Antigen Retention Research Articles

Enhanced model antigen retention in tissue through topical high-frequency ultrasound treatment

Enhanced model antigen retention in tissue through topical high-frequency ultrasound treatment

Controlling the speed of antigens transport in dendritic cells improves humoral and cellular immunity for vaccine

Vaccines are an effective intervention for preventing infectious diseases. Currently many vaccine strategies are designed to improve vaccine efficacy by controlling antigen release, typically involving various approaches at the injection site. Yet, strategies for intracellular slow-release of antigens in vaccines are still unexplored. Our study showed that controlling the degradation of antigens in dendritic cells and slowing their transport from early endosomes to lysosomes markedly enhances both antigen-specific T-cell immune responses and germinal center B cell responses. This leads to the establishment of sustained humoral and cellular immunity in vivo imaging and flow cytometry indicated this method not only prolongs antigen retention at the injection site but also enhances antigen concentration in lymph nodes, surpassing traditional Aluminium (Alum) adjuvants. Additionally, we demonstrated that the slow antigen degradation induces stronger follicular helper T cell responses and increases proportions of long-lived plasma cells and memory B cells. Overall, these findings propose that controlling the speed of antigens transport in dendritic cells can significantly boost vaccine efficacy, offering an innovative avenue for developing highly immunogenic next-generation vaccines.

Antigen persistence following mRNA vaccination and the immune consequence of lymph node stromal cell delivery

Abstract mRNA vaccines have demonstrated their usefulness on a global scale in recent years. They elicit potent humoral and cell mediated responses, but the specific mechanisms by which these responses are produced still require investigation. Our lab described a process termed antigen archiving, in which antigen is retained within lymph node stromal cell (LNSC) subsets following viral infection and subunit vaccination to bolster/prolong memory CD8 T cell responses. Whether mRNA vaccines utilize this mechanism to promote immunity has yet to be determined. Data suggest mRNA-derived antigen persists within LNSC of C57/BL6 mice for at least 3 wks following subcutaneous or intramuscular delivery. We investigate the immune consequence of this prolonged antigen retention using ionizable lipid nanoparticles (LNP) to deliver mRNA constructs encoding the model antigen ovalbumin as well as a fluorescent siinfekl-GFP to monitor antigen-specific T-cell responses and localization of antigen within murine lymph nodes (LNs). We identified LNP formulations facilitating differential uptake/translation within LNSC which also demonstrated robust antigen-specific CD8 T cell responses and persistence of antigen presentation within LNs for at least 3 wks in vivo. We plan to interrogate the immune consequences of enhanced stromal cell targeting and subsequent antigen retention following mRNA vaccination. A better understanding of mRNA and protein delivery and retention will inform future vaccine design.

Size Tuning of Mesoporous Silica Adjuvant for One-Shot Vaccination with Long-Term Anti-Tumor Effect.

Despite recent clinical successes in cancer immunotherapy, it remains difficult to initiate a long-term anti-tumor effect. Therefore, repeated administrations of immune-activating agents are generally required in most cases. Herein, we propose an adjuvant particle size tuning strategy to initiate a long-term anti-tumor effect by one-shot vaccination. This strategy is based on the size-dependent immunostimulation mechanism of mesoporous silica particles. Hollow mesoporous silica (HMS) nanoparticles enhance the antigen uptake with dendritic cells around the immunization site in vivo. In contrast, hierarchically porous silica (HPS) microparticles prolong cancer antigen retention and release in vivo. The size tuning of the mesoporous silica adjuvant prepared by combining both nanoparticles and microparticles demonstrates the immunological properties of both components and has a long-term anti-tumor effect after one-shot vaccination. One-shot vaccination with HMS-HPS-ovalbumin (OVA)-Poly IC (PIC, a TLR3 agonist) increases CD4+ T cell, CD8+ T cell, and CD86+ cell populations in draining lymph nodes even 4 months after vaccination, as well as effector memory CD8+ T cell and tumor-specific tetramer+CD8+ T cell populations in splenocytes. The increases in the numbers of effector memory CD8+ T cells and tumor-specific tetramer+CD8+ T cells indicate that the one-shot vaccination with HMS-HPS-OVA-PIC achieved the longest survival time after a challenge with E.G7-OVA cells among all groups. The size tuning of the mesoporous silica adjuvant shows promise for one-shot vaccination that mimics multiple clinical vaccinations in future cancer immunoadjuvant development. This study may have important implications in the long-term vaccine design of one-shot vaccinations.

Therapeutic antibodies: technical points to consider in tissue cross-reactivity studies.

Tissue cross-reactivity (TCR) studies for the development of therapeutic antibodies are conducted to estimate any possible binding sites within the human body that can be affected by the antibody when assessing safety in humans. Any possible binding sites include specific binding sites of the antibody to its target antigen and nonspecific or off-target binding sites. In TCR studies the therapeutic antibodies and immunohistochemistry (IHC) of frozen tissues must be applied in assays. However, there are technical issues with applying a therapeutic antibody or test article to IHC, such as human-on-human staining, difficulty in applying the test article to IHC, and retention of the target antigen in frozen sections. In the current review, we introduce three case studies in which these technical issues were addressed, and propose a practical scheme for points to consider when conducting a TCR study. Information on the target antigen distribution obtained through robust assays and case-by-case strategies were found to be useful for understanding and assessing the relevance of toxic effects between animals and humans. Thus, we anticipate that by considering the points discussed in the current review and combining the data with information on the biological features of the target antigens and therapeutic antibodies, it will be possible to predict safety risks in humans with higher accuracy.

Enhanced Cellular Immunity for Hepatitis B Virus Vaccine: A Novel Polyinosinic-Polycytidylic Acid-Incorporated Adjuvant Leveraging Cytoplasmic Retinoic Acid-Inducible Gene-Like Receptor Activation and Increased Antigen Uptake.

Conventional aluminum adjuvants exhibit limited cellular immunity. Polyinosinic-polycytidylic acid (poly I:C) activates cytoplasmic retinoic acid-inducible gene-like receptor (RLR), triggering strong T cell activation and cellular responses. However, when applied as an adjuvant, its limited endocytosis and restricted cytoplasmic delivery diminish its effectiveness and increase its toxicity. Hybrid polymer-lipid nanoparticle (PLNP) possesses numerous benefits such as good stability, reduced drug leakage, simple fabrication, easy property modulation, and excellent reproducibility compared to the lipid nanoparticle or the polymeric vector. Here, we developed a novel cationic polymer-lipid hybrid adjuvant capable of incorporating poly I:C to enhance cellular immunity. The hepatitis B surface antigen (HBsAg) was immobilized onto poly I:C-incorprated PLNP (PPLNP) via electrostatic interactions, forming the HBsAg/PPLNP vaccine formulation. The PPLNP adjuvant largely enhanced the cellular endocytosis and cytoplasmic transport of poly I:C, activating RLR followed by promoting type I interferon (IFN) secretion. Meanwhile, PPLNP obviously enhanced the antigen uptake, prolonged antigen retention at the site of administration, and facilitated enhanced transport of antigens to lymph nodes. The HBsAg/PPLNP nanovaccine led to amplified concentrations of antigen-specific immunoglobulin G (IgG), IFN-γ, granzyme B, and an enhanced IgG2a/IgG1 ratio, alongside the FasL+/CD8+ T cell activation, favoring a T helper 1 (TH1)-driven immune response. PPLNP, distinguished by its biocompatibility, ease of fabrication, and effectiveness in augmenting cellular immunity, holds significant promise as a new adjuvant.

Potentiating cancer vaccination by adjuvant-loaded cryo-shocked tumor cells

Cancer vaccine holds vast promise in potentiating tumor immunotherapy. Here, we developed a simple cancer vaccine based on the liquid nitrogen-treated cells (LNT cells) that engineered by one-shot shocking of the live tumor cells in liquid nitrogen. In this vaccine, the obtained LNT cells served as both tumor antigens and delivery vehicles to load the adjuvant imiquimod (R837). This design could achieve efficient co-uptake of antigen/adjuvant by antigen presenting cells (APCs) and prolong in vivo retention of tumor antigens and adjuvants. This adjuvant-loaded LNT cells augmented in vivo antitumor responses and enhanced survival in melanoma-bearing mouse model compared with conventional whole-cell vaccine of the mixture of tumor lysis and adjuvant.

Modeling the kinetics of lymph node retention and exposure of a cargo protein delivered by biotin-functionalized nanoparticles

Generation of protective immunity through vaccination arises from the adaptive immune response developed primarily in the lymph nodes drained from the immunization site. Relative to the intramuscular route, subcutaneous administration allows for direct and rapid access to the lymphatics, but accumulation of soluble protein antigens within the lymph nodes is limited. Subunit vaccines also require immune stimulating adjuvants which may not accumulate in the same lymph nodes simultaneously with antigen. Herein we report the use of biotinylated poly (lactic-co-glycolic acid) nanoparticles (bNPs) to enhance delivery of a model protein antigen to the lymphatics. bNPs provide dual functionality as adjuvant and vehicle to localize antigens with stimulated immune cells in the same draining lymph node. Using streptavidin as a model antigen, which can be loaded directly onto the bNP surface, we evaluated the kinetics of lymph node occupancy and adaptive immune responses in wildtype C57BL/6 mice. Antigen exposure in vivo was significantly improved through surface loading onto bNPs, and we developed a working kinetic model to account for the retention of both particles and antigen in draining lymph nodes. We observed enhanced T cell responses and antigen-specific B cell response in vivo when antigen was delivered on the particle surface. This work highlights the advantage of combining intrinsic adjuvant and antigen loading in a single entity, and the utility of kinetic modeling in the understanding of particle-based vaccines. Statement of significanceDevelopment of safe and effective subunit vaccines depends on effective formulations that render optimized exposure and colocalization of antigens and adjuvants. In this work, we utilize a nanoparticle system which features self-adjuvanting properties and allows for surface loading of recombinant protein antigens. Using in vivo imaging, we demonstrated prolonged co-localization of the antigen and adjuvant particles in draining lymph nodes and provided evidence of B cell activation for up to 21 days following subcutaneous injection. A pharmacokinetic model was developed as a step towards bridging the translational gap between particulate-based vaccines and observed outcomes. The results have implications for the rational design of particle-based vaccines.

Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network.

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

Vaccine antigen archiving by lymphatic endothelial cells boosts archived antigen-specific CD8 T cell memory responses during a pathogenic challenge

Abstract Numerous studies have shown viral antigens can persist in lymph nodes after the resolution of the infection. We demonstrated that lymphatic endothelial cells (LEC), which comprise the lymphatic vasculature necessary for antigen drainage from the tissue, are the predominant cell type required for the persistence of both vaccine and viral antigens within the lymph node. We termed this process antigen archiving due to the ability of LEC to actively archive antigens to which an immune response has occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells (DC), resulting in the presentation of the archived antigens to archived antigen-specific CD8 memory T cells. In this study, we evaluated the effect of LEC archived antigen on archived antigen-specific memory T cells during a pathogenic challenge. We predicted that a pathogenic challenge would result in the release of the archived antigen and subsequent archived antigen presentation. Following a pathogenic challenge, we found a significant increase in archived antigen-specific endogenous or transferred memory CD8 T cells, compared to bystander activation seen by memory T cells of an unrelated specificity. The phenotype of the archived antigen-specific memory CD8 T cells was more effector-like and led to a significant increase in protection against Listeria monocytogenes (LM)-expressing the archived antigen, in the local area. In conclusion, these findings suggest that archived antigen may be released during a pathogenic challenge. As such, the archived antigen may be acquired by migratory DCs as they traffic through the lymphatics in order to present the archived antigen to memory CD8 T cells acting as an effective boost. NIH R01 AI121209

Don't FRET-Antigens in the follicle are protected from degradation.

Low protease activity in the FDC network is crucial for intact antigen retention and has translational potential for more effective vaccination strategies.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity.

Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa. Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention. Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen. Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.

NIR-II Light Powered Asymmetric Hydrogel Nanomotors for Enhanced Immunochemotherapy.

Nanomotors are appealing drug carriers, and the strength of the propelling force is important for their motion capability. Though high motion efficiency has been achieved with 808 nm light driven Janus-structured noble metal nanomotors, the NIR-I light penetration depth and material biocompatibility limit their broad application. Herein, we develop a 1064 nm NIR-II light driven asymmetric hydrogel nanomotor (AHNM) with high motion capability and load it with doxorubicin for enhanced immunochemotherapy. Magnetic field assisted photopolymerization generates an asymmetric distribution of Fe3 O4 @Cu9 S8 nanoparticles in the AHNM, producing self-thermophoresis as driving force under NIR-II irradiation. The AHNM is also functionalized with dopamine for the capture and retention of tumor-associated antigens to boost immune activation. The as-obtained NIR-II light driven AHNM has a high tumor tissue penetration capability and enhances immunochemotherapy, providing a promising strategy for cancer therapy.

Nanoadjuvants Actively targeting lymph node conduits and blocking tumor invasion in lymphatic vessels.

Great efforts have been made to manipulate nanoparticles (NPs) with a diameter of 10-100nm to passively target lymph nodes (LNs) to magnitude anti-tumor activity of T cells. However, no attention has been paid to increasing the retention of NPs with active affinity in order to induce a prolonged release of antigens or molecular adjuvants in the LNs mattering the immune response. Here, we formulated two NPs encapsulated with imiquimod (IMQ), a TLR7/8 agonist, and paclitaxel (PTX) and further modified them with tannic acid (TA), respectively, to generate IMQ NP and PTX NP with a final diameter of approximately 40nm. Attributing a strong affinity of TA molecules to the elastin of LN conduits, the TA modified IMQ NPs can bypass the gaps in the layer of lymphatic endothelial cells and enter the paracortex through the lymph node capsule-associated (LNC) conduits. Similarly, the TA modified PTX NPs increased delivery of PTX to the metastatic tumor site in LNs, where the tumor-associated antigens were released and presented by conduits-lining dendritic cells to activate T cells. Thus, the NPs with deposition to LN conduits showed excellent performance in preventing lymphovascular invasion of triple-negative breast cancer cells and lung metastasis thereafter. On the contrary, the NPs without TA flowed through the subcutaneous sinus existing LNs directly by efferent lymphatic vessels showing relatively poor therapeutic outcomes. This study reveals that TA may mediate the long retention of antigens and molecular adjuvants to be delivered to deep LNs for developing potent vaccination technology.

Dynamics of antigen retention by follicular dendritic cells.

Dynamics of antigen retention by follicular dendritic cells.

Transcutaneous immunization via dissolving microneedles protects mice from lethal influenza H7N9 virus challenge

Avian influenza H7N9 virus has first emerged in 2013 and since then has spread in China in five seasonal waves. In humans, influenza H7N9 virus infection is associated with a high fatality rate; thus, an effective vaccine for this virus is needed. In the present study, we evaluated the usefulness of dissolving microneedles (MNs) loaded with influenza H7N9 vaccine in terms of the dissolution time, insertion capacity, insertion depth, and structural integrity of H7N9 virus in vitro. Our in vitro results showed MNs dissolved within 6 mins. The depth of skin penetration was 270 µm. After coating with a matrix material solution, the H7N9 proteins were agglomerated. We detected the H7N9 delivery time and humoral immune response in vivo. In a mouse model, the antigen retention time was longer for MNs than for intramuscular (IM) injection. The humoral response showed that similar to IM administration, MN administration increased the levels of functional and systematic antibodies and protection against the live influenza A/Anhui/01/2013 virus (Ah01/H7N9). The protection level was determined by the analysis of pathological sections of infected lungs. MN and IM administration yielded results superior to those in the control group. Taken together, these findings demonstrate that the use of dissolving MNs to deliver influenza H7N9 vaccines is a promising immunization approach.

IgG Subclass-Dependent Pulmonary Antigen Retention during Acute IgG-Dependent Systemic Anaphylaxis in Mice.

Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG-allergen immune complexes that induce IgG receptor-expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG-allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.

Amoeba-inspired magnetic microgel assembly assisted by engineered dextran-binding protein for vaccination against life-threatening systemic infection.

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.Electronic Supplementary MaterialSupplementary material (AFM image and zeta potential of MG; TEM, FT-IR, DLS, and zeta potential of MNP−DexBP; zeta potential of MG+CaAg and MG+MNP−DexBP+CaAg; antigen release profile of MG+CaAg and MG+MNP−DexBP+CaAg; aggregation and dispersion of dendritic cells induced by MG+MNP−DexBP+CaAg; uptake of FITC-labeled CaAg (fCaAg) and intracellular distribution of fCaAg in the dendritic cells; antigen retention and dendritic cell activation in lymph nodes; and serum anti-CaAg antibody levels on day 3 after C. albicans infection in the mice pre-immunized by PBS (control), CaAg, MG+CaAg, and MG+MNP−DexBP+CaAg) is available in the online version of this article at 10.1007/s12274-022-4809-1.