A recombinant conjugated pneumococcal vaccine that protects against murine infections with a similar efficacy to Prevnar-13

Abstract

The pneumococcal conjugate vaccine (PCV) strongly protects against vaccine serotypes, but the rapid expansion of non-vaccine serotype disease and the vaccine's high expense has reduced its overall impact. We have developed Protein Glycan Coupling Technology (PGCT) as a flexible methodology for making low-cost polysaccharide/protein glycoconjugates recombinantly in Escherichia coli. We have used PGCT to make a recombinant PCV containing serotype 4 capsular polysaccharide linked to the Streptococcus pneumoniae proteins NanA, PiuA, and Sp0148. The introduction of the Campylobacter jejuni UDP-glucose 4-epimerase gene GalE (gne) into E. coli improved the yield of the resulting glycoprotein. PGCT glycoconjugate vaccination generated strong antibody responses in mice to both the capsule and the carrier protein antigens, with the PiuA/capsule glycoconjugate inducing similar anti-capsular antibody responses as the commercial PCV Prevnar-13. Antibody responses to PGCT glycoconjugates opsonised S. pneumoniae and Streptococcus mitis expressing the serotype 4 capsule and promoted neutrophil phagocytosis of S. pneumoniae to a similar level as antisera generated by vaccination with Prevnar-13. Vaccination with the PGCT glycoconjugates protected mice against meningitis and septicaemia with the same efficacy as vaccination with Prevnar-13. In addition, vaccination with the protein antigen components from PGCT glycoconjugates alone provided partial protection against septicaemia and colonisation. These data demonstrate that a vaccine made by PGCT is as effective as Prevnar-13, identifies PiuA as a carrier protein for glycoconjugate vaccines, and demonstrates that linking capsular antigen to S. pneumoniae protein antigens has additional protective benefits that could provide a degree of serotype-independent immunity.

Fig. 1

Analysis of recombinant proteins from glycoprotein expression strains. a-c Immunoblot analysis of decreasing concentrations of recombinant glycoproteins from galE⁺ and galE^- E. coli strains. Fluorescent intensity of the signal from the glycosylated protein bands (green channel) was measured by densitometry using the LI-COR odyssey fluorescent imaging system and are displayed as mean ± SEM from three replicate experiments. Red channel: mouse anti-His IgG; Green channel: rabbit anti-Type 4 antiserum. d-h Anti-capsular polysaccharide antibody levels in antiserum measured from mice (n = 8) vaccinated with recombinant glycoproteins (closed circles) or cognate unglycosylated antigens (open squares). Antiserum from Prevnar-13 (closed circles) and PBS vaccinated (open squares) animals were included as controls. Results displayed as mean ± SEM from technical replicates

Fig. 2

Vaccination with recombinant glycoproteins generates antibodies that recognise homologous and heterologous pneumococcal isolates. a-c Anti-carrier protein antibodies were measured from mice vaccinated with Combo(Sp4) (closed circles) or Combo (open squares) by sandwich ELISA using a monoclonal anti-His capture antibody and recombinant, unglycosylated carrier proteins. Data are displayed as mean ± SEM from technical replicates. d, e Anti-ST4 d and anti-ST2 e antibodies were measured by whole-cell ELISA using pooled antiserum from the glycosylated (black bars) and unglycosylated (white bars) vaccine groups. Data are displayed as mean ± SEM from three separate cultures. *p < 0.05 vs PBS, ^#p < 0.05 protein vs glycoprotein, ^†p < 0.05 vs Prevnar-13 one-way ANOVA with Bonferroni’s post-test. f Immunoblot analysis of concentrated lysate from ST4 and ST2 overnight cultures using pooled antiserum from the glycosylated and unglycosylated vaccine groups. Molecular mass markers are given in kilodaltons

Fig. 3

Flow cytometry analysis of antibody deposition on streptococcal species. a Representative histograms for antibody deposition on S. mitis(SpT4) in 10% antiserum from glycosylated (red shading) and unglycosylated (grey shading) vaccine groups. PBS vaccinated serum (dashed line) was included as a control. b Antibody deposition measured using a flow cytometry assay on S. mitis(SpT4) in 10% murine antiserum (n = 8) from glycosylated (closed circles) and unglycosylated (open squares) vaccine groups. Red dots indicate reactions containing reduced antiserum concentrations (5 vs 10%) in high titre samples. *p < 0.05 Kruskal–Wallis with Dunn’s post-test (vs PBS). c Examples of flow cytometry histograms for antibody deposition on the TIGR4 S. pneumoniae strain in 2% (red shading), 0.2% (grey shading), and 0.02% (dashed line) antiserum from glycosylated vaccine groups. d Antibody deposition on TIGR4 in pooled antiserum from mice vaccinated with glycosylated or unglycosylated vaccines. Deposition titres were determined using bacteria incubated with decreasing concentrations of Prevnar-13 antiserum to generate a standard curve. Results displayed as mean ± SEM from technical replicates

Fig. 4

Antibody deposition on non-serotype 4 pneumococci. a-d Representative histograms and antibody deposition on homologous and heterologous pneumococcal isolates in 1% pooled antiserum from mice vaccinated with glycosylated NanA (grey shading), Sp0148 (red shading), or PiuA (blue shading) or normal mouse serum (dashed line). Black bars represent the percentage of PE⁺ bacteria and grey bars represent the gMFI of the positive population. Gates were set such that 5–10% of events were PE⁺ in the normal mouse serum (NMS) reactions to account for strain specific differences in auto fluorescence. Data are displayed as mean ± SEM from technical replicates. e Immunofluorescent staining of homologous and heterologous pneumococcal isolates using antiserum from mice vaccinated with the combination vaccine (green channel) and pneumococcal Omni serum (red channel). Length of scale bar is equal to 5 µm

Fig. 5

Effect of antiserum samples on interaction of S. pneumoniae with human neutrophils. a Examples of flow cytometry histograms for fresh human neutrophils incubated with FAM-SE labelled TIGR4 when opsonised in 20% antiserum from glycosylated (red shading) or unglycosylated (grey shading) vaccine groups and 5% baby rabbit complement. PBS vaccinated serum (dashed line) was included as a control. b Percent association of fresh human neutrophils with TIGR4 when opsonised in 20% antiserum (n = 8) from glycosylated (closed circles) or unglycosylated (open squares) vaccine groups and 5% baby rabbit complement. Antiserum from Prevnar-13 (closed circles) and PBS vaccinated (open squares) animals were included as controls. *p < 0.05 Kruskal–Wallis with Dunn’s post-test (vs PBS). c-e Percent association of fresh human neutrophils with non-type 4 pneumococci when opsonised in 20% antiserum (n = 6) from glycosylated (closed circles) or unglycosylated (open squares) vaccine groups and 5% baby rabbit complement. Antiserum from Prevnar-13 (closed circles) and PBS vaccinated (open squares) animals were included as controls

Fig. 6

Vaccination with recombinant glycoconjugates provides protection against several pneumococcal disease aetiologies. Mice were vaccinated with Combo(Sp4), Combo, Prevnar-13, or PBS+ adjuvant as outlined in the methods. a Mice challenged intranasally with 1 × 10⁷ CFU of TIGR4 were culled seven days post-infection and bacterial loads were assessed by nasal washing. b, c Mice were infected intranasally with 1 × 10⁷ CFU of TIGR4 b or 2 × 10⁶ CFU of WCH43 c and culled after 24 or 48 h respectively. Bacterial burdens in the blood and organ homogenates were determined by serial dilution and plating. *p < 0.05 Kruskal–Wallis with Dunn’s post-test (vs PBS)