The importance of rational design for diagnostic tests is predicated by diversity in the immune responses produced by infected individuals. The use of multiplex testing with cytometric bead-based technology has expanded vigorously in recent years and has become a platform for serological testing. In the case of Lyme disease, the C6 peptide has demonstrated superior reliability as a standalone antigen for diagnostic testing. However, the inclusion of multiple antigens into a serologic test may increase sensitivity. This must be accomplished without compromising test specificity.
To gain insight into antigens that may offer the potential for broad (encompassing all phases of disease) sensitivity, we examined longitudinal responses: (i) in the most appropriate animal model for human Lyme disease, the rhesus macaque, and (ii) both pre- and postantibiotic treatment to include assessment of responses that may be linked to disease resolution. In doing so, we discerned a combination of antigens that would be appropriate for inclusion in a multiantigen serologic test.
In mice, DbpA is known to be a T-cell-independent antigen to which a high proportion of the antibody response is directed (9
). It has been tested as a vaccine immunogen (11
) and as a diagnostic antigen (35
). All NHP infected with either B. burgdorferi
strain in this study also produced staunch antibody responses to DbpA. It is likely that this is a T-cell-independent antigen for NHP as well. BBK32 is another extracellular matrix binding protein that has been tested both as a vaccine (11
) and as a diagnostic antigen (35
). Though the titer dropped substantially for one treated B31-infected monkey, only three of the five macaques produced veritable antibody responses to this protein. Due to the strong and sustained responses to DbpA in all animals tested to date, it appears a valuable diagnostic antigen. BBK32 has a lower potential for sensitivity and may not add value to a multiplex test.
Despite the artificial induction of primary anti-OspA responses by inoculation of in vitro
cultured spirochetes, we found the fluctuations in response over time compelling. We detected ospA
transcript from persistently infected macaques (15
), so we reasoned that their anti-OspA antibody responses may not decline predictably over time. In addition, OspA antibodies have been associated with arthritis during disseminated infection (3
) and have more recently been shown to be elevated in patients with posttreatment Lyme disease syndrome (12
). For its potential to detect infection at the late disseminated phase, inclusion of OspA may benefit a multiplex test. OspC is a well-characterized, highly immunogenic B. burgdorferi
lipoprotein. Heterogeneity in ospC
among different isolates and strains has been well documented. However, this does not appear to result in type-specific responses to OspC (23
), so it remains a good candidate diagnostic antigen. Antibody responses to OspC may be expected to arise early and steadily decline, as this antigen's expression is known to be shut off after the advent of the humoral response (28
). This is indeed what we observed.
transcript was detected in heart tissue of mice postantibiotic treatment (8
). Over a decade ago, two-dimensional electrophoresis and immunoblotting revealed antibody responses of infected individuals that targeted an ABC transporter protein of Borrelia garinii
). Human patients are known to produce antibodies to the oligopeptide permease A-type protein from B. burgdorferi
). The OppA-2 protein was also demonstrated to be antigenic in rabbits (13
) and in mice (7
). The gene is on the chromosome, whereas the gene from which C6 is derived is located on a plasmid that can be lost during infection (39
) and possibly also posttreatment (10
). OppA-2 transcript was shown to be produced by B. burgdorferi
in mice postantibiotic treatment, as an indicator of their metabolic viability. Given the broad reactivity of macaques to OppA-2 and the decline in response, albeit moderate, posttreatment, this protein could prove to have utility as a diagnostic antigen both before and after treatment. While we did not see a response to OppA-2 using preimmune serum, the modest homology (27
) that this protein may have with similar bacterial proteins necessitates its careful screening with multiple samples for specificity.
Continuous IgM responses beyond primary introduction of antigen are peculiar but have been observed in B. burgdorferi
infection previously (1
). Primarily through the use of immunoblotting, the continual detection of B. burgdorferi
antigen-specific IgM is well documented; however, fluctuations in specific antibody levels over time have not generally been presented. The induction of IgM-producing cells, particularly by T-cell-independent antigens (42
), may provide a mechanism for such fluctuations and persistent IgM titers.
The results of this study indicate that the antigens OspA, OspC, DbpA, and OppA-2 may offer distinct benefits when they are combined with the C6 peptide into a multiantigen diagnostic test. However, there are limitations to this study, including the inoculum dose, route, and the small number of animals tested. While the inoculum dose used here may not accurately reflect the dose that naturally infected humans would receive, the dynamic responses to particular antigens may be comparable in a general sense. Use of an outbred animal model and two different B. burgdorferi strains yielded patterns of antibody response that were remarkably similar per antigen. We cannot assert that these responses reflect those which may be observed in humans, but future studies are aimed at testing the responses to these specific antigens from a broad panel of Lyme disease patients.
For a multiantigen quantitative diagnostic test, specifically the inclusion of OspC could allow for improved sensitivity in the localized phase and that of OspA has the potential to alert a physician about the possibility of development of late arthritis. Detection of OppA-2 increases in antibody levels posttreatment may be a sign of persistent spirochetes or recrudescence as all three treated animals in the strain B31-inoculated experimental group had indications of persistent B. burgdorferi
). The early (~4 weeks p.i.) and strong response to DbpA by a majority of animals indicates that DbpA may also lend diagnostic value to a multiplex test. Observation of dynamic longitudinal responses to various antigens over time may provide insight for optimal diagnostic antigen combination in developing diagnostic tests for other infections with pathogens that possess multiple candidate diagnostic antigens.