Mapping of an Antigenic Site on the NucleocapsidProtein of Human Parainfluenza Virus Type 3

Aurelija Zvirbliene,1 Indre Sezaite,1 Milda Pleckaityte,1 Indre Kucinskaite-Kodze,1

Mindaugas Juozapaitis,2 and Kestutis Sasnauskas2

Abstract

Human parainfluenza virus type 3 (hPIV3) is a respiratory tract pathogen. The current study aimed to investigateimmunodominant regions of hPIV3 nucleocapsid (N) protein by using monoclonal antibodies (mAbs) raised againstrecombinant N protein and human serum specimens from hPIV3-infected individuals. A panel of murine mAbs wasgenerated following immunization with yeast-expressed hPIV3 N protein self-assembled to nucleocapsid-likeparticles. All mAbs recognized native viral nucleocapsids in hPIV3-infected cells as confirmed by an indirectimmunofluorescence analysis. Antigenic sites recognized by the mAbs were mapped using recombinant over-lapping N protein fragments. One major immunodominant site was identified in the carboxy-terminal region(amino acids [aa] 397–486) of hPIV3 N protein. Further analysis with smaller N protein fragments and a syntheticpeptide revealed one linear epitope representing aa 437–446 of the N protein located within this antigenic site. Thisepitope was reactive with 46% of hPIV3 IgG-positive sera. These results suggest that the above antigenic site on the Nprotein is important in eliciting a humoral immune response against hPIV3.

Introduction

Human parainfluenza viruses (hPIVs) are nonseg-mented RNA viruses that belong to the family Para-

myxoviridae. HPIV-1, -2, and -3 are important respiratorypathogens and are major causes of croup, bronchiolitis, andpneumonia in infants and very young children (11,20). Theyhave been estimated to be the cause of 40% of acute respi-ratory tract illnesses in children from which a virus is re-coverable, and 20% of respiratory illnesses in hospitalizedchildren (15).

Human parainfluenza virus type 3 (hPIV3) is an envelopedRNA virus containing a single-stranded RNA genome ofnegative polarity. HPIV3 is a representative of the genusRespirovirus of the subfamily Paramyxovirinae (5). Two sur-face glycoproteins are found in hPIV3: the hemaglutinin-neuraminidase (HN) and the fusion protein (F). The hPIV3HN and F proteins are the only significant neutralizationantigens and the major protective antigens (19). The matrixprotein (M) is strongly associated with and found just beneaththe viral membrane. Phosphoprotein (P), the ‘‘large’’ (L) pro-tein (RNA-dependent RNA polymerase) and the nucleocap-sid (N) protein are closely associated with the viral RNA (5).The N protein is highly conserved with regard to amino acid(aa) sequence and associates with genomic and antigenomic

RNA to form highly stable, ribonuclease-resistant helicalnucleocapsid. N protein can participate in nucleocapsid for-mation only when it is available in a soluble complex withP. The amino-terminal region of hPIV3 N protein (aa 1–400) isthe most conserved part involved in forming soluble complexwith P, and subsequently associating with other N proteinmonomers and viral RNA to form nucleocapsid. The morevariable carboxy-terminal region of N protein is supposed tobe essential for the interaction of assembled nucleocapsidwith P and M (1–3,22). Despite structural and serologicalsimilarity between hPIV3 and human parainfluenza virustype 1 (hPIV1), another member of the Respirovirus genus,the overall aa sequence identity between their N proteinsis 59% (9).

The knowledge of the regions representing immuno-dominant antigenic sequences of hPIV3 N protein is limited,although the aa sequence of hPIV3 N protein was determineddecades ago (4). Attempts were made to characterize theantigenic structure of hPIV3 N protein by using N protein-specific monoclonal antibodies (mAbs) from a panel ofmAbs raised against the major structural proteins of hPIV3;however, the epitopes were localized only in a competitivemanner (7,13,16).

Recent studies indicated that recombinant yeast-expressedN protein of hPIV3 forms nucleocapsid-like particles (NLPs)

1Laboratory of Immunology and Cell Biology, and 2Laboratory of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius,Lithuania.

VIRAL IMMUNOLOGYVolume 22, Number 3, 2009ª Mary Ann Liebert, Inc.Pp. 181–188DOI: 10.1089=vim.2008.0106

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that share structural and antigenic features with viral nucle-ocapsids, and can be used for the detection of virus-specificantibodies induced by a natural hPIV3 infection (6).

In the current study we have used a panel of mAbs raisedagainst recombinant NLPs to investigate the antigenicstructure of hPIV3 N protein in more detail. We have iden-tified an antigenic site within the carboxy-terminus of hPIVN protein and mapped a linear epitope that was recognizedboth by mAbs and serum antibodies from convalescenthPIV3 patients.

Materials and Methods

Recombinant antigens and synthetic peptide

Recombinant full-length hPIV3 N protein used for theimmunization of mice and mAb screening was expressed inthe yeast Saccharomyces cerevisiae and purified as describedpreviously (6). The recombinant hPIV3 N protein formedNLPs as demonstrated by electron microscopy. RecombinantN proteins of hPIV1, measles virus, and human respiratorysyncytial virus (hRSV) used for testing of mAb specificitywere produced at the Institute of Biotechnology as describedpreviously (6,18). Identification of the linear epitope wasperformed using biotin-labeled synthetic peptide (Biosyntan,Berlin, Germany) GEPQSSIIQY spanning the sequence be-tween aa 437 and 446 of hPIV3 N protein.

Expression of GST-fused N protein fragments in E. coli

Vector pGEX-KT (Amersham, Helsinki, Finland) was usedfor the construction of glutathione S-transferase-(GST)-fusedoverlapping hPIV3 N fragments. For the initial localizationof mAb epitopes, three GST-fused N protein fragmentscomprised of aa 1–103 (fragment no. 1), aa 121–486 (fragmentno. 2), and aa 397–515 (fragment no. 3) of hPIV3 N proteinwere constructed. Fine mapping of antigenic sites was per-formed by constructing three additional GST-fused over-lapping fragments comprised of aa 397–436 (fragment no. 4),aa 417–456 (fragment no. 5), and aa 447–486 (fragment no. 6)of hPIV3 N protein. All GST-fused hPIV3 N protein frag-ments were expressed in E. coli. The expression of GST-fusedhPIV3 N protein fragments was confirmed by Western blotanalysis of total cell lysates with goat anti-GST polyclonalantibodies (GE Healthcare, Munich, Germany) as describedbelow.

Human serum specimens

Serum specimens from children with confirmed hPIV in-fection were received from the National Center of Micro-biology (ISCIII, Majadahonda, Madrid, Spain) and the Centerof Pediatrics, Clinic of Children’s Diseases, Vilnius University(Vilnius, Lithuania). Diagnosis of hPIV infection was per-formed directly on the cells of the respiratory secretions ofchildren with acute respiratory illness by indirect immuno-fluorescence staining as described previously (6). Serumspecimens were tested for the presence of IgG antibodiesagainst hPIV1=2=3 using a commercial HPIV1=2=3 IgG ELISAkit (IBL, Hamburg, Germany) in accordance with the manu-facturer’s protocol, and further tested for the presence of IgGantibodies against hPIV3 N protein as reported previously (6).The panel of human sera included 42 specimens collected

from hPIV-infected patients on days 7–30 after the onset ofdisease.

Production of monoclonal antibodies

Hybridomas producing mAbs against hPIV3 N proteinwere generated essentially as described by Kohler and Mil-stein (8). Eight-week-old female BALB=c mice (obtained froma breeding colony at the Institute of Immunology, Vilnius,Lithuania) were immunized at days 0, 28, and 56 by a sub-cutaneous injection of 50 mg of recombinant hPIV3 N proteinwithout an adjuvant. Three days after the final injection,mouse spleen cells were fused with Sp2=0-Ag 14 mouse my-eloma cells using polyethylene glycol 1500 (PEG=DMSO so-lution HybriMax; Sigma, St. Louis, MO). Hybrid cells wereselected in growth medium supplemented with hypoxan-thine, aminopterin, and thymidine (50�HAT media supple-ment; Sigma). Samples of supernatant from wells with viableclones were screened by indirect enzyme-linked immuno-sorbent assay (ELISA) using the hPIV3 N protein as describedbelow. Hybridomas secreting specific antibodies to hPIV3 Nprotein were subcloned twice by limiting dilution assay.Hybridoma cells were maintained in complete Dulbecco’smodified Eagle’s medium (Biochrom, Berlin, Germany) con-taining 15% fetal calf serum (Biochrom) and antibiotics. An-tibodies in hybridoma culture supernatants were isotypedusing the Mouse Monoclonal Antibody Isotyping kit (ISO-2,Sigma) in accordance with the manufacturer’s protocol.

Indirect ELISA

Microtiter plates (Nunc MaxiSorp, Roskilde, Denmark)were coated with 100 mL of recombinant purified hPIV3 Nprotein (5 mg=mL in 0.05 M Na-carbonate buffer, pH 9.5) andincubated overnight at 48C. The plates were blocked with 2%bovine serum albumin in PBS and washed with PBST (PBSand 0.05% Tween 20). Undiluted hybridoma supernatants orhuman convalescent patient sera diluted 1:100 in PBST wereadded to the wells and incubated for 1 h at 208C. The plateswere washed and incubated with 100mL of either horserad-ish peroxidase (HRP)-labeled anti-mouse IgG (BioRad, Her-cules, CA) or HRP-labeled anti-human IgG (BioRad) diluted1:2000 in PBST for 1 h at 208C. Antibody complexes weredetected using tetramethylbenzidine (TMB) substrate (Sig-ma), and the reactions were stopped with H2SO4 and theoptical density was read at 450 nm (OD450) by a microtiterplate reader (Tecan, Groedig, Austria). To test the reactivityof mAbs and human sera with a biotinylated syntheticpeptide, the plates were coated with 100mL of avidin (Am-resco, Solon, OH) at 2mg=mL in deionized water. Afterwashing, 100mL of the biotinylated peptide (Biosyntan) at aconcentration of 10 mg=mL were added to the avidin-coatedwells and incubated for 1 h at 378C. The plates were thenincubated with hybridoma supernatants or human sera anddeveloped as above.

For the peptide-based ELISA of human serum specimens,the cut-off value was defined as the mean OD450 value ob-tained with hPIV3 IgG-negative serum specimens (diluted1:100) plus 3 standard deviations. Seven human serumspecimens found to be negative both for hPIV1=2=3 IgG by acommercial assay and hPIV3 N protein-specific IgG by anin-house ELISA were used for cut-off calculation.

182 ZVIRBLIENE ET AL.

Western blot

Protein samples were boiled in a reducing sample bufferand subjected to sodium dodecyl sulfate-12% polyacrylamidegel electrophoresis (SDS-PAGE). After SDS-PAGE the pro-teins were transferred to a polyvinyldifluoride (PVDF)membrane (Millipore, Bedford, MA). The membranes wereblocked with 5% milk powder (Biochrom) in PBS for 1 hand then incubated with undiluted hybridoma supernatantsfor 1 h at 208C, or goat anti-GST polyclonal antibodies (GEHealthcare, Munich, Germany) at a dilution of 1:1000 inPBST. HRP-labeled anti-mouse IgG (BioRad) and donkeyanti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA)were used for the detection of specific antibody binding. Afterwashing, the membranes were developed with ready-to-use TMB-blotting substrate (Sigma).

Indirect immunofluorescence

To investigate the reactivity of mAbs with native viralnucleocapsids, biochip slides with hPIV1- and hPIV3-infectedcells were used (Euroimmun, Lubeck, Germany). The slideswere incubated with undiluted hybridoma supernatants for30 min at 208C, flushed for 1 sec, washed with PBST for5 min, and then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (BD Biosciences, FranklinLakes, NJ) for 30 min at 208C. The slides were flushed for 1 secand washed with PBST for 5 min, then examined with aninverted microscope (Olympus IX-70 (Olympus, Tokyo,Japan). As a positive control, human serum containing IgGagainst hPIV1 and hPIV3 was used. As a negative control,human serum non-reactive with hPIV1=hPIV3 was used. Thenegative and positive controls were provided by the manu-facturer (Euroimmun).

Results

Production and characterization of mAbsagainst hPIV3 N protein

Recombinant hPIV3 N protein expressed in yeast cells wasused here as the immunogen for production of mAbs. Elec-tron microscopy analysis revealed that purified hPIV3 Nprotein was self-assembled to herring bone–like structuressimilar to native viral nucleocapsids. Splenocytes from

BALB=c mice immunized with purified hPIV3 N proteinwere fused with murine myeloma cells to generate hybrid-omas. Five hybridomas giving initial ELISA reactivities(OD450) against hPIV3 N protein >2.0 were identified andsubcloned. Four stable hybridoma cell lines producing mAbsof IgG isotype were obtained. Three mAbs were of IgG1subtype (5F12, 2H1, and 11H7) and one was of IgG3 subtype(5C9).

As a first step in examining the specificity of the mAbs, theywere analyzed by indirect ELISA and Western blot for cross-reactivity with different yeast-expressed antigens. All mAbsreacted specifically with hPIV3 N protein and did not cross-react with hPIV1 N protein or recombinant yeast-expressedhRSV N protein used as a negative control (Fig. 1). The mAbsdid not cross-react with recombinant measles virus N protein(data not shown). As a next step, the mAbs were tested forreactivity with native nucleocapsids of hPIV3 by indirect im-munofluorescence. All mAbs showed strong reactivities withmammalian cells infected with wild-type hPIV3 (Fig. 2A).These results demonstrate that the epitopes recognized by themAbs are exposed in viral N protein thus confirming the an-tigenic similarity between yeast-expressed NLPs and viralnucleocapsids. As expected, the mAbs did not cross-react withhPIV1- infected cells (Fig. 2B).

Epitope mapping

To determine the epitope specificities of the mAbs, a seriesof GST fusion proteins containing partially overlappingfragments of hPIV3 N protein were constructed (Fig. 3) andtested with each mAb in Western blots. For the initial lo-calization of mAb epitopes, three GST-fused hPIV3 N proteinfragments were employed: aa 1–103 (fragment no. 1), aa 121–486 (fragment no. 2), and aa 397–515 (fragment no. 3). AllmAbs were reactive with overlapping fragments 2 and 3,which suggests that they recognize epitopes in the region ofaa 397–486 of hPIV3 N protein (Fig. 4B, lanes 2 and 3). Inagreement with these data, none of the mAbs recognizedfragment 1 representing the amino-terminal part of hPIV3N protein (Fig. 4B, lane 1). Fine mapping of mAb epitopeswas performed by Western blot using three additional GST-fused overlapping hPIV3 N protein fragments comprisedof aa 397–436 (fragment no. 4), aa 417–456 (fragment no. 5),and aa 447–486 (fragment no. 6). All mAbs were reactive

FIG. 1. Reactivity of monoclonal antibodies raised against hPIV3 N protein with yeast-expressed N proteins of hPIV3 (lane 1),hPIV1 (lane 2), and hRSV (lane 3) in Western blot. Proteins were separated in a 12% SDS-PAGE and transferred to PVDFmembranes. The membranes were incubated with hybridoma supernatants and developed using HRP-labeled anti-mouse IgGand ready-to-use TMB-blotting substrate. (A) SDS-PAGE. (B–E) Western blot with monoclonal antibodies of clone 2H1 (B),clone 5C9 (C), clone 5F12 (D), and clone 11H7 (E). Lane M shows a prestained protein molecular mass marker.

HPIV3 N PROTEIN EPITOPE MAPPING 183

exclusively with fragment 5 (Fig. 4B, lane 5). As the mAbsdid not react with other partially overlapping fragments(fragments 4 and 6), it was concluded that the mAbs aredirected against a short non-overlapping sequence compris-ing aa 437–446 of hPIV3 N protein. To prove this, the mAbswere tested by an indirect ELISA with a biotinylated syn-thetic peptide GEPQSSIIQY corresponding to this sequence(N437–446). Two mAbs (5C9 and 11H7) were reactive withthe peptide. Thus these mAbs are directed against a linearepitope located within the hPIV3 N protein sequence of aa437–446. The lack of reactivity of the other two mAbs (2H1and 5F12) with the synthetic peptide suggests that theirepitopes are in the same antigenic region, but most likelysome additional aa residues adjacent to the sequence N437–446 contribute to the formation of these epitopes.

Analysis of human sera

To compare the epitope specificities of mAbs raised ag-ainst recombinant hPIV3 N protein with the specificities ofhuman serum IgG induced by a natural hPIV3 infection, weperformed indirect ELISAs with a panel of 42 serum speci-mens collected from hPIV-infected patients. Human serumspecimens were analyzed for the presence of IgG antibodiesagainst hPIV1=2=3 using a commercial ELISA kit, and thenanalyzed for reactivity with recombinant hPIV3 N protein andwith synthetic peptide N437–446 representing the antigenicsequence of hPIV3 N protein. From 42 serum specimens ana-lyzed, 35 were reactive with hPIV3 N protein (Table 1). Tenserum specimens negative for hPIV1=2=3-specific IgG by acommercial assay were found to be positive for hPIV3 N

FIG. 2. Indirect immunofluorescence staining of hPIV-infected cells with monoclonal antibodies raised against hPIV3 Nprotein. Biochip slides containing hPIV3- (A) and hPIV1-infected (B) Vero cells were incubated with hybridoma supernatantsand then stained with FITC-conjugated anti-mouse IgG. Monoclonal antibody codes are indicated on the bottom of eachimage. NEG, negative control (slides incubated with human serum negative for hPIV1=hPIV3); POS, positive control (slidesincubated with human serum containing IgG against hPIV1=hPIV3).

FIG. 3. Schematic representation of GST-fused hPIV3 N protein fragments used for epitope mapping.

184 ZVIRBLIENE ET AL.

protein-specific IgG. The discrepancy between the resultsobtained with a commercial kit and the NLP-based assaymight be explained by the fact that the commercial kit is basedon the use of total viral proteins with a relatively low contentof viral nucleocapsids (6). Eleven hPIV3 IgG-positive serumspecimens showed a cross-reactivity with hPIV1 N protein.The cross-reactivity of serum antibodies with hPIV1 andhPIV3 N proteins might be explained by a similarity in their aasequence (6). The possibility of a double hPIV3=hPIV1 infec-tion also cannot be excluded. Further analysis of the sera usingsynthetic peptide N437–446 revealed that from 35 serumspecimens containing IgG antibodies against hPIV3 N protein,16 were reactive with N437–446 peptide. Thus, 46% of hPIV3IgG-positive serum specimens were shown to contain anti-bodies directed to this linear epitope. These data suggest thatthe epitope N437–446 is immunodominant in the convalescenthumoral response to hPIV3 infection.

Comparison of immunodominant linear epitopesof hPIV3 N protein and measles virus N protein

Previously we have studied the antigenic structure of the Nprotein of measles virus and identified a linear immuno-dominant epitope N440–448 within the carboxy-terminus ofmeasles virus N protein (23). As the nucleocapsids of para-myxoviruses share structural features we analyzed whetherthere is a similarity between the immunodominant epitopes ofN proteins of hPIV3 and measles virus. Therefore, we per-formed sequence alignment of the carboxy-termini of N pro-teins of hPIV3 and measles virus (Fig. 5A). Although there islittle similarity in the regions downstream of aa 397 of Nprotein sequences (11% of identical aa residues), we havedetermined that there are several homologous aa residueswithin the epitopes identified as immunodominant: N440–448 and N437–446 in measles virus N protein and hPIV 3 Nprotein, respectively. From 10 aa residues that constitute theimmunodominant epitope N437–446 of hPIV3, two aa resi-dues are identical and three are similar to those of measlesvirus epitope N440–448. Despite some sequence similarity

between the epitopes, the mAbs directed against hPIV3 N437–446 did not show any cross-reactivity with measles virusN440–448 (data not shown). We have also performed se-quence alignment of carboxy-termini of N proteins of hPIV3and hPIV1 (Fig. 5B). In the regions downstream of aa 397 of Nprotein sequences only 24% of identical aa residues weredetermined. No sequence similarity was determined in thesequence N437–446, which explains the lack of mAb cross-reactivity with hPIV1 N protein.

Discussion

The 515 aa-long N protein of hPIV3 is the most abundantviral structural protein that encapsidates the genomic RNA(5,22). In hPIV3-infected cells, the biosynthesis of N proteinstarts earlier than that of the other viral proteins (21). Analysisof human sera by N protein-specific immunoassays showsthat N protein of hPIV3 induces a strong antibody responseduring virus infection (6). However, immunodominant anti-genic regions of the N protein were not identified. By usingpanels of mAbs raised against structural proteins of hPIV3,localization of B-cell epitopes of hPIV3 N protein was ana-lyzed previously but not in a precise aa sequence manner. Theimmunodominant antigenic regions of N protein were de-fined only as clusters of epitopes (7,13,16).

The availability of recombinant viral antigens provides theability to study their antigenic structure in more detail. Therecombinant yeast-expressed hPIV3 N protein was shown toself-assemble into nucleocapsid-like structures similar to na-tive viral nucleocapsids. Moreover, the antigenic similarity ofrecombinant hPIV3 N protein and hPIV3 N protein fromwild-type virus strains was confirmed by an indirect ELISAand Western blot by testing human serum samples (6). In thecurrent study, four mAbs against recombinant hPIV3 N pro-tein were employed to study the antigenic structure of hPIV3N protein. Although the mAbs were raised against recombi-nant NLPs, they recognized native viral nucleocapsids inhPIV3-infected mammalian cells. As the mAbs react with epi-topes that are well exposed on the surface of nucleocapsids,

FIG. 4. Reactivity of monoclonal antibody 2H1 with GST-fused hPIV3 N protein fragments expressed in E. coli. (A) Westernblot with anti-GST polyclonal antibody. (B) Western blot with monoclonal antibody 2H1 against hPIV3 N protein. Lane M,prestained protein molecular mass marker; lane 1, fragment no. 1 (aa 1–103); lane 2, fragment no. 2 (aa 121–486); lane 3,fragment no. 3 (aa 397–515); lane 4, fragment no. 4 (aa 397–436); lane 5, fragment no. 5 (aa 417–456); lane 6, fragment no. 6 (aa447–486); lane 7, GST; lane 8, purified yeast-expressed full-length hPIV3 N protein.

HPIV3 N PROTEIN EPITOPE MAPPING 185

the reactivity with virus-infected cells confirms the antigenicsimilarity between recombinant N protein and native nucle-ocapsids. Thus, the mAbs were suitable to study the antigenicstructure of hPIV3 N protein. Using a combination of two

different mapping strategies—overlapping N protein frag-ments and synthetic peptide—we have located the majorantibody binding site at the carboxy-terminus of the N pro-tein, between aa 397 and 486. We have further demonstrated

Table 1. Reactivity of Human Sera Collected from Patients at the Convalescent Phase of hPIV Infection

with Synthetic Peptide N437–446 Representing the Antigenic Sequence of hPIV3 N Protein

Serumcodea

Reactivity forhPIV1=2=3

IgG (IBL kit)b

Reactivity in the in-house ELISAusing recombinant N proteins

Serologicalclassification

Reactivity withpeptide N437–446hPIV3 hPIV1

1-a þ=� 3.32 þþþ 3.39 þþþ Cross-reactive 0.22 �1-c þþþ 3.19 þþþ 3.29 þþþ Cross-reactive 0.12 �3-a � 0.29 � 0.22 � hPIV-negative 0.18 �3-c þ=� 2.35 þþþ 0.23 � hPIV3-positive 0.07 �4-a þ=� 2.73 þþþ 0.82 þ hPIV3-positive 0.02 �4-c þ=� 2.45 þþþ 1.21 þþ Cross-reactive 0.11 �5-a � 0.10 � 0.07 � hPIV-negative 0.23 �5-c � 0.10 � 0.07 � hPIV-negative 0.11 �6-a þ 0.79 þ 0.35 � hPIV3-positive 0.47 þ7-a þ=� 0.98 þ 0.29 � hPIV3-positive 0.16 �9-c � 0.47 þ 0.29 � Probable hPIV3 0.42 þ

10-c � 0.48 þ 0.39 � Probable hPIV3 0.21 �11-c � 0.49 þ 0.25 � Probable hPIV3 0.48 þ38 þ 0.92 þ 0.36 � hPIV3-positive 0.18 �39 þ 0.54 þ 0.28 � hPIV3-positive 0.52 þ40 þ 1.71 þþþ 1.72 þþþ Cross-reactive 0.08 �42 þ 0.94 þ 0.38 � hPIV3-positive 0.12 �43 þ 1.18 þþ 0.73 þ Cross-reactive 0.77 þ46 þ 0.52 þ 0.25 � hPIV3-positive 0.28 �47 þ 1.01 þþ 0.26 � hPIV3-positive 0.25 �50 þ 0.79 þ 0.40 � hPIV3-positive 0.08 �51 þ 1.01 þþ 0.38 � hPIV3-positive 0.48 þ52 � 0.61 þ 0.37 � hPIV3-positive 0.51 þ53 þ 0.58 þ 0.28 � hPIV3-positive 0.52 þ54 þ 1.77 þþþ 0.52 þ Cross-reactive 0.22 �55 � 0.10 � 0.08 � hPIV-negative 0.09 �56 � 1.77 þþþ 0.40 � hPIV3-positive 0.56 þ57 � 1.23 þþ 0.32 � hPIV3-positive 0.64 þ58 � 0.64 þ 0.30 � hPIV3-positive 0.57 þ59 þ 0.58 þ 0.30 � hPIV3-positive 0.12 �60 þ 1.13 þþ 0.62 þ Cross-reactive 0.23 �62 � 0.89 þ 0.24 � hPIV3-positive 0.21 �63 � 0.24 � 0.14 � hPIV-negative 0.02 �64 þ 1.03 þþ 0.40 � hPIV3-positive 0.02 �65 þ 1.68 þþþ 1.02 þþ Cross-reactive 0.48 þ66 þ 1.22 þþ 2.06 þþþ Cross-reactive 1.03 þ67 � 1.37 þþ 0.83 þ Cross-reactive 1.06 þ68 � 1.27 þþ 0.70 þ Cross-reactive 0.54 þ70 þ 1.03 þþ 0.40 � hPIV3-positive 0.16 �71 � 0.40 � 0.18 � hPIV-negative 0.20 �72 � 0.29 � 0.18 � hPIV-negative 0.22 �74 þ 0.89 þ 0.30 � hPIV3-positive 0.55 þControl serum specimens from the commercial HPIV1=2=3 IgG ELISA kit (IBL)Positive 2.39 (þþþ) 3.06 þþþ 2.93 þþþ 0.38 �Negative 0.06 (�) 0.08 � 0.08 � 0.02 �

aThe immunoreactivities of serum specimens 1–11 with recombinant hPIV1=hPIV3 N proteins were studied previously (6). In four cases,paired serum specimens collected at a 2-week interval were available (marked a and c).

bAccording to the manufacturer’s recommendations, the interpretation of results obtained with a commercial ELISA kit is as follows:�¼OD450 value <0.3; þ=�¼OD450 value 0.3–0.5; þ¼OD450 value 0.5–1.0; þþþ¼OD450 value >1.5.

Human serum specimens (diluted 1:100) were tested for the presence of IgG antibodies against: (1) hPIV1=2=3 using a commercial ELISAkit (IBL); (2) hPIV3 and hPIV1 N proteins using our in-house ELISA method (6); (3) synthetic peptide N437–446.

For the in-house ELISAs based on the use of hPIV3 and hPIV1 N proteins, sera with OD450 values >0.4 were considered positive.For the peptide-based ELISA, the calculated OD450 cutoff value was 0.38 (mean OD450 obtained with 7 hPIV3 IgG-negative serum

specimensþ 3 SD).

186 ZVIRBLIENE ET AL.

that this antigenic region involving a linear epitope of aa 437–446 is not only present on the surface of recombinant NLPsand elicits epitope-specific mAbs, but is strongly immuno-dominant in the humoral immune response of convalescenthPIV3 patients.

Previous studies led to the conclusion that hPIV3 andhPIV1 N proteins share not only sequence but also antigenicsimilarity at the amino-terminus domain (9). According to thesequence alignment data, 70% of the first 396 aa residues fromthe amino-termini of hPIV3 and hPIV1 N proteins are identi-cal. Little sequence similarity is observed in the distal carboxy-termini: only 24% of aa residues are identical. The analysis ofthe sequence N437–446 representing the immunodominantlinear epitope of hPIV3 N protein revealed no similarity withthe hPIV1 N protein sequence. In line with these data, themAbs directed against the carboxy-terminus of hPIV3 Nprotein did not show any cross-reactivity with hPIV1 N pro-tein. This provides the possibility to use these mAbs as highlyspecific diagnostic reagents for the direct detection of hPIV3 insaliva and airway epithelium specimens.

The mAbs directed against the carboxy-terminus of hPIV3N protein might be also useful for its structure-functionanalysis. The epitopes recognized by the mAbs lie within adomain of N protein that is supposed to interact with P whenforming a complex between the nucleocapsid, L, and P(1,2,22). Furthermore, previous studies of Sendai virus, amurine parainfluenza virus that is highly related to hPIV1,suggest that the carboxy-terminal region of N protein (aa420–466) is responsible for the interaction of the nucleo-capsid with M, which is the central organizer of virusassembly (3).

Interestingly, the immunodominant epitope N437–446 ofhPIV3 is localized in the same N protein region as the im-munodominant linear epitope N440–448 of measles virus,another member of the subfamily Paramyoxvirinae (23).Moreover, the sequence alignment revealed some structural

similarity between these antigenic sites of hPIV3 and measlesvirus N proteins. These data suggest that different paramy-xoviruses may contain highly conserved surface-exposedstructures within the carboxy-termini of N proteins that areeasily accessible to B cells and elicit an antibody response.

In conclusion, the current study enhances the knowledgeof the antigenic structure of hPIV3 N protein and may fa-cilitate the development of better diagnostic methods forhPIV3 infection.

Acknowledgments

We thank Dr. M. Coiras (National Center of Microbiology,Majadahonda, Madrid, Spain) and Dr. I. Narkeviciute (theCenter of Pediatrics, Clinic of Children’s Diseases, VilniusUniversity, Vilnius, Lithuania) for human serum specimens.The work was supported by the Lithuanian Science andStudy Foundation (grant no. B-11).

Disclosure Statement

No conflicting financial interests exist.

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FIG. 5. Alignment of the aa sequence of the hPIV3 N protein C terminal region (GeneBank accession no. D10025; AB012132[4,12]) with that of (A) measles virus (GeneBank accession no. U03668, AF266291 [14,17]) and (B) hPIV1 (GeneBank accessionno. M62850; D01070 [9,10]). Identical and similar aa residues are marked by asterisks and dots, respectively. Sequencesrepresenting linear immunodominant epitopes are highlighted in black.

HPIV3 N PROTEIN EPITOPE MAPPING 187

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Address reprint requests to:Dr. Aurelija Zvirbliene

Institute of BiotechnologyV. Graiciuno 8,

LT-02241 Vilnius, Lithuania;

E-mail: azvirb@ibt.lt

Received December 17, 2008; accepted February 6, 2009.

188 ZVIRBLIENE ET AL.

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