Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and time scale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is a critical need for improved adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improves the magnitude and duration of antibody responses when compared to soluble CpG, and results in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhances the spatiotemporal control over codelivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates humoral responses comparable to those of a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.


Figure S1 :
Figure S1: Influence of DBCO-CpG molar excess on the click reaction conversion.Three equivalents of DBCO-CpG result in reaction conversions higher than 90%.

Figure S2 :
Figure S2: Gel electrophoresis of CpG-NPs.Gel electrophoresis after purification of the CpG-NPs demonstrates complete removal of free CpG from the NPs suspension.Soluble CpG runs through the gel and confirms the 20 base pair length.Unpurified CpG-NPs show presence of both, CpG-NPs in the wells (top) and soluble CpG migrating in the gel.Purified CpG-NPs stay in the well of the agarose gel, consistent with NPs conjugation and purification.

Figure S3 :
Figure S3: GPC traces of 30% CpG-NPs before (3 molar excess of DBCO-CpG) and after purification through a SEC column.Disappearance of the DBCO-CpG peak at 23 mins confirms complete removal of the unconjugated soluble CpG.

Figure S5 :
Figure S5: Calibration curves of CpG-NPs.CpG-NPs absorbance was measured at λ = 280 nm using soluble CpG.Calibration curves are used to measure the exact concentration of CpG on the NPs with different valencies.CpG concentration is meant the total concentration of CpG conjugated to the NPs in 60 μL of CpG-NPs solution.

Figure S6 :
Figure S6: In vitro activity of CpG functionalized NPs.(A) Incubation of Raw-Blue macrophage cells (APCs) with either free CpG or different valencies of CpG-NPs (10%, 20%, 30%, 50%) induces the activation of NF-kB and AP-1.The magnitude of activation is quantified via calorimetric output using QUANTI-Blue solution.(B) Raw value activation curves across a range of CpG concentrations (3.1-29 µg/mL) delivered on CpG-NPs at different densities to 100,000 Raw-Blue cells.The absorbance at 655 nm corresponds to TLR activation.

Figure S7 :
Figure S7: Analysis of systemic toxicity.ELISA analysis of (A) IFN-α and (B) TNF-α serum at 0 h, 3 h, and 24 of CpG adjuvanted vaccines with CpG being either in soluble form (CpG), tethered to the NPs (CpG-NP) or tethered to the NPs and encapsulated in the hydrogel (CpG-NP gel) (n= 5).

Figure S8 :
Figure S8: In vivo humoral response to COVID-19 subunit vaccine.(A) Timeline of mouse immunizations and blood collection for different assays.Control PEG-PLA NP soluble vaccine group was immunized with a prime dose of 10 µg spike antigen and 20 µg PEG-PLA NP at day 0 and received a booster injection of the same treatment at day 21.CpG Gel group was immunized with a prime dose of 20 μg spike antigen and 40 μg of soluble CpG and did not received a boost.Serum was collected over time to determine cytokine levels and IgG titers.IgG1, IgG2b, and IgG2c titers were quantified and neutralization assays were conducted on day 21 and day 35 serum.(B) Anti-spike total IgG ELISA endpoint titer of the vaccine before and after boosting.(C) Area under the curve (AUC) of anti-spike titers from (B). (D) Anti-spike IgG ELISA titers from serum collected week 6.Titers were determined for wildtype spike as well as Beta (B.1.351),Delta (B.1.617.2, and Omicron (B.1.1.529)variants of spike protein.Each point represents an individual mouse (n = 5).Data are shown as mean +/-s.d.

Figure S9 :
Figure S9: Ratio of Anti-Variant Spike IgG titers to Anti-WT spike IgG titers shown in Figure 7D.A ratio closer to 1 indicates a better antibody protection against emerging COVID-19 variants.

Figure S10 :
Figure S10: Anti-spike IgG1 (A) and IgG2c (B) titers from serum collected on week 5, 2 weeks after boosting the PEG-PLA NP control group.(C) The ratio of Anti-spike IgG2c to IgG1 post-boost titers.Lower values (below 1) suggest a Th2 response or humoral response, and higher values (above 1) suggest a Th1 response or cellular response.