DNA Immunization: Basic Mechanisms of the DNA-Raised Antibody Response Using an Influenza Hemagglutinin-Expressing Plasmid: A Dissertation

Abstract

In DNA immunization a plasmid expressing an antigen of interest is inoculated into an animal and antigen-specific humoral and cellular immune responses are raised. In this dissertation we sought to further our understanding of antibody responses raised following DNA inoculation. Specifically, we examined the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, the role of CD4+ and CD8+ T cells in the DNA-raised antibody response, the longevity of functional antigen expression, and the nature of the antigen presenting cell. In all of these studies mice were immunized with an influenza hemagglutinin-expressing plasmid and plasmid was delivered by either the gene gun or intramuscular routes of inoculation. To examine the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, responses raised in gene gun immunized mice were compared to responses raised in mice primed with an influenza infection. Antibody and antibody secreting cell (ASC) responses were analyzed at various times following immunization or sublethal infection for as long as 1.5 years. We found that the antibody response raised with a single gene gun immunization was similar in longevity to that raised in infection-primed mice. The long-term maintenance of the antibody response was associated with the localization of the majority of antibody secreting cells to the bone marrow. The kinetics of ASC bone marrow localization was 4-to-8 weeks slower in DNA-immunized than infection primed mice. This corresponded to a slower rise in the antibody response to plateau levels in DNA-immunized mice. We hypothesize that it is possible that the difference in kinetics may be linked to differences in the time course and dose of antigen expression following DNA immunization and a natural infection. Antibody and ASC responses were also compared following a challenge influenza virus infection. We found that DNA-immunized and infection-primed mice responded similarly in the acute post challenge phase with increases in antibody secreting cells in the mediastinal lymph nodes. While only DNA-immunized mice had post challenge increases in antibody, the antibody response remained 3-to-4 fold lower than post challenge responses in infection primed mice. We suggest that despite post challenge increases in these responses in DNA-immunized mice that the immune response raised with DNA immunization efficiently limited replication of the challenge virus and thus limited the post challenge antibody response. We also addressed the role that CD4+ and CD8+ T cells played in the ability to prime and boost the DNA-raised antibody response. To answer this question mice were in vivo depleted of CD4+ or CD8+ T cells for 3 weeks prior to through 2 weeks following DNA immunization or boost. Antibody responses were measured 4 and 8 weeks after DNA prime and 2 weeks after DNA boost. For both the gene gun and intramuscular routes of inoculation, the antibody response was independent of CD8+ T cells, but dependent on CD4+ T cells. The presence of CD4+ T cells was required at the time of DNA immunization, but not at the time of DNA boost. The absence of CD4+ T cells at the time of DNA delivery resulted in a four week delay in the appearance of antibody. Since influenza hemagglutinin has been characterized as a T-dependent antigen the requirement for CD4+ T cells at the time of DNA prime was not surprising, but the appearance of a delayed H1-specific antibody response suggested that DNA-expressed antigen had continued to be available to prime CD4+ T cells as they reappeared following the disappearance of depleting antibody. The independence of the antibody response on the presence of CD8+ T cells suggested that DNA-primed H1-specific CD8+ T cells did not limit the plateau level of response or the ability to boost a suboptimal response. The results from our CD4+ T cell depletion experiment suggested that DNA-expressed antigen continued to be available for an extended period of time following immunization. To examine the duration of functional antigen expression for raising an antibody response, mice lacking α/β T cells (TCR-/-) were immunized with DNA or immunized with hemagglutinin protein. Naive T cells from TCR+/+ mice were transferred into the immunized TCR-/- mice on various days post DNA or protein immunization. The results from these studies show that antigen is available to raise antibody longer following DNA immunization than following a protein immunization. This result is likely due to continued expression of plasmid DNA. We found differences in the longevity of antigen expression following gene gun and intramuscular routes of inoculation. For gene gun immunizations, not intramuscular immunizations, approximately 90% of functional antigen was lost within one week of immunization. We suggest that this is consistent with a role for antigen expression by transfected cells within the target site, the epidermis, which is largely lost by 1-2 weeks following gene gun immunization. We also found that following intramuscular immunization the dominant IgG isotype changed with time of TCR+/+ T cell transfer. By contrast, there was no change in the dominant isotype following gene gun immunizations. These results suggest that the factor(s) that contribute to the development of the Th1-bias seen following intramuscular DNA immunizations are lost early. To examine the nature of the antigen presenting cell following DNA immunization, dendritic cells were sorted from the inguinal lymph nodes and spleens of gene gun or intramuscularly immunized mice on various days following DNA delivery. The dendritic cell (CD11c+) and non-dendritic cell (CD11c-) populations were used in restimulation assays with H1-specific T cell clones. Despite similar titers of raised antibody in gene gun and intramuscularly immunized mice, H1-specific antigen presenting dendritic cells were isolated from the inguinal lymph nodes and spleens of gene gun, but not intramuscularly immunized mice. Antigen presentation by dendritic cells was detected for as long as 21 days following gene gun delivery. We hypothesize that the inability to detect dendritic cell presentation of antigen following intramuscular DNA delivery may be due to a more broad distribution of antigen presenting cells, different properties of antigen presenting cells, and/or the contribution of other non-dendritic cells to antigen presentation following intramuscular, but not gene gun, immunizations. We present our results within a model for the initiation and maintenance of DNA-raised antibody responses. Within this model our data specifically contribute to understanding the initiation and generation of the DNA-raised antibody response within lymphoid tissue and the maintenance of the DNA-raised antibody response.