The low immunogenicity exhibited by most soluble proteins is generally due to the absence of molecular signatures that are recognized by the immune system as dangerous. In this study, we show that electrostatic binding of synthetic branched cationic or anionic lipopeptides that contain the TLR-2 agonist Pam2Cys markedly enhance a protein’s immunogenicity. Binding of a charged lipopeptide to oppositely charged protein Ags resulted in the formation of stable complexes and occurs at physiologic pH and salt concentrations. The induction of cell-mediated responses is dependent on the electrostatic binding of lipopeptide to the protein, with no CD8+ T cells being elicited when protein and lipopeptide possessed the same electrical charge. The CD8+ T cells elicited after vaccination with lipopeptide–protein Ag complexes produced proinflammatory cytokines, exhibited in vivo lytic activity, and protected mice from challenge with an infectious chimeric influenza virus containing a single OVA epitope as part of the influenza neuraminidase protein. Induction of a CD8+ T cell response correlated with the ability of lipopeptide to facilitate Ag uptake by DCs followed by trafficking of Ag-bearing cells into draining lymph nodes. Oppositely charged but not similarly charged lipopeptides were more effective in DC uptake and trafficking. Very high protein-specific Ab titers were also achieved by vaccination with complexes composed of oppositely charged lipopeptide and protein, whereas vaccination with similarly charged constituents resulted in significant but lower Ab titers. Regardless of whether similarly or oppositely charged lipopeptides were used in the induction of Ab, vaccination generated dominant IgG1 isotype Abs rather than IgG2a.

The Wellcome Trust and Innovative Medicines Initiative 2 Joint Undertaking.

Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20+ B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27hiCD21lo activated memory and CD27+CD21+ resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.

MotivationFlow, hyperspectral and mass cytometry are experimental techniques measuring cell marker expressions at the single cell level. The recent increase of the number of markers simultaneously measurable has led to the development of new automatic gating algorithms. Especially, the SPADE algorithm has been proposed as a novel way to identify clusters of cells having similar phenotypes in high-dimensional cytometry data. While SPADE or other cell clustering algorithms are powerful approaches, complementary analysis features are needed to better characterize the identified cell clusters.ResultsWe have developed SPADEVizR, an R package designed for the visualization, analysis and integration of cell clustering results. The available statistical methods allow highlighting cell clusters with relevant biological behaviors or integrating them with additional biological variables. Moreover, several visualization methods are available to better characterize the cell clusters, such as volcano plots, streamgraphs, parallel coordinates, heatmaps, or distograms. SPADEVizR can also generate linear, Cox or random forest models to predict biological outcomes, based on the cell cluster abundances. Additionally, SPADEVizR has several features allowing to quantify and to visualize the quality of the cell clustering results. These analysis features are essential to better interpret the behaviors and phenotypes of the identified cell clusters. Importantly, SPADEVizR can handle clustering results from other algorithms than SPADE.Availability and ImplementationSPADEVizR is distributed under the GPL-3 license and is available at https://github.com/tchitchek-lab/SPADEVizR.Supplementary information Supplementary data are available at Bioinformatics online.

A conserved 15 amino-acid residue sequence of the ectodomain of the M2 protein of influenza A virus (M2e) induces a strong antibody (Ab) response when incorporated into a synthetic lipopeptide vaccine candidate containing a T-helper epitope from influenza A hemagglutinin and the dendritic cell-targeting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Abs elicited by the truncated M2e sequence were specific for the M2 protein of influenza A virus and were also capable of binding to cells that were infected with influenza A viruses of different subtypes. The Ab titres against the lipopeptide were similar in magnitude to those elicited by the full-length (23 residue) M2e peptide when administered in Freund's adjuvant. Abs to the truncated M2e sequence were also able to significantly reduce the viral load in airways of BALB/c mice after challenge with live influenza virus.

Vaccination is one of the most efficient public healthcare measures to fight infectious diseases. Nevertheless, the immune mechanisms induced in vivo by vaccination are still unclear. The route of administration, an important vaccination parameter, can substantially modify the quality of the response. How the route of administration affects the generation and profile of immune responses is of major interest. Here, we aimed to extensively characterize the profiles of the innate and adaptive response to vaccination induced after intradermal, subcutaneous, or intramuscular administration with a modified vaccinia virus Ankara model vaccine in non-human primates. The adaptive response following subcutaneous immunization was clearly different from that following intradermal or intramuscular immunization. The subcutaneous route induced a higher level of neutralizing antibodies than the intradermal and intramuscular vaccination routes. In contrast, polyfunctional CD8+ T-cell responses were preferentially induced after intradermal or intramuscular injection. We observed the same dichotomy when analyzing the early molecular and cellular immune events, highlighting the recruitment of cell populations, such as CD8+ T lymphocytes and myeloid-derived suppressive cells, and the activation of key immunomodulatory gene pathways. These results demonstrate that the quality of the vaccine response induced by an attenuated vaccine is shaped by early and subtle modifications of the innate immune response. In this immunization context, the route of administration must be tailored to the desired type of protective immune response. This will be achieved through systems vaccinology and mathematical modeling, which will be critical for predicting the efficacy of the vaccination route for personalized medicine.

The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8+ T cell response. Therapeutic protection required IFN-γ and CD8+ T cells, whereas NK and CD4+ T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.

Dendritic cells (DCs) that drain the gut and skin are known to favor the establishment of T cell populations that home to the original site of DC-antigen (Ag) encounter by providing soluble “imprinting” signals to T cells in the lymph node (LN). To study the induction of lung T cell-trafficking, we used a protein-adjuvant murine intranasal and intramuscular immunization model to compare in vivo -activated Ag + DCs in the lung and muscle-draining LNs. Higher frequencies of Ag + CD11b + DCs were observed in lung-draining mediastinal LNs (MedLN) compared to muscle-draining inguinal LNs (ILN). Ag + CD11b + MedLN DCs were qualitatively superior at priming CD4 + T cells, which then expressed CD49a and CXCR3, and preferentially trafficked into the lung parenchyma. CD11b + DCs from the MedLN expressed higher levels of surface podoplanin, Trem4, GL7, and the known co-stimulatory molecules CD80, CD86, and CD24. Blockade of specific MedLN DC molecules or the use of sorted DC and T cell co-cultures demonstrated that DC surface phenotype influences the ability to prime T cells that then home to the lung. Thus, the density of dLN Ag + DCs, and DC surface molecule signatures are factors that can influence the output and differentiation of lung-homing CD4 + T cells.