Identification of Sulfavant A as the First Synthetic TREM2 Ligand Discloses a Homeostatic Response of Dendritic Cells After Receptor Engagement

The immune response arises from a fine balance of cellular and molecular mechanisms that provide for surveillance, tolerance, and elimination of dangers as pathogens. Improving the quality of the immune response remains a major goal in immunotherapy and vaccine development. Sulfavant A (SULF A) is a sulfolipid that has shown promising adjuvant activity in a cancer vaccine model. Here we report that SULF A is the first synthetic small molecule binding to the Triggering Receptor Expressed on Myeloid cells-2 (TREM2). The receptor engagement initiates an unconventional maturation of Dendritic cells (DCs) leading to upregulation of the Major Histocompatibility Complex class II (MHC Class II) and costimulatory molecules (CD83, CD86, DC54) without release of T helper type 1 (Th1) or 2 (Th2) cytokines. According to a TREM2 mechanism, this response is mediated by SYK-NFAT axis and is compromised by blockade and gene silencing of the receptor. Activation by SULF A preserved the DC functions to excite the allogeneic T cell response, and induced interleukin-10 (IL-10) release after lipopolysaccharide (LPS) stimulation. These results well support the adjuvant effect of SULF A and offer novel insights into the role of TREM2 in the differentiation of an unprecedented DC phenotype (homeDCs) that contributes to the maintenance of immune homeostasis without compromising lymphocyte activation and immunogenic response. The biological function of SULF-A may be of interest in various physiological and pathological processes involving the immune system.

S100A4 exerts robust mucosal adjuvant activity for co-administered antigens in mice

The lack of clinically applicable mucosal adjuvants is a major hurdle in designing effective mucosal vaccines. We hereby report that the calcium-binding protein S100A4, which regulates a wide range of biological functions, is a potent mucosal adjuvant in mice for co-administered antigens, including the SARS-CoV-2 spike protein, with comparable or even superior efficacy as cholera toxin but without causing any adverse reactions. Intranasal immunization with recombinant S100A4 elicited antigen-specific antibody and pulmonary cytotoxic T cell responses, and these responses were remarkably sustained for longer than six months. As a self-protein, S100A4 did not stimulate antibody responses against itself, a quality desired of adjuvants. S100A4 prolonged nasal residence of intranasally delivered antigens and promoted migration of antigen-presenting cells. S100A4-pulsed dendritic cells potently activated cognate T cells. Furthermore, S100A4 induced strong germinal center responses revealed by both microscopy and mass spectrometry, a novel technique for measuring germinal center activity. In conclusion, S100A4 may be a promising adjuvant in formulating mucosal vaccines, including vaccines against pathogens that infect via the respiratory tract, such as SARS-CoV-2.

Caspase-1-Dependent Pyroptosis Mediates Adjuvant Activity of Platycodin D as an Adjuvant for Intramuscular Vaccines

Platycodin D (PD) is a potent adjuvant with dual Th1 and Th2 potentiating activity, but its mechanisms of action remain unclear. Here, the C2C12 myoblast cell line and mice were used as in vitro and in vivo models to identify potential signaling pathways involved in the adjuvant activity of PD. PD induced a transient cytotoxicity and inflammatory response in the C2C12 cells and in mouse quadricep muscles. A comparative analysis of microarray data revealed that PD induced similar gene expression profiles in the C2C12 cells and in the quadricep muscles, and triggered rapid regulation of death, immune, and inflammation-related genes, both in vivo and in vitro. It was further demonstrated that caspase-1-dependent pyroptosis was involved in the PD-induced cytotoxicity and inflammatory response in the C2C12 cells via the Ca2+–c-jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK)–NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. Consistently, the in vivo analysis revealed that a local blockage of NLRP3 and caspase-1 inhibited PD-induced cytokine production and immune cell recruitment at the injection site, and impaired the adjuvant activity of PD on antigen-specific immune responses to model antigen ovalbumin (OVA) in mice. These findings identified the caspase-1-dependent adjuvanticity of PD and expanded the current knowledge on the mechanisms of action of saponin-based adjuvants.

Adjuvant Activity of CpG-Oligonucleotide Administered Transcutaneously in Combination with Vaccination Using a Self-Dissolving Microneedle Patch in Mice

In this study, we investigated the mechanism of transcutaneous adjuvant activity of the CpG-oligonucleotide (K3) in mice. Transcutaneous immunization (TCI) with an ovalbumin-loaded self-dissolving microneedle patch (OVA-sdMN) and K3-loaded hydrophilic gel patch (HG) increased OVA-specific Th2- and Th1-type IgG subclass antibody titers more rapidly and strongly than those after only OVA-sdMN administration. However, the antigen-specific proliferation of OVA-specific CD4+ T cells was similar between the OVA-only and the OVA+K3 groups. Population analysis of various immune cells in draining lymph nodes (dLNs) in the primary immune response revealed that the OVA+K3 combination doubled the number of dLN cells, with the most significant increase in B cells. Phenotypic analysis by flow cytometry revealed that B-cell activation and maturation were promoted in the OVA+K3 group, suggesting that direct B-cell activation by K3 largely contributed to the rapid increase in antigen-specific antibody titer in TCI. In the secondary immune response, a significant increase in effector T cells and effector memory T cells, and an increase in memory B cells were observed in the OVA+K3 group compared with that in the OVA-only group. Thus, K3, as a transcutaneous adjuvant, can promote the memory differentiation of T and B cells.

Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

Tannic Acid Exhibits Adjuvant Activity by Enhancing Humoral and Cell-Mediated Immunity Against BSA as a Protein Antigen

Background: Immunization or vaccination is the process of inducing artificial immunity against an antigen taking advantage of the mechanisms of immunological memory. Current vaccines include substances known as adjuvants, which tend to improve the immunogenicity of the antigen, reduce the antigen quantity employed, and boost the immune response in weak responders. Unfortunately, only a few vaccine adjuvants are approved for human use. Objective: Thus, the objective of this study was to investigate the effect of Tannic acid on humoral and cell-mediated immunity against bovine serum albumin (BSA) as a protein antigen in Wistar rats. Method: In order to establish the Tannic acid concentration to test it as an adjuvant, the lethal dose 50 and maximum non-toxic dose were calculated through cytotoxicity and hemolytic assays with J774 A.1 cell line and rat erythrocytes by resazurin reduction method and UV/vis spectrophotometry. Thirty Wistar rats were divided into 5 groups that included two controls without antigen and three treatment groups of adjuvants plus BSA as a protein antigen. The rats were immunized in a 30-day scheme. Blood samples were collected for humoral immunity analysis by means of immunoglobulin quantification, isotyping and antigen-antibody precipitation inhibition analysis. Rat peritoneal macrophages and splenocytes were isolated for cell-mediated immunity analysis by means of nitric oxide quantification from adjuvant stimulated peritoneal macrophages and lymphocytes proliferation assay. Results: Tannic acid was capable of increasing the immunogenicity of the antigen; besides, it was able to stimulate cell-mediated immunity by means of increased lymphocyte proliferation. Moreover, Tannic acid improved the humoral response by means of increased specific antibodies titers. These activities may be attributed to pattern recognition receptors stimulation. Conclusion: Tannic acid was considered biocompatible when tested in vivo because the concentration tested did not show cytotoxicity or hemolytic effect, and there was no detrimental effect observed on the animals’ health. These results show Tannic acid as a promising candidate for vaccine adjuvant.

In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize it’s in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC 50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.