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Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine
Vaccine 35 (2017) 938–944
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Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine Jun Luo 1, Boyue Zhang 1, Yuting Wu 1, Qin Tian 1, Jing Zhao, Ziyu Lyu, Qiong Zhang, Mingzhu Mei, Yongwen Luo ⇑, Xiaofeng Guo ⇑ College of Veterinary Medicine, South China Agricultural University, Guangzhou, China Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, China
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Article history: Received 16 November 2016 Received in revised form 20 December 2016 Accepted 20 December 2016 Available online 12 January 2017 Keywords: Rabies virus HEP-Flury IL6 Vaccine Adjuvant
a b s t r a c t Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEPCaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8+ T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Rabies is an ancient zoonosis and a central nervous system (CNS) disease, causing mortality in more than 55,000 humans annually, with most cases occurring in the developing regions of Asia and Africa, where canine rabies is the main source of infection [1]. Several developed countries have successfully eradicated human rabies by implementing programs to control canine rabies [1]. Although rabies is associated with a high mortality rate in both humans and dogs, rabies vaccines can effectively be used to control the disease. Although many rabies vaccines, most of which are inactivated, have been approved for use in dogs, the costs of these vaccines limit their use in developing countries. Therefore, attenuated live rabies vaccines are appropriate because of their low cost and effectiveness. ⇑ Corresponding authors at: College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Street, Guangzhou 510642, China. E-mail addresses: [email protected] (Y. Luo), [email protected] (X. Guo). 1 Jun Luo, Boyue Zhang, Yuting Wu and Qin Tian contributed equally to the work. http://dx.doi.org/10.1016/j.vaccine.2016.12.069 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Foreign proteins have been expressed between nucleoprotein (N) and phosphoprotein (P), P and matrix protein (M) as well as between glycoprotein (G) and RNA-dependent RNA polymerase (L) in rabies virus (RABV) [2–5]. Furthermore, previous studies reported that a pseudogene sequence between G and L showed no detectable effect on the transcription and growth of RABV [6–8]. Several studies have confirmed the feasibility of enhancing immunogenicity and the viral attenuation of recombinant RABV (rRABV) strains by inserting and expressing genes encoding for immunomodulatory molecules [9–11]. In this context, interleukin-6 (IL6) has been shown to mediate various effects in the immune system. IL6 stimulates the differentiation and maturation of B cells to antibody-producing plasma cells [12], enhances mucosal immune responses [13], and stimulates T cell proliferation [14]. Accordingly, IL6 has been used as a molecular adjuvant expressed in eukaryotic expression vectors to enhance immune responses. For instance, mice challenged with influenza virus after receiving an immunization with an influenza virus DNA vaccine showed better protection if IL6 was used as an adjuvant [15,16]. It was also confirmed that antibodies against porcine circovirus
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type 2 (PCV2) and percentages of CD3+CD8+ peripheral blood T lymphocytes were significantly higher in mice immunized with a PCV2 DNA vaccine and IL6 than in animals that did not receive IL6 [17]. Furthermore, levels of antibodies against classical swine fever virus, IgG, IgA, IFN-c, and CD8+ T cells in peripheral blood were higher if the vaccine used to immunize piglets was combined with IL6 [18]. Previous research implied that IL6 might also fulfil its function in heterologous tissues and animals of other species [14,15]. In this study, the effects of rRABV carrying canine IL6 (rHEPCaIL6) between G and L was investigated in mice. Our results indicate that rHEP-CaIL6 enhances the immunogenicity of RABV but has a good safety profile when compared with the parent strain HEP-Flury. In addition, HEP-Flury was previously shown to induce an immune response in dogs [19]. Therefore, rHEP-CaIL6 might be a rabies vaccine candidate.
2. Materials and methods 2.1. Viruses and animals rHEP-CaIL6 was rescued based on the HEP-Flury strain and confirmed (Supplementary material 2). HEP-Flury and rHEP-CaIL6 were propagated in BHK-21 cells. CVS-24 was propagated in suckling mouse brains. Female Kunming (KM) and BALB/c mice were purchased from the Center for Laboratory Animal Science of the Southern Medical University (Guangzhou, China). Mice were housed in the Laboratory Animal Center of the South China Agricultural University. All animal experiments were carried out in compliance with specific pathogen-free requirements that were previously approved by the ethics committee for animal experiments of the South China Agricultural University. All possible efforts were made to minimize the suffering of laboratory animals. Procedures were based on the national standard Laboratory Animal Requirements of Environment and Housing Facilities (CALAS, GB 14925-2001) as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Investigation of body weight changes and clinical symptoms in mice BALB/c mice (6–7 weeks of age) were infected intracerebrally (i.c.) with 3.3 104 FFU of HEP-Flury, rHEP-CaIL6 or with 30 ll medium (mock infection). Each group consisted of eight mice. Body weight and clinical symptoms were recorded daily for 31 days. Changes in body weight are presented as the mean value ± standard error (SE). Regarding clinical symptoms observed in mice, scores were assigned as follows: 0, no clinical symptoms observed; 1, loss of initial body weight > 5%; and 2, piloerection. 2.3. Immunization and in vivo challenge Groups of 10 KM mice (6–7 weeks of age) were immunized by the intramuscular injection of 1.0 103 FFU, 1.0 104 FFU, 1.0 105 FFU HEP-Flury or rHEP-CaIL6, respectively. Medium was used for mock infections. Serum was obtained at days 7, 14, and 21 after immunization and used to determine VNA levels by means of fluorescent antibody virus neutralization (FAVN) tests as described previously [20]. Concentrations/titres of anti-RABV antibodies were assessed by ELISA (Synbiotics, USA) according to the manufacture’s protocols. For challenge experiments, 23 days after immunization, mice were i.c. challenged with 50 mouse intracerebral lethal doses 50 (MILD50) of CVS-24. The number of survivors was recorded daily for 3 weeks.
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2.4. Flow cytometry KM mice of 6–7 weeks of age were infected intramuscularly with 100 ll (equivalent to 106 FFU/ml) of rHEP-CaIL6, HEP-Flury or DMEM, respectively. Mouse inguinal lymph nodes were harvested at days 4, 7, and 10 post-infection. Single-cell suspensions were obtained as described previously [10], and stained with antibodies against markers of T cells (CD3e-FITC, CD4-PE, CD8a-PerCPCy5.5) or B cells (CD19-FITC, CD40-PE) (all antibodies purchased from Affymetrix eBioscience, USA) by incubation for 30 min on ice. A minimum of 100,000 events were counted using a Beckman FC 500 flow cytometer (Beckman Coulter, USA). Data were analyzed using FCS Express 4 flow cytometry (De Novo Software, USA). 2.5. Quantitative real-time PCR IL6-induced gene expression at days 6, 9, and 12 after infection in CNS tissues was investigated as described in Supplementary material 2. Primers used to amplify target and reference genes are listed in Supplementary material 2. 2.6. Statistical analysis Experiments were carried out in triplicate. Data were analyzed using GraphPad Prism 6 software (GraphPad Software, USA). The statistical significance was determined using the Student’s t-test or Log-rank (Mantel-Cox) Test. P < 0.05 was considered to indicate statistically significant differences. 3. Results 3.1. Safety of rHEP-CaIL6 in mice The safety of rHEP-CaIL6 was determined in BALB/c mice. As shown in Fig. 1A, the body weight of mice infected with rHEPCaIL6 recovered faster compared with HEP-Flury and regained their initial body weight at day 9 post-infection. Clinical symptoms observed are shown in Fig. 1B. Mice infected with either rHEPCaIL6 or HEP-Flury showed similar clinical symptoms during the early stages of infection. However, at day 10 post-infection, mice infected with HEP-Flury started to develop more severe symptoms that persisted for a longer period (P < 0.05 at several time points) than those mice infected with rHEP-CaIL6. In summary, mice infected with rHEP-CaIL6 showed an earlier onset of recovery (increased body weights) compared with HEP-Flury. 3.2. Immunogenicity of rHEP-CaIL6 KM mice were immunized intramuscularly with different doses of rHEP-CaIL6 or HEP-Flury. Increasing inoculating doses of vaccine were designed to investigate whether the low dose vaccine induced high antibody titres when IL6 was over expressed and the lowest dose of rHEP-CaIL6 that would provide sufficient protection from a lethal challenge. Peripheral blood samples were collected at 7, 14, and 21 days post-immunization (dpi) and serum was used to determine the concentrations of anti-RABV antibodies and VNA. As shown in Fig. 2A, at 7 dpi, levels of anti-RABV antibodies were <0.6 EU, independent of the strain and dose administered. After two or three weeks, the anti-RABV antibody concentrations had increased to >0.6 EU, which is considered protective against RABV infection [21]. Interestingly, at 14 and 21 dpi, significantly higher levels of anti-RABV antibodies were detected in mice immunized with rHEP-CaIL6 compared with HEP-Flury group. VNA levels >0.5 international units (IU) are considered protective against RABV infection. As shown in Fig. 2B, concentrations of VNA in mice
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Fig. 1. Safety of RABV strains in mice. BALB/c mice (6–7 weeks of age; n = 8 per group) received i.c. injections of 3.3 104 FFU of HEP-Flury, rHEP-CaIL6 or medium alone (mock infection) to investigate the safety profile of rHEP-CaIL6. Body weight was monitored daily for 31 days (A). Changes of body weight were shown as ratio to body weight at day 0 and data were presented as mean value ± standard error (SE). Clinical symptoms presented by infected mice were scored according to severity on ascale from 0 to 2 and are presented here as mean value ± standard error (SE). Asterisks indicate significant differences among groups, as calculated by Student’s t-test (**P < 0.01).
Fig. 2. Immunogenicity of RABV strains in vivo and survival rates of mice challenged with CVS-24. Groups of ten KM mice (6–8 weeks of age) were immunized i.m. with 1.0 103 FFU, 1.0 104 FFU, or 1.0 105 FFU of HEP-Flury, rHEP-CaIL6 or medium alone (mock infection). Peripheral blood was obtained and concentrations of anti-RABV antibodies (A) in serum were determined using the ELISA (Synbiotics, USA) according to the manufacture’s protocols while serum VNA (B) were ascertained using FAVN as described in Materials and Methods. (C) Then, mice were challenged i.c. with 50 LD50 of CVS-24 at day 23 after immunization and were observed for 16 days. Survival rates were recorded daily. Data were analyzed using GraphPad Prism 6. anti-RABV antibodies and FAVN of asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Comparison of survival curves was calculated by Log-rank (Mantel-Cox) Test (**P < 0.01; ***P < 0.001).
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immunized with HEP-Flury or rHEP-CaIL6 were dose-dependent. Significantly higher levels of VNA were detected in mice immunized with rHEP-CaIL6 at 7, 14, or 21 dpi, compared with animals that received the same dose of HEP-Flury. At 7 dpi, the breakpoint of 0.5 IU was reached by immunization with 1.0 104 FFU or 1.0 105 FFU of rHEP-CaIL6, but not with the same doses of the parent HEP-Flury. Furthermore, the induction of VNA levels >0.5 IU was proven for rHEP-CaIL6 at all doses tested at 14 and 21 dpi. However, the level of VNA in mice immunized with 1.0 103 FFU of HEP-Flury remained <0.5 IU at 14 and 21 dpi. To investigate whether VNA concentrations correlated with protection from infection, immunized mice were then challenged with virulent CVS-24. As shown in Fig. 2C, significantly more mice immunized with rHEP-CaIL6 survived lethal challenges than those immunized with HEP-Flury. After application of the protective doses of 1.0 104 FFU, survival rates were 90 and 40% in each of the respective groups. A similar trend was observed after immunization with 1.0 103 FFU and 1.0 105 FFU, but the differences did not reach statistical significance. Comparative survivors (more than 80%) were observed when mice were immunized with rHEPCaIL6 at doses of 1.0 104 FFU and 1.0 105 FFU per mouse, suggesting low doses of rHEP-CaIL6 may confer protection against lethal RABV infections. 3.3. Effects of rHEP-CaIL6 on the recruitment of B and T cells To investigate whether rHEP-CaIL6 affected the recruitment of immune cells, flow cytometry was conducted to determine the counts of CD19+CD40+ B cells, CD3+CD4+ T cells, and CD3+CD8+ T cells in the lymph nodes at 4, 7, and 10 dpi in mice. Fig. 3A and B show flow cytometric plots representing B cells (CD19+ and/or CD40+) and T cells (CD4+ or CD8+, values based on expression of CD3+ for all T cells) in the inguinal lymph nodes. As shown in Fig. 3C and D, higher numbers of immune cells (CD19+CD40+ B cells and CD8+ T cells) were detected in mice infected with rHEP-CaIL6 compared with mice infected with HEP-Flury at 4 or 7 dpi. At 10 dpi, more CD19+CD40+ B cells had been recruited by rHEP-CaIL6 than by HEP-Flury, while the opposite was observed for CD8+ T cells, which may be explained by the different duration of infection caused by RABVs. Regarding CD4+ T cells, no differences were detected at any time point studied (data not shown). Thus, our studies confirm that rHEP-CaIL6 may recruit more immune cells (B cells and CD8+ T cells) compared with the parental strain HEP-Flury. This may indicate an enhanced immune response to rHEP-CaIL6. 3.4. Viral growth and induction of an immune response in the brain As an attenuated live vaccine, possible invasion to the CNS must be considered. To investigate virus replication and the induction of an immune response in the brain, KM mice were infected intranasally. with either rHEP-CaIL6 or HEP-Flury. Brains were harvested at time points after infection and assessed for the copy numbers of RABV, expression of RABV N, RABV G, canine IL6, CD4, CD8, CD11b, MHC-I, MHC-II, IFN-a, IFN-b, IFN-c, IFN-c-induced protein 10 (IP-10), MIP-1a, RANTES, TNF-a and j-L-chain (IgG) using qPCR. As shown in Fig. 4, the copy numbers of rHEP-CaIL6 and HEP-Flury as well as RABV N and RABV G expression did not differ at 6 dpi. rHEP-CaIL6 copy numbers did not increase drastically at 9 dpi and could not be detected at 12 dpi while HEP-Flury copy numbers were augmented these time points. The expression of RABV N and RABV G in brains of mice infected with rHEP-CaIL6 decreased and could not be detected at 9 and 12 dpi, while expression levels remained unchanged over time in the brains of mice infected with HEP-Flury. In addition the expression of canine IL6 decreased timedependently in mice infected with rHEP-CaIL6. These data indicate
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that rHEP-CaIL6 could be cleared from infected brains faster than HEP-Flury indicating. stronger immune responses might be induced by infection with rHEP-CaIL6. Several studies have indicated that CNS immune responses involve the opening of the blood-brain barrier (BBB) and an increase in BBB permeability that coincides with an enhanced expression of inflammatory cytokines and chemokines [22,23]. In this context, pro-inflammatory cytokine IL6 might enhance the expression of TNF-a [24], which further stimulates inflammatory processes. Chemokines may also be involved in pathogen clearance from the brain. As depicted in Fig. 4, at 6 dpi, the mRNA levels of chemokines IP-10, MIP-1a, and RANTES as well as TNF-a were significantly higher in the CNS of mice infected with rHEP-CaIL6 than in those that were exposed to HEP-Flury. At 9 and 12 dpi, the expression of IP-10, MIP-1a, RANTES, and TNF-a decreased in rHEP-CaIL6-treated mice and reached values similar to those obtained in the control group infected with HEP-Flury. Because an increase in permeability of the BBB may lead to the infiltration of T and B cells, we examined the expression of CD4, CD8, CD11b, CD19, MHC-I, MHC-II, IFN-a, IFN-b, and IFN-c in brain tissue. No differences were observed for the expression of CD4 and CD19 (Fig. 4), but infection with rHEPCaIL6 was associated with higher CD8 and CD11b mRNA levels at 6 and 9 dpi compared to the HEP-Flury group. Additionally, the expression of MHC-I was enhanced in mice infected with rHEPCaIL6 at 6 dpi. Regarding MHC-II, no difference between groups was observed at all-time points. Types I and II IFNs are also implied in antiviral defence mechanisms. Our data demonstrate that rHEPCaIL6 induced a much stronger IFN-related immune response at early stages (6 dpi) compared with HEP-Flury. Antibody production in CNS tissues was determined by IgG-j-L-chain mRNA expression. As shown in Fig. 4, higher levels of IgG were detected at all-time points after immunization with rHEP-CaIL6 compared with HEPFlury. This indicates that IL6 may induce a persistent immune response despite its relatively fast clearance.
4. Discussion Previous studies have shown the G-mediated induction of VNA is critical to prevent animals or humans from wild-type RABV infection [19,25]. In addition, as an attenuated vaccine candidate of RABV which may compromise the CNS, the induction of an immune response is important to permit pathogen clearance from the CNS [22,26]. This study aimed to provide preliminary information for the development of an effective, safe, and inexpensive RABV vaccine candidate. We selected IL6 as an adjuvant because it enhances the immune response [17] and modulates inflammation and BBB permeability [27,28]. All these processes are involved in the clearance of invasive RABV from the CNS [22,23,26]. RABV HEP-Flury is an attenuated strain that does not cause fatal infections after intracerebral or peripheral inoculation [29]. The HEPFlury strain has been used as a vaccine for humans in Japan [30], and for animals in China. Furthermore, an attenuated live RABV vaccine based on the HEP-Flury strain has been licensed and is used in animals in China. Murine cells and mice are sensitive to canine IL6 (provided in Supplementary material 1). rHEP-CaIL6 showed high viral titres at the early stages compared with HEPFlury in NA cells which may provide the foundations of virus production (provided in Supplementary material 2). In this study, we characterized rHEP-CaIL6, derived from HEP-Flury and expressing canine IL6, and compared its effects on immunology and its safety profile. Our study showed that rHEP-CaIL6 more effectively stimulated the production of anti-RABV antibodies and VNA, and thus conferred better protection to mice after a challenge with CVS-24 compared with the parental strain HEP-Flury. Our results are in
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Fig. 3. Effects on the recruitment and activation of B cells and CD8+ T cells in the inguinal lymph nodes. Female KM mice of 6–8 weeks of age were infected i.m. with 1.0 105 FFU of HEP-Flury, rHEP-CaIL6, or medium alone (mock infection). At days 4, 7, and 10 dpi, inguinal lymph nodes were harvested and single-cell suspensions were prepared and stained with antibodies against B cell (CD19-FITC and CD40-PE) or T cell markers (CD3-FITC, CD4-PE, and CD8-PerCP-Cy5.5). Data were collected and analyzed with a Beckman FC 500 flow cytometer (Beckman Coulter) and FCS Express 4 flow cytometry (De Novo Software). (A) Representative flow cytometric plots, showing mature B cells (CD19+/CD40+) in the upper right quadrants of each scatter plot. (B) In the upper left quadrant of each scatter plot, CD3+CD8+ T cells are represented. (C) Percentages of CD19+CD40+ B cells and (D) CD3+CD8+ T cells in inguinal lymph nodes at different time points (n = 3 per group). Values are presented as mean ± standard error. Asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; ****P < 0.0001).
agreement with a recently published study showing that the HEPFlury strain induced VNA in dogs [19], and we speculate that rHEPCaIL6 may also protect dogs from infection with virulent RABV. Thus, IL6 might be used as an adjuvant of RABV vaccines. Because rHEP-CaIL6 induced protective antibodies at lower doses, it might allow for the administration of lower doses without losing infection protection and might contribute to lowering the costs of RABV vaccine production. Recent studies [15,16] suggested IL6 enhanced the serum levels of IgG and favoured an immune response mediated by cytotoxic T cells, which express CD8. In this context, we showed that rHEPCaIL6 augmented the recruitment of B cells and CD8+ T cells in the lymph nodes, and this finding corroborates previous publications. CD19 is a pre-B cell marker and CD40 is mainly expressed by mature B cells after activation [31]. Because B cell activation plays a major role in humoral immunity, we assessed rRABVmediated B cell recruitment and characterized this population of lymphocytes. According to our findings, rHEP-CaIL6 recruited more CD19+ and CD40+ B cells in the lymph nodes compared with HEPFlury, which may explain why rHEP-CaIL6 induced higher levels of anti-RABV antibodies and VNA. CD8+ T cells are involved in the control of acute infections and the clearance of pathogens
[32,33]. Regarding the in vivo evaluations of immune responses triggered by rHEP-CaIL6, an enhanced CD8+ T cells response may be the cause of a faster clearance of resident RABV from host cells. Because our study aimed to develop a candidate attenuated rabies vaccine, the safety of rRABV rHEP-CaIL6 was investigated in mice. It has been confirmed that RABV infections of animals or humans are fatal mainly because of CNS invasion [26]. We showed that mice infected i.c. with rHEP-CaIL6 showed a rapid recovery (increase in body weight) and mild clinical symptoms. These observations may indicate that rHEP-CaIL6 had a good safety profile. Furthermore, rHEP-CaIL6 was cleared rapidly from the brain of infected mice while the HEP-Flury viral multiplication increased time dependently. To identify the mechanisms of the early clearance of rHEP-CaIL6, the expression of inflammatory mediators in the brain was investigated. Our data showed that the mRNA levels of pro-inflammatory cytokines including TNF-a (which may be induced by IL6) and chemokines such as IP-10, MIP-1a, and RANTES were present in higher concentrations in the CNS following immunization with rHEP-CaIL6. Pro-inflammatory cytokines and chemokines may attract immune cells to the site of infection, and these cells may favour the removal of pathogens. Accordingly, the induction of pro-inflammatory mediators and recruitment of
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Fig. 4. Expression of viral genes, inflammatory mediators and immune response-related molecules in the CNS. Female KM mice of 6–8 weeks of age were infected i.n. with 3.3 104 FFU of distinct virus strains, and at days 6, 9, and 12, brains were harvested following the perfusion with PBS. Expression of viral genes and immune responserelated molecules in the brains were investigated by quantitative real-time PCR in a CFX384 Real-Time System. Copy numbers of RABV, expression of RABV N, RABV G, canine IL6, IP-10, MIP-1a, RANTES, TNF-a, CD4, CD8, CD11b, CD19, MHC-I, MHC-II, IFN-a, IFN-b, IFN-c, and IgG j-L chain were normalized to the expression level of the housekeeping gene GAPDH. Expression levels of immune response-related molecules were presented as fold changes over those detected in mock-infected controls. Data were analyzed using BioRad CFX Manager and GraphPad Prism 6. Asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001).
immune cells may be an important mechanism of RABV attenuation [23,34]. This might explain the better safety profile of rHEPCaIL6 compared with HEP-Flury. Previous studies reported that infection of the CNS by laboratory-attenuated RABV was cleared by infiltrating immune cells, which corresponds to an enhanced innate and adaptive immune response [35,36]. Pro-inflammatory cytokines and chemokines may enhance BBB permeability [37] and thus allow immune cells to enter the CNS. We speculate that BBB permeability was increased and stronger cell-mediated immune responses occurred in the CNS after immunization with rHEP-CaIL6. To gain a further insight into the role of immune cells in pathogen clearance from the CNS, the expression of CD4, CD8, CD11b and CD19 was evaluated. Increased concentrations of pro-inflammatory cytokines and chemokines at 6 dpi coincided with an enhanced expression of CD8 and CD11b in mice immunized with rHEP-CaIL6. However, expression levels of CD8 and CD11b remained elevated in these animals when pro-inflammatory mediator concentrations had already decreased (9 dpi). This may be explained by the duration of the enhanced immune response. Both HEP-Flury and rHEP-CaIL6 similarly induced CD4 and CD19 expression in the CNS. This may indicate that rRABV
expressing IL6 did not affect CNS infiltration by CD4+ T cells or CD19+ B cells. Furthermore, no significant differences were observed regarding the mRNA levels of MHC-I, which is expressed by all karyocytes, whereas mRNA levels of MHC-II, expressed by B cells, dendritic cells, macrophages, and subpopulations of activated T cells, were significantly higher in the brains of mice infected with rHEP-CaIL6 than in samples obtained from the HEP-Flury group. This may also be related to the strong IL6mediated recruitment of immune cells to the CNS. IFNs plays important roles in host defence against RABV and affect the degree of attenuation displayed by an individual RABV strain [9]. The expression of IFN-a, IFN-b, and IFN-c was assessed to investigate immune responses in the CNS. At 6 dpi, the mRNA levels of IFN-c were significantly higher in the brains of mice immunized with rHEP-CaIL6. This is in agreement with a previous report of the IL6-induced expression of IFN-c [18]. IFN-c, in turn, may stimulate the expression of type I IFNs [9]. rHEP-CaIL6 increased the expression of both type I IFNs, as measured at 9 dpi. The mRNA levels of IgG were augmented in the brains of mice immunized with rHEP-CaIL6. In summary, the increased safety displayed by rHEP-CaIL6 may be explained by an enhanced immune response induced by the adjuvant IL6.
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To develop a rabies vaccine that may be approved for use in dogs, further studies regarding the duration of immunity and the safety of the product are required. In conclusion, rHEP-CaIL6 enhanced RABV immunogenicity and conferred good protection from infections with virulent RABV. Moreover, IL6 expression by rRABV resulted in increased safety by the induction of a strong immune response in the CNS. rHEP-CaIL6 may have the potential to be developed as an attenuated RABV vaccine candidate. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Contributors Experimental design was conducted by Xiaofeng Guo and Jun Luo. Data analysis and writing article was finished by Jun Luo, Xiaofeng Guo, and Yongwen Luo. Animal experiments were finished by Jun Luo, Boyue Zhang, Yuting Wu, Jing Zhao, Ziyu Lv. Flow cytometry was conducted by Jun Luo, Qin Tian, Boyue Zhang, Yuting Wu, and Mingzhu Mei. Quantitative real-time PCR was conducted by Jun Luo, Qin Tian, Boyue Zhang and Qiong Zhang. Acknowledgments We thank the Guangdong Haid Institute of Animal Husbandry and Veterinary for the support of instruments. This study was partially supported by the National Key Research and Development Program of China (No. 2016YFD0500400), Research of Guangdong Province and Ministry of Education (No. 2014B090901046) and Special Fund for Agro-scientific Research in the Public Interest (No. 201103032). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2016.12. 069. References [1] Fu ZF. Rabies and rabies research: past, present and future. Vaccine 1997;15 (Suppl):S20–4. [2] Luo J, Zhao J, Tian Q, Mo W, Wang Y, Chen H, et al. A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro. Virus Genes 2016;52(3):379–87. [3] Zhao L, Toriumi H, Wang H, Kuang Y, Guo X, Morimoto K, et al. Expression of MIP-1alpha (CCL3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and B cells. J Virol 2010;84(18):9642–8. [4] McGettigan JP, Naper K, Orenstein J, Koser M, McKenna PM, Schnell MJ. Functional human immunodeficiency virus type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and env expressed from a single rhabdovirus-based vaccine vector genome. J Virol 2003;77(20):10889–99. [5] Wu X, Rupprecht CE. Glycoprotein gene relocation in rabies virus. Virus Res 2008;131(1):95–9. [6] Mebatsion T, Schnell MJ, Cox JH, Finke S, Conzelmann KK. Highly stable expression of a foreign gene from rabies virus vectors. Proc Natl Acad Sci USA 1996;93(14):7310–4. [7] Ceccaldi PE, Fayet J, Conzelmann KK, Tsiang H. Infection characteristics of rabies virus variants with deletion or insertion in the pseudogene sequence. J Neurovirol 1998;4(1):115–9. [8] Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. Embo J 1994;13(18):4195–203. [9] Barkhouse DA, Garcia SA, Bongiorno EK, Lebrun A, Faber M, Hooper DC. Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon. J Virol 2015;89(1):312–22.
[10] Wen Y, Wang H, Wu H, Yang F, Tripp RA, Hogan RJ, et al. Rabies virus expressing dendritic cell-activating molecules enhances the innate and adaptive immune response to vaccination. J Virol 2011;85(4):1634–44. [11] Wang Y, Tian Q, Xu X, Yang X, Luo J, Mo W, et al. Recombinant rabies virus expressing IFNalpha1 enhanced immune responses resulting in its attenuation and stronger immunogenicity. Virology 2014;468–470:621–30. [12] Muraguchi A, Hirano T, Tang B, Matsuda T, Horii Y, Nakajima K, et al. The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J Exp Med 1988;167(2):332–44. [13] Ramsay AJ, Husband AJ, Ramshaw IA, Bao S, Matthaei KI, Koehler G, et al. The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science 1994;264(5158):561–3. [14] Van Snick J. Interleukin-6: an overview. Annu Rev Immunol 1990;8:253–78. [15] Larsen DL, Dybdahl-Sissoko N, McGregor MW, Drape R, Neumann V, Swain WF, et al. Coadministration of DNA encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice. J Virol 1998;72 (2):1704–8. [16] Lee SW, Youn JW, Seong BL, Sung YC. IL-6 induces long-term protective immunity against a lethal challenge of influenza virus. Vaccine 1999;17 (5):490–6. [17] Guo XQ, Wang LQ, Qiao H, Yang XW, Yang MF, Chen HY. Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin-6 in mice. Microbiol Immunol 2015;59(3):174–80. [18] Li D, Chen JL, Zhang H, Yang X, Wan XP, Cheng C, et al. Improvement of the immunity of pig to Hog cholera vaccine by recombinant plasmid with porcine interleukin-6 gene and CpG motifs. Vaccine 2011;29(22):3888–94. [19] Liu X, Yang Y, Sun Z, Chen J, Ai J, Dun C, et al. A recombinant rabies virus encoding two copies of the glycoprotein gene confers protection in dogs against a virulent challenge. PLoS ONE 2014;9(2):e87105. [20] Cliquet F, Aubert M, Sagne L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods 1998;212(1):79–87. [21] Luo J, Shi H, Tan Y, Niu X, Long T, Zhao J, et al. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus. Vaccine 2016;34 (37):4392–8. [22] Phares TW, Kean RB, Mikheeva T, Hooper DC. Regional differences in bloodbrain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J Immunol 2006;176 (12):7666–75. [23] Kuang Y, Lackay SN, Zhao L, Fu ZF. Role of chemokines in the enhancement of BBB permeability and inflammatory infiltration after rabies virus infection. Virus Res 2009;144(1–2):18–26. [24] Lin HW, Levison SW. Context-dependent IL-6 potentiation of interferongamma-induced IL-12 secretion and CD40 expression in murine microglia. J Neurochem 2009;111(3):808–18. [25] Koraka P, Bosch BJ, Cox M, Chubet R, Amerongen G, Lovgren-Bengtsson K, et al. A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice. Vaccine 2014;32(36):4644–50. [26] Gomme EA, Wirblich C, Addya S, Rall GF, Schnell MJ. Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression. PLoS Pathog 2012;8(10):e1002971. [27] Gadient RA, Otten UH. Interleukin-6 (IL-6)–a molecule with both beneficial and destructive potentials. Prog Neurobiol 1997;52(5):379–90. [28] Paul R, Koedel U, Winkler F, Kieseier BC, Fontana A, Kopf M, et al. Lack of IL-6 augments inflammatory response but decreases vascular permeability in bacterial meningitis. Brain 2003;126(Pt 8):1873–82. [29] Takayama-Ito M, Inoue K, Shoji Y, Inoue S, Iijima T, Sakai T, et al. A highly attenuated rabies virus HEP-Flury strain reverts to virulent by single amino acid substitution to arginine at position 333 in glycoprotein. Virus Res 2006;119(2):208–15. [30] Morimoto K, Kawai A, Sato Y, Ohkubo A. A unique transcription mode of rabies virus high egg passage-Flury strain detected in infected baby hamster kidney21 cells. Microbiol Immunol 2011;55(8):558–64. [31] Castigli E, Young F, Carossino AM, Alt FW, Geha RS. CD40 expression and function in murine B cell ontogeny. Int Immunol 1996;8(3):405–11. [32] Zhang N, Bevan MJ. CD8(+) T cells: foot soldiers of the immune system. Immunity 2011;35(2):161–8. [33] Carty M, Reinert L, Paludan SR, Bowie AG. Innate antiviral signalling in the central nervous system. Trends Immunol 2014;35(2):79–87. [34] Chai Q, He WQ, Zhou M, Lu H, Fu ZF. 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Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine Jun Luo 1, Boyue Zhang 1, Yuting Wu 1, Qin Tian 1, Jing Zhao, Ziyu Lyu, Qiong Zhang, Mingzhu Mei, Yongwen Luo ⇑, Xiaofeng Guo ⇑ College of Veterinary Medicine, South China Agricultural University, Guangzhou, China Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 16 November 2016 Received in revised form 20 December 2016 Accepted 20 December 2016 Available online 12 January 2017 Keywords: Rabies virus HEP-Flury IL6 Vaccine Adjuvant
a b s t r a c t Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEPCaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8+ T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Rabies is an ancient zoonosis and a central nervous system (CNS) disease, causing mortality in more than 55,000 humans annually, with most cases occurring in the developing regions of Asia and Africa, where canine rabies is the main source of infection [1]. Several developed countries have successfully eradicated human rabies by implementing programs to control canine rabies [1]. Although rabies is associated with a high mortality rate in both humans and dogs, rabies vaccines can effectively be used to control the disease. Although many rabies vaccines, most of which are inactivated, have been approved for use in dogs, the costs of these vaccines limit their use in developing countries. Therefore, attenuated live rabies vaccines are appropriate because of their low cost and effectiveness. ⇑ Corresponding authors at: College of Veterinary Medicine, South China Agricultural University, No. 483 Wushan Street, Guangzhou 510642, China. E-mail addresses: [email protected] (Y. Luo), [email protected] (X. Guo). 1 Jun Luo, Boyue Zhang, Yuting Wu and Qin Tian contributed equally to the work. http://dx.doi.org/10.1016/j.vaccine.2016.12.069 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Foreign proteins have been expressed between nucleoprotein (N) and phosphoprotein (P), P and matrix protein (M) as well as between glycoprotein (G) and RNA-dependent RNA polymerase (L) in rabies virus (RABV) [2–5]. Furthermore, previous studies reported that a pseudogene sequence between G and L showed no detectable effect on the transcription and growth of RABV [6–8]. Several studies have confirmed the feasibility of enhancing immunogenicity and the viral attenuation of recombinant RABV (rRABV) strains by inserting and expressing genes encoding for immunomodulatory molecules [9–11]. In this context, interleukin-6 (IL6) has been shown to mediate various effects in the immune system. IL6 stimulates the differentiation and maturation of B cells to antibody-producing plasma cells [12], enhances mucosal immune responses [13], and stimulates T cell proliferation [14]. Accordingly, IL6 has been used as a molecular adjuvant expressed in eukaryotic expression vectors to enhance immune responses. For instance, mice challenged with influenza virus after receiving an immunization with an influenza virus DNA vaccine showed better protection if IL6 was used as an adjuvant [15,16]. It was also confirmed that antibodies against porcine circovirus
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type 2 (PCV2) and percentages of CD3+CD8+ peripheral blood T lymphocytes were significantly higher in mice immunized with a PCV2 DNA vaccine and IL6 than in animals that did not receive IL6 [17]. Furthermore, levels of antibodies against classical swine fever virus, IgG, IgA, IFN-c, and CD8+ T cells in peripheral blood were higher if the vaccine used to immunize piglets was combined with IL6 [18]. Previous research implied that IL6 might also fulfil its function in heterologous tissues and animals of other species [14,15]. In this study, the effects of rRABV carrying canine IL6 (rHEPCaIL6) between G and L was investigated in mice. Our results indicate that rHEP-CaIL6 enhances the immunogenicity of RABV but has a good safety profile when compared with the parent strain HEP-Flury. In addition, HEP-Flury was previously shown to induce an immune response in dogs [19]. Therefore, rHEP-CaIL6 might be a rabies vaccine candidate.
2. Materials and methods 2.1. Viruses and animals rHEP-CaIL6 was rescued based on the HEP-Flury strain and confirmed (Supplementary material 2). HEP-Flury and rHEP-CaIL6 were propagated in BHK-21 cells. CVS-24 was propagated in suckling mouse brains. Female Kunming (KM) and BALB/c mice were purchased from the Center for Laboratory Animal Science of the Southern Medical University (Guangzhou, China). Mice were housed in the Laboratory Animal Center of the South China Agricultural University. All animal experiments were carried out in compliance with specific pathogen-free requirements that were previously approved by the ethics committee for animal experiments of the South China Agricultural University. All possible efforts were made to minimize the suffering of laboratory animals. Procedures were based on the national standard Laboratory Animal Requirements of Environment and Housing Facilities (CALAS, GB 14925-2001) as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2. Investigation of body weight changes and clinical symptoms in mice BALB/c mice (6–7 weeks of age) were infected intracerebrally (i.c.) with 3.3 104 FFU of HEP-Flury, rHEP-CaIL6 or with 30 ll medium (mock infection). Each group consisted of eight mice. Body weight and clinical symptoms were recorded daily for 31 days. Changes in body weight are presented as the mean value ± standard error (SE). Regarding clinical symptoms observed in mice, scores were assigned as follows: 0, no clinical symptoms observed; 1, loss of initial body weight > 5%; and 2, piloerection. 2.3. Immunization and in vivo challenge Groups of 10 KM mice (6–7 weeks of age) were immunized by the intramuscular injection of 1.0 103 FFU, 1.0 104 FFU, 1.0 105 FFU HEP-Flury or rHEP-CaIL6, respectively. Medium was used for mock infections. Serum was obtained at days 7, 14, and 21 after immunization and used to determine VNA levels by means of fluorescent antibody virus neutralization (FAVN) tests as described previously [20]. Concentrations/titres of anti-RABV antibodies were assessed by ELISA (Synbiotics, USA) according to the manufacture’s protocols. For challenge experiments, 23 days after immunization, mice were i.c. challenged with 50 mouse intracerebral lethal doses 50 (MILD50) of CVS-24. The number of survivors was recorded daily for 3 weeks.
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2.4. Flow cytometry KM mice of 6–7 weeks of age were infected intramuscularly with 100 ll (equivalent to 106 FFU/ml) of rHEP-CaIL6, HEP-Flury or DMEM, respectively. Mouse inguinal lymph nodes were harvested at days 4, 7, and 10 post-infection. Single-cell suspensions were obtained as described previously [10], and stained with antibodies against markers of T cells (CD3e-FITC, CD4-PE, CD8a-PerCPCy5.5) or B cells (CD19-FITC, CD40-PE) (all antibodies purchased from Affymetrix eBioscience, USA) by incubation for 30 min on ice. A minimum of 100,000 events were counted using a Beckman FC 500 flow cytometer (Beckman Coulter, USA). Data were analyzed using FCS Express 4 flow cytometry (De Novo Software, USA). 2.5. Quantitative real-time PCR IL6-induced gene expression at days 6, 9, and 12 after infection in CNS tissues was investigated as described in Supplementary material 2. Primers used to amplify target and reference genes are listed in Supplementary material 2. 2.6. Statistical analysis Experiments were carried out in triplicate. Data were analyzed using GraphPad Prism 6 software (GraphPad Software, USA). The statistical significance was determined using the Student’s t-test or Log-rank (Mantel-Cox) Test. P < 0.05 was considered to indicate statistically significant differences. 3. Results 3.1. Safety of rHEP-CaIL6 in mice The safety of rHEP-CaIL6 was determined in BALB/c mice. As shown in Fig. 1A, the body weight of mice infected with rHEPCaIL6 recovered faster compared with HEP-Flury and regained their initial body weight at day 9 post-infection. Clinical symptoms observed are shown in Fig. 1B. Mice infected with either rHEPCaIL6 or HEP-Flury showed similar clinical symptoms during the early stages of infection. However, at day 10 post-infection, mice infected with HEP-Flury started to develop more severe symptoms that persisted for a longer period (P < 0.05 at several time points) than those mice infected with rHEP-CaIL6. In summary, mice infected with rHEP-CaIL6 showed an earlier onset of recovery (increased body weights) compared with HEP-Flury. 3.2. Immunogenicity of rHEP-CaIL6 KM mice were immunized intramuscularly with different doses of rHEP-CaIL6 or HEP-Flury. Increasing inoculating doses of vaccine were designed to investigate whether the low dose vaccine induced high antibody titres when IL6 was over expressed and the lowest dose of rHEP-CaIL6 that would provide sufficient protection from a lethal challenge. Peripheral blood samples were collected at 7, 14, and 21 days post-immunization (dpi) and serum was used to determine the concentrations of anti-RABV antibodies and VNA. As shown in Fig. 2A, at 7 dpi, levels of anti-RABV antibodies were <0.6 EU, independent of the strain and dose administered. After two or three weeks, the anti-RABV antibody concentrations had increased to >0.6 EU, which is considered protective against RABV infection [21]. Interestingly, at 14 and 21 dpi, significantly higher levels of anti-RABV antibodies were detected in mice immunized with rHEP-CaIL6 compared with HEP-Flury group. VNA levels >0.5 international units (IU) are considered protective against RABV infection. As shown in Fig. 2B, concentrations of VNA in mice
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Fig. 1. Safety of RABV strains in mice. BALB/c mice (6–7 weeks of age; n = 8 per group) received i.c. injections of 3.3 104 FFU of HEP-Flury, rHEP-CaIL6 or medium alone (mock infection) to investigate the safety profile of rHEP-CaIL6. Body weight was monitored daily for 31 days (A). Changes of body weight were shown as ratio to body weight at day 0 and data were presented as mean value ± standard error (SE). Clinical symptoms presented by infected mice were scored according to severity on ascale from 0 to 2 and are presented here as mean value ± standard error (SE). Asterisks indicate significant differences among groups, as calculated by Student’s t-test (**P < 0.01).
Fig. 2. Immunogenicity of RABV strains in vivo and survival rates of mice challenged with CVS-24. Groups of ten KM mice (6–8 weeks of age) were immunized i.m. with 1.0 103 FFU, 1.0 104 FFU, or 1.0 105 FFU of HEP-Flury, rHEP-CaIL6 or medium alone (mock infection). Peripheral blood was obtained and concentrations of anti-RABV antibodies (A) in serum were determined using the ELISA (Synbiotics, USA) according to the manufacture’s protocols while serum VNA (B) were ascertained using FAVN as described in Materials and Methods. (C) Then, mice were challenged i.c. with 50 LD50 of CVS-24 at day 23 after immunization and were observed for 16 days. Survival rates were recorded daily. Data were analyzed using GraphPad Prism 6. anti-RABV antibodies and FAVN of asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Comparison of survival curves was calculated by Log-rank (Mantel-Cox) Test (**P < 0.01; ***P < 0.001).
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immunized with HEP-Flury or rHEP-CaIL6 were dose-dependent. Significantly higher levels of VNA were detected in mice immunized with rHEP-CaIL6 at 7, 14, or 21 dpi, compared with animals that received the same dose of HEP-Flury. At 7 dpi, the breakpoint of 0.5 IU was reached by immunization with 1.0 104 FFU or 1.0 105 FFU of rHEP-CaIL6, but not with the same doses of the parent HEP-Flury. Furthermore, the induction of VNA levels >0.5 IU was proven for rHEP-CaIL6 at all doses tested at 14 and 21 dpi. However, the level of VNA in mice immunized with 1.0 103 FFU of HEP-Flury remained <0.5 IU at 14 and 21 dpi. To investigate whether VNA concentrations correlated with protection from infection, immunized mice were then challenged with virulent CVS-24. As shown in Fig. 2C, significantly more mice immunized with rHEP-CaIL6 survived lethal challenges than those immunized with HEP-Flury. After application of the protective doses of 1.0 104 FFU, survival rates were 90 and 40% in each of the respective groups. A similar trend was observed after immunization with 1.0 103 FFU and 1.0 105 FFU, but the differences did not reach statistical significance. Comparative survivors (more than 80%) were observed when mice were immunized with rHEPCaIL6 at doses of 1.0 104 FFU and 1.0 105 FFU per mouse, suggesting low doses of rHEP-CaIL6 may confer protection against lethal RABV infections. 3.3. Effects of rHEP-CaIL6 on the recruitment of B and T cells To investigate whether rHEP-CaIL6 affected the recruitment of immune cells, flow cytometry was conducted to determine the counts of CD19+CD40+ B cells, CD3+CD4+ T cells, and CD3+CD8+ T cells in the lymph nodes at 4, 7, and 10 dpi in mice. Fig. 3A and B show flow cytometric plots representing B cells (CD19+ and/or CD40+) and T cells (CD4+ or CD8+, values based on expression of CD3+ for all T cells) in the inguinal lymph nodes. As shown in Fig. 3C and D, higher numbers of immune cells (CD19+CD40+ B cells and CD8+ T cells) were detected in mice infected with rHEP-CaIL6 compared with mice infected with HEP-Flury at 4 or 7 dpi. At 10 dpi, more CD19+CD40+ B cells had been recruited by rHEP-CaIL6 than by HEP-Flury, while the opposite was observed for CD8+ T cells, which may be explained by the different duration of infection caused by RABVs. Regarding CD4+ T cells, no differences were detected at any time point studied (data not shown). Thus, our studies confirm that rHEP-CaIL6 may recruit more immune cells (B cells and CD8+ T cells) compared with the parental strain HEP-Flury. This may indicate an enhanced immune response to rHEP-CaIL6. 3.4. Viral growth and induction of an immune response in the brain As an attenuated live vaccine, possible invasion to the CNS must be considered. To investigate virus replication and the induction of an immune response in the brain, KM mice were infected intranasally. with either rHEP-CaIL6 or HEP-Flury. Brains were harvested at time points after infection and assessed for the copy numbers of RABV, expression of RABV N, RABV G, canine IL6, CD4, CD8, CD11b, MHC-I, MHC-II, IFN-a, IFN-b, IFN-c, IFN-c-induced protein 10 (IP-10), MIP-1a, RANTES, TNF-a and j-L-chain (IgG) using qPCR. As shown in Fig. 4, the copy numbers of rHEP-CaIL6 and HEP-Flury as well as RABV N and RABV G expression did not differ at 6 dpi. rHEP-CaIL6 copy numbers did not increase drastically at 9 dpi and could not be detected at 12 dpi while HEP-Flury copy numbers were augmented these time points. The expression of RABV N and RABV G in brains of mice infected with rHEP-CaIL6 decreased and could not be detected at 9 and 12 dpi, while expression levels remained unchanged over time in the brains of mice infected with HEP-Flury. In addition the expression of canine IL6 decreased timedependently in mice infected with rHEP-CaIL6. These data indicate
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that rHEP-CaIL6 could be cleared from infected brains faster than HEP-Flury indicating. stronger immune responses might be induced by infection with rHEP-CaIL6. Several studies have indicated that CNS immune responses involve the opening of the blood-brain barrier (BBB) and an increase in BBB permeability that coincides with an enhanced expression of inflammatory cytokines and chemokines [22,23]. In this context, pro-inflammatory cytokine IL6 might enhance the expression of TNF-a [24], which further stimulates inflammatory processes. Chemokines may also be involved in pathogen clearance from the brain. As depicted in Fig. 4, at 6 dpi, the mRNA levels of chemokines IP-10, MIP-1a, and RANTES as well as TNF-a were significantly higher in the CNS of mice infected with rHEP-CaIL6 than in those that were exposed to HEP-Flury. At 9 and 12 dpi, the expression of IP-10, MIP-1a, RANTES, and TNF-a decreased in rHEP-CaIL6-treated mice and reached values similar to those obtained in the control group infected with HEP-Flury. Because an increase in permeability of the BBB may lead to the infiltration of T and B cells, we examined the expression of CD4, CD8, CD11b, CD19, MHC-I, MHC-II, IFN-a, IFN-b, and IFN-c in brain tissue. No differences were observed for the expression of CD4 and CD19 (Fig. 4), but infection with rHEPCaIL6 was associated with higher CD8 and CD11b mRNA levels at 6 and 9 dpi compared to the HEP-Flury group. Additionally, the expression of MHC-I was enhanced in mice infected with rHEPCaIL6 at 6 dpi. Regarding MHC-II, no difference between groups was observed at all-time points. Types I and II IFNs are also implied in antiviral defence mechanisms. Our data demonstrate that rHEPCaIL6 induced a much stronger IFN-related immune response at early stages (6 dpi) compared with HEP-Flury. Antibody production in CNS tissues was determined by IgG-j-L-chain mRNA expression. As shown in Fig. 4, higher levels of IgG were detected at all-time points after immunization with rHEP-CaIL6 compared with HEPFlury. This indicates that IL6 may induce a persistent immune response despite its relatively fast clearance.
4. Discussion Previous studies have shown the G-mediated induction of VNA is critical to prevent animals or humans from wild-type RABV infection [19,25]. In addition, as an attenuated vaccine candidate of RABV which may compromise the CNS, the induction of an immune response is important to permit pathogen clearance from the CNS [22,26]. This study aimed to provide preliminary information for the development of an effective, safe, and inexpensive RABV vaccine candidate. We selected IL6 as an adjuvant because it enhances the immune response [17] and modulates inflammation and BBB permeability [27,28]. All these processes are involved in the clearance of invasive RABV from the CNS [22,23,26]. RABV HEP-Flury is an attenuated strain that does not cause fatal infections after intracerebral or peripheral inoculation [29]. The HEPFlury strain has been used as a vaccine for humans in Japan [30], and for animals in China. Furthermore, an attenuated live RABV vaccine based on the HEP-Flury strain has been licensed and is used in animals in China. Murine cells and mice are sensitive to canine IL6 (provided in Supplementary material 1). rHEP-CaIL6 showed high viral titres at the early stages compared with HEPFlury in NA cells which may provide the foundations of virus production (provided in Supplementary material 2). In this study, we characterized rHEP-CaIL6, derived from HEP-Flury and expressing canine IL6, and compared its effects on immunology and its safety profile. Our study showed that rHEP-CaIL6 more effectively stimulated the production of anti-RABV antibodies and VNA, and thus conferred better protection to mice after a challenge with CVS-24 compared with the parental strain HEP-Flury. Our results are in
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Fig. 3. Effects on the recruitment and activation of B cells and CD8+ T cells in the inguinal lymph nodes. Female KM mice of 6–8 weeks of age were infected i.m. with 1.0 105 FFU of HEP-Flury, rHEP-CaIL6, or medium alone (mock infection). At days 4, 7, and 10 dpi, inguinal lymph nodes were harvested and single-cell suspensions were prepared and stained with antibodies against B cell (CD19-FITC and CD40-PE) or T cell markers (CD3-FITC, CD4-PE, and CD8-PerCP-Cy5.5). Data were collected and analyzed with a Beckman FC 500 flow cytometer (Beckman Coulter) and FCS Express 4 flow cytometry (De Novo Software). (A) Representative flow cytometric plots, showing mature B cells (CD19+/CD40+) in the upper right quadrants of each scatter plot. (B) In the upper left quadrant of each scatter plot, CD3+CD8+ T cells are represented. (C) Percentages of CD19+CD40+ B cells and (D) CD3+CD8+ T cells in inguinal lymph nodes at different time points (n = 3 per group). Values are presented as mean ± standard error. Asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; ****P < 0.0001).
agreement with a recently published study showing that the HEPFlury strain induced VNA in dogs [19], and we speculate that rHEPCaIL6 may also protect dogs from infection with virulent RABV. Thus, IL6 might be used as an adjuvant of RABV vaccines. Because rHEP-CaIL6 induced protective antibodies at lower doses, it might allow for the administration of lower doses without losing infection protection and might contribute to lowering the costs of RABV vaccine production. Recent studies [15,16] suggested IL6 enhanced the serum levels of IgG and favoured an immune response mediated by cytotoxic T cells, which express CD8. In this context, we showed that rHEPCaIL6 augmented the recruitment of B cells and CD8+ T cells in the lymph nodes, and this finding corroborates previous publications. CD19 is a pre-B cell marker and CD40 is mainly expressed by mature B cells after activation [31]. Because B cell activation plays a major role in humoral immunity, we assessed rRABVmediated B cell recruitment and characterized this population of lymphocytes. According to our findings, rHEP-CaIL6 recruited more CD19+ and CD40+ B cells in the lymph nodes compared with HEPFlury, which may explain why rHEP-CaIL6 induced higher levels of anti-RABV antibodies and VNA. CD8+ T cells are involved in the control of acute infections and the clearance of pathogens
[32,33]. Regarding the in vivo evaluations of immune responses triggered by rHEP-CaIL6, an enhanced CD8+ T cells response may be the cause of a faster clearance of resident RABV from host cells. Because our study aimed to develop a candidate attenuated rabies vaccine, the safety of rRABV rHEP-CaIL6 was investigated in mice. It has been confirmed that RABV infections of animals or humans are fatal mainly because of CNS invasion [26]. We showed that mice infected i.c. with rHEP-CaIL6 showed a rapid recovery (increase in body weight) and mild clinical symptoms. These observations may indicate that rHEP-CaIL6 had a good safety profile. Furthermore, rHEP-CaIL6 was cleared rapidly from the brain of infected mice while the HEP-Flury viral multiplication increased time dependently. To identify the mechanisms of the early clearance of rHEP-CaIL6, the expression of inflammatory mediators in the brain was investigated. Our data showed that the mRNA levels of pro-inflammatory cytokines including TNF-a (which may be induced by IL6) and chemokines such as IP-10, MIP-1a, and RANTES were present in higher concentrations in the CNS following immunization with rHEP-CaIL6. Pro-inflammatory cytokines and chemokines may attract immune cells to the site of infection, and these cells may favour the removal of pathogens. Accordingly, the induction of pro-inflammatory mediators and recruitment of
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Fig. 4. Expression of viral genes, inflammatory mediators and immune response-related molecules in the CNS. Female KM mice of 6–8 weeks of age were infected i.n. with 3.3 104 FFU of distinct virus strains, and at days 6, 9, and 12, brains were harvested following the perfusion with PBS. Expression of viral genes and immune responserelated molecules in the brains were investigated by quantitative real-time PCR in a CFX384 Real-Time System. Copy numbers of RABV, expression of RABV N, RABV G, canine IL6, IP-10, MIP-1a, RANTES, TNF-a, CD4, CD8, CD11b, CD19, MHC-I, MHC-II, IFN-a, IFN-b, IFN-c, and IgG j-L chain were normalized to the expression level of the housekeeping gene GAPDH. Expression levels of immune response-related molecules were presented as fold changes over those detected in mock-infected controls. Data were analyzed using BioRad CFX Manager and GraphPad Prism 6. Asterisks indicate significant differences among groups, as calculated by Student’s t-test (*P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001).
immune cells may be an important mechanism of RABV attenuation [23,34]. This might explain the better safety profile of rHEPCaIL6 compared with HEP-Flury. Previous studies reported that infection of the CNS by laboratory-attenuated RABV was cleared by infiltrating immune cells, which corresponds to an enhanced innate and adaptive immune response [35,36]. Pro-inflammatory cytokines and chemokines may enhance BBB permeability [37] and thus allow immune cells to enter the CNS. We speculate that BBB permeability was increased and stronger cell-mediated immune responses occurred in the CNS after immunization with rHEP-CaIL6. To gain a further insight into the role of immune cells in pathogen clearance from the CNS, the expression of CD4, CD8, CD11b and CD19 was evaluated. Increased concentrations of pro-inflammatory cytokines and chemokines at 6 dpi coincided with an enhanced expression of CD8 and CD11b in mice immunized with rHEP-CaIL6. However, expression levels of CD8 and CD11b remained elevated in these animals when pro-inflammatory mediator concentrations had already decreased (9 dpi). This may be explained by the duration of the enhanced immune response. Both HEP-Flury and rHEP-CaIL6 similarly induced CD4 and CD19 expression in the CNS. This may indicate that rRABV
expressing IL6 did not affect CNS infiltration by CD4+ T cells or CD19+ B cells. Furthermore, no significant differences were observed regarding the mRNA levels of MHC-I, which is expressed by all karyocytes, whereas mRNA levels of MHC-II, expressed by B cells, dendritic cells, macrophages, and subpopulations of activated T cells, were significantly higher in the brains of mice infected with rHEP-CaIL6 than in samples obtained from the HEP-Flury group. This may also be related to the strong IL6mediated recruitment of immune cells to the CNS. IFNs plays important roles in host defence against RABV and affect the degree of attenuation displayed by an individual RABV strain [9]. The expression of IFN-a, IFN-b, and IFN-c was assessed to investigate immune responses in the CNS. At 6 dpi, the mRNA levels of IFN-c were significantly higher in the brains of mice immunized with rHEP-CaIL6. This is in agreement with a previous report of the IL6-induced expression of IFN-c [18]. IFN-c, in turn, may stimulate the expression of type I IFNs [9]. rHEP-CaIL6 increased the expression of both type I IFNs, as measured at 9 dpi. The mRNA levels of IgG were augmented in the brains of mice immunized with rHEP-CaIL6. In summary, the increased safety displayed by rHEP-CaIL6 may be explained by an enhanced immune response induced by the adjuvant IL6.
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To develop a rabies vaccine that may be approved for use in dogs, further studies regarding the duration of immunity and the safety of the product are required. In conclusion, rHEP-CaIL6 enhanced RABV immunogenicity and conferred good protection from infections with virulent RABV. Moreover, IL6 expression by rRABV resulted in increased safety by the induction of a strong immune response in the CNS. rHEP-CaIL6 may have the potential to be developed as an attenuated RABV vaccine candidate. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Contributors Experimental design was conducted by Xiaofeng Guo and Jun Luo. Data analysis and writing article was finished by Jun Luo, Xiaofeng Guo, and Yongwen Luo. Animal experiments were finished by Jun Luo, Boyue Zhang, Yuting Wu, Jing Zhao, Ziyu Lv. Flow cytometry was conducted by Jun Luo, Qin Tian, Boyue Zhang, Yuting Wu, and Mingzhu Mei. Quantitative real-time PCR was conducted by Jun Luo, Qin Tian, Boyue Zhang and Qiong Zhang. Acknowledgments We thank the Guangdong Haid Institute of Animal Husbandry and Veterinary for the support of instruments. This study was partially supported by the National Key Research and Development Program of China (No. 2016YFD0500400), Research of Guangdong Province and Ministry of Education (No. 2014B090901046) and Special Fund for Agro-scientific Research in the Public Interest (No. 201103032). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2016.12. 069. References [1] Fu ZF. Rabies and rabies research: past, present and future. Vaccine 1997;15 (Suppl):S20–4. [2] Luo J, Zhao J, Tian Q, Mo W, Wang Y, Chen H, et al. A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro. Virus Genes 2016;52(3):379–87. [3] Zhao L, Toriumi H, Wang H, Kuang Y, Guo X, Morimoto K, et al. Expression of MIP-1alpha (CCL3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and B cells. J Virol 2010;84(18):9642–8. [4] McGettigan JP, Naper K, Orenstein J, Koser M, McKenna PM, Schnell MJ. Functional human immunodeficiency virus type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and env expressed from a single rhabdovirus-based vaccine vector genome. J Virol 2003;77(20):10889–99. [5] Wu X, Rupprecht CE. Glycoprotein gene relocation in rabies virus. Virus Res 2008;131(1):95–9. [6] Mebatsion T, Schnell MJ, Cox JH, Finke S, Conzelmann KK. Highly stable expression of a foreign gene from rabies virus vectors. Proc Natl Acad Sci USA 1996;93(14):7310–4. [7] Ceccaldi PE, Fayet J, Conzelmann KK, Tsiang H. Infection characteristics of rabies virus variants with deletion or insertion in the pseudogene sequence. J Neurovirol 1998;4(1):115–9. [8] Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. Embo J 1994;13(18):4195–203. [9] Barkhouse DA, Garcia SA, Bongiorno EK, Lebrun A, Faber M, Hooper DC. Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon. J Virol 2015;89(1):312–22.
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