Shukla Anshuman, Khatri Kapil, Gupta Prem N, Goyal Amit K, Mehta Abhinav, Vyas Suresh P
Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, India.
J Pharm Pharm Sci. 2008;11(1):59-66. doi: 10.18433/j3k01m.
Most of the presently available vaccines including hepatitis B vaccines administered through parenteral route, fail to induce a mucosal antibody response. Therefore, oral immunization appears to be an effective and attractive alternative to parenteral immunization. However, the problem of degradation of antigen in the harsh and hostile environment of the gastrointestinal tract consequently requires larger doses and more frequent dosing of antigen. Furthermore, much larger doses can induce antigen tolerance. Therefore the purpose of the present study was firstly to overcome these problems by the use of bile salt stabilized vesicles (bilosomes) and HBsAg as the model antigen,which could provide both protection to the antigen as well as enable transmucosal uptake and subsequent immunization. Another purpose of this study was to determine the dose that could produce serum antibody titres against hepatitis B via the oral route compared to those following intramuscular immunization.
In the present study bilosomes containing recombinant hepatitis B surface antigen were prepared by a lipid cast film method. HBsAg loaded bilosomeswere characterized in vitro for their shape, size, percent antigen entrapment and stability. Fluorescence microscopy was carried out to confirm the uptake of bilosomes by gut associated lymphoid tissues (GALT). The in vivo part of the study comprised estimation of anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions using specific ELISA techniques following oral immunization with low dose loaded bilosomes (B1, 10 microg), intermediate dose loaded bilosomes (B2, 20 microg) and high dose loaded bilosomes (B3, 50 microg) in BALB/c mice.
Fluorescence microscopy suggested that there was an increase in fluorescence intensity following the uptake of bilosomes entrapped FITC-BSA in gut associated lymphoid tissues. The high dose HBsAg bilosomes (B3, 50 microg) produced comparable anti-HBsAg IgG levels in serum to those observed in the case of intramuscular administration of alum adsorbed HBsAg (10 microg). In addition, the bilosomal preparations elicited measurable sIgA in mucosal secretions, where the highest responses were observed with high dose HBsAg bilosomes (B3,50 microg) and as expected, intramuscular administered alum adsorbed HBsAg (10 microg) failed to elicit such responses.
HBsAg loaded bilosomes produced both systemic as well as mucosal antibody responses upon oral administration. Furthermore, bilosomes with a five times higher dose upon oral administration produced comparable serum antibody titres to those obtained after intramuscular immunization without the induction of systemic tolerance.
目前可用的大多数疫苗，包括通过肠胃外途径接种的乙肝疫苗，都无法诱导黏膜抗体反应。因此，口服免疫似乎是肠胃外免疫的一种有效且有吸引力的替代方法。然而，在胃肠道恶劣的环境中抗原会降解，这就需要更大剂量且更频繁地给予抗原。此外，大得多的剂量会诱导抗原耐受性。因此，本研究的目的首先是通过使用胆盐稳定的囊泡（双分子层脂质体）和乙肝表面抗原（HBsAg）作为模型抗原来克服这些问题，这种囊泡既能保护抗原，又能实现跨黏膜摄取并随后引发免疫反应。本研究的另一个目的是确定与肌肉注射免疫相比，通过口服途径能产生抗乙肝血清抗体滴度的剂量。
在本研究中，通过脂质浇铸膜法制备了含有重组乙肝表面抗原的双分子层脂质体。对负载HBsAg的双分子层脂质体进行体外表征，观察其形状、大小、抗原包封率和稳定性。进行荧光显微镜检查以确认肠道相关淋巴组织（GALT）对双分子层脂质体的摄取。该研究的体内部分包括，在BALB/c小鼠中口服低剂量负载双分子层脂质体（B1，10微克）、中等剂量负载双分子层脂质体（B2，20微克）和高剂量负载双分子层脂质体（B3，50微克）后，使用特异性ELISA技术评估血清中抗HBsAg IgG反应以及各种身体分泌物中抗HBsAg sIgA反应。
荧光显微镜检查表明，肠道相关淋巴组织摄取包裹异硫氰酸荧光素标记牛血清白蛋白（FITC-BSA）的双分子层脂质体后，荧光强度增加。高剂量HBsAg双分子层脂质体（B3，50微克）在血清中产生的抗HBsAg IgG水平与肌肉注射明矾吸附的HBsAg（10微克）时观察到的水平相当。此外，双分子层脂质体制剂在黏膜分泌物中引发了可测量的sIgA，其中高剂量HBsAg双分子层脂质体（B3，50微克）引发的反应最高，正如预期的那样，肌肉注射明矾吸附的HBsAg（10微克）未能引发此类反应。
口服负载HBsAg的双分子层脂质体可产生全身和黏膜抗体反应。此外，口服剂量高出五倍的双分子层脂质体产生的血清抗体滴度与肌肉注射免疫后获得的滴度相当，且未诱导全身耐受性。
