Rubella virus nonstructural protein 2 is a minor ... - Semantic Scholar

Virus Research 68 (2000) 155 – 160 www.elsevier.com/locate/virusres

Rubella virus nonstructural protein 2 is a minor immunogen J. Hofmann a, S. Gerstenberger a, I. Lachmann a,b, C.D. Atreya c, U.G. Liebert a,* a

Institut fu¨r Virologie, Uni6ersita¨t Leipzig, Johannisallee 30, 04103 Leipzig, Germany b Institut fu¨r Zoologie, Uni6ersita¨t Leipzig, Talstrasse 33, 04103 Leipzig, Germany c Food and Drug Administration, Bethesda, MD, USA

Received 1 February 2000; received in revised form 24 May 2000; accepted 25 May 2000

Abstract The full-length nonstructural protein P90 of rubella virus (RV) was expressed as recombinant protein in Escherichia coli bacteria, as well as in Vero cells. Monoclonal antibodies raised against the protein specifically reacted with the protein in both P90-transfected and RV infected Vero cells. Ninety human sera obtained from reconvalescents, vaccinees and patients with acute RV infection were tested for reactivity against the P90 protein. A weak immune reaction was detected only in a small minority (8%), indicating that P90 is minor immunogen for RV and is not suitable for diagnostic purposes. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Rubella; Nonstructural protein; Immunity; Monoclonal antibodies

1. Introduction Rubella virus (RV) is a single-stranded RNA virus of positive polarity. The genome contains two open reading frames coding for precursor proteins that are proteolytically cleaved into the nonstructural (150 kDa P150 and 90 kDa P90) and structural (C, E2 and E1) proteins. Rubella virus capsid and envelope proteins are highly immunogenic for both T and B cells (Chaye et al., 1993; McCarthy et al., 1993) and can be used for * Corresponding author. Tel.: + 49-341-9714300; fax: +49341-9714309. E-mail address: [email protected] (U.G. Liebert).

the diagnosis of rubella infections or immunity. Recombinant RV structural proteins may serve as antigens in various serological techniques (Hobman et al., 1994; Pustowoit and Liebert, 1998), and molecular methods including sequence analysis have been introduced into the diagnostics of RV infections particularly during pregnancy (Hofmann et al., 2000). Nonstructural proteins of several viruses are important T and B cell antigens that may play a role in the pathogenesis and can be used as diagnostic tools. Examples include polio virus (Ehrenfeld et al., 1995), hepatitis A (Stewart et al., 1997) and C (Beld et al., 1999), rota virus (Colomina et al., 1998), parvovirus B19 (Gigler et al., 1999; Hemauer et al., 2000) or dengue virus type 2

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(Se-Thoe et al., 1999). However, little is known about the RV nonstructural proteins as targets for an immune response. Although the genome coding for RV-P90 has been cloned (Atreya et al., 1998), no data regarding its immunogenicity have been obtained so far. This report describes attempts to investigate the immunogenicity of RVP90 protein and its usefulness in RV diagnostics. Therefore, pro- and eukaryotic expressed RV-P90 was used as antigen for both the generation of monoclonal antibodies and to search for antibody reactivity in a panel of sera collected from patients with various types of RV infection.

2. Materials and methods

2.1. Full length RV-P90 containing plasmids 2.1.1. Construction of pMal-P90 for bacterial expression The complete coding sequence for RV-P90 (amino acids 1310–2206) was amplified using RV specific PCR primers and cloned into a pMal-c2 vector (New England Biolabs). The 5% primer contained an EcoRI site and the 3% primer a HindIII site. Ligation with T4 ligase (Boehringer Mannheim, Germany) and transformation into DH5 alpha LE cells (Life Technologies, USA) were all performed according to the supplier’s instructions. The resulting RV-P90 has been expressed as a fusion protein with the maltose binding protein (MBP, molecular weight 41 kDa, total molecular weight 131 kDa) and purified by affinity chromatography using amylose resin columns according to the supplier’s protocol (New England Biolabs, USA). 2.2. Construction of pcDNA3 -P90 for eukaryotic expression and transfection of animal cells The P90 gene was PCR amplified and cloned into a pcDNA3 plasmid (Invitrogen, CA) as described earlier (Atreya et al., 1998). The plasmid was used in a T7 polymerase-driven TNT in vitro translation system (Promega, USA) in the presence of 35S-labeled methionine to produce radiolabeled protein according to the suppliers’ protocol.

Vero cells were stabely transfected using lipofectamine and 5 mg linearized (PvuI digest) pcDNA3P90 plasmid resulting in P90-Vero. Clonal selection (neomycine resistance) was achieved by the addition of 800 mg/ml geniticine sulphate (G418). Vero cells transfected with empty pcDNA3 vector served as negative control (V32Vero).

2.3. Cell culture, Western blot, immunoprecipitation and -fluorescence P90-Vero as well as V32-Vero were grown in DMEM supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, antibiotics and 200 mg/ ml G418. For Western blot, total cell protein lysates were prepared, separated on a 10% SDS gel (20 mg of cell lysate or 5 mg of recombinant MBP-P90) and electroblotted onto immobilon membrane (Millipore) as described earlier (Pogue et al., 1996). The blocked membranes were cut into strips and incubated with patient sera diluted from 1:20 to 1:100 in TBST, followed by incubation with anti human IgG conjugated with alkaline phosphatase (diluted 1:3000, DAKO, Denmark). BCIP/NBT was used as substrate to visualize immunoreaction. Immunoprecipitation of P90 from transfected cells was performed to confirm specificity of monoclonal antibodies using 150 mg of proteins per sample. The P90-anti P90 complexes were precipitated by protein A coated to sepharose 4B beads (Pharmacia), washed, subjected to SDS-PAGE 10%, followed by silver staining. For immunofluorescence assays, confluent Vero76 cell cultures grown on Permslides (Heraeus, Germany) were used. P90-Vero cells served as antigen for the detection of anti RV-P90 antibodies and RV M33 infected cells as the target for determination of the immune status to rubella.

2.4. Immunization schedule and hybridoma generation The RV-P90 preparation (50 mg) was mixed with GERBU adjuvant (GERBU Biotechnik GmbH, Germany) and used to immunize female Balb/c mice. After ten days, single cell suspensions were prepared from popliteal lymph nodes


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Fig. 1. Organization of RV genome and cloning strategy for RV-P90 in bacterial and eukaryotic expression plasmids. Since RV-P90 is derived in vivo from a precursor protein and thus does not include an ATG start codon for translation this codon was introduced into the pcDNA3 plasmid by modified 5% primer.

according to Holmdahl et al. (1985). Hybridoma generation and antibody production were performed as described earlier (Lachmann et al., 1998) and immunoglobulin isotypes were determined by a mouse-hybridoma-subtyping kit (Boehringer Mannheim, Germany).

2.5. Human sera Well characterized patient sera (regarding total anti rubella IgG, immunoblot and IgG avidity (Pustowoit and Liebert, 1998) were used for P90antibody screening, including 40 sera from people with naturally acquired rubella more than 2 years ago (20 sera with lower titers ranging from 25 to 150 IU/ml and 20 with higher titers from 150 to 501 IU/ml, panel A), ten sera from patients with fresh rubella infection (approximately 4 weeks post infection (pi), panel B), 20 sera from five vaccinees collected at defined time-points post vaccination (2, 4, 26 and 52 weeks post vaccination titers less than 166 IU/ml, panel C), five samples from patients with congenital rubella syndrome (CRS, titers from 280 to 458 IU/ml, panel D), and 15 samples from patients seronegative for rubella (specific IgGB4 IU/ml, panel E).

was used to immunize Balb-c mice for the generation of monoclonal antibodies. Two monoclonal antibodies of IgM type, two of IgG1, designated as 1G7 and 2B5, and one of IgG2 type (5D6) were obtained. The monoclonal antibodies recognize the fusion protein MBP-P90 in EIA and immunoblot but not the MBP protein alone indicating their specificity for RV-P90. The IgG antibodies immunoprecipitate RV-P90 protein from cell lysates of P90-Vero and from cells acutely infected with RV strain M33 (Fig. 2). All monoclonal antibodies exhibit reactivity in enzyme immuno assays using RV infected cells or P90 transfected cells as target (Table 1). The in vitro synthesis of RV-P90 (aa 13102206) from the pcDNA3-P90 plasmid was demon-

3. Results Two plasmids containing the entire sequence coding for the RV P90 (Fig. 1) for either pro- or eukaryotic expression, were generated by PCR cloning. The fusion protein MBP-P90 expressed from the pMal-P90 plasmid in Escherichia coli

Fig. 2. Detection of RV-P90 using monoclonal antibodies 5D6. Lanes 1 – 3, immunoblot (IB) on P90 Vero cell protein lysate (lane 1), V32 Vero cell protein lysate (lane 2) and MBP-P90 fusion protein (lane 3). Lanes 4 – 7, immunoprecipitation (IP) of RV-P90 from P90-Vero (lane 4), V32-Vero (lane 5), RV-M33 infected (lane 6) and mock infected cells (lane 7).

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Table 1 Reactivity of monoclonal antibodies to RV-P90 Antibody designation Immunoglobulin type

1E7 IgM

5D6 IgG 2b

1G7 IgG 1

2B5 IgG 1

2G6 IgM

Western blota EIAb Immunoprecipitationc

+ + n.a.d

+ + +

+ + +

+ + +

+ + n.a.d

a

Antigens used were MBP-P90 and P90-Vero cell lysate. Antigens used were MBP-P90, RV-infected Vero cell lysate, and P90-Vero cell lysate. c Antigens used were RV-infected Vero and P90-Vero cell lysate, respectively. In addition, each experiment was controlled by using MBP alone or V32-Vero as the control antigen. d n.a., Not applicable. b

strated in a transcription-coupled translation system using 35S-labeled methionine while the empty pcDNA3 vector did not result in RV-specific protein synthesis (data not shown). Transfection of the pcDNA3-P90 plasmid into Vero cells resulted in stable expression of RV-P90 as determined by immunoblot using the monoclonal antibody 5D6. Initial attempts to detect anti RV-P90 antibodies in human sera were performed by immunofluorescence assays on P90-Vero cells. Of the 90 sera tested, none exhibited any reactivity at dilutions from 1:10 to 1:1000. With the exception of the negative sera (panel E) all were determined to be positive on cells acutely infected with the Rubella virus wild type strain M33. Western blot analysis using the MBP-P90 fusion protein as antigen was performed, six sera of panel A and one sample of panel D weakly stained the 131 kDa MBP-P90 fusion protein (Fig. 3). Since no reactivity in the region of 41 kDa was found using MBP alone the 131 kDa protein is obviously detected by anti P90 antibodies. Preincubation of a serum with a protein lysate of P90-Vero leads to the loss of its ability to detect the 131 kDa fusion protein. Anti-P90 activity has been detected exclusively in sera with high anti rubella IgG titers ranging from 256 to 501 IU/ml (mean 330 IU/ml). As summarized in Table 2 none of the samples with lower anti rubella IgG titers exhibited any reactivity to RV-P90. However, high total anti rubella

IgG does not result in presence of anti P90 antibodies necessarily as demonstrated by only six anti P90 positive sera out of the 20 of panel A (Fig. 4) and one positive sample out of the five of panel D.

Fig. 3. Detection of RV-P90 expressed as fusion protein with MBP by representative patient sera. The strips 1 – 4 were four of six high titer RV sera while strip 5 was incubated with a low titer RV serum of panel A, repectively. MBP alone obtained from the pMal-c vector was used in strip 6 and incubated with the sample that were used on strip 3. The faint band at 131 kDa represents the immune reaction to MBP-P90 fusion protein while the lower band at approx. 75 kDa could result from a putative degradation product of RV-P90. The dark band at approximately 80 kDa is probably caused by nonspecific binding to bacterial protein (E. coli ). Note, p41 (MBP) was not detected.


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Table 2 Reactivity of patient sera to RV structural and nonstructural proteins determined by western blot analysis Description of sera

Number of sera tested

Percentage of sera detecting P90

Immune (panel A, high titers) Immune (panel A, low titers) Freshly infected (panel B) Vaccinees (panel C) CRS (panel D) Negative controls (panel E) Total a

20 20 10 20 5 15 90

30 0 0 0 20 0 8

C 100 100 100 100 100 0 83

E2 100 100 0 50a 100 0 61

E1 100 100 100 100 100 0 83

The samples obtained 2 and 4 weeks after vaccination were still negative for anti RV-E2.

4. Discussion Structure, composition and overall genome organization of rubella virus is similar to that of alphaviruses. However, its nearest relatives, Sindbis (SIN) and Semliki Forest (SFV) virus contain four nonstructural proteins. Replicase (GlyAspAsp) and helicase (GlyLysThr) motifs are located within the P90 protein of rubella virus, but in reverse order as compared with SIN and SFV (Dominguez et al., 1990). Due to low level viral replication and the fact that RV does not shut-off host cell macromolecular synthesis, the detection of virus specific nonstructural proteins in infected cells against the host cell background is difficult. There is only one report that describes RV proteins (Bowden and Westaway, 1984) that were immunoprecipitated from infected cell lysate using a single convalescent serum. Despite the use of radiolabeled cells and immunoprecipitation, the nonstructural proteins were detected as faint bands, suggesting the low expression levels of RV-P90 in infected cells. Additionally, chase experiments revealed that RV-P90 underwent rapid degradation (Forng and Frey, 1995). Nonstructural proteins can serve as antigen against which a humoral immune response is directed. The response to these proteins may appear simultaneously with antibodies to structural proteins. This study demonstrated that RV-P90 is immunogenic, since monoclonal antibodies were raised against the protein. The RV-P90 monoclonal antibodies described in this report detect

RV-P90 in different assays, i.e. in Vero cells either transfected with an expression plasmid or infected with RV by ELISA, immunoblot and immunoprecipitation. However, the protein-specific antibodies were only present in a minority of RV-infected subjects. There was no correlation between the presence of anti RV-P90 IgG and the type of infection or the quantity of total anti Rubella IgG present in a given individual. On one hand, none of the sera obtained from vaccinees or primary infected patients (low total anti RV IgG) contains anti RV-P90 IgG, on the other hand, it is only in a minority of sera even with high titers of total anti rubella IgG that do contain anti RV-P90

Fig. 4. P90 reactivity of rubella reconvalescent sera of panel A, correlation to higher anti rubella IgG () and lower anti rubella IgG () content.

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IgG. Taken into account that P90 appears early in the virus replication cycle and is expressed in small amounts, the rather weak humoral immune response is conceivable.

Acknowledgements Expert technical assistance by Marion Kortung is gratefully acknowledged.

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