The sexually transmitted disease gonorrhea is a global health concern, with increasing infection rates in many parts of the world [1
]. The WHO estimates that there are more than 62 million new cases of gonorrhea per year [2
]. In 2004, the CDC reported approximately 330,000 new cases of gonorrhea in the U.S. [3
]. Both of the above approximations are suggested to be underestimated by almost half due to inadequate reporting measures, as well as the prevalence of asymptomatic infection [1
]. In men, hallmark symptoms include urethral mucopurulent discharge and dysuria. Often women suffer asymptomatic infections, with no overt signs or symptoms of the disease [4
]. Symptomatic infections in women can include purulent vaginal discharge, dysuria, intermenstrual bleeding, and menorrhagia [5
]. Complications in women are common and can include ascension into the upper genital tract resulting in pelvic inflammatory disease (PID), which can lead to fallopian tube scarring. This can cause sterility or decreased fertility, and/or ectopic pregnancy.
Gonorrhea can be effectively treated with antibiotics. However, as with most bacteria under antibiotic pressure, gonococcal antibiotic resistance has emerged [6
]. This has resulted in the use of newer and more expensive antibiotics for treating this disease. In addition to multiple-drug resistant organisms, a disturbing finding concerning HIV and gonococcal co-infection has been reported. Studies have demonstrated an increase in HIV titers in the mucosal secretions of both males and females during gonococcal/HIV co-infection [7
]. Co-infected individuals thus increase the probability of infecting other sexual partners with HIV. For these reasons, the search for an effective gonococcal vaccine has become more imperative.
There have been a number of attempts to develop vaccines to prevent gonococcal disease. Vaccines tested in humans using partially-lysed gonococci, pilin, and porin all failed, likely due to antigenic variation of these or surrounding surface structures [9
]. These failed attempts have prompted researchers to look for surface antigens that are conserved in sequence from strain to strain, and not subject to high frequency variation. The transferrin binding proteins fit these criteria [12
]. Vaccine studies using meningococcal Tbps have demonstrated elicitation of antibodies that are cross-reactive against heterologous strains, are bactericidal, and can block transferrin utilization [14
]. Furthermore, in a meningococcal mouse model, mice immunized with TbpA or TbpA and TbpB were completely protected from lethal challenge [16
]. These studies suggest that the neisserial transferrin binding proteins could serve as protective antigens to prevent neisserial diseases. A mutant lacking the transferrin binding proteins was unable to colonize the urethra or cause symptoms of urethritis in a human male challenge model of gonococcal infection [18
]. These data, in conjunction with the vaccine studies in meningococcal models, represent strong evidence that the transferrin binding proteins could be an ideal target in the development of a protective gonococcal vaccine.
We demonstrated previously that intranasal immunization with the gonococcal transferrin-binding proteins chemically conjugated to the cholera toxin B subunit (Ctb) induced systemic and vaginal antibodies against both TbpA and TbpB [19
]. Furthermore, we demonstrated bactericidal activity of immune sera from mice immunized with these Tbp-Ctb chemical conjugates [19
]. One of the drawbacks to using the chemical conjugation of two proteins however is the heterologous nature of the vaccine preparation due to differential cross-linking of two proteins. The adjuvanticity of Ctb is dependent on its ability to bind GM1
]. One potential pitfall of a Ctb chemically conjugated vaccine is that a portion of the vaccine preparation may not be able to bind to its receptor due to steric hindrance caused by a large co-conjugated protein such as a transferrin binding protein. Furthermore, incorporation of full-length proteins in a vaccine antigen preparation has the potential to generate a diverse antibody response, comprising both protective and diversional antibodies. Immunogenic epitopes tend to vary in sequence between strains, due to immunologic pressure, and to result in non-functional antibody deposition on the cell surface. Thus, identification of the protective epitopes within full-length antigens, to be administered with an immune-stimulating adjuvant, may be a productive path towards efficacious vaccine development.
In an effort to determine whether two specific domains of the transferrin-binding proteins could elicit protective antibodies, we made genetic chimeras linked to the non-toxic A2 subunit of cholera toxin. Native cholera toxin is an AB5
exotoxin composed of one catalytic A subunit (CtA) and 5 surface binding B subunits (Ctb) [21
]. The A and B subunits spontaneously combine in a non-covalent fashion in the periplasm to form the holotoxin [22
]. The 5 individual B subunits combine to form a ring-like structure (Ctb). The non-toxic A2 domain of CtA passes through the central pore of the B subunit which allows for the tethering of the activity domain to Ctb [21
]. Previous investigators have demonstrated that the replacement of the toxic A1 moiety of CtA with heterologous proteins genetically fused with the A2 subunit, allowed for production of holotoxin-like chimeras [23
]. Employing this approach for TbpA, we focused on the surface-exposed loop 2 (L2). L2 has a predicted molecular mass of 9 kDa, and the sequence is well conserved among gonococcal isolates [12
]. For TbpB, we focused on the N-terminal transferrin-binding domain (NB), which was demonstrated to be the smallest truncation that retained the ability to bind transferrin by Western blot [13
]. Portions of this domain are also relatively well conserved among gonococcal isolates [13
Because L2 is relatively small in size, and from a protein that is not especially immunogenic [19
], we constructed a double genetic chimera with this peptide. The strategy we employed expressed NB and L2 together in anticipation that the larger NB would be more immunogenic and could augment antibody responses to L2. To this end, we genetically linked the L2 region in frame, immediately downstream of NB to make an NB-L2 chimera. This approach also afforded us the opportunity to determine what effects the inclusion of both epitopes would have on bactericidal killing and growth inhibition compared to mice immunized with NB only. We immunized mice intranasally and parenterally with the chimeric proteins and demonstrated that both chimeras were immunogenic, eliciting Tbp-specific serum antibodies. Furthermore, both chimeras induced bactericidal and growth inhibitory antibodies. This study demonstrates the feasibility of using epitopes instead of full-length Tbps in eliciting protective immune responses.