In Vitro Assessment of Antiborrelial Activity of OspA Vaccine Sera

Abstract

Prevention of Lyme disease by the recombinant OspA-based vaccine reportedly works by preventing transmission of spirochetes from ticks to humans. We report on an in vitro microculture assay, which can be used to provide an indicator of the need for booster doses of vaccine.To date, there is no commercially available assay for assessing whether a recipient of the recombinant OspA-based vaccine for Lyme disease possesses adequate antiborrelial activity to prevent transmission of Borrelia burgdorferi from ticks (7). Efficacy trials and marketing of the vaccine were recently suspended by the manufacturer; however, interest in sublicensing of the product and patents covering potential use of variants of the OspA lipoprotein suggest that a need for monitoring antiborrelial status of vaccine recipients will continue to be clinically relevant. We describe an in vitro microculture assay that can be easily established in and performed by any laboratory equipped to grow B. burgdorferi in culture. This method is similar to the borreliacidal and other in vitro assays for detecting antibodies with antiborrelial activity that have been previously reported (1,8,13). Unlike those assays, this method is intended solely as a means of determining the need for booster doses of vaccine to maintain efficacy. The design of our method was directed by the novel mechanism of intratick killing of B. burgdorferi by which this vaccine works (6). The implications of this mechanism on assay design include the following. (i) Use of high serum dilutions to provide an index of the titer is probably unnecessary, since the volume of blood entering a feeding tick greatly exceeds the volume of the tick's fluids in which the blood is "diluted." (ii) It has been reported that both the tick and the spirochete itself possess anticomplement activity (9, 14); therefore, an assay to assess antiborrelial factors resulting from vaccination with OspA should be capable of detecting complement-independent antiborrelial activity.The in vitro assessment of antiborrelial activity was performed using a microculture system. The bacteria (B. burgdorferi ATCC strain B31) were grown to log phase in modified BSK-H medium (Sigma) at 29°C. Aliquots (150 l) of the borrelia were then transferred to microculture wells in 48-well plates. An equal volume of test sera was added, and cultures were then incubated overnight at 29°C. Following incubation, samples from each culture were prepared as thin-film wet preps and examined microscopically using a 40ϫ phase-contrast objective on a Zeiss axioplan photomicroscope.Antiborrelial effects of serum were determined by scoring each culture for motility, aggregation, bleb formation, and lysis of spirochetes using a scale of 0 to 4, where 0 corresponds to uninfected, healthy appearance and 4 corresponds to extensive evidence of lysis, bleb formation, aggregation, or loss of motility. The scoring was assessed by comparison of blind readings by two individuals. Justification for the scoring system was based on results obtained for healthy,...