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In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by... more
In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by intracellular pathogens. Conversely, in humans, the question of a role for NO as an antimicrobial defense mechanism has been the subject of much debate. Recently, however, iNOS expression by human Mphi and formation of NO or its derivatives have been reported both in vivo and in vitro, strongly suggesting that human Mphi are indeed capable of inducible NO synthesis. However, the conditions allowing NO production by human Mphi in culture remain poorly defined, rendering more difficult the study of the effector functions of NO in these cells. To alleviate this problem, cells of the U937 monocytoid line were engineered to express iNOS by transfection with human hepatic iNOS (DFGiNOS), leading to production of NO on supplementation with the cofactor tetrahy...
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In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by... more
In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by intracellular pathogens. Conversely, in humans, the question of a role for NO as an antimicrobial defense mechanism has been the subject of much debate. Recently, however, iNOS expression by human Mphi and formation of NO or its derivatives have been reported both in vivo and in vitro, strongly suggesting that human Mphi are indeed capable of inducible NO synthesis. However, the conditions allowing NO production by human Mphi in culture remain poorly defined, rendering more difficult the study of the effector functions of NO in these cells. To alleviate this problem, cells of the U937 monocytoid line were engineered to express iNOS by transfection with human hepatic iNOS (DFGiNOS), leading to production of NO on supplementation with the cofactor tetrahy...
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Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under... more
Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine ...
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In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was... more
In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.
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Background. Recent advances in rational adjuvant design and antigen selection have enabled a new generation of vaccines with potential to treat and prevent infectious disease. The aim of this study was to assess whether therapeutic... more
Background. Recent advances in rational adjuvant design and antigen selection have enabled a new generation of vaccines with potential to treat and prevent infectious disease. The aim of this study was to assess whether therapeutic immunization could impact the course of Mycobacterium tuberculosis infection with use of a candidate tuberculosis vaccine antigen, ID93, formulated in a synthetic nanoemulsion adjuvant, GLA-SE, administered in combination with existing first-line chemotherapeutics rifampicin and isoniazid.Methods. We used a mouse model of fatal tuberculosis and the established cynomolgus monkey model to design an immuno-chemotherapeutic strategy to increase long-term survival and reduce bacterial burden, compared with standard antibiotic chemotherapy alone.Results. This combined approach induced robust and durable pluripotent antigen-specific T helper-1-type immune responses, decreased bacterial burden, reduced the duration of conventional chemotherapy required for surviv...
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Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included... more
Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-gamma recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine ca...
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Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin... more
Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.
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Specialized APC such as macrophages, B cells and especially DC have the capacity to capture, process and present in a MHC class-I restricted manner various exogenous cell associated antigens, including minor histocompatibility antigens,... more
Specialized APC such as macrophages, B cells and especially DC have the capacity to capture, process and present in a MHC class-I restricted manner various exogenous cell associated antigens, including minor histocompatibility antigens, tumor antigens, or antigens derived from apoptotic, necrotic and/or virus-infected cells (1-4), a process referred to as cross-presentation. Cross-presentation can be extended to a wide range of
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Innate immunity confers an immediate nonspecific mechanism of microbial recognition through germ line-encoded pattern recognition receptors (PRRs). Of these, Toll-like receptors (TLRs) and nucleotide-binding and oligomerization domain... more
Innate immunity confers an immediate nonspecific mechanism of microbial recognition through germ line-encoded pattern recognition receptors (PRRs). Of these, Toll-like receptors (TLRs) and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) have shaped our current understanding of innate regulation of adaptive immunity. It is now recognized that PRRs are paramount in instructing an appropriate adaptive immune response. Their ligands have been the focus of adjuvant research with the goal of generating modern vaccine combinations tailored to specific pathogens. In this review we will highlight the recent findings in the field of adjuvant research with a particular focus on the potential of TLR and NLR ligands as adjuvants and their influence on adaptive immune responses.
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We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of... more
We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of the P. falciparum 7G8 isolate CS protein expressed on the sporozoite surface. They comprise what is believed to be the mature protein, except for the central repetitive B cell domain. BALB/c (H-2d) mice were immunized s.c. with 50 micrograms soluble CS polypeptides emulsified in IFA. After a single immunization, CS-specific helper and cytotoxic T lymphocytes (CTLs) could be obtained. The resultant CTLs obtained by in vitro restimulation of primed lymph node (LN) cells recognized H-2Kd target cells in the presence of short synthetic peptides defined in the present study. These epitopes are contained within the N- and C-terminal regions of the CS protein, and correspond to sequences 39-47 and 333-342. In addition, these CTLs can specifically lyse H-2...
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Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or... more
Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-γ, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequent...
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A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing... more
A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.
Research Interests: Malaria, Senegal, Biological Sciences, Humans, Mice, and 15 moreAnimals, Follow-up studies, Vaccine, Peptides, Protein structure, T lymphocytes, Longitudinal Studies, Protein Secondary Structure Prediction, Plasmodium falciparum, Western blot, Biological activity, Structure activity Relationship, Epitopes, Malaria Vaccines, and Peripheral Blood Mononuclear Cells
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Research Interests: Flow Cytometry, Virology, Calcium, Apoptosis, Cell Culture, and 17 moreWestern blotting, Biological Sciences, Cell line, Mice, Animals, Cell Death, Endoplasmic Reticulum, P-glycoprotein, Rubella Virus, Programmed cell death, Capsid, Amino Acid Profile, Transfection, Rabbits, Capsid Protein, RNA Virus, and Birth Defect
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Research Interests: Cytokines, Biological Sciences, Humans, Female, Male, and 5 moreYoung Adult, Vaccine, Middle Aged, Adult, and immunoglobulin G
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The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic... more
The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.
Research Interests: Macrophages, Multidisciplinary, Nature, Ubiquitin, Escherichia coli, and 14 moreMice, Animals, Salmonella Typhimurium, Endoplasmic Reticulum, Antigen Presentation, MHC class II, Phagocytosis, Major histocompatibility complex, Multienzyme complexes, Cysteine endopeptidases, Amino Acid Sequence, Antigens, MHC class I, and Brucella abortus
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Research Interests: RNA, Flow Cytometry, Fluorescence Microscopy, Cell Cycle, Calcium, and 17 moreBiological Chemistry, Cytoskeleton, Western blotting, Biological Sciences, Phylogeny, Animals, Biological, Plasmids, CHEMICAL SCIENCES, Molecular cloning, Time Factors, Transfection, Protein Conformation, Amino Acid Sequence, Base Sequence, Calcium Binding Proteins, and Immunoblotting
In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and... more
In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms. However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms. The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway. Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.
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We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional... more
We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum-infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.