Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of protein antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore we evaluated the humoral response to these antigens in mice when flagellin was used as an adjuvant. Flagellin-L1R and flagellin-B5R fusion proteins were more potent than flagellin, L1R, and B5R as separate proteins. At least three immunizations with flagellin-L1R and flagellin-B5R fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Immune mice exhibited only limited signs of disease following challenge. Additionally, virus neutralization titers correlated with protection. Depletion of complement using cobra venom factor resulted in a marked decrease in the survival of immunized mice after challenge with vaccinia virus. Our results are consistent with the conclusion that flagellin-L1R and flagellin-B5R fusion proteins are effective in eliciting protective immunity against vaccinia virus that is dependent, in large part, on complement.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Activating the anti-tumor immune response in the characteristically immune-suppressive peritoneal environment presents a potential strategy to treat this disease. In this study, we show that a toll-like receptor (TLR) and C-type lectin receptor (CLR) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits tumor growth and ascites development in a mouse model of aggressive mammary cancer-induced peritoneal carcinomatosis. MPL/TDCM treatment similarly inhibited peritoneal EL4 tumor growth and ascites development. These effects were not observed in mice lacking B cells or mice lacking CD19, which are deficient in B-1a cells, an innate-like B cell population enriched in the peritoneal cavity. Remarkably, adoptive transfer of B-1a cells, but not splenic B cells from WT mice restored MPL/TDCM-induced protection in mice with B cell defects. Treatment induced B-1 cells to rapidly produce high levels of natural IgM reactive against tumor-associated carbohydrate antigens. Consistent with this, we found significant deposition of IgM and C3 on peritoneal tumor cells as early as 5 days post-treatment. Mice unable to secrete IgM or complement component C4 were not protected by MPL/TDCM treatment, indicating tumor killing was mediated by activation of the classical complement pathway. Collectively, our findings reveal an unsuspected role for B-1 cell-produced natural IgM in providing protection against tumor growth in the peritoneal cavity, thereby highlighting potential opportunities to develop novel therapeutic strategies for the prevention and treatment of peritoneal metastases.

Flagellin is a highly effective adjuvant for CD4+ T cell and humoral immune responses. However, there is conflicting data in the literature regarding the ability of flagellin to promote a CD8+ T cell response. In this article, we report that immunization of wild-type, TLR5−/−, and MyD88−/− adoptive transfer recipient mice revealed the ability of flagellin fusion proteins to promote OVA-specific CD8+ T cell proliferation independent of TLR5 or MyD88 expression by the recipient animal. Wild-type and TLR5−/− APCs were able to stimulate high levels of OVA-specific CD8+ T cell proliferation in vitro in response to a flagellin fusion protein containing full-length OVA or the SIINFEKL epitope and 10 flanking amino acids (OVAe), but not to OVA and flagellin added as separate proteins. This effect was independent of the conserved regions of flagellin and occurred in response to OVAe alone. Comparison of IFN-g production by CD8+ effector cells revealed higher levels of SIINFEKL peptide–MHC I complexes on the surface of APCs that had been pulsed with OVAe–flagellin fusion proteins than on cells pulsed with OVA. Inhibition of the proteasome significantly reduced Ag-specific proliferation in response to OVAe fusion proteins. In summary, our data are consistent with the conclusion that flagellin–OVA fusion proteins induce an epitope-specific CD8+ T cell response by facilitating Ag processing and not through stimulatory signaling via TLR5 and MyD88. Our findings raise the possibility that flagellin might be an efficient Ag carrier for Ags that are poorly processed in their native state.

Protection against encapsulated bacteria can be elicited using polysaccharide vaccines. These antigens often behave as T-cell-independent type 2 antigens (TI-2 Ags). However, TI-2 Ags, including pneumococcal polysaccharides, often elicit weak immunoglobulin G (IgG) responses and are refractive to boosting. Conjugate vaccines have not completely overcome this challenge and hence, alternative strategies are required to enhance polysaccharide vaccine responses. Herein, we describe an adjuvant consisting of a Toll-like receptor and C-type lectin receptor agonist pairing that significantly increases primary immunoglobulin M and IgG responses to TI-2 Ags as well as enables significant boosting when coadministered with polysaccharide vaccines. Consistent with this, the adjuvant significantly increased the generation of both TI-2 memory B cells and long-lived antibody secreting cells. Adjuvant effects were highly dependent on B-cell-intrinsic MyD88, but not Trif expression. Importantly, coadministration of the adjuvant with the Pneumovax vaccine significantly increased the protective efficacy of vaccination in a lethal challenge mouse model of pneumococcal respiratory infection. Collectively, these data provide evidence that B-cell-directed adjuvants have promise in significantly improving the quality and quantity of serologic and B-cell memory responses to clinically relevant polysaccharide vaccines.

CD22 (Siglec-2) is a critical regulator of B cell activation and survival. CD22 mice generate significantly impaired Ab responses to T cell-independent type 2 (TI-2) Ags, including haptenated Ficoll and pneumococcal polysaccharides, Ags that elicit poor T cell help and activate BCR signaling via multivalent epitope crosslinking. This has been proposed to be due to impaired marginal zone (MZ) B cell development/maintenance in CD22 mice. However, mice expressing a mutant form of CD22 unable to bind sialic acid ligands generated normal TI-2 Ab responses, despite significantly reduced MZ B cells. Moreover, mice treated with CD22 ligand-binding blocking mAbs, which deplete MZ B cells, had little effect on TI-2 Ab responses. We therefore investigated the effects of CD22 deficiency on B-1b cells, an innate-like B cell population that plays a key role in TI-2 Ab responses. B-1b cells from CD22 mice had impaired BCR-induced proliferation and significantly increased intracellular Ca concentration responses following BCR crosslinking. Ag-specific B-1b cell expansion and plasmablast differentiation following TI-2 Ag immunization was significantly impaired in CD22 mice, consistent with reduced TI-2 Ab responses. We generated CD22 mice with reduced CD19 levels (CD22CD19) to test the hypothesis that augmented B-1b cell BCR signaling in CD22 mice contributes to impaired TI-2 Ab responses. BCR-induced proliferation and intracellular Ca concentration responses were normalized in CD22CD19 B-1b cells. Consistent with this, TI-2 Ag-specific B-1b cell expansion, plasmablast differentiation, survival, and Ab responses were rescued in CD22CD19 mice. Thus, CD22 plays a critical role in regulating TI-2 Ab responses through regulating B-1b cell signaling thresholds.