Temperature-mediated recombinant anthrax protective antigen aggregate development: Implications for toxin formation and immunogenicity |
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Institution: | 1. Technische Thermodynamik, Universität Bremen, Badgasteiner Str. 1, 28359 Bremen, Germany;2. MAPEX Center of Materials and Processes, Universität Bremen, Germany;3. School of Engineering, University of Aberdeen, Fraser Noble Building, Aberdeen AB24 3UE, UK;4. Erlangen Graduate School in Advanced Optical Technologies (SAOT), Universität Erlangen-Nürnberg, Germany |
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Abstract: | Anthrax vaccines containing recombinant PA (rPA) as the only antigen face a stability issue: rPA forms aggregates in solution after exposure to temperatures ?40 °C, thus losing its ability to form lethal toxin (LeTx) with Lethal Factor. To study rPA aggregation’s impact on immune response, we subjected rPA to several time and temperature combinations. rPA treated at 50 °C for 30 min formed high mass aggregates when analyzed by gel electrophoresis and failed to form LeTx as measured by a macrophage lysis assay (MLA). Aggregated rPA-formed LeTx was about 30 times less active than LeTx containing native rPA. Mice immunized with heat-treated rPA combined with Al(OH)3 developed antibody titers about 49 times lower than mice immunized with native rPA, as measured by a Toxicity Neutralization Assay (TNA). Enzyme Linked Immunosorbent Assay (ELISA) of the same immune sera showed anti-rPA titers only 2–7 times lower than titers elicited by native rPA. Thus, rPA’s ability to form LeTx correlates with its production of neutralizing antibodies, and aggregation significantly impairs the protein’s antibody response. However, while these findings suggest MLA has some value as an in-process quality test for rPA in new anthrax vaccines, they also confirm the superiority of TNA for use in vaccine potency. |
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Keywords: | Anthrax Vaccine Stability Protective antigen Aggregation PA"} {"#name":"keyword" "$":{"id":"k0035"} "$$":[{"#name":"text" "_":"protective antigen rPA"} {"#name":"keyword" "$":{"id":"k0045"} "$$":[{"#name":"text" "_":"recombinant PA LeTx"} {"#name":"keyword" "$":{"id":"k0055"} "$$":[{"#name":"text" "_":"lethal toxin MLA"} {"#name":"keyword" "$":{"id":"k0065"} "$$":[{"#name":"text" "_":"macrophage lysis assay TNA"} {"#name":"keyword" "$":{"id":"k0075"} "$$":[{"#name":"text" "_":"Toxicity Neutralization Assay LF"} {"#name":"keyword" "$":{"id":"k0085"} "$$":[{"#name":"text" "_":"lethal factor EF"} {"#name":"keyword" "$":{"id":"k0095"} "$$":[{"#name":"text" "_":"edema factor RT"} {"#name":"keyword" "$":{"id":"k0105"} "$$":[{"#name":"text" "_":"room temperature |
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