Ramos-Nieves Riseilly, Armina-Rodriguez Albersy, Figueroa-Gispert Maria Del Mar, Figueroa-Quiñones Ghalib, Ocasio-Malavé Carlimar, Espino Ana M
Department of Microbiology and Medical Zoology, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936, USA.
Department of Biology, University of Puerto Rico, Arecibo Campus, Arecibo, PR 00614, USA.
Vaccines (Basel). 2025 May 20;13(5):545. doi: 10.3390/vaccines13050545.
BACKGROUND/OBJECTIVES: In the past, FhSAP-2, an 11.5 kDa recombinant protein belonging to the saposin-like/NK-lysin family, has been shown to induce over 60% partial protection in immunized rabbits and mice when challenged with metacercariae. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic nature has made its purification a challenging process. The present study aimed to determine whether a modified 9.8 kDa variant of protein (mFhSAP-2), lacking a string of 16 hydrophobic amino acids at the amino terminus and a dominant Th1 epitope, could retain its immunogenic and Th1-inducing properties.
RAW264.7 cells were stimulated with mFhSAP-2, and TNFα levels were determined. C57BL/6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Total anti-mFhSAP-2 IgG subtypes, along with their avidity and titers, were measured using ELISA. The T cell proliferation index and levels of CD4+/CD8+ and IFNγ/IL-4 ratios were determined.
In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 developed high-avidity IgG2a and IgG2c antibodies at levels that were significantly higher than IgG1 antibody levels. However, the mFhSAP-2+ISA50 formulation induced higher and more homogenous antibody titers than mFhSAP-2, suggesting that an adjuvant may be required to enhance mFhSAP-2 immunogenicity. Immunization with mFhSAP-2+ISA50 also induced significantly higher activated CD4+/CD8+ T cell ratios and IFNγ/IL-4 ratios compared to naïve mice.
Our results demonstrate that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by the Montanide ISA50 adjuvant. The present study highlights the feasibility of inducing Th1-associated immune responses in mice using mFhSAP-2 as an antigen. Further studies are required to assess the potential application of the mFhSAP-2+ISA50 formulation as a vaccine against in natural hosts such as cattle and sheep, which could contribute to improved control and aid in the prevention and eradication of infection.
背景/目的：过去的研究表明，FhSAP - 2是一种属于类鞘脂激活蛋白/NK - 溶素家族的11.5 kDa重组蛋白，在用尾蚴攻击时，能使免疫的兔和小鼠产生超过60%的部分保护作用。然而，尽管FhSAP - 2是一种很有前景的疫苗候选物，但其疏水性使其纯化成为一个具有挑战性的过程。本研究旨在确定一种修饰的9.8 kDa蛋白变体（mFhSAP - 2），该变体在氨基末端缺少一串16个疏水氨基酸和一个主要的Th1表位，是否能保留其免疫原性和诱导Th1的特性。
用mFhSAP - 2刺激RAW264.7细胞，并测定肿瘤坏死因子α（TNFα）水平。将C57BL/6小鼠单独用mFhSAP - 2免疫或与Montanide ISA50乳化后免疫。使用酶联免疫吸附测定法（ELISA）测量总抗mFhSAP - 2 IgG亚型及其亲和力和滴度。测定T细胞增殖指数以及CD4 + /CD8 +和干扰素γ（IFNγ）/白细胞介素4（IL - 4）比值水平。
在体外，mFhSAP - 2在RAW264.7细胞中诱导出剂量依赖性的TNFα产生。在体内，用mFhSAP - 2或mFhSAP - 2 + ISA50免疫的小鼠产生了高亲和力的IgG2a和IgG2c抗体，其水平显著高于IgG1抗体水平。然而，mFhSAP - 2 + ISA50制剂诱导的抗体滴度比mFhSAP - 2更高且更均匀，这表明可能需要佐剂来增强mFhSAP - 2的免疫原性。与未免疫的小鼠相比，用mFhSAP - 2 + ISA50免疫还诱导出显著更高的活化CD4 + /CD8 + T细胞比值和IFNγ/IL - 4比值。
我们的结果表明，mFhSAP - 2保留了其免疫原性和Th1极化特性，Montanide ISA50佐剂增强了这些特性。本研究突出了使用mFhSAP - 2作为抗原在小鼠中诱导Th1相关免疫反应的可行性。需要进一步研究评估mFhSAP - 2 + ISA50制剂作为针对牛和羊等自然宿主感染的疫苗的潜在应用，这可能有助于改善防控并有助于预防和根除感染。
