Bueno Da Costa Maria Helena, Quintilio Wagner, Tanizaki Martha Massako, Sant'Anna Osvaldo Augusto, Schwendener Reto Albert, de Araujo Pedro Soares
Lab de Microesferas e Liposomos, C Biotecnologia, Av Vital Brasil, São Paulo, SP.
J Liposome Res. 2002 Feb-May;12(1-2):29-35. doi: 10.1081/lpr-120004773.
The modern vaccinology encompasses the recombinant DNA technology, protein and carbohydrate chemistry to obtain safe molecularly defined vaccines. Nevertheless most of the vaccines are poorly immunogenic because a large number of antigens are membrane proteins and consequently they are not present in their active conformation in the vaccine. Others are not as potent because they contain only B epitopes and therefore, cannot stimulate cellular memory. We have been studying the characteristics of the recombinant heat shock protein 18kDa-hsp from Mycobacterium leprae as an alternative carrier protein with a T epitope source to enhance the activity of these second generation vaccines. Here we proved that the 18kDa-hsp acted as carrier, without masking the activity of the carried antigen, with similar immune stimulatory effect when compared with ODN1668. Supramolecular aggregates of 18kDa-hsp and Mice serum albumin (MSA) were obtained using glutaraldehyde as cross linker. The Neisseria meningitides serogroup C polysaccharide (PSC, a B epitope) and the carrier protein 18kDa-hsp were co-encapsulated within Soybean phosphatidylcholine liposomes (SPC: Cho : alpha-Toc, 22 : 5 : 0.18 molar ratio, respectively). These liposomes were prepared in MPB buffer (20 mM phosphate, 295 mM mannitol pH 7.2) in the presence or absence of the ODN1668, TCCATGACGTTCCTGATGCT. When mice were injected with 18kDa-hsp-MSA no antibody against the MSA was observed. This means that the 18kDa-hsp acted as carrier, without masking the carried protein immune activity. Stable liposomes of 150 nm were obtained using mannitol as a cryoprotector. Genetically selected mice when injected with liposomes containing PSC and 18kDa-hsp displayed an antibody titer of 12. In contrast, in those mice injected with free PSC there was no response. The 18kDa-hsp adjuvant effect on the PSC liposomal formulation was comparable to that observed when ODN1668 was co-encapsulated with PSC. Confirming our expectations we observed that the formulation containing 18kDa-hsp conferred a memory response to the carried antigen--the Neisseria meningitidis serogroup C polysaccharide.
现代疫苗学涵盖重组DNA技术、蛋白质和碳水化合物化学，以获得安全的分子定义疫苗。然而，大多数疫苗的免疫原性较差，因为大量抗原是膜蛋白，因此它们在疫苗中不以其活性构象存在。其他疫苗则效力不足，因为它们只包含B表位，因此不能刺激细胞记忆。我们一直在研究来自麻风分枝杆菌的重组热休克蛋白18kDa-hsp的特性，将其作为一种具有T表位来源的替代载体蛋白，以增强这些第二代疫苗的活性。在这里，我们证明了18kDa-hsp作为载体，不会掩盖所携带抗原的活性，与ODN1668相比，具有相似的免疫刺激作用。使用戊二醛作为交联剂获得了18kDa-hsp和小鼠血清白蛋白（MSA）的超分子聚集体。将C群脑膜炎奈瑟菌多糖（PSC，一种B表位）和载体蛋白18kDa-hsp共同包裹在大豆磷脂酰胆碱脂质体（SPC:Cho:α-生育酚，摩尔比分别为22:5:0.18）中。这些脂质体在MPB缓冲液（20 mM磷酸盐，295 mM甘露醇，pH 7.2）中制备，存在或不存在ODN1668，即TCCATGACGTTCCTGATGCT。当给小鼠注射18kDa-hsp-MSA时，未观察到针对MSA的抗体。这意味着18kDa-hsp作为载体，不会掩盖所携带蛋白质的免疫活性。使用甘露醇作为冷冻保护剂获得了150 nm的稳定脂质体。基因选择的小鼠注射含有PSC和18kDa-hsp的脂质体后，抗体滴度为12。相比之下，注射游离PSC的小鼠没有反应。18kDa-hsp对PSC脂质体制剂的佐剂作用与ODN1668与PSC共同包裹时观察到的作用相当。正如我们所期望的，我们观察到含有18kDa-hsp的制剂赋予了对所携带抗原——C群脑膜炎奈瑟菌多糖的记忆反应。
