Harnessing galactose oxidase in the development of a chemoenzymatic platform for glycoconjugate vaccine design

Abstract

In the preparation of commercial conjugate vaccines, capsular polysaccharides (CPSs) must undergo chemical modification to generate the reactive groups necessary for covalent attachment to a protein carrier. One of the most common approaches employed for this derivatization is sodium periodate (NaIO₄) oxidation of vicinal diols found within CPS structures. This procedure is largely random and structurally damaging, potentially resulting in significant changes in the CPS structure and therefore its antigenicity. Additionally, periodate activation of CPS often gives rise to heterogeneous conjugate vaccine products with variable efficacy. Here, we explore the use of an alternative agent, galactose oxidase (GOase) isolated from Fusarium sp. in a chemoenzymatic approach to generate a conjugate vaccine against Streptococcus pneumoniae. Using a colorimetric assay and NMR spectroscopy, we found that GOase generated aldehyde motifs on the CPS of S. pneumoniae serotype 14 (Pn14p) in a site-specific and reversible fashion. Direct comparison of Pn14p derivatized by either GOase or NaIO₄ illustrates the functionally deleterious role chemical oxidation can have on CPS structures. Immunization with the conjugate synthesized using GOase provided a markedly improved humoral response over the traditional periodate-oxidized group. Further, functional protection was validated in vitro by measure of opsonophagocytic killing and in vivo through a lethality challenge in mice. Overall, this work introduces a strategy for glycoconjugate development that overcomes limitations previously known to play a role in the current approach of vaccine design.

Keywords: Streptococcus pneumoniae; capsular polysaccharide; galactose oxidase; glycoconjugate vaccine; pneumonia; vaccine development.

Figure 1

Highlighting the structural differences between the chemical and chemoenzymatic conjugation strategies.A, in the traditional chemical approach harnessing sodium periodate, poorly controlled activation along the CPS structure results in the permanent opening of the vicinal diols prior to conjugation to carrier. The varying formation of aldehydes results in random conjugations with the carrier protein further contributing to structural heterogeneity of the glycoconjugate vaccine. B, harnessing GOase as an enzymatic approach selectively modifies the C6 hydroxyl on branching galactose residues in a manner that does not change the core structure prior to conjugation. After reductive amination, any nonconjugated aldehydes would be converted back to the hydroxyl with sodium borohydride treatment maintaining native structural identity. CPS, capsular polysaccharide; GOase, galactose oxidase.

Figure 2

NMR characterization of reversible GOase activation of Pn14p.A, 1D ¹H spectra of three samples corresponding to Pn14p, GOase oxidized Pn14p, and GOase oxidized Pn14p that has been treated with NaBH₄. B, 2D TOCSY and NOESY spectra of the GOase oxidized Pn14p highlighting the 6-oxogalacto configuration. GOase, galactose oxidase.

Figure 3

Functional comparison between GOase and periodate oxidation of Pn14p. Pn14p was treated with either GOase or one of two different concentrations of sodium periodate, relating to 0.25 or 1 mol equivalent of NaIO₄ to 1 mol of CPS repeat unit. A, a dot blotting assay was performed on the polysaccharides in quadruplicate using Anti-Pn14p IgG with the optical density of the samples measured using the NIH ImageJ software. B, the polysaccharides were then used in a competition ELISA in which samples were incubated in quadruplicate with the anti-Pn14p polyclonal IgG prior to introduction to a Pn14p coated plate. Values represent the mean ± SD of the optical density or percentage of inhibition, respectively. ∗∗∗∗p < 0.0001 and ∗∗p < 0.01 represent significant differences between groups determined using one-way ANOVA with Dunnett's multiple comparisons test. CPS, capsular polysaccharide; GOase, galactose oxidase.

Figure 4

The effect activation strategy has on IgM and IgG antibodies generated. Mice (n = 5) were primed (Day 0) and boosted on day 14 and 28, with sera collected 2 weeks after each vaccine administration to calculate kinetics of the humoral response generated with each sample tested in quadruplicate. Pn14p (A and C) and fixed whole-bacteria S. pneumoniae expressing the type 14 capsule (B and D) specific antibody responses from each time point were measured using ELISA. Regression curves based on individual serum dilutions were used to determine IgG and IgM titer levels at an OD of 0.5. Shown are calculated mouse titers as the reciprocal dilution at OD of 0.5 at 405 nm in the ELISA assay. Values represent the mean ± SD of the IgM or IgG titers, respectively. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, and ∗p < 0.05 represent significant differences between groups determined using two-way ANOVA with Dunnett's multiple comparisons test.

Figure 5

Functional protection elicited through glycoconjugate vaccination.A, using serum from each sample group at day 42, opsonic capacity of antibodies was determined against live Spn14, with the correlated degree of bacteria killed from an OPA shown. B, survival of vaccinated mice (n = 6) challenged on day 49, 3 weeks after the final boost, by virulent Spn14. Values (A) represent the mean percent ±SD of the bacteria killed or (B) murine survival. ∗∗∗∗p < 0.0001 and ∗∗p < 0.01 represent significant differences between groups determined using one-way ANOVA with Dunnett's multiple comparisons test or survival curve comparison using Mantel–Cox Log-rank test.