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Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin.

ABSTRACT: The defining property of core streptavidin (cSA) is not only its high binding affinity for biotin but also its pronounced thermal and chemical stability. Although potential applications of these properties including therapeutic methods have prompted much biological research, the high immunogenicity of this bacterial protein is a key obstacle to its clinical use. To this end, we have successfully constructed hypoimmunogenic cSA muteins in a previous report. However, the effects of these mutations on the physicochemical properties of muteins were still unclear. These mutations retained the similar electrostatic charges to those of wild-type (WT) cSA, and functional moieties with similar hydrogen bond pattern. Herein, we performed isothermal titration calorimetry, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to gain insight into the physicochemical properties and functions of these modified versions of cSA. The results indicated that the hypoimmunogenic muteins retained the biotin-binding function and the tetramer structure of WT cSA. In addition, we discuss the potential mechanisms underlying the success of these mutations in achieving both immune evasion and retention of function; these mechanisms might be incorporated into a new strategy for constructing hypoimmunogenic proteins.

SUBMITTER: Yumura K

PROVIDER: S-EPMC3588917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Therapeutic proteins are yielding ever more advanced and efficacious new drugs, but the biological origins of these highly effective therapeutics render them subject to immune surveillance within the patient's body. When recognized by the immune system as a foreign agent, protein drugs elicit a coordinated response that can manifest a range of clinical complications including rapid drug clearance, loss of functionality and efficacy, delayed infusion-like allergic reactions, more serious anaphylactic shock, and even induced auto-immunity. It is thus often necessary to deimmunize an exogenous protein in order to enable its clinical application; critically, the deimmunization process must also maintain the desired therapeutic activity.To meet the growing need for effective, efficient, and broadly applicable protein deimmunization technologies, we have developed the EpiSweep suite of protein design algorithms. EpiSweep seamlessly integrates computational prediction of immunogenic T cell epitopes with sequence- or structure-based assessment of the impacts of mutations on protein stability and function, in order to select combinations of mutations that make Pareto optimal trade-offs between the competing goals of low immunogenicity and high-level function. The methods are applicable both to the design of individual functionally deimmunized variants as well as the design of combinatorial libraries enriched in functionally deimmunized variants. After validating EpiSweep in a series of retrospective case studies providing comparisons to conventional approaches to T cell epitope deletion, we have experimentally demonstrated it to be highly effective in prospective application to deimmunization of a number of different therapeutic candidates. We conclude that our broadly applicable computational protein design algorithms guide the engineer towards the most promising deimmunized therapeutic candidates, and thereby have the potential to accelerate development of new protein drugs by shortening time frames and improving hit rates.

Project description:BACKGROUND:Eradication of Plasmodium falciparum malaria will likely require a multivalent vaccine, but the development of a highly efficacious subunit-based formulation has been challenging. We previously showed that production and immunogenicity of two leading vaccine targets, PfMSP119 (blood-stage) and Pfs25 (sexual stage), could be enhanced upon genetic fusion to merozoite surface protein 8 (PfMSP8). Here, we sought to optimize a Pfs25-based formulation for use in combination with rPfMSP1/8 with the goal of maintaining the immunogenicity of each subunit. METHODS:Comparative mouse studies were conducted to assess the effects of adjuvant selection (Alhydrogel vs. glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE)) and antigen dose (2.5 vs. 0.5 ?g) on the induction of anti-Pfs25 immune responses. The antibody response (magnitude, IgG subclass profile, and transmission-reducing activity (TRA)) and cellular responses (proliferation, cytokine production) generated in response to each formulation were assessed. Similarly, immunogenicity of a bivalent vaccine containing rPfMSP1/8 and rPfs25/8 was evaluated. RESULTS:Alum-based formulations elicited strong and comparable humoral and cellular responses regardless of antigen form (unfused rPfs25 or chimeric rPfs25/8) or dose. In contrast, GLA-SE based formulations elicited differential responses as a function of both parameters, with 2.5 ?g of rPfs25/8 inducing the highest titers of functional anti-Pfs25 antibodies. Based on these data, chimeric rPfs25/8 was selected and tested in a bivalent formulation with rPfMSP1/8. Strong antibody titers against Pfs25 and PfMSP119 domains were induced with GLA-SE based formulations, with no indication of antigenic competition. CONCLUSIONS:We were able to generate an immunogenic bivalent vaccine designed to target multiple parasite stages that could reduce both clinical disease and parasite transmission. The use of the same PfMSP8 carrier for two different vaccine components was effective in this bivalent formulation. As such, the incorporation of additional protective targets fused to the PfMSP8 carrier into the formulation should be feasible, further broadening the protective response.

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Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin.

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