c mice

Biochimica et Biophysica Acta 1760 (2006) 1884 – 1893 www.elsevier.com/locate/bbagen

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice Jennifer Kovacs-Nolan, Yoshinori Mine ⁎ Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received 28 February 2006; received in revised form 4 July 2006; accepted 27 July 2006 Available online 29 July 2006

Abstract The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M1ASLIYRQLL10, a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1–10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection. © 2006 Published by Elsevier B.V. Keywords: Human rotavirus; VP8; Neutralization epitope; Peptide antigen; B- and T-cell epitope; Antibody response

1. Introduction Human rotavirus (HRV) is the major cause of severe acute gastroenteritis among infants and young children, accounting for as many as 600,000 deaths per year, mostly in developing countries [1]. In developed countries HRV remains the most common cause of hospitalizations for acute gastroenteritis in children. Furthermore, individuals who are immunocompromised, due to aging, malnutrition, immune deficiencies, or cancer treatment, often develop persistent and recurring rotavirus infections [2]. Although clinical trials to assess the efficacy of two live oral attenuated HRV vaccines are currently under way, vaccination is often less effective in immunocompromised patients [3], and alternative therapies are necessary. The oral administration of pathogen-specific antibodies has been suggested as a method of

⁎ Corresponding author. Tel.: +1 519 824 4124x52901; fax: +1 519 824 6631. E-mail address: [email protected] (Y. Mine). 0304-4165/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.bbagen.2006.07.015

establishing passive immunity against rotavirus gastroenteritis [4], and would require the production of HRV-neutralizing antibodies. Rotavirus is a member of the Reoviridae family of viruses, and is a non-enveloped icosahedral virus. The virus particle contains multiple protein layers surrounding a double-stranded RNA genome [5]. The outermost layer is composed of two proteins, VP7 and VP4. VP7 forms the smooth external surface of the outer shell. VP4 is present as a series of dimeric spikes which project outward from the VP7 shell [6], and is important in the determination of virulence, host cell tropism, receptor binding and cell penetration. Viral infectivity is enhanced by the cleavage of VP4, by trypsin, into two proteins, VP5 and VP8 [7], and a number of studies have demonstrated the successful induction of rotavirus-neutralizing antibodies using VP8 subunit vaccines [8–11]. Previously, we described the presence of five of linear B cell epitopes on the VP8 subunit protein of the Wa strain of HRV, three of which were found to be involved in virus neutralization [12]. The epitope comprising amino acids 1–10 of VP8

J. Kovacs-Nolan, Y. Mine / Biochimica et Biophysica Acta 1760 (2006) 1884–1893

(M1ASLIYRQLL10) is highly conserved among strains of human rotavirus. Thus, focusing the antibody response towards this epitope could result in the production of highly specific, and potentially cross reactive, HRV-neutralizing antibodies. The use of peptide antigens allows the production of antibodies against a defined region of a protein, including antigenic sites that may not be easily accessible to the immune system, or that otherwise might be subdominant in the pathogen [13]. They can be designed to stimulate an appropriate protective immune response against specific sites on an antigen, and by using a mixture of peptides or by use of conserved sequences, they can stimulate broad immunity to protect against different strains or serotypes of a pathogen [14]. When using the strategy of peptide-based vaccines, it is important to consider the specificity of antigen processing, and the presence of both B cell and T cell epitopes [15,16]. B cell epitopes, such as amino acids 1–10 of VP8, cannot induce epitope-specific immune responses by themselves. The introduction of a T cell epitope is necessary in order to activate specific T cells to produce certain cytokines necessary for T and B cell activation, interaction and proliferation [17]. Several universally immunogenic T cell epitopes have been identified in tetanus toxin [18–20], a number of which, including the epitope P2 (aa. 830–844), have been found to modulate and enhance the immune responses to various proteins and peptides [13,21–23]. Furthermore, factors such as the peptide sequence, epitope density (i.e. number of peptides), conformation and orientation of the epitopes have been shown to influence the effectiveness of a peptide antigen in eliciting an immune response [24]. We describe here the production of recombinant peptide antigen constructs displaying the neutralization epitope M1 ASLIYRQLL10 of the VP8 subunit protein of HRV. The effect of epitope number, as well as the requirement for covalent attachment of the peptide to a carrier protein and T-cell epitope was examined by immunizing BALB/c mice, and the specific anti-peptide and virus-neutralizing antibody responses were examined.

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2. Materials and methods 2.1. Construction of plasmids encoding peptide antigens Expression plasmids encoding the P2 epitope of tetanus toxin (aa. 830–844 QYIKANSKFIGITEL; GenBank X04436) and residues 1–10 of the VP8 subunit protein of the Wa strain of human rotavirus (MASLIYRQLL; GenBank L34161) were constructed using the pET32b thioredoxin (Trx) fusion expression plasmid (Novagen, Darmstadt, Germany). Complementary oligonucleotide pairs, as shown in Table 1, were synthesized (Sigma-Genosys, TX, USA) and inserted into the pET32b plasmid (shown schematically in Fig. 1), to produce plasmids encoding TrxP2, TrxP2VP81–10, and TrxP2(VP81–10)3, which contained no copies, one copy, or three tandem copies of residues 1–10 of VP8, respectively. Synthetic oligonucleotides were first annealed by heating to 95 °C, followed by cooling to 20 °C, according to supplier's instructions. Oligonucleotides encoding the P2 epitope were ligated into the SalI/NotI site of the pET32b plasmid to produce the plasmid pETP2. Oligonucleotides encoding a single copy of residues 1–10 of the VP8 subunit protein of HRV were then ligated into the NotI/XhoI site of the pETP2 plasmid to produce the plasmid pETP2VP81–10. Oligonucleotides encoding three tandem copies of residues 1–10 of the VP8 subunit protein of HRV were also ligated into the NotI/XhoI site of the pETP2 plasmid to produce the plasmid pETP2 (VP81–10)3. Ligation reactions were carried out at a vector to insert ratio of 1:3, using T4 DNA ligase (Roche Diagnostics Corporation, IN, USA), and were used to transform Escherichia coli strain DH5α, according to the supplier's instructions (Invitrogen, CA, USA). Plasmids containing the correct inserts were verified by restriction digestion, and confirmed by DNA sequencing.

2.2. Transformation of expression host Competent E. coli Rosetta-gami™ B(DE3)pLysS cells (Novagen) were prepared by the calcium chloride method, using a modification of the method of Cohen et al. [25]. Briefly, Luria Bertani (LB) broth (BD Biosciences, CA, USA) was inoculated with E. coli Rosetta-gami™ B(DE3)pLysS, and grown at 37 °C to an OD600 of approximately 0.3. The culture was chilled on ice, and pelleted by centrifuging at 2000×g, for 10 min at 4 °C. The pellet was washed with 25 mM Tris–HCl, pH 7.5, containing 10 mM NaCl, centrifuged, and resuspended in 25 mM Tris–HCl, pH 7.5, containing 10 mM NaCl and 50 mM CaCl2. Following a final centrifugation step, the cells were resuspended in 25 mM Tris–HCl, pH 7.5, containing 10 mM NaCl and 50 mM CaCl2. Plasmid DNA (20 ng) was added to 50 μL of the competent E. coli cells. The cells and DNA were kept on ice for 30 min, followed by

Table 1 Complementary synthetic oligonucleotide pairs used to construct the pETP2, pETP2VP81–10, and pETP2(VP81–10)3 plasmids Product

Oligonucleotide sequence

P2 P2c

5′-tcgacggggggcagtacatcaaagctaactccaaattcatcggtatcaccgaactggggggggc-3′ a 5′-ggccgccccccccagttcggtgataccgatgaatttggagttagctttgatgtactgccccccg-3′a

VP81–10 VP8c1–10

5′-ggccgcggggctccttcagagatatattctctcagctatggggc-3′ b 5′-tcgagccccatagctgagagaatatatctctgaaggagccccgc-3′b

VP81–10−1 VP81–10−1c VP81–10−2 VP81–10−2c VP81–10−3 VP81–10−3c

5′-ggccgcggggctccttcagagatatattctctcag-3′ c 5′-ccatagctgagagaatatatctctgaaggagccccgc-3′c 5′-ctatggggctccttcagagatatattctctcagctatggg-3′ d 5′-aggagccccatagctgagagaatatatctctgaaggagcc-3′d 5′-gctccttcagagatatattctctcagctatggggc-3′ e 5′-tcgagccccatagctgagagaatatatctctga-3′e

a b c d e

Nucleotides in boldface ensured re-creation of a 5′ SalI site and a 3′ NotI site. Nucleotides in boldface ensured re-creation of a 5′ NotI site and a 3′ XhoI site. Nucleotides in boldface ensured re-creation of a 5′ NotI site. Oligonucleotides were 5′ phosphorylated. Nucleotides in boldface ensured re-creation of a 3′ XhoI site.

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Fig. 1. Schematic representation of the construction of the pETP2, pETP2VP81–10, and pETP2(VP81–10)3 expression plasmids. Complementary synthetic oligonucleotides corresponding to the tetanus toxin P2 epitope, a single copy of the VP81–10 epitope, and three tandem copies of VP81–10 were inserted into a pET32b expression vector. heat shock at 42 °C for 60 s. The cells were grown for 1 h, at 37 °C in 950 μL S.O.C. Medium (Invitrogen), and plated onto LB plates containing 50 μg/mL ampicillin (Roche) and 34 μg/mL chloramphenicol (Sigma Chemical Co., MO, USA).

PBS, and concentrated by ultrafiltration using a molecular weight cut-off of 10 kDa (YM 10; Millipore Corporation, MA, USA). Protein concentrations were determined using a DC protein assay (Bio-Rad Laboratories, CA, USA).

2.3. Expression and purification of recombinant peptide antigens

2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

LB broth (BD Biosciences) containing ampicillin (Roche) and chloramphenicol (Sigma) was inoculated with transformed E. coli Rosetta-gami™ B(DE3) pLysS cells, and grown to an OD600 of 0.5. Protein expression was induced by the addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) (Roche) for 3 h at 37 °C. The cells were pelleted by centrifugation at 5000×g for 10 min, and resuspended in 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, and 0.5% Triton X-100, to give a 50× concentrated cell lysate. Cells were disrupted by sonication, and centrifuged at 10,000×g for 10 min to pellet the insoluble fraction. Cell lysates were incubated with Ni–NTA agarose (QIAGEN Sciences, MD, USA), which had been equilibrated with 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 20 mM imidazole (Fisher Scientific, NJ, USA), for 1 h at 4 °C. The agarose was washed with 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 20 mM imidazole, and impurities were removed by washing with 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 100 mM imidazole. The recombinant Trx fusion proteins were eluted using 50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl and 250 mM imidazole. Fractions containing the pure protein were pooled, diafiltered against

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [26]. Samples were resolved using 10% acrylamide. Gels were stained using 0.02% (w/v) Coomassie Brilliant Blue R-250 and destained using a 40% (v/v) methanol, 7% (v/v) acetic acid solution.

2.5. Immunoblotting Proteins were separated by SDS-PAGE, using 10% acrylamide, and transferred electrophoretically to a PVDF membrane (Pall Corporation, MI, USA) in buffer containing 25 mM Tris–HCl, 192 mM glycine, and 20% (v/v) methanol. Membranes were blocked with Tris-buffered saline (TBS) containing 5% (w/v) bovine serum albumin (BSA) (Fisher Scientific), and then probed with mouse anti-Trx antibodies or chicken anti-VP8 antibodies [27], diluted 1/1000 in TBS containing 5% (w/v) BSA. Following washing with TBS containing 0.05% (v/v) Tween-20 (TBST), the membranes were incubated with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma) or rabbit antichicken IgG (Sigma), diluted 1/20 000 in TBS containing 5% BSA. Bound

J. Kovacs-Nolan, Y. Mine / Biochimica et Biophysica Acta 1760 (2006) 1884–1893 antibody was visualized using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche) and nitroblue tetrazolium chloride (NBT) (Roche) in 0.1 M Tris–HCl, pH 9.5, containing 0.1 M NaCl and 0.05 M MgCl2, according to manufacturer's instructions.

2.6. Animals and immunization Female BALB/c mice, aged 6–8 weeks, were obtained from Charles River Canada (Saint-Constant, Quebec, Canada) and were housed at the Central Animal Facility, University of Guelph, in accordance with the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. Groups of 5 mice were immunized subcutaneously with 50 μg of TrxP2VP81–10 or TrxP2(VP81–10)3 in PBS, emulsified with an equal volume of Freund's Complete Adjuvant (FCA) (Sigma). A group of mice were also co-immunized with 50 μg of a mixture of TrxP2 and chemically synthesized VP81–10 peptide (Nucleic Acids Protein Services Unit, UBC Biotechnology Laboratory, University of British Columbia). A schematic of the peptide antigens used for immunization is shown in Fig. 2. PBS emulsified with adjuvant was used as a negative control, and recombinant VP8, which had been shown previously to induce HRV-neutralizing antibodies [27], was used as a positive control. Booster immunizations of antigen in PBS emulsified with an equal volume of Freund's Incomplete Adjuvant (FIA) (Sigma) were administered 28 days following primary immunizations. Blood was collected at days 0 and 35, and serum was removed and stored at − 80 °C until required.

2.7. Assessment of antigen-specific antibody responses Serum antibody levels were measured using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well microwell plates (Corning Costar Corp., MA, USA) were coated with 0.25 μg/well of antigen in 0.1 M sodium carbonate buffer, pH 9.6, and incubated at 4 °C overnight. The plates were washed with TBST, and blocked with 2% (w/v) BSA (Fisher Scientific) in 0.1 M sodium carbonate buffer. The plates were then washed with TBST, and incubated at 37 °C, for 1 h, with 100 μL/well of mouse sera, serially diluted in TBS. The plates were washed again, and 100 μL/well of alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma), diluted 1/20 000 in TBS containing 1% (w/v) BSA, was added and incubated for 1 h, at 37 °C. The plates were washed and developed using 100 μL/well of p-nitrophenol phosphate (Sigma) in 1 M diethanolamine buffer, pH 9.8. The reaction was stopped using 3 N NaOH, and absorbances were read at 405 nm using a BioRad model 550 plate reader. Antibody titres were determined using all three Trx-containing antigens as coating antigen. Reciprocal titres were calculated as the serum dilution corresponding to an absorbance value of 0.2 units above background levels, and are expressed as the mean reciprocal titres for each group of anti-sera. Antigen-specific IgG1 and IgG2a titres were determined as described above, using rat anti-mouse IgG1 and IgG2a antibodies (BD Biosciences, CA, USA) at a concentration of 1 μg/mL, and horseradish peroxidase (HRP)-conjugated antirat IgG (BD Biosciences), diluted 1/2000. Detection was carried out using

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3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma), and absorbances were read at 450 nm.

2.8. Peptide-specific antibody response Residues 1–10 of the VP8 subunit protein of the Wa strain of human rotavirus (MASLIYRQLL) were synthesized using the SPOTs (Simple Precise Original Test System; Sigma-Genosys) synthesis method, as described by Frank and Overwin [28]. Peptides were synthesized by coupling fluorenylmethyoxycarbonyl (Fmoc) amino acids (Sigma-Genosys) to a cellulose membrane derivatized with a dimer of β-alanine-NH2 groups (Sigma-Genosys), according to the manufacturer's instructions. Following synthesis, membranes were washed with TBS, and blocked overnight in SPOTs blocking buffer (Sigma-Genosys), diluted 10× in TBST containing 5% (w/v) sucrose. The membranes were then washed with TBST and incubated overnight, at room temperature, with pooled mouse sera diluted 1/50 in SPOTs blocking buffer. Antibodies bound to the peptide were detected with alkaline phosphatase-conjugated anti-mouse IgG (Sigma), diluted 1/20 000 in SPOTs blocking buffer. Following washing with TBST, the bound antibodies were visualized using the chemiluminescent substrate CDP-Star (Boehringer Mannheim, Germany) and enhancer Nitro-Block II (Tropix, MA, USA), both diluted 1/100 in 0.1 M Tris–HCl, pH 9.5, containing 0.1 M NaCl, and the chemiluminescent reaction was measured with an EG and G BERTHOLD Molecular Light Imager (Bad Wildbad, Germany). Relative antibody binding for each group was reported as counts per second. Counts obtained from negative control (PBS) sera were considered background, and were subtracted from all samples to obtain the relative spot intensities for each antigen.

2.9. Virus neutralization assay Virus neutralization titres were determined by the fluorescent focus reduction neutralization (FFN) assay, using a modification of the method of Coulson and Masendycz [29]. A total 25 μl of serially diluted mouse serum was mixed with an equal volume of Wa strain HRV (2018-VR) (ATCC, MD, USA) suspension containing 5.0 × 104 focus forming units in Dulbecco's modified Eagle medium (DMEM) (Gibco, NY, USA), followed by incubation at 37 °C for 1 h. The mixtures of serum and virus were inoculated onto MA-104 (African green monkey cells) (ATCC) cells (2.5 × 105 cells/well) in 96-well tissue culture plates (Corning Costar), and after an additional 1 h of incubation at 37 °C, 100 μl of fresh DMEM was added, followed by overnight cultivation at 37 °C in 5% CO2. The cells were then washed four times with cold PBS to remove unbound virus. The cells were fixed in the cold (− 80 °C), and reaction with fluorescein isothiocyanate (FITC)-labelled anti-HRV antibodies (1:1000 dilution) was performed accordingly [29]. The presence of virus-infected cells was detected using a Fluoview™ 1000 Confocal Microscope (Olympus, Tokyo, Japan). Neutralization titres were expressed as the reciprocal of the dilution giving a 50% reduction in the number of fluorescing cells. The neutralization assays were performed in duplicate and repeated at least twice.

Fig. 2. Schematic representation of peptide antigens used for immunization of BALB/c mice.

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2.10. Statistical analysis Statistical analysis was carried out using SigmaStat 3.1 for Windows (Systat Software Inc., CA, USA). Analysis of variance (one-way ANOVA) and the Mann–Whitney U-test were used to determine the significance in antibody response. Groups were considered significantly different at P < 0.05. Unless stated, results were reported as the means of at least two independent determinations ± S.D.

3. Results 3.1. Expression and purification of TrxP2, TrxP2VP81–10, and TrxP2(VP81–10)3 Complementary synthetic oligonucleotide pairs corresponding to the tetanus toxin P2 T-cell epitope, as well as a single copy or three tandem copies of the B-cell epitope VP81–10 of HRV, were inserted into the multiple cloning site of the pET32b expression vector. This produced the

expression plasmids pETP2, pETVP81–10, and pETP2 (VP81–10)3, which were designed to co-linearly express the P2 and VP81–10 peptides, separated by glycine spacers, located at the C-terminus of the thioredoxin (Trx) fusion protein. Expression of the Trx fusion proteins was confirmed by SDS-PAGE and Western blotting analysis (Fig. 3A and B). No basal expression of the recombinant proteins was observed in the un-induced cultures. Following cell lysis, the Trx fusion proteins were found in the soluble fraction, and were purified from the cell lysate by Ni2+ ion affinity chromatography. Contaminant proteins were eluted with 100 mM imidazole, and successive elution steps with 250 mM imidazole resulted in pure fusion protein, as shown in Fig. 3C, at yields of approximately 5–8 mg/L of culture. The presence of the VP81–10 epitope was confirmed by Western blotting (Fig. 3D) using antibodies previously produced against recombinant VP8 [27].

Fig. 3. Expression of recombinant TrxP2, TrxP2VP81–10, and TrxP2(VP81–10)3. Recombinant antigens were expressed in E. coli Rosetta-Gami B(DE3)pLysS, and expression was visualized by SDS-PAGE (A) and Western blotting (B) analysis of un-induced (U) cultures and cultures induced for 3 h with 1 mM IPTG (I). Recombinant antigens were purified from the soluble fraction (cell lysate) by Ni2+ affinity chromatography. Purified proteins (10 μg), TrxP2 (1), TrxP2VP81–10 (2), and TrxP2(VP81–10)3 (3) were visualized by SDS-PAGE (C), and the presence of the VP81–10 epitope was confirmed by Western blotting, by probing with anti-VP8 antibodies (D).

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Fig. 4. Serum antibody titres of TrxP2 + VP81–10, TrxP2VP81–10, and TrxP2(VP81–10)3 anti-sera. Titres were measured by ELISA using TrxP2 ( ), TrxP2VP81–10 (□), and TrxP2(VP81–10)3 ( ) as coating antigen, and expressed as the mean reciprocal titres ± S.D. for each group. * Statistically significant (P < 0.05).

3.2. Immune response to VP81–10 peptide antigens

binding (P < 0.05) of specific anti-VP81–10 antibodies was observed with TrxP2(VP81–10)3 and TrxP2 + VP81–10 anti-sera.

Prior to immunization, sera from all mice were screened for non-specific binding to each of the antigens used. No antibody response was observed against any of the antigens, nor was there any detectable antibody response against any of the antigens in the negative control sera (mice immunized with only PBS). Following immunization, serum was collected from each mouse and antigen-specific antibody levels against all three antigens were measured by ELISA (Fig. 4). Antibody titres were highest in mice immunized with TrxP2(VP81–10)3, followed by VP81–10 co-administered with TrxP2, and then TrxP2VP81–10. Significantly higher titres (P < 0.05) were observed in anti-sera from all three antigen groups when TrxP2(VP81–10)3 was used as the coating antigen, when compared to coating with either TrxP2 or TrxP2VP81–10. Immunization with VP8 induced high IgG1/IgG2a ratios (Table 2), indicative of a Th2-biased immune response, while the peptide antigens, and TrxP2(VP81–10)3 in particular, induced a relatively more balanced immune response. To examine the specific anti-peptide antibody levels elicited by each peptide antigen, the anti-VP81–10 antibody response was evaluated by synthesizing the VP81–10 peptide on a solid support and probed with pooled anti-sera from each group (Fig. 5). The results indicated that all of the antigens used were capable of generating antibodies directed specifically against the VP81–10 region of VP8, however significantly higher

Table 2 Antigen-specific IgG1/IgG2a subclass ratios in mouse serum Antigen

IgG1/IgG2a

VP8 Trx + VP81–10 TrxVP81–10 Trx(VP81–10)3

31.67 ± 9.00 6.07 ± 4.10 3.77 ± 1.18 1.12 ± 0.15

3.3. HRV-neutralizing activity of anti-VP81–10 antibodies The ability of the anti-peptide antibodies to prevent viral infection in vitro was examined by a virus neutralization assay, using pooled sera from each group of mice. Neutralization titres are reported as the means of replicate measurements for each group (Fig. 6). The resulting virus neutralization titres were significantly higher (P < 0.05) than that of the of the negative control (PBS) sera, which was <8, and as expected, neutralizing activity was demonstrated by the anti-VP8 (positive control) sera. The anti-TrxP2(VP81–10)3 sera produced significantly higher neutralization titres (P < 0.05) than all other anti-peptide sera, including that from mice immunized with VP8. 4. Discussion Synthetic peptides provide the opportunity for producing highly specific immune responses, and introduce the possibility of producing cross-protective responses against pathogens where antigenic variation presents problems, by targeting conserved regions of the protein [30]. The amino acid region represented by amino acids 1–10 of the VP8 subunit protein of HRV was selected from the three previously identified linear neutralization epitopes [12] as it is highly conserved among HRV strains belonging to different serotypes. In order for a peptide to be used successfully as an antigen, it must stimulate the appropriate B-cells in order to elicit antibodies that will recognize the native protein in the pathogen and neutralize its infectivity. The design of a peptide antigen is therefore very important, and a number of factors have been shown to influence its overall success at eliciting an immune response against the desired peptide sequence, and more importantly, to induce neutralizing antibodies against the

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Fig. 5. Detection of specific anti-VP81–10 antibodies. Membrane-bound synthetic peptide VP81–10 was probed with anti-sera from mice immunized with each antigen, and bound antibody was measured by chemiluminescent detection. *Statistically significant (P < 0.05).

pathogen [31]. As peptides themselves are generally poor immunogens, it is common practice to couple peptides to a carrier protein in order to elicit an effective immune response. Although inherent T-cell epitopes are generally provided by the carrier protein, the inclusion of an additional T-cell epitope has been shown to enhance the immunogenicity of the attached antigen [21,32,33]. Studies have shown that residues 830–844 (P2) of the tetanus toxin represent a universal T-cell epitope in mice, and its inclusion in antigens has been demonstrated to strongly influence the antibody responses to poorly immunogenic B-cell epitopes [22,34–36]. The P2

sequence was therefore chosen to provide additional T-cell help for the VP81–10 B-cell epitope. The orientation and number of copies of the epitope can influence the way in which they are processed and presented, thereby affecting the specificity, levels and affinities of the antibodies produced following immunization with such constructs [31,37]. Several studies have illustrated the importance of orientation of the T-cell epitope with respect to the B-cell epitope. Constructs in which the T-cell epitope was N-terminal to a B-cell epitope have been found to induce a strong and specific antibody response to the B-cell epitope, whereas the C-

Fig. 6. HRV-neutralizing activity of anti-peptide antibodies. Neutralization titres for pooled anti-sera were determined against the Wa strain of HRV, and expressed as the reciprocal of the highest serum dilution resulting in a 50% reduction in fluorescent cell forming units. *Statistically significant (P < 0.05).

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terminal orientation of the T-cell epitope failed to induce such a response [31,38–41]. It has also been suggested that the Nterminal placement of the T-cell epitope in the overall construct may result in the peptide adopting a conformation that favours a more efficient binding of the B-cell epitope by peptide-specific surface immunoglobulin of B-cells, and has been shown to be associated with the production of increased levels of neutralizing antibodies [38]. In the present study, the peptide VP81–10, composed of amino acids 1–10 of the VP8 subunit protein of HRV, was colinearly expressed with the P2 T-cell epitope of tetanus toxin as a fusion protein with thioredoxin (Trx). The effect of epitope number was examined, comparing constructs containing one copy of the VP81–10 sequence versus three tandem copies of the epitope. The expression plasmids were designed such that the recombinant VP81–10 peptides were located C-terminally relative to the P2 epitope. The Trx fusion protein and P2 epitope were also co-administered with free peptide VP81–10, to examine the requirement for covalent attachment of the B- and T-cell epitopes in inducing a peptide-specific immune response. The constructs containing three tandem repeats of the VP81–10 epitope produced significantly higher antibody titres than those containing a single epitope or the co-administered B- and T-cell epitopes. Because the observed serum antibody titres included antibodies produced against not only the VP81–10 region of the protein, but also TrxP2, sera were screened using TrxP2, TrxP2VP81–10 and TrxP2(VP81–10)3 as ELISA coating antigens, to examine relative binding to TrxP2 versus the increasing number of VP81–10 epitopes. The apparent increase in antibody titre of all samples when TrxP2VP81–10 and TrxP2 (VP81–10)3 were used as coating antigens, as compared to TrxP2 alone, suggested that antibodies were indeed produced in response to the VP81–10 region of the protein. However, with anti-sera from all three groups, titres appeared to be significantly increased when the protein containing tandem repeats of the epitope was used as the coating antigen, suggesting that while all three antigens induced anti-peptide antibodies, the VP81–10 peptide region may have been more accessible to the antibodies in this particular peptide antigen construct, thereby facilitating antibody binding. Determination of IgG subclass ratios indicated that the antigen containing three tandem repeats induced a balanced Th1/Th2 response, which has been suggested to play a role in protection from HRV infection [42], and suggests the activation of both a humoral and cell-mediated immune response. In order to examine the peptide-specific antibody response, the VP81–10 peptide was synthesized on a solid support and probed with anti-serum from each group. The results indicated that all of the antigens used were capable of generating antibodies directed specifically against the VP81–10 region of VP8. However, the TrxP2(VP81–10)3, and more surprisingly TrxP2 + VP81–10 anti-sera, demonstrated significantly higher binding of specific anti-VP81–10 antibodies, greater even than the native VP8. The observation that the construct containing three tandem copies of the VP81–10 epitope could elicit higher levels of antipeptide antibodies is supported by a number of other studies,

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which have shown that an increase in the peptide copy number can increase the immunogenicity of a peptide [30,37,43,44]. Pillai et al. [33] suggested that the coupling of a single peptide to a larger carrier protein could induce conformational changes in B-cell epitopes, thereby reducing immunogenicity, or sterically inhibiting B-cell receptor/antibody binding to the peptide. Longer stretches of peptides, on the other hand, may result in the peptide being more accessible, and adopting a conformation favouring a more effective binding of the B-cell epitope to the surface immunoglobulin of the B-cell [41,43,45,46]. Liu et al. [47] expressed various numbers of tandem copies of a peptide attached to the recombinant fusion protein glutathione S-transferase (GST), and found that high epitope density could effectively enhance the peptide-specific immune response and enhance in vivo protective immunity. They also found that higher epitope density induced the production of higher affinity antibodies, and suggested that an increase in epitope repeats may amplify the average avidity (number of potential binding sites) of the peptide, which might in turn enhance their recognition and interaction with the antibody molecules on the surface of B-cells [48]. In the case of the co-administered TrxP2 and VP81–10 peptide, since aqueous mixtures of the peptides were emulsified in FCA, individual droplets may have co-entrapped both the fusion protein and the VP81–10 peptide, facilitating the uptake of both TrxP2 and VP81–10 by the same B-cells [49], thereby leading to the anti-peptide antibody levels observed here. It has been suggested that a covalent linkage between the B- and T-cell epitopes is not compulsory, and a number of studies have demonstrated that co-immunization with peptides representing B- and T-cell epitopes can result in the production of antibody to a B-cell epitope, without the requirement for the covalent linkage [13,31,40,49,50]. The successful induction of antipeptide antibodies following co-immunization of B- and T-cell epitopes may have arisen from the generation of “bystander help” [50], whereby cytokines secreted by T-cells in response to TrxP2 may have indirectly activated B-cells binding the free VP81–10 peptide, resulting in the production of VP81–10specific antibodies. However, it has been found that during such bystander help, insufficient co-stimulatory signals resulting from insufficient contact between the B- and T-cells preferentially activates low affinity B-cells, resulting in antibodies of lower titre and affinity than those produced when the epitopes are covalently attached [49,51]. The presence of anti-peptide antibodies does not always correlate with binding to the native pathogen proteins and subsequent neutralization. The ability of the anti-peptide antibodies to prevent viral infection in vitro was examined using a virus neutralization assay. As expected, neutralizing activity was demonstrated by the anti-VP8 sera, similar to previous results obtained using anti-VP8 antibodies [27]. However, the anti-TrxP2(VP81–10)3 sera demonstrated significantly higher neutralization titres than all other anti-peptide sera, including that from mice immunized with VP8. The high specific anti-peptide antibody levels which had been observed in the mice co-immunized with TrxP2 and free peptide did not, however, correlate with the observed neutralization titres, which

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were low. The differences observed in the neutralizing activities of TrxP2(VP81–10)3 and TrxP2 + VP81–10, despite both demonstrating high levels of anti-peptide antibodies, could be due to differences in their relative antibody affinities. Antibody affinity can vary depending on the level of help provided by the T-cells during the induction of an antibody response [51], and the effect of multiple tandem epitopes and covalently linked T-cell epitopes versus co-administered B- and T-cell epitopes on antibody affinity was discussed above. This may suggest that antibodies produced by the co-immunization of TrxP2 and the VP81–10 peptide had lower affinity for the VP81–10 epitope in the native VP8 protein, and therefore, could not neutralize the virus as effectively. We have demonstrated here that peptide antigens incorporating the amino acid sequence M1ASLIYRQLL10 of the HRV subunit protein VP8 were capable of eliciting anti-peptide antibodies. However, only the construct containing three tandem copies of the VP81–10 epitope resulted in HRVneutralizing antibodies, with neutralization titres significantly higher than those produced by free peptide, a single copy of the epitope, or the VP8 subunit protein. These results suggest that repeating VP81–10 epitopes are a more potent inducer of HRVneutralizing antibodies than VP8, and may be a more effective antigen for the production of HRV-specific antibodies for passive immunotherapy of HRV infection. Acknowledgements This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), Ontario Egg Producers' Marketing Board, Agriculture and Agri-Food Canada, and the Ontario Ministry of Agriculture and Food. We would also like to thank the staff at the University of Guelph Central Animal Facility for their assistance with the animal studies. References [1] M.A. Miller, L. McCann, Policy analysis of the use of hepatitis B, Haemophilus influenzae type B-, Streptococcus pneumoniae-conjugate, and rotavirus vaccines in national immunization schedules, Health Econ. 9 (2000) 19–35. [2] M.-A. Widdowson, J.S. Bresee, J.R. Gentsch, R.I. Glass, Rotavirus disease and its prevention, Curr. Opin. Gastroenterol. 21 (2004) 26–31. [3] K. Higo-Moriguchi, Y. Akahori, Y. Iba, Y. Kurosawa, K. Taniguchi, Isolation of monoclonal antibodies that neutralize human rotavirus, J. Virol. 78 (2004) 3325–3332. [4] R.M. Reilly, R. Domingo, J. Sandhu, Oral delivery of antibodies—Future pharmacokinetic trends, Clin. Pharmacokinet. 4 (1997) 313–323. [5] A.Z. Kapikian, Y. Hoshino, R.M. Chanock, in: D.M. Knipe, P.M. Howley (Eds.), Field's Virology, 4th Ed., Lippincott Williams and Wilkins, Philadelphia, 2001, pp. 1787–1833. [6] B.V.V. Prasad, G.J. Wang, J.P.M. Clerx, W. Chiu, Three-dimensional structure of rotavirus, J. Mol. Biol. 199 (1988) 269–275. [7] J.E. Ludert, A.A. Krishnaney, J.W. Burns, P.T. Vo, H.B. Greenberg, Cleavage of rotavirus VP4 in vivo, J. Gen. Virol. 77 (1996) 391–395. [8] J. Lee, L.A. Babiuk, R. Harland, E. Gibbons, Y. Elazhary, D. Yoo, Immunological response to recombinant VP8* subunit protein of bovine rotavirus in pregnant cattle, J. Gen. Virol. 76 (1995) 2477–2483. [9] D. Yoo, J. Lee, R. Harland, E. Gibbons, Y. Elazhary, L.A. Babiuk,

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