NYMC Student Theses and Dissertations

Date of Award

8-31-2020

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Paul Arnaboldi, Ph.D.

Second Advisor

Dana G. Mordue, Ph.D.

Third Advisor

Alison A. McCormick, Ph.D.

Abstract

Yersinia pestis, the causative agent of plague, has killed millions throughout human history. Today, it remains a significant threat for use as a bioweapon. Naturally occurring antibiotic resistance has been observed in Y. pestis isolates, and resistant strains have been engineered for use in biological warfare. Vaccines remain our best means of protection against plague, as well as countless other global contagions. We have developed a vaccine consisting of two Y. pestis virulence proteins, LcrV (V) and F1, conjugated to Tobacco Mosaic Virus (TMV), a safe, non-replicating plant virus that can be administered mucosally, providing complete protection against pneumonic plague, the deadliest form of the disease and the one most likely to be seen in a bioterror attack. The goal of this dissertation was to understand the mechanism(s) by which intranasal (i.n.) administration of TMV-F1 and TMV-V conjugates enhanced vaccine-specific immunity in a mouse model of pneumonic plague. I hypothesized that the recombinant TMV (rTMV) virion was enhancing the immune response to the associated subunit proteins through direct uptake of the virus by APCs and could induces responses analogous to VLP vaccines. The resulting high-density display of antigens on the virus acts as an immunostimulant when compared to their soluble forms, without the need for adjuvants. My research was divided into three overarching aims: (1) APC uptake of TMV conjugates, (2) T and B cell responses from TMV conjugate immunization, and (3) use of TMV conjugates in a single dose plague vaccine and its efficacy in controlling inflammation and bacterial dissemination. Challenge experiments utilized BSL-2 Y. pestis strains with the chromosomal pgm locus deleted (CO92pgm−) or mutated (KIM5caf1A). We found TMV conjugation to F1 and V increased their uptake into splenic and mucosal B-cells, and xvi BMDCs in vitro, as well lung DCs in vivo, and was not due to any TMV-specific mechanism. We could not show that these vaccines were immunostimulants, as both conjugates failed to upregulate MHC II, CD80, or CD86 over that of unstimulated CD11c+ DCs in vitro. We also demonstrated that generation of antibody‐based, but not T cell-based, immunity could be attributed to vaccination with TMV conjugates. Mice immunized i.n. with TMV-F1, specifically, predictably developed high F1-specific serum IgG titers after single and multiple administrations. When applied to an infection model using F1+ Y. pestis CO92pgm-, we observed that the protective memory response induced by TMV vaccination did not require CD4+ or CD8+ T cells. This protection is lost against F1- Y. pestis, where mice vaccinated with TMV-F1+TMV-V demonstrated poor protection. Finally, TMV-F1+TMV-V vaccination showed significant reduction in histopathology, bacterial burden, and inflammatory cytokine production in the days following challenge with 100 LD50 Y. pestis CO92pgm-. Pneumonic challenge with this strain resulted in systemic dissemination of the bacteria in all groups, but only TMV-F1+TMV-V immunized mice rapidly cleared bacteria from the spleen and liver within 5 days. There was a direct correlation between pre-challenge serum F1 titers and protection in all immunized mice, strongly suggesting a role for anti-F1 antibody in the neutralization and/or opsonization of Y. pestis in this model. Furthermore, the TMV-F1+TMV-V i.n. vaccine was capable of mediating single-dose protection. Taken together, my data indicates the usefulness of rTMV – an inactivated virus – as a carrier for VLP immunogenic presentation and mucosal delivery of whole protein antigens. This particulate vaccine has the ability to target antigens to DCs at mucosal surfaces, which elicits a potent antiviral-like immune response to the bound proteins in the form of xvii neutralizing IgG antibodies. T follicular helper (TFH) cells may be required in the activation of naïve B cells and/or activation and maintenance of memory B cells in secondary lymphoid organs. Further work should identify where vaccine-specific IgG+ memory B cells reside (lymph nodes or spleen) after i.n. immunization with the TMV conjugates, the duration of B cell memory in this model, and reasons for why TMV-V immunization cannot generate 100% protection against plague.

Download

Share

COinS