Nanoencapsulation of inactivated-viral vaccine using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in olive flounder (Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection
Nanoencapsulation of inactivated-viral vaccine using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in olive flounder (Paralichthys olivaceus) against viral haemorrhagic septicaemia virus (VHSV) infection
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DOI：
10.1016/j.fsi.2019.05.017
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发表时间：
2019-08-01
影响因子：
4.7
通讯作者：
Jung, Sung-Ju
中科院分区：
文献类型：
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作者：
Kole, Sajal;Qadiri, Syed Shariq Nazir;Jung, Sung-Ju
病毒性出血性败血症病毒（viral haemorrhagic septicaemia virus，VHSV）是弹状病毒科（Rhabdoviridae）新弹状病毒属（Novirhabdovirus）的一种单链RNA病毒，是引起病毒性出血性败血症（viral haemorrhagic septicaemia virus，VHS）的病原体。以前，我们开发了一种灭活疫苗，即，福尔马林灭活的VHSV与角鲨烯作为佐剂混合，其可有效地赋予针对VHSV的保护性免疫（58-76%相对存活百分比），但施用模式是腹腔内注射，这对于小型鱼种不可行。为了克服这一局限性，我们目前专注于用口服和浸泡途径代替疫苗递送的注射途径。在这种情况下，我们用壳聚糖纳米粒（CNPs-IV）通过油包水（W/O）乳化法包裹灭活的VHSV疫苗。包封后，进行两组体内接种试验，即，初步试验I和最终试验II。在初步试验I中，通过不同的递送策略（包括口服和浸泡途径（单次/加强剂量））用CNP-IV接种橄榄比目鱼鱼种（10.5 +/- 1.7 g），然后用VHSV（1 x 10（6）TCID 50病毒/鱼）攻毒，以评价不同应用的递送策略中的有效方法。随后，进行了最终试验II，以更好地理解所采用的递送策略的功效背后的免疫机制，并进一步改进具有初次加强（初次浸泡和口服加强）组合的递送机制，以改善宿主中的瞬时抗VHSV应答。试验I中RPS分析的评价显示CNPs-IV中RPS更高，分别为46.7%和53.3%（浸泡）和CNPs-IV分别与CNPs-IV（口服）组中的0%RPS和CNPs-IV（口服）组中的20%RPS相比，对照NVC中100%累积死亡率百分比计算时，（口服/口服）组在试验II中，CNPs-IV（浸泡/浸泡）和CNPs-IV（浸泡/经口）组分别获得60%和66.6%的RPS。免疫组的鱼血清、皮肤粘液和肠粘液中特异性（抗VHSV）抗体滴度在免疫后显著增强（p < 0.05）。此外，与对照相比，在免疫后以及攻击后宿主的全身（肾）和粘膜（皮肤和肠）免疫区室中，CNPs-IV免疫的鱼显示出不同免疫基因转录物（IgM、IgT、pIgR、MHC-I、MHC-II、IFN-γ和胱天蛋白酶3）的显著（p < 0.05）上调。综上所述，CNPs-IV疫苗的粘膜免疫可以在橄榄比目鱼中组织针对VHSV的协调免疫应答中协调有效的免疫策略，从而在最小的应激下对宿主表现出更高的保护效力。
Viral haemorrhagic septicaemia virus (VHSV), a (-) ssRNA virus belonging to the genus Novirhabdovirus of rhabdoviridae family, is the aetiological agent of viral haemorrhagic septicaemia (VHS) disease which causes huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. Previously, we developed an inactivated vaccine viz., formalin-inactivated VHSV mixed with squalene as adjuvant which was effective in conferring protective immunity (58-76% relative percentage survival) against VHSV but the mode of administration was intraperitoneal injection which is not feasible for small sized fingerling fish. To overcome this limitation, we presently focused on replacing the injection route of vaccine delivery by oral and immersion routes. In this context, we encapsulated the inactivated VHSV vaccine with chitosan nanoparticles (CNPs-IV) by water-in-oil (W/O) emulsification method. After encapsulation, two sets of in vivo vaccination trials were conducted viz., preliminary trial-I and final trial-II. In preliminary trial-I, olive flounder fingerlings (10.5 +/- 1.7 g) were vaccinated with CNPs-IV by different delivery strategies involving oral and immersion routes (single/booster dose) followed by challenge with VHSV (1 x 10(6) TCID50 virus/fish) to evaluate an effective method amongst different applied delivery strategies. Subsequently, a final trial-II was conducted to better understand the immune mechanism behind the efficacy of the employed delivery strategy and also to further improvise the delivery mechanism with prime boost (primary immersion and oral boosting) combination in order to improve the transient anti-VHSV response in the host. Evaluation of RPS analysis in trial-I revealed higher RPS of 46.7% and 53.3% in the CNPs-IV (immersion) and CNPs-IV (immersion/immersion) groups, respectively compared to 0% RPS in the CNPs-IV (oral) group and 20% RPS in the CNPs-IV (oral/oral) group when calculated against 100% cumulative mortality percentage in the NVC (non-vaccinated challenged) control group, whereas, in the trial-II, RPS of 60% and 66.6% were obtained for CNPs-IV (immersion/immersion) and CNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, CNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-gamma, and Caspase3) compared to control, in both the systemic (kidney) and mucosal (skin and intestine) immune compartments of the host post immunization as well as post challenge. To conclude, mucosal immunization with CNPs-IV vaccine can orchestrate an effective immunization strategy in organizing a co-ordinative immune response against VHSV in olive flounder thereby exhibiting higher protective efficacy to the host with minimum stress.