A novel microporous biomaterial vaccine platform for long-lasting antibody mediated immunity against viral infection

Abstract

Current antigen delivery platforms, such as alum and nanoparticles, are not readily tunable, thus may not generate optimal adaptive immune responses. We created an antigen delivery platform by loading lyophilized Microporous Annealed Particle (MAP) with aqueous solution containing target antigens. Upon administration of antigen loaded MAP (VaxMAP), the biomaterial reconstitution forms an instant antigen-loaded porous scaffold area with a sustained release profile to maximize humoral immunity. VaxMAP induced CD4⁺ T follicular helper (Tfh) cells and germinal center (GC) B cell responses in the lymph nodes similar to Alum. VaxMAP loaded with SARS-CoV-2 spike protein improved the magnitude, neutralization, and duration of anti-receptor binding domain antibodies compared to Alum vaccinated mice. A single injection of Influenza specific HA1-loaded-VaxMAP enhanced neutralizing antibodies and elicited greater protection against influenza virus challenge than HA1-loaded-Alum. Thus, VaxMAP is a platform that can be used to promote adaptive immune cell responses to generate more robust neutralizing antibodies, and better protection upon pathogen challenge.

Keywords: Antigen delivery platform; Biomaterial; Sustained release; Vaccine platform.

Fig. 1.

VaxMAP scaffold provides a rapid way to load an immunogen at the desired concentration with a two-stage release profile. The hydrogel microspheres that constitute MAP are provided lyophilized for longer shelf-life stability and are reconstituted with an immunogen at time of use. While the hydrogel microspheres are swelling, part of the antigen is passively diffusing within the hydrogel microspheres, whereas some of the antigen is remaining within the interstitial space between particles. VaxMAP has pores that are large enough for the immune cells to immediately migrate into the scaffold prior to its degradation. Its double-stage release profile provides an initial burst release for antigen exposure to prime immune responses followed by a slower and sustained release for long-term memory antibody and cellular responses.

Fig. 2.

(A) MAP hydrogel microsphere fabrication and chemical annealing. The hydrogel microspheres are obtained by a crosslinking reaction between the vinyl sulfone (VS) groups on the PEG polymer (PEG-VS) and the thiol (SH) groups on the degradable peptide through Michael addition in a water-in-oil emulsion. Immediately prior to use, MAP is mixed with a PEG-dithiol crosslinker leading the excess VS groups on neighboring microspheres to react with the thiol groups (chemical annealing) to form an interconnected porous network. (B)) Proliferation of 3 T3 fibroblasts after treatment with varying concentrations of PEG-dithiol (PEG-(SH)2, 3.4 kDa) via a WST-8 assay. Data are expressed as Mean ± SD (n = 3). (C) Particle size distribution (volume weighted) after lyophilization, reconstitution and swelling of the hydrogel microspheres in PBS (pH 7.4). (D) Annealing kinetic of MAP at different pHs. Increasing pH accelerates the annealing reaction. (E) Compressive elastic modulus before and after annealing the hydrogel microspheres using PEG-dithiol (final concentration: 0.2 mM) at pH 7.4 and at a volume fraction of 80%. Data are expressed as Mean ± SD (n = 4). (F) Confocal microscopy images of fluorescent MAP loaded with IgG-CF568 (single confocal slices). The hydrgogel microspheres (particles) were labeled with a CF647 maleimide fluorescent dye (red) and the IgG protein was labeled with a CF568 maleimide fluorescent dye (blue). Top images: non-diluted MAP. Bottom images: MAP diluted 1:100 in PBS. Quantification of fluorescence intensity from IgG-CF568 in the particles and pores. Data are expressed as Mean ± SD (n = 20 for pores and n = 30 for particles).

Fig. 3.

(A) Release profile of IgG-CF568 from MAP. The red arrow indicates the addition of trypsin to accelerate cargo release. (B–C) B6 mice were immunized with MAP loaded with IgG-CF568 and iLN were harvested 5 and 15 days post injection. (B) CD11b cells were stained for flow cytometry Representative facs plot at day (left) and quantification (right) of CD11b + IgG-CF568+ cells from each group. (C) CD11c cells were stained for flow cytometry Representative facs plot at day (left) and quantification (right) of CD11c + IgG-CF568+ cells from each group. Data are representative of two independent experiments with 3–5 mice per group. * = p ≤ 0.05, ** = p ≤ 0.01.

Fig. 4.

VaxMAP elicits adaptive immune responses. (A) B6 mice were immunized with VaxMAP(S) at different doses of spike antigen (0, 10, 25 and 50 μg). ELISA quantification of serum Anti-RBD IgG antibody of immunized mice 30 days post-immunization. (B–I) B6 mice were immunized with VaxMAP(S), Alum(S) or PBS(S) using a 50 μg dose of spike antigen. (B) Tfh cells were stained for flow cytometry and quantified at days 10, 28 and 60 post immunization. Representative facs plot at day 10 (left) and quantification (right) Tfh cells from each group. (C) GC B cells were stained for flow cytometry and quantified. Representative facs plot from day 10 (left) and quantification (right) GC B cells from each group. (D) ELISA quantification of serum Anti-RBD IgG antibody of immunized mice at days 10, 28 and 60. Graphs of titers (top) and area under the curve (bottom). (E) Anti-RBD IgG antibodies from each group were directly compared over time using a 1:300 titer for each sample from the three time points. (F) Representative anti-Spike IgG ELISPOT with number of cells plated per well of B cells from bone marrow of immunized mice 60 days post-immunization. (G-H) ELISA quantification of serum Anti-RBD IgG antibody of immunized mice at days 28 or 60 compared to an mRNA vaccine. Data are representative of three independent experiments with 3–8 mice per group. (I) SARS-CoV-2 pseudovirus neutralization assay: relative 50% inhibitory concentration (IC50) of sera from immunized mice at day 60 post-immunization. Positive control is from pooled human serum from individuals vaccinated with an mRNA vaccine. * = p ≤ 0.05, ** = p ≤ 0.01, **** p ≤ 0.0001.

Fig. 5.

VaxMAP-immunized mice demonstrate a robust recall response. B6 mice were immunized with VaxMAP(S), Alum(S) or PBS(S) and then rechallenged with Alum(S) 65 days later. Draining lymph nodes and sera were collected 7 days after re-immunization. (A) Tfh cells were stained for flow cytometry and quantified. Representative facs plot (left) and quantification (right) Tfh cells from each group. (B) GC B cells were stained for flow cytometry and quantified. Representative facs plot (left) and quantification (right) GC B cells from each group. (C) ELISA quantification of serum Anti-RBD IgG antibody of re-immunized mice. Graphs of titers (left) and area under the curve (right). Data are representative of three independent experiments with 5–8 mice per group. ** = p ≤ 0.01.

Fig. 6.

VaxMAP elicits protection against influenza infection. (A) B6 mice were immunized with VaxMAP(HA1), Alum(HA1), PBS(HA1), or empty VaxMAP and ELISA quantification of serum Anti-RBD IgG antibody of immunized mice at days 21, 45 and 55. Graphs of titers (top) and area under the curve (bottom). (B) Sera from day 55 immunized mice was used in a HA agglutination assay with different dilutions of PR8. (C–D) Mice were given a high dose of PR8 60 days after immunization (C) Daily weight loss based on the percentage of the initial weight of each mouse out to 20 days post infection. (D) Survival curve of mice following infection. Data are representative of 2 independent experiments with 5–8 mice per group. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** p ≤ 0.0001.