Design and Biological Assembly of Polyester Beads Displaying Pneumococcal Antigens as Particulate Vaccine

Abstract

Streptococcus pneumoniae can cause life-threatening infections mostly in infants, children and elderly people. Capsular polysaccharide conjugate vaccines provide serotype-dependent protection against S. pneumoniae infections but fail to protect against new emerging serotypes. To overcome these limitations, pneumolysin (Ply), a serotype-independent and conserved protein was selected. As such subunit vaccines lack immunogenicity, we engineered Ply to be attached to self-assembled polyester beads in order to boost immunogenicity. To display Ply at the surface of these polyester beads, it was translationally fused to the N-terminus of the polyhydroxybutyrate (PHB) synthase (PhaC), which mediates PHB bead assembly inside recombinant Escherichia coli. We also chemically conjugated the capsular polysaccharide (CPS) 19F to isolated PHB beads to further assess their antigen carrier properties. CPS conjugated to soluble tetanus toxoid served as control. Balb/c mice immunized with Ply-PhaC beads and 19F-PhaC beads induced specific and higher IgG levels than the respective soluble counterparts. The induced IgG antibodies recognized Ply in whole cell lysates of six different serotypes of S. pneumoniae. Additionally, restimulated splenocytes from animals immunized with Ply-PhaC beads produced a balanced INF-ɣ/IL-17A profile unlike animals immunized with soluble Ply. The 19F-PhaC beads induced production of antibodies showing high opsonophagocytic titers against the homologous strain, serotype 19F, while CPS 19F only mixed with PhaC beads did not elicit any detectable immune response. This study provided insight into the design of PHB beads as a carrier of proteinaceous antigens and CPS in order to induce immune responses for the prevention of pneumococcal infections.

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“…González-Miró et al, 2018 [90] produced PHB beads by using an inducible expression of a fusion protein composed by a PhaC and a modified, non-toxic pneumolysin equivalent of a virulence factor produced by Streptococcus pneumoniae. This system induced a better immunogenic response, and also increased a broad range immune response towards pneumolysin of different serotypical origin.…”

“…PLGA-PEG nanoparticles, containing two drugs, cisplatin and paclitaxel, as an aid for chemoradiotherapy against non-small cell lung cancers, showed greater tumour inhibition compared to the single-loaded nanoparticle or the drug administration in a free form [96]. Human oral squamous carcinoma cell lines, such as PE/CA-PJ15, were targeted in vitro by a study performed by González-Miró et al, 2018 [90] produced PHB beads by using an inducible expression of a fusion protein composed by a PhaC and a modified, non-toxic pneumolysin equivalent of a virulence factor produced by Streptococcus pneumoniae. This system induced a better immunogenic response, and also increased a broad range immune response towards pneumolysin of different serotypical origin.…”

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“…Briefly, cells were subjected to mechanical disruption using a microfluidizer M-110P (Microfluidics, Westwood, MA, USA). Particles were sedimented and sequentially washed 3 times as previously described and stored at 4°C for further analysis (31,34). In addition, soluble recombinant vaccines His6-H4 and His6-H28 were also prepared as previously described by Chen et al (33).…”

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“… [ 138 ] PHB-core protein-shell particle Fusion antigenic epitopes AlgE, OprF and OprI Alum s.c. Mouse IgG1 and IgG2c, with opsonophagocytic antibody titer Th1 N/E Without the adjuvant, Th1 immune response can be induced [ 156 ] Streptococcus pneumoniae cCHP nanogel PspA – i.n. Rhesus Macaque PspA-specific bronchoalveolar fluid IgG and nasal wash IgA antibodies, with neutralizing antibody titer Th2 and Th17 N/E The mice injected intraperitoneally with the pooled sera of macaques nasally immunized with the nanogels were protected from the challenge for at least 2 weeks [ 157 ] PHB-core protein-shell particle Ply and 19F CPS – s.c. Mouse IgG with the dominant IgG1 and IgG2b, and opsonophagocy-tic antibody titer Th2 N/E The IgG was persistent for up to 6 months and recognized Ply in whole cell lysates of six different S. pneumoniae serotypes [ 158 ] PHB-core protein-shell particle PsaA – s.c. Mouse IgG with the dominant IgG1 and IgG2b Th2 N/E The elicited IgG recognized PsaA in whole cell lysate of seven different serotypes of S. pneumoniae [ 159 ] Neisseria meningtidis PHB-core protein-shell particle NadA and MenC – …”