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The protein II (P.II) outer membrane proteins of Neisseria gonorrhoeae, which have been implicated in gonococcal pathogenesis, have been previously shown to undergo a type of phase variation in which expression of any of several different forms of the proteins may be switched on or off. We identified six electrophoretically distinct forms of P.II proteins (designated P.IIa through P.IIf) within strain FA1090, and we isolated colonial variants of FA1090 that expressed only one of the six different P.II protein forms. Two monoclonal antibodies that bound specifically and differentially to P.II proteins were produced. One antibody bound to proteins P.IIb and P.IId and was bactericidal for all colonial variants expressing P.IIb. The second antibody bound to P.IIa and was bactericidal for colonial variants expressing P.IIa. P.II protein profiles of survivors of antibody killing indicated that multiple P.II protein species may be expressed on a single bacterium and that P.II protein switching in the gonococcus is nonrandom.

Monoclonal antibodies were produced against the HAR-13 strain (A) of Chlamydia trachomatis. By an indirect immunofluorescence technique, three clones (1-7, 2-8, 9F) were found to produce antibody that reacted only with serotype A strains. Clone 3-5 produced antibody that cross-reacted with serotypes A, C, H, I, and J. Using an indirect immunoferritin technique, we examined the binding of these antibodies to strain HAR-13 by electron microscopy. Antibodies from all four clones were shown to bind to the outer membrane surface of both reticulate and elementary bodies. Two distinct binding patterns were demonstrated. Antibodies from clones 1-7 and 9F bound to the outer membrane surface in a homogeneous pattern, whereas antibodies 2-8 and 3-5 bound to the outer membrane surface in an irregular, patchy distribution. There was a direct correlation between the distribution of antigen on the outer membrane surface and neutralization of C. trachomatis HAR-13 infectivity in vitro. Neutralization of HAR-13 infectivity by antibodies 1-7 and 9F was complement dependent, whereas antibodies 2-8 and 3-5 did not neutralize under any conditions.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.

This study showed that a protein II (PII) of Neisseria gonorrhoeae FA1090 appeared to act as a mediator of attachment to HeLa cells. Two colony variants of FA1090 were selected. Both gonococcal variants were nonpiliated, but one contained a PII and the other did not. A monoclonal antibody (1090-10.1), which was directed against the PII, inhibited the apparent PII-mediated attachment to HeLa cells. Antibodies produced from clone 1035-4, which had no PII specificity, did not inhibit the attachment and were used as controls. Inhibition of gonococcal attachment by the 1090-10.1 monoclonal antibodies was demonstrated by fluorescent microscopy analysis. Monoclonal antibody 1090-10.1 appeared to cause agglutination of the PII-containing organism. To block the clumping caused by the PII-specific monoclonal antibodies, Fab fragments of goat anti-mouse IgG were incubated with gonococci and the 1090-10.1 monoclonal antibodies. The results showed that the goat anti-mouse IgG Fab fragments partially blocked the agglutination caused by the PII-specific monoclonal antibody. The effect of the 1090-10.1 antibodies on attachment was also determined by monitoring the HeLa cells with attached iodinated gonococci. The monoclonal antibody appeared to inhibit the PII-mediated attachment.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.