Antigen delivery format variation and formulation stability through use of a hybrid vector

Abstract

A hybrid biological-biomaterial antigen delivery vector comprised of a polymeric shell encapsulating an Escherichia coli core was previously developed for in situ antigen production and subsequent delivery. Due to the engineering capacity of the bacterial core, the hybrid vector provides unique opportunities for immunogenicity optimization through varying cellular localization (cytoplasm, periplasm, cellular surface) and type (protein or DNA) of antigen. In this work, three protein-based hybrid vector formats were compared in which the pneumococcal surface protein A (PspA) was localized to the cytoplasm, surface, and periplasmic space of the bacterial core for vaccination against pneumococcal disease. Furthermore, we tested the hybrid vector's capacity as a DNA vaccine against Streptococcus pneumoniae by introducing a plasmid into the bacterial core to facilitate PspA expression in antigen presenting cells (APCs). Through testing these various formulations, we determined that cytoplasmic accumulation of PspA elicited the strongest immune response (antibody production and protection against bacterial challenge) and enabled complete protection at substantially lower doses when compared to vaccination with PspA + adjuvant. We also improved the storage stability of the hybrid vector to retain complete activity after 1 month at 4 °C using an approach in which hybrid vectors suspended in a microbial freeze drying buffer were desiccated. These results demonstrate the flexibility and robustness of the hybrid vector formulation, which has the potential to be a potent vaccine against S. pneumoniae.

Keywords: APCs, antigen presenting cells; AS, aqueous storage; CDM, chemically defined bacterial growth medium; CFA, Complete Freund's Adjuvant; CHV, cytoplasmic hybrid vector; CPSs, capsular polysaccharides; ClyA, cytolysin A; DNA vaccine; DS, desiccated storage; EHV, empty hybrid vector; IN, intranasal; IP, intraperitoneal; LBVs, live bacterial vectors; LLO, listeriolysin O; NVT, non-vaccine type; PAMPs, pathogen-associated molecular patterns; PCVs, pneumococcal conjugate vaccines; PHV, periplasmic hybrid vector; PcpA, pneumococcal choline-binding protein A; PhtD, histidine triad protein D; Pneumococcal disease; Pneumococcal surface protein A (PspA); PspA, pneumococcal surface protein A; SC, subcutaneous; SHV, surface hybrid vector; Streptococcus pneumoniae; Vaccine delivery; pHV, plasmid hybrid vector.

Fig. 1

Schematic of protein and DNA hybrid vector formulations. (A) Transformation of bacterial expression plasmids (protein production and localization) and mammalian expression plasmid (DNA delivery) into the hybrid vector bacterial core. (B) Three plasmids were used to localize PspA in various bacterial cellular locations including the periplasm (1), cytoplasm (2), and surface (3). (C) A proposed pathway for the uptake and processing of hybrid vectors containing the mammalian expression plasmid encoding PspA.

Fig. 2

Evaluation of the periplasmic hybrid vector storage. (A) Relative antibody titers from mice immunized with periplasmic hybrid vector stored in PBS, trehalose, and storage buffer in both aqueous and desiccated formats for varied lengths of time relative to freshly prepared vector. Error bars represent 95% confidence intervals of three replicates. (B) Pneumonia challenge model in mice vaccinated with various hybrid vector formulations with PBS (sham) controls. (C) Pneumonia challenge model in mice vaccinated with various hybrid vector formulations with sham controls. The 4 Wk samples in trehalose performed identical to the 2 Wk samples. Aqueous storage (AS), desiccated storage (DS).

Fig. 3

Panels for periplasmic hybrid vector (pUAB055-pspA) showing (A) SDS-PAGE gels for whole-cell and soluble PspA at 22 °C across varied IPTG concentrations and induction times. (B) Standard curves for quantity of PspA per well versus SDS-PAGE gel band density using linear (red) and logarithmic (blue) regressions. (C) Yield of soluble vs insoluble (difference between whole cell and soluble protein values) PspA across varied induction temperatures (at 100 µM IPTG and 8 h post-induction incubation). Error bars represent the 95% confidence interval of three replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Immunogenicity and efficacy of the protein hybrid vector variants. (A) Relative antibody titers from mice immunized with 10⁷ cells/dose of various hybrid vectors. **P < 0.01, *P < 0.05, relative to control (CFA/IFA – 15 µg PspA) on associated days. Error bars represent 95% confidence intervals of three replicates. Complete protection is represented by a time to death of ≥14 and 21 days for pneumonia and sepsis challenge models, respectively. (B) Sepsis challenge model in mice vaccinated with various hybrid vector formulations, PspA + adjuvant, and an empty hybrid vector (EHV) control. (C) Pneumonia challenge model in mice vaccinated with various hybrid vector formulations, PspA + adjuvant, and EHV.

Fig. 5

Effect of PspA localization on hybrid vector immunogenicity. (A) Relative antibody titers from mice immunized with 1 µg of soluble PspA for each hybrid vector formulation. ***P < 0.001, *P < 0.05, relative to control (CFA/IFA – 15 µg PspA) on associated days. Error bars represent 95% confidence intervals of three replicates. Complete protection for sepsis challenge models is represented by a time to death of ≥21 days. (B) Sepsis challenge model in mice vaccinated with various hybrid vector formulations compared to EHV control. (C) Antibody class distribution in serum from mice immunized with each hybrid vector formulation at days 14 and 28. Antibody class percentages were calculated by comparing individual antibody class titers with total antibody titers.

Fig. 6

DNA hybrid vector immunogenicity. (A) Relative antibody titers from mice immunized with DNA hybrid vectors (Plasmid and Plasmid + LLO). *P < 0.05, relative to control (CFA/IFA – 15 µg PspA) on associated days. Error bars represent 95% confidence intervals of three replicates. Complete protection is represented by a time to death of ≥14 and 21 days for pneumonia and sepsis challenge models, respectively. (B) Antibody class distributions from mice immunized with DNA hybrid vectors at days 14 and 28. Antibody class percentages were calculated by comparing individual antibody class titers with total antibody titers. (C) Sepsis challenge model in mice vaccinated with various hybrid vector formulations, PspA + adjuvant, and EHV. (D) Pneumonia challenge model in mice vaccinated with various hybrid vector formulations, PspA + adjuvant, and EHV.