Semisynthetic Pneumococcal Glycoconjugate Nanovaccine

Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.


Endotoxin testing
The quantification of endotoxins in the samples used for DC cell studies was performed using the Pierce Chromogenic Endotoxin Quant Kit (A39552).Briefly, 2 micrograms of antigen (PsaA or GC) or 50 micrograms of chemicals (Chitosan, Kolliphor-188 or TPP) were suspended in water and tested for the presence of endotoxin according to the manufacturer's instructions. 1e calibration curve (0.1-8 EU/mL) was constructed using a stock solution of 10 EU/mL.To avoid external endotoxin interference, all the dilutions were prepared in endotoxin-free water, which was also used as blank.All the tubes and tips used to perform this quantification were endotoxin-free and the entire procedure was performed inside of a laminar flow cabinet.
Endotoxin content was lower than 0.5 EU/μg in all the cases (Figure S2), which are far below the maximum recommended endotoxin levels for recombinant subunit vaccines (20 EU/mL). 2 The colloidal stability of the nanoparticles was evaluated by measuring their size and PDI in the R10 medium and was found to be concentration dependent.The aggregation of the CNPs was observed when the concentrations were above 500 μg/ml in R10 media after 24 h.Below this concentration, the NPs showed no signs of aggregation.As can be seen, all of them shows levels below 0.5 EU/µg.

Internalization of GC-CNPs in DC2.4 cell lines after different time periods of incubation
The DC2.4 cells (25,000 per well) were seeded on Lab-Tek plates with 0.2 mL of R10 media.
Following that cells were incubated with the Cy5 labelled GC-CNPs (0.5 µg of NPs) for different time periods (0.5, 1, 2, 4 and 24h).After the incubation time, the DCs were washed with PBS.
Afterwards, the cells were fixed with 4% PFA for 15 min.For imaging, the cells were stained using DAPI and wheat germ agglutinin (WGA, at 0.2 µg/ml) for 10 min, and the DCs were washed twice with PBS after incubation.The cells were suspended in 20-30 µl of STORM buffer.

The study on internalization of nanoparticles by DCs by cytometry and laser confocal scanning microscopy (LCSM)
To evaluate the time-dependent uptake of CNPs by human MoDCs, iDCs (5x10 5 per well) were plated into a 24-well plate with 0.5 mL of R10 media.Immediately, Cy5-labelled GC-CNPs (Cy5-GC-CNPs) were added at a concentration of 50 μg/mL.At different time intervals (0, 0.5, 1, 2 and 4 h of incubation), cells were collected for uptake analysis.The collected cells were washed immediately with PBS and fixed in a flow cytometry tube using 200 μL of PBS S5 containing 1% paraformaldehyde (1% PFA).As a control, the MoDCs treated with an equal amount of CNPs at 4 °C was used.The samples were diluted with 500 μL of PBS, and the suspension was analysed by flow cytometry (BD FACS Calibur™ cytometer).
As can be seen in Figure S3, the pattern of GC-CNPs (50 μg/ml) internalization in DCs at 37 °C shows greater uptake with the increase in incubation time.However, only 10% increase in the GC-CNPs uptake was observed between 2 h and 4 h of incubation at 37 °C.While the DCs incubated at 4 °C as a control (treated with 50 µg/ml of GC-CNPs), did not show any significant uptake of GC-CNPs even after 4 h, showing a specific energy-dependent nanoparticle uptake at 37 °C.For LCSM analysis, the DCs (5x10 5 per well) were plated into a 24-well plate with 0.5 mL of R10 media and incubated for 30 min.Following that the DCs were incubated with the Cy5 S6 labelled GC-CNPs for 1 h.After the incubation time, the DCs were washed with PBS and seeded at a density of 1 x 10 5 onto the poly-L-lysine coated coverslips and allowed to adhere for 15 min.Afterwards, the cells were fixed with 4% PFA for 15 min.The DCs were permeabilized/ fixed using 100 μL of BD Citofix / Citoperm™ (15 min, RT, in the dark) and were washed twice with PBS.The cells were stained using DAPI and phalloidin-488 and fixed on the glass slide using mounting medium (Vectashield® Antifade Mounting Medium, Vector Labs, USA).The image acquisition was made using CLSM (Leica SP5, Mannheim, Germany).Excitation wavelengths were 670 nm for Cy5 and 488 nm for Alexa 488.
In the Figure S5

Figure S1 .
Figure S1 S2 Endotoxin testing S2 Figure S2 S3 Internalization of GC-CNPs in DC2.4 cell lines after different time periods of incubation S3 Figure S3 S4 The study on internalization of nanoparticles by DCs by cytometry and laser confocal scanning microscopy (LCSM) S4 Figure S4 S5 Figure S5 S7 References S7

Figure S2 .
Figure S2.Endotoxin analysis of CNPs formulation components.A) The calibration curve used to quantify the endotoxin level in the formulation components.B) Endotoxin quantification in the different CNPs components.

FigureFigure S3
FigureS3clearly displays that NPs internalization increases with time.With the increase in time, the NPs concentration increased both on the surface and the centre of the cells, which indicates the NPs were continuously internalized throughout the incubation time and not just during the initial hours of incubation.The images from the mid-section of the cells show the NPs concentration in the cytoplasm increases with the time, and the NPs were present around the nucleus and were not observed inside.The results obtained from these studies are identical to that of nanoparticle uptake by MoDCs (Manuscript, Figure6).