The passive immunization findings presented in this study are best considered in the context of previous passive protection studies in rabbits. Turner et al. (45
), Perine et al. (38
), and Bishop and Miller (5
) demonstrated lesion delays ranging from 3 to 6 days following either a prechallenge passive IRS immunization or the cessation of daily administration of IRS. The meaning of lesion delays in the rabbit after injection of T. pallidum
was considered by Turner et al. Studies determining the relationship between T. pallidum
inoculum size and incubation time (32
) estimated a generation time of 30 to 33 h for T. pallidum.
A 10-fold difference in inoculum size resulted in a 4-day difference in the incubation period (46
). In another study, a 3-day delay in lesion appearance was interpreted to represent a 10-fold reduction in the numbers of T. pallidum
cells inoculated (45
Knowing that M131 had potent in vitro bactericidal activity, we chose to conduct passive immunization experiments with M131 because it provided a means of assessing the protective potential of its target epitope without having it available as an immunogen. We were aware that administration of a mouse monoclonal antibody into rabbits would result in an antibody response that might clear M131 from blood and tissues. However, we reasoned that M131 administered to rabbits the day before challenge and immediately following challenge would still be available to reduce treponemal numbers before the development of anti-M131 antibodies.
Rabbits that received IRS developed lesions at a mean time of 13.2 days, as did rabbits that received the control monoclonal antibody. Rabbits that received NRS developed lesions at all sites at day 11. These times to lesion appearance were all within the range expected following an injection of 103 T. pallidum
). Although the administration of IRS resulted in no significant delay in time to lesion appearance, the lesions in this group remained atypical compared to those in controls in that they were much smaller, flatter, less erythematous, and nonulcerative. In this context, the findings in the group passively immunized with M131 were striking. At 17 out of 24 sites, lesions developed with an approximately 8-day delay compared with the control MAb group. At the remaining seven sites on one of these animals, no lesions ever developed. These findings represent a higher level of protection than that achieved with IRS in the previously reported experiments using the rabbit model (5
The following considerations provide a hypothesis of how lesions developed at some sites but not others in the group that received M131. Given that 103 T. pallidum
cells were injected at each site, the 8-day delay observed to lesion appearance is consistent with killing 99% of the organisms injected at a site. Thus, lesions appeared in the time frame expected if 10 organisms had been injected into that site. It has been reported that when one to five treponemes are injected intradermally, lesions may not develop (27
). Also pertinent to our findings, Magnuson et al. (32
) reported that when intradermal sites were each inoculated with 20 treponemes, lesions appeared at only 70% of the sites. Therefore, the failure of lesions to appear at 29% of the injection sites (7/24) in animals passively immunized with M131 is again consistent with a 99% killing of the 1,000-organism challenge inoculum that was used per site.
In an effort to relate lesion appearance to numbers of treponemes, we used real-time PCR to quantitate T. pallidum DNA copy numbers. Real-time PCR of control lesions over a 55-day period showed that at day 10 postchallenge, there were approximately 3 × 104 T. pallidum DNA copies per μg of rabbit DNA. Maximal copy numbers of approximately 5 × 106 were seen at day 20. This information is relevant to interpreting the significance of delays in lesion appearance conferred by passive immunization with M131. At day 20 postchallenge, which is approximately the mean time to lesion appearance in the group that received M131 (day 21), the numbers of T. pallidum DNA copies approximated those in the control animals at days 10 through 15. Further, these atypical lesions at day 20 postchallenge, compared to 20-day-postchallenge lesions from control animals that received control MAb or NRS, showed a three- to sixfold decrease in DNA copy numbers. However, by day 25 postchallenge, the DNA copy numbers in these atypical lesions were similar to those in the controls, even though these lesions were significantly smaller in appearance. These considerations do not take lesion volume into account, and these atypical smaller lesions may contain fewer total spirochetes than the normal typical larger lesions. Given this measurable decrease in the DNA copy number at 20 days postchallenge in animals passively immunized with M131, it is reasonable to infer that these differences might be even greater at earlier time points during lesion development. In support of this idea, we found that at sites where lesions did not develop in the rabbit passively immunized with M131, a greater than 4-log difference in DNA copy number was detected.
While lesion development and the numbers of treponemes in these developing lesions were clearly affected by passive immunization with M131, we did not observe an alteration in disseminated infection as determined by real-time PCR analysis and infectivity testing of popliteal lymph nodes. One explanation for the lack of protection from disseminated infection might be the requirement for a prolonged presence of killing antibody, whereas in our study, passive immunizations were administered only before, the day of, and shortly after the time of challenge.
The binding of M131 to the surfaces of T. pallidum
cells was demonstrated by IEM, by indirect immunofluorescence of organisms in gel microdroplets, and by dot blot analysis of whole intact organisms. Treponemes incubated in the absence of complement with M131 for IEM were observed to be actively motile at the end of the incubation, confirming that organisms maintained structural integrity and that M131 binding was to surface outer membrane targets. Treponemes encapsulated in gel agarose microdroplets, a sensitive technique that preserves organism integrity (16
), also showed specific M131 surface binding that was observed to be in a beaded pattern, suggesting antibody aggregation. Surface antibody aggregation has been proposed to be an important factor in the activation of complement and killing of T. pallidum
). Treponemes whose outer membranes were removed by Triton X-100 treatment showed greater M131 binding by IEM, by the gel microdroplet assay, and by dot blot analysis, indicating that the target for M131 was both surface and prominently subsurface located on T. pallidum
In our initial attempts to identify the target of M131, we speculated that it was one of the T. pallidum
lipoproteins previously reported to be both outer membrane and subsurface located (9
). However, the findings presented in this study showed that the target for M131 is not a protein but rather phosphorylcholine. The phospholipids phosphatidylcholine and sphingomyelin, both of which possess a phosphorylcholine polar head group, reacted with M131. In contrast, none of the other phospholipid species associated with T. pallidum
), which do not possess a phosphorylcholine head group, were reactive with M131. The suggestion that phosphorylcholine is the specific target of M131 is further supported by the RP-HPLC fractionation of total T. pallidum
lipid, which shows that all reactive fractions contain a phosphorylcholine-containing lipid. Of further interest was the finding that M131 binding to phosphatidylcholine, sphingomyelin, or T. pallidum
-extracted lipid requires a liposomal form, suggesting that conformation or possibly membrane packing of these phospholipid polar head groups is required to generate the M131 binding epitope. This was further indicated by experiments showing that liposomes made in combination with phosphatidylcholine and either phosphatidylethanolamine or phosphatidylserine, two phospholipids that do not react with M131, require phosphatidylcholine concentrations of at least 60% and 90%, respectively, in order to maintain M131 reactivity. This suggests that the composition and perhaps distribution of T. pallidum
phosphorylcholine are critical factors in generating this epitope.
Phosphatidylcholine has been previously shown to be the predominant phospholipid species in T. pallidum
membranes and in the membranes of other members of the genus Treponema
). Both T. pallidum
and Treponema denticola
possess a licCA
fusion gene, suggesting that both utilize a CDP-choline pathway for the biosynthesis of phosphatidylcholine (22
). However, our finding that “T. phagedenis
” biotype Reiter did not react with M131 further demonstrates the specificity of this epitope and indicates that the mere presence of a phosphorylcholine-containing phospholipid, like phosphatidylcholine, in a biological membrane is by itself not sufficient to generate the M131 binding epitope. It is pertinent to note that “T. phagedenis
” and the other cultivatable treponemes, but not T. pallidum
, possess lipopolysaccharide in their outer membranes. It is possible that lipopolysaccharide may inhibit the formation of this epitope in the outer membranes of these other treponeme species.
Phosphorylcholine has been shown to be an important pathogenesis-related surface molecule for several bacterial pathogens including Haemophilus influenzae
), Streptococcus pneumoniae
), Pseudomonas aeruginosa
), and Neisseria
). With several of these pathogens, a common phosphorylcholine epitope has been demonstrated using monoclonal antibodies, including MAb TEPC-15 that is specific for phosphorylchloline (48
). In our studies, however, TEPC-15 did not react with T. pallidum
or to phosphatidycholine liposomes and M131 did not react with P. aeruginosa
or to phosphorylcholine-conjugated KLH, indicating that T. pallidum
does not possess this common phosphorylchloline epitope and that the T. pallidum
epitope defined by M131 is not common to these pathogens that possess phosphorylcholine.
VDRL antigen has been used for many decades as a serological screening test for the diagnosis of syphilitic infection. VDRL antigen contains lecithin, which is composed of approximately 23% phosphatidylcholine. Baker-Zander et al. (3
) previously showed that immunization of rabbits with VDRL antigen elicited partial protection evidenced by both delays and the absence of lesions following challenge, similar to the findings presented here. However, as shown in this study, M131 did not react with VDRL antigen. Further, we have tested the anti-VDRL serum from the Baker-Zander study (kindly provided by Sheila Lukehart) and have found no bactericidal activity against T. pallidum
. Taken together, these results suggest that the immunological target for the partial protection demonstrated in the Baker-Zander study is different from the target defined by M131.
We have also tested whether M131 would react with other biological membranes that contain phosphatidylcholine. Red blood cells are known to contain as much as 23.4% phosphatidylcholine in the exterior membrane leaflet (28
). However, red blood cells also did not react with M131. These findings again suggest that the target of M131 is unique and possibly conferred by the phosphorylcholine composition and conformation in the membranes of T. pallidum
We have previously reported that IRS binding to the surface of T. pallidum
, evidenced by killing activity, cannot be demonstrated by immunoelectron microscopy (21
). However, the binding of M131 to the T. pallidum
surface was easily detected by this technique, indicating that the titer of antibodies in IRS to this phosphorylcholine epitope must not be high. Nonetheless, the reaction of phosphatidylcholine liposomes with all IRS specimens tested suggests that the phosphorylcholine epitope that M131 recognizes may contribute to the protective immunity that develops during syphilitic infection, although this hypothesis must be directly tested.
In an effort to elicit protective immunity, past studies have been conducted that have examined the course of experimental syphilis in the rabbit after active immunization with individual T. pallidum
proteins. Immunizations with endoflagella (15
), 4D (10
), glycerophosphodiester phosphodiesterase (11
), Tp92 (12
), the 15-kDa lipoprotein (13
), and recently, the T. pallidum
repeat protein K (TprK) (14
) have been conducted. Challenge results of these studies have shown the development of atypical lesions at most sites, appearing in either the time frame expected for control lesions or in an accelerated time frame, but delays in lesion appearance were not observed. While several of these T. pallidum
proteins have been suggested to be surface exposed, based in part upon antibodies that were either opsonophagocytic (11
) or had some killing activity (10
), none of these proteins have been physically visualized on the surface of the spirochete by using these antibodies. Indeed, the microdroplet assay failed to demonstrate surface exposure of the recently studied TprK protein (25
), a member of a T. pallidum
orthologous gene family that has been suggested to be surface exposed, based in part upon opsonophagocytic activity (14
). By comparison, binding of M131 to the surfaces of structurally intact T. pallidum
cells, as demonstrated by both the microdroplet assay and immunoelectron microscopy, represents the first physical demonstration of a T. pallidum
surface antigen. The ability of the M131-defined epitope to serve as a protective immunogen is currently under study in our laboratory.