Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-α and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required

Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture

We present an optical approach for intracellular delivery of molecules contained within oxidation-sensitive polymersomes. The photosensitizer ethyl eosin is associated with the polymersome membrane to oxidatively increase the hydrophilicity of the hydrophobic block under optical excitation. This optofluidic interaction induces rapid polymersome rupture and payload release <i>via</i> the reorganization of the aggregate structure into smaller diameter vesicles and micelles. When the particles are endocytosed by phagocytes, such as RAW macrophages and dendritic cells, the polymersomes’ payload escapes the endosome and is released in the cell cytosol within a few seconds of illumination. The released payload is rapidly distributed throughout the cytosol within milliseconds. The presented optofluidic method enables fast delivery and distribution throughout the cytosol of individual cells, comparable to photochemical internalization, but a factor of 100 faster than similar carrier mediated delivery methods (<i>e.g.</i>, liposomes, polymersomes, or nanoparticles). Due to the ability to simultaneously induce payload delivery and endosomal escape, this approach can find applications in detailed characterizations of intra- and intercellular processes. As an example in quantitative cell biology, a peptide antigen was delivered in dendritic cells and MHC I presentation kinetics were measured at the single cell and single complex level

Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture

We present an optical approach for intracellular delivery of molecules contained within oxidation-sensitive polymersomes. The photosensitizer ethyl eosin is associated with the polymersome membrane to oxidatively increase the hydrophilicity of the hydrophobic block under optical excitation. This optofluidic interaction induces rapid polymersome rupture and payload release <i>via</i> the reorganization of the aggregate structure into smaller diameter vesicles and micelles. When the particles are endocytosed by phagocytes, such as RAW macrophages and dendritic cells, the polymersomes’ payload escapes the endosome and is released in the cell cytosol within a few seconds of illumination. The released payload is rapidly distributed throughout the cytosol within milliseconds. The presented optofluidic method enables fast delivery and distribution throughout the cytosol of individual cells, comparable to photochemical internalization, but a factor of 100 faster than similar carrier mediated delivery methods (<i>e.g.</i>, liposomes, polymersomes, or nanoparticles). Due to the ability to simultaneously induce payload delivery and endosomal escape, this approach can find applications in detailed characterizations of intra- and intercellular processes. As an example in quantitative cell biology, a peptide antigen was delivered in dendritic cells and MHC I presentation kinetics were measured at the single cell and single complex level

'De metallis in genere'

We present an optical approach for intracellular delivery of molecules contained within oxidation-sensitive polymersomes. The photosensitizer ethyl eosin is associated with the polymersome membrane to oxidatively increase the hydrophilicity of the hydrophobic block under optical excitation. This optofluidic interaction induces rapid polymersome rupture and payload release <i>via</i> the reorganization of the aggregate structure into smaller diameter vesicles and micelles. When the particles are endocytosed by phagocytes, such as RAW macrophages and dendritic cells, the polymersomes’ payload escapes the endosome and is released in the cell cytosol within a few seconds of illumination. The released payload is rapidly distributed throughout the cytosol within milliseconds. The presented optofluidic method enables fast delivery and distribution throughout the cytosol of individual cells, comparable to photochemical internalization, but a factor of 100 faster than similar carrier mediated delivery methods (<i>e.g.</i>, liposomes, polymersomes, or nanoparticles). Due to the ability to simultaneously induce payload delivery and endosomal escape, this approach can find applications in detailed characterizations of intra- and intercellular processes. As an example in quantitative cell biology, a peptide antigen was delivered in dendritic cells and MHC I presentation kinetics were measured at the single cell and single complex level

Synthetic vaccine particles for durable cytolytic T lymphocyte responses and anti-tumor immunotherapy

<div><p>We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8⁺ T cell effector memory cells locally and centrally, which persisted for at least 1–2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13–14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.</p></div

CTL induction and anti-tumor activity of two SVP formulations.

<p>A-D. Analysis of T lymphocyte populations after immunization with SVP. Animals (4 mice/group/time-point) have been injected with SVP and antigen-specific T cells evaluated. A, B–cells from draining lymph nodes (A) or spleens (B) stained for CD8 and SIINFEKL-specific TCR (CD19⁺ cells gated out). C, D. CD8⁺SIINFEKL-specifc cells are stained for CD62L and CD44 T markers. C–popliteal lymph nodes; D–spleens. Summary of two independent experiments is shown. E. SVP induction of antigen-specific cytotoxicity. Animals (3–6 per time-point in each group) were injected with SVP[OVA]-PLA or SVP[OVA]-PLGA combined with SVP[R848] and CTL activity measured in vivo at times indicated. F-H. Anti-tumor effect of SVP immunization. Animals inoculated with EG.7-OVA cells were treated with SVP[OVA]-PLA or SVP[OVA]-PLGA combined with SVP[R848] at days 1, 4, 11, and 18 (F) or 3, 7, 14, and 21 (G) by s.c. administration at a tumor-distant site. H. SVP-treated animals surviving EG.7-OVA challenge were re-challenged with the same cells without additional treatment. Summary of two (F, H) or five (G) independent experiments is shown. * p <0.05, ** p <0.01, *** p<0.001, **** p<0.0001.</p

Immunogenicity of SVP-entrapped HPV-16 antigens and their therapeutic efficacy in vivo.

<p>A, B. Antigen-specific cytotoxicity at 7 days after immunization with SVP[E7.I.49], SVP[E7*] or SVP[E7/E6*]. Target cells were pulsed by E7.I.49 peptide (A) or by a pool of subdominant E7 peptides (B). C. Treatment of TC-1 tumors by SVP-entrapped E.I.49, E7* or E7/E6* combined with SVP[R848], SVP administered on days 10, 14, 21 and 28 after tumor inoculation. Number of mice in each group is shown in parentheses. Summary of four independent experiments is shown. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.</p

Experimental scheme for NHP immunization and sample analysis.

<p>Times of SVP vaccinations (thick arrows) are shown above the general timeline, analysis time-points (serum and PBMC isolation) are shown by thin arrows below the timeline.</p

Treatment of TC-1 tumors by SVP[E7.I.49] combined with different adjuvants.

<p>Survival proportions are shown on each graph. A, B. SVP-entrapped R848 vs. PS-1826 CpG. Treatments administered on days 3, 7, 14 and 21 (A) or days 6, 10, 17 and 24 (B) after tumor inoculation. Summary of three (A) or four (B) independent experiments shown. C, D. SVP-entrapped or free CpG ODN; PS-1826 (C) or PO-1826 (D). Treatments administered on days 6, 10, 17 and 24 after tumor inoculation. Summary of three (C) or two (D) independent experiments is shown. ** p <0.01, *** p<0.001, **** p < 0.0001.</p