Sheth H B, Glasier L M, Ellert N W, Cachia P, Kohn W, Lee K K, Paranchych W, Hodges R S, Irvin R T
Protein Engineering Network Centres of Excellence, University of Alberta, Edmonton, Canada.
Biomed Pept Proteins Nucleic Acids. 1995;1(3):141-8.
This study describes the development of passive and active vaccines directed at the Pseudomonas aeruginosa pilus adhesin. Passive immunization studies were carried out with P. aeruginosa strain K pilus-specific (PK3B, PK99H) and cross-reactive (PAK-13) monoclonal antibodies (MAbs). When A.BY/SnJ mice were passively immunized with a pilus-specific MAb (PK99H), which inhibited pilus-mediated adherence to respiratory epithelial cells, mice challenged with 5 x LD 50 of P. aeruginosa were completely protected while mice were not protected when animals were passively immunized with a pilus specific MAb (PK3B), which did not inhibit pilus adherence to epithilial cells. MAb PAK-13 was found to cross-react with the C-terminal portion of pili of different strains of P. aeruginosa. When mice were passively immunized with MAb PAK-13, subsequent challenge with KB7 (3 x LD50), PAO (8 x LD50) and PAK (3 x LD50) strains of P. aeruginosa resulted in a 70%, 60% and 90% protection of the mice, respectively. MAb PK99H has been previously shown to recognize a linear antigenic epitope consisting of the sequence DEQFIPK. This epitopic peptide was conjugated to protein carriers using different coupling strategies. Use of an appropriate adjuvant and the correct conjugation strategy were critical for raising high affinity antipeptide antisera. In a comparison of Freund's, alum, and Adjuvax, as adjuvants for a peptide-tetanus toxoid conjugate vaccine, highest titers for the synthetic peptide component of the conjugate were obtained with Adjuvax, while highest titers for the carrier protein components were obtained with Freund's. Of the four peptide-conjugates used in this study, only the C-terminal conjugated peptide failed to produce antibodies that bind to native antigen and did not protect mice in active immunization experiments (no survivors at 80 h in the mouse infection model). Conformationally restricted peptide conjugates in which the peptide was conjugated to the carrier at both ends provided better protection in mice challenged with lethal doses of P. aeruginosa than either N- or C-terminal linked peptide-conjugates. The pilus adhesin plays a critical role in P. aeruginosa pathogenesis and this is an excellent vaccine target for either active or passive immunization strategies.
本研究描述了针对铜绿假单胞菌菌毛黏附素的被动和主动疫苗的研发情况。使用铜绿假单胞菌K菌株菌毛特异性（PK3B、PK99H）和交叉反应性（PAK - 13）单克隆抗体（MAb）进行了被动免疫研究。当用抑制菌毛介导的对呼吸道上皮细胞黏附的菌毛特异性单克隆抗体（PK99H）对A.BY/SnJ小鼠进行被动免疫时，用5倍半数致死剂量（LD50）的铜绿假单胞菌攻击的小鼠得到了完全保护，而当用不抑制菌毛对上皮细胞黏附的菌毛特异性单克隆抗体（PK3B）对动物进行被动免疫时，小鼠未得到保护。发现单克隆抗体PAK - 13与不同铜绿假单胞菌菌株菌毛的C末端部分发生交叉反应。当用单克隆抗体PAK - 13对小鼠进行被动免疫时，随后用KB7（3倍LD50）、PAO（8倍LD50）和PAK（3倍LD50）铜绿假单胞菌菌株攻击，分别使70%、60%和90%的小鼠得到保护。先前已证明单克隆抗体PK99H识别由序列DEQFIPK组成的线性抗原表位。使用不同的偶联策略将该表位肽与蛋白质载体偶联。使用合适的佐剂和正确的偶联策略对于产生高亲和力抗肽抗血清至关重要。在比较弗氏佐剂、明矾和Adjuvax作为肽 - 破伤风类毒素偶联疫苗的佐剂时，Adjuvax使偶联物的合成肽成分获得最高滴度，而弗氏佐剂使载体蛋白成分获得最高滴度。在本研究中使用的四种肽偶联物中，只有C末端偶联肽未能产生与天然抗原结合的抗体，并且在主动免疫实验中未保护小鼠（在小鼠感染模型中80小时无存活者）。肽在两端与载体偶联的构象受限肽偶联物，在用致死剂量铜绿假单胞菌攻击的小鼠中提供的保护比N末端或C末端连接的肽偶联物更好。菌毛黏附素在铜绿假单胞菌致病过程中起关键作用，这是主动或被动免疫策略的一个极佳疫苗靶点。
