Date of Award

1-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmaceutics

Research Advisor

Maria Gomes-Solecki, DVM

Committee

Santosh Kumar, PhD; Maureen A. McGargill, PhD; Michael M. Meagher, PhD; Marko Radic, PhD; Ae-Kyung Yi, PhD

Abstract

Introduction. Lyme disease, otherwise called Lyme borreliosis, is a vector-borne infectious disease caused by the spirochete Borrelia burgdorferi (Bb). Studying the increase in overall Lyme disease incidence requires understanding the role of the vector Ixodes scapularis (I. scapularis) as the agent that carries the spirochete which is important to limit the occurrence of Lyme disease. Once we understand the vector-pathogen response, it also becomes important to determine the remedies for Lyme disease. Lyme disease can be treated with antibiotics if detected early, but the major obstacle lies in the early detection process. Therefore, it is important to investigate alternative preventive measures. Although vaccines could serve that purpose, there are no vaccines currently available for Lyme disease in humans. A few Outer surface protein A (OspA) based compositions are currently undergoing clinical trials for human use. Outer surface protein C (OspC) is an alternative in the same line because it induces a significant antibody response and provides strain-specific protection after the tick challenge. However, due to OspC heterogeneity, there are limitations to using OspC as a broad-spectrum vaccine candidate. To this end, our lab designed a cocktail vaccine (OspCCt) comprised of 8 different OspC types selected for their ability to disseminate to target human organs. OspA and OspC are two important outer surface proteins of Bb, and they are genetically dissimilar. Despite the antigenic differences, we observed cross-reactivity between the OspC antigen and OspA serum antibodies. Studies have shown OspA to be an effective transmission-blocking vaccine. Therefore it is also important to investigate the transmission-blocking properties of the OspCCt vaccine observed in the ticks recovered from infected and vaccinated mice. Objective. Our first goal is to investigate whether I. scapularis nymphs kept in a prolonged questing state under optimal environmental conditions are fit to fulfill their transmission role in the enzootic cycle of Bb. We wanted to check for differences in Bb infection prevalence between the questing (actively moving and looking for hosts on which to feed) and diapausal (in suspended development) nymphs over a year. Our second objective is to design a vaccine candidate utilizing OspC. Finally, we analyzed whether there is any cross-reactivity between OspC and OspA, to define the function of OspC alone in blocking Bb transmission. Research Design. To understand the transmission competence of I. scapularis during prolonged questing, we first generated infected I. scapularis nymphs by allowing larvae to feed on infected mice. Firstly, the mice were infected through sub-cutaneous needle inoculation with cultured Bb (105) recovered from frozen stock. After needle inoculation, it takes three weeks for Bb to disseminate in naive mice fully. Then, I. scapularis larvae are placed on the infected mice and allowed to engorge and fall off naturally fully. Infected engorged larvae are collected and kept in glass vials for six weeks to allow them to molt into nymphs. In another six weeks after molting, the infected I. scapularis nymphs are ready for their nymphal blood meal. During this study, we analyzed differences in Bb infection prevalence between questing and diapausal nymphs after six weeks post-molting (prime questing). Additionally, we checked for differences in the infection prevalence of these nymphs when kept in a prolonged questing state for over 12 months. Lastly, we analyzed the ability of nymphal ticks to transmit Bb into naive mice at certain time points during the prolonged questing phase. Our second objective was to determine the efficacy of our OspCCt vaccine. To design this vaccine, we utilized eight full-length OspC recombinant proteins predominantly expressed by Bb when disseminated to secondary sites in humans (blood and cerebrospinal fluid). The cocktail vaccine is comprised of OspC types A, B, C, D, H, I, K, and N, in equal concentrations, and 50 µg of the cocktail was delivered subcutaneously. The vaccination schedule includes a prime vaccination and two boosts. The efficacy of the OspCCt was determined by analyzing serum antibodies against Bb and by checking Bb dissemination in different sites of vaccinated mice after the tick challenge. Serology was also performed to investigate the source of cross-reactivity between OspA and OspC. Results. Regarding our first objective, we determined that Bb-infected unfed I. scapularis nymphs survived for a year in prolonged questing under optimal environmental conditions in the laboratory and maintained a nymphal infection prevalence sufficient to effectively fulfill the enzootic cycle of Bb. In pursuit of our second objective, we confirmed the absence of Bb in ticks recovered from 55% of the mice vaccinated with rOspCCt, suggesting a partial transmission-blocking mechanism. The cocktail vaccine also generated antigen-specific antibodies in serum and partially protected the tick-challenged mice from borrelial dissemination. Additionally, serum from the challenged mice (55%) was shown to contain effective neutralizing antibodies against Bb. In addition, our study provided ample evidence that antibodies to OspC do not cross-react directly with OspA and vice versa, which further validates the transmission-blocking function of OspC independently of OspA. Conclusion. We investigated the transmission competence of I. scapularis for an extended period, and this study raises important questions regarding the prolonged survival of Bb-infected host-seeking nymphal I. scapularis ticks. Infected ticks can potentially increase the risk of Lyme disease if environmental conditions such as temperature and humidity become favorable to the enzootic cycle of Bb in regions currently classified as non-endemic. The 55% efficacy observed in the OspCCt-derived vaccine can be explained by the difference between OspC types present in the cocktail and those present in the ticks used for the challenge. Although OspC remains a potential vaccine candidate for Lyme disease, future studies must account for additional OspC types and challenges, including field-caught infected ticks. Our study also shows that OspA and OspC are two entirely different antigens with no antigen-antibody cross-reactivity. Antibodies to OspC alone are responsible for the observed transmission-blocking mechanism, independently of OspA.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0001-6456-601X

DOI

10.21007/etd.cghs.2023.0614

Additional Files
2023-001=Samanta-DOA.pdf (171 kB)
Declaration of Authorship
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