Hydrogel‐Based Slow Release of a Receptor‐Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS‐CoV‐2

Abstract

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID‐19. The receptor‐binding domain (RBD) of the SARS‐CoV‐2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically‐relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime‐boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer‐nanoparticle (PNP) hydrogel elicited potent anti‐RBD and anti‐spike antibody titers, providing broader protection against SARS‐CoV‐2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically‐relevant adjuvant systems. Notably, a SARS‐CoV‐2 spike‐pseudotyped lentivirus neutralization assay revealed that hydrogel‐based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

SciScore for 10.1101/2021.03.31.437792: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	8-10 week-old female mice were used.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Mouse Serum ELISAs: Anti-RBD and Anti-spike trimer antibody titers were measured using an end-point ELISA.	Anti-spike suggested: None
One of the following goat-anti-mouse secondary antibodies was used: IgG Fc-HRP (1:10,000, Invitrogen A16084)	secondary suggested: (Thermo Fisher Scientific Cat# A16084, RRID:AB_2534758)
For flow cytometry analysis, cells were blocked with anti-CD16/CD38 (clone: 2.4G2) and then stained with fluorochrome …

SciScore for 10.1101/2021.03.31.437792: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	8-10 week-old female mice were used.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Mouse Serum ELISAs: Anti-RBD and Anti-spike trimer antibody titers were measured using an end-point ELISA.	Anti-spike suggested: None
One of the following goat-anti-mouse secondary antibodies was used: IgG Fc-HRP (1:10,000, Invitrogen A16084)	secondary suggested: (Thermo Fisher Scientific Cat# A16084, RRID:AB_2534758)
For flow cytometry analysis, cells were blocked with anti-CD16/CD38 (clone: 2.4G2) and then stained with fluorochrome conjugated antibodies: CD19, GL7, CD95, CXCR4, CD86, IgG1, CD4, CXCR5, and PD1.	anti-CD16/CD38 suggested: None CD19 suggested: (Diaclone Cat# 873.039.050, RRID:AB_1587088) GL7 suggested: None CD95 suggested: None CXCR4 suggested: None CD86 suggested: None IgG1 suggested: None CD4 suggested: None CXCR5 suggested: None PD1 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Viral Neutralization Assay: Neutralization assays were conducted as described previously.39 Briefly, SARS-CoV-2 spike-pseudotyped lentivirus was produced in HEK239T cells.	HEK239T suggested: None
For the neutralization assay, ACE2/HeLa cells were plated 1-2 days prior to infection.	ACE2/HeLa suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Mice and Vaccination: C57BL/6 mice were purchased from Charles River and housed at Stanford University.	C57BL/6 suggested: None
Software and Algorithms
Sentences	Resources
For RBD retention, the points were fit with a linear fit in GraphPad Prism and the half-life of release was determined (n=3).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Data were analyzed with FlowJo 10 (FlowJo LLC).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
P values listed were determined in GraphPad Prism software using a one-way ANOVA with Tukey’s multiple comparison test.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

By combining the promising antigen, RBD, and well-studied adjuvants, CpG and Alum, with our hydrogel platform, we have created a vaccine that overcomes many of the current limitations and is suitable for broader distribution. In addition to the choice of antigen and adjuvant, there is a growing need for novel and efficient drug delivery systems like our PNP hydrogel platform.45 The hydrogel is injectable and forms a depot that allows for slow release of the vaccine over several days to weeks, which leads to greater affinity maturation and a more potent, durable humoral response.24 This is critical for eliminating the need for a booster shot, in-turn increasing the feasibility of vaccinating billions of people across the globe. In this work we validated that, unlike other hydrogel or microneedle vaccine platforms, the PNP hydrogel is readily loaded with diverse molecular cargo, allowing for the antigen and adjuvants to be co-presented to immune cells. As discussed in previous work from our lab, the hydrogel likely acts through two main mechanisms: as a local stimulatory niche where infiltrating cells experience high local concentrations of adjuvant and antigen and as a slow release depot that allows for prolonged vaccine exposure.24 By delivering RBD with the clinically de-risked adjuvants, CpG and Alum, in our PNP hydrogel, we were able to achieve titers and neutralization levels that greatly exceeded those of the relevant clinical controls, Alum and MF59, as well as the bolu...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Hydrogel‐Based Slow Release of a Receptor‐Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS‐CoV‐2

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