BIOLOGICAL-ACTIVITY OF SERUM ANTIBODIES TO A NONACYLATED FORM OF LIPOPROTEIN-D OF HAEMOPHILUS-INFLUENZAE

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well cons erved among both type b and non-typeable Haemophilus influenzae strain s, and is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of prote in D (PDm) from the periplasmic space of Escherichia coli overexpressi ng PDm. Screening of human sera for levels of antibodies to PDm demons trated that the immunoglobulin G (IgG) antibody level is above backgro und levels in infants less than 6 months of age. Following a drop to b ackground values in the age group 6 months to 1 year, IgG antibody lev els start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month to 1-year-old infants whose IgG antibody levels have dropped to the post natal background level. Affinity-purified antibodies from humans and f rom PDm-immunized rats detected epitopes of protein D which are normal ly exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. in fluenzae. Loss of bactericidal activity may occur in this buffer, as w as demonstrated in pooled human sera with high bactericidal activity a fter incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and s ignificant bactericidal activity against homologous and heterologous H . influenzae strains.