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. 2015 Jun 17:2:15021.
doi: 10.1038/mtm.2015.21. eCollection 2015.

Structure-based redesign of lysostaphin yields potent antistaphylococcal enzymes that evade immune cell surveillance

Kristina Blazanovic  1 , Hongliang Zhao  2 , Yoonjoo Choi  3 , Wen Li  1 , Regina S Salvat  1 , Daniel C Osipovitch  4 , Jennifer Fields  5 , Leonard Moise  6 , Brent L Berwin  7 , Steven N Fiering  7 , Chris Bailey-Kellogg  3 , Karl E Griswold  8
Affiliations

Structure-based redesign of lysostaphin yields potent antistaphylococcal enzymes that evade immune cell surveillance

Kristina Blazanovic et al. Mol Ther Methods Clin Dev. .

Abstract

Staphylococcus aureus infections exert a tremendous burden on the health-care system, and the threat of drug-resistant strains continues to grow. The bacteriolytic enzyme lysostaphin is a potent antistaphylococcal agent with proven efficacy against both drug-sensitive and drug-resistant strains; however, the enzyme's own bacterial origins cause undesirable immunogenicity and pose a barrier to clinical translation. Here, we deimmunized lysostaphin using a computationally guided process that optimizes sets of mutations to delete immunogenic T cell epitopes without disrupting protein function. In vitro analyses showed the methods to be both efficient and effective, producing seven different deimmunized designs exhibiting high function and reduced immunogenic potential. Two deimmunized candidates elicited greatly suppressed proliferative responses in splenocytes from humanized mice, while at the same time the variants maintained wild-type efficacy in a staphylococcal pneumonia model. Overall, the deimmunized enzymes represent promising leads in the battle against S. aureus.

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Figures

Figure 1
Figure 1
Epitope map of wild-type LSTCAT catalytic domain and positions of deimmunizing mutations. A map of predicted epitopes in wild type LSTCAT is shown as a bar plot, where amino acid positions are indicated on the x-axis and the number of MHC II alleles predicted to bind a given nonamer peptide are shown on the y-axis. Epitopes are indicated by bars at the first residue of the nonamer peptide. Epitopes cluster into five distinct regions shown as purple, blue, green, orange, and red bars, respectively. The positions of deimmunizing mutations are indicated with black triangles on the x-axis, and wild type residues are noted in red text. The mutational compositions of the seven characterized variants are shown below in black text, and the fraction of putative epitopes deleted by each mutation is indicated in parenthesis next to the mutant residue (i.e., “3/10” indicates deletion of 3 out of 10 putative epitopes in that cluster).
Figure 2
Figure 2
Two stage computationally-driven development of LSTCAT variants: Stage 1-left; stage 2-right. A structural map of putative LSTCAT epitope content bridges the Design Priors for both stages. Thick red tubes indicate dense, overlapping MHC II binding peptides, and thin white tubes indicate no predicted MHC II binding. (a) Priors for the initial design calculations identified allowed mutations based on their frequencies in a multiple sequence alignment of LSTCAT homologs, represented here as a logo plot. (b) Initial EpiSweep calculations generated an undominated Pareto optimal frontier, and near-optimal frontiers, at each mutational load. Mutational loads are: red = 8, blue = 7, purple = 6, green = 5, orange = 4, dark blue = 3, yellow = 2, black = wild type. X-axis is reduction in predicted epitope content and y-axis is computed molecular energy in arbitrary units, with wild type reference at 0. (c, top) Characterization of 15 individual point mutations that appeared frequently in designs from b. Expression level is shown in blue on left y-axis and specific lytic activity towards S. aureus in green on the right y-axis. (c, bottom) Predicted number of epitopes deleted by each point mutation. Validated substitutions selected for stage 2 protein design are highlighted in green boxes. (d) Priors for stage 2 design calculations were constrained to twelve specific mutations validated in c. Mutation S122D was included in all designs. (e) Final EpiSweep calculations generated a new set of Pareto optimal and near-optimal designs at mutational loads of 2–8 per enzyme, and fourteen candidates were selected for experimental analysis (indicated as filled diamonds). (f, top) Functional characterization of seven high expressing constructs. Apparent melting temperature is shown in purple on the left y-axis, and specific lytic activity in green on the right y-axis. (f, bottom) Measured MHC II binding for the seven engineered variants. Five corresponding constituent peptides from each LST design were evaluated for binding to eight different human MHC II proteins. Peptides were categorized as strong – red (IC50 < 0.1 µmol/l), moderate – orange (0.1 µmol/l ≤ IC50 < 1 µmol/l), weak – yellow (1 µmol/l ≤ IC50 < 10 µmol/l), or nonbinders (IC50 ≥ 10 µmol/l), and total counts of binders are shown for each variant. Variants F4a and F8a, highlighted in green boxes, were chosen for further analysis.
Figure 3
Figure 3
In vivo efficacy and immunogenicity analysis. (a) Bacterial burden in the lungs of C57Bl/6 mice following infection with S. aureus and treatment with wild type LST (red), variant F4a (dark blue), variant F8a (light blue), or a PBS control (black). Shown are mean values and standard deviations (n = 6 per group). (b) BLT mice (all humanized from a single donor) were immunized subcutaneously with either wild type LST, variant F4a, or variant F8a, and splenocytes were harvested and restimulated ex vivo with the same protein or DMSO. Proliferation was measured as uptake of tritiated thymidine. Shown are mean values and standard deviations (n = 4 per group, pooled and measured in triplicate). (c) Transgenic DR4 mice were immunized with multiple subcutaneous injections of wild-type LST. Following the final boost, mice were allowed to recover for 20 weeks, divided into two groups, and rechallenged with either wild type LST or variant F4a. Splenocytes were harvested and restimulated ex vivo with the rechallenge protein or DMSO, and proliferation was measured as uptake of tritiated thymidine. Shown are mean values and standard deviations (n = 5 per group, pooled and measured in triplicate). Statistical significance was assessed by one-way analysis of variance (panel a) or two-way analysis of variance (panels b and c). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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