Hepatitis E Virus Capsid as a Carrier of Exogenous Antigens for the Development of Chimeric Virus-Like Particles.

INTRODUCTION: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. METHODS: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. RESULTS: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. CONCLUSION: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2.

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis.

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs.

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

Intranasal Vaccination With Salmonella-Derived Serodominant Secreted Effector Protein B Associated With Gas-Filled Microbubbles Partially Protects Against Gut Infection in Mice.

Salmonella infection is an increasingly important public health problem owing to the emergence of multidrug resistance and the lack of broadly efficient vaccines. Novel strategies of vaccination are required to induce protective immune responses at mucosal surfaces and in the circulation, to limit bacteria entry and dissemination. To this aim, intranasal anti-Salmonella vaccination with an innovative formulation composed of gas-filled microbubbles and the pathogen-derived protective protein serodominant secreted effector protein B (SseB-MB) was evaluated in a mouse infection model. Intranasal application of SseB-MB induced gut and systemic immunoglobulin A, T-helper type 17 cell (Th17), and Th1 responses, all of which are associated with natural immunity against Salmonella In vaccinated mice, a significant reduction in bacterial load was observed in intestinal tissues and the spleen after an otherwise lethal oral infection. Therefore, MB serve as an efficient carrier for nasal delivery of a Salmonella antigen that results in protection upon activation of the common mucosal immune system.

Synthesis and Characterization of Thiolated Nanoparticles Based on Poly (Acrylic Acid) and Algal Cell Wall Biopolymers for the Delivery of the Receptor Binding Domain from SARS-CoV-2.

The COVID-19 pandemic required great efforts to develop efficient vaccines in a short period of time. However, innovative vaccines against SARS-CoV-2 virus are needed to achieve broad immune protection against variants of concern. Polymeric-based particles can lead to innovative vaccines, serving as stable, safe and immunostimulatory antigen delivery systems. In this work, polymeric-based particles called thiolated PAA/Schizo were developed. Poly (acrylic acid) (PAA) was thiolated with cysteine ethyl ester and crosslinked with a Schizochytrium sp. cell wall fraction under an inverse emulsion approach. Particles showed a hydrodynamic diameter of 313 ± 38 nm and negative Zeta potential. FT-IR spectra indicated the presence of coconut oil in thiolated PAA/Schizo particles, which, along with the microalgae, could contribute to their biocompatibility and bioactive properties. TGA analysis suggested strong interactions between the thiolated PAA/Schizo components. In vitro assessment revealed that thiolated particles have a higher mucoadhesiveness when compared with non-thiolated particles. Cell-based assays revealed that thiolated particles are not cytotoxic and, importantly, increase TNF-α secretion in murine dendritic cells. Moreover, immunization assays revealed that thiolated PAA/Schizo particles induced a humoral response with a more balanced IgG2a/IgG1 ratio. Therefore, thiolated PAA/Schizo particles are deemed a promising delivery system whose evaluation in vaccine prototypes is guaranteed.

Positively Charged-Amylose-Entangled Au-Nanoparticles Acting as Protein Carriers and Potential Adjuvants to SARS-CoV-2 Subunit Vaccines.

The COVID-19 pandemic continues to spread worldwide. To protect and control the spread of SARS-CoV-2, varieties of subunit vaccines based on spike (S) proteins have been approved for human applications. Here, we report a new subunit vaccine design strategy that functions as both an antigen carrier and an adjuvant in immunization to elicit high-level immune responses. The complex of 2-hydroxypropyl-trimethylammonium chloride chitosan and amylose entangles Au nanoparticles (HTCC/amylose/AuNPs) forming 40 nm nanocarriers with a positive charge. The obtained positively charged nanoparticles reveal many merits, including the larger S protein loading capacity in PBS buffer, higher cellular uptake ability, and lower cell cytotoxicity, supporting their potential as safe vaccine nanocarriers. Two functionalized nanoparticle subunit vaccines are prepared via loading full-length S proteins derived from SARS-CoV-2 variants. In mice, both prepared vaccines elicit high specific IgG antibodies, neutralize antibodies, and immunoglobulin IgG1 and IgG2a. The prepared vaccines also elicit robust T- and B-cell immune responses and increase CD19+ B cells, CD11C+ dendritic cells, and CD11B+ macrophages at the alveoli and bronchi of the immunized mice. Furthermore, the results of skin safety tests and histological observation of organs indicated in vivo safety of HTCC/amylose/AuNP-based vaccines. Summarily, our prepared HTCC/amylose/AuNP have significant potential as general vaccine carriers for the delivery of different antigens with potent immune stimulation.

Enhanced Systemic Humoral Immune Response Induced in Mice by Generalized Modules for Membrane Antigens (GMMA) Is Associated with Affinity Maturation and Isotype Switching.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen-GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Synthesis and Characterization of Innovative Microgels Based on Polyacrylic Acid and Microalgae Cell Wall and Their Potential as Antigen Delivery Vehicles.

In this study, hybrid polyacrylic acid and Schizochytrium sp. microalgae (PAA/Schizo) microgels were synthesized by inverse emulsion assisted by ultrasound using the cell wall fraction as crosslinker. Physicochemical characterization of PAA/Schizo microgels revealed polymeric spherical particles (288 ± 39 nm) and were deemed stable and negatively charged. The produced microgels are not inherently toxic as cell viability was sustained above 80% when mice splenocytes were exposed to concentrations ranging 10-900 µg/mL. PAA/Schizo microgels were evaluated as antigen delivery nanovehicle by adsorbing bovine serum albumin (BSA); with a loading efficiency of 72% and loading capacity of 362 µg/mg. Overall, intranasally-immunized BALB/c mice showed null IgG or IgA responses against PAA/Schizo microgel-BSA, whereas soluble BSA induced significant humoral responses in systemic and mucosal compartments. Splenocytes proliferation assay upon BSA stimulus revealed positive CD4+ T cells-proliferation response in PAA/Schizo microgels-BSA group. Thus, PAA/Schizo microgels constitute functional antigen delivery vehicles of simple and ecofriendly synthesis. Moreover, the use of cell wall fraction as cross-linker agent provides an alternative use for the generation of high-value products using residual algae biomass from the oil industry. Our data suggests that the PAA/Schizo microgels are potential antigen delivery vehicles for immunotherapy development.

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines.

Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.

Analysis of the Efficacy and Safety of PEGylated Interferon-α2b Treatment in Inactive Hepatitis B Surface Antigen Carriers.

INTRODUCTION: Hepatitis B virus (HBV) infection is associated with the onset of several major liver diseases. Inactive hepatitis B surface antigen (HBsAg) carriers (IHCs) may be successfully treated with PEGylated interferon-α2b (PEG-IFNα2b)-based antiviral therapy; however, studies on this treatment have been insufficient. In this study, we evaluated the efficacy and safety of PEG-IFNα2b treatment in IHCs. METHODS: Nineteen IHCs were treated with subcutaneous PEG-IFNα2b (180 µg/week) for 48 weeks (treatment group). Patients were followed up for 24 weeks after treatment discontinuation. Twenty untreated control patients were observed for 72 weeks (control group). HBsAg clearance (HBsAg < 0.05 IU/mL), HBsAg seroconversion, and alanine aminotransferase levels were monitored. RESULTS: Of the 19 patients treated with PEG-IFNα2b, 16 showed HBsAg loss (84.2%), and 13 showed HBsAg seroconversion (68.4%) at 72 weeks. All patients in the treatment group exhibited virological response (serum HBV DNA level < 10 IU/mL) at the time of drug withdrawal. In the control group, no patients experienced HBsAg loss during the observational period. There were no serious adverse events during treatment, and the therapy was well tolerated. CONCLUSIONS: Short PEG-IFNα2b therapy in IHCs produced a high functional cure rate and good safety profile, suggesting that PEG-IFNα2b treatment may be the best choice for clinical cure of some IHCs.

Design of Outer Membrane Vesicles as Cancer Vaccines: A New Toolkit for Cancer Therapy.

Cancer vaccines have been extensively studied in recent years and have contributed to exceptional achievements in cancer treatment. They are some of the most newly developed vaccines, although only two are currently approved for use, Provenge and Talimogene laherparepvec (T-VEC). Despite the approval of these two vaccines, most vaccines have been terminated at the clinical trial stage, which indicates that although they are effective in theory, concerns still exist, including low antigenicity of targeting antigens and tumor heterogeneity. In recent years, with new understanding of the biological function and vaccine potential of outer membrane vesicles (OMVs), their potential application in cancer vaccine design deserves our attention. Therefore, this review focuses on the mechanisms, advantages, and prospects of OMVs as antigen-carrier vaccines in cancer vaccine development. We believe that OMV-based vaccines present a safe and effective cancer therapeutic option with broad application prospects.

Displaying Whole-Chain Proteins on Hepatitis B Virus Capsid-Like Particles.

The highly immunogenic icosahedral capsid of hepatitis B virus (HBV) can be exploited as a nanoparticulate display platform for heterologous molecules. Its constituent core protein (HBc) of only ~180 amino acids spontaneously forms capsid-like particles (CLPs) even in E. coli. The immunodominant c/e1 epitope in the center of the HBc primary sequence comprises a solvent-exposed loop that tolerates insertions of flexible peptide sequences yet also of selected whole proteins as long as their 3D structures fit into the two acceptor sites. This constraint is largely overcome in the SplitCore system, where the sequences flanking the loop are expressed as two separate but self-complementing entities, with the foreign sequence fixed to the carrier at one end only. Both the contiguous and the split type of CLP strongly enhance immunogenicity of the displayed sequence but also nonvaccine applications can easily be envisaged. After a brief survey of the basic features of the two HBc carrier forms, we provide conceptual guidelines concerning which foreign proteins are likely to be presentable, or not, on either carrier type. We describe generally applicable protocols for CLP expression in E. coli, cell lysis and CLP enrichment by sucrose gradient velocity sedimentation, plus a simple but meaningful gel electrophoretic assay to assess proper particle formation.

Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo.

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.