Localization of a new neutralizing epitope on the mumps virus hemagglutinin–neuraminidase protein

Virus Research 74 (2001) 133 – 137 www.elsevier.com/locate/virusres

Localization of a new neutralizing epitope on the mumps virus hemagglutinin–neuraminidase protein Maria Grazia Cusi a,*, Susanne Fischer b, Reinhard Sedlmeier b, Marcello Valassina a, Pier Egisto Valensin a, Marco Donati a, Wolfgang Jens Neubert b a

Department of Molecular Biology, Section of Microbiology, Uni6ersity of Siena, Via Laterina, 8 -53100 Siena, Italy b Max-Planck-Institut fu¨r Biochemie, MolekulareVirologie, 82152 Martinsried, Germany Received 16 August 2000; received in revised form 30 November 2000; accepted 30 November 2000

Abstract Four protein fragments which span the entire hemagglutinin– neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213–372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329 – 340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response. © 2001 Elsevier Science B.V. All rights reserved.

1. Introduction Mumps virus is a human pathogen, which can infect the glands, the central nervous system, the respiratory tract and possibly also muscle and connective tissue (Wolinsky and Waxham, 1990). This spectrum of susceptible organs indicates the occurrence of a wide variation of host cells permissive for viral infection. Studies with monoclonal antibodies (mAbs) suggest that the hemagglutinin–neuraminidase protein (HN) is the * Corresponding author. Tel.: +39-0577-233850; fax: +390577-233870. E-mail address: [email protected] (M.G. Cusi).

major target for the humoral immune response upon mumps virus infection (Wolinsky et al., 1985). However, few data are available concerning the mapping of important HN epitopes, in particular of epitopes inducing the synthesis of neutralizing antibodies. Virus escape mutants induced by selection pressure through neutralizing monoclonal antibodies (mAbs) have been employed to identify regions of HN protein responsible for protection against mumps virus. One putative region for an epitope that is recognized by a selection of neutralizing mAbs was mapped in a hydrophilic region encompassing amino acids 352–360 (aa) of the HN protein (Ko¨vamees et al., 1990).

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The 3D structure of the paramyxovirus hemagglutinin–neuraminidase protein has not been defined yet, however, synthetic peptides or protein fragments expressed in mammalian cells have often been exploited for a first screening of epitopes capable to induce neutralizing antibodies (Van der Werf et al., 1994; Anderson et al., 1995; Zamorano et al., 1995; Rigby et al., 1996; Jurkiewicz et al., 1997, Van Regenmortel and Muller, 1998; Plotnicky-Gilquin et al., 1999). This approach appears to be a valid method to localize important viral epitopes involved in virus neutralization and allows the study of the immunogenic profile of viral antigens. We, therefore, applied this methodology to define the immunogenicity of HN protein. The aim of our study was to characterize further putative neutralizing epitopes by using sera of mice previously immunized with fragments of HN protein and to confirm and localize those putative epitopes on HN protein by using synthetic peptides. Four fragments of HN were synthesized in eukaryotic cells via the vaccinia virus expression system. For cloning of the HN gene, RNA was extracted from Vero cells infected by MuV wild type (isolated in the Virology lab., Siena Cusi et al., 1998), as shown using guanidine isothiocyanate (Chomczynsky and Sacchi, 1987). Reverse transcription of HN-RNA was carried out with primer Mu1 (nt 78 – 99 of the HN gene) and subsequent PCR amplification was performed with the primers Mu1 and Mu2 (nt 1826 – 1849). The amplification product was purified (QIAGEN) and cloned into the pTM1 expression vector at the unique NcoI site (Moss et al., 1990). The construct pTM HN was confirmed by DNA sequencing with a Sequenase kit (United States Biochemical Corp.). Four selected areas of the MuV HN gene encoding the protein fragments HN1: aa 1 – 80, HN2: aa 81–212, HN3: aa 213 – 372 and HN4: aa 373 – 582, were then amplified by PCR using the plasmid pTM HN as template and the subfragments were cloned into the NcoI site of pTM1. The constructs were confirmed by restriction analysis and DNA sequencing. For eukaryotic expression of the subfragments, HeLa cells were infected with the recombinant Vaccinia virus vTF7-3 ex-

pressing the T7 polymerase (multiplicity of infection= 8) and incubated for 1 h at 33°C. After removal of the virus suspension, cells were transfected with the plamids pTMHN1, pTMHN2, pTMHN3 or pTMHN4, respectively, using lipofectamine (Gibco BRL) for 4 h. pTM1-plasmid without viral genes was included as a negative control. After 24 h of incubation, cell supernatants were removed, cells were lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris; pH 7.5) and sonicated for 30 s. All HN fragments expressed with the vaccinia T7 system lack a signal sequence for synthesis and transport in the endoplasmatic reticulum and the Golgi apparatus. The protein fragments are, therefore, non-glycosylated and any humoral immune response upon application will be directed against the peptide chain and not against any sugar moieties. However, in the cytoplasm the peptides can be folded spontaneously depending on their the primary amino acid sequence and when one of the fragments elicits a neutralizing antibody response, this strategy allows to narrow down the immunogenic domains using synthetic peptides. Total extracts of transfected HeLa cells were tested in immunoblots with and without renaturation of the proteins (Homann et al., 1991) using rabbit polyclonal anti-MuV serum (produced in our laboratory) or anti MuV HN monoclonal antibodies HN 11 (kindly provided by Professor Wolinsky). Briefly, renaturation was achieved by a modified western blot transfer protocol and a subsequent renaturation step (for details see Homann et al., 1991). After this treatment not all proteins or protein fragment recover their ability to bind antibodies. The missing reactivity of the protein fragments HN 1–2 and 4 could be due to an incorrect folding. However, fragment HN3 reacted with rabbit polyclonal serum or the mAb HN 11, which had neutralizing activity against the wild type virus (Kilham strain) but only after renaturation of the protein (Fig. 1). This result indicates that conformational epitopes present on the HN3 fragment have to be restored after the separation and blotting procedures in order to allow a recognition by the MuV specific antibodies.

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Fig. 1. Detection of the renatured HN3 fragment by immuno blotting. Extracts from HeLa cells (30 mg proteins per well) containing the mumps virus HN3 fragment (lane 2) were separated by 15% SDS – PAGE and transferred to nitrocellulose. Specific anti-MuV monoclonal (mAbs), or polyclonal (pAbs) antibodies were tested after renaturation of the proteins. Extracts from HeLa cells transfected with empty pTM1 vector (lanes 1), and MuV proteins (lane 3). The relative mobilities of protein standards (kDa) are indicated.

In order to test the immunogenicity of the expressed HN fragments, five 8-week-old female BALB/c mice (Charles Rivers) were immunized intraperitoneally (ip) four times at 15 day intervals with 100 ml of the sonicated RIPA extracts ( $ 5× 106 cells) containing the expressed HN1, HN2, HN3 or HN4 fragments. The same schedule of immunization was applied to a group of five mice immunized with lysates of cells transfected with the empty pTM1 plasmid (negative control, anti-pTM1). A positive control was represented

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by a group of mice immunized intramuscularly (im) twice with 105 pfu of live MuV (Urabe Am9 vaccine). Sera were collected 7 days after the last immunization and tested by ELISA (O8 rvell, 1984) against purified mumps virus proteins. All sera (anti-HN1-4) drawn from mice immunized with HN1, HN2 HN3 or HN4, respectively, revealed the presence of anti-MuV antibodies, indicating that these protein fragments maintained some MuV specific immunogenic epitopes and were able to elicit an humoral immune response (Table 1). Using the hemagglutination-inhibiting assay (HAI) (O8 rvell, 1976), and the plaque reduction neutralization assay (PRNT) as described by O8 rvell et al. (1997), anti MuV antibodies with hemagglutination-inhibiting (HAI, 1/20) and neutralizing (PRNT, 1/16) activity were detected in the anti-HN3 serum. The anti-HN3 serum showed neutralizing activity to the Urabe strain (E/K at aa 335) and to a wild type strain (K at aa 335) of the mumps virus isolated in our laboratory (Table 1). In order to verify the neutralizing activity induced by HN3 and to define a first amino acid sequence corresponding to a neutralizing epitope, a synthetic peptide spanning aa 329–340 NSTLGVKSAREF was employed. By computer analysis, this peptide shows a high surface probability (Chou and Fasman, 1974), and it is supposed to be part of a new putative antigenic domain. One

Table 1 Titers of ELISA-, HAI-, and neutralizing antibodies in sera of mice immunized with the HN1-4 fragments and the synthetic peptide a Serum

ELISA

HAI

PRNT versus wt

PRNT versus Urabe

Anti-HN1 Anti-HN2 Anti-HN3 Anti-HN4 Anti-peptide Anti-MuV Anti-pTM1

1/800 1/1600 1/600 1/800 1/300 1/5120 Neg

Neg Neg 1/20 Neg 1/20 1/160 Neg

Neg Neg 1/16 Neg 1/16 1/64 Neg

Neg Neg 1/16 Neg Neg 1/64 Neg

a

HAI, hemagglutination-inhibiting assay with wild type, and Urabe virus strains. NT titre was evaluated as the highest dilution of serum capable to reduce the number of plaques by 50%; anti-MuV, polyclonal serum; neg, negative all experiments were performed independently three times.

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of the escape mutants (M11) analyzed by Ko¨vamees et al., showed a non-conservative amino acid substitution at position 329 (D instead of N), thus destroying a putative glycosylation site (NST, aa 329–331), which should have a high surface probability (Kaumaya et al., 1995). Furthermore, an important mutation responsible for the attenuation of the MuV Urabe vaccine strain has been located at the adjacent amino acid 335 (Brown et al., 1996, 1997; Afzal et al., 1998). The Urabe Am9 vaccine is a mixture of viruses differing at aa 335 (K/E) where the strain containing E at position 335 seems to be less neuropathogenic (Brown and Wright, 1998). Moreover, the HN protein of mumps wildtype viruses shows a lysine (K) at aa 335 (Cusi et al., 1998). This part of the HN protein spanning aa 329 – 335 could, therefore, not only be relevant for the virulence of mumps virus but also for the induction of neutralizing antibodies. Another group of five mice was immunized ip with the synthetic peptide NSTLGVKSAREF (aa 329 – 340; MedProbe) coupled to KLH (50 mg per dose each mouse) (Briand et al., 1985) in complete Freund’s adjuvant. The dodecapeptide proved competent to evoke a measurable humoral antibody response (anti-peptide serum) (Table 1). In the ELISA assay, the anti-HN1-4 sera reacted stronger with the MuV proteins than the anti-peptide serum (1/1600-800 vs. 1/300, Table 1). The anti-peptide sera contained anti MuV antibodies with hemagglutination-inhibiting activity (HAI, 1/ 20) to the wildtype and the Urabe strains, just like the anti-HN3 serum. While the HAI titre was similar versus the two strains tested, the neutralizing activity was different. In fact, the neutralizing activity of the anti-peptide serum was able to neutralize the mumps wildtype virus (with K at aa 335) isolated in our laboratory, but it was unable to neutralize the Urabe Am9 virus (Table 1). This could be due to the fact that the peptide selected in our study contains a lysine (position 7) instead of a glutamic acid which is present only in the HN sequence of the attenuated variant of the Urabe Am9 mumps vaccine (Brown et al., 1996). This result supports the hypothesis that the amino acid substitution at aa 335 could also be involved in antiviral protection. Analysis of meningitis

mumps isolates following Urabe Am9 vaccination showed that the clinical isolates were homogeneous and possessed the wild type K335 in HN protein (Brown and Wright, 1998). The E335 mutation seems, therefore, to be a crucial aminoacid within a neutralizing epitope of HN protein. When escape mutants of the Urabe strain were isolated under the negative selection pressure of neutralizing MAbs, two of them showed an identical exchange E335 to K335 in addition to other mutations (Yates et al., 1996). In fact, a Urabe specific monoclonal antibody was described that neutralizes MuV with E335 (Urabe) but not wild type virus with K335 (Afzal et al., 1998). However, the fact that anti-HN3 serum is able to neutralize both the attenuated and the wild type viruses demonstrates that in addition to the epitope aa 329–340 at least another epitope(s) such as aa 352–360 exists that is involved in virus neutralization. Nonetheless, the ability of the sera to neutralize the infectivity of MuV confirmed the capacity of the HN3 fragment and, in particular, of the peptide to induce a population of antibodies reactive to viral surface epitopes. The region aa 329–340 could be one of the important epitopes involved in the virulence and antiviral protection. Studies are now in progress in order to analyze systematically the whole aa 213–372 HN region including the role of lysine at residue 7 of the synthetic peptide and to define the precise sequences capable to induce a neutralizing antibody response. The lack of an animal model suitable to show the MuV pathogenesis has slackened the molecular study of this virus. However, since the HN protein represents the major target to induce a protective immune response to mumps virus (Houard et al., 1995), these results could be a step towards a genetically engineered vaccine against mumps virus infections.

Acknowledgements The authors would like to thank Dr J. Wolinsky for the gift of anti-HN monoclonal antibodies. This work was supported by the EU grant BIOMED BMH1CT941600.

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