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1.  Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design 
Rappuoli et al. illustrate how new breakthroughs in the field of human immunology can be combined with insights from structural and computational biology for the rational design of novel broadly protective immunogens, potentiating the development of new vaccines against infectious diseases that still present an important unmet medical need.
Traditionally, vaccines have been developed by cultivating infectious agents and isolating the inactivated whole pathogen or some of its purified components. 20 years ago, reverse vaccinology enabled vaccine discovery and design based on information deriving from the sequence of microbial genomes rather than via the growth of pathogens. Today, the high throughput discovery of protective human antibodies, sequencing of the B cell repertoire, and the increasing structural characterization of protective antigens and epitopes provide the molecular and mechanistic understanding to drive the discovery of novel vaccines that were previously impossible. We are entering a “reverse vaccinology 2.0” era.
PMCID: PMC4821650  PMID: 27022144
2.  Novel adjuvant Alum-TLR7 significantly potentiates immune response to glycoconjugate vaccines 
Scientific Reports  2016;6:29063.
Although glycoconjugate vaccines are generally very efficacious, there is still a need to improve their efficacy, especially in eliciting a strong primary antibody response. We have recently described a new type of vaccine adjuvant based on a TLR7 agonist adsorbed to alum (Alum-TLR7), which is highly efficacious at enhancing immunogenicity of protein based vaccines. Since no adjuvant has been shown to potentiate the immune response to glycoconjugate vaccines in humans, we investigated if Alum-TLR7 is able to improve immunogenicity of this class of vaccines. We found that in a mouse model Alum-TLR7 greatly improved potency of a CRM197-MenC vaccine increasing anti-MenC antibody titers and serum bactericidal activity (SBA) against MenC compared to alum adjuvanted vaccine, especially with a low dose of antigen and already after a single immunization. Alum-TLR7 also drives antibody response towards Th1 isotypes. This adjuvant was also able to increase immunogenicity of all polysaccharides of a multicomponent glycoconjugate vaccine CRM197-MenACWY. Furthermore, we found that Alum-TLR7 increases anti-polysaccharide immune response even in the presence of a prior immune response against the carrier protein. Finally, we demonstrate that Alum-TLR7 adjuvant effect requires a functional TLR7. Taken together, our data support the use of Alum-TLR7 as adjuvant for glycoconjugate vaccines.
PMCID: PMC4954951  PMID: 27439378
3.  A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells 
Human Vaccines & Immunotherapeutics  2015;11(8):2038-2050.
Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8+ T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8+ T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8+ T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8+ T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.
PMCID: PMC4635931  PMID: 26024409
B cells; cross presentation/priming; cytotoxic T cells; dendritic cells; vaccination
4.  Different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens 
Scientific Reports  2016;6:19570.
The majority of vaccine candidates in clinical development are highly purified proteins and peptides relying on adjuvants to enhance and/or direct immune responses. Despite the acknowledged need for novel adjuvants, there are still very few adjuvants in licensed human vaccines. A vast number of adjuvants have been tested pre-clinically using different experimental conditions, rendering it impossible to directly compare their activity. We performed a head-to-head comparison of five different adjuvants Alum, MF59®, GLA-SE, IC31® and CAF01 in mice and combined these with antigens from M. tuberculosis, influenza, and chlamydia to test immune-profiles and efficacy in infection models using standardized protocols. Regardless of antigen, each adjuvant had a unique immunological signature suggesting that the adjuvants have potential for different disease targets. Alum increased antibody titers; MF59® induced strong antibody and IL-5 responses; GLA-SE induced antibodies and Th1; CAF01 showed a mixed Th1/Th17 profile and IC31® induced strong Th1 responses. MF59® and GLA-SE were strong inducers of influenza HI titers while CAF01, GLA-SE and IC31® enhanced protection to TB and chlamydia. Importantly, this is the first extensive attempt to categorize clinical-grade adjuvants based on their immune profiles and protective efficacy to inform a rational development of next generation vaccines for human use.
PMCID: PMC4726129  PMID: 26791076
5.  Development and Application of a Novel SPE-Method for Bioassay-Guided Fractionation of Marine Extracts 
Marine Drugs  2015;13(9):5736-5749.
The biological diversity of marine habitats is a unique source of chemical compounds with potential use as pharmaceuticals, cosmetics and dietary supplements. However, biological screening and chemical analysis of marine extracts pose specific technical constraints and require adequate sample preparation. Here we report an improved method on Solid Phase Extraction (SPE) to fractionate organic extracts containing high concentration of salt that hampers the recovery of secondary metabolites. The procedure uses a water suspension to load the extracts on a poly(styrene-divynylbenzene)-based support and a stepwise organic solvent elution to effectively desalt and fractionate the organic components. The novel protocol has been tested on MeOH-soluble material from three model organisms (Reniera sarai, Dendrilla membranosa and Amphidinium carterae) and was validated on a small panel of 47 marine samples, including sponges and protists, within discovery programs for identification of immuno-stimulatory and anti-infective natural products.
PMCID: PMC4584351  PMID: 26378547
drug discovery; bioactive marine natural products; SPE fractionation methods
6.  Modulation of Endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by Genetic Lipid A Modifications 
The Journal of Biological Chemistry  2014;289(36):24922-24935.
Background: GMMA from Gram-negative bacteria are an attractive vaccine technology, but lipopolysaccharide (LPS) reactogenicity limits use.
Results: Genetic LPS modification resulting in penta-acylation reduced Shigella GMMA reactogenicity to a TLR2-mediated limit. Modifications resulting in palmitoleoylated hexa-acylated LPS triggered higher TLR4-mediated reactogenicity.
Conclusion: Use of GMMA as vaccines will likely require LPS penta-acylation.
Significance: Understanding the relative contribution of TLR activation guides GMMA vaccine development.
Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.
PMCID: PMC4155660  PMID: 25023285
Cytokine; Endotoxin; Lipopolysaccharide (LPS); Toll-like Receptor (TLR); Vaccine; GMMA; Outer Membrane Vesicles; Shigella; htrB; msbB
7.  DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells 
Immunome Research  2010;6:10.
The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).
Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.
The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.
PMCID: PMC3000836  PMID: 21092113
8.  Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists 
PLoS ONE  2010;5(7):e11491.
Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC.
Principal Findings
In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation.
We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.
PMCID: PMC2901334  PMID: 20634889
9.  Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein 
The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in ∼170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2–mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.
PMCID: PMC2196014  PMID: 11781363
natural killer cells; inhibitory signaling; immune evasion; chronic viral infection; tetraspanin

Results 1-9 (9)