Lipid-based vaccine nanoparticles for induction of humoral immune responses against HIV-1 and SARS-CoV-2.

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.

Sugar-Nanocapsules Imprinted with Microbial Molecular Patterns for mRNA Vaccination.

Innate immune cells recognize and respond to pathogen-associated molecular patterns. In particular, polysaccharides found in the microbial cell wall are potent activators of dendritic cells (DCs). Here, we report a new class of nanocapsules, termed sugar-capsules, entirely composed of polysaccharides derived from the microbial cell wall. We show that sugar-capsules with a flexible polysaccharide shell and a hollow core efficiently drain to lymph nodes and activate DCs. In particular, sugar-capsules composed of mannan (Mann-capsule) carrying mRNA (mRNA) promote strong DC activation, mRNA translation, and antigen presentation on DCs. Mann-capsules elicit robust antigen-specific CD4+ and CD8α+ T-cell responses with antitumor efficacy in vivo. The strategy presented in this study is generally applicable for utilizing pathogen-derived molecular patterns for vaccines and immunotherapies.

Extracellular Trap-Mimicking DNA-Histone Mesostructures Synergistically Activate Dendritic Cells.

Extracellular traps (ETs), such as neutrophil extracellular traps, are a physical mesh deployed by immune cells to entrap and constrain pathogens. ETs are immunogenic structures composed of DNA, histones, and an array of variable protein and peptide components. While much attention has been paid to the multifaceted function of these structures, mechanistic studies of ETs remain challenging due to their heterogeneity and complexity. Here, a novel DNA-histone mesostructure (DHM) formed by complexation of DNA and histones into a fibrous mesh is reported. DHMs mirror the DNA-histone structural frame of ETs and offer a facile platform for cell culture studies. It is shown that DHMs are potent activators of dendritic cells and identify both the methylation state of DHMs and physical interaction between dendritic cells and DHMs as key tuning switches for immune stimulation. Overall, the DHM platform provides a new opportunity to study the role of ETs in immune activation and pathophysiology.

Self-healing encapsulation and controlled release of vaccine antigens from PLGA microparticles delivered by microneedle patches.

There is an urgent need to reduce reliance on hypodermic injections for many vaccines to increase vaccination safety and coverage. Alternative approaches include controlled release formulations, which reduce dosing frequencies, and utilizing alternative delivery devices such as microneedle patches (MNPs). This work explores development of controlled release microparticles made of poly (lactic-co-glycolic acid) (PLGA) that stably encapsulate various antigens though aqueous active self-healing encapsulation (ASE). These microparticles are incorporated into rapid-dissolving MNPs for intradermal vaccination. PLGA microparticles containing Alhydrogel are loaded with antigens separate from microparticle fabrication using ASE. This avoids antigen expsoure to many stressors. The microparticles demonstrate bi-phasic release, with initial burst of soluble antigen, followed by delayed release of Alhydrogel-complexed antigen over approximately 2 months in vitro. For delivery, the microparticles are incorporated into MNPs designed with pedestals to extend functional microneedle length. These microneedles readily penetrate skin and rapidly dissolve to deposit microparticles intradermally. Microparticles remain in the tissue for extended residence, with MNP-induced micropores resealing readily. In animal models, these patches generate robust immune responses that are comparable to conventional administration techniques. This lays the framework for a versatile vaccine delivery system that could be self-applied with important logistical advantages over hypodermic injections.

Vaccine nanoparticles displaying recombinant Ebola virus glycoprotein for induction of potent antibody and polyfunctional T cell responses.

The recent outbreaks of Ebolavirus (EBOV) in West Africa underscore the urgent need to develop an effective EBOV vaccine. Here, we report the development of synthetic nanoparticles as a safe and highly immunogenic platform for vaccination against EBOV. We show that a large recombinant EBOV antigen (rGP) can be incorporated in a configurational manner into lipid-based nanoparticles, termed interbilayer-crosslinked multilamellar vesicles (ICMVs). The epitopes and quaternary structure of rGP were properly maintained on the surfaces of ICMVs formed either with or without nickel nitrilotriacetic acid (NTA)-functionalized lipids. When administered in mice, rGP-ICMVs without NTA-lipids efficiently generated germinal center B cells and polyfunctional T cells while eliciting robust neutralizing antibody responses. This study suggests the potential of vaccine nanoparticles as a delivery platform for configurational, multivalent display of large subunit antigens and induction of neutralizing antibody and T cell responses.

Dendritic Cell Membrane Vesicles for Activation and Maintenance of Antigen-Specific T Cells.

Cell membranes have recently gained attention as a promising drug delivery system. Here, dendritic cell membrane vesicles (DC-MVs) are examined as a platform to promote T cell responses. Nanosized DC-MVs are derived from DCs pretreated with monophosphoryl lipid A (MPLA), a FDA-approved immunostimulatory adjuvant. These "mature" DC-MVs activate DCs in vitro and increase their expression of costimulatory markers. DC-MVs also promote cross-priming of antigen-specific T cells in vitro, increasing their survival and CD25 expression. In addition, these mature DC-MVs potently augment the expansion of adoptively transferred CD8+ T cells in vivo, generating twofold to fourfold higher frequency of antigen-specific T cells, compared with other control formulations, including "immature" DC-MVs obtained without the MPLA pretreatment. Taken together, these results suggest that DC-MVs are an effective delivery platform for T cell activation and may serve as a potential delivery system for improving adoptive T cell therapy.

Interrogation of Antigen Display on Individual Vaccine Nanoparticles for Achieving Neutralizing Antibody Responses against Hepatitis C Virus.

Elicitation of neutralizing antibody responses against hepatitis C virus (HCV) has been a challenging goal. While the E2 subunit of the HCV envelope glycoprotein complex is a promising target for generating cross-genotype neutralizing antibodies, vaccinations with soluble E2 immunogens generally induce weak neutralizing antibody responses. Here, E2 immunogens (i.e., E2.661 and E2c.661) were loaded into lipid-based nanovaccines and examined for induction of neutralizing antibody responses. Compared with soluble E2 immunogens, E2 nanoparticles elicited 6- to 20-fold higher E2-specific serum IgG titers in mice. Importantly, E2 vaccine nanoparticles analyzed at a single particle level with a flow cytometry-based method revealed interesting dynamics between epitope display on the surfaces of nanoparticles in vitro and induction of neutralizing antibody responses in vivo. E2c.661 nanoparticles that are preferentially bound by a broadly neutralizing antibody, HCV1, in vitro elicit neutralizing antibody responses against both autologous and heterologous HCV virions in vivo. In stark contrast, E2.661 nanoparticles with reduced HCV1-antibody binding in vitro mainly induce autologous neutralizing antibody responses in vivo. These results show that rationale antigen design coupled with interrogation of epitope display on vaccine nanoparticles at a single particle level may aid in vaccine development toward achieving neutralizing antibody responses in vivo.

PEGylated tumor cell membrane vesicles as a new vaccine platform for cancer immunotherapy.

Despite the promise and advantages of autologous cancer cell vaccination, it remains challenging to induce potent anti-tumor immune responses with traditional immunization strategies with whole tumor cell lysate. In this study, we sought to develop a simple and effective approach for therapeutic vaccination with autologous whole tumor cell lysate. Endogenous cell membranes harvested from cancer cells were formed into PEGylated nano-vesicles (PEG-NPs). PEG-NPs exhibited good serum stability in vitro and draining efficiency to local lymph nodes upon subcutaneous administration in vivo. Vaccination with PEG-NPs synthesized from murine melanoma cells elicited 3.7-fold greater antigen-specific cytotoxic CD8+ T lymphocyte responses, compared with standard vaccination with freeze-thawed lysate in tumor-bearing mice. Importantly, in combination with anti-programmed death-1 (αPD-1) IgG immunotherapy, PEG-NP vaccination induced 4.2-fold higher frequency of antigen-specific T cell responses (P < 0.0001) and mediated complete tumor regression in 63% of tumor-bearing animals (P < 0.01), compared with FT lysate + αPD-1 treatment that exhibited only 13% response rate. In addition, PEG-NPs + αPD-1 IgG combination immunotherapy protected all survivors against a subsequent tumor cell re-challenge. These results demonstrate a general strategy for eliciting anti-tumor immunity using endogenous cancer cell membranes formulated into stable vaccine nanoparticles.

Dual TLR agonist nanodiscs as a strong adjuvant system for vaccines and immunotherapy.

Recent studies have shown that certain combinations of Toll-like receptor (TLR) agonists can induce synergistic immune activation. However, it remains challenging to achieve such robust responses in vivo in a manner that is effective, facile, and amenable for clinical translation. Here, we show that MPLA, a TLR4 agonist, and CpG, a TLR9 agonist, can be efficiently co-loaded into synthetic high-density lipoprotein nanodiscs, forming a potent adjuvant system (ND-MPLA/CpG) that can be readily combined with a variety of subunit antigens, including proteins and peptides. ND-MPLA/CpG significantly enhanced activation of dendritic cells, compared with free dual adjuvants or nanodiscs delivering a single TLR agonist. Importantly, mice immunized with physical mixtures of protein antigens ND-MPLA/CpG generated strong humoral responses, including induction of IgG responses against protein convertase subtilisin/kexin 9 (PCSK9), leading to 17-30% reduction of the total plasma cholesterol levels. Moreover, ND-MPLA/CpG exerted strong anti-tumor efficacy in multiple murine tumor models. Compared with free adjuvants, ND-MPLA/CpG admixed with ovalbumin markedly improved antigen-specific CD8+ T cell responses by 8-fold and promoted regression of B16F10-OVA melanoma (P < 0.0001). Furthermore, ND-MPLA/CpG admixed with E7 peptide antigen elicited ~20% E7-specific CD8+ T cell responses and achieved complete regression of established TC-1 tumors in all treated animals. Taken together, our work highlights the simplicity, versatility, and potency of dual TLR agonist nanodiscs for applications in vaccines and cancer immunotherapy.

Chemo-photothermal therapy combination elicits anti-tumor immunity against advanced metastatic cancer.

Photothermal therapy (PTT) is a promising cancer treatment modality, but PTT generally requires direct access to the source of light irradiation, thus precluding its utility against disseminated, metastatic tumors. Here, we demonstrate that PTT combined with chemotherapy can trigger potent anti-tumor immunity against disseminated tumors. Specifically, we have developed polydopamine-coated spiky gold nanoparticles as a new photothermal agent with extensive photothermal stability and efficiency. Strikingly, a single round of PTT combined with a sub-therapeutic dose of doxorubicin can elicit robust anti-tumor immune responses and eliminate local as well as untreated, distant tumors in >85% of animals bearing CT26 colon carcinoma. We also demonstrate their therapeutic efficacy against TC-1 submucosa-lung metastasis, a highly aggressive model for advanced head and neck squamous cell carcinoma (HNSCC). Our study sheds new light on a previously unrecognized, immunological facet of chemo-photothermal therapy and may lead to new therapeutic strategies against advanced cancer.

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.

Self-encapsulating Poly(lactic-co-glycolic acid) (PLGA) Microspheres for Intranasal Vaccine Delivery.

Herein we describe a formulation of self-encapsulating poly(lactic-co-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.

Toward a Single-Dose Vaccination Strategy with Self-Encapsulating PLGA Microspheres.

Poly(lactic-co-glycolic acid) (PLGA) microspheres have been widely examined for vaccine applications due to their attractive features of biocompatibility, biodegradability, ability to be internalized by antigen-presenting cells, and long-term antigen release. However, one of the major challenges for PLGA particle vaccines is the potential for antigen instability and loss of antigenicity and immunogenicity. To address this challenge, we have developed a new method of "self-healing" encapsulation in PLGA microspheres, where pre-made PLGA microspheres are loaded with protein antigens under aqueous conditions with minimal impact on their antigenicity and immunogenicity. In this report, we show that mice immunized with self-encapsulating PLGA microspheres in a prime-boost regimen generated significantly enhanced antigen-specific CD8α+ T cell and antibody responses, compared with mice immunized with free, soluble protein admixed with calcium phosphate gel, a widely used adjuvant. Furthermore, a single-dose of microspheres designed for >40 day sustained antigen release elicited robust cellular and humoral immune responses as efficiently as the prime-boost vaccinations with calcium phosphate gel. Overall, these results suggest excellent potential of our self-encapsulating PLGA microspheres as a vaccine platform for multiple-dose as well as single-dose vaccinations.

Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens.

Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation - the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials.

Designer vaccine nanodiscs for personalized cancer immunotherapy.

Despite the tremendous potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent innovations in tumour exome sequencing have signalled the new era of personalized immunotherapy with patient-specific neoantigens, but a general methodology for stimulating strong CD8α+ cytotoxic T-lymphocyte (CTL) responses remains lacking. Here we demonstrate that high-density lipoprotein-mimicking nanodiscs coupled with antigen (Ag) peptides and adjuvants can markedly improve Ag/adjuvant co-delivery to lymphoid organs and sustain Ag presentation on dendritic cells. Strikingly, nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and even 31-fold greater than perhaps the strongest adjuvant in clinical trials (that is, CpG in Montanide). Moreover, multi-epitope vaccination generated broad-spectrum T-cell responses that potently inhibited tumour growth. Nanodiscs eliminated established MC-38 and B16F10 tumours when combined with anti-PD-1 and anti-CTLA-4 therapy. These findings represent a new powerful approach for cancer immunotherapy and suggest a general strategy for personalized nanomedicine.

Whole-animal imaging and flow cytometric techniques for analysis of antigen-specific CD8+ T cell responses after nanoparticle vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a "pathogen-mimicking" nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

Cationic liposome-hyaluronic acid hybrid nanoparticles for intranasal vaccination with subunit antigens.

Here we report the development of a new cationic liposome-hyaluronic acid (HA) hybrid nanoparticle (NP) system and present our characterization of these NPs as an intranasal vaccine platform using a model antigen and F1-V, a candidate recombinant antigen for Yersinia pestis, the causative agent of plague. Incubation of cationic liposomes composed of DOTAP and DOPE with anionic HA biopolymer led to efficient ionic complexation and formation of homogenous liposome-polymer hybrid NPs, as evidenced by fluorescence resonance energy transfer, dynamic light scattering, and nanoparticle tracking analyses. Incorporation of cationic liposomes with thiolated HA allowed for facile surface decoration of NPs with thiol-PEG, resulting in the formation of DOTAP/HA core-PEG shell nanostructures. These NPs, termed DOTAP-HA NPs, exhibited improved colloidal stability and prolonged antigen release. In addition, cytotoxicity associated with DOTAP liposomes (LC50~0.2mg/ml) was significantly reduced by at least 20-fold with DOTAP-HA NPs (LC50>4mg/ml), as measured with bone marrow derived dendritic cells (BMDCs). Furthermore, NPs co-loaded with ovalbumin (OVA) and a molecular adjuvant, monophosphoryl lipid A (MPLA) promoted BMDC maturation and upregulation of co-stimulatory markers, including CD40, CD86, and MHC-II, and C57BL/6 mice vaccinated with NPs via intranasal route generated robust OVA-specific CD8(+) T cell and antibody responses. Importantly, intranasal vaccination with NPs co-loaded with F1-V and MPLA induced potent humoral immune responses with 11-, 23-, and 15-fold increases in F1-V-specific total IgG, IgG1, and IgG2c titers in immune sera by day 77, respectively, and induced balanced Th1/Th2 humoral immune responses, whereas mice immunized with the equivalent doses of soluble F1-V vaccine failed to achieve sero-conversion. Overall, these results suggest that liposome-polymer hybrid NPs may serve as a promising vaccine delivery platform for intranasal vaccination against Y. pestis and other infectious pathogens.

Biomaterials for nanoparticle vaccine delivery systems.

Subunit vaccination benefits from improved safety over attenuated or inactivated vaccines, but their limited capability to elicit long-lasting, concerted cellular and humoral immune responses is a major challenge. Recent studies have demonstrated that antigen delivery via nanoparticle formulations can significantly improve immunogenicity of vaccines due to either intrinsic immunostimulatory properties of the materials or by co-entrapment of molecular adjuvants such as Toll-like receptor agonists. These studies have collectively shown that nanoparticles designed to mimic biophysical and biochemical cues of pathogens offer new exciting opportunities to enhance activation of innate immunity and elicit potent cellular and humoral immune responses with minimal cytotoxicity. In this review, we present key research advances that were made within the last 5 years in the field of nanoparticle vaccine delivery systems. In particular, we focus on the impact of biomaterials composition, size, and surface charge of nanoparticles on modulation of particle biodistribution, delivery of antigens and immunostimulatory molecules, trafficking and targeting of antigen presenting cells, and overall immune responses in systemic and mucosal tissues. This review describes recent progresses in the design of nanoparticle vaccine delivery carriers, including liposomes, lipid-based particles, micelles and nanostructures composed of natural or synthetic polymers, and lipid-polymer hybrid nanoparticles.