All ETDs from UAB

Advisory Committee Chair

David E Briles

Advisory Committee Members

Scott R Barnum

William Benjamin

Shaper Mirza

Alexander J Szalai

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Current pneumococcal vaccines containing pneumococcal capsular polysaccharide antigens protect against infection by pneumococci. However, this protection is limited to strains expressing the capsule types included in the vaccine. Since the introduction of these vaccines, the increase in incidence of infection by serotypes not targeted by the vaccine, a process called "serotype replacement," and the growing antibiotic resistance of replacement strains both stress the need for vaccines with broader protection. The alpha-helical region of a protein present on all clinical isolates of pneumococci, Pneumococcal Surface Protein A (PspA), elicits protective immunity. Downstream of the alpha-helical region is a highly conserved region containing a proline residue every 2 to 3 amino acids called the proline-rich region (PR). In certain serotypes, the PR can be interrupted by a conserved block of 27 amino acids lacking proline residues, termed the non-proline block (NPB). Herein we demonstrate that protective immunity against pneumococcal sepsis can be achieved by vaccination with both the proline repeats and the NPB of the PR. Further, we show that monoclonal antibodies to these epitopes passively protect mice against infection. The currently accepted method for gauging the protective capacity of the immune response of humans vaccinated with PspA is to measure the ability of human immune sera to passively protect mice against pneumococcal infection. In contrast, an Opsonization Phagocytosis Killing Assay (OPKA) is used to measure the immune response the protective capacity of antisera raisied against polysaccharide vaccines. Historically, this OPKA was not able to measure the ability of anti-PspA sera to mediate killing of pneumococci. By modifying the OPKA procedure and by employing a target strain sensitive to complement-deposition, we achieved killing in an OPKA that correlates with passive protection of mice using human anti-PspA sera. Furthermore, rabbit sera against both the alpha-helical and proline-rich regions of PspA mediated killing in this modified OPKA. In conclusion, we have demonstrated that the proline-rich region of PspA when used as a vaccine is capable of eliciting antibody-mediated protection and that capacity of antisera raised against both the alpha helical and proline rich regions of PspA can be measured in a modified OPKA.

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