CSIRO Division of Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC 3220, Australia
Correspondence
John R. White
John.White{at}csiro.au
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ABSTRACT |
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Present address: The Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, VIC 3002, Australia.
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INTRODUCTION |
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HeV and NiV possess 83 % amino acid identity and extensive serological cross-reactivity (Chua et al., 2000; Harcourt et al., 2000
; Wang et al., 2001
). Neither shows perceptible serological cross-reactivity with other Paramyxoviridae (Wang et al., 2001
). Cross-neutralization tests using polyclonal antisera have shown a consistent and reciprocal four to eightfold difference in titres between the viruses (Harcourt et al., 2000
). Additionally, Tamin et al. (2002)
found that vaccinia virus-expressed NiV envelope glycoproteins induced neutralizing antibodies to both viruses, and a soluble form of the HeV attachment protein (HeV G) elicited a pronounced cross-reactive neutralizing antibody response to NiV (Bossart et al., 2005
). Therefore, we were interested in the nature and location of virus neutralization epitopes on HeV G and the extent of their conservation on NiV G. Characterization of henipavirus neutralization sites will assist studies of host cell tropism, the development of diagnostic assays and potentially provide reagents for vaccine development and validation.
The attachment proteins of members of the family Paramyxoviridae appear to possess a similar conformation (Colman et al., 1993; Langedijk et al., 1997
; Pitt et al., 2000
; Vongpunsawad et al., 2004
). Langedijk et al. (1997)
have proposed a three-dimensional model for the structure of haemagglutinin/neuraminidase (HN) and haemagglutinin (H) proteins of viruses within this family. This model generated a hypothetical structure for HeV G that closely resembled respiroviruses and rubulaviruses more than morbilliviruses (Yu et al., 1998
). We wished to determine whether the location of virus neutralization related amino acid sites in HeV resembled those determined for other Paramyxovirinae and how these sites presented within the proposed model. We selected neutralization-escape HeV variants using anti-HeV G monoclonal antibodies (mAbs), determined the location and nature of amino acid substitutions in individual variants and mapped them onto the HeV G globular head model (Yu et al., 1998
). Potential spatial and conformational relationships between individual epitopes were investigated by determining the extent of individual mAb binding to and neutralization of, homologous and heterologous variants. These data and information on the relative ability of the mAbs to compete for binding to HeV G, provided support for the proposed structural model and revealed new information about the antigenic relationships between the G proteins of HeV and NiV.
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METHODS |
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Antisera.
Human and equine anti-HeV antisera were obtained from the 1994 HeV outbreak (Selvey et al., 1995; Murray et al., 1995
). Antisera to HeV were also produced in experimentally infected horses and rabbits under BSL-4 conditions. Swine and equine anti-NiV antisera were obtained during the NiV outbreak in 1999 (Chua et al., 1999
; Mohd Nor et al., 2000
) and flying fox sera possessing antibodies to HeV were collected in Queensland (Halpin et al., 2000
). All sera raised to infectious virus were gamma irradiated at 0·06 MGy prior to use.
Serum neutralization test (SNT).
As previously described (Crameri et al., 2002), 50 µl aliquots of serially diluted antibody was mixed with an equal volume of EMEM containing 100 TCID50 units of HeV and incubated for 30 min at 37 °C. Vero cells were added and the mixture incubated in the wells of a 96-well plate at 37 °C. The cells were monitored for cytopathic effect (CPE) over 5 days. Dilutions were tested in quadruplicate. Neutralization titres were expressed as the reciprocal of the dilution of antibody that completely blocked development of a CPE.
G protein expression.
The full-length HeV G gene was isolated by PCR from cDNA synthesized from total genomic RNA using random hexamer primers, and cloned into the pZErO vector (Invitrogen) for sequencing analysis. The gene was then cut out of the pZErO clone by digestion with BamHI (partial) and EcoRI, and subcloned into the pFastBac1 vector (Invitrogen) to form the expression plasmid pFB-HeV-G. The gene was then inserted into bacmid DNA using the Bac-to-Bac system (Invitrogen) and the resultant recombinant (HeV-G-Bac) was transfected into Spodoptera frugiperda (Sf21) cells using CELLfectin (Invitrogen). A vaccinia recombinant virus that expressed HeV G (VV-326) was also obtained (Stevens, 2001). This construct used the Western Reserve strain of vaccinia virus and the late promoter of the plasmid pMJ602 (Davison & Moss, 1990
). For baculovirus production, Sf21 cells were infected with the recombinant virus at an m.o.i. of 1·0 and infected cells were recovered at 4872 h post-infection (p.i.). Cell pellets were washed twice and resuspended in an equal volume of 0·1 M PBS (pH 7·2). Following three freezethaw cycles at 20 °C, the preparation was sonicated on ice (3x10 s pulses) in a bath sonicator (Branson Sonifier 250) set at 80 % output. The preparation was centrifuged for 10 min at 3000 g and the supernatant retained for use as ELISA antigen. For VV-326 virus production, CV-1 cells were infected at an m.o.i. of 510 and the cell layer harvested 2448 h later. Processing of this pellet was as described for the recombinant baculovirus preparation. Recombinant vaccinia virus-produced HeV G was used for both ELISA and mAb production. The level of HeV G expression from each construct was determined by Western blotting and indirect ELISA (I-ELISA) using hyperimmune rabbit antiserum to purified HeV.
mAb production and characterization.
Murine mAbs were prepared as previously described (Eaton et al., 1987) with some variations. For the first cell fusion, 20 µg gamma-irradiated, purified HeV in 100 µl PBS was mixed with an equal volume of Montanide ISA50V adjuvant (SEPPIC) and inoculated intraperitoneally into female BALB/c mice older than 10 weeks of age. Mice were further inoculated with 10 µg purified, inactivated virus alone at 2 and 4 weeks p.i. and at 4 days prior to fusion. A second fusion used 10 µg VV-326-expressed HeV G, prepared as described above, for the second and subsequent inoculations. Hybridoma supernatants were screened against both VV-326- and HeV-G-Bac-expressed HeV G in an I-ELISA. Positive supernatants were tested in an SNT and selected neutralizing mAbs were then grown to a high concentration in a bioreactor system (miniPERM classic; Vivascience AG) as previously described (Bruce et al., 2002
). Individual mAb isotypes and post-bioreactor concentrations were simultaneously determined using a bead-based isotyping kit (Beadlyte; Upstate) run on a Bio-Plex protein array system (Bio-Rad) as specified by the manufacturers.
Competitive-binding assay.
The ability of one mAb to affect the binding of another was measured using an ELISA-based additivity assay as previously described (Friguet et al., 1983; Choumet et al., 1992
), with some modifications. Briefly, mAbs were used at concentrations that saturated the binding capacity of a limiting amount of purified virus (Wong et al., 1992
). Pairs of mAbs were analysed with the pre-determined saturating concentration of each being maintained in each case. Results were expressed as a percentage inhibition level rather than an additivity index score. The mean percentage level of maximum potential competition between each mAb pair was determined as follows:
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Variant virus production.
HeV was diluted to 5x106 TCID50 ml1 in EMEM. Aliquots of 100 µl were incubated with an equal volume of undiluted mAb for 30 min at 37 °C, prior to adsorption onto Vero cells grown to 80 % confluency in 25 cm2 flasks. Following incubation for 30 min at 37 °C, 5 ml of mAb diluted 1/50 in EMEM was added and incubation continued. Where discrete syncytial foci formed (after 35 days) and after the subsequent development of a significant CPE, the supernatant was removed and titrated in a plaque assay. Between 4 and 13 plaques were picked for each mAb and the virus in each plaque was designated by the mAb name followed by V1 to V13. Virus from each original plaque was mixed directly with an equal volume of homologous, undiluted mAb and neutralization resistant variants were grown in Vero cells and plaque picked as before. This process was repeated. Individual clones were tested for neutralization resistance to the homologous mAb and stocks prepared by infecting Vero cells at an m.o.i. of 0·01 TCID50 per cell in the absence of mAb.
Normalized variant virus-binding assay.
Antigen generated by wild-type and selected variant viruses was prepared 24 h after infection of Vero cells in 150 cm2 flasks at an m.o.i. of 0·01 TCID50 per cell. Cells were scraped from the flask, lysed by Dounce homogenization in the presence of 1 % NP40, and the nuclear and cytoskeletal components removed by centrifugation at 500 g for 15 min. Antigen was inactivated by gamma irradiation and bound to Maxisorp microplates for analysis by I-ELISA. Non-neutralizing, HeV G-specific mAbs were screened for their ability to bind excess amounts of each variant antigen. mAb 30.6 gave equivalent optical density values with all variant preparations and was consequently included in the mAb panel tested against each variant. The optical density of each neutralizing mAb was normalized in each case to the binding level of mAb 30.6 to yield comparative binding data (White, 1994).
Nucleotide sequencing.
Purified, gamma-irradiated HeV was subjected to reverse transcription and PCR amplification using the one-step RT-PCR kit (Qiagen). Five sets of primers were designed to amplify overlapping HeV G gene fragments of between 600 and 800 nt each. Amplified DNA fragments were purified using a QIAquick kit (Qiagen), and sequenced twice in each direction using the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and the automated DNA sequencer ABI PRISM 377 (Applied Biosystems). Sequence analysis was conducted using the Lasergene software package (DNASTAR).
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RESULTS |
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Site III (aa 289) and site IV (aa 324) identified in 8H4 variants (highlighted in pink in Fig. 2) both mapped to the bottom of the
2-sheet, at the end of the S1 and at the start of the S4 strands, respectively. Amino acid 289 was located in the middle of a postulated small loop created by a cysteine bridge joining the top of the
2S1 strand to the base of the
2S2 strand (Yu et al., 1998
). This may bring aa 289 closer to aa 324. Thus, mAb 8H4 defined a second epitope located on the base of the globular head and on the opposite side to the H1/H2.1 epitope. In support of an association between these epitopes, mAbs 8H4 and H1 also exhibited the most significant level of mutual binding inhibition (91 %) in the competition ELISA (Table 3
).
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Despite the respective locations of the mAb 3A5.D2 defined epitope (on top of the 3-sheet) and mAb 8H4 defined epitope (at the bottom of the
2-sheet), these mAbs showed an unexpectedly high level of mutual binding inhibition (48 %). However, given that these epitopes resided on adjacent
-sheets, steric hindrance factors or some level of conformational inter-dependency may have prevented optimal binding levels of either or each mAb.
Ability of mAbs to bind and neutralize variants
To determine how particular amino acid substitutions contributed to the presentation of individual mAb-defined epitopes, variants were tested for their ability to bind and be neutralized by the mAb used in their selection and all remaining mAbs (Table 4). All mAbs were compromised in their ability to neutralize variants regardless of the mAb used in their selection. Thus, alterations at any one site induced conformational changes to the globular head of HeV G that influenced the involvement of all the other sites in virus neutralization. Seven variants selected using mAb H1 or H2.1 also displayed an inability to bind to either of these mAbs or be neutralized by mAb H2.1 (mAb H1 was not tested in the SNT) consistent with the hypothesis that mAbs H1 and H2.1 bound the same epitope. All other mAbs bound these variants near, or better than, wild-type levels which suggested their respective epitopes were distinct from the H1/H2.1 site.
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mAb 17A5 bound its three homologous variants at, or near to, wild-type levels. Thus, rather than escaping virus neutralization by impairment of mAb binding, the amino acid changes in these variants apparently affected the way the bound mAb interacted with a critical neutralization site somewhere else on the virus. This mAb showed significantly reduced binding to 8H4 variants and also failed to neutralize them and mAb 8H4 exhibited a similarly reduced ability to neutralize 17A5 variants (Table 4). These observations reaffirmed the suggestion that the epitopes associated with these mAbs existed in close proximity to one another.
Level of amino acid sequence similarity between NiV G and HeV G at the sites of neutralization epitopes located on HeV G
The nucleotide sequences in the immediate region of the identified HeV G neutralization sites were compared to the analogous regions in NiV G (Chua et al., 2002) (Fig. 2
). Of the four sites associated with the H1/H2.1 epitope (sites I, VI, VII and VIII) only site VIII showed pronounced differences in the amino acid sequence of each virus immediately adjacent to the site of amino acid substitution (aa 570). These differences alone might have contributed to the much reduced ability of the mAbs H1 and H2.1 to bind NiV, compared with HeV (Table 1
). Alternatively, the discontinuous nature of this epitope and the potentially unique overall conformation of NiV G, may result in it being present in an altered state compared with HeV G. Significant differences in sequence similarity were apparent at the mAb 8H4-related regions site III and IV, the mAb 17A5-related site II and the mAb 3A5.D2-related site V and thus provided an explanation for the decreased ability of these mAbs to bind to NiV G (Table 1
). In particular, mAb 3A5.D2 failed to bind NiV at all and there were two adjacent amino acid that differed in the HeV and NiV G sequences at site V (-Ile-His- compared with -Thr-Lys-). These 2 aa were critical in the related HeV variants (Table 4
) and most likely represented key residues in this epitope. The lack of analogous residues in NiV G probably prevented the binding of mAb 3A5.D2.
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DISCUSSION |
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Amino acid substitutions in 17A5 variants (site II, aa 191195) mapped to the top of the postulated 6S4-sheet and the
1L0,1 loop. In spite of its unique location, site II related variants resemble their mAb H1/H2.1 and mAb 8H4-selected counterparts in having amino acid changes that appear to influence the conformation of a distal virus neutralization-associated site. Variants selected by mAb 17A5 still bound the homologous mAb and mAb 8H4 at, or near, wild-type level and mAb 3A5.D2 also bound two of the three 17A5 variants at normal level (Table 4
). Thus, the reduced ability of mAbs 17A5, 8H4 and 3A5.D2 to neutralize the 17A5 variants would appear to be due to a site II orchestrated conformational alteration that influences virus neutralization more profoundly than mAb binding at specific epitopes.
Separate discontinuous virus neutralization epitopes are on the underside of the globular head region of HeV G
The alignment of amino acid alterations in variants selected by mAbs H1/H2.1 (sites I, VI, VII and VIII) and mAb 8H4 (sites III and IV) with the proposed three-dimensional model for HeV G has identified two discontinuous virus neutralizing epitopes, involving the base of the postulated -sheets
46 and
2, respectively (Table 3
, Figs 1 and 2
). Discontinuous domains related to host-cell attachment and/or HN activity also occur on the globular head of the attachment proteins of many other Paramyxoviridae (van Wyke Coelingh et al., 1987
; Makela et al., 1989a
, b
; Lyn et al., 1991
; Iorio et al., 1991
, 2001
; Ray et al., 1992
; Baty & Randall, 1993
; Hu et al., 1993
; Langedijk et al., 1997
; Moeller et al., 2001
; Masse et al., 2002
, 2004
; Vongpunsawad et al., 2004
). These though, are frequently located on the top of the globular head once the relevant amino acid locations are integrated into the postulated model (Langedijk et al., 1997
; Crennell et al., 2000
; Vongpunsawad et al., 2004
). However, also according to the model, some point mutations and assumed linear epitopes defined by other paramyxovirus neutralization-escape variant studies include amino acid changes at sites that closely approximate some of the component sites of the H1/H2.1 epitope. A major epitope of RPV mapped to the region aa 587592 of the attachment protein, within 17 aa of the C terminus of the protein (Sugiyama et al., 2002
). This would place it at the bottom of strand
6S4 at a similar location to site I (aa 183185). An analogous epitope has also been characterized for MeV using synthetic peptides (Makela et al., 1989a
, b
). The RPV study also identified a mutation at aa 556 that, it was implied, influenced the conformation of the true-binding site of the mAb used in selection of the relevant variant (Sugiyama et al., 2002
). This site is analogous to site VIII (aa 570) and is located in the
6L1,2 loop. A substitution at aa 541 on the HN molecule of a variant of Simian virus 5 also occupied a similar location to site VIII (Baty & Randall, 1993
), and a substitution at aa 552 on the H protein was related to neutralization-resistance in MeV (Hu et al., 1993
). Thus, HeV resembles other members of the subfamily Paramyxovirinae in having critical amino acid sites related to virus neutralization on the underside of the globular head of the attachment protein involving the base of
-sheets 46.
Structural relationships between neutralizing epitopes and their potential influence on HeV G cell attachment
The base of the globular head is unlikely to be the location of the host cell receptor, a structure more likely to present on the more accessible upper surface of the protein (Colman et al., 1993; Langedijk et al., 1997
; Crennell et al., 2000
; Iorio et al., 2001
; Masse et al., 2004
). However, antibody binding to the underside of the protein head could induce conformational changes to the head region that affects its ability to bind a host cell receptor or facilitate cell fusion (Iorio et al., 1992
; Deng et al., 1999
; Hu et al., 2004
; Masse et al., 2004
; Vongpunsawad et al., 2004
). Alternatively, a channel or groove may be present through the head that permits access from the top of the protein to regions located deep within the structure. A funnel structure on top of the globular head has in fact been proposed to be associated with neuraminidase (N) activity in paramyxoviruses (Langedijk et al., 1997
; Vongpunsawad et al., 2004
). A link between the epitopes mapped to the top of the globular head (related to mAbs 3A5.D2 and 17A5) and to the bottom (mAbs H1/H2.1 and 8H4) in this study was implied by the impaired ability of heterologous mAbs to neutralize H1/H2.1 variants (Table 4
). An interaction between the H1/H2.1 and 3A5.D2 sites was particularly indicated by the greatly reduced ability of mAbs H1 and H2 to bind to 3A5.D2 variants. A cross-linkage between one of the three cysteine residues (aa 396) in the loop
3L2,3 and a cysteine residue in the middle of the large loop connecting the top of the
4-sheet to that of the
5-sheet (
4L1,0) has been previously proposed (Langedijk et al., 1997
). Thus, the top of the
3-sheet in the L2,3 loop (the postulated site of the 3A5.D2 epitope) could directly influence the underside of the globular head in sheets
46, which appears to include the conformation-dependent H1/2.1 epitope. Of further interest was the observation that the H1/H2.1 epitope related site VI (aa 447, postulated to lie on the
4S1 strand near the base of the globular head) is analogous to a region on MeV H (aa 429438), which has been shown to be associated with binding to the known host cell receptor, signalling lymphocyte activation molecule (SLAM) (Hu et al., 2004
). In addition, site VIII (aa 570) maps relatively closely to specific amino acids (546, 548 and 549) recognized to be within a region of MeV H (approx.
5S4 to
6S1 in the postulated model) that has been implicated in haemadsorption and cell receptor (CD46) binding (Masse et al., 2002
, 2004
). Therefore, similarly to MeV, sites VI and VIII, although associated with the base of the globular head of HeV G, may still have an involvement in receptor binding.
Immunological relationships between HeV and NiV G proteins
The binding of all five mAbs used in this study was significantly inhibited by field-derived human, bat and horse antiserum to HeV and also pig antiserum to NiV, when tested in a competitive ELISA format incorporating expressed HeV G (J. R. White, V. Boyd, G. S. Crameri & C. J. Duch, unpublished results). Considerable cross-neutralization also occurs between these viruses (Harcourt et al., 2000; Wang et al., 2001
) and between their G proteins (Bossart et al., 2005
) using polyvalent antisera. However, mAbs that neutralized both HeV and NiV were not isolated in this study. Rather, there seemed very little conservation of neutralization epitopes between HeV G and NiV G (Table 1
), with only one (HeV1/2.1) exhibiting relatively high levels of conserved amino acid similarity at three of four sites related to this epitope (Fig. 2
). Thus, other neutralizing epitopes remain to be identified on HeV G. Alternatively, because the mAbs possessed a reduced affinity for NiV (Table 1
), the multi-hit model for virus neutralization (Burton et al., 2001
) might explain how individual neutralizing mAbs, each with reduced affinity for a heterologous virus, may still achieve heterologous neutralization when acting together, as would occur in a polyclonal antiserum response.
Structural relationships between henipaviruses and other Paramyxovirdae
Despite the proposed folding pattern of the globular head region of HeV G appearing most similar to respiroviruses (Langedijk et al., 1997; Yu et al., 1998
; Wang et al., 2001
; Eaton et al., 2004
), the location of the epitopes described in this study resembled those found on at least two morbilliviruses (Makela et al., 1989a
, b
; Hu et al., 1993
, 2004
; Hu & Norrby, 1994
; Ziegler et al., 1996
; Cusi et al., 2001
; Moeller et al., 2001
; Sugiyama et al., 2002
; Putz et al., 2003
; Masse et al., 2002
, 2004
). Recent publications of revised three-dimensional models for the general structure of the H protein of morbilliviruses (Masse et al., 2004
; Vongpunsawad et al., 2004
), based upon the known crystal structure of Newcastle disease virus HN protein (Crennell et al., 2000
), also indicate the similarities between HeV and respiroviruses are less remarkable than previously implied, particularly for sites on the
6-sheet (Langedijk et al., 1997
; Masse et al., 2004
; Vongpunsawad et al., 2004
). Henipaviruses, like morbilliviruses but unlike most other Paramyxoviridae, possess no N activity (Murray et al., 1995
; Wang et al., 2001
) and indeed, some of the virus neutralization related amino acid sites identified in this study appear to be in similar locations to sites implicated in MeV cell receptor binding (Masse et al., 2002
, 2004
; Hu et al., 2004
; Vongpunsawad et al., 2004
). It is hoped the data and hypotheses presented in this study will assist with ongoing efforts to characterize further the nature of Henipavirus neutralization by host antisera and help to locate the host cell receptor site(s) on HeV G that will ultimately facilitate identification of potential host cell receptor molecules (Bossart et al., 2002
; Eaton et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 30 May 2005;
accepted 11 July 2005.