially overlapping epitopes. Antibody pairs with an index of < 25% were considered to recognize identical or entirely overlapping epitopes.
Competitive ELISA A comparison of the abilities of NE patient sera to compete with biotinylated anti-glycoprotein MAbs in binding to the antigen was performed by ELISA. The anti-G 2 MAbs 4G2 and 5B7, affinity purified on protein A-Sepharose columns and diluted to 10 gg MAb/ ml in 0.05 M carbonate buffer pH 9.6, were coated to microtiter plates over night at 4 °C. Ascitic fluid containing the anti-Hantaan-G 1 mouse MAb 8B6 ([8]; kindly provided by Dr. Connie Schmaljohn), cross reactive with PUU virus, was diluted 1 : 50 and coated in the same way. Unsaturated protein binding sites were blocked with 3% bovine serum albumin in PBS. Detergent treated PUU virus antigen, prepared as described below for cell separation, diluted 1 : 20, was added and plates were incubated for 1 h. Unlabelled sera diluted 1:40 from 15 serologically confirmed NE-patients [15]; 5 acute phase sera (drawn 1-8 days post-infection), 5 late convalescent sera (drawn 2 years post-infection) and 5 sera drawn > 10 years post-infection, were incubated in duplicate for I h. Serum from a noninfected human was used as an internal negative-standard in all tests. Each anti-glycoprotein MAb (0.1 gg/ml), conjugated to biotin as described elsewhere [16], was incubated for 1 h followed by incubation for 1 h with Extravidin-alkaline phosphatase or streptavidin-peroxidase (Sigma, St. Louis, MO), diluted 1 : 5,000 and 1 : 1,000, respectively. Washing was performed after each step and the plates were developed by addition of p-nitrophenylphosphate (Sigma) or 3, Y, 5, 5'-tetradimethyl benzidine (ICN Biochemicals, Cleveland, OH) according to the manufacturer's instructions.
Antigen-spec~'c selection of cells by antigen-coated magnetic beads Confluent Vero E 6 cells in roller-bottles were infected with PUU virus (strain 82-223 L). Fourteen days post infection, the cells were harvested by centrifugation and washed once in PBS. The cell-pellets were dissolved in 1.5 ml/roller-bottle of RIPA-buffer (0.01 M TrisHC1 pH7.8, 2% Triton X-100, 0.15M NaC1, 0.6M KC1, 5raM EDTA, 1% aprotinin (Sigma), and 1 mM PMSF). Remaining cell-debris was removed by centrifugation. The virus antigen was stored at - 20 °C until used. 100 ~tg of each affinity purified anti-G t and -G 2 MAbs 5A2 and 5B7 were incubated with 108 magnetic beads coupled with goat anti-mouse IgG according to the manufacturer's instructions (Dynal, Osto, Norway). Non-saturated binding sites were blocked with normal mouse IgG (Sigma) for 2 h. The beads were then incubated with virus antigen preparations diluted 1 : 2 in PBS over night followed by 5 washes in PBS with 1% FCS. The specificity of the antigen coupled beads was investigated by using the anti-G 2 MAb producing hybridoma, 4G2, in cell separation experiments. Three hybridoma clones secreting MAbs against N of PUU virus and a non-secreting human-mouse heterohybridoma [17] were used as negative controls. The cell-separations were performed as described elsewhere [18] with minor modifications. Briefly, cells were washed twice with cold RPMI1640 (Gibco) containing 10% FCS before incubation with the beads. Rosetting was performed for 2 h at 4 °C in the same medium during gentle agitation of the suspension in an end-over-end rotator. The rosette cells were separated from non-rosetted cells by placing the tube in a magnetic device (MPC-1, Dynal) for 60 s. The magnet bound cells were washed
Neutralizing MAbs against Puumala virus
97
5 times with 10 ml cold medium by resuspension followed by further magnetic separation. The percentage of magnet-separable rosettes was determined by microscopic cell counts. Results
Generation and characterization of bank vole MAbs RIPA revealed that 4 supernatants from 30 cryopreserved polyclonal heterohybridomas contained detectable antibodies against G 1 of the P U U virus (strain 83-223 L). Two of these were shown to have neutralizing activity. Of the remaining 26 supernatants, 22 precipitated a band correlating to either N or G 2 in RIPA, not distinguishable due to insufficient gel-separation. Adequate separation was later achieved by use of gradient gels. The supernatants were further tested by IFA against the PUU-related virus strains 82-223 L, 83-L20, CG1820, and K-27. Most of the supernatants reacted with all the tested strains, however, 4 gave different IFA-patterns; negative to K-27 and/or negative to CG-1820. Eight polyclonal heterohybridomas were selected for further singlecell clonations: 4 precipitating G t, 2 IFA-negative against K-27 and CG-1820 and 2 IFA-positive against all tested virus strains. Six of the 8 heterohybridomas could successfully be recloned with maintained stable antibody production. One MAb, designated 5A2, precipitated G 1 in RIPA (Fig. 1). The MAbs 4G2 and 5B7 both precipitated G2, although 4G2 also showed a weak reaction with G 1. MAbs 4E5, 5A3, and 5El showed specific reactivity with N. The heterohybridomas have to date remained stable antibody secretors for over 9 months in continuous cell culture.
Immunofluorescence patterns of viral antigens in acetone fixed cells The IFA-patterns in P U U virus-infected, acetone-fixed Vero E 6 cells, showed that different viral components could be distinguished by the MAbs. All anti-
Fig. 1. [3SS]methionine-labelledvirus (83-223 L) antigen was immunoprecipitated with: 1 human negative control serum; 2 NE patient serum, 3-7 bank vole MAbs 4E5, 4G2, 5A2, 5A3, and 5B7, respectively. 8 Polyclonal supernatant of heterohybridoma 5B7, containing antibodies against N and G 2, prior to recloning. The immunoprecipitates were separated by gradient gels ranging from 6 to 15% acrylamide and 0.08 to 0.30% N, N'-methylenebisacrylamide
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~. Lundkvist and B. Niklasson
N MAbs showed a similar speckled pattern of small to large granules or inclusions in the cytoplasm (Fig. 2 A). The anti-G 1 MAb 5A2 showed a staining pattern of mainly large, distinct dots (Fig. 2 B). The two anti-G 2 MAbs (4G2 and 5B7) showed similar smooth diffuse staining patterns (Fig. 2 C and D).
Virus neutralization activity of the MAbs The eight anti-N MAbs described previously [12] together with the six MAbs described in the present study were tested for neutralizing activity against PUU virus (strain 83-223 L). Only 4G2 and 5A2 showed neutralizing activity when tested as unconcentrated supernatants (approx. 10 gg MAb/ml) with reciprocal titers of 16 and 256, respectively. Serum from the splenectomized bank vole used for fusion showed a neutralizing titer of 5120.
Fig. 2. IFA staining pattern obtained by bank vole MAbs in PUU virus infected, acetone fixed, Vero E 6 cells. The fluorescent signal was enhanced for photography by using biotinlabelled rabbit immunoglobulins to mouse IgG followed by FITC-labelled Extravidin. A The anti-N MAb 5Et reacted with antigen found in the cytoplasm of infected cells. B The anti-G 1 MAb 5A2 gave a staining with mainly large, distinct dots. The anti-G 2 MAbs C 4G2 and D 5B7 showed a more diffuse staining pattern
Neutralizing MAbs against Puumala virus
99
Epitope mapping Additivity tests indicated that the anti-G 1 and the two anti-G 2 MAbs recognized unique epitopes (AI > 50%). The anti-N MAb 5El reacted with a unique N-epitope, whereas MAbs 4E5 and 5A3 in pair showed an additivity index of < 25%, indicating reactivities with identical or entirely overlapping epitopes. MAb 5El was further compared to the previously described MAb 3H9 [t2], which according to IFA-reactivity also recognized an epitope suggested to be specific for PUU-related virus strains. When paired, these two MAbs gave an AI of 67%, indicating reactivities with two different epitopes.
Competitive binding assay of MAbs and sera from NE-patients The assay was used to determine whether serum antibodies from NE-patients recognized epitopes similar to or different from those recognized by the bank vole anti-glycoprotein MAbs. The results demonstrated that antibodies in 5 acute phase sera competed with the 2 neutralizing MAbs 4G2 and 5A2 at less than 5% at a dilution of 1 : 40. All sera, however, were reactive with P U U virus in an IFA (Table 2). In contrast, 5 late convalescent sera and 5 sera drawn > 10 years after disease showed a mean competition of 64% at the same dilution. This competition can be interpreted as either reaction with similar, or spatially close, epitopes on the glycoproteins. Similar results were obtained for the nonneutralizing MAb 5B7. Acute phase sera showed a mean competition of 7% and convalescent sera a mean competition of 30%.
Antigenic comparbson of eleven hantavirus strains The MAbs were serially diluted 2-fold and assayed by IFA against eleven virus strains in the genus Hantavirus. No significant variation (i> 4-fold) in titers against the different virus strains were detected (data not shown). For this reason only the IFA-reactivities at the lowest dilution (1:2) are presented (Table 3). When the IFA-reactivities of the 8 anti-N MAbs previously described [12] were included, the variation in reactivity allowed a classification of the MAbs into six groups. The neutralizing anti-G 1 and -G2 MAbs (5A2 and 4G2) showed an exclusive specificity for PUU-related virus strains. The nonneutralizing anti-G 2 MAb (5B7) showed cross-reactivity with Seoul virus. Five different reactivity patterns were observed among the anti-N MAbs; i.e., those that only recognized the eight PUU-related strains; all PUU-related strains and Prospect Hill virus; all PUU-related strains and Hantaan virus; or all of the eleven virus strains. Three anti-N MAbs were reactive with all virus strains except two of the PUU-related virus strains; K-27 and CG-1820, isolated in western Russia.
Specific selection of anti-glycoprotein antibody producing hybridomas The antigen specificity of the magnetic beads, indirectly coated with the viral glycoproteins, and their applicability for selection of specific antibody secreting
100
~. Lundkvist and B. Niklasson Table 2. Summary of competitive ELISA
NE sera a
IFA titer b
Clone 5A2 (G 1)
4G2 (G2)
5B7 (G2)
Acute phase (1-8 days) 1
8
0°
0
6
2 3 4 5
32 16 128 1024
4 4 0 0
0 3 4 0
0 6 16 7
256 512 1024 1024 512
54 58 62 69 76
48 73 86 75 77
17 34 60 47 38
128 32 128 128 128
54 51 62 73 56
92 81 34 26 67
46 16 14 2 28
2 years 6 7 8 9 10 > 10 years 11 12 13 14 15
a PUU virus-positive patient sera, previously confirmed by ELISA and IFA b Reciprocal IFA titers to strain 83-223 L. Data previously published [15] ° Percentage inhibition by unlabelled polyclonal serum (competitor). Each figure represents the mean of duplicate of two replicate assay determinations
cells, were investigated by cell separation trials. One million cells o f the antiG 2 M A b secreting clone 4G2 were incubated with 3 x 106 beads for two hours. The anti-N M A b secreting clones 4C3 [12], 4E5 a n d 5E1 and the non-secreting h u m a n - m o u s e h y b r i d o m a SPAM-8 were used in parallel as negative controls. 10% (11% a n d 9%, respectively in two independent experiments) o f the 4G2 cells were still attached to the magnetic beads after 5 washes. In the negative controls, no rosetted cells could be detected after washing. The separated 4G2cells were grown in cell culture flasks together with the attached beads without any visible influence on the cell growth. Specific M A b secretion f r o m the separated h y b r i d o m a cells was confirmed by ELISA. Discussion
This paper describes, for the first time, the generation o f m o n o c l o n a l antibodies specific for the envelope glycoproteins of P U U virus. In a previous study, we reported the establishment o f eight b a n k vole M A b s directed to the nucleocapsid
+ a + + + + + + + . .
.
.
-
+ + + + + + + +
.
. + .
+ + + + + + + + . .
.
.
+ + + + + + + + .
5El
. . +
+ + + + + + + +
3G5 b
+
+ + + + + + + +
5F4 b
+ + + + + + + + + (+) (-)
4E5
+ + + + + + + + (+) -
5B5 b
o n l y a f e w infected cells stained weakly; - negative
+ + + + + + + + . .
3H9 b
5B7
5A2
4G2
anti-N
anti-G 1 anti-G2
Monoclonal antibody
a + Positive; ( + ) w e a k l y positive; ( - ) b D a t a previously p u b l i s h e d [ 1 2 ]
Puumala Sotkamo 83-223 L 83-L20 CG-13891 CG-1820 K-27 H-45 P-360 H a n t a a n 76-118 Seoul Prospect Hill
Virus strain
Table 3. M A b reactivity w i t h h a n t a v i r u s strains in I F A
+ + + + (-) + + + (+) +
1C12 b
+ + + + (-) + + + + +
3 E l 1b
+ + + + (-) + + + + +
5A3
+
(+) (+)
+ + + + + + + +
2E12 b
+ + + + +
(+)
+ + + + +
4C3 b
gD
o'o
s.
Z
102
A. Lundkvist and B. Niklasson
protein of this virus [12]. We speculated that our ELISA-screening system, which is based on virus antigen bound via polyclonal rabbit antibodies to PUU virus, was more sensitive for detection of anti-N than anti-glycoprotein antibodies. This problem was circumvented in the present study by additional screening by RIPA and NT. When the established MAbs were compared in the ELISA used for screening, we indeed found the test to be substantially less sensitive for detection of antibodies against the glycoproteins; the absorbances obtained with saturating amounts of the anti-glycoprotein MAbs were only 24 times the background signal. This should be compared to the absorbances of more than 20 times the background signal obtained with all anti-N MAbs. Therefore RIPA and NT, although cumbersome and laborious, seem to be preferable screening assays for selective production of MAbs specific for different PUU virus antigens. Two MAbs (5A2 and 4G2) showed a neutralizing activity against PUU virus. MAb 4G2 precipitated, in addition to G 2, a weak G 1-band in RIPA, which made the definition of a neutralizing G2-epitope by this MAb questionable. The neutralizing activity of MAb 4G2 may hypothetically be due to a weak cross-reactivity with G 1. However, when examined by ELISA, no reactivity with G 1 could be detected, indicating that the reactivity with G 1 is extremely weak and therefore not likely to be responsible for neutralization (data not shown). The smooth diffuse IFA-staining pattern obtained with the 2 anti-G 2 MAbs, was consistent with similar observations previously described for Hantaan virus and other hemorrhagic fever viruses [11, 19--21]. The anti-G 1 MAb 5A2, in contrast, showed an IFA-pattern of mainly large, distinct dots. The relevance of the disparate IFA-patterns obtained for the two glycoproteins remains to be elucidated. Altogether 14 anti-PUU virus MAbs have been produced, 3 against the glycoproteins and 11 against the nucleocapsid. None of the MAbs specific for the nucleocapsid showed any neutralizing activity. These results agree with investigations performed for Hantaan and Seoul viruses [11, 19, 22, 23], where only MAbs against epitopes on the envelope proteins were able to block the infectivity of these viruses. It was also reported that both glycoproteins of these two viruses contained neutralizing epitopes. The IFA against 11 hantavirus strains suggested that the 2 defined neutralizing epitopes are unique for PUU-related virus strains, since these epitopes were expressed on all 8 PUU-related virus strains tested, but not on any of the virus strains representing 3 of the other recognized serotypes. The IFA, in combination with additivity assays, also indicated the existence of two different N-epitopes specific for PUU-related virus strains, as defined by MAbs 5El and 3H9. Two virus strains isolated in western Russia, K-27 and CG-1820, reacted differently as compared to the other PUU-related virus strains. The differing nature of strain CG-1820 was also seen by cross-neutralization tests [13]. The results suggest that the large group of PUU-related hantaviruses now isolated
Neutralizing MAbs against Puumala virus
103
may be more complex than previously believed when convalescent sera were used in IFA or immunoprecipitation. Competitive binding assays indicated that the neutralizing and the nonneutralizing glycoprotein epitopes, defined by the MAbs, are immunogenic also in man. Since all tested convalescent sera inhibited all 3 anti-glycoprotein MAbs, it is suggested that these epitopes are immunodominant in humans. Although the sensitivity of the assays for the 3 MAbs may not be identical, it is interesting to note the differences in inhibition of the two neutralizing MAbs 4G2 and 5A2 as compared to the non-neutralizing MAb 5B7. All 10 convalescent sera inhibited MAb 5A2 at more than 50% and 7 of these sera also inhibited MAb 4G2 at more than 50%. Five convalescent sera inhibited MAb 5B7 at less than 30% and only 1 serum at more than 50%. These differences might depend on higher levels of antibodies directed to neutralizing epitopes in late convalescent sera. The results suggest that the defined neutralizing epitopes may represent common immunodominant neutralization sites, shared by humans and bank voles, which may be of biological importance in the induction of protective immunity. The results are of special interest for further investigations of neutralizing domains. The cell-separation experiments were performed to investigate the suitability of these reagents for a "one-step" affinity purification of crude virus antigen preparations, combined with specific separation of cells secreting anti-glycoprotein antibodies. Large amounts of P U U viral antigens are difficult to obtain in vitro, which makes purification with common methods, e.g., uttracentrifugation or affinity chromatography difficult. The described procedure is timesaving and excludes any influence on the antigen due to rough elution environments. The separation of cells secreting anti-G 2 antibodies, and the absence of binding to irrelevant cells, showed that the method is highly specific and efficient. Our intention is to adapt the method for separation of human B-cells, in order to establish human MAbs against viral antigens. Preliminary results indicate that specific isolation of Epstein-Barr virus-immortalized human Bcells can be achieved. We believe that this technique will be most useful, since isolation of specific antibody secreting cells is one of the major obstacles in production of human monoclonal antibodies.
Acknowledgements We thank Marona Engvall and Angelica Fatouros for excellent technical support. This project was supported by the U.S. Army Medical Research and Development Command DAMD 17-89-Z-9010.
References 1. Yanagihara R, Gajdusek DC (1988) Hemorrhagic fever with renal syndrome: a historical perspective and review of recent advances. In: Gear JHS (ed) Handbook on viral and rickettsial hemorrhagic fevers. CRC Press, Boca Raton, pp 151-188
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Authors' address: A. Lundkvist, Department of Virology, National Bacteriological Laboratory, S-10521 Stockholm, Sweden. Received October 28, 1991
