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Mar 9, 1988 - deaths of infants and young children, predominantly in developing ..... 136-141. 13. Gunn, P. R., F. Sato, K. F. H. Powell, A. R. Bellamy, J. R..

JOURNAL OF VIROLOGY, Nov. 1988, p. 4265-4269

Vol. 62, No. 11

0022-538X/88/114265-05$02.00/0 Copyright © 1988, American Society for Microbiology

A Synthetic Peptide Corresponding to the Cleavage Region of VP3 from Rotavirus SAll Induces Neutralizing Antibodies HANS-JURGEN STRECKERT,1* HARALD BRUSSOW,2 AND HERMANN WERCHAU' Abteilung fur Medizinische Mikrobiologie und Virologie der Ruhr-Universitat Bochum, Postfach 10 21 48,

D4630 Bochum 1, Federal Republic of Germany,' and Nestle Research Center, Nestec Ltd., Vers chez les Blanc, CH-J000 Lausanne 26, Switzerland2 Received 9 March 1988/Accepted 1 August 1988

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-ArgAsn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SAil in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SAil particles was observed.

Rotavirus infections cause an annual estimated 1 million deaths of infants and young children, predominantly in developing countries (37). To reduce the morbidity and mortality of this pathogen, a safe vaccine protecting against all known serotypes would be desirable. Synthetic peptides are of increasing importance for such a task (18, 35, 36). Prerequisite for the development of a synthetic vaccine is knowledge of the neutralizing epitopes of the virus. Rotavirus-neutralizing antibodies are directed against two components of the outer capsid, VP7 and VP3 (2, 3, 11, 12, 14, 23, 27, 34). The application of synthetic antigens is limited to continuous epitopes, at least until efficient prediction methods have been developed to characterize discontinuous epitopes. In the case of VP7, nearly all epitopes of suitable hydropathic character (17) have been examined (13, 29, 30). Only one antiserum raised against peptides 247 to 259 exhibited a low neutralizing titer (serum dilution, 1:32) (13). Noncontinuous or assembled determinants (4, 5) may be associated with this protein. VP3 is the rotavirus hemagglutinin (15, 16). In the presence of trypsin, VP3 is cleaved into two fragments, VP5* and VP8*, of 60 and 28 kilodaltons, respectively (6). Infectivity and hemagglutinating activity are influenced by this cleavage (7, 16, 19). From the amino acid sequence of VP3 (10, 19), it can be deduced that the C-terminal part of VP8* may be associated with a neutralizing determinant because it contains hydrophilic residues and shows variations. In this report, we describe the induction of antibodies by the peptide GlnAsn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3, and the reactivity of the induced antipeptide antibodies.

described previously (12). The virus was purified further by centrifugation (Beckman SW40 Ti rotor; 30,000 rpm, 3 h) using a preformed CsCl gradient (density, 1.3 to 1.4 g/ml). Peptide synthesis and induction of antipeptide antibodies. Peptide was synthesized and synthetic antigen was prepared as reported previously (28), with one exception; instead of bovine serum albumin, the homologous protein rabbit serum albumin was used as a carrier protein. Rabbits were immunized with 1 mg of peptide carrier conjugate emulsified in 0.3 ml of sodium phosphate buffer (pH, 7.5) and 0.3 ml of complete Freund adjuvant. On day 21, the procedure was repeated by using incomplete adjuvant. For the second booster injection, on day 42, 1 mg of glutaraldehyde-crosslinked peptide was used. Animals were bled on day 56. Monitoring the immune response by ELISA. The enzymelinked immunosorbent assay (ELISA) was performed exactly as described previously (31). Plates were coated with 5 ,ug of peptide OVA conjugate or 50 to 100 ng of purified SAl1 per well. Determination of protein specificity by protein electroblotting. Proteins were separated on sodium dodecyl sulfate15% polyacrylamide gels and electrophoretically transferred to nitrocellulose. A semi-dry blotting equipment was used (Fast Blot; Biometra, Gottingen, Federal Republic of Germany). Nonspecific binding sites were blocked in phosphate buffer containing 0.3% Tween 20 and 3% milk powder. Antigen-antibody complexes were enzymatically stained by using a biotinylated anti-rabbit antibody and extravidin peroxidase conjugate (Sigma Chemical Co., St. Louis, Mo.). 4-Chloro-1-naphthol was used for substrate reaction. Virus neutralization assay. The virus neutralization assay was performed with MA104 cells grown in Dulbecco modified Eagle medium with 5% basal medium supplement (Seromed, Berlin, Federal Republic of Germany) in tissue culture microdilution plates. Virus activation was done with 20 ,il of trypsin per ml (30 min, 37° C). Equal volumes of virus dilutions in medium (approximately 200 focus-forming units per 0.1 ml) and appropriate antibody dilutions were incubated for 90 min at 37° C. A 100-p.l portion of this suspension was mixed with 104 cells in 100 ,ul of medium containing 7.5 ,ug of trypsin per ml and seeded into the wells. Cell monolayers were fixed with ethanol and stained with 0.5% crystal violet. Alternatively, rotavirus proteins were detected with an

MATERIALS AND METHODS Growth and concentration of virus. Simian rotavirus strain SAl was originally obtained from T. H. Flewett and has been cultivated in our laboratory since 1979. Simian rotavirus strain SAl1, human rotavirus strains Wa, S2, YO, and Hochi, and bovine strains NCDV and UK were grown in MA104 cells, concentrated by centrifugation at 90,000 x g for 2.5 h, and suspended in a solution containing 10 mM Tris hydrochloride (pH 7.4) and 10 mM calcium chloride, as *

Corresponding author. 4265



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6 a _

FIG. 1. Binding of antipeptide mune serum

(x) to CsCl-purified





(0) and appropriate preimbound to ELISA plates.

enzymatic staining procedure (9). For some experiments, SAil was grown without trypsin activation and without trypsin supplementation of the growth medium. In this case, the virus titer was reduced by a factor of 103. Immune electron microscopy. Immune electron microscopy was performed essentially as described previously (28) on a Philips EM 300 electron microscope at 80 kV acceleration voltage. Viral particles were negatively stained with 2% phosphotungstic acid (pH 7.0). RESULTS Specificity and selectivity of the antipeptide serum. The synthetic peptide induced an antibody response in rabbits that could be monitored in an ELISA experiment up to a serum dilution of at least 1:200,000. The reactivity of the antipeptide serum raised against rotavirus SA1l is shown in Fig. 1. Plate-bound SAil was recognized up to a serum dilution of 1:200,000. Rotavirus strains Wa, S2, and Hochi were recognized to a significantly lesser extent when applied to the ELISA plate in equivalent amounts. The antipeptide serum recognized rotavirus strains Wa, S2, and Hochi in a dilution of 1:10,000, 1:20,000, and 1:50,000, respectively. Controls with preimmune serum were used in all experiments.

The antipeptide serum demonstrated its reactivity towards denatured SA11-VP3 in a blotting experiment. VP3, with an apparent molecular size of 88 kilodaltons, was recognized (Fig. 2, lane c) by the antipeptide serum. Weak crossreactivity was observed with the three amino acids Ile-ValPro in positions 211 to 213 in SA11-VP6 (1). Specificity of protein recognition was confirmed by inhibition experiments with the peptide-rabbit serum albumin conjugate used as



I c



FIG. 2. Immunoblotting experiment performed with purified SAil. Separation was achieved by means of sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Electroblotting on nitrocellulose was done with a semi-dry blotting equipment. Nitrocellulose strips were incubated with SAll-hyperimmune serum at a dilution of 1:500 (lane a), with preimmune serum (lane b), and with antipeptide serum (lane c) diluted 1:500. Lane d, Inhibition of protein recognition by preincubation of the antipeptide serum (diluted 1:500) with 10 pLg of peptide-rabbit serum albumin conjugate per ml of dilution.








test sera from 1: 100

(row A)


to 1: 12,800

Serial (row H)

twofold dilutions were


incubated with


with MA104 appropriate amounts of virus. Suspensions were Cell monolayers indicating virus neutralization were stained with crystal violet. Lanes 1 to 4, Preimmune serum (lanes 1 and 2) and antipeptide serum (lanes 3 and 4) incubated with rotavirus strain Wa; lanes 5 to 8, Preimmune serum displaying no neutralizing activity against SAil (lanes 5 and is evident in 6). Neutralization of SAil by the antipeptide 7 and 8. Controls demonstrating the effect of pure diluted virus (SAil, lane 9; Wa, lane 10) growth of uninfected cells (lanes 11 cells and seeded into the wells.









VOL. 62, 1988 TABLE 1. Neutralizing activity of the antipeptide serum and appropriate preimmune serum (approximately 100 focusforming units were applied for each test)" Virus strain"

SAll SAllntt SAllpit Wa S2 YO Hochi UK NCDV

Reciprocal neutralizing titer of:

Antipeptide serum

Preimmune serum

800 800 '3,200

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