Theilacker Christian, Coleman Fadie T, Mueschenborn Simone, Llosa Nicolas, Grout Martha, Pier Gerald B
Channing Laboratory, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115-5804, USA.
Infect Immun. 2003 Jul;71(7):3875-84. doi: 10.1128/IAI.71.7.3875-3884.2003.
Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.
囊性纤维化（CF）患者的肺功能恶化与黏液型铜绿假单胞菌的慢性肺部感染密切相关。已证明铜绿假单胞菌的黏液样胞外多糖（MEP）可在小鼠体内诱导调理抗体，这些抗体对这种慢性感染具有保护作用。在缺乏可检测到的铜绿假单胞菌感染的老年CF患者血清中也普遍存在MEP特异性调理抗体。然而，在一项人体疫苗试验中，MEP仅能极少量地诱导调理抗体。为了评估MEP与载体蛋白偶联是否能提高其免疫原性，我们使用琥珀酰亚胺基-4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（SMCC）作为连接剂，将硫醇化的MEP与钥孔血蓝蛋白（KLH）结合。与天然MEP聚合物不同，MEP-KLH偶联疫苗在C3H-HeN小鼠和兔子体内诱导出高滴度的MEP特异性免疫球蛋白G（IgG）。用MEP-KLH偶联物免疫的小鼠血清显示出调理杀伤活性，而用相当剂量天然MEP免疫的动物血清则未显示。用MEP-KLH偶联物进行疫苗接种可诱导出对铜绿假单胞菌异源黏液样菌株具有广泛交叉反应性的调理抗体。在正常人血清中，包括年轻CF患者，都存在预先存在的针对MEP的非调理抗体，它们的存在会阻碍调理抗体的诱导。通过腹腔注射MEP或注射或喂食交叉反应性抗原海藻酸盐诱导非调理抗体后，会降低后续用MEP-KLH偶联物免疫所引发的总体IgG水平。然而，只有在预先存在由MEP诱导的抗体而非海藻酸盐诱导的抗体的MEP-KLH偶联物免疫的小鼠血清中，调理活性才较低。用MEP-KLH进行免疫可引发相当比例的针对涉及O-乙酰基残基表位的特异性抗体，并且这一亚群抗体在体外介导了对黏液型铜绿假单胞菌的调理杀伤作用。这些结果表明，MEP与KLH偶联可显著增强其在小鼠和兔子体内的免疫原性以及调理抗体的产生，该偶联物在预先存在非调理抗体的情况下仍能诱导调理抗体，并且针对MEP的调理抗体针对的是包括糖醛酸聚合物上乙酸酯残基的表位。
