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Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine
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Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine Shengtao Fan a, Yun Liao a, Guorun Jiang a, Li Jiang b, Lichun Wang a, Xingli Xu a, Min Feng a, Erxia Yang b, Ying Zhang a, Wei Cui b, Qihan Li a,⇑ a Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming 650118, China b Aimei Convac BioPharm (Jiangsu) Co., Ltd., Taizhou 225300, Jiangsu, China
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Article history: Received 31 July 2019 Received in revised form 28 November 2019 Accepted 19 December 2019 Available online xxxx Keywords: Enterovirus type 71 (EV71) Coxsackievirus A 16 (CA16) Bivalent vaccine Rhesus macaques
a b s t r a c t Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are recognized as the major pathogens responsible for human hand-foot-mouth disease. To develop a bivalent EV71-CA16 vaccine, rhesus macaques immunized with two doses of this vaccine via the intradermal route were challenged with EV71 or CA16, and their clinical symptoms, viral shedding, neutralizing antibodies, IFN-c-specific ELISpots, and tissue viral load were examined longitudinally. Specific immunity against EV71 and CA16 was observed in the macaques, which exhibited controlled proliferation of the EV71 and CA16 viruses and upregulated expression of immune-related genes compared with the controls. Furthermore, broad protection against EV71 and CA16 challenge without immunopathological effects was observed in all the immunized macaques. These studies suggest that the bivalent EV71-CA16 inactivated vaccine was effective against wildtype EV71 or CA16 viral challenge in rhesus macaques. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Numerous epidemics of human hand-foot-mouth disease (HFMD), which is a viral infectious disease, have occurred in children, and due to its clinical pathologic outcomes, HFMD has become a concern for many families [1–5]. The identified pathogenic agents of this disease include enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16), which are responsible for severe neurogenic cases and deaths [6,7]. These two viruses have similar capsid structures and present comparable biological characteristics but exhibit no cross-reactivity in serological tests [8]. In 2015, the first EV71 inactivated vaccine was licensed and applied in a child population [9–12]. However, no CA16 vaccine has been developed for use in clinical trials because evaluation of the immunized rhesus model after challenge with the wild-type virus failed to provide evidence of clinically protective immunity [8,13], even though this test was positive for the EV71 vaccine [9]. We previously investigated this issue and reported several notable findings. First, compared with the VP1 protein of EV71, which is expressed in epithelial cells, the CA16 protein might not function as a viral ⇑ Corresponding author. E-mail address: [email protected] (Q. Li).
pathogen-associated molecular pattern (PAMP) by interacting with cellular innate immune molecules, such as pattern recognition receptors (PRR), to induce further activation of NF-ƙB and thus the transcription of immune-related signaling molecules [14]. Second, CA16, similarly to EV71, is capable of infecting CD11+ dendritic cells (DCs) and modulating their immunological phenotype [13,15] but provides a weaker stimulus to induce the expression of various immune-related signaling molecules in infected DCs than EV71 [16]. In this case, augmenting the immunogenicity of the CA16 antigen might determine not only the design of the vaccine but also the most effective immunization procedure. Based on this hypothesis and our knowledge of innate and adaptive immunity, particularly the activation of T cells by DCs and other cells [15,17], an innate immune signal should be transferred to T cells based on the systemic process integrating antigen presentation with the signaling network composed of various cytokines [18]. In the present study, we designed a bivalent EV71-CA16 inactivated vaccine for the immunization of animals through the intradermal route, and its administration was expected to lead to integrated activation of innate immunity and the initiation of a specific T cell response and to provide effective clinical protection during viral infection. This hypothesis was investigated using mice and was confirmed by the induction of a favorable immune
https://doi.org/10.1016/j.vaccine.2019.12.057 0264-410X/Ó 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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response, which showed the production of specific neutralizing antibodies and cytotoxic T lymphocytes (CTLs), as well as a protective effect on adult and postnatal mice challenged with the viruses [17]. However, the limitations of the mouse model resulted in a failed pathogenic process of CA16 infection; thus, rhesus macaques, which exhibit sensitivity to CA16 infection and a close genetic relationship to humans [13,19], were used in this study. Our description focused on the immune response induced in rhesus macaques immunized with a bivalent EV71-CA16 inactivated vaccine via the intradermal route and the clinical efficacy of immune protection provided by the vaccine against challenge with the EV71 and/or CA16 wild-type strains. All data obtained in this study indicate that this vaccine exhibits the efficacy and safety required for a clinical trial.
2. Materials and methods 2.1. Study design Prior to the formal experiment, preliminary immunological observations of this bivalent EV71-CA16 vaccine was obtained from four rhesus macaques immunized via the muscular route; in this preliminary analysis, neutralizing antibodies and specific CTLs against EV71 and CA16 were first detected 28 days after the administration of two immunization doses at an interval of 4 weeks (each dose contained 100 U/100 U of EV71/CA16 inactivated vaccine antigens and 0.25 mg of aluminum in 0.5 ml of buffer; Fig. S1). At day 56, each two this bivalent vaccine immunized
macaques were challenged with the wild-type EV71 or CA16 strain, respectively, and were then clinically monitored for pathological manifestations, particularly vesicles in oral and/or limb skin (Fig. S1). At days 4 and 8 post-challenge, all the macaques were euthanized for viral load detection and histopathological observation, respectively (Fig. S1). Based on preliminary data, the following formal experiment was designed: 16 rhesus macaques aged 6–12 months were divided into groups A and B, with eight animals in each group. All the macaques were isolated for 2 weeks and confirmed to be negative for anti-EV71/CA16 antibodies. Four macaques in each group served as a negative control for viral challenge, and the other four were immunized with the bivalent EV71-CA16 vaccine via the intradermal route at the same time on days 0 and 28. Briefly, the back skin of the rhesus monkeys was shaved, and a MicronJet 600 needle (NanoPass Technologies, Ltd.) was used for vaccine delivery according to the manufacturer’s instructions. Each dose vaccine contained 100 U/100 U EV71/CA16 antigens and 0.125 mg of aluminum adjuvant in 250 ml of buffer. At 12 and 24 h after the first inoculation, the animals were administered topical anesthesia, and a minimal sample of local skin tissue was collected for detection of the gene profile. On days 28 and 56 after immunization, blood samples were collected from all the macaques for neutralizing antibody and IFN-c-specific enzymelinked immunospot (ELISpot) assays (Fig. 1). All the animals in group A were subjected to viral challenge using the following protocol: two immunized animals and two control animals were challenged with the wild-type EV71 strain (FY23, 5 104 CCID50 for each animal), and the other two were challenged with the wildtype CA16 strain (G20, 5 104 CCID50 for each animal; Fig. 1).
Fig. 1. Schematic depicting the experimental process. Four macaques from groups A (#18005, #18001, #18007 and #18009) and B (#18181, #18027, #18021 and #18025) were immunized with the EV71-CA16 vaccine. The control macaques were treated with aluminum adjuvant alone. On days 28 and 56, serum samples from all the immunized animals were collected for neutralizing antibody assays and IFN-c-specific ELISpot assays. On day 60, two immunized and two control macaques from groups A and B were challenged with wild-type EV71 or CA16. All the challenged animals from group A were observed for clinical symptoms and sacrificed on day 64 or 68 for further analysis. On day 100, the macaques in group B that were initially challenged with EV71 were challenged with CA16, whereas the macaques in group B that were initially challenged with CA16 were challenged with EV71. On day 160, all the macaques from group B were challenged with a combination of EV71 and CA16, and the immunized and control animals were sacrificed on day 164 or 168 for further analysis.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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All the challenged animals were observed for clinical symptoms. Each immunized and control animal infected with EV71 or CA16 was sacrificed under anesthesia on days 4 and 8 after challenge for further analyses (Fig. 1). The immunized macaques from group B were challenged with the two viruses using the same protocol and monitored for clinical symptoms (Fig. 1). On day 40 after the first viral challenge, a second viral challenge was administered using a crisscross approach: the animals previously challenged with EV71 were now challenged with the wild-type CA16 strain, and the animals previously challenged with CA16 were now challenged with the wild-type EV71 strain. The two control animals in group B were infected with EV71 or CA16. The clinical symptoms were monitored for 10 days after the second challenge. On day 60 after the second challenge, all immunized and control animals were challenged with a mixture of the wild-type EV71 and CA16 strains (5 104 CCID50/5 104 CCID50 for each animal), and the symptoms of all the animals were monitored. On days 4 and 8 after the last challenge, all the immunized and control animals infected with EV71 or CA16 were sacrificed under anesthesia, and samples of various tissues were collected for pathological detection. 2.2. Ethics statement The rhesus macaque experiments were designed based on the Guidelines for the Care and Use of Laboratory Animals from the National Research Council of the National Academies and the Guidance for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of the People’s Republic of China (2006). The animal experimental protocols were approved by The Yunnan Provincial Experimental Animal Management Association (approval No. SYXK [Dian] K 2015-0006) and the institutional Experimental Animal Ethics Committee (approval No. DWSP201803013-2). 2.3. Animal administration and care Rhesus macaques obtained from the Primate Research Center of the Institute of Medical Biology (IMB) and the Chinese Academy of Medicine Science (CAMS) were housed in cages (BSL-2 conditions) with an ambient temperature of 25 °C, provided adequate water and food, and received appropriate veterinarian care daily. 2.4. Viruses and cells The FY23 (EV71), G20 (CA16) and KM/M08 (CA16) strains were used in this study. Human diploid cells (KMB-17 strain) and Vero cells were used for viral proliferation and titration, respectively. 2.5. Production of the bivalent EV71-CA16 inactivated vaccine and quality standards The FY23 strain of EV71 and KM/M08 strain of CA16 were inoculated into KMB-17 cells grown in cell factory, respectively. The virus was harvested once 80% typical cytopathic effects (CPEs) observed. The virus stock was inactivated by incubation with a 1:4000 dilution of formaldehyde at 37 °C for 72 h and then concentrated 50-fold through ultrafiltration. The viral protein was purified by Sepharose 6 Fast Flow (Amersham, USA) chromatography [20]. The viral purity was assessed by RP-HPLC according to the manufacturer’s recommended protocol (Waters, USA). Equal amounts of the purified EV71 and CA16 antigens were emulsified in 0.5 mg/ml Al(OH)3 adjuvant. EV71 and CA16 standard antigens (Lot.201507001) were purchased from National Institutes for Food and Drug Control as references. The antigen content was determined by ELISAs [21].
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2.6. Detection of the viral load by q-RT-PCR Various tissues collected from the immunized macaques were homogenized using a TissueLyser II system (Qiagen, Hilden, Germany). Swab and blood samples were continuously collected from the infected macaques for 14 days postchallenge. The viral RNA from the tissue and swab samples was extracted using the Simply P Total RNA Extraction kit according to the manufacturer’s recommended protocol. The q-RT-PCR was performed using the One Step PrimeScript RT-PCR Kit with a CFX Connect real-time system (BioRad, USA). The primers and probes for EV71 and CA16 are shown in Table S1. 2.7. Expression profile of the mRNAs encoding immune-related signaling molecules in local skin tissues obtained from the inoculation site Total RNA from the inoculated skin tissues of the macaques collected 12 and 24 h after immunization was extracted using the TRIzol-A+ Reagent (Cat # DP421, Tiangen, China), according to the manufacturer’s recommended protocol. The RNA was amplified using the One Step TB GreenTM Prime ScriptTM PLUS RT-PCR Kit (Cat # RR096A, TaKaRa, Japan). The primers used to quantify the LIGHT, IKKa, IKKb, CD160, TLIA, NIK, IFN-a, IFN-b, IFN-k, GITRL, 4-IBBL, IL5, IL-13 and IL-22 mRNA levels are shown in Table S2. 2.8. Expression profile of mRNAs in PBMCs from immunized macaques after challenge PBMCs were collected from the macaques 4 days after viral challenge, and total RNA from the PBMCs was extracted and purified using the miRNeasy Mini Kit (Cat # 217004, QIAGEN, GmbH, Germany) according to the manufacturer’s instructions. The RNA integrity was assessed by determining the RIN number using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). mRNA sequencing libraries were generated from the PBMC samples using the mRNA Sample Pre Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s recommended protocol. HiSeq/MiSeq was used for sequencing. Based on the expression of 63 genes, 16 genes were randomly selected to validate the expression profiles by q-RT-PCR using a CFX Connect real-time system (Bio-Rad, USA). The primers used to validate these results are shown in Table S3. 2.9. Neutralizing antibody assay The macaque serum samples were subjected to 2-fold serial dilutions and incubated with 100 TCID50 of the FY23 strain (EV71) or KM/M08 strain (CA16) for 1 h at 37 °C. The samples were then inoculated into Vero cells and incubated at 37 °C in a 5% CO2 incubator for CPE assessment. The end-point neutralization titers were defined through 50% plaque reduction assays. 2.10. ELISpot assays and ELISA An ELISpot assay was performed according to the manufacturer’s instructions (Cat # 3421 M-4HPW-2, Mabtech, Sweden). Briefly, PBMCs were collected from immunized macaques, and EV71 or CA16 antigen (20 U/well) was added to the PBMC suspension (5 105 cells/well) in a 96-well plate. The mixture of antigen and cells was incubated at 37 °C in a 5% CO2 atmosphere for 36 h. The plate was then washed four times with PBS and sequentially incubated with primary antibody, secondary antibody and substrate solution. The spots were counted using an automated ELISpot reader (CTL, OH, USA). Colorimetric sandwich ELISA kits were used to quantify the serum IL-6 and TNF-a levels. Briefly,
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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diluted serum and standard samples were added to microwell plates that had been precoated with specific antibody. After washing, secondary antibody was added to detect the captured primary antibody. Horseradish peroxidase (HRP)-conjugated antibody and tetramethyl-benzidine (TMB) reagent were sequentially added for signal development. The stop solution was then added, and the absorbance at a wavelength of 450 nm was read using a microplate reader.
2.11. Histopathological observations Tissues from euthanized macaques were fixed with 10% formaldehyde for 7 days, dehydrated with ethanol, cleared with
xylene, embedded in paraffin and then cut into 4-lm-thick sections for hematoxylin and eosin (HE) staining. The pathological changes were examined using a light microscope (ECLIPSE Ti-s, Nikon).
2.12. Evaluation of clinical symptoms The clinical symptoms of the rhesus macaques, including their mental state, temperature changes, and the presence of vesicular lesions on the lips, hands and feet, were examined after challenge. If symptoms were observed during the observation period, the following clinical scoring criteria were employed: 0, asymptomatic; 1, redness; 2, lesion; 3, bullae; 4, multiple bullae; and 5, ulceration.
Fig. 2. A specific immune response is induced in rhesus macaques by the bivalent EV71-CA16 vaccine. (A) The neutralization of EV71 and CA16 by serum samples from control and immunized animals collected on days 28 and 56 was tested. (B) Levels of IFN-c secreted from PBMCs from all macaques stimulated with the EV71 and CA16 antigens on days 28 and 56. (C) The LIGHT, NIK, CD160, TLIA IKKa, IKKb, (D) GITRL, 4-IBBL, (E) IFN-a, IFN-b, IFN-k, (F) IL-5, IL-13 and IL-22 mRNA levels in samples collected from the site of inoculation in the immunized macaques were detected by q-RT-PCR. All the data obtained from the control group served as reference values for calculating the relative fold changes. The statistical significance was assessed using unpaired t-tests (***p < 0.001).
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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2.13. Statistical analysis The data are presented as the means ± SDs. The differences between two groups were evaluated using Student’s t-tests (GraphPad Prism software; GraphPad Software, San Diego, CA, USA), and p < 0.05 was regarded as indicating a significant difference.
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ilar to previous data [4,8]. This finding demonstrates the immune response induced by the bivalent EV71-CA16 vaccine in macaques. The mRNA profiles of some innate immune-related signaling molecules in the local tissue 12 and 24 h after vaccine inoculation suggest the activation of innate immunity (Fig. 2C–F), consistent with the upregulation of some molecules in related signaling pathways [22,23].
3. Results 3.1. Bivalent EV71-CA16 inactivated vaccine enables stimulation of specific immunity against EV71 and CA16 A preliminary experiment involving immunization via the muscular route suggested inefficient and efficient immune responses against CA16 and EV71 challenge, respectively, in macaques (Fig. S2A); specifically, a no clinical protective effect was detected in the animals challenged with CA16, which presented various clinical pathological features, including oral vesicles and viremia (Fig. S2B), whereas complete control of the EV71 challenge was observed (Fig. S2B). Based on these results combined with data from a previous mouse experiment [17], a bivalent EV71-CA16 inactivated vaccine was designed for the immunization of rhesus macaques in groups A and B based on the schematic presented in Fig. 1. The immunization induced the production of neutralizing antibodies against EV71 and CA16, and lower and higher titers were observed on day 28 after the first and second immunizations, respectively (Fig. 2A). The titers of CA16-neutralizing antibodies were lower than those of EV71-neutralizing antibodies, as observed in a previous study [8]. ELISpot detection of IFN-c suggested the generation of specific CTL responses to EV71 and CA16 antigens on days 28 and 56 after immunization (Fig. 2B), sim-
3.2. Clinical immunoprotective effect induced by the bivalent EV71CA16 inactivated vaccine after viral challenge To investigate the efficacy of the CA16 inactivated vaccine in macaques [19], the validity of the bivalent EV71-CA16 vaccine was assessed based on clinical pathological observations from immunized macaques after challenge with the two viruses. Our study was designed to first identify the clinical symptoms of the immunized animals in groups A and B after challenge with the two viruses compared with the control animals. All immunized macaques in groups A and B were asymptomatic after challenge with either EV71 or CA16 (Fig. S3), whereas the control animals in both groups showed typical vesicles in the oral mucosa and skin of the limbs as well as body temperature variations after both challenges (Fig. S3). Importantly, the two viral challenges led to typical viremia in the control animals in both groups, with a peak at approximately day 6 (Fig. 3A), but the immunized animals consistently showed negative results (Fig. 3A). The detection of viral shedding in the mouth, nose, urine and feces revealed generally similar trends (Fig. 3B–E). Specifically, little or no trace of the virus was detected in the immunized macaques, whereas virus was detected in the control animals; in addition, the copies of the EV71 genome in the oral cavity showed slight increases at day 8,
Fig. 3. Immunoprotective effect of the bivalent EV71-CA16 vaccine on rhesus macaques after viral challenge. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from the animals in group A. The samples were collected every two days for 8 days after challenge.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 4. The bivalent EV71-CA16 vaccine controls EV71 or CA16 proliferation in rhesus macaques. Detection of the viral load in the main organs (A), nervous tissues (B), intestinal tissues (C) and lymph nodes (D) of the macaques in group A on days 4 and 8 after challenge with wild-type EV71. Detection of the viral load in the main organs (E), nervous tissues (F), intestinal tissues (G) and lymph nodes (H) of the macaques in group A on days 4 and 8 after challenge with wild-type CA16.
but this increase might be due to the q-RT-PCR detection limit (Fig. 3B–E). These data suggest that immunization with the bivalent vaccine via the intradermal route leads to a clinical immunoprotective effect against challenge with either EV71 or CA16. 3.3. Immunity induced by the bivalent EV71-CA16 vaccine enables control of the proliferation of both viruses in vivo To better assess the protective efficacy of this vaccine, the viral load was detected by measuring the EV71 and CA16 genomes in various tissues collected from the euthanized macaques, including both the immunized and control individuals in group A on days 4 and 8 after viral challenge. Lower or even trace viral loads were detected in the organs of all immunized macaques 4 and 8 days challenged with either EV71 or CA16, whereas higher loads were found in the organs of the euthanized control animals (Fig. 4A and E). Further experiments revealed similar results in the nervous tissues (Fig. 4B and F), intestinal tissues (Fig. 4C and G) and lymph nodes of various tissues (Fig. 4D and H). Pathological observations of these tissues revealed few nonspecific inflammatory sites in most tissues from the immunized macaques (Table S4) and less inflammatory pathological changes or lesions in the infected control animals, similar to the results obtained in our previous study [9,13]. These results suggest that the EV71-CA16 vaccine can induce protective immunity that can enable inhibition of both the proliferation of infecting EV71 or CA16 and the pathological process associated with clinical outcomes. 3.4. Challenge upregulates mRNAs related to immune regulation in immunized macaques compared with controls In our previous work, the mRNA profiles in immunized individuals were used for the evaluation of immunity [10], and this reference indicator reflects the systemic process of generating an
immune response and the associated homeostasis of the inflammatory reaction [24,25]. Here, PBMCs from the immunized and control macaques in group A were collected at day 4 after viral challenge to analyze the mRNA profile related to the immune response. Lower expression of mRNAs related to immune regulation was observed in the CA16- compared with the EV71infected controls (Fig. 5A–C). However, the expression of many mRNAs related to immune regulation and signal transduction showed a larger fold change in the immunized macaques after challenge with either EV71 or CA16 (Fig. 5D–F). Comparisons of the data presented in Fig. 5A–F revealed that immunization with the bivalent vaccine elicits an immune response that is capable of systemically and effectively combating viral infection, particularly CA16 infection. The expression levels of 16 selected genes were identified using q-RT-PCR with their specific primers to be consistent with the results shown in the heat map (Fig. S4).
3.5. Immunogenicity induced by the bivalent EV71-CA16 vaccine enables breadth protection against EV71 and CA16 infection The designed bivalent EV71-CA16 vaccine aims to induce immunity capable of resisting EV71 or/and CA16 infection. In this study, a second crisscross viral challenge was administered to the immunized and control macaques in group B at day 40 after the first challenge. At this stage, the immunized and control macaques previously challenged with EV71 were now infected with CA16, and those that were first challenged with CA16 were now infected with EV71. Within 10 days after the second challenge, all the control animals showed vesicles in the oral mucosa and limbs, variations in body temperature, and other signs similar to those observed in the control animals in group A (Fig. S5A and S5B), but none of the immunized macaques presented these signs (Fig. S5A and S5B). Importantly, detection of the viral load in the blood over the 10 days postchallenge revealed no viremia in any
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 5. mRNA expression profiles in skin samples from rhesus macaques collected on day 4 after viral challenge. Expression of mRNAs related to the DC surface signature (A), chemokines, interferon regulatory factor (B), interleukins, tumor necrosis factor, and TNF receptor-associated factor (C) in the EV71-challenged and CA16-challenged control groups. Expression of mRNAs related to the DC surface signature (D), chemokines, interferon regulatory factor (E), interleukins, tumor necrosis factor, and TNF receptor-associated factor (F) in the EV71-challenged and CA16-challenged immunized groups. Each row indicates one gene. The values are presented as log2-fold changes. All the data from normal rhesus macaques served as reference values for calculating the relative fold changes.
of the immunized macaques and typical viremia, with a peak at day 4, in all the control animals (Fig. 6A). Similar trends were obtained from the detection of viral shedding in the mouth, nose, urine and feces (Fig. 6B–E). The increased titer of neutralizing antibodies against both viruses in the immunized macaques observed at day 10 after the second cross-challenge also indicates the breadth-protection elicited by the vaccine (Fig. 6F). 3.6. Immunity elicited by the bivalent EV71-CA16 vaccine enables protection against challenge with two viruses with no immunopathological effects To obtain more data on the safety of the bivalent EV71-CA16 vaccine, we observed various characteristics of the immune response in the immunized macaques after a third viral challenge with both EV71 and CA16. Specifically, we aimed to investigate not
only the immunoprotection against both viruses but also the safety of the vaccine with respect to the immunopathological process after combined infection with the two viruses. In this experiment, all the macaques, including the immunized and control animals in group B, were challenged with EV71/CA16 (5 104 CCID50/5 104 CCID50) on day 60 after the second viral challenge and were monitored for clinical symptoms. The results indicated that all the immunized macaques were asymptomatic, similar to the findings after the first and second challenges (Fig. S5C and S5D). In contrast, all the controls presented various clinical manifestations, particularly typical vesicles in the oral mucosa and limbs (Fig. S5C and S5D), which might reflect the inability of macaques to control CA16 reinfection observed in our previous work [13]. Detection of the viral load in the blood using EV71- and CA16-specific primers revealed that all the immunized macaques were negative for both viral genomes (Fig. 7A), whereas all the control macaques
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 6. The bivalent EV71-CA16 vaccine enables broad protection against EV71 and CA16 challenge in rhesus macaques. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from animals in group B. The samples were collected every two days for 10 days after challenge. The neutralization of EV71 and CA16 by serum samples from control and immunized animals collected on day 28 after the second challenge was tested (F). ‘‘EV711st-CA162nd” indicates that EV71 was used in the first challenge and CA16 was used in the second challenge. ‘‘CA161st-EV712nd” indicates that CA16 was used in the first challenge and EV71 was used in the second challenge.
showed positive results for the CA16 genome (Fig. 7A). Similar trends were obtained from the analysis of viral shedding in oral, nasal, urine and fecal samples (Fig. 7B–E). On days 4 and 8 after the third infection, two immunized animals and two controls were sacrificed under anesthesia to observe the pathological changes and variations in the levels of inflammatory factors in the blood. Histopathological staining of the main organs from the immunized macaques did not reveal specific inflammatory pathological lesions related to challenge with the two viruses (Table S5). Slight changes in the levels of inflammatory factors in blood samples collected on days 4 and 8 after the third challenge were regarded as signs of immune homeostasis in these individuals (Fig. 7F and G). The neutralizing antibody assay also indicated increasing titers (Fig. 7H). All these data support the conclusion that the bivalent EV71CA16 vaccine possesses value for further development.
4. Discussion Although the inactivated EV71 vaccine has been administered to children [26–28], no CA16 vaccine or EV71-CA16 combined vaccine is available for clinical trials [29–32]. Our previous work revealed that compared with EV71, CA16 stimulates weaker expression of some important immune signaling molecules in CD11+ dendritic cells [15], which means that immunization with the CA16 vaccine using the traditional protocol might not easily elicit effective immunity [8]. Therefore, a new design was proposed based on the following facts: (1) EV71 and CA16 present the same structural and biologic characteristics [33], (2) an EV71 inactivated
antigen is capable of activating innate immunity in a nonspecific manner [17], and (3) no interference effect between specific immunity against both viruses has been observed [8]. We hypothesized that immunization with the bivalent EV71-CA16 vaccine via the intradermal route could activate innate immunity and lead to effective adaptive immunity against the two viruses, and this hypothesis is partly supported by the application of IPV through the intradermal route, as recommended by the WHO [34]. This design was confirmed in our previous study using a mouse model [17]. In particular, our tentative investigation suggested that immunization with this bivalent vaccine via the muscular route elicits inefficient immune protection against CA16 challenge in rhesus macaques, which is likely due to incomplete stimulation of the CA16 antigen in innate immunity due to its inoculation via the muscular route [17]. In this study, based on our previous data on validity of macaques as a model for EV71 and CA16 infection, our design was extended through a study of the immunity elicited by the vaccine via the intradermal route, with a particular focus on the immune-mediated protection obtained in rhesus macaques against challenge with both viruses [11,13]. This study reveals that although the CA16 inactivated vaccine induces weaker immunogenicity, its association with the EV71 antigen results in increased immunogenicity that enables elicitation of protective immunity in macaques. This finding was likely obtained due to the activation of innate immunity by the combination of both antigens via the intradermal route, which leads to effective stimulation of the T cell response depending on the systematic response of innate immune cells in epithelial tissue. Our detection of the mRNA profile of some immune-regulatory signal-
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 7. The bivalent EV71-CA16 vaccine protects rhesus macaques against challenge with a mixture of EV71 and CA16. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from animals in group B. The samples were collected every two days for 10 days after challenge. The levels of the inflammatory factors IL-6 (F) and TNF-a (G) were detected at 0, 4 and 8 days after infection with a mixture of EV71 and CA16. The EV71- and CA16-neutralizing ability of serum samples from the control and immunized animals collected on days 4 and 8 after infection with a mixture of EV71 and CA16 was assessed (H). ‘‘(EV711st*CA162nd**)3rd***” indicates that EV71 was used in the first challenge, CA16 was used in the second challenge, and a mixture of EV71 and CA16 was used in the third challenge. ‘‘(CA161st*-EV712nd**)3rd***” indicates that CA16 was used in the first challenge, EV71 was used in the second challenge, and a mixture of EV71 and CA16 was used in the third challenge. The statistical significance was assessed using unpaired t-tests (*p < 0.01).
ing molecules in inoculated local skin suggests the activation of innate immune cells, which is the first step in the process of antigen stimulation of the immune system after vaccination [35]. These results further suggest that macaques also provide data
regarding the activation of innate immunity in intradermal tissues [17] and that the immunity induced by the bivalent vaccine via this route does exert a systemic protective effect, which enables resistance to infection with either or both of the viruses by controlling
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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their proliferation in vivo. Therefore, no obvious clinical manifestation was observed in any of the immunized macaques after three viral challenges. Importantly, although slight nonspecific infiltration of inflammatory cells was observed in some organs, including the nervous, lymphoid and spleen tissues, after viral challenge, no available evidence links this effect to any possible immunopathological process. All of the data obtained in this and previous studies suggest the potential for the development of this bivalent EV71CA16 vaccine. However, further studies on the mechanism and safety of this vaccine are needed. 5. Conclusions The bivalent EV71-CA16 inactivated vaccine enables the stimulation of specific immunity and protective effects against EV71 and CA16 challenge in rhesus macaques. Funding This study was founded by the PUMC Youth Fund, Fundamental Research Funds for the Central Universities (3332016115), the CAMS Initiative for Innovative Medicine (2016-I2M-1-019), the State Project for Essential Drug Research and Development (2017ZX09307013-2 and 2016ZX09101120-2 and 2018ZX09737011-002), the National Science and Technology Major Project (2018ZX10101001), the National Natural Science Foundation (31700931), and funds from the Yunnan Key Laboratory of Vaccine Research & Development in Severe Infectious Diseases and Major Science and Technology Special Projects of Yunnan Province (2019FB101, 2017ZF020 and 2017FB018). This study is also supported by Technical innovation talents of Yunnan Province and Medical Reserve Talents plan of Yunnan health and Health Committee (H-2017035). This study was also supported by Aimei Convac BioPharm (Jiangsu) Co., Ltd. CRediT authorship contribution statement S.F., Y.L., and W.C. performed the experiments. G.J. and X.X. analyzed the data. S.F. and L.J. generated the figures and tables. Y.L. and Y.Z. performed the q-PCR and ELISpot analyses. E.Y., M.F. and L.W. performed the neutralizing antibody assays. S.F. and Q.L. designed the study and wrote the paper. All authors read and approved the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We thank Qinfang Jiang, Yun Li and Kaili Ma for the assistance provided with the animal handling and Qingling Wang and Dong Shen for analyzing the pathological observations. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material The following are available online at www.mdpi.com/xxx/s1, Fig. S1: Schematic depicting the preliminary experimental process; Fig. S2: A specific immune response via the muscular route and
clinical symptoms after viral challenge; Fig. S3: Gene expression levels in PBMCs; Fig. S4: Clinical symptoms of the immunized and control animals after the EV71 or CA16 challenge; Fig. S5: Clinical symptoms of immunized and control animals after viral challenge; Table S1: Primers and probes used for detection of EV71 and CA16 antigens; Table S2: Primers used for quantitative RTPCR in this study; Table S3: Primers used in the RT-PCR assays performed for validation; Table S4: Pathological changes observed in control and immunized macaques after viral challenge; Table S5: Pathological changes observed in control and immunized macaques after mixed viral challenge. Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.12. 057. References [1] Esposito S, Principi N. Hand, foot and mouth disease: Current knowledge on clinical manifestations, epidemiology, aetiology and prevention. 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Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine Shengtao Fan a, Yun Liao a, Guorun Jiang a, Li Jiang b, Lichun Wang a, Xingli Xu a, Min Feng a, Erxia Yang b, Ying Zhang a, Wei Cui b, Qihan Li a,⇑ a Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming 650118, China b Aimei Convac BioPharm (Jiangsu) Co., Ltd., Taizhou 225300, Jiangsu, China
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Article history: Received 31 July 2019 Received in revised form 28 November 2019 Accepted 19 December 2019 Available online xxxx Keywords: Enterovirus type 71 (EV71) Coxsackievirus A 16 (CA16) Bivalent vaccine Rhesus macaques
a b s t r a c t Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are recognized as the major pathogens responsible for human hand-foot-mouth disease. To develop a bivalent EV71-CA16 vaccine, rhesus macaques immunized with two doses of this vaccine via the intradermal route were challenged with EV71 or CA16, and their clinical symptoms, viral shedding, neutralizing antibodies, IFN-c-specific ELISpots, and tissue viral load were examined longitudinally. Specific immunity against EV71 and CA16 was observed in the macaques, which exhibited controlled proliferation of the EV71 and CA16 viruses and upregulated expression of immune-related genes compared with the controls. Furthermore, broad protection against EV71 and CA16 challenge without immunopathological effects was observed in all the immunized macaques. These studies suggest that the bivalent EV71-CA16 inactivated vaccine was effective against wildtype EV71 or CA16 viral challenge in rhesus macaques. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Numerous epidemics of human hand-foot-mouth disease (HFMD), which is a viral infectious disease, have occurred in children, and due to its clinical pathologic outcomes, HFMD has become a concern for many families [1–5]. The identified pathogenic agents of this disease include enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16), which are responsible for severe neurogenic cases and deaths [6,7]. These two viruses have similar capsid structures and present comparable biological characteristics but exhibit no cross-reactivity in serological tests [8]. In 2015, the first EV71 inactivated vaccine was licensed and applied in a child population [9–12]. However, no CA16 vaccine has been developed for use in clinical trials because evaluation of the immunized rhesus model after challenge with the wild-type virus failed to provide evidence of clinically protective immunity [8,13], even though this test was positive for the EV71 vaccine [9]. We previously investigated this issue and reported several notable findings. First, compared with the VP1 protein of EV71, which is expressed in epithelial cells, the CA16 protein might not function as a viral ⇑ Corresponding author. E-mail address: [email protected] (Q. Li).
pathogen-associated molecular pattern (PAMP) by interacting with cellular innate immune molecules, such as pattern recognition receptors (PRR), to induce further activation of NF-ƙB and thus the transcription of immune-related signaling molecules [14]. Second, CA16, similarly to EV71, is capable of infecting CD11+ dendritic cells (DCs) and modulating their immunological phenotype [13,15] but provides a weaker stimulus to induce the expression of various immune-related signaling molecules in infected DCs than EV71 [16]. In this case, augmenting the immunogenicity of the CA16 antigen might determine not only the design of the vaccine but also the most effective immunization procedure. Based on this hypothesis and our knowledge of innate and adaptive immunity, particularly the activation of T cells by DCs and other cells [15,17], an innate immune signal should be transferred to T cells based on the systemic process integrating antigen presentation with the signaling network composed of various cytokines [18]. In the present study, we designed a bivalent EV71-CA16 inactivated vaccine for the immunization of animals through the intradermal route, and its administration was expected to lead to integrated activation of innate immunity and the initiation of a specific T cell response and to provide effective clinical protection during viral infection. This hypothesis was investigated using mice and was confirmed by the induction of a favorable immune
https://doi.org/10.1016/j.vaccine.2019.12.057 0264-410X/Ó 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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response, which showed the production of specific neutralizing antibodies and cytotoxic T lymphocytes (CTLs), as well as a protective effect on adult and postnatal mice challenged with the viruses [17]. However, the limitations of the mouse model resulted in a failed pathogenic process of CA16 infection; thus, rhesus macaques, which exhibit sensitivity to CA16 infection and a close genetic relationship to humans [13,19], were used in this study. Our description focused on the immune response induced in rhesus macaques immunized with a bivalent EV71-CA16 inactivated vaccine via the intradermal route and the clinical efficacy of immune protection provided by the vaccine against challenge with the EV71 and/or CA16 wild-type strains. All data obtained in this study indicate that this vaccine exhibits the efficacy and safety required for a clinical trial.
2. Materials and methods 2.1. Study design Prior to the formal experiment, preliminary immunological observations of this bivalent EV71-CA16 vaccine was obtained from four rhesus macaques immunized via the muscular route; in this preliminary analysis, neutralizing antibodies and specific CTLs against EV71 and CA16 were first detected 28 days after the administration of two immunization doses at an interval of 4 weeks (each dose contained 100 U/100 U of EV71/CA16 inactivated vaccine antigens and 0.25 mg of aluminum in 0.5 ml of buffer; Fig. S1). At day 56, each two this bivalent vaccine immunized
macaques were challenged with the wild-type EV71 or CA16 strain, respectively, and were then clinically monitored for pathological manifestations, particularly vesicles in oral and/or limb skin (Fig. S1). At days 4 and 8 post-challenge, all the macaques were euthanized for viral load detection and histopathological observation, respectively (Fig. S1). Based on preliminary data, the following formal experiment was designed: 16 rhesus macaques aged 6–12 months were divided into groups A and B, with eight animals in each group. All the macaques were isolated for 2 weeks and confirmed to be negative for anti-EV71/CA16 antibodies. Four macaques in each group served as a negative control for viral challenge, and the other four were immunized with the bivalent EV71-CA16 vaccine via the intradermal route at the same time on days 0 and 28. Briefly, the back skin of the rhesus monkeys was shaved, and a MicronJet 600 needle (NanoPass Technologies, Ltd.) was used for vaccine delivery according to the manufacturer’s instructions. Each dose vaccine contained 100 U/100 U EV71/CA16 antigens and 0.125 mg of aluminum adjuvant in 250 ml of buffer. At 12 and 24 h after the first inoculation, the animals were administered topical anesthesia, and a minimal sample of local skin tissue was collected for detection of the gene profile. On days 28 and 56 after immunization, blood samples were collected from all the macaques for neutralizing antibody and IFN-c-specific enzymelinked immunospot (ELISpot) assays (Fig. 1). All the animals in group A were subjected to viral challenge using the following protocol: two immunized animals and two control animals were challenged with the wild-type EV71 strain (FY23, 5 104 CCID50 for each animal), and the other two were challenged with the wildtype CA16 strain (G20, 5 104 CCID50 for each animal; Fig. 1).
Fig. 1. Schematic depicting the experimental process. Four macaques from groups A (#18005, #18001, #18007 and #18009) and B (#18181, #18027, #18021 and #18025) were immunized with the EV71-CA16 vaccine. The control macaques were treated with aluminum adjuvant alone. On days 28 and 56, serum samples from all the immunized animals were collected for neutralizing antibody assays and IFN-c-specific ELISpot assays. On day 60, two immunized and two control macaques from groups A and B were challenged with wild-type EV71 or CA16. All the challenged animals from group A were observed for clinical symptoms and sacrificed on day 64 or 68 for further analysis. On day 100, the macaques in group B that were initially challenged with EV71 were challenged with CA16, whereas the macaques in group B that were initially challenged with CA16 were challenged with EV71. On day 160, all the macaques from group B were challenged with a combination of EV71 and CA16, and the immunized and control animals were sacrificed on day 164 or 168 for further analysis.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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All the challenged animals were observed for clinical symptoms. Each immunized and control animal infected with EV71 or CA16 was sacrificed under anesthesia on days 4 and 8 after challenge for further analyses (Fig. 1). The immunized macaques from group B were challenged with the two viruses using the same protocol and monitored for clinical symptoms (Fig. 1). On day 40 after the first viral challenge, a second viral challenge was administered using a crisscross approach: the animals previously challenged with EV71 were now challenged with the wild-type CA16 strain, and the animals previously challenged with CA16 were now challenged with the wild-type EV71 strain. The two control animals in group B were infected with EV71 or CA16. The clinical symptoms were monitored for 10 days after the second challenge. On day 60 after the second challenge, all immunized and control animals were challenged with a mixture of the wild-type EV71 and CA16 strains (5 104 CCID50/5 104 CCID50 for each animal), and the symptoms of all the animals were monitored. On days 4 and 8 after the last challenge, all the immunized and control animals infected with EV71 or CA16 were sacrificed under anesthesia, and samples of various tissues were collected for pathological detection. 2.2. Ethics statement The rhesus macaque experiments were designed based on the Guidelines for the Care and Use of Laboratory Animals from the National Research Council of the National Academies and the Guidance for Experimental Animal Welfare and Ethical Treatment by the Ministry of Science and Technology of the People’s Republic of China (2006). The animal experimental protocols were approved by The Yunnan Provincial Experimental Animal Management Association (approval No. SYXK [Dian] K 2015-0006) and the institutional Experimental Animal Ethics Committee (approval No. DWSP201803013-2). 2.3. Animal administration and care Rhesus macaques obtained from the Primate Research Center of the Institute of Medical Biology (IMB) and the Chinese Academy of Medicine Science (CAMS) were housed in cages (BSL-2 conditions) with an ambient temperature of 25 °C, provided adequate water and food, and received appropriate veterinarian care daily. 2.4. Viruses and cells The FY23 (EV71), G20 (CA16) and KM/M08 (CA16) strains were used in this study. Human diploid cells (KMB-17 strain) and Vero cells were used for viral proliferation and titration, respectively. 2.5. Production of the bivalent EV71-CA16 inactivated vaccine and quality standards The FY23 strain of EV71 and KM/M08 strain of CA16 were inoculated into KMB-17 cells grown in cell factory, respectively. The virus was harvested once 80% typical cytopathic effects (CPEs) observed. The virus stock was inactivated by incubation with a 1:4000 dilution of formaldehyde at 37 °C for 72 h and then concentrated 50-fold through ultrafiltration. The viral protein was purified by Sepharose 6 Fast Flow (Amersham, USA) chromatography [20]. The viral purity was assessed by RP-HPLC according to the manufacturer’s recommended protocol (Waters, USA). Equal amounts of the purified EV71 and CA16 antigens were emulsified in 0.5 mg/ml Al(OH)3 adjuvant. EV71 and CA16 standard antigens (Lot.201507001) were purchased from National Institutes for Food and Drug Control as references. The antigen content was determined by ELISAs [21].
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2.6. Detection of the viral load by q-RT-PCR Various tissues collected from the immunized macaques were homogenized using a TissueLyser II system (Qiagen, Hilden, Germany). Swab and blood samples were continuously collected from the infected macaques for 14 days postchallenge. The viral RNA from the tissue and swab samples was extracted using the Simply P Total RNA Extraction kit according to the manufacturer’s recommended protocol. The q-RT-PCR was performed using the One Step PrimeScript RT-PCR Kit with a CFX Connect real-time system (BioRad, USA). The primers and probes for EV71 and CA16 are shown in Table S1. 2.7. Expression profile of the mRNAs encoding immune-related signaling molecules in local skin tissues obtained from the inoculation site Total RNA from the inoculated skin tissues of the macaques collected 12 and 24 h after immunization was extracted using the TRIzol-A+ Reagent (Cat # DP421, Tiangen, China), according to the manufacturer’s recommended protocol. The RNA was amplified using the One Step TB GreenTM Prime ScriptTM PLUS RT-PCR Kit (Cat # RR096A, TaKaRa, Japan). The primers used to quantify the LIGHT, IKKa, IKKb, CD160, TLIA, NIK, IFN-a, IFN-b, IFN-k, GITRL, 4-IBBL, IL5, IL-13 and IL-22 mRNA levels are shown in Table S2. 2.8. Expression profile of mRNAs in PBMCs from immunized macaques after challenge PBMCs were collected from the macaques 4 days after viral challenge, and total RNA from the PBMCs was extracted and purified using the miRNeasy Mini Kit (Cat # 217004, QIAGEN, GmbH, Germany) according to the manufacturer’s instructions. The RNA integrity was assessed by determining the RIN number using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). mRNA sequencing libraries were generated from the PBMC samples using the mRNA Sample Pre Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s recommended protocol. HiSeq/MiSeq was used for sequencing. Based on the expression of 63 genes, 16 genes were randomly selected to validate the expression profiles by q-RT-PCR using a CFX Connect real-time system (Bio-Rad, USA). The primers used to validate these results are shown in Table S3. 2.9. Neutralizing antibody assay The macaque serum samples were subjected to 2-fold serial dilutions and incubated with 100 TCID50 of the FY23 strain (EV71) or KM/M08 strain (CA16) for 1 h at 37 °C. The samples were then inoculated into Vero cells and incubated at 37 °C in a 5% CO2 incubator for CPE assessment. The end-point neutralization titers were defined through 50% plaque reduction assays. 2.10. ELISpot assays and ELISA An ELISpot assay was performed according to the manufacturer’s instructions (Cat # 3421 M-4HPW-2, Mabtech, Sweden). Briefly, PBMCs were collected from immunized macaques, and EV71 or CA16 antigen (20 U/well) was added to the PBMC suspension (5 105 cells/well) in a 96-well plate. The mixture of antigen and cells was incubated at 37 °C in a 5% CO2 atmosphere for 36 h. The plate was then washed four times with PBS and sequentially incubated with primary antibody, secondary antibody and substrate solution. The spots were counted using an automated ELISpot reader (CTL, OH, USA). Colorimetric sandwich ELISA kits were used to quantify the serum IL-6 and TNF-a levels. Briefly,
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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diluted serum and standard samples were added to microwell plates that had been precoated with specific antibody. After washing, secondary antibody was added to detect the captured primary antibody. Horseradish peroxidase (HRP)-conjugated antibody and tetramethyl-benzidine (TMB) reagent were sequentially added for signal development. The stop solution was then added, and the absorbance at a wavelength of 450 nm was read using a microplate reader.
2.11. Histopathological observations Tissues from euthanized macaques were fixed with 10% formaldehyde for 7 days, dehydrated with ethanol, cleared with
xylene, embedded in paraffin and then cut into 4-lm-thick sections for hematoxylin and eosin (HE) staining. The pathological changes were examined using a light microscope (ECLIPSE Ti-s, Nikon).
2.12. Evaluation of clinical symptoms The clinical symptoms of the rhesus macaques, including their mental state, temperature changes, and the presence of vesicular lesions on the lips, hands and feet, were examined after challenge. If symptoms were observed during the observation period, the following clinical scoring criteria were employed: 0, asymptomatic; 1, redness; 2, lesion; 3, bullae; 4, multiple bullae; and 5, ulceration.
Fig. 2. A specific immune response is induced in rhesus macaques by the bivalent EV71-CA16 vaccine. (A) The neutralization of EV71 and CA16 by serum samples from control and immunized animals collected on days 28 and 56 was tested. (B) Levels of IFN-c secreted from PBMCs from all macaques stimulated with the EV71 and CA16 antigens on days 28 and 56. (C) The LIGHT, NIK, CD160, TLIA IKKa, IKKb, (D) GITRL, 4-IBBL, (E) IFN-a, IFN-b, IFN-k, (F) IL-5, IL-13 and IL-22 mRNA levels in samples collected from the site of inoculation in the immunized macaques were detected by q-RT-PCR. All the data obtained from the control group served as reference values for calculating the relative fold changes. The statistical significance was assessed using unpaired t-tests (***p < 0.001).
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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2.13. Statistical analysis The data are presented as the means ± SDs. The differences between two groups were evaluated using Student’s t-tests (GraphPad Prism software; GraphPad Software, San Diego, CA, USA), and p < 0.05 was regarded as indicating a significant difference.
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ilar to previous data [4,8]. This finding demonstrates the immune response induced by the bivalent EV71-CA16 vaccine in macaques. The mRNA profiles of some innate immune-related signaling molecules in the local tissue 12 and 24 h after vaccine inoculation suggest the activation of innate immunity (Fig. 2C–F), consistent with the upregulation of some molecules in related signaling pathways [22,23].
3. Results 3.1. Bivalent EV71-CA16 inactivated vaccine enables stimulation of specific immunity against EV71 and CA16 A preliminary experiment involving immunization via the muscular route suggested inefficient and efficient immune responses against CA16 and EV71 challenge, respectively, in macaques (Fig. S2A); specifically, a no clinical protective effect was detected in the animals challenged with CA16, which presented various clinical pathological features, including oral vesicles and viremia (Fig. S2B), whereas complete control of the EV71 challenge was observed (Fig. S2B). Based on these results combined with data from a previous mouse experiment [17], a bivalent EV71-CA16 inactivated vaccine was designed for the immunization of rhesus macaques in groups A and B based on the schematic presented in Fig. 1. The immunization induced the production of neutralizing antibodies against EV71 and CA16, and lower and higher titers were observed on day 28 after the first and second immunizations, respectively (Fig. 2A). The titers of CA16-neutralizing antibodies were lower than those of EV71-neutralizing antibodies, as observed in a previous study [8]. ELISpot detection of IFN-c suggested the generation of specific CTL responses to EV71 and CA16 antigens on days 28 and 56 after immunization (Fig. 2B), sim-
3.2. Clinical immunoprotective effect induced by the bivalent EV71CA16 inactivated vaccine after viral challenge To investigate the efficacy of the CA16 inactivated vaccine in macaques [19], the validity of the bivalent EV71-CA16 vaccine was assessed based on clinical pathological observations from immunized macaques after challenge with the two viruses. Our study was designed to first identify the clinical symptoms of the immunized animals in groups A and B after challenge with the two viruses compared with the control animals. All immunized macaques in groups A and B were asymptomatic after challenge with either EV71 or CA16 (Fig. S3), whereas the control animals in both groups showed typical vesicles in the oral mucosa and skin of the limbs as well as body temperature variations after both challenges (Fig. S3). Importantly, the two viral challenges led to typical viremia in the control animals in both groups, with a peak at approximately day 6 (Fig. 3A), but the immunized animals consistently showed negative results (Fig. 3A). The detection of viral shedding in the mouth, nose, urine and feces revealed generally similar trends (Fig. 3B–E). Specifically, little or no trace of the virus was detected in the immunized macaques, whereas virus was detected in the control animals; in addition, the copies of the EV71 genome in the oral cavity showed slight increases at day 8,
Fig. 3. Immunoprotective effect of the bivalent EV71-CA16 vaccine on rhesus macaques after viral challenge. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from the animals in group A. The samples were collected every two days for 8 days after challenge.
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 4. The bivalent EV71-CA16 vaccine controls EV71 or CA16 proliferation in rhesus macaques. Detection of the viral load in the main organs (A), nervous tissues (B), intestinal tissues (C) and lymph nodes (D) of the macaques in group A on days 4 and 8 after challenge with wild-type EV71. Detection of the viral load in the main organs (E), nervous tissues (F), intestinal tissues (G) and lymph nodes (H) of the macaques in group A on days 4 and 8 after challenge with wild-type CA16.
but this increase might be due to the q-RT-PCR detection limit (Fig. 3B–E). These data suggest that immunization with the bivalent vaccine via the intradermal route leads to a clinical immunoprotective effect against challenge with either EV71 or CA16. 3.3. Immunity induced by the bivalent EV71-CA16 vaccine enables control of the proliferation of both viruses in vivo To better assess the protective efficacy of this vaccine, the viral load was detected by measuring the EV71 and CA16 genomes in various tissues collected from the euthanized macaques, including both the immunized and control individuals in group A on days 4 and 8 after viral challenge. Lower or even trace viral loads were detected in the organs of all immunized macaques 4 and 8 days challenged with either EV71 or CA16, whereas higher loads were found in the organs of the euthanized control animals (Fig. 4A and E). Further experiments revealed similar results in the nervous tissues (Fig. 4B and F), intestinal tissues (Fig. 4C and G) and lymph nodes of various tissues (Fig. 4D and H). Pathological observations of these tissues revealed few nonspecific inflammatory sites in most tissues from the immunized macaques (Table S4) and less inflammatory pathological changes or lesions in the infected control animals, similar to the results obtained in our previous study [9,13]. These results suggest that the EV71-CA16 vaccine can induce protective immunity that can enable inhibition of both the proliferation of infecting EV71 or CA16 and the pathological process associated with clinical outcomes. 3.4. Challenge upregulates mRNAs related to immune regulation in immunized macaques compared with controls In our previous work, the mRNA profiles in immunized individuals were used for the evaluation of immunity [10], and this reference indicator reflects the systemic process of generating an
immune response and the associated homeostasis of the inflammatory reaction [24,25]. Here, PBMCs from the immunized and control macaques in group A were collected at day 4 after viral challenge to analyze the mRNA profile related to the immune response. Lower expression of mRNAs related to immune regulation was observed in the CA16- compared with the EV71infected controls (Fig. 5A–C). However, the expression of many mRNAs related to immune regulation and signal transduction showed a larger fold change in the immunized macaques after challenge with either EV71 or CA16 (Fig. 5D–F). Comparisons of the data presented in Fig. 5A–F revealed that immunization with the bivalent vaccine elicits an immune response that is capable of systemically and effectively combating viral infection, particularly CA16 infection. The expression levels of 16 selected genes were identified using q-RT-PCR with their specific primers to be consistent with the results shown in the heat map (Fig. S4).
3.5. Immunogenicity induced by the bivalent EV71-CA16 vaccine enables breadth protection against EV71 and CA16 infection The designed bivalent EV71-CA16 vaccine aims to induce immunity capable of resisting EV71 or/and CA16 infection. In this study, a second crisscross viral challenge was administered to the immunized and control macaques in group B at day 40 after the first challenge. At this stage, the immunized and control macaques previously challenged with EV71 were now infected with CA16, and those that were first challenged with CA16 were now infected with EV71. Within 10 days after the second challenge, all the control animals showed vesicles in the oral mucosa and limbs, variations in body temperature, and other signs similar to those observed in the control animals in group A (Fig. S5A and S5B), but none of the immunized macaques presented these signs (Fig. S5A and S5B). Importantly, detection of the viral load in the blood over the 10 days postchallenge revealed no viremia in any
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 5. mRNA expression profiles in skin samples from rhesus macaques collected on day 4 after viral challenge. Expression of mRNAs related to the DC surface signature (A), chemokines, interferon regulatory factor (B), interleukins, tumor necrosis factor, and TNF receptor-associated factor (C) in the EV71-challenged and CA16-challenged control groups. Expression of mRNAs related to the DC surface signature (D), chemokines, interferon regulatory factor (E), interleukins, tumor necrosis factor, and TNF receptor-associated factor (F) in the EV71-challenged and CA16-challenged immunized groups. Each row indicates one gene. The values are presented as log2-fold changes. All the data from normal rhesus macaques served as reference values for calculating the relative fold changes.
of the immunized macaques and typical viremia, with a peak at day 4, in all the control animals (Fig. 6A). Similar trends were obtained from the detection of viral shedding in the mouth, nose, urine and feces (Fig. 6B–E). The increased titer of neutralizing antibodies against both viruses in the immunized macaques observed at day 10 after the second cross-challenge also indicates the breadth-protection elicited by the vaccine (Fig. 6F). 3.6. Immunity elicited by the bivalent EV71-CA16 vaccine enables protection against challenge with two viruses with no immunopathological effects To obtain more data on the safety of the bivalent EV71-CA16 vaccine, we observed various characteristics of the immune response in the immunized macaques after a third viral challenge with both EV71 and CA16. Specifically, we aimed to investigate not
only the immunoprotection against both viruses but also the safety of the vaccine with respect to the immunopathological process after combined infection with the two viruses. In this experiment, all the macaques, including the immunized and control animals in group B, were challenged with EV71/CA16 (5 104 CCID50/5 104 CCID50) on day 60 after the second viral challenge and were monitored for clinical symptoms. The results indicated that all the immunized macaques were asymptomatic, similar to the findings after the first and second challenges (Fig. S5C and S5D). In contrast, all the controls presented various clinical manifestations, particularly typical vesicles in the oral mucosa and limbs (Fig. S5C and S5D), which might reflect the inability of macaques to control CA16 reinfection observed in our previous work [13]. Detection of the viral load in the blood using EV71- and CA16-specific primers revealed that all the immunized macaques were negative for both viral genomes (Fig. 7A), whereas all the control macaques
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 6. The bivalent EV71-CA16 vaccine enables broad protection against EV71 and CA16 challenge in rhesus macaques. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from animals in group B. The samples were collected every two days for 10 days after challenge. The neutralization of EV71 and CA16 by serum samples from control and immunized animals collected on day 28 after the second challenge was tested (F). ‘‘EV711st-CA162nd” indicates that EV71 was used in the first challenge and CA16 was used in the second challenge. ‘‘CA161st-EV712nd” indicates that CA16 was used in the first challenge and EV71 was used in the second challenge.
showed positive results for the CA16 genome (Fig. 7A). Similar trends were obtained from the analysis of viral shedding in oral, nasal, urine and fecal samples (Fig. 7B–E). On days 4 and 8 after the third infection, two immunized animals and two controls were sacrificed under anesthesia to observe the pathological changes and variations in the levels of inflammatory factors in the blood. Histopathological staining of the main organs from the immunized macaques did not reveal specific inflammatory pathological lesions related to challenge with the two viruses (Table S5). Slight changes in the levels of inflammatory factors in blood samples collected on days 4 and 8 after the third challenge were regarded as signs of immune homeostasis in these individuals (Fig. 7F and G). The neutralizing antibody assay also indicated increasing titers (Fig. 7H). All these data support the conclusion that the bivalent EV71CA16 vaccine possesses value for further development.
4. Discussion Although the inactivated EV71 vaccine has been administered to children [26–28], no CA16 vaccine or EV71-CA16 combined vaccine is available for clinical trials [29–32]. Our previous work revealed that compared with EV71, CA16 stimulates weaker expression of some important immune signaling molecules in CD11+ dendritic cells [15], which means that immunization with the CA16 vaccine using the traditional protocol might not easily elicit effective immunity [8]. Therefore, a new design was proposed based on the following facts: (1) EV71 and CA16 present the same structural and biologic characteristics [33], (2) an EV71 inactivated
antigen is capable of activating innate immunity in a nonspecific manner [17], and (3) no interference effect between specific immunity against both viruses has been observed [8]. We hypothesized that immunization with the bivalent EV71-CA16 vaccine via the intradermal route could activate innate immunity and lead to effective adaptive immunity against the two viruses, and this hypothesis is partly supported by the application of IPV through the intradermal route, as recommended by the WHO [34]. This design was confirmed in our previous study using a mouse model [17]. In particular, our tentative investigation suggested that immunization with this bivalent vaccine via the muscular route elicits inefficient immune protection against CA16 challenge in rhesus macaques, which is likely due to incomplete stimulation of the CA16 antigen in innate immunity due to its inoculation via the muscular route [17]. In this study, based on our previous data on validity of macaques as a model for EV71 and CA16 infection, our design was extended through a study of the immunity elicited by the vaccine via the intradermal route, with a particular focus on the immune-mediated protection obtained in rhesus macaques against challenge with both viruses [11,13]. This study reveals that although the CA16 inactivated vaccine induces weaker immunogenicity, its association with the EV71 antigen results in increased immunogenicity that enables elicitation of protective immunity in macaques. This finding was likely obtained due to the activation of innate immunity by the combination of both antigens via the intradermal route, which leads to effective stimulation of the T cell response depending on the systematic response of innate immune cells in epithelial tissue. Our detection of the mRNA profile of some immune-regulatory signal-
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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Fig. 7. The bivalent EV71-CA16 vaccine protects rhesus macaques against challenge with a mixture of EV71 and CA16. Detection of viral shedding in blood samples (A), oral swabs (B), nasal swabs (C), urine (D) and feces (E) from animals in group B. The samples were collected every two days for 10 days after challenge. The levels of the inflammatory factors IL-6 (F) and TNF-a (G) were detected at 0, 4 and 8 days after infection with a mixture of EV71 and CA16. The EV71- and CA16-neutralizing ability of serum samples from the control and immunized animals collected on days 4 and 8 after infection with a mixture of EV71 and CA16 was assessed (H). ‘‘(EV711st*CA162nd**)3rd***” indicates that EV71 was used in the first challenge, CA16 was used in the second challenge, and a mixture of EV71 and CA16 was used in the third challenge. ‘‘(CA161st*-EV712nd**)3rd***” indicates that CA16 was used in the first challenge, EV71 was used in the second challenge, and a mixture of EV71 and CA16 was used in the third challenge. The statistical significance was assessed using unpaired t-tests (*p < 0.01).
ing molecules in inoculated local skin suggests the activation of innate immune cells, which is the first step in the process of antigen stimulation of the immune system after vaccination [35]. These results further suggest that macaques also provide data
regarding the activation of innate immunity in intradermal tissues [17] and that the immunity induced by the bivalent vaccine via this route does exert a systemic protective effect, which enables resistance to infection with either or both of the viruses by controlling
Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057
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their proliferation in vivo. Therefore, no obvious clinical manifestation was observed in any of the immunized macaques after three viral challenges. Importantly, although slight nonspecific infiltration of inflammatory cells was observed in some organs, including the nervous, lymphoid and spleen tissues, after viral challenge, no available evidence links this effect to any possible immunopathological process. All of the data obtained in this and previous studies suggest the potential for the development of this bivalent EV71CA16 vaccine. However, further studies on the mechanism and safety of this vaccine are needed. 5. Conclusions The bivalent EV71-CA16 inactivated vaccine enables the stimulation of specific immunity and protective effects against EV71 and CA16 challenge in rhesus macaques. Funding This study was founded by the PUMC Youth Fund, Fundamental Research Funds for the Central Universities (3332016115), the CAMS Initiative for Innovative Medicine (2016-I2M-1-019), the State Project for Essential Drug Research and Development (2017ZX09307013-2 and 2016ZX09101120-2 and 2018ZX09737011-002), the National Science and Technology Major Project (2018ZX10101001), the National Natural Science Foundation (31700931), and funds from the Yunnan Key Laboratory of Vaccine Research & Development in Severe Infectious Diseases and Major Science and Technology Special Projects of Yunnan Province (2019FB101, 2017ZF020 and 2017FB018). This study is also supported by Technical innovation talents of Yunnan Province and Medical Reserve Talents plan of Yunnan health and Health Committee (H-2017035). This study was also supported by Aimei Convac BioPharm (Jiangsu) Co., Ltd. CRediT authorship contribution statement S.F., Y.L., and W.C. performed the experiments. G.J. and X.X. analyzed the data. S.F. and L.J. generated the figures and tables. Y.L. and Y.Z. performed the q-PCR and ELISpot analyses. E.Y., M.F. and L.W. performed the neutralizing antibody assays. S.F. and Q.L. designed the study and wrote the paper. All authors read and approved the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We thank Qinfang Jiang, Yun Li and Kaili Ma for the assistance provided with the animal handling and Qingling Wang and Dong Shen for analyzing the pathological observations. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material The following are available online at www.mdpi.com/xxx/s1, Fig. S1: Schematic depicting the preliminary experimental process; Fig. S2: A specific immune response via the muscular route and
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Please cite this article as: S. Fan, Y. Liao, G. Jiang et al., Study of integrated protective immunity induced in rhesus macaques by the intradermal administration of a bivalent EV71-CA16 inactivated vaccine, Vaccine, https://doi.org/10.1016/j.vaccine.2019.12.057