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CD8 + cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8 + T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. Fender stratocaster serial number z95. To induce a robust antigen-specific CD8 + T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8 + T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant. DRelative potency in comparison to αGalCer; mo mouse, hu human Several studies have reported that αGalCer is an effective adjuvant for the elicitation of epitope-specific CD8 + T cells against the model protein antigen ovalbumin [, –], or for enhancing antigen-specific CD8 + T cell responses upon immunization with live viral vaccines [, ].

We recently demonstrated the potency of αGalCer as a CD8 + T cell adjuvant for in vivo immunogenicity and protection studies which exploited pathogen-derived protein antigens ([] and unpublished data). It is noteworthy that the activation of NKT cells by αGalCer results in the production of proinflammatory as well as immunoregulatory cytokines and chemokines (reviewed in []).

Since DC activation and licensing as well as CD8 + T cell differentiation require proinflammatory cytokines and chemokines, efforts to synthesize and identify αGalCer analogues that elicit proinflammatory activity are being sought but are not discussed here ( see; reviewed in Ref. Herein, we describe protocols for the preparation of protein antigens and αGalCer adjuvant, mouse immunization, and assessment of antigen-specific CD8 + T cell response, while analysis of antigen-specific antibody response and protective immunity is not described. Experiments that assess immunogenicity require a large quantity of pure protein antigens, which, unlike model antigens, are not typically available commercially. Instead, the desired proteins are produced by recombinant DNA methods in Escherichia coli and then purified to a high degree of purity through engineered tags with the use of affinity resin.

We designed, cloned, and produced nine recombinant VACV-derived protein subunits (rA3L 62–319, rD1R 565–844, rD5R 330–470, rE2L 26–301, rF4L 1–319, rJ6R 188–466, and rL4R 33–249, as well as epitope-engineered subunits L4R/B8R 70–79 and L4R/A34R 32–90), all of which contain a C-terminal histidinetag to facilitate purification (; []). Upon immunization of mice, αGalCer-formulated antigens induced robust epitope-specific CD8 + T cell and antibody responses that were detectable in blood and in various tissues (). Vaccination with adjuvant and microbial antigen induced protective immunity against subsequent microbial challenge ( and unpublished data). Our immunization approach is suitable for elicitation of antigen-specific CD8 + T cell and antibody responses to recombinant proteins that are produced as insoluble inclusion bodies (IB). Results that have emerged from the use of approaches described herein demonstrate the efficacy of the proposed immunization strategy for generating high-frequency antigen-specific CD8 + T cells against microbial protein antigens that can be exploited for immunologic and vaccine studies. Design of recombinant VACV-derived protein antigen subunits that contain immune CD8 + T cell epitopes. Schematic chart of engineered proteins showing nine VACV antigens with amino acid positions selected for the design of a truncated recombinant subunit.

Shown are the location and sequences of immune CD8 + T cell epitopes, and their N- and C-terminal tag sequences introduced to facilitate expression and purification of recombinant proteins in E. Soluble protein rL4R 33–249 was engineered with B8R 70–79 and A34R 82–90 immune epitopes to facilitate expression of soluble antigens by E.