pestis(10 g/ml) was used with or without PBC micelles (10 g/ml), Pluronic F127 micelles (10 g/ml), or PBC unimers (10 g/ml)

pestis(10 g/ml) was used with or without PBC micelles (10 g/ml), Pluronic F127 micelles (10 g/ml), or PBC unimers (10 g/ml). of host immune responses to vaccines, requiring the development of novel adjuvants. A case in point is the coronavirus disease 2019 (COVID-19) pandemic, which has, for example, disproportionately affected older adults (1). Efficient preventive steps for such threats require the design of vaccine adjuvants that can balance competing and complex requirements. To achieve rational design of vaccines for older adults, it is important to generate vaccine formulations that account for immunosenescence and judiciously balance the competition between the exacerbated inflammation associated with aged individuals and the induction of efficacious immune responses. Traditional adjuvants [e.g., Toll-like receptor (TLR) agonists] typically induce strong immune responses but can also exacerbate the chronic inflammatory environment, which is known to diminish immune responses in aged individuals (2,3). Because a strong B cell response is critical for providing antibody-based protection against many infectious diseases (including COVID-19) (46), mechanistic studies exploring the conversation of adjuvants and immunogens with B cells have important implications for the development of new and more effective vaccines. Self-assembling micellar systems of different sizes and chemistries have been extensively used for vaccine delivery (7). Their small size (<100 nm), flexibility for functionalization, and ability to deliver antigens (Ag) to Ag-presenting cells (APCs) make them promising platforms for vaccine adjuvants. There are multiple reports on the use of micelle-based adjuvants (810). Their immune-enhancing characteristics R112 are similar to that of the aforementioned traditional adjuvants in that they activate APCs, induce pro-inflammatory cytokine secretion, and lead to antibody responses with comparable kinetics (1113). However, little is known about their mechanism of action in the context of B cell activation, differentiation, and antibody production. We recently reported on the synthesis of a new class of amphiphilic pentablock copolymers (PBCs) based on the U.S. Food and Drug Administration (FDA)approved temperature-responsive (14) Pluronic F127 and cationic end-group blocks of poly(diethylaminoethylmethacrylate) (PDEAEM), which undergo both heat- and pH-responsive self-assembly to form micelles (15). The cationic PBC micelles have been shown to associate with Ag to form PBC micelleAg complexes and enhance Ag delivery to APCs (16). They have also been demonstrated to induce rapid and short-term enhancement in Rabbit Polyclonal to Bax (phospho-Thr167) antibody responses in mice (16). However, the underlying mechanism of action of these PBC micelles with respect to B cell activation is usually unknown. In addition, studies indicate that this PBC micelles neither activate APCs (e.g., cell surface marker expression) nor lead to the induction of innate effector molecules such as nitric R112 oxide, reactive oxygen species, or pro-inflammatory cytokines (16). This enhanced humoral immune response induced by the PBC micelles while inducing less inflammation is especially beneficial for the development of vaccine formulations for older adults (17,18). This is because traditional adjuvants such as TLR agonists, alum, and MF59 may exacerbate chronic inflammation, which leads to diminished immune responses in aged individuals (1921). We hypothesize that this dissimilarity in characteristics of the immune response generated between the PBC micelles and traditional adjuvants may be attributed to the material properties of the PBC micelles and the mechanisms involved in their interactions with and activation of B cells. In this work, we delineated the mechanism underlying the Ag-specific B cell responses induced by PBC micelles and identified key attributes contributing to this behavior. By observing increased expression of Nur77 [a marker for AgB cell receptor (BCR) engagement], we exhibited that cross-linking of BCRs by PBC micelles leads to B cell activation (22). After confirming the engagement of BCRs with the PBC micelleAg complexes, we show that BCR cross-linking induces B cell proliferation in vitro, leading to the production of Ag-specific antibodies. Last, we demonstrate that BCR cross-linking by PBC micelles can be exploited for in vitro production of therapeutic antibodies to a diverse array of Ag associated withYersinia pestis(F1-V) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [spike (S) protein]. == RESULTS == == Material characterization == The purity and molecular mass of R112 the synthesized PBC were decided using1H nuclear magnetic resonance (NMR). The spectrum showed multiple characteristic peaks (fig. S1, A and B), consistent with previous work (23), and the average molecular weight was calculated to be 14,600 g/mol. The mean diameter of the PBC micelles (fig. S1C) determined using dynamic light scattering was 30.4 nm (2.7 nm), and the zeta potential was +5.82 mV (0.89 mV) with R112 a critical micellar.