Self-adjuvanting Vaccine Research Articles

Programmable Macrophage Vesicle Based Bionic Self-Adjuvanting Vaccine for Immunization against Monkeypox Virus.

The emergence of monkeypox has become a global health threat after the COVID-19 pandemic. Due to the lack of available specifically treatment against MPV, developing an available vaccine is thus the most prospective and urgent strategy. Herein, a programmable macrophage vesicle based bionic self-adjuvanting vaccine (AM@AEvs-PB) is first developed for defending against monkeypox virus (MPV). Based on MPV-related antigen-stimulated macrophage-derived vesicles, the nanovaccine is constructed by loading the mature virion (MV)-related intracellular protein (A29L/M1R) and simultaneously modifying with the enveloped virion (EV) antigen (B6R), enabling them to effectively promote antigen presentation and enhance adaptive immune through self-adjuvant strategy. Owing to the synergistic properties of bionic vaccine coloaded MV and EV protein in defensing MPV, the activation ratio of antigen-presenting cells is nearly four times than that of single antigen in the same dose, resulting in stronger immunity in host. Notably, intramuscular injection uptake of AM@AEvs-PB demonstrated vigorous immune-protective effects in the mouse challenge attempt, offering a promising strategy for pre-clinical monkeypox vaccine development.

Minimalist Adjuvant-Free Nano-Vaccine Based on Antigen Self-Assembled Amyloid-Like Fibrils to Induce Potent Immune Response.

The development of cancer vaccines is at the forefront of cancer immunotherapy. Most existing strategies to induce an efficient anti-tumor immune response rely on molecular adjuvants and the incorporation of complex synthetic vectors into vaccine formulations. In contrast, this study introduces a one-step engineering technique to assemble the model antigen, Ovalbumin (OVA), into amyloid aggregates, leveraging biomimetic folding and aggregation to create non-fibrillar OVA globular aggregates and OVA amyloid-like fibrils as single-component, adjuvant-free vaccines. Notably, the OVA amyloid-like fibrils induced stronger immune responses compared to the native form, as evidenced by robust humoral immune reactions and the establishment of immune memory. These enhanced responses can be attributed to the self-adjuvant effect of the unique assembled structure, which preserves antigenic epitopes, improves antigen stability, facilitates antigen internalization, prolongs retention at the injection site, enhances antigen trafficking to the lymphoid organs, and promotes increased secretion of antibodies and cytokines. Furthermore, the efficacy of the vaccine was validated in a high OVA-expressing tumor model, demonstrating the potential of OVA amyloid-like fibrils as an effective vaccine for cancer immunoprevention. This minimalist self-adjuvant vaccine strategy holds promising implications for cancer immunotherapy and can inform the design of other protein antigen-based vaccines.

A self-adjuvanted VLPs-based Covid-19 vaccine proven versatile, safe, and highly protective

Vaccination has played a critical role in mitigating COVID-19. Despite the availability of licensed vaccines, there remains a pressing need for improved vaccine platforms that provide high protection, safety, and versatility, while also reducing vaccine costs. In response to these challenges, our aim is to create a self-adjuvanted vaccine against SARS-CoV-2, utilizing Virus-Like Particles (VLPs) as the foundation. To achieve this, we produced bacteriophage (Qβ) VLPs in a prokaryotic system and purified them using a rapid and cost-effective strategy involving organic solvents. This method aims to solubilize lipids and components of the cell membrane to eliminate endotoxins present in bacterial samples. For vaccine formulation, Receptor Binding Domain (RBD) antigens were conjugated using chemical crosslinkers, a process compatible with Good Manufacturing Practice (GMP) standards. Transmission Electron Microscopy (TEM) confirmed the expected folding and spatial configuration of the QβVLPs vaccine. Additionally, vaccine formulation assessment involved SDS-PAGE stained with Coomassie Brilliant Blue, Western blotting, and stereomicroscopic experiments. In vitro and in vivo evaluations of the vaccine formulation were conducted to assess its capacity to induce a protective immune response without causing side effects. Vaccine doses of 20 µg and 50 µg stimulated the production of neutralizing antibodies. In in vivo testing, the group of animals vaccinated with 50 µg of vaccine formulation provided complete protection against virus infection, maintaining stable body weight without showing signs of disease. In conclusion, the QβVLPs-RBD vaccine has proven to be effective and safe, eliminating the necessity for supplementary adjuvants and offering a financially feasible approach. Moreover, this vaccine platform demonstrates flexibility in targeting Variants of Concern (VOCs) via established conjugation protocols with VLPs.

Fully Synthetic TF-Based Self-Adjuvanting Vaccine Simultaneously Triggers iNKT Cells and Mincle and Protects Mice against Tumor Development.

The Thomsen-Friedenreich (TF) antigen has proven to be a promising target for developing novel therapeutic cancer vaccines. Here, a new strategy that TF antigen covalently coupled with KRN7000 and vizantin was developed. The resulting three-component vaccine KRN7000-TF-vizantin simultaneously triggers invariant natural killer T (iNKT) cells and macrophage-inducible C-type lectin (Mincle) signaling pathways, eliciting much stronger TF-specific immune responses than glycoprotein vaccine TF-KLH/alum and the corresponding two-component conjugate vaccines TF-KRN7000 and TF-vizantin. The analysis of IgG isotypes and the secretion of cytokines revealed that KRN7000-TF-vizantin induced Th1/Th2 mixed immune responses, where Th1 was dominant. In vivo experiments demonstrated that KRN7000-TF-vizantin increased the survival rate and survival time of tumor-challenged mice, and surviving mice rejected further tumor attacks without any additional treatment. This work demonstrates that covalently coupled KRN7000 and vizantin could serve as a promising TF-based vaccine carrier for antitumor immune therapy, and KRN7000-TF-vizantin features great potential to be a vaccine candidate.

Intranasal Self-Adjuvanted Lipopeptide Vaccines Elicit High Antibody Titers and Strong Cellular Responses against SARS-CoV-2.

Despite concerted efforts to tackle the COVID-19 pandemic, the persistent transmission of SARS-CoV-2 demands continued research into novel vaccination strategies to combat the virus. In light of this, intranasally administered peptide vaccines, particularly those conjugated to an immune adjuvant to afford so-called "self-adjuvanted vaccines", remain underexplored. Here, we describe the synthesis and immunological evaluation of self-adjuvanting peptide vaccines derived from epitopes of the spike glycoprotein of SARS-CoV-2 covalently fused to the potent adjuvant, Pam2Cys, that targets toll-like receptor 2 (TLR2). When administered intranasally, these vaccines elicited a strong antigen-specific CD4+ and CD8+ T-cell response in the lungs as well as high titers of IgG and IgA specific to the native spike protein of SARS-CoV-2. Unfortunately, serum and lung fluid from mice immunized with these vaccines failed to inhibit viral entry in spike-expressing pseudovirus assays. Following this, we designed and synthesized fusion vaccines composed of the T-cell epitope discovered in this work, covalently fused to epitopes of the receptor-binding domain of the spike protein reported to be neutralizing. While antibodies elicited against these fusion vaccines were not neutralizing, the T-cell epitope retained its ability to stimulate strong antigen-specific CD4+ lymphocyte responses within the lungs. Given the Spike(883-909) region is still completely conserved in SARS-CoV-2 variants of concern and variants of interest, we envision the self-adjuvanting vaccine platform reported here may inform future vaccine efforts.

Conjugation of TLR7 and TLR7/8 agonists onto weak protein antigen via versatile oxime ligation for enhanced vaccine efficacy

Protein-based subunit vaccines are weakly immunogenic, and developing self-adjuvanting vaccines with adjuvant conjugated to antigen is a promising approach for generating optimal immune responses. Here, we report a novel adjuvant-protein conjugate vaccine based on versatile oxime ligation technique. Firstly, the adjuvant properties of a series of TLR7 and TLR7/8 small molecule agonists in self-adjuvanting vaccines were systematically compared by coupling them to proteins in consistent ratio via p-carboxybenzaldehyde (p-CBA) for the first time. All conjugate vaccines induced cytokine secretion in murine and human macrophages in vitro, and promoted specific antibody production in vivo. Notably, a conjugate containing imidazoquinoline TLR7/8 agonist (TLR7/8a1) showed the greatest enhancement in Th1/2 balanced antibody response. To minimize the interference with the protein antigenic integrity, we further developed a systematic glycoconjugation strategy to conjugate this TLR7/8a1 onto the glycan chains of SARS-CoV-2 S1 glycoprotein via oxime ligation, in which S1 containing different numbers of aldehyde groups were obtained by differential periodate oxidation. The resulting TLR7/8a1-S1 conjugate triggered a potent humoral and cellular immunity in vivo. Together these data demonstrate the promise of these TLR7 and TLR7/8 agonists as effective built-in adjuvants, and the versatile oxime ligation strategy might broaden potential applications in designing different conjugate vaccines.

The synthesis and preliminary immunological evaluation of a dual-adjuvant SARS-CoV-2 RBD vaccine: Covalent integration of TLR7/8 and iNKT cell agonists

Covalently linking an adjuvant to an antigenic protein enhances its immunogenicity by ensuring a synergistic delivery to the immune system, fostering a more robust and targeted immune response. Most adjuvant-protein conjugate vaccines incorporate only one adjuvant due to the difficulties in its synthesis. However, there is a growing interest in developing vaccines with multiple adjuvants designed to elicit a more robust and targeted immune response by engaging different aspects of the immune system for complex diseases where traditional vaccines fall short. Here, we pioneer the synthesis of a dual-adjuvants protein conjugate Vaccine 1 by assembling a toll-like receptor 7/8 (TLR7/8) agonist, an invariant natural killer T cell (iNKT) agonist with a clickable bicyclononyne (BCN). The BCN group can bio-orthogonally react with azide-modified severe acute respiratory syndrome coronavirus-2 receptor-binding domain (SARS-CoV-2 RBD) trimer antigen to give the three-component Vaccine 1. Notably, with a mere 3 μg antigen, it elicited a balanced subclass of IgG titers and 20-fold more IgG2a than control vaccines, highlighting its potential for enhancing antibody-dependent cellular cytotoxicity. This strategy provides a practicable way to synthesize covalently linked dual immunostimulants. It expands the fully synthetic self-adjuvant protein vaccine that uses a single adjuvant to include two different types of adjuvants.

Synergistic Glutathione Depletion and STING Activation to Potentiate Dendritic Cell Maturation and Cancer Vaccine Efficacy.

Dendritic cell (DC) maturation and antigen presentation are key factors for successful vaccine-based cancer immunotherapy. This study developed manganese-based layered double hydroxide (Mn-LDH) nanoparticles as a self-adjuvanted vaccine carrier that not only promoted DC maturation through synergistically depleting endogenous glutathione (GSH) and activating STING signaling pathway, but also facilitated the delivery of model antigen ovalbumin (OVA) into lymph nodes and subsequent antigen presentation in DCs. Significant therapeutic-prophylactic efficacy of the OVA-loaded Mn-LDH (OVA/Mn-LDH) nanovaccine was determined by the tumor growth inhibition in the mice bearing B16-OVA tumor. Our results showed that the OVA/Mn-LDH nanoparticles could be a potent delivery system for cancer vaccine development without the need of adjuvant. Therefore, the combination of GSH exhaustion and STING pathway activation might be an advisable approach for promoting DC maturation and antigen presentation, finally improving cancer vaccine efficacy.

Synthesis and immunological evaluation of TLR1/2 ligand-conjugated RBDs as self-adjuvanting vaccine candidates against SARS-CoV-2.

We synthesized and evaluated Pam3CSK4-conjugated receptor binding domain (RBD)/deglycosylated RBD as potential anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidates. Our investigation revealed the critical importance of limiting the number of introduced Pam3CSK4 molecules to the RBD in order to preserve its antigenicity. We also confirmed the harmonious integration of the adjuvant-conjugation strategy with the glycan-shield removal strategy.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

The L-DBF vaccine cross protects mice against different Shigella serotypes after prior exposure to the pathogen.

Shigellosis (bacillary dysentery) is a severe diarrheal disease caused by members of the genus Shigella, which results in 90 million cases annually around the world. The Shigella type III secretion system (T3SS) is a specialized secretion system that is the primary virulence factor it uses to infect the colonic mucosa. The type III secretion apparatus (T3SA) proteins IpaB and IpaD, as well as the genetic fusion, DBF, have been demonstrated to protect mice from Shigella spp. infection in a lethal pulmonary model. In a previous study, we fused LTA1, the active moiety of lethal toxin from enterotoxigenic Escherichia coli to DBF to produce a self-adjuvanting vaccine candidate L-DBF, which cross-protected mice against four serotypes of Shigella flexneri and Shigella sonnei. Here, we exposed mice with one or two sublethal doses of S. flexneri 2a to identify whether the immune response induced by L-DBF in the host would be affected by prior infection by homologous or heterologous Shigella serotypes. We demonstrate that pre-infection with two sublethal doses of S. flexneri 2a did not elicit cross-protection against S. sonnei, while vaccination with L-DBF did. Our results indicate that L-DBF is a feasible vaccine candidate that offers cross-protection against Shigella's different serotypes even after prior exposure to the pathogen. This work provides a proof of concept that a novel subunit vaccine can not only protect a naïve host from Shigella challenge, but also can protect against challenge after prior infection by the same or different Shigella serotypes. IMPORTANCE Shigellosis is endemic to low- and middle-income regions of the world where children are especially vulnerable. In many cases, there are pre-existing antibodies in the local population and the effect of prior exposure should be considered in the development and testing of vaccines against Shigella infection. Our study shows that L-DBF-induced immune responses are not adversely affected by prior exposure to this pathogen. Moreover, somewhat different cytokine profiles were observed in the lungs of vaccinated mice not having been exposed to Shigella, suggesting that the immune responses elicited by Shigella infection and L-DBF vaccination follow different pathways.

An RNA-Scaffold Protein Subunit Vaccine for Nasal Immunization.

Developing recombinant proteins as nasal vaccines for inducing systemic and mucosal immunity against respiratory viruses is promising. However, additional adjuvants are required to overcome the low immunogenicity of protein antigens. Here, a self-adjuvanted protein-RNA ribonucleoprotein vaccine was developed and found to be an effective nasal vaccine in mice and the SARS-CoV-2 infection model. The vaccine consisted of spike RBD (as an antigen), nucleoprotein (as an adaptor), and ssRNA (as an adjuvant and RNA scaffold). This combination robustly induced mucosal IgA, neutralizing antibodies and activated multifunctional T-cells, while also providing sterilizing immunity against live virus challenge. In addition, high-resolution scRNA-seq analysis highlighted airway-resident immune cells profile during prime-boost immunization. The vaccine also possesses modularity (antigen/adaptor/RNA scaffold) and can be made to target other viruses. This protein-RNA ribonucleoprotein vaccine is a novel and promising approach for developing safe and potent nasal vaccines to combat respiratory virus infections.

Self-Adjuvanting Calcium-Phosphate-Coated Microcrystal-Based Vaccines Induce Pyroptosis in Human and Livestock Immune Cells.

Successful vaccines require adjuvants able to activate the innate immune system, eliciting antigen-specific immune responses and B-cell-mediated antibody production. However, unwanted secondary effects and the lack of effectiveness of traditional adjuvants has prompted investigation into novel adjuvants in recent years. Protein-coated microcrystals modified with calcium phosphate (CaP-PCMCs) in which vaccine antigens are co-immobilised within amino acid crystals represent one of these promising self-adjuvanting vaccine delivery systems. CaP-PCMCs has been shown to enhance antigen-specific IgG responses in mouse models; however, the exact mechanism of action of these microcrystals is currently unclear. Here, we set out to investigate this mechanism by studying the interaction between CaP-PCMCs and mammalian immune cells in an in vitro system. Incubation of cells with CaP-PCMCs induced rapid pyroptosis of peripheral blood mononuclear cells and monocyte-derived dendritic cells from cattle, sheep and humans, which was accompanied by the release of interleukin-1β and the activation of Caspase-1. We show that this pyroptotic event was cell-CaP-PCMCs contact dependent, and neither soluble calcium nor microcrystals without CaP (soluble PCMCs) induced pyroptosis. Our results corroborate CaP-PCMCs as a promising delivery system for vaccine antigens, showing great potential for subunit vaccines where the enhancement or find tuning of adaptive immunity is required.

A novel self-adjuvanted mRNA vaccine expressing the Antigen-85B-p25 epitope of Mycobacterium tuberculosisprotects mice against tuberculosis

Abstract Mycobacterium tuberculosis (Mtb) is a leading cause of death due to infections. BCG is the most used vaccine despite having partial efficacy against the tuberculosis. We have developed a synthetic mRNA vaccine, SelmRp25-C5, which expresses the p25 epitope of Mtb-derived Antigen-85B and a TLR-2 stimulating C5 adjuvant peptide from CFP-10 protein. We evaluated its efficacy both ex vivo and in mice. Methods: Mouse and human macrophages (MΦ) and mouse dendritic cells (DCs) were treated with SelmRp25-C5 vaccine or EGFP-mRNA control followed by antigen presentation assays and assessment of cytokine secretion. C57Bl/6 mice were vaccinated subcutaneously with two doses (5μg per mouse) of SelmRp25-C5 vaccine, or EGFP-mRNA and one dose of subcutaneous BCG (10 6CFU per mouse) as a control, followed by aerosol infection with Mtb. Four weeks after Mtb challenge, mice were sacrificed, and organs were analyzed for Mtb CFU, RNAseq, Ag85B-p25 tetramer-specific CD4 T cells and memory T cells. Results: SelmRp25-C5 vaccine induced robust antigen presentation to T cells in both mouse MΦs and DCs and in human MΦs, and enhanced Th1 cytokine secretion. Two doses of SelmRp25-C5 vaccine protected mice by reducing Mtb burden by 1log 10. Protection induced by SelmRp25-C5 vaccine was associated with an increase in Ag85B-p25-specific CD4 T cells, increased IFN-γ and IL-2 secreting T cells and CD4 effector memory T cells. Lung and spleens of protected mice showed an upregulation of Spleen tyrosine kinase (Syk) and genes of the C-type lectin receptor-pathway compared to control mice. Conclusions: We propose that the new generation self-adjuvanted mRNA vaccine strengthen both innate and adaptive immunity to provide protection against tuberculosis. Supported by NIAID, NIH AI122070, AI138587; AI161015.

In silico designed mRNA vaccines targeting CA-125 neoantigen in breast and ovarian cancer.

Somatic mutation-derived neoantigens are associated with patient survival in breast and ovarian cancer. These neoantigens are targets for cancer, as shown by the implementation of neoepitope peptides as cancer vaccines. The success of cost-effective multi-epitope mRNA vaccines against SARS-Cov-2 in the pandemic established a model for reverse vaccinology. In this study, we aimed to develop an in silico pipeline designing an mRNA vaccine of the CA-125 neoantigen against breast and ovarian cancer, respectively. Using immuno-bioinformatics tools, we predicted cytotoxic CD8+T cell epitopes based on somatic mutation-driven neoantigens of CA-125 in breast or ovarian cancer, constructed a self-adjuvant mRNA vaccine with CD40L and MHC-I -targeting domain to enhance cross-presentation of neoepitopes by dendritic cells. With an in silico ImmSim algorithm, we estimated the immune responses post-immunization, showing IFN-γ and CD8+T cell response. The strategy described in this study may be scaled up and implemented to design precision multi-epitope mRNA vaccines by targeting multiple neoantigens.

Development of synthetic, self-adjuvanting, and self-assembling anticancer vaccines based on a minimal saponin adjuvant and the tumor-associated MUC1 antigen.

The overexpression of aberrantly glycosylated tumor-associated mucin-1 (TA-MUC1) in human cancers makes it a major target for the development of anticancer vaccines derived from synthetic MUC1-(glyco)peptide antigens. However, glycopeptide-based subunit vaccines are weakly immunogenic, requiring adjuvants and/or additional immunopotentiating approaches to generate optimal immune responses. Among these strategies, unimolecular self-adjuvanting vaccine constructs that do not need coadministration of adjuvants or conjugation to carrier proteins emerge as a promising but still underexploited approach. Herein, we report the design, synthesis, immune-evaluation in mice, and NMR studies of new, self-adjuvanting and self-assembling vaccines based on our QS-21-derived minimal adjuvant platform covalently linked to TA-MUC1-(glyco)peptide antigens and a peptide helper T-cell epitope. We have developed a modular, chemoselective strategy that harnesses two distal attachment points on the saponin adjuvant to conjugate the respective components in unprotected form and high yields via orthogonal ligations. In mice, only tri-component candidates but not unconjugated or di-component combinations induced significant TA-MUC1-specific IgG antibodies able to recognize the TA-MUC1 on cancer cells. NMR studies revealed the formation of self-assembled aggregates, in which the more hydrophilic TA-MUC1 moiety gets exposed to the solvent, favoring B-cell recognition. While dilution of the di-component saponin-(Tn)MUC1 constructs resulted in partial aggregate disruption, this was not observed for the more stably-organized tri-component candidates. This higher structural stability in solution correlates with their increased immunogenicity and suggests a longer half-life of the construct in physiological media, which together with the enhanced antigen multivalent presentation enabled by the particulate self-assembly, points to this self-adjuvanting tri-component vaccine as a promising synthetic candidate for further development.

Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development.

We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule. Using Tn antigen as the model, a three-component vaccine (MPLA-Tn-KRN7000) containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized. This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers. The corresponding two-component conjugate vaccines Tn-MPLA, Tn-KRN7000 and Tn-CRM197 were also synthesized, as controls. The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity. The antibodies specifically recognized, bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells. In addition, MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice, and surviving mice reject further tumor attacks without any additional treatment. Compared to the glycoprotein vaccine Tn-CRM197, the two-component conjugate vaccines, Tn-MPLA and Tn-KRN7000, and the physical mixture of Tn-MPLA and Tn-KRN7000, MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both invitro and invivo. The comparison of immunological studies in wild-type and TLR4 knockout mice, along with the test of binding affinity to CD1d protein suggests that the covalently linked MPLA-KRN7000 immunostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn. This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.

A modular and self-adjuvanted multivalent vaccine platform based on porcine circovirus virus-like nanoparticles

BackgroundVirus-like particles (VLPs) are supramolecular structures composed of multiple protein subunits and resemble natural virus particles in structure and size, making them highly immunogenic materials for the development of next-generation subunit vaccines. The orderly and repetitive display of antigenic epitopes on particle surface allows efficient recognition and cross-link by B cell receptors (BCRs), thereby inducing higher levels of neutralizing antibodies and cellular immune responses than regular subunit vaccines. Here, we present a novel multiple antigen delivery system using SpyCatcher/Spytag strategy and self-assembled VLPs formed by porcine circovirus type 2 (PCV2) Cap, a widely used swine vaccine in solo.ResultsCap-SC, recombinant Cap with a truncated SpyCatcher polypeptide at its C-terminal, self-assembled into 26-nm VLPs. Based on isopeptide bonds formed between SpyCatcher and SpyTag, classical swine fever virus (CSFV) E2, the antigen of interest, was linked to SpyTag and readily surface-displayed on SpyCatcher decorated Cap-SC via in vitro covalent conjugation. E2-conjugated Cap VLPs (Cap-E2 NPs) could be preferentially captured by antigen presenting cells (APCs) and effectively stimulate APC maturation and cytokine production. In vivo studies confirmed that Cap-E2 NPs elicited an enhanced E2 specific IgG response, which was significantly higher than soluble E2, or the admixture of Cap VLPs and E2. Moreover, E2 displayed on the surface did not mask the immunodominant epitopes of Cap-SC VLPs, and Cap-E2 NPs induced Cap-specific antibody levels and neutralizing antibody levels comparable to native Cap VLPs.ConclusionThese results demonstrate that this modularly assembled Cap-E2 NPs retains the immune potential of Cap VLP backbone, while the surface-displayed antigen significantly elevated E2-induced immune potency. This immune strategy provides distinctly improved efficacy than conventional vaccine combination. It can be further applied to the development of dual or multiple nanoparticle vaccines to prevent co-infection of PCV2 and other swine pathogens.

ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells.

Simple SummaryPeptide-based cancer vaccines require adjuvants to increase their immunogenicity to mount an antigen-specific reliable immune response. We have previously shown that immunization with self-adjuvanting peptide-based vaccines issued from our KISIMA vaccine platform, and targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. The aim of our study was to assess the molecular mechanism of action of the first human candidate vaccine derived from the KISIMA platform, ATP128. By using an in vitro (THP-1 cell line) and ex vivo model system (human monocyte-derived dendritic cells), we were able to demonstrate that ATP128 relies on both the cell-penetrating peptide and TLR agonist domain present in the construct to activate the NF-κB and IRF3 pathways, in a self-adjuvanting manner. Importantly, we demonstrated that ATP128 is the first therapeutic vaccine able to activate human DCs through TLR2 and TLR4.The use of cancer vaccines is a promising therapeutic strategy able to stimulate anti-tumor immunity by inducing both humoral and cellular immunity. In this study, antigen presenting cells play a key role by inducing a strong activation of the T cell-mediated adaptive immune response, essential for the anti-tumor potential of cancer vaccines. The first human candidate vaccine created from the KISIMA platform, ATP128, bears three tumor-associated antigens highly expressed in colorectal cancer tissues. At the N-terminus, the cell-penetrating peptide allows the antigen delivery inside the cell and, together with the TLR agonist-derived peptide at the C-terminus, ensures the activation of the monocyte-derived dendritic cells. Here, we show that ATP128 leads to both NF-κB and IRF3 pathway activation, with subsequent pro-inflammatory cytokines and type I Interferon release, as well as an increase in the expression of costimulatory molecules, alongside an upregulation of MHC class I molecules. This cellular immune response involves TLR2 and TLR4, for both membrane and intracellular signaling. We demonstrated an endocytic component in ATP128’s activity by combining the use of a variant of ATP128 lacking the cell-penetrating peptide with endocytosis inhibitors. Importantly, this internalization step is detemined essential for the activation of the IRF3 pathway. This study validates the design of the self-adjuvanting ATP128 vaccine for cancer immunotherapy.

CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity

Safe, effective, and economical vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and end the pandemic. We constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains immunodominant peptides screened from the receptor-binding domain (RBD) and is fully chemically synthesized. It has been formulated in an optimized nanoemulsion formulation and is stable at 40 °C for 1 month. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of protective neutralizing antibodies against multiple RBD mutations, SARS-CoV-2 original strain, and variants (B.1.1.7 and B.1.617.2). Specific peptides booster immunization against the B.1.351 variant has also been shown to be effective in improving protection against B.1.351. Meanwhile, CoVac501 elicited the increase of memory T cells, antigen-specific CD8+ T-cell responses, and Th1-biased CD4+ T-cell immune responses in NHPs. Notably, at an extremely high SARS-CoV-2 challenge dose of 1 × 107 TCID50, CoVac501 provided near-complete protection for the upper and lower respiratory tracts of cynomolgus macaques.