associated with infection by Hantaan (HTN), Seoul (SEO), ... diagnostic tools. ... 373A automated DNA sequencer (ABI) using a Taq fluorescent dideoxy.
Journal of General Virology (1998), 79, 659–665. Printed in Great Britain ...................................................................................................................................................................................................................................................................................
Human recombinant Puumala virus antibodies : cross-reaction with other hantaviruses and use in diagnostics Eeva-Marjatta Salonen,1, 2 Paul W. H. I. Parren,1 Yvo F. Graus,1 AI ke Lundkvist,3 Paola Fisicaro,1 Olli Vapalahti,2 Hannimari Kallio-Kokko,2 Antti Vaheri2 and Dennis R. Burton1 1
Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road (IMM2), La Jolla, CA 92037, USA Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland 3 Swedish Institute for Infectious Disease Control, Stockholm, Sweden 2
A panel of seven human monoclonal Fabs against Puumala virus (PUU) nucleocapsid protein (N) was obtained by panning an antibody phage-display library prepared from the spleen of a PUU-immune individual. Three antibodies reacted in immunoblotting and cross-reacted strongly with Tula and Sin Nombre virus recombinant N proteins. These antibodies mapped to the amino terminus of the N protein. One PUU glycoprotein 2 (G2)-specific Fab
Introduction Hantaviruses are a genus of serologically related, negativesense RNA viruses of the family Bunyaviridae (Elliott, 1990 ; Lundkvist & Niklasson, 1994 ; Plyusnin et al., 1996). The genome of a hantavirus is composed of three segments : L, encoding the RNA polymerase ; M, encoding the envelope glycoproteins G1 and G2 ; and S, encoding the nucleocapsid protein (N). Each hantavirus is maintained in a reservoir of a specific persistently infected rodent species. Infection is spread by aerosolized excreta and transmission to humans seems to occur under conditions of close contact with the rodent host. Several hantaviruses are known to cause disease in humans. Haemorrhagic fever with renal syndrome (HFRS) has been associated with infection by Hantaan (HTN), Seoul (SEO), Dobrava (DOB) and Puumala (PUU) viruses, the latter usually causing a milder form of disease (Brummer-Korvenkontio et al., 1980 ; Lundkvist & Niklasson, 1994 ; Lundkvist et al., 1997). Hantavirus pulmonary syndrome (HPS), with a mortality of approximately 50 %, is caused mainly by Sin Nombre virus (SN) (Duchin et al., 1994 ; Nichol et al., 1993 ; Zaki et al., 1995). In the last 3 years, there have also been outbreaks of HPS in Author for correspondence : Dennis R. Burton. Fax 1 619 784 8360. e-mail burton!scripps.edu
0001-5146 # 1998 SGM
obtained against a novel epitope (G2c) crossreacted with Khabarovsk virus but not with the other hantavirus serotypes. An N protein-specific Fab was successfully used as capture antibody to detect PUU-specific serum IgG and IgM antibodies in an enzyme immunoassay. The result demonstrates the usefulness of recombinant human Fabs as potential diagnostic tools.
several different geographical regions of South America (Hjelle et al., 1996 ; Levis et al., 1995, 1997 ; Lopez et al., 1996 ; Weissenbacher et al., 1996). Since the implication of SN in the outbreak of a severe respiratory distress syndrome in the Four Corners region of the United States in the summer of 1993, additional pathogenic hantaviruses have been recognized in the USA, including Black Creek Canal (Rollin et al., 1995), Bayou (Morzunov et al., 1995) and New York viruses (Hjelle et al., 1995). Mirroring the phylogeny of the host rodents, SN and related viruses are more closely homologous to PUU and Prospect Hill virus (PH) than to HTN or SEO. The presence of hantaviruses in many rodent species has not yet been studied and the emergence of novel hantavirus serotypes is therefore a possibility. The factors influencing hantavirus virulence are unknown and no vaccine or specific therapy is currently available against HPS. This indicates the need to identify broadly cross-reactive (human) antibodies for diagnostic and therapeutic purposes and to aid in vaccine design. From combinatorial antibody libraries displayed on a filamentous phage, monoclonal human recombinant Fabs can be selected and affinity-purified using immobilized target molecules as binding ligands (Burton et al., 1991). The phagedisplay technology permits production of specific antibodies in bacteria without the need for animal experimentation.
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E.-M. Salonen and others
Here we describe the isolation of hantavirus cross-reactive human anti-PUU Fabs from a phage-display library.
Methods + Antibody phage-display library. Human spleen cells were obtained from a 60-year-old female patient splenectomized because of idiopathic thrombocytopenic purpura. The donor was chosen because she had a high titre of serum IgG reactive with the N and envelope proteins of PUU, and monoclonal antibodies (MAbs) to PUU had previously been isolated from her spleen cells using a cellular approach (Lundkvist et al., 1993). RNA was isolated from the spleen lymphocyte fraction and a cDNA library of expressed antibody genes was prepared by PCR amplification of IgG1 heavy chain Fd regions and whole λ and κ light chains in the phage-display vector pComb3H, in essence as described previously (Burton et al., 1991 ; Persson et al., 1991). + Selection and production of Fabs. The library was first panned against recombinant PUU N expressed by using the baculovirus system in Sf9 cells (bac-PUU-N) (Vapalahti et al., 1996) coated to microtitre plates (10 µg}ml in 0±01 M PBS, pH 7±4). The library was also panned against a β-octylglycoside (0±1 %) lysate of sucrose gradient purified PUU (Sotkamo strain) grown in Vero E6 cells (Schmaljohn et al., 1985 ; Vapalahti et al., 1992). The lysate was diluted 1 : 5 in PBS and coated to the wells of a microtitre plate. To obtain glycoprotein-specific Fabs, the library was panned against PUU Sotkamo lysate captured by bank vole MAbs specific for G1 (5A2 ; neutralizing MAb) and G2 (5B7 ; non-neutralizing MAb) (Lundkvist & Niklasson, 1992). The MAbs were coated overnight at 10 µg}ml in PBS and, after washing, bound glycoprotein during an overnight incubation at 4 °C from undiluted lysate. + Sequence analysis. Nucleic acid sequencing was carried out on a 373A automated DNA sequencer (ABI) using a Taq fluorescent dideoxy terminator cycle sequencing kit (ABI). The DNA sequences of the Fab heavy and light chains are accessible in GenBank under the following numbers, respectively : puu41, AF029329, AF029337 ; puu58, AF029330, AF029338 ; puu68, AF029331, AF029334 ; puu74, AF029332, AF029335 ; pul69, AF029326, AF029339 ; pul75, AF029327, AF029336 ; pul77, AF029328, AF029340 ; puuA1, AF029333, AF029341. + Immunofluorescence (IFA). Vero E6 cells infected with PUU (Sotkamo strain), Khabarovsk virus (KBR) (MF-43), PH (PH-1), DOB (Dobrava), Tula virus (TUL) (Moravia), SN (CC107), SEO (80-39) or HTN virus (76-118) were fixed with cold acetone and stained using indirect immunofluorescence as described (Hedman et al., 1991 ; Lundkvist et al., 1991). + Immunoblotting. Recombinant N proteins, expressed in insect cells for PUU (see above), TUL (Plyusnin et al., 1994 ; Lundkvist et al., 1996), SEO (Yoshimatsu et al., 1993 a) and HTN (Schmaljohn et al., 1988) and in E. coli for SN (Feldmann et al., 1993), were run in SDS–PAGE for immunoblotting as described (Towbin et al., 1979). + Binding of Fabs to immobilized PUU-N. Polystyrene microtitre wells (Nunc) were used to immobilize 100 µl PUU N diluted 1 : 400 (100 µl per well in PBS). The incubation took place overnight at room temperature, followed by washing three times with PBS containing 0±05 % Tween 20 (PBS-T ; Fluka). To block nonspecific binding sites 100 µl of PBS-T supplemented with 3 % BSA was added per well. Blocking was carried out overnight at room temperature followed by three washes. For reaction with PUU-N, the Fab was diluted in PBS-T supplemented with 3 % BSA for 1 h at 37 °C and the wells were washed as above. The Fab was detected using 100 µl of horseradish peroxidase
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(HRPO)-labelled goat anti-human F(ab«) conjugate (Pierce), incubation # for 1 h at 37 °C and washing. The three washes were repeated. A 100 µl volume of substrate (0±2 % disodium p-nitrophenyl phosphate in diethanolamine buffer) was added per well and incubated for 1 h at room temperature. The reaction was stopped with 100 µl 1 M NaOH and the absorbance recorded at 405 nm. + Diagnostic assay of PUU-N-specific antibodies by using human Fab as capture antibody in enzyme immunoassay (EIA). Fab puu58 (see Results) was immobilized from a concentration of 300 ng}ml PBS (100 µl per well) overnight at room temperature, followed by blocking of nonspecific binding sites with buffer containing 3 % BSA, and overnight incubation at room temperature. The coated wells were washed and then received 100 µl bac-PUU-N protein diluted 1 : 2000 in binding buffer (PBS-T supplemented with 0±5 % BSA). The wells were incubated for 1 h at 37 °C and washed. Serum specimens (n ¯ 21 ; paired sera) from patients with PUU infection (nephropathia epidemica) were obtained from the diagnostic routine of the Department of Virology, University of Helsinki. Serum specimens (n ¯ 36) from healthy controls were collected from the laboratory personnel of the Department. The serum specimens were diluted 1 : 50 and 100 µl was added to the wells and incubated for 1 h at room temperature. The washes were repeated, and 100 µl of HRPO-labelled rabbit anti-human IgG or IgM conjugate (Dako), diluted 1 : 5000 for IgG and 1 : 3000 for IgM were added per well, and incubated for 1 h at 37 °C. o-Phenylenediamine was used as substrate, and after the reaction was stopped with sulphuric acid absorbance was recorded at 492 nm.
Results Characterization of N-specific Fabs
By panning against bac-PUU-N we obtained four antibodies termed puu41 (γ1, λ), puu58 (γ1, λ), puu68 (γ1, κ) and puu74 (γ 1, κ) after four rounds of selection. Sequence analysis revealed the clones to have unique heavy and light chains, and puu41 was found to be the predominant clone. From a further panning using native viral antigen and puu41 as a masking antibody, we obtained three Fabs termed pul69 (γ1, λ), pul75 (γ1, κ) and pul77 (γ1, λ). These Fabs were also found to bind to N in EIA, but with a lower signal than puu41 and puu58. These Fabs (pul69, pul75 and pul77) reacted strongly in immunoblotting with recombinant PUU-N in contrast to two of the Fabs obtained by panning against recombinant N, namely Fabs puu68 and puu74 (Fig. 1 A, B). Interestingly, pul69, pul75 and pul77 showed a strong cross-reaction with recombinant N proteins of SN (Fig. 1 B) and TUL but not HTN or SEO (Fig. 1 C ; SEO not shown). The seven anti-N Fabs were analysed by IFA against eight hantavirus serotypes (Table 1). Five of the antibodies reacted with the prototype PUU strain (Sotkamo). Three Fabs cross-reacted with PH, TUL, KBR and SN. None of the Fabs reacted with DOB, SEO or HTN. Phylogenetic analysis has shown that PUU is most closely related to KBR, PH, TUL and SN, whereas there is a greater divergence from HTN and SEO (Plyusnin et al., 1996). The reaction pattern of the Fabs is consistent with this analysis. To map the epitope(s) recognized by Fabs pul69, pul75 and pul77, immunoblots were performed with N-deletion mutants expressed in E. coli as glutathione S-transferase fusion proteins
Human recombinant PUU antibodies
Fig. 1. Cross-reaction of human anti-N Fabs with hantaviruses in immunoblotting. The samples were run in 10 % SDS–PAGE and transferred onto nitrocellulose. Similar amounts of protein were present in each lane as determined by Ponceau S staining after transfer. The blot was reacted with anti-PUU-N control MAbs (1C12 and 4C3) (1 : 400) followed by HRPO-labelled antimouse IgG (1 : 1000) or recombinant human Fabs (2 µg/ml) followed by HRPO-labelled anti-human F(ab«)2 conjugate (1 : 1000). Panels A and B show the reaction of control antigen (lanes 1, E. coli cell lysate) and recombinant N protein of SN (lanes 2) with control MAbs 4C3 and 1C12 (panel A), and Fabs pul69, pul75 and pul77 (panel B). Panel C shows the reaction of baculovirus-produced recombinant N protein of PUU (lane 1), TUL (lane 2) and HTN (lane 3) with Fabs pul69, pul75 and pul77.
Table 1. Immunofluorescence antibody reactivity of recombinant human PUU N-specific Fabs to Vero E6 cells infected with different hantaviruses
et al., 1996), and map these epitopes within the first 61 amino acids of N.
The antibodies were used at 5 µg}ml (except for puuA1 which was used at 205 µg}ml) ; , not done.
Characterization of a G2-specific Fab
Hantavirus Antibody N-specific puu41 puu58 puu68 pul69 puu74 pul75 pul77 MAb 1C12 G2-specific puuA1
PUU
KBR
PH
TUL
SN
DOB SEO HTN
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(Lundkvist et al., 1996). The epitopes were found to reside in a fragment containing the first 79 amino-terminal residues of PUU or 61 amino-terminal residues of TUL N proteins. The binding was also verified using the fusion proteins immobilized in EIA (data not shown). These findings further confirm that the human antibody response towards PUU is strongest to the amino terminus of the N protein (Elgh et al., 1996 ; Lundkvist
To obtain G-specific Fabs, the library was panned against PUU-infected cell lysate captured by bank vole MAbs specific for G1 (5A2 ; neutralizing MAb) or G2 (5B7 ; non-neutralizing MAb). After four rounds of panning one Fab clone, termed puuA1, was retrieved from the 5B7-capture antibody, while none were retrieved by 5A2 capture. Of the PUU proteins, Fab puuA1 reacted in antigen-capture EIA only with G2 but not with G1 or N protein (Table 2). The Fab puuA1 did not neutralize PUU as measured in a focus reduction neutralization test (FRNT) (Niklasson et al., 1991 ; Lundkvist & Niklasson, 1992). The Fab puuA1 did not compete for binding with other MAbs that recognized two previously defined epitopes on G2, i.e. MAb 4G2 (G2a1) and MAb 5B7 (G2b), and thus bound to a novel epitope on G2, designated G2c (Table 2 and data not shown). When tested for cross-reactivity with other hantaviruses in IFA, the Fab reacted with PUU and KBR, but not with the other hantavirus serotypes tested, i.e. PH, DOB, SEO and HTN (Table 1). Recombinant Fab as capture antibody in EIA
We further studied the applicability of the Fabs for use in EIA for the detection of PUU-specific IgM and IgG antibodies and for the serological diagnosis of PUU infection in humans. Fab puu58 was selected as a capture antibody because it was PUU specific and was one of the strongest binding Fabs
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Table 2. EIA using PUU proteins captured by MAbs Values are mean absorbance (405 nm) of duplicate samples (background obtained against control antigen has been subtracted) ; , not tested. Indirectly coated antigen (capture MAb) Antibody Puu-A1 (2 µg}ml) G1-1E7-1E5 (anti-G1-b) 1C9 (anti-G2-a2) M (Pos. serum, 1 : 500) C (Neg. serum, 1 : 500)
Species
N (MAb 1C12)
G1 (MAb 5A2)
G2 (MAb 4G2)
G2 (MAb 5B7)
Human
0±000
0±020
1±693
1±250
Bank vole Bank vole Human Human
3±424 0±023
0±694 0±000 2±466 0±007
1±498 0±000
0±000 3±420 1±394 0±000
Fig. 2. Binding of Fab puu58 to immobilized PUU-N. Different concentrations of Fab puu58 (E) or Fab puu74 (D), diluted in PBS-T supplemented with 0±5 % BSA were incubated with immobilized PUU-N (300 ng/ml) for 1 h at 37 °C. The bound antibodies were detected with alkaline phosphatase-labelled goat anti-human F(ab«)2 conjugate.
patients with acute PUU infection (nephropathia epidemica) are shown in Table 3. A total of 19 patients had IgG-class antibodies to PUU-N at the time the patient had symptoms of infection and the first serum specimen was drawn for analysis. The concentration of PUU-N-specific IgG antibodies also increased, as expected, with time from the onset of infection in these 19 cases. Two patients, IgG-negative in EIA at the onset of the infection but with moderate (patient no. 13) or low (patient no. 20) positive antibody titres in IFA, had detectable levels of IgG when their sera were analysed 41 or 17 days later. In the first case (no. 13) we failed to demonstrate IgM-class antibodies, although the patient had a seroconversion in IgGEIA. In IgM-EIA, 20 patients had PUU-N-specific antibodies in the first serum sample, 18 showing a distinct decrease in antibody titre specimens taken during the later stage of the disease, as was expected.
Discussion against N in EIA. An irrelevant antibody (Fab 74) showed only a weak background binding to solid-phase bound PUU-N. From the binding curve (Fig. 2) a concentration of 300 ng Fab puu58}ml was selected for immobilization. The bac-PUU-N antigen was used in a constant dilution (1 : 400) of insect cell lysate (selected on the basis of a 1 : 50 dilution of a high-titre serum giving maximal binding with immobilized Fab puu58– PUU-N complexes). A total of 36 PUU IgG-antibody-negative sera (according to IFA) were assayed for IgG- and IgM-class antibody binding to solid-phase Fab58–PUU-N complexes in an EIA procedure. These results were used to determine the cut-off for positive values in the EIA at 10 enzyme immunoassay units (EIU)}ml, which equalled the mean absorbance2SD, with the absorbance obtained with the positive control sample set at 100 EIU}ml (Table 3). PUU-specific IgG and IgM antibody assay results for 21
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These results show that human Fabs reactive with PUU N and G2 proteins obtained from an antibody phage-display library can be useful tools for viral diagnostics, both in immunoblotting, IFA and EIA. We show that one of the Fabs with the strongest binding against N can be conveniently used in a diagnostic antigen-capture immunoassay. Advantages of antigen capture over direct coating to the well is that antigen capture in general may better preserve native antigen conformation and epitope presentation and further non-specific components present in the preparation can be washed away. Human monoclonal Fabs provide some advantage as capture antibodies over conventional poly- or monoclonal antibodies, because, in addition to their specificity, they lack the Fc fragment of the Ig molecule and are therefore not detected by conventional enzyme conjugates against heavy-chain. Crossreactivity with the capture antibody due to cross-species reactions of labelled heavy-chain-specific anti-human Ig anti-
Human recombinant PUU antibodies
Table 3. EIA results on sera from patients with acute PUU infection, compared with the routine diagnostic IFA test Samples were considered positive in both IgG and IgM tests if the signal was greater than 10 EIU}ml, which equals the mean absorbance 2SD obtained from 36 negative sera.
Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Time from onset of infection (days)
IFA
1 8 1 6 2 10 4 12 4 10 39 4 20 1 16 5 21 5 20 4 31 6 32 3 20 6 39 4 53 5 13 10 18 3 11 53 4 12 1 12 1 18 1 5
640 2560 80 320 20 640 640 5120 640 640 640 640 640 640 640 320 640 320 2560 320 2560 2560 1280 640 1280 640 640 320 320 160 320 320 640 80 1280 2560 2560 1280 640 1280 40 640 60 640
IgG class IgM class antibody antibody (EIU/ml) (EIU/ml) 22 50 32 39 29 54 35 52 52 53 50 53 57 33 45 36 54 31 48 35 48 40 51 27 40 2 43 32 51 40 39 44 45 25 48 54 45 50 42 49 7 38 42 43
46 29 37 27 44 35 37 20 20 22 16 27 15 36 31 44 19 47 31 40 23 33 19 46 34 4 4 27 8 30 29 32 25 50 27 15 23 18 18 8 31 18 22 21
bodies used for detection of antigen-bound sample antibody, or due to the presence of rheumatoid factors in the patient serum (Salonen et al., 1980) can thus be avoided.
Potential uses for the Fabs include the purification of viral components from crude extracts, which is simplified by the use of Fabs coupled to affinity matrix. Fabs coupled to peroxidase or other labels may be conveniently used for direct detection of antigen in cases where a secondary antibody gives background. The Fabs do not give background staining in virus-infected cells and may therefore be most valuable reagents for antigen detection in tissues of, e.g., patients with HPS and HFRS or in hantavirus-infected rodents. Of the two Fabs with the strongest reactivity against PUU-N in EIA (puu58 and puu41), puu58 was tested and found useful as a capture antibody in serological diagnosis of acute infection (IgM) and in immunity screening (IgG). The panel of N-specific Fabs further offers a tool for antigenic classification of hantaviruses. Two Fabs were reactive only with PUU, while three reacted with all the hantaviruses carried by arvicoline rodents (PUU, TUL, PH, KBR) and with a sigmodontine rodent (SN), but not with any of the viruses carried by murine rodents, which is in line with the different phylogenetic lineages of hantaviruses, mirroring the carrier rodents. Two of the Fabs reactive in capture assays were not reactive in IFA, indicating that these epitopes were conformational and sensitive to the fixation process. One G2-specific Fab (puuA1) was obtained. This Fab identified a novel epitope designated G2c which is shared by PUU and KBR, but not PH. KBR is a recently discovered hantavirus isolated from Microtus fortis in far-eastern Russia and has been identified as distinct but closely related to PH and PUU on the basis of serological data and genetic analysis (Dzagurova et al., 1995 ; Ho$ rling et al., 1996). Since PUU is pathogenic to humans, in contrast to PH, further characterization of such epitopes may give clues to hantavirus pathogenicity. The G2 Fab may prove useful as G2-specific antibodies from two different species (human and bank vole) with different epitopes are now available for use in immunoassays. The presence of distinct antigenic regions on PUU N, which are recognized by human antibodies, has been demonstrated previously. Our results, however, identify for the first time the presence of specific human MAb}Fab epitopes in a hantaviral N protein. Different degrees of cross-reactivity with other hantavirus serotypes were shown by IFA. There are at least three distinct epitope clusters recognized by the panel of anti-N Fabs based on three different reaction and crossreactivity patterns observed in IFA (Table 1) : epitopes recognized by Fabs puu41}58, Fabs puu68}74 and Fabs pul69}75}77, respectively. Moreover, these three epitopes are distinct from that recognized by MAb 1C12. The epitopes of puu41}58 were not expressed by KBR, thereby defining epitopes recognized by the human immune response that distinguish these two antigenically closely related hantaviruses. The data suggested a fine-tuned specificity of these human Fabs against the infective (homologous) agent and will provide most valuable reagents for characterization of the
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E.-M. Salonen and others
rapidly growing number of recognized hantaviruses. The present results also confirm that the human IgG response against N is mainly directed against the amino-terminal region of the protein (aa 1–61) (Vapalahti et al., 1992, 1995 ; Elgh et al., 1996 ; Lundkvist et al., 1996). Previous studies have shown that recombinant HTN and PUU N proteins, or fragments representing the amino-terminus (aa 1–118 or aa 1–267) of PUU, are sufficient to induce protective immunity against virus infection in hamsters or bank voles (Schmaljohn et al., 1990 ; Lundkvist et al., 1996). Although the protective immunity evoked by immunization with recombinant N proteins is likely to be mediated by cellular responses, antibodies may play a role in protection. Several bank vole MAbs, reactive with different aminoterminal epitopes on PUU N, induced partial protection when passively transferred alone to bank voles (A. Lundkvist and others, unpublished results). Similar observations have been reported for HTN infection where MAbs directed to N protein were able to partially protect in a mouse model (Yoshimatsu et al., 1993 b). It may be interesting to repeat some of these experiments with antibodies against N derived from human infection as isolated in this study. This work was supported by National Institutes of Health grant AI39808 (D. R. B.) and is a contribution from The Scripps Research Institute Emerging Diseases Research Center. This work was further supported by grants from the Academy of Finland, Finnish Culture Foundation and Sigrid Juse! lius Foundation, Helsinki, and the Swedish Medical Research Council (Project no. 12177).
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