Coxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all-trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide-based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.
柯薩奇病毒B3（CVB3）是腸病毒屬的成員，與多種疾病有關(guān)，包括心肌炎，心肌炎可進(jìn)展為擴(kuò)張型心肌病。盡管正在進(jìn)行臨床前研究，但沒有臨床批準(zhǔn)
目前已有針對CVB3的疫苗，這突顯了對有效預(yù)防方案的迫切需求。本研究介紹了一種基于蜘蛛小安瓿絲蛋白（MiSp）的納米疫苗平臺。該平臺利用嵌合蛋白工程化的蛋白質(zhì)納米顆粒，將CVB3抗原肽摻入定制的MiSp中，隨后負(fù)載全反式視黃酸（RA）。這些功能性納米疫苗能夠在皮下給藥后引發(fā)粘膜和全身免疫反應(yīng)，并對小鼠CVB3感染顯示出顯著的保護(hù)作用。這項(xiàng)研究標(biāo)志著一種基于肽的腸外疫苗策略的方法，利用工程化的MiSp納米顆粒與RA相結(jié)合
這項(xiàng)研究標(biāo)志著基于肽的腸外注射的一種疫苗策略，利用工程化的MiSp納米顆粒與RA相結(jié)合。該方法通過利用蜘蛛絲蛋白和RA的獨(dú)特免疫調(diào)節(jié)特性有效對抗這些病原體，為預(yù)防腸道病毒感染提供了一條有前景的途徑。
Protein Preparation: The chimeric protein sequences encoding NM-VP1T and NM-VP1B, respectively, were synthesized and cloned into the pET-32a plasmid using NdeI and XhoI restriction sites. These plas-mids were subsequently transformed into E. coli BL21 (DE3) competent cells, which were cultured at 37 °C in LB medium supplemented with100 μg mL. ampicillin. Growth continued until OD600 reached ≈0.8, after which the temperature was lowered to 25 °C and 1 mm Isopropyl ??-D-Thiogalactoside (IPTG) was added to induce protein expression. The cells were incubated for an additional 12 h. For protein purification, the cells were harvested via centrifugation and lysed using a high-pressure homogenizer (PhD Technology LLC, USA). The resulting insoluble pellets were thoroughly washed three times with 30 mL of washing buffer (20 mm Tris, 300 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 m urea, pH 8.0). To remove any residual detergent, the purified inclusion bodies were finally rinsed with 20 mm Tris (pH 8.0) and solubilized using a freeze-thaw method.
蛋白質(zhì)制備：編碼NM的嵌合蛋白序列-VP1T和NM-VP1B分別被合成并克隆到pET-32a質(zhì)粒使用NdeI和XhoI限制性位點(diǎn)。隨后將這些質(zhì)粒轉(zhuǎn)化到大腸桿菌BL21（DE3）感受態(tài)細(xì)胞中，在37°C下在添加了100μg mL氨芐青霉素的LB培養(yǎng)基中培養(yǎng)。生長持續(xù)到OD600達(dá)到≈0.8，之后溫度降至25°C，加入1mm異丙基-D硫代半乳糖苷（IPTG）以誘導(dǎo)蛋白質(zhì)表達(dá)。將細(xì)胞再孵育12小時。為了純化蛋白質(zhì)，通過離心收集細(xì)胞，并使用高壓均質(zhì)機(jī)（美國PhD Technology LLC）破碎細(xì)胞。用30mL洗滌緩沖液（20mm Tris、300mm NaCl、1mm EDTA、1%Triton X-100、1m尿素，pH 8.0）徹底洗滌所得不溶性顆粒3次。為了去除任何殘留的洗滌劑，最終用20mm Tris（pH 8.0）沖洗純化的包涵體，并使用凍融法溶解。
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