Induction of long-lived humoral immunity to poxvirus infections
Creators: name: Eldi, Preethi
Publisher: name: The Australian National University
Year: 2015
Subjects: Somatic Hypermutation
Antibody
Poxvirus
Ectromelia
Vaccination
T‐follicular helper cells
T‐follicular regulatory cells
Germinal centre B cells

description: Smallpox eradication through the use of a live-virus vaccine is one of the most successful public health endeavors of modern medicine. Humoral immunity against the smallpox virus, characterized by neutralizing antiviral antibody response, is stable, lasts for decades, and is considered a valuable benchmark of the functional attributes of a good vaccine. Indeed, neutralizing antibody is the best correlate of long-term protective immunity for all the currently licensed clinically effective viral vaccines. In this thesis, we investigated the contribution of host and viral factors that are responsible for the induction of long-lived antiviral humoral immunity. Crosstalk between T follicular helper (TFH) cells, T follicular regulatory (TFR) cells and B cells is essential for induction of optimal germinal center (GC) responses and subsequent high affinity antibody generation. We hypothesized that the escalating concentrations of replicating virus (antigens) provide important cues to these immune cell subsets in the GC response, and these are critical for induction and maintenance of robust, long-lived humoral immunity. To test this, we used mousepox, the disease caused by ectromelia virus (ECTV) in mice and an excellent surrogate small animal model for smallpox and generalized viral infections. We found that replication-competent wild type ECTV induced more robust TFH and GC responses, contemporaneous with significantly higher neutralizing antibody titers, compared with a poorly replicating ECTV strain. We were able to demonstrate via passive transfer-challenge experiments, that antibodies generated following infection with the poorly replicating strain were not as effective in controlling a secondary challenge as compared to antibodies generated following infection with a replicationcompetent virus. Similar differences were observed between replicating and non-replicating strains of both Vaccinia virus (VACV), vaccine-strain Modified Vaccinia Ankara (MVA) and influenza A virus. Additionally, strategies to enhance the immunogenicity of the replicating-inefficient virus by changes in antigen availability, via alterations in the dose and route of administration were evaluated. Importantly, we have discussed the ability to prospectively predict the magnitude and potency of longlived neutralizing antibody induction using immunological biomarkers in blood, which would particularly aid the field of vaccine biology. Using replication-competent and replication-poor recombinant ECTV expressing mutant versions of hen egg lysozyme (HEL) in combination with SWHEL transgenic B cells, we extended our studies to antigen-specific B cell responses in poxvirus infection. To date, affinity maturation has been studied extensively using protein or protein-hapten immunization and the current study is the first, to our knowledge, to systematically examine antigen-specific B cell responses and affinity maturation to a virus-expressed antigen during infection of a natural host under physiological conditions. In mice transferred with SWHEL B cells and infected with recombinant ECTV encoding HEL, replicating virus induced massive proliferation of SWHEL B cells, somatic hypermutations (SHM) in the variable region exon of SWHEL Ig heavy chain, and affinity maturation. In contrast, poorly replicating viruses induced minimal proliferation of SWHEL B cells with little or no SHM events. Significantly, robust SWHEL B cell responses, associated with augmented proliferation, induction of SHM events and secretion of antibody, occurred when the dose and route of administration of poorly replicating ECTV encoding HEL was altered. Collectively, the data presented in this thesis provide valuable insights into the rational design of poxviral vaccine vectors to achieve an efficacious induction of long-lasting immune memory.