Identifying Key Drivers of Efficient B Cell Responses: On the Role of T Help, Antigen-Organization, and Toll-like Receptor Stimulation for Generating a Neutralizing Anti-Dengue Virus Response.

T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed "Immune-tag" technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein-nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses.

Intranodal Injection of Immune Activator Demonstrates Antitumor Efficacy in an Adjuvant Approach.

The tumor-draining lymph nodes (tdLN) are the initial site of metastases and are the prime site for generating robust antitumor responses. In this study, we explored the efficacy of a universal immune activator (ImmAct) targeted to the tdLN. This approach can be viewed as an attempt to turn a cold, unresponsive tdLN into a hot, responsive site. The adjuvant antitumor efficacy of our novel intranodal injection was evaluated in an aggressive metastatic mammary carcinoma murine model. The cancer cells were inoculated subcutaneously in the lower quadrant of the mouse to provoke the tdLN (inguinal lymph node). The study encompasses a range of methodologies, including in vivo and in vitro assays and high-dimensional flow cytometry analysis. Our findings demonstrated that intranodal administration of ImmAct following the dissection of the primary tumor led to improved tumor-free survival and minimized weight loss. ImmAct led to both local and systemic alterations in the cellular and humoral immunity. Additionally, after ImmAct treatment, non-responders showed a higher rate of exhausted CD8+ T cells compared to responders. Indeed, our innovative approach surpassed the gold standard surgery of sentinel lymph node excision. Overall, intranodal administration of ImmAct yielded a robust antitumor immune response, offering protection against micrometastases and relapse.

Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens.

Virus-like particles (VLPs) are virus-derived artificial nanostructures that resemble a native virus-stimulating immune system through highly repetitive surface structures. Improved safety profiles, flexibility in vaccine construction, and the ease of VLP production and purification have highlighted VLPs as attractive candidates for universal vaccine platform generation, although exploration of different types of expression systems for their development is needed. Here, we demonstrate the construction of several simple Escherichia coli expression systems for the generation of eggplant mosaic virus (EMV) VLP-derived vaccines. We used different principles of antigen incorporation, including direct fusion of EMV coat protein (CP) with major cat allergen Feld1, coexpression of antigen containing and unmodified (mosaic) EMV CPs, and two coexpression variants of EMV VLPs and antigen using synthetic zipper pair 18/17 (SYNZIP 18/17), and coiled-coil forming peptides E and K (Ecoil/Kcoil). Recombinant Fel d 1 chemically coupled to EMV VLPs was included as control experiments. All EMV-Feld1 variants were expressed in E. coli, formed Tymovirus-like VLPs, and were used for immunological evaluation in healthy mice. The immunogenicity of these newly developed vaccine candidates demonstrated high titers of Feld1-specific Ab production; however, a comparably high immune response against carrier EMV was also observed. Antibody avidity tests revealed very specific Ab production (more than 50% specificity) for four out of the five vaccine candidates. Native Feld1 recognition and subclass-specific antibody tests suggested that the EMV-SZ18/17-Feld1 complex and chemically coupled EMV-Feld1 vaccines may possess characteristics for further development.

Comparison of Bacterial Expression Systems Based on Potato Virus Y-like Particles for Vaccine Generation.

Plant-based virus-like particle (VLP) vaccines have been studied for years, demonstrating their potential as antigen-presenting platforms. In this paper, we describe the development of, and compare between, simple Escherichia coli-based antigen display platforms for the generation of potato virus Y (PVY) VLP-derived vaccines, thus allowing the production of vaccines from a single bacterial cell culture. We constructed four systems with the major cat allergen Fel d 1; namely, direct fusion with plant virus PVY coat protein (CP), mosaic PVY VLPs, and two coexpression variants of conjugates (SpyTag/SpyCatcher) allowing coexpression and conjugation directly in E. coli cells. For control experiments, we included PVY VLPs chemically coupled with Fel d 1. All constructed PVY-Fel d 1 variants were well expressed and soluble, formed PVY-like filamentous particles, and were recognized by monoclonal Fel d 1 antibodies. Our results indicate that all vaccine variants induced high titers of anti-Fel d 1 antibodies in murine models. Mice that were immunized with the chemically coupled Fel d 1 antigen exhibited the highest antibody titers and antibody-antigen interaction specificity, as detected by binding avidity and recognition of native Fel d 1. IgG1 subclass antibodies were found to be the dominant IgG class against PVY-Fel d 1. PVY CP-derived VLPs represent an efficient platform for the comparison of various antigen presentation systems to help evaluate different vaccine designs.

The impact of size on particle drainage dynamics and antibody response.

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMVTT-VLPs successfully form icosahedral particles T = 3, with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMVTT-VLPs results in the formation of Rod-shaped VLPs, ~1 µm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMVTT-VLPs and Rod-shaped CCMVTT-VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMVTT-VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMVTT-VLPs, Round-shaped CCMVTT-VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMVTT-VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size.