Curr Microbiol (2014) 68:503–509 DOI 10.1007/s00284-013-0498-z

Neutralizing Activity Induced by the Attenuated Coxsackievirus B3 Sabin3-like Strain Against CVB3 Infection Nadia Jrad-Battikh • Amira Souii • Leila oueslati • Mahjoub Aouni • Didier hober • Jawhar Gharbi • Manel Ben M’hadheb-Gharbi

Received: 26 July 2013 / Accepted: 7 October 2013 / Published online: 10 December 2013 Ó Springer Science+Business Media New York 2013

Abstract Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. In the present work, we assessed whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and CVB4 E2 strains in mice by both oral and intraperitoneal (IP) routes. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their anti-CVB3 neutralizing activity. CVB3 Sabin3-like was highly attenuated in vivo and was able to induce an antiCVB3 activity of the serum. However, at 4 days post-CVB3 challenge, significant increased titers of CVB3 neutralizing antibodies were detectable in the sera of immunized mice over the next 6 days. Non-immunized mice challenged with CVB3 Nancy had no anti-CVB3 activity in their sera until 10 days post-infection. CVB3 Nancy induced higher viral titers than did the mutant strain. There was no variation of the neutralizing activity of serum taken from mice immunized with CVB3 Sabin3-like and challenged with CVB4 E2, compared to non-immunized mice. Despite the fact that CVB3 and CVB4 are closely related viruses, virus-neutralizing activity clearly distinguish between these viruses. A variable and limited amount of pancreatic inflammation was N. Jrad-Battikh  A. Souii  L. oueslati  M. Aouni  J. Gharbi Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives (LR99-ES27), Faculte´ de Pharmacie de Monastir, Avenue Avicenne, 5000 Biotola, Tunisia D. hober Univeriste´ Lille 2, Faculte´ de Me´decine, CHU Lille, Laboratoire de Virologie EA3610, 59120 Loos-lez-Lille, France J. Gharbi (&)  M. Ben M’hadheb-Gharbi Institut Supe´rieur de Biotechnologie de Monastir, Avenue Tahar Hadded BP 74, 5000 Biotola, Tunisia e-mail: [email protected]

seen in some mice 10 days after Sabin3-like inoculation by IP route, whereas there was no evidence of pancreatic damage in mice inoculated by oral route. All immunized mice were protected from myocarditis and pancreatitis at 8 days post-challenge with CVB3 or CVB4 E2. These findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.

Introduction Coxsackievirus B3 (CVB3), a member of the picornavirus family and the genus enterovirus, is a commonly associated factor of acute and fulminant myocarditis in humans [1]. Myocarditis is a major cardiovascular cause of sudden death in men less than 40 years of age and can lead to the development of dilated cardiomyopathy in which mortality reached 60 % at 10 years [2]. Despite the well characterized molecular structure of CVB3 [3, 4] and the successful use of common vaccination strategies in animal models [5– 8], no virus-specific preventive procedures against CVB3 are in clinical use today. The first CVB-specific vaccine was a temperature-sensitive mutant virus, which produced neutralizing anti-CVB3 antibodies after vaccination [9]. Afterward, several types of candidate vaccine against CVB3 have been developed [10–12] and several murine models have been established [13, 14]. In a previous study, we have addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the CVB3. Accordingly, they have described that Sabin3-like mutation (U473 ? C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency.

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Serum neutralizing antibodies are major markers of enterovirus infection and protective immunity [15]. The neutralization test based on inhibition of cytopathic effect is a standard method recommended by the World Health Organization (WHO) for measuring the level of neutralizing antibodies against polioviruses [16] and was also applied widely to determine the neutralizing antibody titers against Echovirus 71 [17–19]. On the basis of these data, we assessed, in the present work, whether the Sabin3-like mutant of CVB3 could induce a protective immunity against virulent CVB3 Nancy and Coxsackievirus B4 (CVB4) E2 strains in mice by both oral and IP routes. The B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections as mentioned above. The CVB4 is a virus that can trigger an autoimmune reaction which results in destruction of the insulin-producing beta cells of the pancreas, which is one of several different etiologies of diabetes mellitus. Serum samples, taken from mice inoculated with Sabin3-like, were assayed in vitro for their antiCVB3 neutralizing activity.

Materials and Methods Virus The CVB3 Nancy prototype strain, and the Sabin3-like mutant of CVB3 used as a ‘‘vaccine candidate’’, were propagated in Vero cells (African green monkey kidney cells) (Bio Whittaker). The CVB4 E2 diabetogenic strain, isolated in 1979 from the pancreas of a child who died from diabetic ketoacidosis was propagated in HEp-2 cells (BioWhittaker, Verviers, Belgium) in Eagle’s minimum essential medium (MEM) supplemented with 10 % heat-inactivated fetal calf serum (FCS) (Sigma), 1 % L-glutamine, 50 lg/ml of streptomycin, 50 UI/ml of penicillin (Bio Whittaker), 1 % nonessential amino acids (Gibco BRL), and 0.05 % Fongizone (Amphotericin B, Apothecon). Supernatants were collected at 3 days after inoculation, clarified at 2.000 g for 10 min, divided into aliquots and stored at -80 °C. Virus titers in stocks were determined on Vero cells (CVB3) and HEp-2 cells (CVB4 E2) by limiting dilution assays for 50 % tissue culture infectious doses (TCID50) by the method of Reed and Muench [20]. Infection Protocol Swiss Albinos female mice (3–4 weeks old) (Pasteur Institute, Tunis, Tunisia), were divided into six groups. Three groups of three mice each were orally inoculated using a rigid canula, with 1.26 9 106 TCID50/ml of CVB3 Nancy contained in 100 ll culture supernatant of infected cells for the first group, 107 TCID50/ml of CVB4 E2 contained in 100 ll

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culture supernatant of infected cells for the second group, and 1.11 9 106 TCID50/ml of CVB3 Sabin 3-like mutant contained in 100 ll culture supernatant of infected cells for the third group, respectively. Negative control mice received 100 ll of PBS (uninfected). For the IP infection, mice were divided into different groups as described for oral infection and received the same virus doses. Mice were treated according to general ethic rules and maintained under specific pathogen-free conditions with unlimited access to food and water. Mouse blood was collected from the tail on days 0, 7, and 10 for antibody assays (Fig. 1a). Mice were sacrificed 10 days after inoculation. Immunization and Challenge Experiments Five-week-old female Swiss Albinos mice were distributed into four groups of 4 mice each. Groups 1 and 2 were immunized by a single oral or IP inoculation with the attenuated strain CVB3 Sabin3-like (1.11 9 106 TCID50/ml) at day 0 and challenged by oral or IP inoculation at day 16, with CVB3 (1.26 9 106 TCID50/ml) and CVB4 E2 (107 TCID50/ml), respectively. The same route of inoculation was used for immunization and infection (challenge). Control naı¨ve mice received 100 ll of PBS (uninfected controls). Mouse blood was collected from the tail on days 0, 7, 13, 16, 17, 20, and 24 for specific antibody assays (Fig. 1a, b). All animals were sacrificed 8 days post-challenge. Neutralizing Antibody Titers All serum samples (n = 3) were tested in triplicate and were serially diluted 1:10 before adding 103 TCID/ml of the virus (25 ll) and incubated for 2 h at 37 °C in 96-well microtiter plates. 100 ll of HEp-2 cells (containing105cells/well) were added to each well and incubated at 37 °C. After 3 days of growth in 2 % serum, the titer was read as the highest dilution that resulted in more than 50 % cytopathic effect and expressed as the reciprocal of the last serum dilution to neutralize 103 TCID/ml of CVB3 in 50 % of the wells. Histopathology and Image Analyses Serial 3-5 lm thick sections of formalin-fixed and paraffinembedded samples of the hearts, pancreas, and intestines were stained with hematoxylin and eosin in a standard way and examined by light microscopy for the presence of inflammatory lesions, cellular infiltration, necrosis, and muscle damage. Statistical Analysis All data were expressed as means ± standard deviations (SD). ANOVA test using STATVIEW statistical software package served for statistical analysis. Normality and

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Fig. 1 a Infection protocol of control mice. Swiss Albinos female were inoculated, with CVB3 Nancy (1.26 9 106 TCID50/ml) or CVB4 E2 (107 TCID50/ml). Mouse blood was collected from the tail on days 0, 7, and 10 for antibody assays. Mice were sacrificed 10 days after inoculation. b Protocol of immunization of mice. Attenuated strain CVB3 Sabin3like (1.11 9 106 TCID50/ml) was administered to Swiss Albinos female mice then they were challenged with CVB3 Nancy (1.26 9 106 TCID50/ml) or CVB4 E2 (107 TCID50/ml). Mouse blood was collected from the tail on days 0, 7, 13, 16, 17, 20, and 24 for specific antibody assays. All animals were sacrificed 8 days after challenge

homogeneity of data were confirmed before the statistical test, and data were considered statistically significant at P \ 0.05 level.

Results Anti-CVB3 Neutralizing Activity of Serum from Mice Inoculated with CVB3 Sabin3-like Strain Prior to inoculation, serum samples (dilution 1/10) from every mouse were not neutralizing toward CVB3. Whereas, at 10 days post-inoculation with CVB3 Nancy, the mean titers of neutralizing activity of sera were 280 by oral and 320 by IP routes (Fig. 2a). Serum samples of mice inoculated with CVB3 Sabin3-like have had an anti-CVB3 neutralizing activity. 7 days after CVB3 Sabin3-like inoculation (postimmunization), and before challenge, at day 16 with CVB3 Nancy (Fig. 1A), the mean titers of neutralizing activity of sera were 33 at day 7 and 55 at day 13 by IP route (Fig. 3a). According to ANOVA statistical data, the anti-CVB3 neutralizing titer induced by CVB3 Nancy (Fig. 2a) was higher at day 10 (P \ 0.05) than the one induced by CVB3 Sabin3-like mutant at day 13 (Fig. 3a). CVB3 Sabin3-like Can Protect Mice against CVB3 Nancy and CVB4 E2 In the present work, we assessed whether CVB3 Sabin3like strain could induce a protective immunity against

subsequent virulent CVB3 Nancy and CVB4 E2 strains in mice by oral and IP routes. Five-week-old female Swiss Albinos mice were inoculated at day 0 with PBS or the attenuated CVB3 Sabin3-like strain via oral or IP route, and then challenged 16 days later, with virulent CVB3 Nancy or CVB4 E2 strains. Mouse blood was collected from the tail on days 0, 7, 13, 16, 17, 20, and 24 for specific seroneutralization assays. Upon challenge with a specific dose of virulent strain CVB3 Nancy, all of the immunized mice survived without any symptom (in hearts, pancreas or intestines), and the antibody titers in serum were 690 (IP route) and 450 (oral route) by day 8 post-challenge (Fig. 3a) in comparison to control mice (320 IP/280 oral) (P \ 0.05) (Fig. 2a). At 10 days post-inoculation with CVB4 E2, the mean titers of neutralizing activity of sera were 35 by oral route and 960 by IP route (Fig. 2b). In serum taken from mice immunized with CVB3 sabin3-like at day 0 and challenged with CVB4 E2 at day 16, the titer of anti-CVB4 E2 antibodies on day 8 post-challenge was 970 by IP route and 50 by oral route (Fig. 2b). Titers of the neutralizing activity of serum taken from mice at day 13 after immunization with CVB3 Sabin3-like and before challenge with CVB4 E2 were 35 by IP and 30 by oral routes (Fig. 3b). Effects of CVB3 Sabin3-like Inoculation in Mice Mice were inoculated with Sabin3-like mutant virus by oral or IP route. Hearts of these animals were indistinguishable from hearts of uninfected control mice (Fig. 4). A variable

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Fig. 2 Neutralizing antibodies in serum of mice inoculated with Coxsackievirus B. Anti-CVB3 neutralizing antibody titers after CVB3 Nancy infection (a) or CVB4 E2 infection (b) by oral (O) or IP routes. Serum samples were collected from three animals. The titers are expressed as the reciprocal of the last serum dilution to neutralize 103 TCID50/ml of CVB3 in 50 % of the wells. Control mice (c) were inoculated with PBS

and limited amount of pancreatic inflammation was seen in some mice 10 days after CVB3 Sabin3-like inoculation by IP route (Fig. 4b), whereas there was no evidence of pancreatic damage in mice inoculated by oral route (Fig. 4a). During this study, none of the Swiss Albinos mice inoculated with either virulent CVB3 Nancy or CVB4 E2 or attenuated CVB3 Sabin3-like died after oral or IP infection. In marked contrast, both virulent parental CVB3 and CVB4 E2 strains induced inflammatory lesions with extension areas of infiltration and necrosis in heart muscle at 10 days after infection by oral (Fig. 4a) and IP (Fig. 4b) routes as well as widespread inflammation in acinar tissue in pancreas. Histology of intestine revealed no evidence for inflammation after oral or IP infection with CVB3 Sabin3like (data not shown). A single inoculation of the mutant CVB3 Sabin3-like by oral (Fig. 4c) and IP (Fig. 4d) routes protected all mice challenged 16 days later with a dose of virulent CVB3 Nancy and CVB4 E2 that induced heart disease and severe pancreatic disease in the infected control mice.

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Fig. 3 Neutralizing antibodies in serum of immunized mice and challenged with Coxsackievirus B. a Evolution of anti-CVB3 neutralizing antibody after immunization by the attenuated strain at day 0 and challenge by CVB3 Nancy or CVB4 E2 b by oral (O) or IP routes at day 16. Sera were collected from day 0 to day 16 after immunization and from day 16 to day 24 after challenge, and serially diluted 1:10 to determine the highest dilution able to completely inhibit cytopathic effect by CVB3 Nancy. C control mice (PBS inoculated mice)

Discussion Numerous examples in CVB3 and other picornaviruses prove that mutations in the 50 non translated region (50 NTR) markedly decrease multiplication efficiency, alter cell tropism and attenuate virulence [21]. In the best-known example, each of the three attenuated Sabin vaccine strains for poliovirus, consider the prototype picornavirus, contains nucleotide substitutions in domain V of the 50 NTR that are responsible for attenuation. In a previous study, Ben M’hadheb-Gharbi [10, 22] and collaborators constructed an attenuated CVB3 virus designed Sabin3-like by site-specific direct mutagenesis carrying out equivalent mutations to that of Sabin vaccine strains of PV, and reported the limited efficiency of the translation of the Sabin3-like mutant of CVB3. Many studies demonstrated that an attenuated CVB3 can induce a stronger immune response than inactivated viral strains and it involves all viral proteins and hence has the maximum set of epitopes for immune responses.

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Fig. 4 Histological aspect of heart and pancreas of mice inoculated by oral route a and IP route b with CVB3 Sabin3-like, CVB3 Nancy, and CVB4 E2. 10 days after infection, mice were sacrificed; hearts and pancreas were fixed in formalin, sectioned, and stained with hematoxylin and eosin. Black arrows indicate inflammation area. c Histological aspect of heart and pancreas of mice that were

immunized and challenged 16 days later by oral route and d IP route with CVB3 Nancy and CVB4 E2. 8 days after challenge, mice were sacrificed; hearts and pancreas were fixed in formalin, sectioned, and stained with hematoxylin and eosin. Black arrows indicate inflammation area

We analyzed, in vivo, the phenotype of CVB3 Sabin3like strain in Swiss mice inoculated by oral or IP routes. In fact, we have chosen the oral route to mimic the natural route of infection. The IP route represents the general systemic route used in vaccinology. The oral infection results in systemic spreading of the viral RNA, as IP route, from intestine to different organs’ tissues. Oral vaccination

is safe and easily administered, making it particularly suitable for protecting against infectious agents. Thus, we explored the capacity of this attenuated mutant to act as a vaccine vector in experimental animals after challenge with virulent strains CVB3 Nancy or CVB4 E2. CVB3 Sabin3-like was found to be attenuating for the hearts of mice inoculated with both oral and IP routes,

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although minor amounts of pancreatic inflammatory disease (pancreatitis) in some mice inoculated with CVB3 Sabin3-like examined on day 10 by IP route were noticed. In order to clarify the processes of humoral immune responses to the CVB3 Sabin3-like strain, we determined in this study, whether Sabin3-like mutant was able to elicit CVB3-Neutralizing activity induced by virulent CVB3 Nancy and CVB4 E2 infections. For this experiment, we used the cytopathic effect-based neutralization test known to be a standard method for measurement of protective antibodies, for laboratory diagnosis of enterovirus infection, and for the evaluation of vaccine-induced immunity. The absence of an inflammation in the heart and liver of immunized mice regardless the route of immunization, and the levels of serum neutralizing activity would suggest the involvement of another protective factor as the leading role of the cellular immune response in eliminating the virus. Development of vaccines to protect against enterovirus disease is a public health priority in certain countries. Similar to vaccines developed against poliovirus, neutralizing antibody responses will be an important indicator for evaluation of the immunogenicity of different enterovirus vaccine candidates. Although CVB3 has not yet been used as a viral vector, some DNA vaccines for CVB3, which included encoding VP1, VP3, and the middle region of VP1, were characterized [23]. Despite the fact that these vaccines protect mice against viral challenge, they did not induce neutralizing antibodies against CVB3 in mouse serum [23]. It was reported that some new constructed mutants induced a generation of neutralizing antibodies against CVB3 and could protect mice immunized with a lethal wild-type CVB3 strain [17, 24]. In the present work, we demonstrated that immunization procedure induced anti- CVB3 antibodies, and 4 days after the challenge, significantly increased titers of CVB3-specific antibodies were detectable in the sera of Sabin3-like immunized mice, indicating that the immunization procedure induced an immunologic memory that was accompanied by a secondary antibody response. This may then lead to reduced viral replication in the heart tissue detected in the immunized mice. CVB3 Nancy induced higher viral titers than did the mutant strain, because CVB3 strain replication was higher than the one of CVB3 Sabin3-like. Therefore, it may have induced a stronger and faster immune response than did CVB3 Sabin3-like. Until 10 days post-challenge, the antibody titer in sera of the vaccinated groups remained always higher than the one in control groups, especially when mice were inoculated by IP route. In conclusion, the inoculation of CVB3 Sabin3-like induced the highest virus-specific antibody concentrations in sera of Swiss Albinos mice after IP inoculation in comparison with oral route. Following experiments were focused on

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the characterization of the vaccine-mediated protection. Ten days after the challenge, CVB3 Sabin3-like immunized mice revealed higher virus-specific serum antibody titers, reduced viral replication in pancreas and heart, and less myocardial damage; indicating an induced immunologic memory in immunized mice prior challenge in comparison to control mice. Taking together, these data suggest that the Sabin3-like mutant exhibits a little capacity for replication in pancreatic tissues. While the attenuated strain is capable of replicating in mice, it induces little or no disease in hearts or pancreas of susceptible mice. A single inoculation by oral or IP routes of mice with CVB3 Sabin3-like protected mice from disease when challenged by both oral and IP route with either of the two virulent strains CVB3 and CVB4 E2, demonstrating that CVB3 Sabin3-like rapidly induces a protective immune response in the murine host.

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Neutralizing activity induced by the attenuated coxsackievirus B3 Sabin3-like strain against CVB3 infection.

Coxsackievirus B3 (CVB3) causes viral myocarditis, and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use...
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