Department of Microbiology and Immunology, The University of Melbourne, Royal Parade, Parkville, Australia 3010.
Mol Pharm. 2012 Jan 1;9(1):81-90. doi: 10.1021/mp200264m. Epub 2011 Dec 16.
It has become increasingly recognized that polymer particle size can have a profound effect on the interactions of particle-based vaccines with antigen presenting cells (APCs) thereby influencing and modulating ensuing immune responses. With the aim of developing chitosan particle-based immunocontraceptive vaccines, we have compared the use of chitosan-based nanoparticles and chitosan-based microparticles as vaccine delivery vehicles for vaccine candidates based on luteinizing hormone-releasing hormone (LHRH). Particles, functionalized with chloroacetyl groups, which allows the covalent attachment of thiol-containing antigens, were able to adsorb ~60-70% of their weight of peptide-based antigen and 10-20% of their weight of protein-based antigen. Quantitation by amino acid analysis of antigen associated with particles demonstrated a correlation between associated antigen and the degree of chloracetylation of particles. Visualization of fluorescently labeled antigen-loaded particles by confocal microscopy indicated that the majority of antigen was localized at the particle surface with a smaller amount located in the interior. We also found that uptake of both fluoresceinated nanoparticles and microparticles by dendritic cells occurred in a manner dependent on particle concentration. Nanoparticles trafficked from the injection site to draining lymph nodes faster than microparticles; high numbers of nanoparticle-bearing cells appeared in draining lymph nodes on day 3 and microparticles on day 4. This difference in trafficking rate did not, however, appear to have any significant impact on the ensuing immune response because inoculation with both peptide-conjugated and protein-conjugated particles induced high levels of LHRH-specific antibodies. In the case of protein-conjugated particles, the levels of antibodies elicited were similar to those elicited following inoculation with antigen emulsified with complete Freund's adjuvant. The approach to vaccine design that we have described here could represent another useful method for inducing immune responses against microbial, viral and tumorigenic protein antigens.
人们越来越认识到，聚合物颗粒大小会对基于颗粒的疫苗与抗原呈递细胞（APCs）的相互作用产生深远影响，从而影响和调节随后的免疫反应。为了开发壳聚糖颗粒为基础的免疫避孕疫苗，我们比较了壳聚糖纳米颗粒和壳聚糖微球作为基于黄体生成激素释放激素（LHRH）的候选疫苗的疫苗传递载体的用途。颗粒用氯乙酰基官能化，允许含有巯基的抗原的共价连接，能够吸附其重量的~60-70%的肽基抗原和其重量的 10-20%的蛋白基抗原。通过对与颗粒结合的抗原进行氨基酸分析定量，证明了与颗粒结合的抗原与颗粒的氯乙酰化程度之间存在相关性。通过共焦显微镜观察荧光标记的抗原负载颗粒，表明大多数抗原定位于颗粒表面，少量位于内部。我们还发现，树突状细胞对荧光标记的纳米颗粒和微球的摄取方式取决于颗粒浓度。纳米颗粒从注射部位运送到引流淋巴结的速度比微球快；在第 3 天和第 4 天，携带大量纳米颗粒的细胞出现在引流淋巴结中。然而，这种运输速率的差异似乎并没有对随后的免疫反应产生任何重大影响，因为接种肽缀合和蛋白缀合颗粒都能诱导高水平的 LHRH 特异性抗体。对于蛋白缀合颗粒，诱导的抗体水平与用完全弗氏佐剂乳化抗原接种后诱导的水平相似。我们在这里描述的疫苗设计方法可能代表了另一种诱导针对微生物、病毒和肿瘤蛋白抗原的免疫反应的有用方法。
