Nasal immunization of mice with a rotavirus DNA vaccine that induces protective intestinal IgA antibodies

Vaccine 23 (2004) 489–498

Nasal immunization of mice with a rotavirus DNA vaccine that induces protective intestinal IgA antibodies Ana Garc´ıa-D´ıaz, Pilar L´opez-And´ujar, Jes´us Rodr´ıguez D´ıaz, Rebeca Montava, Clara Torres Barcel´o, Juan M. Ribes, Javier Buesa∗ Departament de Microbiologia, Facultat de Medicina, Hospital Cl´ınic Universitari, Universitat de Val`encia, Avda. Blasco Ib´an˜ ez 17, 46010 Valencia, Spain Received 10 October 2003; received in revised form 8 June 2004; accepted 24 June 2004 Available online 25 July 2004

Abstract DNA vaccination using a plasmid encoding the rotavirus inner capsid VP6 has been explored in the mouse model of rotavirus infection. BALB/c mice were immunized with a VP6 DNA vaccine by the intramuscular, nasal and oral routes. VP6 DNA vaccination by the nasal and oral routes induced the production of anti-VP6 IgA antibodies by intestinal lymphoid cells. Intramuscular DNA injection stimulated the production of serum anti-VP6 IgG but not serum anti-VP6 IgA antibodies. Protection against shedding of rotaviruses in stools after oral challenge with the murine EDIM rotavirus strain was investigated in the immunized mice. A significant reduction in the level of rotavirus antigen shedding was demonstrated in those mice immunized at mucosal surfaces, both orally and nasally, with the VP6 DNA vaccine. Intramuscular DNA immunization, which elicited serum anti-VP6 IgG responses but not virus-specific intestinal IgA antibodies, did not provide significant protection against rotavirus challenge. © 2004 Elsevier Ltd. All rights reserved. Keywords: Rotavirus; DNA vaccine; Intestinal IgA antibody

1. Introduction The antibody response is considered to be the main effector mechanism that mediates protection against rotavirus infection [1–3]. Protection against oral murine rotavirus infection has been correlated with total serum and stool rotavirus IgA titers [4,5]. Murine rotavirus-specific CD8+ cells also have a direct antiviral effect, are involved in the resolution of primary rotavirus infection and can mediate partial protection against reinfection [6]. The protective effect of rotavirusspecific immunoglobulin A (IgA) against naturally acquired rotavirus infection has been proven in humans [7]. It has also been shown that CD4+ cells are essential for the development of more than 90% of the rotavirus-specific intestinal IgA [1]. Intestinal immunity can be induced by intranasal (i.n.) inoculation with non-replicating immunogens, with or without ∗

Corresponding author. Tel.: +34 96 386 46 58; fax: +34 96 386 46 58. E-mail address: [email protected] (J. Buesa).

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.06.018

mucosal adjuvants [8–11]. Purified rotavirus proteins [8], inactivated rotavirus [9] and rotavirus virus-like particles (VLPs) [10,11] administered by the nasal route have all been shown to elicit fecal antibodies or protection against viral challenge in mice. In contrast, oral inoculation with inactivated rotavirus may require high doses of viral protein or multiple inoculations [9]. The use of DNA vaccines is a new approach to immunization that may provide more effective rotavirus vaccines. VLPs and DNA vaccines may constitute a third generation of rotavirus vaccines [12]. It was previously reported that parenteral immunization with rotavirus VP4, VP6 and VP7 DNA vaccines induces high levels of serum rotavirus IgG but fails to protect mice against viral challenge [13,14]. However, other studies reported protection against rotavirus infection in mice by DNA vaccination [12,15–17]. In this study we report the efficient immunization and subsequent protection against rotavirus challenge in adult BALB/c mice achieved by the administration of a DNA vaccine encoding the rotavirus VP6 protein by both the intranasal

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and the oral routes. Mucosal immunization with a pcDNA3.1 plasmid vector carrying rotavirus SA11 genome segment 6 (VP6) evokes fecal IgA antibody responses. In addition, rotavirus-specific IgA production has been demonstrated in Peyer’s patches and mesenteric lymph nodes cells cultured in vitro after intranasal and oral administration of the VP6 DNA vaccine but not after intramuscular inoculation. Immunized adult mice challenged with the murine rotavirus strain EDIM showed significant protection as measured by the reduction in viral shedding in comparison to non-immunized controls.

2. Materials and methods 2.1. Vaccine plasmid construction SA11 rotavirus VP6 cDNA was obtained by a reverse transcriptase reaction/polymerase chain reaction (RT/PCR) procedure from viral RNA extracted as previously described [13]. The primers used were designed according to the genomic sequences of the RNA segment 6 of rotavirus SA11 [18]. Oligonucleotide sequences included the restriction endonucleases sites BamHI and XhoI to facilitate the cloning of the inserts in the poly-linker of the plasmid vector. The VP6 primers’ sequences were 5 -AACGGATCCTTCAACATGGATGTCC-3 (13–38) and 5 -AAAGCTCGAGACCAAGTTGTTAGC-3 (1248–1224). Underlined areas indicate the BamHI and XhoI sites, respectively. VP6 cDNA was cloned using these restriction sites into the poly-linker of plasmid pcDNA3.1(+) (Invitrogen Life Technologies, Paisley, Scotland, UK), downstream of the CMV immediate/early promoter. Correct insertion of the VP6 gene was confirmed by DNA sequencing in both directions with the T7 forward and pcDNA3.1/BHG reverse primers (Invitrogen) using the ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an automated sequencer (Applied Biosystems ABI PRISM 377). The nucleotide sequence for VP6 was determined to correspond to the sequence of the simian rotavirus strain SA11 in GenBank Acc. No. X00421. The recombinant plasmid and the control plasmid (pcDNA3.1 vector without the viral cDNA insert) were purified using a plasmid purification kit (UltraClean Endotoxin Free Midi Plasmid Prep Kit, MoBio Laboratories, Solana Beach, CA) following the manufacturer’s instructions. 2.2. Viruses and cell cultures SA11 rotavirus strain (ATCC VR-899) (G3, P5B [2]) was used to generate cDNA encoding VP6 and to produce virus antigen stock to be applied as the antigen in the ELISA tests. The murine EDIM strain of rotavirus (G3 P10 [16]) was originally provided by Dr. R.L. Ward (Children’s Hospital Medical Center, Cincinnati, OH). Both viral strains were grown in confluent MA104 cells maintained in Earle’s minimum essential medium (EMEM) without serum and with 1 ␮g/ml

trypsin. Passage-10 preparation of the EDIM strain was used for virus challenge of mice in this study. CV-1 cells were grown in Dulbecco minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and were used to verify the expression of rotavirus VP6 by the DNA vaccine plasmid in vitro. Transfection experiments were carried out on CV-1 cells maintained with Opti-MEM cell culture medium (Gibco Invitrogen). 2.3. Transfection of CV-1 cells CV-1 cells were transfected with the recombinant plasmid pcDNA3.1-VP6 and with the control plasmid pCMV-Sport␤gal, which expresses ␤-galactosidase, using Lipofectamine reagent (Invitrogen Life Technologies) according to the protocol provided by the manufacturer. 2.4. Immunoprecipitation In vitro expression of VP6 by the pcDNA3.1-VP6 vector was confirmed by immunoprecipitation of 35 S-labeled CV-1 transfected cells with an anti-VP6 monoclonal antibody (Chemicon International, Temecula, CA) using methods previously described [19]. Immunoprecipitation of the VP6 protein of the RF strain of rotavirus produced by a recombinant baculovirus (a gift from Dr. J. Cohen, CNRS-INRA, Gif sur Yvette, France) was used as a control. 2.5. Immunization of mice BALB/c mice (Charles River Laboratories) were previously confirmed to be negative for anti-rotavirus antibodies by enzyme-linked immunosorbent assay (ELISA). They were housed in plastic microisolator cages before and after immunization. A group of 20 female BALB/c mice (4–5 weeks of age) were anesthetized by halothane inhalation and inoculated intramuscularly using a 1 ml syringe and a 28-gauge needle into the pretibial muscle of the hind leg with 50 ␮l of pcDNA3.1-VP6 plasmid suspension (75 ␮g DNA per dose). As a control, a group of 10 mice were given the same dosage of pcDNA3.1 lacking the insert. Animals were boosted twice at 2-week intervals. A group of 15 adult female BALB/c mice were intranasally administered 75 ␮g pcDNA3.1-VP6 prepared with Cellfectin (Invitrogen), a cationic liposome formulation. Plasmid DNA was previously mixed with Cellfectin reagent at a DNA:lipid (w/w) ratio of 1:1, incubated at room temperature for 30 min and gently instilled into the nostrils of the mice with a micropipette. Two boosters were given at 2-week intervals. Another group of 15 mice were given the same preparation by the oral route following the same immunization protocol. As controls, two groups of five mice received equal doses of vector lacking the insert by the nasal and oral routes. Another group of five mice was orally infected with 5 × 104 focus-forming units (FFUs) of the EDIM strain. Blood samples were collected from a tail vein of each mouse on the day mice were boosted and 2 weeks

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after the last immunization. Four dams from each group were mated once it was confirmed that they were immunized. 2.6. Measurement of anti-rotavirus antibodies in serum and fecal samples by enzyme-linked immunosorbent assay (ELISA)

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SA11 rotavirus (5 ␮g of purified virus) for in vitro cell restimulation. Cells were incubated at 37 ◦ C with 5% CO2 for 5 days and supernatants were collected at day 5 for IgA antibody detection. 2.8. Challenge experiments

IgG and IgA anti-rotavirus antibody titers in serum samples were determined by ELISA as previously described [20]. The SA11 rotavirus preparation used as the coating antigen was purified by fluorocarbon extraction and CsCl gradient centrifugation from virus-infected MA-104 cells showing 90% cytopathic effect as previously described with some modifications [21]. The visible bands containing tripleand double-layered virus particles were collected, mixed, dialyzed and concentrated by ultracentrifugation. The pellets were resuspended in TNC buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2 ) and stored at −70 ◦ C. Microtiter ELISA plates (Nunc Immuno I, Nunc, Roskilde, Denmark) were coated for 2 h at 37 ◦ C with 0.2 ␮g/well of the purified virus preparation previously treated with 1.5 M NaSCN for 15 min and diluted with 0.06 M carbonatebicarbonate buffer (pH 9.6). Serum samples were serially diluted with PBS–0.1% Tween 20 (PBS-T) containing 1% BSA, starting from 1 to 50 for IgG and from 1 to 25 for IgA, and added to antigen-coated wells. Ten per cent (w/v) fecal suspensions, regarded as 1:10 dilutions, were prepared in PBS and stored frozen (−30 ◦ C) until tested. After a 2-h incubation at 37 ◦ C, plates were washed and peroxidase-conjugated goat anti-mouse IgG or IgA (Sigma Immunochemicals, St. Louis, MO) in 1% BSA–PBS-T were added and incubated for 2 h at 37 ◦ C. After three washes the reactions were developed using o-phenylenediamine and stopped with 0.5 M H2 SO4 . Optical density (OD) was determined at 492 nm. Optimal dilutions of reagents were determined by checkerboard titration. A sample was considered to be positive when the OD was greater than or equal to three times the negative control (mock-infected MA104 cells).

To investigate whether immunization with the VP6 DNA vaccine protects mice against rotavirus infection both the adult mouse model developed by Ward et al. [23] and the newborn mouse model were used. In the adult mouse model, infection rather than disease is investigated, because diarrhea is generally limited to infant mice younger than 15 days of age. Immunized adult mice were orally given 5 × 104 FFUs of EDIM rotavirus 2 weeks after the last immunization. Litters of BALB/c mouse pups born to the immunized dams were allowed to suckle for 6–9 days after birth and were then orally inoculated with 100 DD50 of EDIM rotavirus strain. The DD50 of the stock virus was the 50% diarrhea-inducing dose as determined in infected newborn mice by the method of Reed and Muench [24]. Inoculated mice were housed in separate isolation units and were examined daily for diarrhea by gentle palpation of the abdomen. All procedures were conducted in accordance with the regulations established by the European Community Council on the protection of animals with experimental and scientific applications (86/609/CEE).

2.7. Lymphoid cell cultures

Statistical analysis was carried out using SPSS for Windows, release 11.5.1 (LEAD Technologies Inc., USA). Analysis of variance was used to determine the significance of the differences in the antibody responses and in the level of virus shedding among the mouse groups. All possible pairwise comparisons were performed using the Mann–Whitney U-test and significance level (initially P < 0.05) was corrected by means of Bonferroni adjustment to compensate for type 1 error.

Spleen, bronchial lymph node and intestinal lymphoid cells were cultured to assess the production of anti-VP6 IgA antibodies by lymphoid tissues using previously described techniques [22]. IgA antibodies to VP6 were investigated in the supernatant of lymphoid cell cultures from spleen, bronchial lymph nodes (BLN), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) obtained from mice inoculated 10 days earlier with the final boost of the VP6 DNA vaccine. Lymphoid tissues were aseptically removed and mechanically dissociated. Cells were washed with RPMI medium (Gibco Invitrogen) supplemented with 2 mM lglutamine, 20 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 ␮g/ml streptomycin. The cells were resuspended in RPMI medium plus 10% FBS (4 × 106 cells/ml) and placed into wells of 24-well plates in the presence of

2.9. Detection of rotavirus shedding Stools were collected into 0.5 ml of TNC buffer (0.05 M Tris–HCl, 0.15 M NaCl, 0.01 M CaCl2 ) on the day of the EDIM rotavirus challenge and daily over 10 days following the challenge. Rotavirus antigen shedding in the fecal samples was determined by an ELISA test for rotavirus antigen as previously described, basing antigen shedding on optical density values (A492 ) [4]. 2.10. Statistical analysis

3. Results 3.1. In vitro expression of the recombinant VP6 protein To ensure that gene 6 subcloned into the eukaryotic expression vector pcDNA3.1 could express VP6 in vitro,

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Fig. 1. Detection of VP6 by immunoprecipitation with an anti-VP6 monoclonal antibody in 35 S-methionine labeled lysates of CV-1 cells transfected with the pcDNA3.1-VP6 vector. A 42 kDa band, corresponding to the molecular weight of VP6, was demonstrated. The identity of this band was confirmed by immunoprecipitation of the VP6 protein produced by a recombinant baculovirus expressing VP6. CV-1 cell lysates transfected with the vector pCMV-Sport-␤gal were precipitated with the same monoclonal antibody as the negative control.

CV-1 cells were transfected with the vector developed in our laboratory (pcDNA3.1-VP6) and the results were analyzed by immunoprecipitation. Radiolabeled lysates of CV-1 cells transfected with the pcDNA3.1-VP6 vector were precipitated with an anti-VP6 monoclonal antibody and a 42 kDa band was obtained corresponding to the molecular weight of VP6 (Fig. 1). The VP6 protein produced by a recombinant baculovirus grown on Sf9 cells was used as a control. CV-1 cell lysates transfected with pcDNA3.1 and with pCMV-Sport-␤gal were precipitated with the same antibody as the negative controls. The percentage of transfected cells was calculated as 10%, based on the in situ expression of ␤-galactosidase from positive control cells transfected with pCMV-Sport-␤gal (result not shown). 3.2. Serum anti-VP6 IgG and IgA antibodies production To determine whether the expressed recombinant protein was immunogenic or not, BALB/c mice were first immunized intramuscularly with pcDNA3.1-VP6 (75 ␮g per dose, three doses). A single intramuscular immunization was sufficient to elicit a week anti-VP6 IgG response in all the injected mice. No substantial increase was observed after the second dose (Fig. 2). The VP6 DNA vaccine administered by the mucosal routes (both orally and nasally) induced low serum IgA responses but did not elicit serum IgG antibodies (Fig. 3). VP6specific serum IgA antibodies were significantly stimulated

after intranasal immunization (P < 0.05) and after oral immunization (P < 0.001) compared to the serum IgA response induced by the intramuscular route. 3.3. Fecal anti-VP6 IgA antibodies detection Rotavirus-specific IgA antibodies were detected in the fecal samples collected from those animals inoculated by the intranasal and the oral routes with pcDNA3.1-VP6 before the virus challenge (Fig. 3). Mice inoculated intramuscularly did not develop a detectable fecal anti-VP6 IgA response. The results of the ELISA tests for rotavirus-specific serum IgG, serum IgA and fecal IgA analysis in the mice immunized by the different routes are shown in Fig. 3. We found no differences in the anti-rotavirus antibody titers triggered by the VP6 DNA vaccine given by the nasal and oral routes (P > 0.05). 3.4. Induction of anti-VP6 antibody production by intestinal lymphoid tissues after nasal and oral DNA immunization Having assessed the immunogenic potential of the VP6 DNA vaccine, we examined the utility of intranasal and oral inoculation with the plasmid pcDNA3.1-VP6 (given at three doses of 75 ␮g DNA with liposomes) to elicit the production of rotavirus-specific antibodies by intestinal lymphoid cells.

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Fig. 2. Rotavirus-specific serum IgG antibodies determined by ELISA in mice which were immunized intramuscularly with pcDNA3.1-VP6 (75 ␮g per dose, three doses at 2-week intervals). Control mice were either inoculated i.m. with pcDNA3.1 (75 ␮g per dose, three doses) or orally inoculated with EDIM rotavirus (5 × 104 FFUs, three doses). A492 values correspond to the 1/100 serum dilutions. Serum samples were collected on day 0, when mice were boosted, and then 2 weeks after the last inoculation. The symbol (*) indicates significant differences (P < 0.001 in the Mann–Whitney U-test) in the absorbance values between pcDNA3.1-VP6-inoculated mice and the negative control group.

Fig. 3. Antibody responses (serum IgG, serum IgA and fecal IgA) elicited by the immunization of BALB/c mice with plasmid pcDNA3.1-VP6. Female mice were given three doses of the plasmid DNA by the nasal, oral or intramuscular routes at 2-week intervals. Plasmid DNA administered mucosally was previously mixed with a cationic liposome formulation (Cellfectin, Invitrogen) at a DNA:lipid (w/w) ratio of 1:1. Results are expressed as mean A492 values + standard error. Serum samples were diluted 1/100 and fecal samples 1/80. The absorbance values of serum and fecal samples from mice DNA-vaccinated by the three routes were compared with the control groups, which received the plasmid lacking the insert. Statistically significant differences are indicated by an asterisk (P < 0.001 in the Mann–Whitney U-test).

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Fig. 4. Rotavirus-specific IgA antibodies, determined by ELISA, produced in the supernatant of cultured lymphoid cells from spleen, bronchial lymph nodes (BLN), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) obtained from mice immunized with the VP6 DNA vaccine by intramuscular, intranasal or oral routes. Lymphoid tissues were aseptically removed and mechanically dissociated. Cells were incubated at 37 ◦ C with 5% CO2 for 5 days and supernatants were collected for antibody detection. Results are expressed as mean A492 values + standard error. Statistically significant differences between lymphoid cell cultures from immunized and non-immunized mice are indicated by an asterisk (P < 0.001 in the Mann–Whitney U-test).

Serum and fecal anti-rotavirus IgA were detected in those mice immunized by the nasal and oral routes, although the amount of IgA appeared to be low (Fig. 3). For this reason, generation of anti-VP6 antibodies was investigated in the supernatant of cultured lymphoid cells from spleen, bronchial lymph nodes (BLN), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) obtained from mice inoculated with the final intranasal boost of the VP6 DNA vaccine 10 days earlier. VP6-specific IgA antibodies were produced at highest levels by MLN lymphocytes cultured in vitro, at lower amounts by PP cells and also by BLN cells from those mice immunized intranasally (Fig. 4). No detectable levels of VP6-specific IgA antibodies were found in the supernatants of mucosal lymphoid cell cultures from mice inoculated intramuscularly with the VP6 DNA vaccine. We did not detect significant production of virus-specific IgA antibodies by spleen cells derived from either parenteral or mucosal immunized mice (Fig. 4).

and 6A). Significant levels of protection were achieved in both mouse groups vaccinated via the oral and nasal routes (74 and 78% reduction in viral shedding, respectively). Protection levels were not significantly different between the two groups (P > 0.05). Mice immunized by the intramuscular route had a non-significant level of reduction in rotavirus shedding relative to the unimmunized control mice (15%, P > 0.05) (Fig. 6A). In addition, all the infant mice breast-fed by dams immunized with the VP6 DNA vaccine suffered diarrhea when they were orally challenged with 100 DD50 of live EDIM virus, irrespectively of the vaccination route employed (Fig. 6B). As in other studies, our results show that the protection against rotavirus infections in mice correlates with the production of local IgA antibodies and not with serum IgG [4,25].

4. Discussion 3.5. Protection against rotavirus challenge Adult mice immunized by intranasal and oral VP6 DNA inoculation were partially protected from rotavirus infections as they demonstrated a significant decrease (P < 0.001) in the shedding of rotavirus in feces after oral challenge with EDIM rotavirus compared to control mice which had been administered pcDNA3.1 vector lacking the insert (Figs. 5

In this study we have demonstrated the stimulation of an anti-VP6 serum IgG antibody response by the intramuscular inoculation of the pcDNA3.1-VP6 plasmid to BALB/c mice. However, these antibodies are not protective, as was demonstrated by the fact that the immunized mice shed viral antigen in stools in similar quantities to the unimmunized controls. More importantly, a protective IgA response has

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Fig. 5. Protection against 5 × 104 focus-forming units (FFUs) of EDIM virus challenge in immunized BALB/c mice. Rotavirus antigen shedding was determined by ELISA in feces diluted 1/80 in TNC buffer following an oral challenge with the EDIM rotavirus of BALB/c mice immunized with plasmid pcDNA3.1-VP6 by the nasal, oral or intramuscular routes. Error bars represent the standard error of the mean for viral shedding for each mouse group (N = 6 mice per group). The control group (N = 5) was orally given pcDNA3.1 lacking the insert. The symbol (*) indicates significant (Mann–Whitney U-test, P < 0.001) differences in the absorbance values reflecting viral antigen shed between pcDNA3.1-VP6 immunized groups by the nasal and oral routes and the control group.

been achieved by both nasal and oral immunization of mice with a VP6 DNA vaccine. Mucosal DNA immunizations, performed by both the nasal and oral routes, generate antirotavirus intestinal VP6 IgA antibodies and these antibodies elicit protection against rotavirus infection in the adult mouse model. Although care was taken to avoid the possibility that mice inoculated nasally with the DNA vaccine would swallow the preparation, we cannot exclude that this event may have occurred to some degree. Therefore, the results of nasal and oral administration of the DNA vaccine to mice were assayed separately to analyse differences in the uptake and immunization capability of the DNA preparation by both routes. However, no differences in the anti-rotavirus antibody titers triggered by the VP6 DNA vaccine given by the nasal and oral routes were found. It has previously been stated that virus-specific IgA detection in supernatant fluids from intestinal organ cultures is considerably more sensitive than its detection in intestinal washes or stools [26]. Our findings reveal higher levels of anti-VP6 IgA in the supernatant of cultured lymphoid cells from mesenteric lymph nodes and Peyer’s patches than in stools from the immunized mice, confirming the results reported by Offit et al. [26]. We have achieved significant protection against murine rotavirus challenge using a simian rotavirus VP6 DNA vaccine. This suggests that heterologous protection against a heterotypic rotavirus can be obtained by immunizing with a VP6

DNA vaccine, an observation also reported by Yang et al. [17]. The amino acid sequences of the simian SA11 rotavirus strain VP6 and the murine EDIM rotavirus strain VP6 are approximately 93% homologous. DNA vaccines against rotavirus infection were previously developed by Herrmann et al. [16] and by Choi et al. [14], who also investigated the protection elicited against murine rotavirus infection in the mouse model. They tested plasmid DNA vaccines encoding for murine rotaviral proteins VP4, VP6 and VP7 and they reported that the strongest immune response was induced by the VP6 DNA vaccine. For this reason we chose VP6 as the viral protein to be expressed by our plasmid DNA vaccine. However, some discrepancies were obtained by Herrmann et al. [16] and by Choi et al. [14] in the immunogenicity of the DNA vaccines employed and in their protective outcome [13]. Our results contrast with those obtained by Choi et al. who reported that the genegun inoculation was very effective in inducing anti-rotavirus IgG serum antibodies, that the intradermal injection was less efective and that no measurable IgG response was detected following intramuscular injection [13]. In the current study we demonstrate that intramuscular immunization with the VP6 DNA vaccine indeed elicits an IgG anti-VP6 response. Choi et al. [13] found that parenteral immunization with VP6 actually triggered large anti-rotavirus IgG responses but did not stimulate protection against murine rotavirus infection in the mouse model, which is confirmed by our results.

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Fig. 6. Protection levels (as percentage of protection) induced by the VP6 DNA vaccine in adult BALB/c mice against viral shedding (A) and in newborn BALB/c mice against diarrhea (B) after challenge with murine rotaviruses. (A) Groups of adult BALB/c mice were immunized three times with 75 ␮g of pcDNA3.1-VP6 by the intranasal, oral or intramuscular routes. Control mice were given plasmid pcDNA3.1 or were orally infected with 5 × 104 FFUs of the EDIM strain. All mouse groups were orally challenged with 5 × 104 FFUs of EDIM rotavirus 2 weeks after the last immunization. Stools were collected on the day of the EDIM rotavirus challenge and daily over 10 days following the challenge. Rotavirus antigen shedding in the fecal samples was determined by ELISA. The average reduction in virus shedding was calculated as a percentage relative to unimmunized control mice. (B) Passive protection of newborns against diarrhea. Dams were immunized three times with 75 ␮g of pcDNA3.1-VP6 by the intranasal, oral or intramuscular routes. A control group of female mice were orally infected with EDIM rotavirus (5 × 104 FFUs, three doses). Breeding was started at week 5 by mating them with seronegative males. Litters of pups born to the immunized dams were allowed to suckle for 6–9 days after birth and then orally inoculated with 100 DD50 of the EDIM strain. Pups were examined daily for diarrhea.

Yang et al. [17] reported that intramuscular immunization of mice with VP6 DNA vaccines induced high levels of VP6-specific serum IgG and IgA antibodies but not fecal IgA antibodies. They also achieved partial protection against EDIM rotavirus challenge in mice immunized with both a bovine and a murine rotavirus VP6 DNA vaccine administered by the intramuscular route. In our study no significant serum IgA antibodies were detected after intramuscular immunization and the mice were not found to be protected against rotavirus infection. These discrepancies may be due to differences in vaccine composition: the VP6 proteins expressed by the vaccine plasmids prepared by Yang et al. were from murine strain EW and from bovine rotavirus strain UK, whereas in this study the VP6 cDNA was derived from the simian rotavirus strain SA11. In addition, the localization and distribution of DNA vaccines after i.m. injection in mice, the amount of antigen delivered to immunologic sites and the type of activated antigen-presenting cells (APCs) may influence the production of virus-specific IgA [27,28]. Coffin et al. [29] demonstrated that migration of Ag-presenting B cells from peripheral lymphoid tissues to gut-associated lymphoid tissues (GALT) contributed to the generation of mucosal IgA responses after parenteral immunization with live, wild-type rotavirus. In that case, B cells derived from peripheral lymph nodes functioned as antigen-presenting cells and stimulated a short-lived virus-specific response in GALT. In our experiments, i.m. DNA immunization probably did not induce activation of B cells as APCs. However, other types of APCs such as macrophages or dendritic cells have been shown to play a critical role in the induction of immune responses by DNA vaccination [30]. The differential induction of virus-specific IgA or IgG by different types of APCs depends on the pattern of cytokines produced by DC4+ T cells which may be influenced by the stimulating APC type [29]. Oral immunization with a rotavirus DNA vaccine was demostrated for the first time by Chen et al. [12], who prepared an encapsulated VP6 DNA vaccine in poly(lactidecoglycolide) (PLG) microparticles and achieved protection against rotavirus infection. Our study confirms their results and demonstrates that the same outcome can be obtained after intranasal administration of a liposome-formulated DNA vaccine. We have previously shown that the immunization of dams with the VP8∗ subunit of the VP4 capsid protein elicits homotypic protection against rotavirus-induced diarrhea in their offspring [19]. Antibodies against VP4, and also against VP7, are known to have viral neutralizing activity. Burns et al. showed that IgA non-neutralizing monoclonal antibodies towards VP6 exhibited a protective effect against rotavirus infection in mice [31]. It has been hypothesized that these VP6-specific IgA antibodies can inactivate virus replication during transcytosis through epithelial cells. IgA-mediated intracellular virus neutralization has been demonstrated for both Sendai virus and influenza A virus [32]. In agreement with this hypothesis, mice genetically knocked out for the J chain of immunoglobulins and therefore unable to

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transcytose were found to be not protected when immunized with VLPs made of VP2 and VP6 [33]. In the adult mice, and probably also in humans, polymeric IgA antibodies can mediate intracellular protection when they interact with virus or with subviral particles in the cytosol of infected enterocytes. In this study we have demonstrated protection against viral shedding following oral challenge with EDIM rotavirus in adult mice orally and nasally immunized with the VP6 DNA vaccine, but not in mice immunized by the intramuscular route. However, pups born to dams immunized by any route with the VP6 DNA vaccine were not protected against diarrhea after having been orally challenged with the EDIM strain. As we have previously demonstrated, protection against rotavirus disease in newborn mice is mediated by neutralizing secretory antibodies present in the milk rather than by serum antibodies transferred through the placenta to the offspring [19]. Antibodies to VP6 do not elicit passive protection to the newborns, suggesting that these antibodies do not neutralize extracellular virus. The route of immunization is critical for generating the correct immune response in order to obtain protection against pathogenic organisms. It is known that vaccines which are delivered by intramuscular or subcutaneous injections induce strong systemic responses but generally no mucosal immunity [34]. Intramuscular injection of DNA in aqueous solution has been found to preferentially induce a Th1 response in mice, with the expansion of the IgG2a isotype [17,27]. Choi et al. achieved the induction of immune responses of BALB/c mice by parenteral administration of DNA vectors expressing rotaviral VP4, VP6 and VP7 proteins. Although they assayed different delivery methods with the three antigens, they failed to protect mice against viral challenge despite the high levels of serum IgG antibodies induced [13]. Mucosal immunization with DNA vaccines by targeting plasmid DNA to mucosal inductive sites associated with the MALT can be an efficient approach for inducing protection against viral enteric infections [35]. To improve DNA immunization of the mucosa a strategy has been developed using an M cell ligand, the reovirus ␴1 protein, to direct the DNA vaccine to mucosal inductive tissues and enhance the IgA and CTL responses [36]. CTL activity and cytokine levels have not been analysed in this study. However, intestinal IgA antibodies are considered pivotal in protection from rotavirus infection in mice [37]. We cannot exclude that CD8+ T lymphocytes are involved in the clearance of rotavirus from the intestinal mucosa after the VP6 DNA vaccination. Other viral proteins (VP7, VP3, NSP1) in addition to VP6 have also been identified in different studies as being the main targets of rotavirus-specific cytotoxic T lymphocytes in rotavirus infections in mice [38–41]. Gamma interferon (IFN-␥) has also been suggested to be partially responsible for protection against rotavirus shedding after i.n. VP6 immunization [42]. However, immunization of adult Stat1−/− mice with VP6 and the mucosal adjuvant Escherichia coli heat-labile toxin LT(R192G) elicited full protection against rotavirus replication [43], suggesting

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that IFN-␥ is not an indispensable effector mechanism for protection. According to our results, mucosal DNA immunization against rotavirus-induced diarrhea elicits effective protection compared to the use of the parenteral route. Stimulation of a secretory local IgA response is clearly involved as a protective mechanism stimulated by the DNA vaccination. Further studies are needed to clarify the role played by VP6-specific CD8+ and CD4+ T cells in this DNA-based immunization. Acknowledgements This work was supported by the Fondo de Investigaci´on Sanitaria (FIS) of the Spanish Ministry of Health Grant Contract No. 00/0592. J. Rodr´ıguez-D´ıaz was the recipient of a fellowship from the Spanish Ministry of Education and Culture (No. AP2000-3623). We thank Dr. A. MacCabe for reviewing the English. References [1] Franco MA, Greenberg HB. Immunity to rotavirus in T cell deficient mice. Virology 1997;238:169–79. [2] Franco MA, Greenberg HB. Role of B cells and cytotoxic T lymphocytes in clearance of and immunity to rotavirus infection in mice. J Virol 1995;69:7800–6. [3] McNeal MM, Barone KS, Rae MN, Ward RL. Effector functions of antibody and CD8+ cells in resolution of rotavirus infection and protection against reinfection in mice. Virology 1995;214:387–97. [4] McNeal MM, Broome RL, Ward RL. Active immunity against rotavirus infection in mice is correlated with viral replication and titers of serum rotavirus IgA following vaccination. Virology 1994;204:642–50. [5] Ward RL. Possible mechanisms of protection elicited by candidate rotavirus vaccines as determined with the adult mouse model. Viral Immunol 2003;16(1):17–24. [6] Franco MA, Tin C, Greenberg HB. CD8+ T cells can mediate almost complete short-term and partial long-term immunity to rotavirus in mice. J Virol 1997;71(5):4165–70. [7] Hjelt K, Grauballe PC, Paerregaard A, Nielsen OH, Krasilnikoff PA. Protective effect of preexisting rotavirus-specific immunoglobulin A against naturally acquired rotavirus infection in children. J Med Virol 1987;21:39–47. [8] Choi AH-C, Basu M, McNeal M, Clements J, Ward R. Antibodyindependent protection against rotavirus infection of mice stimulated by intranasal immunization with chimeric VP4 or VP6 protein. J Virol 1999;73(9):7574–81. [9] McNeal MM, Rae MN, Bean JA, Ward RL. Antibody-dependent and -independent protection following intranasal immunization of mice with rotavirus particles. J Virol 1999;73(9):7565–73. [10] O’Neal CM, Clements JD, Estes MK, Conner ME. Rotavirus 2/6 virus-like particles administered intranasally with cholera toxin, Escherichia coli heat-labile toxin (LT), and LT-R192G induce protection from rotavirus challenge. J Virol 1998;72(4):3390–3. [11] Fromantin C, Jamot B, Cohen J, Piroth L, Pothier P, Kohli E. Rotavirus 2/6 virus-like particles administered intranasally in mice, with or without the mucosal adjuvants cholera toxin and Escherichia coli heat-labile toxin, induce a Th1/Th2-like immune response. J Virol 2001;75(22):11010–6. [12] Chen SC, Jones DH, Fynan EF, Farrar GH, Clegg JCS, Greenberg HB, et al. Protective immunity induced by oral immunization

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