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Shigellosis, an acute gastroenteritis infection caused by Shigella species, remains a public health burden in developing countries. Recently, many outbreaks due to Shigella sonnei multidrug-resistant strains have been reported in high-income countries, and the lack of an effective vaccine represents a major hurdle to counteract this bacterial pathogen. Vaccine candidates against Shigella sonnei are under clinical development, including a Generalized Modules for Membrane Antigens (GMMA)-based vaccine. The mechanisms by which GMMA-based vaccines interact and activate human immune cells remain elusive. Our previous study provided the first evidence that both adaptive and innate immune cells are targeted and functionally shaped by the GMMA-based vaccine. Here, flow cytometry and confocal microscopy analysis allowed us to identify monocytes as the main target population interacting with the S. sonnei 1790-GMMA vaccine on human peripheral blood. In addition, transcriptomic analysis of this cell population revealed a molecular signature induced by 1790-GMMA mostly correlated with the inflammatory response and cytokine-induced processes. This also impacts the expression of genes associated with macrophages' differentiation and T cell regulation, suggesting a dual function for this vaccine platform both as an antigen carrier and as a regulator of immune cell activation and differentiation. [ABSTRACT FROM AUTHOR]

Generalized Modules for Membrane Antigens (GMMA) are outer membrane exosomes purified from Gram-negative bacteria genetically mutated to increase blebbing and reduce risk of reactogenicity. This is commonly achieved through modification of the lipid A portion of lipopolysaccharide. GMMA faithfully resemble the bacterial outer membrane surface, and therefore represent a powerful and flexible platform for vaccine development. Although GMMA-based vaccines have been demonstrated to induce a strong and functional antibody response in animals and humans maintaining an acceptable reactogenicity profile, the overall impact on immune cells and their mode of action are still poorly understood. To characterize the GMMA-induced immune response, we stimulated human peripheral blood mononuclear cells (hPBMCs) with GMMA from Shigella sonnei. We studied GMMA both with wild-type hexa-acylated lipid A and with the corresponding less reactogenic penta-acylated form. Using multicolor flow cytometry, we assessed the activation of immune cell subsets and we profiled intracellular cytokine production after GMMA stimulation. Moreover, we measured the secretion of thirty cytokines/chemokines in the cell culture supernatants. Our data indicated activation of monocytes, dendritic, NK, B, and γδ T cells. Comparison of the cytokine responses showed that, although the two GMMA have qualitatively similar profiles, GMMA with modified penta-acylated lipid A induced a lower production of pro-inflammatory cytokines/chemokines compared to GMMA with wild-type lipid A. Intracellular cytokine staining indicated monocytes and dendritic cells as the main source of the cytokines produced. Overall, these data provide new insights into the activation of key immune cells potentially targeted by GMMA-based vaccines. [ABSTRACT FROM AUTHOR]

CD8+ T cells play a key role in mediating protective immunity after immune challenges such as infection or vaccination. Several subsets of differentiated CD8+ T cells have been identified, however, a deeper understanding of the molecular mechanism that underlies T-cell differentiation is lacking. Conventional approaches to the study of immune responses are typically limited to the analysis of bulk groups of cells that mask the cells' heterogeneity (RNA-seq, microarray) and to the assessment of a relatively limited number of biomarkers that can be evaluated simultaneously at the population level (flow and mass cytometry). Single-cell analysis, on the other hand, represents a possible alternative that enables a deeper characterization of the underlying cellular heterogeneity. In this study, a murine model was used to characterize immunodominant hemagglutinin (HA533-541)-specific CD8+ T-cell responses to nucleic- and protein-based influenza vaccine candidates, using single-cell sorting followed by transcriptomic analysis. Investigation of single-cell gene expression profiles enabled the discovery of unique subsets of CD8+ T cells that co-expressed cytotoxic genes after vaccination. Moreover, this method enabled the characterization of antigen specific CD8+ T cells that were previously undetected. Single-cell transcriptome profiling has the potential to allow for qualitative discrimination of cells, which could lead to novel insights on biological pathways involved in cellular responses. This approach could be further validated and allow for more informed decision making in preclinical and clinical settings. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre‐existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell‐sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F‐protein (cross‐binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross‐binder nAbs used a wide array of gene rearrangements, while preF‐binder nAbs derived mostly from the expansion of B‐cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F‐protein, identifying an important site of pathogen vulnerability. Synopsis: Several RSV vaccines are currently under clinical development and these will impact the pre‐existing immunity of pre‐infected individuals. We performed in depth analyses of the protective antibody response following natural infection to understand the B cells that will be impacted by vaccination. The majority of nAbs elicited following natural infection recognize epitopes shared between the preF and postF form of the RSV F‐protein.The repertoire analyses of neutralizing antibodies reveal that preF specific nAbs derive mostly from the expansion of B cells from the IGHV1 germline.IGHV1‐derived nAbs recognize an important site of pathogen vulnerability on the preF form of the RSV F‐protein. [ABSTRACT FROM AUTHOR]

Introduction: Bacterial outer membrane vesicles (OMVs) are naturally produced by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII. Materials and methods: The 14 amino acid B cell epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of kif18b gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth. Results: Immunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site. Conclusion: OMVs can be decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions. [ABSTRACT FROM AUTHOR]

A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen. [ABSTRACT FROM AUTHOR]

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms. © 2015 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA+ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses. [ABSTRACT FROM AUTHOR]

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4+ T cells broadly reactive with drifted H5. The CD4+ response was dominated by IL-2+ IFN-γ IL-13- T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4+ T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months. later. We suggest that CD4+ T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use. [ABSTRACT FROM AUTHOR]