Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS One. 2012;7(9):e43813. doi: 10.1371/journal.pone.0043813. Epub 2012 Sep 6.
The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1-6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 µg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide.
金黄色葡萄球菌感染的频率、严重程度和对抗生素的耐药性不断增加，使得开发针对这种病原体的免疫疗法比以往任何时候都更加紧迫。以前使用单价抗原进行免疫接种的尝试最多只能对金黄色葡萄球菌感染提供部分保护水平。因此，我们推断，合成由两种广泛表达的金黄色葡萄球菌抗原组成的双价缀合疫苗，将导致针对每种疫苗成分的抗体产生相加/协同作用，和/或增加菌株覆盖率。为此，我们使用还原胺化，将金黄色葡萄球菌抗原凝聚因子 A (ClfA) 和去乙酰化多-N-β-(1-6)-乙酰葡萄糖胺 (dPNAG) 共价连接。用 1、5 或 10 µg 的 dPNAG-ClfA 缀合物免疫的小鼠以剂量依赖性方式对 dPNAG 和 ClfA 产生 IgG 反应，而用 ClfA 和 dPNAG 混合物免疫的小鼠对 ClfA 的抗体滴度显著降低，对 PNAG 没有抗体。dPNAG-ClfA 疫苗在兔、恒河猴和山羊中也具有高度免疫原性。此外，从 dPNAG-ClfA 免疫血清中亲和纯化的 ClfA 抗体可阻断三种金黄色葡萄球菌菌株与固定化纤维蛋白原的结合。在调理吞噬测定 (OPKA) 中，dPNAG-ClfA 疫苗的山羊抗体在补体和多形核细胞存在下，杀死了金黄色葡萄球菌 Newman，并且在较低程度上杀死了金黄色葡萄球菌 NewmanΔclfA。PNAG 阴性同基因突变体未被杀死。此外，PNAG 抗原完全抑制了 dPNAG-ClfA 疫苗抗血清对金黄色葡萄球菌 Newman 的杀伤作用。最后，用 dPNAG-ClfA 或 dPNAG-白喉类毒素缀合物的山羊抗血清被动免疫接种的小鼠在感染三种不同的金黄色葡萄球菌菌株后 2 小时，血液中的细菌数量减少程度与给予正常山羊血清的小鼠相当。总之，ClfA 是一种免疫原性载体蛋白，可引发抗黏附抗体，但不能增强对 PNAG 细胞表面多糖的 OPK 和保护活性。
