Rocco Cantisani

Oil-in-Water Emulsion MF59 Increases Germinal Center B Cell Differentiation and Persistence in Response to Vaccination

Induction of persistent protective immune responses is a key attribute of a successful vaccine formulation. MF59 adjuvant, an oil-in-water emulsion used in human vaccines, is known to induce persistent high-affinity functional Ab titers and memory B cells, but how it really shapes the Ag-specific B cell compartment is poorly documented. In this study, we characterized the Ab- and Ag-specific B cell compartment in wild-type mice immunized with HlaH35L, a Staphylococcus aureus Ag known to induce measurable functional Ab responses, formulated with MF59 or aluminum salts, focusing on germinal centers (GC) in secondary lymphoid organs. Taking advantage of single-cell flow cytometry analyses, HlaH35L-specific B cells were characterized for the expression of CD38 and GL-7, markers of memory and GC, respectively, and for CD80 and CD73 activation markers. We demonstrated that immunization with MF59-, but not aluminum salt–adjuvanted HlaH35L, induced expanded Ag-specific CD73+CD80− GC B cells in proximal- and distal-draining lymph nodes, and promoted the persistence of GC B cells, detected up to 4 mo after immunization. In addition to increasing GC B cells, MF59-adjuvanted HlaH35L also increased the frequency of T follicular helper cells. This work extends previous knowledge regarding adaptive immune responses to MF59-adjuvanted vaccines, and, to our knowledge, for the first time an adjuvant used in human licensed products is shown to promote strong and persistent Ag-specific GC responses that might benefit the rational design of new vaccination strategies.

Vaccine Adjuvant MF59 Promotes Retention of Unprocessed Antigen in Lymph Node Macrophage Compartments and Follicular Dendritic Cells

Ag retention within lymph nodes (LNs) upon vaccination is critical for the development of adaptive immune responses, because it facilitates the encounter of the Ag with cognate lymphocytes. During a secondary exposure of the immune system to an Ag, immune complexes (ICs) that contain the unprocessed Ag are captured by subcapsular sinus macrophages and are transferred onto follicular dendritic cells, where they persist for weeks, facilitating Ag presentation to cognate memory B cells. The impact of adjuvants on Ag retention within the draining LNs is unknown. In this article, we provide the first evidence, to our knowledge, that the oil-in-water emulsion adjuvant MF59 localizes in subcapsular sinus and medullary macrophage compartments of mouse draining LNs, where it persists for at least 2 wk. In addition, we demonstrate that MF59 promotes accumulation of the unprocessed Ag within these LN compartments and facilitates the consequent deposition of the IC-trapped Ag onto activated follicular dendritic cells. These findings correlate with the ability of MF59 to boost germinal center generation and Ag-specific Ab titers. Our data suggest that the adjuvant effect of MF59 is, at least in part, due to an enhancement of IC-bound Ag retention within the LN and offer insights to improve the efficacy of new vaccine adjuvants.

Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans.

CD4+ T follicular helper cells (TFH) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4+IL-21+ICOS1+ T helper (TH) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59®-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4+ TFH1 ICOS+ TFH cells and H1N1-specific CD4+IL-21+ICOS+ CXCR5+ TFH and CXCR5- TH cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4+ T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these TFH cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4+ TFH1 ICOS+ cells and of H1N1-specific CD4+IL-21+ICOS+ CXCR5+, measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4+ TFH subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity. Trial Registration ClinicalTrials.gov NCT01771367

Surface molecules on stimulated plasmacytoid dendritic cells are sufficient to cross-activate resting myeloid dendritic cells.

Human plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are 2 types of antigen-presenting cells that exert complementary roles in innate immune responses. We demonstrated previously that in the presence of suboptimal stimulation or when only 1 dendritic cell type is directly stimulated, contact-dependent crosstalk between mDCs and pDCs leads to the activation of both cell types and thus provides them with the ability to induce an optimal T-cell response. The precise mechanism is currently unknown. Here we demonstrate that pDCs, unable to secrete soluble factors because of previous stimulation, induce optimal mDC maturation, indicating that resting immature mDCs are fully competent to respond to Toll-like receptor-9-engaged pDCs in the absence of soluble factors. Thus, we conclude that immature mDCs already express receptors recognized by ligands that are upregulated on the surface of activated pDCs. Intercellular adhesion molecule-1 upregulated by activated pDCs may play a role in a donor-dependent manner.

A phase I, randomized, controlled, dose-ranging study of investigational acellular pertussis (aP) and reduced tetanus-diphtheria-acellular pertussis (TdaP) booster vaccines in adults

ABSTRACT Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012–June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19+CD20+GM-CSF+ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19+CD20+GM-CSF- B cells. These cells reside within the B cell follicles, are mostly IgM+IgD+, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

Vaccine Adjuvants Confer an Advantage to the Kinetics of Activation of Follicular Dendritic Cells that are Sensitive to Peripheral Tissue's Injury.

To the Editor: Vaccine adjuvants are substances used to enhance the efficacy of vaccines because they are able to increase the immune response to co-administered antigens[1, 2]. MF59 is a safe and effective adjuvant licensed for human influenza vaccine in the European countries[1, 3]. Its mechanism of action is not completely understood yet, but it has been demonstrated that MF59 induces inflammation at injection

Toll-like receptor 9-independent responsiveness of human monocytes to microbial DNA.

To the Editor: Monocytes are important effector cells of the innate immune system equipped with various pattern-recognition receptors to interact with foreign pathogens [1, 2]. Upon microbial infection, monocytes migrate into the infection site where they are involved in the pathogen clearance through either secretion of pro-inflammatory mediators or phagocytic and bactericidal activity. Moreover, monocytes, depending on the tissue where they migrate, generate resident macrophages, whose specific functional features are critical for an optimal immune response against the invading pathogen. As they are also precursors of DCs that prime naive T cell, monocytes have also a primary role in adaptive immune responses [1]. Toll-like receptors (TLRs) are one of the most studied categories of pattern-recognition receptors that are localized on the cell surface or in the endosomal compartment [2, 3]. Human monocytes are commonly considered TLR9 negative and consequently unresponsive to its engagement [2–4]. Nevertheless, two studies challenged this view, showing that monocytes respond to the TLR9 agonist, unmethylated CpG oligodeoxynucleotide (CpG-ODN), upregulating co-stimulatory molecules and producing IL6 to a different extent, depending on the type of CpGODN or promoting transition to DCs [5, 6]. In these two studies, TLR9 expression was not checked. Given the central role of monocytes in immune responses, we decided to compare the TLR9 expression of monocytes with their responsiveness to its agonists. We analysed the TLR transcript level profile of monocytes, isolated by positive selection with magnetic sorting, using quantitative real-time PCR, and we were not able to detect TLR9 transcripts (Fig. 1A), consistent with previous studies [3]. The same PCR primers were used to detect TLR9 transcripts in purified pDCs to ensure that the primers used were working (data not shown). However, the same purified monocytes stimulated overnight with different types of CpG-ODN (types A, B and C) underwent full activation, secreting pro-inflammatory cytokines (Fig. 1B). In addition, purified bacterial DNA, as expected, displayed higher capacity than CpG-ODNs in the stimulation of monocytes, while eukaryotic DNA had not any effect (Fig. 1B), suggesting that only microbial nucleic acids serve as pathogen-associated molecular pattern for monocytes. The stimulator potential of bacterial DNA is not due to lipopeptide or LPS contamination, because the same bacterial DNA did not stimulate the A