Quantitation and Stability of Protein Conjugation on Liposomes for Controlled Density of Surface Epitopes

The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery

Releasable Layer-by-Layer Assembly of Stabilized Lipid Nanocapsules on Microneedles for Enhanced Transcutaneous Vaccine Delivery

Here we introduce a new approach for transcutaneous drug delivery, using microneedles coated with stabilized lipid nanocapsules, for delivery of a model vaccine formulation. Poly(lactide-<i>co</i>-glycolide) microneedle arrays were coated with multilayer films <i>via</i> layer-by-layer assembly of a biodegradable cationic poly(β-amino ester) (PBAE) and negatively charged interbilayer-cross-linked multilamellar lipid vesicles (ICMVs). To test the potential of these nanocapsule-coated microneedles for vaccine delivery, we loaded ICMVs with a protein antigen and the molecular adjuvant monophosphoryl lipid A. Following application of microneedle arrays to the skin of mice for 5 min, (PBAE/ICMV) films were rapidly transferred from microneedle surfaces into the cutaneous tissue and remained in the skin following removal of the microneedle arrays. Multilayer films implanted in the skin dispersed ICMV cargos in the treated tissue over the course of 24 h <i>in vivo</i>, allowing for uptake of the lipid nanocapsules by antigen presenting cells in the local tissue and triggering their activation <i>in situ</i>. Microneedle-mediated transcutaneous vaccination with ICMV-carrying multilayers promoted robust antigen-specific humoral immune responses with a balanced generation of multiple IgG isotypes, whereas bolus delivery of soluble or vesicle-loaded antigen <i>via</i> intradermal injection or transcutaneous vaccination with microneedles encapsulating soluble protein elicited weak, IgG₁-biased humoral immune responses. These results highlight the potential of lipid nanocapsules delivered by microneedles as a promising platform for noninvasive vaccine delivery applications

Immunogenic Cell Death Amplified by Co-localized Adjuvant Delivery for Cancer Immunotherapy

Despite their potential, conventional whole-cell cancer vaccines prepared by freeze–thawing or irradiation have shown limited therapeutic efficacy in clinical trials. Recent studies have indicated that cancer cells treated with certain chemotherapeutics, such as mitoxantrone, can undergo immunogenic cell death (ICD) and initiate antitumor immune responses. However, it remains unclear how to exploit ICD for cancer immunotherapy. Here, we present a new material-based strategy for converting immunogenically dying tumor cells into a powerful platform for cancer vaccination and demonstrate their therapeutic potential in murine models of melanoma and colon carcinoma. We have generated immunogenically dying tumor cells surface-modified with adjuvant-loaded nanoparticles. Dying tumor cells laden with adjuvant nanodepots efficiently promote activation and antigen cross-presentation by dendritic cells in vitro and elicit robust antigen-specific CD8α⁺ T-cells in vivo. Furthermore, whole tumor-cell vaccination combined with immune checkpoint blockade leads to complete tumor regression in ∼78% of CT26 tumor-bearing mice and establishes long-term immunity against tumor recurrence. Our strategy presented here may open new doors to “personalized” cancer immunotherapy tailored to individual patient’s tumor cells

Subcutaneous Nanodisc Vaccination with Neoantigens for Combination Cancer Immunotherapy

While cancer immunotherapy provides new exciting treatment options for patients, there is an urgent need for new strategies that can synergize with immune checkpoint blockers and boost the patient response rates. We have developed a personalized vaccine nanodisc platform based on synthetic high-density lipoproteins for co-delivery of immunostimulatory agents and tumor antigens, including tumor-specific neoantigens. Here we examined the route of delivery, safety profiles, and therapeutic efficacy of nanodisc vaccination against established tumors. We report that nanodiscs administered via the subcutaneous (SC) or intramuscular (IM) routes were well tolerated in mice without any signs of toxicity. The SC route significantly enhanced nanoparticle delivery to draining lymph nodes, improved nanodisc uptake by antigen-presenting cells, and generated 7-fold higher frequency of neoantigen-specific T cells, compared with the IM route. Importantly, when mice bearing advanced B16F10 melanoma tumors were treated with nanodiscs plus anti-PD-1 and anti-CTLA-4 IgG therapy, the combination immunotherapy exerted potent antitumor efficacy, leading to eradication of established tumors in ∼60% of animals. These results demonstrate nanodiscs customized with patient-specific tumor neoepitopes as a safe and powerful vaccine platform for immunotherapy against advanced cancer

VMP001-NP immunization triggers germinal center formation.

<p>(A) C57Bl/6 mice were vaccinated with 0.1 µg VMP001 and 5 µg MPLA in either soluble or VMP001-NP formulations, and on day 21, inguinal dLNs were isolated and analyzed for germinal center formation. The number of isotype-switched germinal center B cells (GL-7⁺PNA⁺), gated on B220⁺IgD^low populations in dLNs was measured with flow cytometric analysis. (B) Inguinal dLNs were cryo-sectioned on day 21 post-immunization and stained with anti-B220, anti-IgD, and anti-GL-7 (markers for B cells, immature B cells, and germinal center, respectively) and examined by confocal microscopy. Scale bars, 50 µm. *, <i>p</i><0.05, analyzed by Student's <i>t</i> test.</p

Synthesis of PLGA NPs with surface-conjugated VMP001 (VMP001-NPs).

<p>(A) Schematic illustration of synthesis of lipid-enveloped PLGA NPs with surface-conjugated VMP001. PLGA NPs were incubated with thiolated VMP001, conjugating the antigen to maleimide-functionalized lipids displayed on the particle membranes. Particles were then PEGylated in a reaction with PEG-thiol. (B) A scanning electron microcopy image of VMP001-NPs. Scale bar, 2 µm. (C) Confocal microscopy image of VMP001-NPs incubated with anti-his-tag and fluorescent secondary antibodies to detect particle surface-conjugated VMP001. Scale bar, 10 µm.</p

VMP001-NP immunization elicits high avidity antibodies capable of agglutinating live sporozoites.

<p>(A) Avidity indices of anti-VMP001 IgG sera obtained from mice immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031472#pone-0031472-g003" target="_blank">Fig. 3</a> with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were characterized over 6 months following vaccination. *, <i>p</i><0.01 and **, <i>p</i><0.001, analyzed by two-way ANOVA, followed by a Bonferroni post-test. (B) Anti-VMP001 IgG antibodies elicited with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were further examined for their affinities against key fragments of VMP001, including peptides representing the Type I repeat, AGDR motif, Region I, Region II, C-terminus, and scrambled negative peptide control. Sera from non-immunized mice were also included as controls (black squares). (C,D) Sera obtain from mice on day 63 post-immunizations with 2.5 µg VMP001 and 25 µg MPLA in either (C) soluble or (D) VMP001-NP formulations were incubated with live VK210 sporozoites, and immunoflurescence assay was performed to assess recognition of native CSP present on the surface of live sporozoites by anti-VMP001 IgG sera. Mice immunized with VMP001-NP vaccines raised sera that agglutinated live VK210 subtype of <i>P. vivax</i>.</p

Immunization with VMP001-NPs elicits Th1/Th2 balanced antibody responses.

<p>(A) C57Bl/6 mice were immunized <i>s.c.</i> on days 0 and 21 with 25 µg of MPLA and 1 µg of VMP001 in either soluble or VMP001-NP formulations, and anti-VMP001 IgG sera were characterized on days 35 and 120 for (A) IgG, (B) IgG₁, (C) IgG_2b, (D) IgG_2c, and (E) IgG₃ titers.</p

Self-encapsulating Poly(lactic-<i>co</i>-glycolic acid) (PLGA) Microspheres for Intranasal Vaccine Delivery

Herein we describe a formulation of self-encapsulating poly­(lactic-<i>co</i>-glycolic acid) (PLGA) microspheres for vaccine delivery. Self-healing encapsulation is a novel encapsulation method developed by our group that enables the aqueous loading of large molecules into premade PLGA microspheres. Calcium phosphate (CaHPO₄) adjuvant gel was incorporated into the microspheres as a protein-trapping agent for improved encapsulation of antigen. Microspheres were found to have a median size of 7.05 ± 0.31 μm, with a w/w loading of 0.60 ± 0.05% of ovalbumin (OVA) model antigen. The formulation demonstrated continuous release of OVA over a 49-day period. Released OVA maintained its antigenicity over the measured period of >21 days of release. C57BL/6 mice were immunized via the intranasal route with prime and booster doses of OVA (10 μg) loaded into microspheres or coadministered with cholera toxin B (CTB), the gold standard of mucosal adjuvants. Microspheres generated a Th2-type response in both serum and local mucosa, with IgG antibody responses approaching those generated by CTB. The results suggest that this formulation of self-encapsulating microspheres shows promise for further study as a vaccine delivery system

GLA and CpG combine to enhance T_H1 responses to ID93.

<p>Mice were immunized with ID93 adjuvanted with GLA, CpG, or GLA+CpG. (A and B) One week after the final immunization ID93-specific CD4⁺ T cells were identified by staining with I-A^b tetramers presenting dominant epitopes from Rv2608 and Rv3619. Cells are gated as singlet, CD4⁺ CD44⁺. Splenic ID93-specific T_H1 CD4⁺ T cells from immunized mice were identified by cytokine production following <i>ex-vivo</i> restimulation with ID93 and analyzed for (C) total cytokine response or (D) poly-functional responses. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083884#pone.0083884.s001" target="_blank">Figure S1</a> for representative cytokine staining. Data are representative of four experiments with similar results with 3–5 animals per group. ^*,^**,^***, and ^**** indicate <i>P</i><0.05, 0.01, 0.001, and 0.0001 respectively, relative to GLA+CpG as determined by ANOVA using the Bonferroni correction for multiple comparisons.</p