Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-γ production and IFN-γ-secreting cells

The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-γ activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-γ-mediated effector function, and a surrogate marker for IFN-γ-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8⁺ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8⁺ T cells and IFN-γ. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-γ ELISPOT, IFN-γ ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-γ spot-forming cells (SFCs) detected by ELISPOT (r²=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-γ activity

Optimal induction of antigen-specific CD8+ T cell responses requires bystander cell participation

Efficient activation of specific immune responses requires a concerted interaction between T cells and antigen-presenting cells. A requirement for bystander participation of CD4 T cells for expansion and maintenance of memory CD8 T cells has been noted in several models, but a role with regard to effector CD8 T responses has not been well-defined. In this report, the requirement of bystander participation for optimal induction of antigen-specific CD8 T cell effector function was determined by directly quantitating antigen-specific interferon- (IFN-) CD8 T cell responses by enzyme-linked immunospot assays, and by indirectly evaluating induction of the chemokine monokine induced by IFN- as a marker for IFN--mediated effector function. Our results demonstrate that bystander cell participation, mediated by CD4 T cell and natural killer (NK) cells, is required for optimal induction of antigen-specific CD8 T cell effector responses. Our data further establish a novel role for NK cells in the activation of antigen-specific immune responses. J. Leukoc. Biol. 72: 1164–1171; 2002

Extended immunization intervals enhance the immunogenicity and protective efficacy of plasmid DNA vaccines

Effective vaccines against infectious diseases and biological warfare agents remain an urgent public health priority. Studies have characterized the differentiation of effector and memory T cells and identified a subset of T cells capable of conferring enhanced protective immunity against pathogen challenge. We hypothesized that the kinetics of T cell differentiation influences the immunogenicity and protective efficacy of plasmid DNA vaccines, and tested this hypothesis in the Plasmodium yoelii murine model of malaria. We found that increasing the interval between immunizations significantly enhanced the frequency and magnitude of CD8+ and CD4+ T cell responses as well as protective immunity against sporozoite challenge. Moreover, the interval between immunizations was more important than the total number of immunizations. Immunization interval had a significantly greater impact on T cell responses and protective immunity than on antibody responses. With prolonged immunization intervals, T cell responses induced by homologous DNA only regimens achieved levels similar to those induced by heterologous DNA prime/ virus boost immunization at standard intervals. Our studies establish that the dosing interval significantly impacts the immunogenicity and protective efficacy of plasmid DNA vaccines

Transcriptionally active PCR for antigen identification and vaccine development: In vitro genome-wide screening and in vivo immunogenicity

We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data

The US capitol bioterrorism anthrax exposures: Clinical epidemiological and immunological characteristics

Background: Bioterrorism-related anthrax exposures occurred at the US Capitol in 2001. Exposed individuals received antibiotics and anthrax vaccine adsorbed immunization. Methods: A prospective longitudinal study of 124 subjects—stratified on the basis of spore exposure, nasopharyngeal culture results, and immunization status from inside and outside an epidemiologically defined exposure zone—was performed to describe clinical outcome and immune responses after Bacillus anthracis exposure. Antibody and cell-mediated immune (CMI) responses to protective antigen (PA) and lethal factor were assayed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. Results: Antibody and CMI dose-exposure responses, albeit generally of low magnitude, were seen for unimmunized subjects from inside, within the perimeter, and outside the exposure zone and in nonexposed control subjects. Anti-PA antibody and CMI responses were detected in 94% and 86% of immunized subjects. No associations were seen between symptoms and exposure levels or immune responses. Conclusions: Anthrax spores primed cellular and possibly antibody immune responses in a dose-dependent manner and may have enhanced vaccine boost and recall responses. Immune responses were detected inside the perimeter and outside the exposure zone, which implies more-extensive spore exposure than was predicted. Despite postexposure prophylaxis with antibiotics, inhalation of B. anthracis spores resulted in stimulation of the immune system and possibly subclinical infection, and the greater the exposure, the more complete the immune response. The significance of low-level exposure should not be underestimated