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.

Recent progress in synthetic self-adjuvanting vaccine development.

Vaccination is a proven way to protect individuals against many infectious diseases, as currently highlighted in the global COVID-19 pandemic. Peptides- or small molecule antigen-based vaccination offer advantages over the classical vaccine approaches. However, peptides or small molecules by themselves are generally not sufficiently immunogenic, and thus require an adjuvant to boost an immune response. Several conjugated systems have been developed in recent years to overcome this obstacle. This review summarises different moieties which, when conjugated to peptide antigens, facilitate a specific immune response. Different classes of self-adjuvant moieties are reviewed, including self-assembly peptides, lipids, glycolipids, and polymers.

Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern.

Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are the best approach to successfully combat the COVID-19 pandemic. The receptor-binding domain (RBD) of the viral spike protein is a major target to develop candidate vaccines. α-Galactosylceramide (αGalCer), a potent invariant natural killer T cell (iNKT) agonist, was site-specifically conjugated to the N-terminus of the RBD to form an adjuvant-protein conjugate, which was anchored on the liposome surface. This is the first time that an iNKT cell agonist was conjugated to the protein antigen. Compared to the unconjugated RBD/αGalCer mixture, the αGalCer-RBD conjugate induced significantly stronger humoral and cellular responses. The conjugate vaccine also showed effective cross-neutralization to all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results suggest that the self-adjuvanting αGalCer-RBD has great potential to be an effective COVID-19 vaccine candidate, and this strategy might be useful for designing various subunit vaccines.

Site-Specific and Stable Conjugation of the SARS-CoV-2 Receptor-Binding Domain to Liposomes in the Absence of Any Other Adjuvants Elicits Potent Neutralizing Antibodies in BALB/c Mice.

Understanding immune responses toward viral infection will be useful for potential therapeutic intervention and offer insights into the design of prophylactic vaccines. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. To understand the complex immune responses toward SARS-CoV-2 infection, here we developed a method to express and purify the recombinant and engineered viral receptor-binding domain (RBD) to more than 95% purity. We could encapsulate RNA molecules into the interior of a virion-sized liposome. We conjugated the purified RBD proteins onto the surface of the liposome in an orientation-specific manner with defined spatial densities. Both the encapsulation of RNAs and the chemical conjugation of the RBD protein on liposome surfaces were stable under physiologically relevant conditions. In contrast to soluble RBD proteins, a single injection of RBD-conjugated liposomes alone, in the absence of any other adjuvants, elicited RBD-specific B cell responses in BALB/c mice, and the resulting animal sera could potently neutralize HIV-1 pseudovirions that displayed the SARS-CoV-2 spike proteins. These results validate these supramolecular structures as a novel and effective tool to mimic the structure of enveloped viruses, the use of which will allow systematic dissection of the complex B cell responses to SARS-CoV-2 infection.

Nanoparticular CpG-adjuvanted SARS-CoV-2 S1 protein elicits broadly neutralizing and Th1-biased immunoreactivity in mice.

The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.

Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant.

The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.

Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Large-Sized Graphene Oxide Nanosheets Increase DC-T-Cell Synaptic Contact and the Efficacy of DC Vaccines against SARS-CoV-2.

Dendritic cell (DC) vaccines are used for cancer and infectious diseases, albeit with limited efficacy. Modulating the formation of DC-T-cell synapses may greatly increase their efficacy. The effects of graphene oxide (GO) nanosheets on DCs and DC-T-cell synapse formation are evaluated. In particular, size-dependent interactions are observed between GO nanosheets and DCs. GOs with diameters of >1 µm (L-GOs) demonstrate strong adherence to the DC surface, inducing cytoskeletal reorganization via the RhoA-ROCK-MLC pathway, while relatively small GOs (≈500 nm) are predominantly internalized by DCs. Furthermore, L-GO treatment enhances DC-T-cell synapse formation via cytoskeleton-dependent membrane positioning of integrin ICAM-1. L-GO acts as a "nanozipper," facilitating the aggregation of DC-T-cell clusters to produce a stable microenvironment for T cell activation. Importantly, L-GO-adjuvanted DCs promote robust cytotoxic T cell immune responses against SARS-CoV-2 spike 1, leading to >99.7% viral RNA clearance in mice infected with a clinically isolated SARS-CoV-2 strain. These findings highlight the potential value of nanomaterials as DC vaccine adjuvants for modulating DC-T-cell synapse formation and provide a basis for the development of effective COVID-19 vaccines.

Carbohydrate-containing nanoparticles as vaccine adjuvants.

Introduction: Adjuvants are essential to vaccines for immunopotentiation in the elicitation of protective immunity. However, classical and widely used aluminum-based adjuvants have limited capacity to induce cellular response. There are increasing needs for appropriate adjuvants with improved profiles for vaccine development toward emerging pathogens. Carbohydrate-containing nanoparticles (NPs) with immunomodulatory activity and particulate nanocarriers for effective antigen presentation are capable of eliciting a more balanced humoral and cellular immune response.Areas covered: We reviewed several carbohydrates with immunomodulatory properties. They include chitosan, ß-glucan, mannan, and saponins, which have been used in vaccine formulations. The mode of action, the preparation methods, characterization of these carbohydrate-containing NPs and the corresponding vaccines are presented.Expert opinion: Several carbohydrate-containing NPs have entered the clinical stage or have been used in licensed vaccines for human use. Saponin-containing NPs are being evaluated in a vaccine against SARS-CoV-2, the pathogen causing the on-going worldwide pandemic. Vaccines with carbohydrate-containing NPs are in different stages of development, from preclinical studies to late-stage clinical trials. A better understanding of the mode of action for carbohydrate-containing NPs as vaccine carriers and as immunostimulators will likely contribute to the design and development of new generation vaccines against cancer and infectious diseases.

Toll-like Receptor-4 (TLR4) Agonist-Based Intranasal Nanovaccine Delivery System for Inducing Systemic and Mucosal Immunity.

Eliciting a robust immune response at mucosal sites is critical in preventing the entry of mucosal pathogens such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This task is challenging to achieve without the inclusion of a strong and safe mucosal adjuvant. Previously, inulin acetate (InAc), a plant-based polymer, is shown to activate toll-like receptor-4 (TLR4) and elicit a robust systemic immune response as a vaccine adjuvant. This study investigates the potential of nanoparticles prepared with InAc (InAc-NPs) as an intranasal vaccine delivery system to generate both mucosal and systemic immune responses. InAc-NPs (∼250 nm in diameter) activated wild-type (WT) macrophages but failed to activate macrophages from TLR4 knockout mice or WT macrophages when pretreated with a TLR4 antagonist (lipopolysaccharide-RS (LPS-RS)), which indicates the selective nature of a InAc-based nanodelivery system as a TLR4 agonist. Intranasal immunization using antigen-loaded InAc-NPs generated ∼65-fold and 19-fold higher serum IgG1 and IgG2a titers against the antigen, respectively, as compared to PLGA-NPs as a delivery system. InAc-NPs have also stimulated the secretion of sIgA at various mucosal sites, including nasal-associated lymphoid tissues (NALTs), lungs, and intestine, and produced a strong memory response indicative of both humoral and cellular immune activation. Overall, by stimulating both systemic and mucosal immunity, InAc-NPs laid a basis for a potential intranasal delivery system for mucosal vaccination.

Designing the Next Generation of Vaccines: Relevance for Future Pandemics.

The development of vaccines is one of the greatest medical interventions in the history of global infectious diseases and has contributed to the annual saving of at least 2 to 3 million lives worldwide. However, many diseases are not preventable through currently available vaccines, and the potential of modulating the immune response during vaccination has not been fully exploited. The first golden age of vaccines was based on the germ theory and the use of live, attenuated, inactivated pathogens or toxins. New strategies and formulations (e.g., adjuvants) with an immunomodulatory capacity to enhance the protective qualities and duration of vaccines have been incompletely exploited. These strategies can prevent disease and improve protection against infectious diseases, modulate the course of some noncommunicable diseases, and increase the immune responses of patients at a high risk of infection, such as the elderly or immunocompromised patients. In this minireview, we focus on how metabolic and epigenetic modulators can amplify and enhance the function of immunity in a given vaccine. We propose the term "amplifier" for such additives, and we pose that future vaccines will have three components: antigen, adjuvant, and amplifier.

Design and application of nanoparticles as vaccine adjuvants against human corona virus infection.

In recent years, some viruses have caused a grave crisis to global public health, especially the human coronavirus. A truly effective vaccine is therefore urgently needed. Vaccines should generally have two features: delivering antigens and modulating immunity. Adjuvants have an unshakable position in the battle against the virus. In addition to the perennial use of aluminium adjuvant, nanoparticles have become the developing adjuvant candidates due to their unique properties. Here we introduce several typical nanoparticles and their antivirus vaccine adjuvant applications. Finally, for the combating of the coronavirus, we propose several design points, hoping to provide ideas for the development of personalized vaccines and adjuvants and accelerate the clinical application of adjuvants.

Molecular Basis of the Therapeutical Potential of Clove (Syzygium aromaticum L.) and Clues to Its Anti-COVID-19 Utility.

The current COronaVIrus Disease 19 (COVID-19) pandemic caused by SARS-CoV-2 infection is enormously affecting the worldwide health and economy. In the wait for an effective global immunization, the development of a specific therapeutic protocol to treat COVID-19 patients is clearly necessary as a short-term solution of the problem. Drug repurposing and herbal medicine represent two of the most explored strategies for an anti-COVID-19 drug discovery. Clove (Syzygium aromaticum L.) is a well-known culinary spice that has been used for centuries in folk medicine in many disorders. Interestingly, traditional medicines have used clove since ancient times to treat respiratory ailments, whilst clove ingredients show antiviral and anti-inflammatory properties. Other interesting features are the clove antithrombotic, immunostimulatory, and antibacterial effects. Thus, in this review, we discuss the potential role of clove in the frame of anti-COVID-19 therapy, focusing on the antiviral, anti-inflammatory, and antithrombotic effects of clove and its molecular constituents described in the scientific literature.

Adjuvanted SARS-CoV-2 spike protein elicits neutralizing antibodies and CD4 T cell responses after a single immunization in mice.

BACKGROUND: SARS-CoV-2 has caused a global pandemic, infecting millions of people. A safe, effective vaccine is urgently needed and remains a global health priority. Subunit vaccines are used successfully against other viruses when administered in the presence of an effective adjuvant. METHODS: We evaluated three different clinically tested adjuvant systems in combination with the SARS-CoV-2 pre-fusion stabilized (S-2P) spike protein using a one-dose regimen in mice. FINDINGS: Whilst spike protein alone was only weakly immunogenic, the addition of either Aluminum hydroxide, a squalene based oil-in-water emulsion system (SE) or a cationic liposome-based adjuvant significantly enhanced antibody responses against the spike receptor binding domain (RBD). Kinetics of antibody responses differed, with SE providing the most rapid response. Neutralizing antibodies developed after a single immunization in all adjuvanted groups with ID50 titers ranging from 86-4063. Spike-specific CD4 T helper responses were also elicited, comprising mainly of IFN-γ and IL-17 producing cells in the cationic liposome adjuvanted group, and more IL-5- and IL-10-secreting cells in the AH group. INTERPRETATION: These results demonstrate that adjuvanted spike protein subunit vaccine is a viable strategy for rapidly eliciting SARS-CoV-2 neutralizing antibodies and CD4 T cell responses of various qualities depending on the adjuvant used, which can be explored in further vaccine development against COVID-19. FUNDING: This work was supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653.

Exploring the possible use of saponin adjuvants in COVID-19 vaccine.

There is an urgent need for a safe, efficacious, and cost-effective vaccine for the coronavirus disease 2019 (COVID-19) pandemic caused by novel coronavirus strain, severe acute respiratory syndrome-2 (SARS-CoV-2). The protective immunity of certain types of vaccines can be enhanced by the addition of adjuvants. Many diverse classes of compounds have been identified as adjuvants, including mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles, and liposomes. Several saponins have been shown to stimulate both the Th1-type immune response and the production of cytotoxic T lymphocytes against endogenous antigens, making them very useful for subunit vaccines, especially those for intracellular pathogens. In this review, we discuss the structural characteristics, mechanisms of action, structure-activity relationship of saponins, biological activities, and use of saponins in various viral vaccines and their applicability to a SARS-CoV-2 vaccine.

Synthetic Biology-derived triterpenes as efficacious immunomodulating adjuvants.

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.

Biotin Functionalized Self-Assembled Peptide Nanofiber as an Adjuvant for Immunomodulatory Response.

Biotinylated peptide amphiphile (Biotin-PA) nanofibers, are designed as a noncovalent binding location for antigens, which are adjuvants to enhance, accelerate, and prolong the immune response triggered by antigens. Presenting antigens on synthetic Biotin-PA nanofibers generated a higher immune response than the free antigens delivered with a cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN) (TLR9 agonist) adjuvant. Antigen attached Biotin-PA nanofibers trigger splenocytes to produce high levels of cytokines (IFN-γ, IL-12, TNF-α, and IL-6) and to exhibit a superior cross-presentation of the antigen. Both Biotin-PA nanofibers and CpG ODN induce a Th-1-biased IgG subclass response; however, delivering the antigen with Biotin-PA nanofibers induce significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin-PA nanofibers also enhance antigen-specific splenocyte proliferation and increase the proportion of the antigen-specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin-PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID-19, or cancer cells to induce humoral and cellular immune responses against the antigens.

Particulate Alum via Pickering Emulsion for an Enhanced COVID-19 Vaccine Adjuvant.

For rapid response against the prevailing COVID-19 (coronavirus disease 19), it is a global imperative to exploit the immunogenicity of existing formulations for safe and efficient vaccines. As the most accessible adjuvant, aluminum hydroxide (alum) is still the sole employed adjuvant in most countries. However, alum tends to attach on the membrane rather than entering the dendritic cells (DCs), leading to the absence of intracellular transfer and process of the antigens, and thus limits T-cell-mediated immunity. To address this, alum is packed on the squalene/water interphase is packed, forming an alum-stabilized Pickering emulsion (PAPE). "Inheriting" from alum and squalene, PAPE demonstrates a good biosafety profile. Intriguingly, with the dense array of alum on the oil/water interphase, PAPE not only adsorbs large quantities of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antigens, but also harbors a higher affinity for DC uptake, which provokes the uptake and cross-presentation of the delivered antigens. Compared with alum-treated groups, more than six times higher antigen-specific antibody titer and three-fold more IFN-γ-secreting T cells are induced, indicating the potent humoral and cellular immune activations. Collectively, the data suggest that PAPE may provide potential insights toward a safe and efficient adjuvant platform for the enhanced COVID-19 vaccinations.