Vol. 65, No. 6

JOURNAL OF VIROLOGY, June 1991, p. 3106-3113

0022-538X/91/063106-08$02.00/0 Copyright © 1991, American Society for Microbiology

Heterotypic Passive Protection Induced by Synthetic Peptides Corresponding to VP7 and VP4 of Bovine Rotavirust M. K.

IJAZ,l S. K. ATTAH-POKU,' M. J. REDMOND,' M. D. PARKER,'

M. I. SABARA,' AND L. A. BABIUKl3* Veterinary Infectious Disease Organization' and Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan,3 Saskatoon, Saskatchewan, Canada S7N OWO, and Praxis Biologics, Inc., Rochester, New York 14623-14932 Received 6 August 1990/Accepted 4 March 1991

We have evaluated the potential of two peptides derived from highly conserved regions of rotavirus outer capsid proteins (VP7 and VP4) to act as a rotavirus vaccine. The capacity of peptides coupled to rotavirus VP6 spherical particles to provide passive protection in a murine model was compared with the protection induced by peptide-keyhole limpet hemocyanin (KLH) conjugates. Female mice were immunized a total of three times before and during pregnancy. Suckling mouse pups were challenged at 7 days of age with either homologous or heterologous rotavirus serotypes. The efficacy of vaccination was determined by analyzing the clinical symptoms and measuring xylose adsorption in the intestine. In this model the VP4 peptide-VP6 conjugate provided protection equal to that obtained using bovine rotavirus (BRV) as the immunogen. The VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate. A mixture of the VP4 peptide-VP6 and VP7 peptide-VP6 conjugates provided better heterologous protection than immunization with BRV. In contrast, KLH-conjugated peptides provided only partial protection. The significance of a synthetic-peptide-based rotavirus vaccine in the prevention of rotavirus infections is discussed.

trypsin into two fragments, VP5 (approximately 60K) and VP8 (approximately 28K). Trypsin-enhanced infectivity is a common feature of all rotavirus serotypes. Sequence analysis of the VP4 gene from several human and animal rotavirus strains shows that the cleavage site is conserved (12). A peptide corresponding to this site was synthesized and was found to mimic the authentic trypsin cleavage site under in vitro conditions (35b). We have previously demonstrated that synthetic peptides corresponding to VP7 amino acid sequence 275-295 and VP6 amino acid sequence 40-60, coupled to keyhole limpet hemocyanin (KLH), elicit passive protection in a murine rotavirus model (14). However, complications arise from carrierinduced suppression when KLH is used as an immunological carrier (14, 18, 39), making these vaccines unsuitable for human or veterinary use. To overcome this problem, we have developed a carrier based on rotavirus VP6 (the major inner capsid protein) assembled in vitro into spherical, viruslike particles (35, 37). Many peptides have been coupled to this carrier and induce a strong humoral immune response without producing carrier-induced suppression (35a). In this communication we report that vaccination with VP7 and VP4 peptides coupled to VP6 particles induces an effective immune response. This effect was demonstrated by the passive protection of suckling neonatal mice against challenge with homologous and heterologous strains of rotavirus. This data shows the potential of using a synthetic peptide vaccine against rotavirus infections of humans and animals.

Rotavirus is an important etiological agent of neonatal enteritis in mammalian species, including infants and young children, throughout the world. It has been estimated that rotavirus infection in developing countries causes between half a million to one million deaths every year (8). Although infection also occurs with a high frequency, in developed countries, the disease is associated with low mortality. In the United States, rotavirus affecting humans in the 1- to 4-year age group has been estimated to annually cause over one million cases of severe diarrhea but results in only 150 deaths (19). Worldwide efforts have now been directed at producing a safe, economical, and effective vaccine against these viruses. Rotaviruses belong to the family Reoviridae and are composed of a double-capsid structure enclosing 11 segments of double-stranded RNA. The outer capsid is composed of two proteins, VP7 and VP4. VP7 is the major glycoprotein, with a molecular weight of 38,000 (38K) in its unreduced form and 41K in its reduced form. It has been shown that VP7 binds to host cells and that certain monoclonal antibodies directed against this protein can neutralize the virus and also inhibit virus attachment to host cells (15, 28, 36). VP7 contains regions that are conserved in a number of human and animal rotaviruses (for a review, see reference 12). A peptide from one such conserved region was found to block the binding of radiolabeled bovine rotavirus (BRV) to MA-104 cells (14), suggesting that if antibodies were produced against this peptide, infection may be prevented. The second outer capsid protein, VP4, has a molecular weight of 80K to 84K. VP4 has been shown to play a major role in the penetration of virus into the host cell (15). The mechanism of penetration involves the cleavage of VP4 by

MATERIALS AND METHODS Animals. Rotavirus-free mice (strain CD-1) purchased from Harlan Sprague-Dawley Inc. (Indianapolis, Ind.) were confirmed as seronegative for antibodies to rotavirus using an enzyme-linked immunosorbent assay (ELISA) (21). The

Corresponding author. t Contribution 102 from the Veterinary Infectious Disease Organization. *

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TABLE 1. Synthetic peptides from VP4, VP7, and VP6 of bovine rotavirus Viral protein

VP4a

VP7b VP6C BP (VP4)

Peptide

Sequence

232-255 275-295 40-60 240-248

c-N-I-A-P-A-S-I-V-S-R-N-I-V-Y-T-R-A-Q-P-N-Q-D-I-A-OH (G)-P-T-T-A-P-Q-T-E-R-M-M-R-I-N-W-K-K-W-W-Q-V-c-OH T-M-N-G-N-E-F-Q-T-G-G-I-G-N-L-P-I-R-N-W-N-(A)-c-OH C-G-A-S-R-N-I-V-Y-T-R-A-(G)

The valines present at positions 4, 6, and 25 on VP4 were replaced by alanines to improve synthetic yields, and a cysteine was added at position 1 to facilitate conjugation of the peptide to a carrier protein (KLH or VP6 particle). R indicates a trypsin cleavage site. b A glycine at the N terminus of VP7 was used as a spacer, and a cysteine at the C terminus was added to facilitate coupling to KLH. C At the N terminus of VP6, an alanine was used as a spacer and a cysteine was added to facilitate coupling to KLH. a

animals were housed in isolation units throughout the experiment. Cells and Virus. MA-104 cells (African green monkey) were cultured in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO Laboratories, Grand Island, N.Y.). BRV isolate C486 was cultured from the feces of diarrheic calves by a method described previously (4). Simian rotavirus strain SA-11 (serotype 3) was obtained from H. Malherbe (San Antonio, Tex.), and human rotavirus strains DS-1 (serotype 2) and strain Wa (serotype 1) were obtained from H. Greenberg (Stanford University, Stanford, Calif.). Rotavirus strains were propagated in confluent MA-104 cells in the presence of 1 jig of trypsin (Difco Laboratories, Detroit, Mich.) per ml without FBS. Cells and supernatant were harvested together, and cells were removed by centrifugation at 500 x g for 20 min. Virus was concentrated from the clarified supernatant fluids by centrifugation through a 40% sucrose gradient (100,000 x g 2.5 h, 15°C). The virus pellet was resuspended in double-distilled water, and the protein concentration was determined by using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, Calif.) (19). Plaque assay. A plaque assay for the quantitation of infectious rotavirus was performed by the method described previously (1). Briefly, 12-well tissue culture plates (Nunc) containing confluent monolayers of MA-104 cells were washed twice with MEM (without FBS). Serial 10-fold dilutions of each rotavirus isolate were prepared in MEM containing trypsin (Difco) to a final concentration of 10 p.g/ml. Following adsorption of the virus at 37°C for 1 h, the inoculum was aspirated and cells were washed with MEM and overlaid with Dulbecco's modified Eagle medium containing 2% Sephadex G-75 beads (Pharmacia), 20 mM glutamine, and 25 ,ug of Pancreatin (GIBCO) per ml. The plates were incubated for 2 days at 37°C, and the overlay was aspirated. The plates were then stained with 0.5% crystal violet-80% methanol-phosphate-buffered saline (PBS) and washed, and plaques were enumerated. ELISA procedure. The ELISA was a modification of a previously described procedure (36). All incubations were performed at room temperature (20°C) for 1 h unless stated otherwise. Polystyrene, 96-well plates (Immunolon 2; Dynatech Laboratories Inc., Alexandria, Va.) were prepared for each of the assays as follows. For the detection of peptide-specific antibody, the plates were coated overnight with the respective peptide (5 pmol per well) diluted in 0.05 M carbonate bicarbonate buffer at pH 9.6. Unabsorbed peptide was removed by extensive washing with distilled water. The uncoated sites on the plate were blocked by overnight treatment with 3% horse serum in 0.01 M PBS, pH 7.2, and then washed with double-distilled H20. The plated antigen was overlaid with mouse antiserum (75 ,ul per well) in 0.01 M PBS containing 1% horse serum and 0.05% Tween

20. Incubation was carried out for 2 h at room temperature after which time the unbound antibody was removed by washing with 0.01 M PBS containing 0.05% Tween 20 (PBST). A 1/5,000 dilution (in PBST plus 1% horse serum) of biotinylated goat anti-mouse immunoglobulin (Zymed Laboratories Inc., San Francisco, Calif.) was then added and incubated for 1 h at room temperature. After the plates were washed with PBST, they were incubated for 1 h with 75 ,ul of streptavidin horseradish peroxidase conjugate diluted in PBST and 1% horse serum. After the plates were washed with PBST, the substrate 2,2'-Azino-di-(3-ethyl-benzthiazoline sulfonate) (ABTS) (Boehringer-Mannheim, Quebec, Canada) was added. The color development was stopped after 10 min by the addition of 10% sodium dodecyl sulfate (SDS). The optical density of the wells was determined at 405 nm by an ELISA reader (Bio-Rad). Titers were expressed as a reciprocal of the highest dilution with an optical density of >2 standard deviations over mean background levels. SDS-PAGE. Viral proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) under both reducing and nonreducing conditions by the procedure described by Laemmli (24). Virus samples were resuspended in sample buffer (0.337 M Tris [pH 6.8], 6% SDS, 30% glycerol, 0.03% bromophenol blue) for electrophoresis under nonreducing conditions; 3.75% mercaptoethanol was added to sample buffer for electrophoresis under reducing conditions. The samples were boiled for 5 min and electrophoretically separated on a 10% polyacrylamide resolving gel with a 3% stacking gel. Western blotting (immunoblotting) of rotavirus polypeptides. Peptide-specific antibodies were detected by the Western blotting technique described by Towbin et al. (43). Viral proteins that had been separated on a 10% polyacrylamide gel were transferred to nitrocellulose paper (pore size, 0.45 ,um) (Bio-Rad) by electroblotting at 100 V for 1 h in a buffer containing 20 mM Tris, 190 mM glycine, and 20% methanol. Replica nitrocellulose strips were stained with amido black to determine the efficiency of protein transfer. After transfer, reactivity of viral proteins with serum samples was determined as described previously (6). Nonspecific reactions were blocked with 3% bovine serum albumin in 0.01 M Tris-buffered saline, pH 7.4. After washes with Tris-buffered saline plus 0.05% Tween 20, the reaction was developed with protein A-gold (Bio-Rad) for 1 h. Following development, the protein bands were intensified by silver enhancement (IntenSe BL, Amersham, England). Synthesis of peptides. The peptides were synthesized (Table 1) on an Applied Biosystems 403A peptide synthesizer using solid-phase methods of Barany and Merrifield (5). Commercially available aminoacylphenylacetamidomethyl resin and appropriately protected tertiary butyloxycarbonylL-aminO acids were used in combination with the recom-

3108

IJAZ ET AL.

mended coupling programs. A cysteine was incorporated at the carboxyl termini of the VP6 and VP7 peptides to facilitate coupling to KLH. Cleavage of the resin-bound peptide and deprotection of the side chains was achieved by solvolysis with anhydrous liquid hydrofluoric acid in the presence of the scavengers anisole, p-thiocresol, and dimethylsulfide. Purification of the crude material by reversed-phase highperformance liquid chromatography on a Vydac protein C-4 semipreparative column (length, 25 cm; inner diameter, 10 mm) yielded a product whose purity was greater than 98% as measured by analytical HPLC. An additional composite peptide composed of the VP7 peptide 275-295 plus amino acid sequence CGASRNIVY TRA(G) was made. This sequence designated "binding peptide" (BP) is derived from the trypsin cleavage site of VP4 and includes amino acids 240 to 248 of BRV strain C486 (33) with the addition of an N-terminal cysteine and three amino acid spacers (glycine and alanine at the N terminus and glycine at the C terminus [Table 1]). It has been reported that this sequence spontaneously binds to the rotavirus VP6 carrier through a covalent disulfide bond (35a). Thus, the BP sequence was synthesized directly onto the amino terminus of the 275-295 (VP7) peptide, and the BP-275-295 complex was then directly coupled to the VP6 carrier. Peptides were tested for immunoreactivity in a dot blot assay following the procedures provided by Bio-Rad Laboratories using a polyclonal antirotavirus serum. Recombinant VP6 production and particle assembly. The construction of recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) containing gene 6 from BRV and assembly of VP6 particles following infection of Spodoptera frugiperda (SF9) cells has been described previously (35a). Briefly, genomic RNA extracted from purified BRV strain C486 was used to produce cDNA. The cDNA was ligated into the PstI site of pBR322 and used to transform Escherichia coli DH1. The resulting colonies were probed with radiolabeled cDNA prepared from purified genomic RNA segment 6 as template. After gene 6 cDNA was appropriately tailored, it was ligated into baculovirus transfer vector pAc373. Integration of the rotavirus gene into the genome of A. californica was then carried out by homologous recombination in S. frugiperda (SF9) cells as outlined by Summers and Smith (41). Recombinants were identified by plaque hybridization using radiolabeled cDNA prepared from purified genomic RNA segment 6. Recombinants were plaque purified and analyzed for expression of recombinant gene 6-produced proteins by SDS-PAGE analysis and Western blotting. The recombinant virus containing gene 6 was used to infect SF9 cells. Following incubation for 72 h at 27°C, the cells were lysed in 2 ml of NaHCO3 buffer (pH 7.5) containing 0.05% Triton X-100 and 0.2 trypsin inhibitor unit per ml. Cellular debris was removed by centrifugation at 1,500 x g. The supernatant was dialyzed against 0.1 M glycine buffer (pH 2.4) for 24 h. The dialysis solution was exchanged for 0.01 M Tris-HCl (pH 7.4). Dialysis was then continued until the protein suspension became clear. The quality of the VP6 spheres produced by this method was determined by electron microscopy, and purity was confirmed by SDS-PAGE. Coupling of peptides to carrier proteins. (i) KLH-peptide conjugates. Two peptides (VP7 275-295; VP6 40-60) were coupled through the cysteine at the carboxyl terminus to KLH by the procedure of Green et al. (16) using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). VP4 232255 was coupled through the cysteine at its N terminus. Briefly, KLH (20.0 mg) in 1 ml of 0.01 M sodium phosphate

J. VIROL.

buffer (pH 7.2) was stirred with MBS (5.1 mg; 25.5 ,umol dissolved in 500 p.1 of dimethylformamide) at room temperature for 1 h. Unreacted MBS was removed by HPLC on a Synchropak GPC 100 gel filtration column (length, 500 mm; inner diameter, 10 mm) that had been equilibrated with 0.1 M KH2PO4 buffer (pH 6.0), and MBS was eluted with the same buffer. The amounts of maleimido groups bound to KLH were estimated graphically by adding aliquots of L-cysteine to the conjugate and reacting the excess cysteine with Ellman's reagent, 5,5'-dithiobis-(2-nitro-benzoic acid). The peptides in 200 pul of 0.1 M PBS, pH 7.2, were added to a solution of the KLH-MBS conjugate (1 ml) containing approximately 0.5 pumol of maleimido groups, and the pH was adjusted to 7.8 with 0.1 N NaOH. The mixture was stirred at room temperature for 12 h and dialyzed against 0.1 M PBS, pH 7.2. (ii) VP6-BP peptide conjugates. The peptide-VP6 conjugates (BP 275-295 or 232-255) were prepared by mixing the peptide and VP6 particles at a 10:1 (wt/wt) peptide/carrier ratio and incubating for 30 min at 37°C. SDS-PAGE using samples prepared under nonreducing conditions was used to confirm binding of the peptide to the carrier. Immunization of dams and challenge of the neonates. For primary immunization, each immunogen (50 ,ug) was emulsified with Freund's complete adjuvant. For the second and third immunizations, each immunogen was emulsified with Freund's incomplete adjuvant. Equal volumes of the immunogen and the adjuvant were used. Nine groups of mice (strain CD-1) were immunized intramuscularly with different peptide-carrier preparations. The first immunization was given when the mice were 7 weeks old and was followed by the second and third immunizations at 2-week intervals. Litters were born when the mice were 12 to 14 weeks old. Pups were allowed to suckle their dams, and on average, 10 pups per group were challenged at 7 days of age with one of four rotavirus isolates. The challenge dose (suspended in MEM in 100-pul volume) for each isolate was approximately 104 PFU per mouse. This dose produces diarrhea in 100% of neonatal mice born to rotavirus antibody-free dams (21, 22). For challenge, the virus preparations were administered by intubation of the stomach with a soft flexible plastic feeding tube. Trypan blue dye (GIBCO) was used as a marker to assess the accuracy of intubation. The appearance of diarrhea was scored clinically for up to 72 h postchallenge, as described previously (21). Xylose absorption test. Xylose absorption tests were performed to measure small intestine dysfunction in neonatal mice following challenge with BRV as described previously (22). Briefly, challenge animals were inoculated with BRV, whereas control animals received distilled water. At different times after virus inoculation, D-xylose was administered orally, and 2 h later, the animals were sacrificed by decapitation. Blood samples were collected using heparinized hematocrit tubes. Plasma was separated by centrifugation and assayed for D-xylose concentration as described previously (22). RESULTS Coupling of the synthetic peptides to recombinant VP6assembled particle. The peptides were coupled to VP6 by incubating the peptide and VP6 (10:1) for 30 min at 37°C. A disulfide bond rapidly formed between a VP6 cysteine and the N-terminal cysteine of the BP, thus linking the peptide and carrier protein. Coupling was confirmed by SDS-PAGE with samples prepared under nonreducing conditions. These

PASSIVE PROTECTION INDUCED BY SYNTHETIC PEPTIDES

VOL. 65, 1991

y-

1 06

y

105 104

L-

4, ,

4,'

Oct f

t [

[

3109

go

l'

10

3

1o2

'200 '92.5

101

-69

101

FIG. 1. Analysis of binding synthetic peptides to VP6. The peptides and VP6 were mixed in a ratio of 1:10 (wt/wt) and incubated for 30 min. Prior to electrophoresis on a 10% polyacrylamide gel, samples were prepared in a sample buffer without reducing agents and boiled for 5 min. Following electrophoresis, the proteins were electroblotted to nitrocellulose and reacted with rabbit polyclonal antisera to rotavirus. Antibody binding was visualized with protein A-gold staining, followed by silver enhancement. An increased molecular weight (103) of VP6, as marked by the brackets, was taken as an indication of peptide binding.

conditions disrupted the VP6 particles while retaining the disulfide bond between the carrier and peptide. Thus, an increase in the apparent molecular weight of VP6 from 45,000 to 49,000 indicated the covalent coupling of peptide to the carrier. Western blots of these preparations with antisera directed against VP6 supported this conclusion (Fig. 1). Immune response to rotavirus. To study the effect of carrier proteins on a peptide-specific immune response, experiments were conducted to compare the immune response to different combinations of peptide-carrier conjugates. An immune response of >103 was elicited to all peptide-carrier conjugates. All of the antipeptide antibodies reacted specifically with the peptide used for immunization, but none reacted with unrelated peptides. In an ELISA using BRV as the antigen, the peptides coupled to the VP6 carrier produced titers of >104 and always outperformed vaccines based on KLH (Fig. 2). As a result of the inherent difficulties in performing rotavirus neutralization assays with mouse serum, the correlations between ELISA, plaque reduction, hemagglutination inhibition, and protection were inconclusive (data not shown). Cross-reactivity of antipeptide antibodies with native viral proteins. In order to determine whether antipeptide antibodies reacted with native viral polypeptides, Western blots of rotavirus polypeptides were probed with antipeptide sera. Antipeptide antisera raised using KLH-peptide conjugates reacted specifically with their respective authentic viral proteins, confirming their specificity and also verifying that the animals used in this study were not primed prior to immunization (Fig. 3). Similarly, when antipeptide antisera were raised using VP6-peptide conjugates, the antisera also reacted specifically with the respective authentic viral proteins. Because VP6 carrier is a rotavirus inner capsid protein, this antipeptide sera showed additional immunoreactivity with viral VP6 in the immunoblot. In general, the intensity of the reaction with anti-VP6-and-VP7 serum was stronger than that with anti-VP4 serum.

8

7

6

4 5 Group

3

2

1

46

9

FIG. 2. ELISA to show the titers of antipeptide antisera against BRV. The analysis was performed using sera obtained 3 weeks after the second immunization. Each titer represents the average taken from 10 mice. The groups were as follows: 1, placebo; 2, VP6; 3, BRV; 4, KLH-VP7 peptide 275-295; 5, KLH-VP4 peptide 232-255; 6, KLH-VP6 peptide 40-60; 7, VP6-VP4 peptide 232-255; 8, VP6VP7 peptide 275-295; 9, mixture of group 7 and 8.

Passive protection of suckling mice from homologous and heterologous challenge. Since all the peptide-carrier conjugates elicited antipeptide antibodies that recognized the viral proteins in a Western blotting assay, studies were conducted to determine whether neonates suckling immune dams were protected against virulent challenge by either homologous or heterologous strains of rotaviruses. In this model, diarrhea and morbidity are apparent within 24 h in nonimmune pups following challenge (challenge dose, i104 PFU per mouse). Morbidity, mortality, and severity of diarrhea were scored clinically on a scale from 0 to 4 and by the D-xylOse adsorption test over a 72-h period following challenge. The results of mouse challenge experiments are shown in Fig. 4 and Table 2. A variable degree of cross protection was obtained with peptide-carrier conjugates. Of the peptides coupled to VP6, the VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate that provided protection equal to that obtained using BRV as the immunogen. In contrast, the KLH conjugates provided only partial protection. The protection induced by either the

1

2

3

4

5

6

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8

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4

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4 30 221.5

FIG. 3. Western blotting of antipeptide mouse sera against BRV proteins. Viral proteins were separated by SDS-PAGE, under nonreducing conditions, and electroblotted onto nitrocellulose. The nitrocellulose sheets were cut into strips and used to analyze the reactivity of antisera with the viral proteins. Reactivity is indicated by the presence of black bands on the nitrocellulose strips. The antisera groups are as described in the legend to Fig. 2 and Table 2. Arrowheads indicate the locations of faintly reacting bands.

J . VlIROL .

IJAZ ET AL.

3110

TABLE 2. Protection of neonates from rotavirus challenge following immunization of dams with synthetic peptidesa Group

a) o

ct C]

Placebo

2

VP6

3

BRV

Protectionb from challenge strain Wa DS-1 Sa-11 BRV

N

N

N

N

0

p

p

N

p

*

C

C

p

C

4

KLH-275-295

C

p

N

p

5

KLH-232-255

C

C

p

p

-rl

6

KLH-40-60

p

p

N

p

C/) 0

7

VP6-232-255

c

c

c

c

x

8

VP6-BP-275-295

p

C

N

p

9

Group 7 + 8

C

C

C

C

c's

._

1

Structure (NH2-*COOH)

0 0 0

a)

a)

Immunogen

E

a-

pW4

a Suckling 7-day-old mice from immunized dams were challenged with rotavirus isolates (104 PFU per mouse). Morbidity and mortality were scored clinically, and intestinal function was assessed by a D-xylose adsorption test. b The protection scale is based on both clinical scores and D-xylose concentrations in plasma. C, complete protection (no clinical score and xylose concentration of >100 ,ug/,ul); p, partial protection (clinical score of 2 and xylose concentration of