Cytosolic Antigen Delivery Research Articles

Peptide-based coacervates for cytosolic delivery of antigens

Peptide-based coacervates for cytosolic delivery of antigens

Preparation of glycopeptide-modified pH-sensitive liposomes for promoting antigen cross-presentation and induction of antigen-specific cellular immunity.

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen. Polyglycidol-based or polysaccharide-based pH-responsive polymers can provide liposomes with delivery performance of antigenic proteins into cytosol via membrane fusion with endosomes responding to acidic pH, leading to induction of cross-presentation. Mannose residue was introduced to pH-responsive polysaccharides to increase uptake selectivity to antigen presenting cells and to improve cross-presentation efficiency. However, direct introduction of mannose residue into pH-responsive polysaccharides suppressed cytoplasmic delivery performance of liposomes. To avoid such interference, for this study, mannose-containing glycans were incorporated separately into pH-responsive polysaccharide-modified liposomes. Soybean agglutinin-derived glycopeptide was used as a ligand for lectins on antigen presenting cells. Incorporation of glycopeptide significantly increased the cellular uptake of liposomes by dendritic cell lines and increased cross-presentation efficiency. Liposomes incorporated both glycopeptide and pH-responsive polysaccharides exhibited strong adjuvant effects in vitro and induced the increase of dendritic cells, M1 macrophages, and effector T cells in the spleen. Subcutaneous administration of these liposomes induced antigen-specific cellular immunity, resulting in strong therapeutic effects in tumor-bearing mice. These results suggest that separate incorporation of glycopeptides and pH-responsive polysaccharides into antigen-loaded liposomes is an effective strategy to produce liposome-based nanovaccines to achieve antigen cross-presentation and induction of cellular immunity towards cancer immunotherapy.

Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells.

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LFN) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LFN showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

A rationally designed cancer vaccine based on NIR-II fluorescence image-guided light-triggered remote control of antigen cross-presentation and autophagy.

Cancer vaccines represent a promising immunotherapeutic treatment modality. The promotion of cross-presentation of extracellular tumor-associated antigens on the major histocompatibility complex (MHC) class I molecules and dendritic cell maturation at the appropriate time and place is crucial for cancer vaccines to prime cytolytic T cell response with reduced side effects. Current vaccination strategies, however, are not able to achieve the spatiotemporal control of antigen cross-presentation. Here, we report a liposomal vaccine loading the second near-infrared window (NIR-II, 1000-1700nm) fluorophore BPBBT with an efficient photothermal conversion effect that offers an NIR-light-triggered endolysosomal escape under the imaging guidance. The NIR-II image-guided vaccination strategy specifically controls the cytosolic delivery of antigens for cross-presentation in the draining lymph nodes (DLNs). Moreover, the photothermally induced endolysosomal rupture initiates autophagy. We also find that the adjuvant simvastatin acts as an autophagy activator through inhibiting the PI3K/AKT/mTOR pathway. The light-induced autophagy in the DLNs together with simvastatin treatment cooperatively increase MHC class II expression by activating autophagy machinery for dendritic cell maturation. This study presents a paradigm of NIR-II image-guided light-triggered vaccination. The approach for remote control of antigen cross-presentation and autophagy represents a new strategy for vaccine development.

Orchestrated Cytosolic Delivery of Antigen and Adjuvant by Manganese Ion-Coordinated Nanovaccine for Enhanced Cancer Immunotherapy.

Cancer vaccines have received tremendous attention in cancer immunotherapy due to their capability to induce a tumor-specific immune response. However, their effectiveness is compromised by the insufficient spatiotemporal delivery of antigens and adjuvants in the subcellular level to induce a robust CD8+ T cell response. Herein, a cancer nanovaccine G5-pBA/OVA@Mn is prepared through multiple interactions of manganese ions (Mn2+), benzoic acid (BA)-modified fifth generation polyamidoamine (G5-PAMAM) dendrimer, and the model protein antigen ovalbumin (OVA). In the nanovaccine, Mn2+ not only exerts a structural function to assist OVA loading as well as its endosomal escape, but works as an adjuvant of stimulator of interferon genes (STING) pathway. These collaboratively facilitate the orchestrated codelivery of OVA antigen and Mn2+ into cell cytoplasm. Vaccination with G5-pBA/OVA@Mn not only shows a prophylactic effect, but also significantly inhibits growth against B16-OVA tumors, indicating its great potential for cancer immunotherapy.

Dual-Responsive Glycopolymers for Intracellular Codelivery of Antigen and Lipophilic Adjuvants.

Traditional approaches to vaccines use whole organisms to trigger an immune response, but they do not typically generate robust cellular-mediated immunity and have various safety risks. Subunit vaccines composed of proteins and/or peptides represent an attractive and safe alternative to whole organism vaccines, but they are poorly immunogenic. Though there are biological reasons for the poor immunogenicity of proteins and peptides, one other key to their relative lack of immunogenicity could be attributed to the poor pharmacokinetic properties of exogenously delivered proteins and peptides. For instance, peptides often aggregate at the site of injection and are not stable in biological fluids, proteins and peptides are rapidly cleared from circulation, and both have poor cellular internalization and endosomal escape. Herein, we developed a delivery system to address the lack of protein immunogenicity by overcoming delivery barriers as well as codelivering immune-stimulating adjuvants. The glycopolymeric nanoparticles (glycoNPs) are composed of a dual-stimuli-responsive block glycopolymer, poly[2-(diisopropylamino)ethyl methacrylate]-b-poly[(pyridyl disulfide ethyl methacrylate)-co-(methacrylamidoglucopyranose)] (p[DPA-b-(PDSMA-co-MAG)]). This polymer facilitates protein conjugation and cytosolic release, the pH-responsive release of lipophilic adjuvants, and pH-dependent membrane disruption to ensure cytosolic delivery of antigens. We synthesized p[DPA-b-(PDSMA-co-MAG)] by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by the formation and physicochemical characterization of glycoNPs using the p[DPA-b-(PDSMA-co-MAG)] building blocks. These glycoNPs conjugated the model antigen ovalbumin (OVA) and released OVA in response to elevated glutathione levels. Moreover, the glycoNPs displayed pH-dependent drug release of the model hydrophobic drug Nile Red while also exhibiting pH-responsive endosomolytic behavior as indicated by a red blood cell hemolysis assay. GlycoNPs coloaded with OVA and the toll-like receptor 7/8 (TLR-7/8) agonist Resiquimod (R848) activated DC 2.4 dendritic cells (DCs) significantly more than free OVA and R848 and led to robust antigen presentation of the OVA epitope SIINFEKL on major histocompatibility complex I (MHC-I). In sum, the dual-stimuli-responsive glycopolymer introduced here overcomes major protein and peptide delivery barriers and could vastly improve the immunogenicity of protein-based vaccines.

Cationic lipid potentiated the adjuvanticity of polysaccharide derivative-modified liposome vaccines

Antigen carriers that can selectively deliver antigens to antigen presenting cells and which can simultaneously activate these cells (adjuvant property) are necessary for efficient cancer immunotherapy or vaccination. Delivery of a model antigen into dendritic cell cytosol has been achieved by pH-responsive polymer-modified liposomes via destabilization of endosomal membranes responding to acidic pH, which impelled antigen-specific cellular immunity. Furthermore, β-glucan-based pH-responsive polysaccharides have shown not only cytosolic antigen delivery performance but also adjuvant property, which further heightened cellular immune responses. Because pH-responsive polysaccharides have anionic carboxy groups, cationic lipid was introduced to liposomes in this study to improve the modification efficiency of pH-responsive polysaccharides and to improve their adjuvanticity and immunity-inducing functions. Introduction of cationic lipids increased the amounts of polysaccharide derivatives on the liposome and increased the cellular association of the liposomes to dendritic cells. Liposomes containing β-glucan-based pH-responsive polysaccharides and cationic lipids increased cytokine production from dendritic cells much more than other polysaccharide derivatives did. Furthermore, through improvement of intra-tumoral immunosuppression and induction of antigen-specific cellular immunity, administering these liposomes impelled tumor suppression even with a small antigen dose. These results suggest that introducing cationic lipids and using pH-responsive polysaccharides having intrinsically adjuvant function are effective for producing liposomal nanovaccines showing strong immunity-inducing function.

Cell Squeeze: driving more effective CD8 T-cell activation through cytosolic antigen delivery.

Cell Squeeze is a novel technology that relies on temporarily disrupting the cell membrane to deliver cargo directly into the cytosol. This approach is applicable to a broad range of cell types (peripheral blood mononuclear cells, red blood cells, hematopoietic stem cells, etc.) and cargos (peptides, proteins, small molecules, nucleic acids, and gene-editing complexes) while minimally disrupting normal cell function. By enabling direct cytosolic delivery, one can use this technology to dramatically enhance major histocompatibility complex (MHC) class I presentation of antigens (Ags) for CD8+ T-cell activation—a longstanding challenge for the therapeutic cancer vaccine field that has generally relied on cross-presentation of endocytosed Ags. In addition, by coupling improved MHC class I presentation with coexpression of additional stimulatory factors or systemic immune modulators, one can further enhance the potential impact of an antitumor CD8 response. Pursuing a more direct cellular engineering strategy, which is independent of viral transduction, genetic manipulation, and expansion steps, enables <24 h manufacturing of autologous cell therapies. Through generation of more sophisticated, multifunctional, cell-based vaccines, clinical testing of this technology will elucidate its potential for impact across multiple tumor types.

Dual-responsive nanovaccine for cytosolic delivery of antigens to boost cellular immune responses and cancer immunotherapy

Cancer vaccine contributing to the success of the treatment and prevention of tumors has attracted a huge attention as a strategy for tumor immunotherapy in recent years. A major challenge of cancer vaccine is to target cytosols of dendritic cells (DCs) in the lymph nodes (LNs) to enhance efficiency of antigen cross-presentation, which elicits high levels of cytotoxic T-lymphocytes to destruct tumor cells. Here, we address this issue by conjugating ovalbumin (OVA) to PEG-PCL using disulfide bond (-ss-), and the degradable pH-responsive polymer-PEI-PCL as delivery carrier. In addition, the mol ratio of PEG-PCL to PEI-PCL in the mixed micelles was tailored to deliver the OVA to LNs. Subsequently, CpG ODN1826, a TLR-9 agonist, was further introduced into a mixed micelle of 30 nm or less as a unique tumor vaccine. Importantly, the results demonstrated the mixed micelles with 1:1 mol of PCL-PEG and PCL-PEI can effectively migrate to distal LNs where antigen were efficiently captured by DCs, meanwhile, OVA was modified to the surface of mixed micelles via disulfide bonds (-ss-) for promotion efficiency of antigen cross-presentation. More surprisingly, combination of tumor vaccine with anti-PD-1, the therapy of ectopic melanoma (B16-OVA) and lung metastasis melanoma (B16-OVA) is excellent therapeutic effect. Taken together, our works offers a novel strategy for the cytosol delivery of antigens to achieve potent cancer immunotherapy.

Virus-Like Particle-Templated Silica-Adjuvanted Nanovaccines with Enhanced Humoral and Cellular Immunity.

Virus-like particles (VLPs) are self-assembled viral proteins that represent a superior form of antigens in vaccine formulations. To enhance immunogenicity, adjuvants, especially the aluminum salts (Alum), are essentially formulated in VLP vaccines. However, Alum only induce biased humoral immune responses that limits further applications of VLP-based vaccines. To stimulate more balanced immunity, we, herein, develop a one-step strategy of using VLPs as the biotemplates to synthesize raspberry-like silica-adjuvanted VLP@Silica nanovaccines. Hepatitis B surface antigen (HBsAg) VLPs and human papillomavirus type 18 (HPV 18) VLPs are selected as model templates. Circular dichroism (CD) and affinity analyses demonstrate that HBsAg VLPs in the nanovaccines maintain their secondary structure and immunogenicity, respectively. VLP@Silica promote silica dissolution-induced lysosomal escape and cytosolic delivery of antigens, and enhance the secretion of both Th1 and Th2 type cytokines in murine bone marrow-derived dendritic cells (BMDCs). Additionally, they could improve antigen trafficking and mediate DC activation in draining lymph nodes (DLNs). Vaccination study demonstrate that both HBsAg VLP@Silica and HPV 18 VLP@Silica nanovaccines induce enhanced antigen-specific antibody productions and T-cell mediated adaptive immune responses. This design strategy can utilize VLPs derived from a diversity of viruses or their variants as templates to construct both prophylactic and therapeutic vaccines with improved immunogenicity.

Elastic Nanovaccine Enhances Dendritic Cell-Mediated Tumor Immunotherapy.

Nanovaccine-based immunotherapy (NBI) has the ability to initiate dendritic cell (DC)-mediated tumor-specific immune responses and maintain long-term antitumor immune memory. To date, the mechanism by which the mechanical properties of nanoparticles alter the functions of DCs in NBI remains largely unclear. Here, a soft mesoporous organosilica-based nanovaccine (SMONV) is prepared and the elasticity-dependent effect of the nanovaccine on the underlying DC-mediated immune responses is studied. It is found that the elasticity results in greater internalization of SMONV by DCs, followed by the induction of substantial cytosolic delivery of antigens via endosomal escape, leading to effective DC maturation and antigen cross-presentation. Impressively, elasticity enables SMONV to enhance lymphatic drainage of antigens in vivo, thus stimulating robust humoral and cellular immunity. The results from therapeutic tumor vaccination further reveal that subcutaneously administered SMONV effectively suppresses tumor growth in tumor-bearing mice by evoking antigen-specific CD8+ T-cell immune responses, mitigating regulatory T-cell-mediated immunosuppression, and increasing central memory and effector memory T-cell populations. Furthermore, combinatorial immunization with SMONV and anti-PD-L1 blocking antibodies results in an amplified therapeutic effect on tumor-bearing mice. These findings reveal the elastic effect of the nanovaccine on DC-mediated immune responses, and the prepared SMONV represents a facile and powerful strategy for antitumor immunotherapy.

Cytosolic delivery of HBsAg and enhanced cellular immunity by pH-responsive liposome

It remains a major challenge to prime the cytotoxic T lymphocytes (CTLs) response for the treatment of Hepatitis B virus (HBV) infection. Inspired by an important natural biological behavior, membrane fusion, we constructed a pH-responsive nanocarrier (HBsAg&CpG@Lip) with a membrane fusion capacity for HBsAg intracellular delivery and subsequent process of CTLs. In the in vitro experiments, HBsAg&CpG@Lip greatly promoted the cellular uptake of HBsAg and the activation of BMDCs. It induced the cytosolic release of HBsAg in BMDCs, which was in conformity with the membrane fusion capacity of HBsAg&CpG@Lip. Such a capacity was improved by 4.4-fold in pH = 5.0 than that in pH = 7.4 at 60 min, which was calculated through fluorescence resonance energy transfer (FRET) efficiency. Furthermore, the cross-presentation activity induced by liposome at 1 μg/mL was equivalent to that by soluble antigen at 2000 μg/mL, which indicated an advantage for the liposome in cytosolic antigen delivery and potent CTLs activation. In respect of the transport of liposomes into draining lymph nodes (DLNs), the recruitment of liposome+migratory DCs at 24 h raised 11-fold than that at 3 h, suggesting that liposomes were mainly carried by migratory DCs. Meanwhile, HBsAg&CpG@Lip also elicited the activation of DCs and the generation of Tfh cells in DLNs. After immunization, HBsAg&CpG@Lip induced a higher anti-HBsAg IgG and ratio of IgG2c/IgG1 than that by HBsAg or Alum+HBsAg group. It also augmented a strong CTLs response as expected. Specifically, stimulation with HBsAg&CpG@Lip increased the number of IFN-γ-secreting splenocytes, the proportion of CD107α+CD8+and FasL+CD8+ T cells and the secretion of Granzyme B, which verified the design of membrane fusion in vivo. In summary, this design for HBsAg cytosolic delivery exhibits great benefits for triggering CTLs, opening an avenue for the development of a potent therapeutic HBV vaccine.

Chondroitin Sulfate-Based pH-Sensitive Polymer-Modified Liposomes for Intracellular Antigen Delivery and Induction of Cancer Immunity.

Induction of cancer-specific cytotoxic T lymphocytes is crucially important to complement therapeutic effects of immune checkpoint inhibitors and to achieve efficient cancer immunotherapy. To induce cancer-specific cytotoxic T lymphocytes, cancer antigen carriers must have multiple functions to deliver cancer antigens to antigen presenting cells, release antigens into cytosol, and promote the maturation of these cells. We earlier achieved cytosolic delivery of antigens and induction of antigen-specific cytotoxic T lymphocytes using carboxylated polyglycidol or polysaccharide derivative-modified liposomes that can induce membrane fusion with endosomes in response to weakly acidic pH. Furthermore, pH-sensitivity and adjuvant properties of these polymers were enhanced strongly by introduction of hydrophobic carboxylated units to dextran. Against our expectations, these polymer-modified liposomes only slightly induce cancer immunity, probably because of the high hydrophobicity of spacer units. This study used a polysaccharide with charged groups (chondroitin sulfate) instead of dextran as a backbone to reduce hydrophobicity. Chondroitin sulfate derivative-modified liposomes showed almost equal pH-sensitivity to that of dextran derivative-modified liposomes and achieved selective delivery to dendritic cells, whereas dextran derivative-modified liposomes were highly taken up by both dendritic cells and fibroblasts. Chondroitin sulfate derivative-modified liposomes delivered model antigenic proteins into cytosol of dendritic cells and promoted cytokine production from the cells, leading to tumor regression on tumor-bearing mice after subcutaneous administration. Results demonstrate that charged groups having polysaccharide as a backbone can be used in an effective strategy to balance strong hydrophobicity of spacer units with their utilization for immunity-inducing systems.

38P - SQZing cells to rapidly generate antigen presenting cells (APC) for solid tumor immune therapies with efficient, scalable manufacturing

Background: CD8 T cells are critical to immunotherapeutic intervention in cancer. However, the ability to efficiently target antigens for MHC-I presentation has limited the efficacy of immunization strategies. Here, we use microfluidics-based SQZ cell therapy platform to deliver antigen to the cytosol of target APCs, resulting in enhanced presentation on MHC-I. Given that no expansion time is required to engineer these APCs, this approach allows for rapid manufacturing of billions of non-conventional APCs capable of priming CD8+ T cell responses. Methods: Protein and peptide antigens were delivered to murine or human T cells with the SQZ cell therapy platform. The response to in vivo immunization of C57BL/6 mice was assessed by flow cytometry. Tumor experiments were conducted with the TC-1 cell line. Human T cells were co-cultured with epitope-reactive human responder CD8+ T cells, and interferon gamma production was quantified to assess antigen-specific responses. Results: In murine systems, we demonstrate that therapeutic immunization with T cells loaded with E7 using the SQZ platform are capable of eliciting strong anti-tumor effects. These anti-tumor responses correlate with an increase in antigen-specific CD8+ tumor infiltrating lymphocytes that is dependent on the co-administration of an adjuvant. In human cells, we demonstrate that primary T cells loaded with CMV and HPV16 antigens using SQZ can stimulate antigen-specific CD8+ T cell responses in vitro. Importantly, delivery to T cells scales from millions up to billions of cells per minute and antigen presentation is retained even after cryo-preservation. Conclusions: Through the direct cytosolic delivery of antigen, the SQZ cell therapy platform enables the use of T cells, a readily accessible and abundant cell type, to function as APCs for immunization. This strategy has demonstrated the ability to generate CD8+ T cell responses in both murine and human systems and has been scaled up for clinical implementation. With the ease and speed of manufacturing, these results have the potential to translate into a cellular immune therapy for oncology patients. Legal entity responsible for the study: SQZ Biotechnologies. Funding: SQZ Biotechnologies. Disclosure: S.M. Loughhead, M.G. Booty, K. Hlavaty, A. Vicente-Suarez, K. Blagovic, M. Myint, B. Stokes, D. Yarar, H. Bernstein, A. Sharei: Employee of SQZ Biotech.

Nanovaccine Incorporated with Hydroxychloroquine Enhances Antigen Cross-Presentation and Promotes Antitumor Immune Responses.

Induction of effective antigen-specific CD8+ T-cell responses is critical for cancer immunotherapy success. Hydroxychloroquine (HCQ) is a widely used classical antimalarial and antirheumatic drug. HCQ is also an endosomal membrane disrupting agent that can lead to vesicular swelling and membrane permeabilization, which likely facilitates the release of therapeutic agents from lysosomes into the cytoplasm. Here, we develop a minimalistic nanovaccine, which is composed of poly(lactide- co-glycolide)acid (PLGA) nanoparticles (NPs) encapsulating a physical mixture of ovalbumin (OVA, a model antigen) and HCQ (HCQ-OVA-PLGA NPs). We tested whether HCQ could spatiotemporally control the cytosolic delivery of antigens, enhance antigen processing and presentation via the major histocompatibility complex (MHC)-I pathway, and thus generate a sufficient antitumor cytotoxic T-cell response. The results of in vitro experiments showed that HCQ-OVA-PLGA NPs significantly enhanced OVA escape from lysosomes into the cytoplasm within bone-marrow-derived dendritic cells. We also observed that HCQ-OVA-PLGA NPs enhanced the expression level of MHC-I on dendritic cells and improved cross-presentation of antigen, compared to free OVA or OVA-PLGA NPs. Results of in vivo experiments confirmed that HCQ initiated Th1-type responses and strong CD8+ T-cell responses that induced tumor cell apoptosis. Moreover, vaccination of mice with HCQ-OVA-PLGA NPs effectively generated memory immune responses in vivo and prevented tumor progression. We conclude that co-encapsulation of HCQ with antigens in nanovaccines can boost antigen-specific antitumor immune responses, particularly through CD8+ T-cells, serving as a simple and effective platform for the treatment of tumors and infectious diseases.

Synergistic effect of dual targeting vaccine adjuvant with aminated β-glucan and CpG-oligodeoxynucleotides for both humoral and cellular immune responses

Presently, clinically approved adjuvants (such as aluminum salts) fail to induce cellular immune responses, which is crucial to defend against intracellular pathogens (including HIV, malaria, tuberculosis and Ebola) and cancer. However, Freund’s complete adjuvant potently stimulates both humoral and cellular immune responses, accompanying by high toxicity and severe side reactions. Here in this work, a CpG-oligodeoxynucleotides (CpG-OND) crosslinked aminated β-glucan-Ovalbumin dual targeting nanoparticle (CpG-OND-AG-OVA) is prepared through a simple and mild ionic complexation method. The aminated β-glucan plays dual roles as antigen presenting cells (APCs) targeted carrier and immunopotentiator (targeting and activating dectin-1 on APCs). Meanwhile, CpG-OND also plays dual roles as ionic crosslinker and immunopotentiator (targeting and activating Toll-like receptor 9 in APCs). The adjuvant activity of the particles is evaluated through in vitro and in vivo experiments. The particles significantly enhance uptake and sustained proteolytic processing of antigens, and result in APCs maturation, inducing robust Th1 and Th2-type immune responses comparable to Freund’s adjuvant without obvious toxicity. The potent adjuvant activity of the nanoparticles may originate from dual targeting synergistic effects between aminated β-glucan and CpG-OND. Accordingly, the dual targeting nanoparticles may be a promising vaccine adjuvant for inducing robust humoral and cellular immune responses against infectious diseases and cancers. Statement of SignificanceAn ideal adjuvant for subunit vaccine should act as both a carrier to enhance the uptake, sustained processing and cytosolic delivery of antigens, and an immunopotentiator to stimulate antigen presenting cells (APCs) for activation of naive T cells. Additionally, it should be easy to obtain and safe with negligible toxicity. Unfortunately, both synthetic and natural polymers that have been developed into antigen delivery system cannot completely fulfill the requirements. In the present study, the authors design nanoparticles with aminated β-glucan and CpG-oligodeoxynucleotides (CpG-OND) through a simple and mild method. β-Glucan (a dectin-1 and TLR2 targeted PAMP) and CpG-OND (a TLR9 targeted PAMP) are readily accessible. Aminated β-glucan plays dual roles in the nanoparticle as APCs targeted carrier and immunopotentiator. Meanwhile, CpG-OND also plays dual roles as crosslinker and APCs targeted immunopotentiator. By making use of synergistic effect of the dual targeting vaccine adjuvant with aminated β-glucan and CpG-OND, the nanoparticles induce robust antigen specific immune responses comparable to Freund’s adjuvant without obvious toxicity.

Photothermally Controlled MHC Class I Restricted CD8+ T-Cell Responses Elicited by Hyaluronic Acid Decorated Gold Nanoparticles as a Vaccine for Cancer Immunotherapy.

Cancer vaccines aim to induce a strong major histocompatibility complex class I (MHC-I)-restricted CD8+ cytotoxic T-cell response, which is an important prerequisite for successful cancer immunotherapy. Herein, a hyaluronic acid (HA) and antigen (ovalbumin, OVA)-decorated gold nanoparticle (AuNPs)-based (HA-OVA-AuNPs) vaccine is developed for photothermally controlled cytosolic antigen delivery using near-infrared (NIR) irradiation and is found to induce antigen-specific CD8+ T-cell responses. Chemical binding of thiolated HA and OVA to AuNPs facilitates antigen uptake of dendritic cells via receptor-mediated endocytosis. HA-OVA-AuNPs exhibit enhanced NIR absorption and thermal energy translation. Cytosolic antigen delivery is then permitted through the photothermally controlled process of local heat-mediated endo/lysosome disruption by laser irradiation along with reactive oxygen species generation, which helps to augment proteasome activity and downstream MHC I antigen presentation. Consequently, the HA-OVA-AuNPs nanovaccine can effectively evoke a potent anticancer immune response in mice under laser irradiation. This NIR-responsive nanovaccine is promising as a potent vaccination method for improving cancer vaccine efficacy.

A nano-liposome vaccine carrying E75, a HER-2/neu-derived peptide, exhibits significant antitumour activity in mice

E75 (HER-2/neu-369–377), is an immunogenic peptide which is highly expressed in breast cancer patients. The purpose of this study was to develop an effective vaccine delivery/adjuvant system by attachment of this peptide to the surface of liposomes consisting of phospholipids including distearoylphosphocholine (DSPC) and distearoyl phosphoglycerol (DSPG) with high transition temperature (Tm) and dioleoylphosphatidylethanolamine (DOPE) (a pH-sensitive lipid for cytosolic antigen delivery) to improve antitumour immune activity against the E75 peptide. For this purpose, the E75 peptide was incorporated into liposomes consisting of DSPC/DSPG/cholesterol (Chol)/DOPE (15/2/3/5 molar ratio) through conjugation with distearoylphosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (maleimide-PEG2000-DSPE). Immunization of BALB/c mice was performed three times with different forms of liposomal formulations at 2-week intervals and antitumour immunity responses were evaluated. Results of ELISpot and flow cytometry analysis showed that mice vaccinated with DSPC/DSPG/Chol/DOPE/E75 have significantly enhanced the antigen-specific IFN-γ response of CD8+ T cells and generated cytotoxic T lymphocytes (CTL) antitumour responses. CTL responses induced by this formulation resulted in inhibition of tumour progression and longer survival time in the mice TUBO tumour model. The results revealed that the liposomes consist of DSPC/DSPG/Chol/DOPE could be suitable candidates for vaccine delivery of E75 peptide for the prevention and therapy of HER2-positive breast cancer and merit further investigation.

Coordinating antigen cytosolic delivery and danger signaling to program potent cross-priming by micelle-based nanovaccine

Although re-activating cytotoxic T-cell (CTLs) response inside tumor tissues by checkpoint blockade has demonstrated great success in tumor immunotherapy, active induction of efficient endogenous CTL response by therapeutic vaccines has been largely hampered by inefficient cytosolic delivery of antigens and coordinated activation of dendritic cells (DCs) in lymph nodes. Here we show that polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles transform soluble peptides into α-helix to enable their efficient cytosolic delivery. The same PEG-PE micelles also serve as chaperon of TLR4 signaling to coordinate its adjuvant effect on the same DCs. Furthermore, these nanovaccines effectively target lymph node DCs. Thus, PEG-PE micelle vaccines program at multiple key aspects for inducing strong CTL responses and build up a foundation for combinational tumor therapy.

Cytosolic Delivery of Liposomal Vaccines by Means of the Concomitant Photosensitization of Phagosomes.

One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and presentation for stimulation of strong CD8(+) T-cell responses. Therefore, photosensitive particulate vaccines may have the potential to add to current vaccine practice a new method of vaccination that, as opposed to current vaccines, can stimulate strong CD8(+) T-cell responses.