Robert D. Gilmore

Relapsing fever Borrelia (RFB) employs antigenic variation to alter its surface protein structure in response to host immune pressure. This process occurs by the single translocation of archived variable major protein (Vmp) pseudogenes into a vmp expression locus. Borrelia miyamotoi , phylogenetically grouped with RFB, has the genetic makeup for antigenic variation, but it has not been determined whether B. miyamotoi can create new variant serotypes in vivo . We inoculated mice with a non-clonal parental B. miyamotoi CT13-2396 strain with a known Vmp majority serotype with spirochete isolation at various days post-infection. The vmp that determined the reisolated variant serotype was identified by PCR of the expression locus followed by DNA sequencing of the amplified product. For each mouse reisolate, new variants replaced the parent majority serotype. Moreover, some mice produced additional variant reisolates days apart, indicative of the presentation seen in relapsing fever infections. Infection of mice with a clonal population resulted in the elimination of the inoculated serotype and isolation of new variants. Mouse serum obtained following infection revealed IgM antibodies reactive to the parent Vmp serotype, suggesting that the immune response eliminated or greatly reduced the majority population. These results demonstrated that B. miyamotoi reisolated from infected mice exhibited serotype populations differing from the inoculated strain, indicating the spirochetes underwent antigenic variation to evade the host’s immune response. However, whether the observed variation occurred by way of outgrowth of minority populations or by translocation of archived pseudogenes to the expression locus creating new variants awaits further study.

The spirochete Borreliella burgdorferi is a causative agent of Lyme disease, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal.

Borrelia miyamotoi is a causative agent of hard tick relapsing fever, was first identified in the early 1990s, and was characterized as a human pathogen in 2011. Unlike other relapsing fever Borrelia species, B. miyamotoi spread by means of Ixodes ticks.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.