Molecular basis for potent B cell responses to antigen displayed on particles of viral size

Abstract

Although it has long been appreciated that multivalent Ags - and particularly viral epitope display - produce extremely rapid, robust, and T-independent humoral immune responses, the biochemical basis for such potency has been incompletely understood. Here we take advantage of a set of neutral liposomes of viral size that are engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme at precisely varied density. We show that particulate Ag display by liposomes induces highly potent B cell responses that are dose- and density-dependent but affinity-independent. Titrating dose of particulate, but not soluble, Ag reveals bimodal Erk phosphorylation and cytosolic calcium increases. Particulate Ag induces signal amplification downstream of the B cell receptor (BCR) by selectively evading LYN-dependent inhibitory pathways, but in vitro potency is independent of CD19. Importantly, Ag display on viral-sized particles signals independently of MYD88 and IRAK1/4, but activates NF-κB robustly in a manner that mimics T cell help. Together, such biased signaling by particulate Ag promotes MYC expression and reduces the threshold required for B cell proliferation relative to soluble Ag. These findings uncover a molecular basis for highly sensitive B cell response to viral Ag display and remarkable potency of virus-like particle vaccines that is not merely accounted for by avidity and BCR cross-linking, and is independent of the contribution of B cell nucleic acid-sensing machinery.

Minimal determinants for lifelong antiviral antibody responses in BALB/c mice from a single exposure to virus-like immunogens at low doses

“…And this antibody response is in the absence of Tolllike receptor signalling (50). Consistent with these results, our studies using the same system of pHEL on Ag-specific B cells in vitro revealed that pHEL alone, without any exogenous adjuvants, can trigger robust B cell proliferation and survival in the absence of MyD88, IRAK1/4 and CD40 ligation (44). Moreover, the nAb response induced by pRBD or pHEL was completely abrogated in B6 mice that were deficient in CD19 (CD19 -/-) (51) (Condition 10).…”

“…Consistent with this, in vitro studies using the same system of pHEL demonstrated that these SVLS without iNA could activate the NF-B signalling pathway in Ag-specific B cells independently of MyD88, IRAK1/4 and CD40 ligation (44). Although the response in vivo towards this ED signal is weaker in B6 mice than BALB/c, in the presence of iNA, both mouse strains mounted strong nAb responses of comparable magnitudes, suggesting that this is an evolutionarily conserved mechanism.…”

“…Thus, neither live infection nor any other adjuvants are necessary to initiate these class switches. B cells use a signalling mechanism that is distinct from that for soluble antigens to sense these structures, notably through evasion of Lyn-dependent inhibitory pathways to initiate proliferation (44). CD19 is an important coreceptor for B cell antigen receptor activation (52)(53)(54).…”

“…In fact, a dose escalation of either sRBD or sHEL by tenfold could not elicit any detectable Ag-specific IgG through the same procedure. The exquisite sensitivity of animals towards pRBD or pHEL suggests that there are distinct signalling mechanisms involved in the sensing of these particulate Ags ( 44 ). In fact, in vitro studies using the same system of pHEL revealed that these SVLS were over 1000-fold more potent than respective soluble proteins in activation of Ag-specific B cells ( 44 ).…”

Antigen-display exosomes provide adjuvant-free protection against SARS-CoV-2 disease at nanogram levels of spike protein

“…However, the success of these and other mRNA vaccines has highlighted the fact that all expression dependent vaccines require, and are affected by, cisacting sorting sequences that govern how the foreign antigen(s) are trafficked and processed by human cells during their biogenesis. These considerations, together with the increasing evidence that B-cells respond best to clustered antigen arrays (18,19), led us to ask whether spike-display exosomes are able to elicit protective immunity at low doses of antigen and without adjuvant, Here we show that systematic elimination of competing sorting information from spike, together with the optimization of the membrane proximal extracellular region (MPERs), transmembrane domain (TMDs), and cytoplasmic carboxy-terminal tail (CTTs) that comprise an exosome membrane anchor domain (EMAD), led to the efficient loading of multiple spikes into most of the exosomes produced by a spike-EMAD13-expressing cell line. Furthermore, we show that injection of spike-EMAD13 exosomes elicited protective immunity against SARS-CoV-2 disease at doses of just 0.5-5 ng of spike protein, without adjuvant.…”