Bacterial Flagellum-Drug Nanoconjugates for Carrier-Free Immunochemotherapy.

The combination of immunotherapy and chemotherapy to ablate tumors has attracted substantial attention due to the ability to simultaneously elicit antitumor immune responses and trigger direct tumor cell death. However, conventional combinational strategies mainly focus on the employment of drug carriers to deliver immunomodulators, chemotherapeutics, or their combinations, always suffering from complicated preparation and carrier-relevant side effects. Here, the fabrication of bacterial flagellum-drug nanoconjugates (FDNCs) for carrier-free immunochemotherapy is described. FDNCs are simply prepared by attaching chemotherapeutics to amine residues of flagellin through an acid-sensitive and traceless cis-aconityl linker. By virtue of native nanofibrous structure and immunogenicity, bacterial flagella not only show long-term tumor retention and highly efficient cell internalization, but also provoke robust systemic antitumor immune responses. Meanwhile, conjugated chemotherapeutics exhibit an acid-mediated release profile and durable intratumoral exposure, which can induce potent tumor cell inhibition via direct killing. More importantly, this combination is able to augment immunoactivation effects associated with chemotherapy-enabled immunogenic tumor cell death to further enhance antitumor efficacy. By leveraging the innate response of the immune system to pathogens, the conjugation of therapeutic agents with self-adjuvant bacterial flagella provides an alternative approach to develop carrier-free nanotherapeutics for tumor immunochemotherapy.

Delivery of mRNA vaccine with a lipid-like material potentiates antitumor efficacy through Toll-like receptor 4 signaling.

Intracellular delivery of messenger RNA (mRNA)-based cancer vaccine has shown great potential to elicit antitumor immunity. To achieve robust antitumor efficacy, mRNA encoding tumor antigens needs to be efficiently delivered and translated in dendritic cells with concurrent innate immune stimulation to promote antigen presentation. Here, by screening a group of cationic lipid-like materials, we developed a minimalist nanovaccine with C1 lipid nanoparticle (LNP) that could efficiently deliver mRNA in antigen presenting cells with simultaneous Toll-like receptor 4 (TLR4) activation and induced robust T cell activation. The C1 nanovaccine entered cells via phagocytosis and showed efficient mRNA-encoded antigen expression and presentation. Furthermore, the C1 lipid nanoparticle itself induced the expression of inflammatory cytokines such as IL-12 via stimulating TLR4 signal pathway in dendritic cells. Importantly, the C1 mRNA nanovaccine exhibited significant antitumor efficacy in both tumor prevention and therapeutic vaccine settings. Overall, our work presents a C1 LNP-based mRNA cancer nanovaccine with efficient antigen expression as well as self-adjuvant property, which may provide a platform for developing cancer immunotherapy for a wide range of tumor types.

A new polysaccharide platform constructs self-adjuvant nanovaccines to enhance immune responses.

BACKGROUND: Nanovaccines have shown the promising potential in controlling and eradicating the threat of infectious diseases worldwide. There has been a great need in developing a versatile strategy to conveniently construct diverse types of nanovaccines and induce potent immune responses. To that end, it is critical for obtaining a potent self-adjuvant platform to assemble with different types of antigens into nanovaccines. RESULTS: In this study, we identified a new natural polysaccharide from the rhizomes of Bletilla striata (PRBS), and used this polysaccharide as a platform to construct diverse types of nanovaccines with potent self-adjuvant property. In the construction process of SARS-CoV-2 nanovaccine, PRBS molecules and RBD protein antigens were assembled into ~ 300 nm nanoparticles by hydrogen bond. For HIV nanovaccine, hydrophobic effect dominantly drove the co-assembly between PRBS molecules and Env expression plasmid into ~ 350 nm nanospheres. Importantly, PRBS can potently activate the behaviors and functions of multiple immune cells such as macrophages, B cells and dendritic cells. Depending on PRBS-mediated immune activation, these self-adjuvant nanovaccines can elicit significantly stronger antigen-specific antibody and cellular responses in vivo, in comparison with their corresponding traditional vaccine forms. Moreover, we also revealed the construction models of PRBS-based nanovaccines by analyzing multiple assembly parameters such as bond energy, bond length and interaction sites. CONCLUSIONS: PRBS, a newly-identified natural polysaccharide which can co-assemble with different types of antigens and activate multiple critical immune cells, has presented a great potential as a versatile platform to develop potent self-adjuvant nanovaccines.

Self-Adjuvant Effect by Manipulating the Bionano Interface of Liposome-Based Nanovaccines.

Nanovaccines are of increasing scrutiny due to their plasticity in size, composition, and surface properties to enhance antigenicity. However, inevitable absorption of plasma proteins affects the in vivo fate of nanovaccines by reshaping biological identity. Herein IgM was validated as a self-adjuvant by regulating antigen-presenting cells recognition of liposome-based nanovaccines. DCDX-modified liposomes with loading of ovalbumin (DCDX-sLip/OVA) heavily absorbed IgM via electrostatic interaction, demonstrating significant splenic B cells targeting. IgM absorbed on DCDX-sLip/OVA enhanced antigen uptake and presentation by both IgM-complement and IgM-FcµR pathways. DCDX-sLip/OVA induced a stronger IgG1 titer than ovalbumin-loaded plain liposomes (sLip/OVA) while maintaining a comparably high level of IgG2a titer with high biosafety, indicating that IgM absorption after DCDX modification could improve the antigenicity by enhancing the Th2-polarized immune response. The present work suggested manipulation of IgM absorption may provide a new impetus to improve in vivo performance of nanovaccines.

Self-adjuvant Astragalus polysaccharide-based nanovaccines for enhanced tumor immunotherapy: a novel delivery system candidate for tumor vaccines.

The study of tumor nanovaccines (NVs) has gained interest because they specifically recognize and eliminate tumor cells. However, the poor recognition and internalization by dendritic cells (DCs) and insufficient immunogenicity restricted the vaccine efficacy. Herein, we extracted two molecular-weight Astragalus polysaccharides (APS, 12.19 kD; APSHMw, 135.67 kD) from Radix Astragali and made them self-assemble with OVA257-264 directly forming OVA/APS integrated nanocomplexes through the microfluidic method. The nanocomplexes were wrapped with a sheddable calcium phosphate layer to improve stability. APS in the formed nanocomplexes served as drug carriers and immune adjuvants for potent tumor immunotherapy. The optimal APS-NVs were approximately 160 nm with uniform size distribution and could remain stable in physiological saline solution. The FITC-OVA in APS-NVs could be effectively taken up by DCs, and APS-NVs could stimulate the maturation of DCs, improving the antigen cross-presentation efficiency in vitro. The possible mechanism was that APS can induce DC activation via multiple receptors such as dectin-1 and Toll-like receptors 2 and 4. Enhanced accumulation of APS-NVs both in draining and distal lymph nodes were observed following s.c. injection. Smaller APS-NVs could easily access the lymph nodes. Furthermore, APS-NVs could markedly promote antigen delivery efficiency to DCs and activate cytotoxic T cells. In addition, APS-NVs achieve a better antitumor effect in established B16-OVA melanoma tumors compared with the OVA+Alum treatment group. The antitumor mechanism correlated with the increase in cytotoxic T cells in the tumor region. Subsequently, the poor tumor inhibitory effect of APS-NVs on the nude mouse model of melanoma also confirmed the participation of antitumor adaptive immune response induced by NVs. Therefore, this study developed a promising APS-based tumor NV that is an efficient tumor immunotherapy without systemic side effects.

Self-adjuvant multiepitope nanovaccine based on ferritin induced long-lasting and effective mucosal immunity against H3N2 and H1N1 viruses in mice.

The influenza A virus (IAV) is a ubiquitous and continuously evolving respiratory pathogen. The intranasal vaccination mimicking natural infections is an attractive strategy for controlling IAVs. Multiepitope vaccines accurately targeting multiple conserved domains have the potential to broaden the protective scope of current seasonal influenza vaccines and reduce the risk of generating escape mutants. Here, multiple linear epitopes from the matrix protein 2 ectodomain (M2e) and the hemagglutinin stem domain (HA2) are fused with the Helicobacter pylori ferritin, a self-assembled nanocarrier and mucosal adjuvant, to develop a multiepitope nanovaccine. Through intranasal delivery, the prokaryotically expressed multiepitope nanovaccine elicits long-lasting mucosal immunity, broad humoral immunity, and robust cellular immunity without any adjuvants, and confers complete protection against H3N2 and H1N1 subtypes of IAV in mice. Importantly, this intranasal multiepitope nanovaccine triggers memory B-cell responses, resulting in secretory immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) levels persisting for more than five months post-immunization. Therefore, this intranasal ferritin-based multiepitope nanovaccine represents a promising approach to combating respiratory pathogens.

Synthetic Self-Adjuvanted Lipopeptide Vaccines Conferred Protection against Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) colonizes the stomach epithelium of half the world's population and is responsible for various digestive diseases and even stomach cancer. Vaccine-mediated protection against H. pylori infection depends primarily on the specific mucosal and T-cell responses. In this study, the synthetic lipopeptide vaccines, Hp4 (Pam2 Cys modified UreB T-cell epitope) and Hp10 (Pam2 Cys modified CagA T/B cell combined epitope), not only induce the bone marrow derived dendritic cells (BMDCs) maturation by activating a variety of pattern-recognition receptors (PRRs) such as Toll-like receptor (TLR), Nod-like receptor (NLR), and retinoic acid-inducing gene (RIG) I-like receptor (RLR), and but also stimulate BMDCs to secret cytokines that have the potential to modulate T-cell activation and differentiation. Although intranasal immunization with Hp4 or Hp10 elicits robust epitope-specific T-cell responses in mice, only Hp10 confers protection against H. pylori infection, possibly due to the fact that Hp10 also induces substantial specific sIgA response at mucosal sites. Interestingly, Hp4 elevates the protective response against H. pylori infection of Hp10 when administrated in combination, characterized by better protective effect and enhanced specific T-cell and mucosal antibody responses. The results suggest that synthetic lipopeptide vaccines based on the epitopes derived from the protective antigens are promising candidates for protection against H. pylori infection.

A Toll-like Receptor-Activating, Self-Adjuvant Glycan Nanocarrier.

The global pandemic of COVID-19 highlights the importance of vaccination, which remains the most efficient measure against many diseases. Despite the progress in vaccine design, concerns with suboptimal antigen immunogenicity and delivery efficiency prevail. Self-adjuvant carriers-vehicles that can simultaneously deliver antigens and act as adjuvants-may improve efficacies in these aspects. Here, we developed a self-adjuvant carrier based on an acetyl glucomannan (acGM), which can activate toll-like receptor 2 (TLR2) and encapsulate the model antigen ovalbumin (OVA) via a double-emulsion process. In vitro tests showed that these OVA@acGM-8k nanoparticles (NPs) enhanced cellular uptake and activated TLR2 on the surface of dendritic cells (DCs), with increased expression of co-stimulatory molecules (e.g. CD80 and CD86) and pro-inflammatory cytokines (e.g. TNF-α and IL12p70). In vivo experiments in mice demonstrated that OVA@acGM-8k NPs accumulated in the lymph nodes and promoted DCs' maturation. The immunization also boosted the humoral and cellular immune responses. Our findings suggest that this self-adjuvant polysaccharide carrier could be a promising approach for vaccine development.

RNA cancer vaccines: developing mRNA nanovaccine with self-adjuvant property for cancer immunotherapy.

Messenger RNA (mRNA)-based cancer vaccine has become a popular approach for developing personalized and effective antitumor immunotherapy. To achieve robust antitumor efficacy, mRNA-encoding tumor antigens needs to be efficiently delivered and translated in dendritic cells for efficient antigen presentation; meanwhile, the vaccine would have adjuvant effect by stimulating innate immune response to boost the full activation of adaptive immunity. Recently, we reported a minimalist nanovaccine by formulating tumor antigen-encoding mRNA with a lipid-like material named C1, which could efficiently deliver mRNA into dendritic cells with simultaneous Toll-like receptor 4 (TLR4) stimulation, together induced T cell activation. Importantly, C1 mRNA nanovaccine exhibited significant antitumor efficacy on several tumor mouse models. Here, we discuss the nanovector-facilitated mRNA delivery and translation in dendritic cells, the self-adjuvant property of nanovectors, the challenges of personalized tumor antigen selection, and the potential strategies for developing efficacious mRNA cancer vaccines targeting the immunosuppressive tumor microenvironment.

Comparison of Fluorinated and Nonfluorinated Lipids in Self-Adjuvanting Delivery Systems for Peptide-Based Vaccines.

Safe immunostimulants (adjuvants) are essential for the development of highly potent peptide-based vaccines. This study examined for the first time whether fluorinated lipids could stimulate humoral immunity in vivo when conjugated to peptide antigen. The impact of fluorination on humoral immunity was tested using a library of peptide-based vaccine candidates against the group A streptococcus (GAS). The fluorinated constructs stimulated similar mouse IgG titers to those elicited by complete Freund's adjuvant (CFA) and were higher than those produced in mice that received the nonfluorinated constructs.

Recent advances in self-assembled peptides: Implications for targeted drug delivery and vaccine engineering.

Self-assembled peptides have shown outstanding characteristics for vaccine delivery and drug targeting. Peptide molecules can be rationally designed to self-assemble into specific nanoarchitectures in response to changes in their assembly environment including: pH, temperature, ionic strength, and interactions between host (drug) and guest molecules. The resulting supramolecular nanostructures include nanovesicles, nanofibers, nanotubes, nanoribbons, and hydrogels and have a diverse range of mechanical and physicochemical properties. These molecules can be designed for cell-specific targeting by including adhesion ligands, receptor recognition ligands, or peptide-based antigens in their design, often in a multivalent display. Depending on their design, self-assembled peptide nanostructures have advantages in biocompatibility, stability against enzymatic degradation, encapsulation of hydrophobic drugs, sustained drug release, shear-thinning viscoelastic properties, and/or adjuvanting properties. These molecules can also act as intracellular transporters and respond to changes in the physiological environment. Furthermore, this class of materials has shown sequence- and structure-dependent impacts on the immune system that can be tailored to non-immunogenic for drug targeting, and immunogenic for vaccine delivery. This review explores self-assembled peptide nanostructures (beta sheets, alpha helices, peptide amphiphiles, amino acid pairing, elastin like polypeptides, cyclic peptides, short peptides, Fmoc peptides, and peptide hydrogels) and their application in vaccine delivery and drug targeting.

Systematic evaluation of self-adjuvanting lipopeptide nano-vaccine platforms for the induction of potent CD8(+) T-cell responses.

AIM: Systematically evaluate lipid core peptide vaccine delivery platforms to identify core features promoting strong CD8(+) T-cell responses. MATERIALS & METHODS: Three different self-adjuvanting lipid core peptide nanovaccines each comprising four copies of the dominant ovalbumin CD8(+) T-cell epitope and varying in the utilization of a polylysine or glucose core with 2-amino-hexadecanoic acid (C16) or 2-amino-dodecanoic acid (C12) lipids were synthesized. Vaccines were tested for ability to induce CD8(+) T-cell responses and inhibit tumor growth in vivo. RESULTS: The construct utilizing C12 lipids and polylysine core induced very robust effector T cells shown to have in vivo effector capability as demonstrated by in vivo cytotoxicity and ability to inhibit tumor growth as well as modulation of dendritic cell activation. CONCLUSION: The C12 polylysine platform was an effective configuration for induction of potent CD8(+) T-cell responses.

Self-assembled or mixed peptide amphiphile micelles from Herpes simplex virus glycoproteins as potential immunomodulatory treatment.

The use of micelle aggregates formed from peptide amphiphiles (PAs) as potential synthetic self-adjuvant vaccines to treat Herpes simplex virus (HSV) infection are reported here. The PAs were based on epitopes gB409-505 and gD301-309, selected from HSV envelope glycoprotein B (gB) and glycoprotein D (gD), that had their N-terminus modified with hydrophobic moieties containing two C18 hydrocarbon chains. Pure and mixed micelles of gB and/or gD peptide epitopes were easily prepared after starting with the synthesis of corresponding PAs by solid phase methods. Structural characterization of the aggregates confirmed that they were sufficiently stable and compatible with in vivo use: critical micelle concentration values around 4.0 ⋅ 10(-7) mol ⋅ Kg(-1); hydrodynamic radii (RH) between 50-80 nm, and a zeta potential (ζ) around - 40 mV were found for all aggregates. The in vitro results indicate that both peptide epitopes and micelles, at 10 µM, triggered U937 and RAW 264.7 cells to release appreciable levels of cytokines. In particular, interleukin (IL)-23-, IL-6-, IL-8- or macrophage inflammatory protein (MIP)-2-, and tumor necrosis factor (TNF)-α-release increased considerably when cells were treated with the gB-micelles or gD-micelles compared with the production of the same cytokines when the stimulus was the single gB or gD peptide.

Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles.

Nano-sized hepatitis B virus core virus-like particles (HBc-VLP) are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10) is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.