Date on Master's Thesis/Doctoral Dissertation

5-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Shirwan, Haval

Author's Keywords

SA-4-1BBL; Adjuvants; Vaccine; Plague; Breast cancer

Subject

Vaccines--Technological innovations; Vaccines--Research

Abstract

Vaccines against infectious diseases are one of the most critical accomplishments in modern medicine. Despite significant progress in vaccinology, there is still a dire need for developing vaccines against various acute and chronic infections and cancer. In general, vaccines are categorized as prophylactic, given to healthy individuals to prevent disease, and therapeutic, administered to people who already have disease. As such, the nature, quality, and quantity of immune responses required for the efficacy of these two types vaccines are different. Prophylactic vaccines against infectious diseases primarily rely on the generation of neutralizing high titers of antibody for their efficacy. These vaccines are generally effective because they target a host with an unaltered and competent immune system. In marked contrast, the efficacy of therapeutic vaccines has been a major challenge since they are administered into a host with a compromised immune system. Therapeutic vaccines need not only to generate effective adaptive cellular, particularly CD8+ T cell, immune responses to chronic infection and cancer, but they also need to overcome various immune evasion mechanisms employed by infection and progressing tumor. For both types of vaccines, the generation of a long-lasting adaptive immunity is the key. Historically, prophylactic vaccines against infections were made from live-attenuated or inactivated forms of the microbes, but there were concerns about stability, side effects and safety of such vaccines. Advancements in molecular biology and DNA technologies led to the development of recombinant subunit vaccine with well-defined antigens. In particular, vaccines based on recombinant proteins present an attractive approach because of their ease of production, storage, distribution, and safety profiles. However, recombinant protein based subunit vaccines are poorly immunogenic and require adjuvants for efficacy. Most of the adjuvants that have been approved for clinical use, and those under development primarily target innate arm of the immune system for the generation of subsequent adaptive immunity. Key to the initiation of adaptive immune responses is the interactions between an APC and T cells and acquisition of 3 distinct singles by T cells. Signal 1 is delivered by the interaction of TCR on T cells with an MHC/peptide complex on APC. This signal is then qualified by costimulatory receptor ligand interaction on the APC and T cells, providing signal 2. Signal 3 is provided by various cytokines elaborated by activated APCs and T cells and critical for the expansion of the immune response. The lack of costimulation during these interactions results in T cell anergy or apoptosis. Costimulation is not only important for the generation of adaptive immunity, but also is involved in the regulation of the various immune evasion mechanisms employed by cancer and chronic infections. Therefore, we hypothesized that costimulatory ligands may serve as the preferred adjuvants for generating effective and long-lasting adaptive immunity. We particularly focused on the natural costimulatory ligands of tumor necrosis factor (TNF) family given their pleiotropic function on cells of innate, adaptive, and regulatory immunity. The TNF family represents a critical group of costimulatory molecules since their receptors (TNFR) are inducibly expressed on activated cells and may serve as preferred targets for antigen specific responses through induction of expansion, survival of T cells and establishment of long term memory. Among these family members, 4-1BB/4-1BBL interaction has received the most attention as signaling through 4-1BB provides essential signals for CD8+ T cell expansion, effector function, and survival. Importantly, this signaling also endows effector CD8+ T cells resistant to suppression by regulatory T cells that are the predominant mechanism of immune evasion used by cancer and chronic infections. Since 4-1BBL has costimulatory function as a cell surface membrane-bound protein and has no function in soluble form, our laboratory has previously generated a novel form of this molecule chimeric with streptavidin, SA-4-1BBL. This molecule was demonstrated to have robust costimulatory activity with a Th1 bias as a soluble protein. The main premise of this PhD thesis is to use SA-4-1BBL as an adjuvant platform to develop adjuvant systems for subunit vaccines with desired immune activities for targeted indications. We particularly focused on subunit vaccines against two indications; Y. pestis and breast cancer for the development of prophylactic and therapeutic vaccines, respectively. First, we tested if SA-4-1BBL can improve the immune efficacy of a lead subunit vaccine, rF1-V (a recombinant Y. pestis fusion protein), adjuvanted with alum with a Th2 bias against plague. Inasmuch as the lead candidate vaccine generates a Th2 response, and Th1 cellular responses have been shown to be important in protection against Y. pestis infection, we hypothesize that SA-4-1BBL as a Th1 adjuvant will improve the immune efficacy of the lead candidate vaccine. Single immunization with a vaccine formulation containing rF1-V as antigen and SA-4-1BBL as single adjuvant generated increased TNFa and IFN signature cytokines for Th1 responses in both CD4+ and CD8+ T cells without detectable antibody titers against rF1-V. This vaccine formulation protected 20% of mice against bubonic plague. However, in a prime-boost setting, SA-4-1BBL and rF1-V generated long lasting high titers of antibodies and protected all mice from bubonic Y. pestis infection. Alum adjuvanted rF1-V vaccine generated high titers of antibodies against rF1-V without a significant Th1 response, and protected 80% of mice against bubonic plague. A combination of SA-4-1BBL and alum as an adjuvant system generated at balanced Th1 cellular and humoral responses that resulted in 100% protection in bubonic plague model. Next, we tested if SA-4-1BBL has efficacy as adjuvant component of a Her-2/neu protein-based subunit vaccine against breast cancer and if the therapeutic efficacy of this subunit vaccine can further be improved by using toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPL) as adjuvant system. We hypothesize that MPL will work in synergy with SA-4-1BBL by targeting antigen presenting cell for activation, antigen presentation to T cells, leading to T cell activation and up regulation of 4-1BB receptor. Activated T cells will then serve as a direct target of SA-4-1BBL for expansion, acquisition of effector function, and establishment of long-term memory. A prime-boost immunization with extracellular domain of the rat Her-2/neu protein and SA-4-1BBL resulted in eradication of established Her-2/neu expressing A2L2 tumors in 10% of BALB/c mice. In contrast, MPL monotherapy did not have a therapeutic effect. However, vaccination with combined adjuvants resulted in eradication of established tumors in 30% of BALB/c mice, and showed better therapeutic efficacy over individual therapies. Furthermore, immunization with combined adjuvants resulted in eradication A2L2 tumors in 20% of tolerogenic BALB/neuT mice. Depletion of Tregs prior to tumor challenge increased the efficacy of combined adjuvants to 40%. The therapeutic efficacy of combined adjuvant platform correlated with increased tumor specific killing response and pro-inflammatory cytokine IFN production. The combination of SA-4-1BBL and MPL achieved therapeutic efficacy in the absence of detectable toxicity as assessed by various indicators of toxicity, including liver enzymes, total number of various lymphocyte populations in several lymphoid tissues, vaccine-induced organ damage, and histological analysis of the liver. Taken together, these data provide scientific rationale for using SA-4-1BBL as a novel adjuvant platform with other adjuvants having synergistic immune activities for the development of subunit vaccines against intracellular infections and cancer.

Download

Share

COinS