Ernesto Oviedo-Orta | Novartis Vaccines (original) (raw)
Books by Ernesto Oviedo-Orta
Plasma membrane-associated channels known as gap junctions, along with their protein building blo... more Plasma membrane-associated channels known as gap junctions, along with their protein building blocks—connexins—have an important functional role in a range of immunological processes, including heart function, cell growth and specialization, and early development. Spanning basic science and potential clinical applications, Connexin Cell Communication Channels: Roles in the Immune System and Immunopathology assembles and synthesizes four decades of the most important research carried out in this field.
The book first provides a historical overview of the discovery of these membrane channels in cells and tissues of the immune system. It describes their general molecular and biological characteristics and examines how they participate in the evolution, organization, function, and regulation of leukocytes, as well as their interaction with other tissues.
The next section examines immunologically related disease scenarios where gap junctions and connexins have been shown to play a fundamental role. The contributors explain how gap junctional communication participates in the establishment and maintenance of immunological properties such as antibody and cytokine production, as well as lymphocyte immune surveillance in both physiological and pathological conditions. The book explores the most important technical approaches used and how they have been specially adapted to answer key biological questions particular to the mobile nature of leukocytes. It also describes the most recent understanding of how gap junctions and connexins participate in antigen recognition, cross-presentation, lymphocyte activation, and in the assembly and function of the immunological synapse.
Finally, the book focuses on the latest progress made on translating the knowledge gained to specific treatment modalities. Topics in this section include approaches for reducing scarring and cardiac arrhythmia, combating inflammation in the central nervous system, and enhancing epithelial tissue repair. A comprehensive view of achievements in this promising field, the book will inform and update specialists, clinical practitioners, and those studying the potential for commercial applications.
Protein degradation and peptide presentation are crucial events in the generation of a wide varie... more Protein degradation and peptide presentation are crucial events in the generation of a wide variety of antigen-specific T cell-mediated immune responses and the focus of study and application of modern proteomic approaches. Here we discuss the potential emergence of immunopeptidomics as a novel approach to understand how the adaptive immune system operates and to describe the potential benefits of these new applications for biomarker and drug discovery. This article emphasises the importance that naturally occurring or processed proteins and peptides have on the establishment of self tolerance and in shaping immune responses against foreign antigens. An overview is given on some of the methodology currently used to study the composition, structure and functional properties of naturally occurring peptides through the use of proteomic based approaches and current peptidomic-based applications. Finally, we provide several examples from the clinical and laboratory practice in which peptidomics approaches have been used or have the potential to be used to diagnose and/or treat immune-based disorders.
Papers by Ernesto Oviedo-Orta
Vaccine, 2015
Recently an investigational meningococcal B vaccine has been used in two college outbreaks in the... more Recently an investigational meningococcal B vaccine has been used in two college outbreaks in the US. This is the first time that a meningococcal B vaccine has been used for outbreak control in the US. However, strain specific vaccines for meningococcal B outbreaks have been developed in Norway, Cuba and to control a large prolonged outbreak in New Zealand. Although meningococcal disease is mostly endemic and baseline rates in the US have fallen over the past decade, outbreaks are not uncommon in the US and globally. In an outbreak, disease risk can rise 1000 fold or more and such outbreaks can last a decade or longer causing significant morbidity and mortality. Here we review the evolution of several serogroup B outbreaks, and, when applicable, the development and impact of meningococcal B vaccines to control these outbreaks. Prior to the availability of "broad spectrum" meningococcal B vaccines, vaccines developed to control meningococcal B outbreaks were strain specific. With the development of two newly licensed meningococcal B vaccines - a four component meningococcal B vaccine (Bexsero(®), Novartis) and the two component fHBP vaccine (Trumenba(®), Pfizer) that target a broad array of meningococcal B strains, there is now the potential to prevent outbreaks and as well as to shorten the delay between identification of an outbreak and availability of a vaccine.
PLOS ONE, 2015
While formal reporting, surveillance, and response structures remain essential to protecting publ... more While formal reporting, surveillance, and response structures remain essential to protecting public health, a new generation of freely accessible, online, and real-time informatics tools for disease tracking are expanding the ability to raise earlier public awareness of emerging disease threats. The rationale for this study is to test the hypothesis that the HealthMap informatics tools can complement epidemiological data captured by traditional surveillance monitoring systems for meningitis due to Neisseria meningitides (N. meningitides) by highlighting severe transmissible disease activity and outbreaks in the United States.
Atherosclerosis, 2010
The vaccinia virus broad-spectrum CC chemokine inhibitory protein, 35K, is a potent inhibitor of ... more The vaccinia virus broad-spectrum CC chemokine inhibitory protein, 35K, is a potent inhibitor of CC chemokine function, which we have previously shown to be potent in reducing atherosclerosis in the ApoE−/− model. We aim to use a cell-associated form of this molecule to probe the role of the CC chemokine family in the trafficking of different leukocyte populations in inflammation. We have engineered a membrane-associated form of the 35K molecule by coupling the 35K molecule to the transmembrane domain of Fas ligand and have tagged the molecule with both a cytosolic GFP molecule and an HA tag (mem35K) to allow detection of expressing cells. We have produced a transgenic mouse that carries the mem35K transgene under control of a floxed stop promoter so expression is restricted to cells harbouring the cre enzyme. Using a Tie2cre driver line, which expresses the cre molecule in a haematopoietic progenitor population, we have shown a cre dependent excision of the stop cassette in primary murine macrophages by PCR. Furthermore we show a cre-dependent expression of the mem35K protein, as detected by western blotting for both the HA tag and the 35K molecule. In addition mem35K expressing macrophages demonstrate increased GFP fluorescence as assessed by both flow cytometry and fluorescence microscopy. Expression of the mem35K molecule is sufficient to cause a significant decrease in in vitro macrophage chemotaxis towards the CC-chemokine RANTES but not the chemoattractant LtB4. This novel mouse model will enable new approaches to testing the cell-specific roles of CCchemokines in vivo.
Journal of Leukocyte Biology, 2010
As expression of Cxs in cells of the immune system increases upon cellular activation, we investi... more As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4 ؉ T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.
Current opinion in immunology, Aug 31, 2013
Modern society is characterized by a steady increase in the aged population. Increasing numbers o... more Modern society is characterized by a steady increase in the aged population. Increasing numbers of elderly people are exposed to infectious diseases in addition to suffering from chronic non-communicable illnesses. Key differences exist between immune responses elicited against infectious agents in the elderly and in the youngest population. Responses in the aged are characterized by a physiological state of impaired immunity. Such state has forced scientist and vaccine manufacturers to re-think the way vaccines are designed and tested in the elderly. Multiple strategies have been used to overcome the consequences of immunosenescence including the use of higher antigen dose, adjuvanted vaccines, and alternative routes of immunization. However, the lack of understanding of the immune regulatory mechanisms underlying immunosenescence in the elderly represents one of the main hurdles in the pathway to produce effective vaccines for seniors. This article reviews in a succinct form the current state of the art on the development of vaccines for the elderly and critically assesses the past and current literature on this topic, while also proposing new avenues for future studies.
Nature communications, 2013
The presence of multiple connexins was recently demonstrated in platelets, with notable expressio... more The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor (37,43)Gap27 on Cx40(-/-) mouse platelets and of the Cx40 inhibitor (40)Gap27 on Cx37(-/-) mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.
Infection and immunity, May 28, 2013
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. diff... more Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
PloS one, Apr 2013
Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively s... more Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE(-/-) mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.
Expert review of vaccines, Mar 13, 2013
This Special Focus Issue on ‘Therapeutic Vaccines and Immunotherapies: Current Challenges and New... more This Special Focus Issue on ‘Therapeutic Vaccines and Immunotherapies: Current Challenges and New Frontiers’ covers a broad range of topics, including: cancer immunotherapy, with particular reference to prostate cancer, non-small-cell lung cancer, HPV therapeutic vaccines and dendritic cells in cancer immunotherapy; immunotherapy for autoimmune diseases and vaccines against atherosclerosis; and vaccines against substance abuse and tobacco addiction. This issue will provide a critical overview of the state of the art of therapeutic vaccines and immunotherapies highlighting both their strengths and weaknesses, and should stimulate reflection on what has been achieved and on the key hurdles ahead.
BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric h... more BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair.
METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function.
CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.
Vaccines and antibiotics have significantly contributed to improve health and also to increase th... more Vaccines and antibiotics have significantly contributed to improve health and also to increase the longevity of human beings. The fast-acting effect of antibiotics makes them indispensable to treat infected patients. Likewise, when the causative agent of the infection is unknown and in cases of superinfections with different species of bacteria, antibiotics appear to be the only therapeutic option. On the contrary, vaccines are usually not efficacious in people already infected and their action is generally limited to a much narrowed range of pathogens. However, vaccines have contributed to the eradication of some of the most deadly infectious agents worldwide, can generate immunity to infections lasting for several years or life-long, and are able to induce herd immunity. Nonetheless, infectious diseases are still among the leading causes of morbidity and mortality worldwide. This is mainly owing to the emergence of bacterial resistance to antibiotics and the lack of efficacious medications to treat several other infectious diseases. Development of new vaccines appears to be a promising solution to these issues. Indeed, with the advent of new discovery approaches and adjuvants, today is possible to make vaccines virtually against every pathogen. In addition, while vaccine-resistant bacteria have never been reported, accumulating literature is providing evidence that vaccination can reduce the raise of antibiotic resistant strains by decreasing their use.
Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparatio... more Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparations to treat atherosclerosis. Such vaccines have been shown to reduce atherosclerotic plaque development by 50 % in experimental animals, and this effect is associated with induction of T helper (Th)2 immune responses. In this study we immunised apolipoprotein E-deficient (apoE(-/-)) mice with apoB-100-derived peptides P2, P45 and P210. Animals received BSA-conjugated peptides or peptide-loaded bone marrow-derived dendritic cells (DCs). We used enzyme-linked immunosorbent assays to assess the synthesis of anti-peptide-specific IgG1 and IgG2a as well as the levels of interleukin (IL-)10 and interferon gamma (IFN-γ) in plasma of immunised animals. We also measured the effect of immunisation on the number of spleen-derived CD4(+) and CD8(+) regulatory T cells (Tregs) in these animals. Peptide and peptide-loaded DC immunisation significantly increased the levels of peptide-specific immunoglobulins and the number of Tregs in apoE(-/-) mice. This was accompanied by a significant increase in the secretion of IL-10 with no effect on IFN-γ levels. The results also show that the peptides can modulate the homing properties of DCs. Altogether, this study provides novel evidence for the immune mechanisms excerpted by apoB-100-derived peptides and their effect on Tregs and DCs relevant to their use in vaccine preparations.
Over the last twenty years research has provided an important insight into the mechanisms respons... more Over the last twenty years research has provided an important insight into the mechanisms responsible for the immunotoxicity of both local and systemic adverse reactions following the use of immunostimulating drugs and adjuvants. In this article we provide an update of the present knowledge relating to the various parameters and reactants of the immune system at the cellular as well as molecular level that are believed to play a key role in reactogenicity. We discuss evidence obtained from observations in vitro, in vivo in animal models and from clinical applications, including adjuvants used in large scale vaccination today. The data discussed are mainly taken from animal models following hyperstimulation of the immune system; either by the use of very powerful adjuvants, like Freund's that are too toxic for use in practical vaccination, by deliberate high dose application of adjuvants or by the in vivo application of cytokines. Although such hyperstimulating regimens are unlikely to find their way into practical vaccination of humans, this information is of great value as it may facilitate the understanding of the toxicity mechanisms, aid the design of standardised models for the assessment of adjuvant safety and the possible application of new adjuvants in vaccines for humans.
The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins... more The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins of Chlamydophila pneumoniae. Although MOMP can elicit strong immune responses it fails to confer long-term protection against infection in animal models. This effect has been attributed, at least in part, to an inadequate induction of protective Th1-mediated immune responses. In an effort to understand the cellular mechanisms associated to the immunomodulatory properties of MOMP we studied the effect of this protein on mouse macrophages and naïve T-lymphocytes. We found that incubation of mouse macrophages with recombinant MOMP (rMOMP) results in an increased secretion of MMP-9 and a down-regulation of MHC class II, CD86 and CD40. This was accompanied by an increase in IL-10 and IFNgamma but not in IL-12 secretion. rMOMP induced a down-regulation of the expression of CD69 and CD154 markers by activated CD4(+) T cells, and enhanced the secretion of IL-2 and IL-10 by these cells. Conversely, rMOMP-treated macrophages up-regulated the expression of CD69 but not CD154, inhibited the synthesis of IL-10 and up-regulated the production of IFNgamma by activated CD8(+) T cells. Immunization of mice with MOMP induced the synthesis only of MOMP-specific IgG1 but no differences in cytokine profile were observed compared to controls. Our results provide new evidence on the role of MOMP in modulating T cell-mediated immune responses.
As expression of Cxs in cells of the immune system increases upon cellular activation, we investi... more As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.
Toxicology letters, Jan 1, 2009
There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydroca... more There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydrocarbons (PAHs) are involved in the pathogenesis of cardiovascular diseases. However, heterocyclic amines (HAs), a class of carcinogenic compounds present in food, which share many biochemical features with PAHs, have not received much attention. Previous reports have shown that the heterocyclic amine 2-amino-9H-pyrido[2,3-b]indole (AalphaC) binds and metabolically affects endothelial cells in animal models suggesting a potential role in vascular remodeling. The present study investigates the effect of exposure to HAs on atherosclerotic plaque development in the apoE(-/-) mice. We observed that animals treated with AalphaC developed atherosclerotic lesions characterized by lower lipid content but richer in inflammatory cells and collagen content when compared with control animals. Moreover, atherosclerotic plaques from AalphaC-treated apoE(-/-) mice were also smaller with a marked reduction in the tunica media thickness. Furthermore, total cholesterol levels were significantly reduced in AalphaC-treated apoE(-/-) mice. In contrast to what has been previously reported for PAHs, we provide for the first time evidence that HAs may protect against cardiovascular disease by inducing stable atherosclerotic plaques and reducing circulating cholesterol levels. These results open new avenues to further investigate the role of these food-borne carcinogens in cardiovascular physiology and pathology.
Journal of …, Jan 1, 2007
Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary ly... more Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4+ Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.
Plasma membrane-associated channels known as gap junctions, along with their protein building blo... more Plasma membrane-associated channels known as gap junctions, along with their protein building blocks—connexins—have an important functional role in a range of immunological processes, including heart function, cell growth and specialization, and early development. Spanning basic science and potential clinical applications, Connexin Cell Communication Channels: Roles in the Immune System and Immunopathology assembles and synthesizes four decades of the most important research carried out in this field.
The book first provides a historical overview of the discovery of these membrane channels in cells and tissues of the immune system. It describes their general molecular and biological characteristics and examines how they participate in the evolution, organization, function, and regulation of leukocytes, as well as their interaction with other tissues.
The next section examines immunologically related disease scenarios where gap junctions and connexins have been shown to play a fundamental role. The contributors explain how gap junctional communication participates in the establishment and maintenance of immunological properties such as antibody and cytokine production, as well as lymphocyte immune surveillance in both physiological and pathological conditions. The book explores the most important technical approaches used and how they have been specially adapted to answer key biological questions particular to the mobile nature of leukocytes. It also describes the most recent understanding of how gap junctions and connexins participate in antigen recognition, cross-presentation, lymphocyte activation, and in the assembly and function of the immunological synapse.
Finally, the book focuses on the latest progress made on translating the knowledge gained to specific treatment modalities. Topics in this section include approaches for reducing scarring and cardiac arrhythmia, combating inflammation in the central nervous system, and enhancing epithelial tissue repair. A comprehensive view of achievements in this promising field, the book will inform and update specialists, clinical practitioners, and those studying the potential for commercial applications.
Protein degradation and peptide presentation are crucial events in the generation of a wide varie... more Protein degradation and peptide presentation are crucial events in the generation of a wide variety of antigen-specific T cell-mediated immune responses and the focus of study and application of modern proteomic approaches. Here we discuss the potential emergence of immunopeptidomics as a novel approach to understand how the adaptive immune system operates and to describe the potential benefits of these new applications for biomarker and drug discovery. This article emphasises the importance that naturally occurring or processed proteins and peptides have on the establishment of self tolerance and in shaping immune responses against foreign antigens. An overview is given on some of the methodology currently used to study the composition, structure and functional properties of naturally occurring peptides through the use of proteomic based approaches and current peptidomic-based applications. Finally, we provide several examples from the clinical and laboratory practice in which peptidomics approaches have been used or have the potential to be used to diagnose and/or treat immune-based disorders.
Vaccine, 2015
Recently an investigational meningococcal B vaccine has been used in two college outbreaks in the... more Recently an investigational meningococcal B vaccine has been used in two college outbreaks in the US. This is the first time that a meningococcal B vaccine has been used for outbreak control in the US. However, strain specific vaccines for meningococcal B outbreaks have been developed in Norway, Cuba and to control a large prolonged outbreak in New Zealand. Although meningococcal disease is mostly endemic and baseline rates in the US have fallen over the past decade, outbreaks are not uncommon in the US and globally. In an outbreak, disease risk can rise 1000 fold or more and such outbreaks can last a decade or longer causing significant morbidity and mortality. Here we review the evolution of several serogroup B outbreaks, and, when applicable, the development and impact of meningococcal B vaccines to control these outbreaks. Prior to the availability of "broad spectrum" meningococcal B vaccines, vaccines developed to control meningococcal B outbreaks were strain specific. With the development of two newly licensed meningococcal B vaccines - a four component meningococcal B vaccine (Bexsero(®), Novartis) and the two component fHBP vaccine (Trumenba(®), Pfizer) that target a broad array of meningococcal B strains, there is now the potential to prevent outbreaks and as well as to shorten the delay between identification of an outbreak and availability of a vaccine.
PLOS ONE, 2015
While formal reporting, surveillance, and response structures remain essential to protecting publ... more While formal reporting, surveillance, and response structures remain essential to protecting public health, a new generation of freely accessible, online, and real-time informatics tools for disease tracking are expanding the ability to raise earlier public awareness of emerging disease threats. The rationale for this study is to test the hypothesis that the HealthMap informatics tools can complement epidemiological data captured by traditional surveillance monitoring systems for meningitis due to Neisseria meningitides (N. meningitides) by highlighting severe transmissible disease activity and outbreaks in the United States.
Atherosclerosis, 2010
The vaccinia virus broad-spectrum CC chemokine inhibitory protein, 35K, is a potent inhibitor of ... more The vaccinia virus broad-spectrum CC chemokine inhibitory protein, 35K, is a potent inhibitor of CC chemokine function, which we have previously shown to be potent in reducing atherosclerosis in the ApoE−/− model. We aim to use a cell-associated form of this molecule to probe the role of the CC chemokine family in the trafficking of different leukocyte populations in inflammation. We have engineered a membrane-associated form of the 35K molecule by coupling the 35K molecule to the transmembrane domain of Fas ligand and have tagged the molecule with both a cytosolic GFP molecule and an HA tag (mem35K) to allow detection of expressing cells. We have produced a transgenic mouse that carries the mem35K transgene under control of a floxed stop promoter so expression is restricted to cells harbouring the cre enzyme. Using a Tie2cre driver line, which expresses the cre molecule in a haematopoietic progenitor population, we have shown a cre dependent excision of the stop cassette in primary murine macrophages by PCR. Furthermore we show a cre-dependent expression of the mem35K protein, as detected by western blotting for both the HA tag and the 35K molecule. In addition mem35K expressing macrophages demonstrate increased GFP fluorescence as assessed by both flow cytometry and fluorescence microscopy. Expression of the mem35K molecule is sufficient to cause a significant decrease in in vitro macrophage chemotaxis towards the CC-chemokine RANTES but not the chemoattractant LtB4. This novel mouse model will enable new approaches to testing the cell-specific roles of CCchemokines in vivo.
Journal of Leukocyte Biology, 2010
As expression of Cxs in cells of the immune system increases upon cellular activation, we investi... more As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4 ؉ T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.
Current opinion in immunology, Aug 31, 2013
Modern society is characterized by a steady increase in the aged population. Increasing numbers o... more Modern society is characterized by a steady increase in the aged population. Increasing numbers of elderly people are exposed to infectious diseases in addition to suffering from chronic non-communicable illnesses. Key differences exist between immune responses elicited against infectious agents in the elderly and in the youngest population. Responses in the aged are characterized by a physiological state of impaired immunity. Such state has forced scientist and vaccine manufacturers to re-think the way vaccines are designed and tested in the elderly. Multiple strategies have been used to overcome the consequences of immunosenescence including the use of higher antigen dose, adjuvanted vaccines, and alternative routes of immunization. However, the lack of understanding of the immune regulatory mechanisms underlying immunosenescence in the elderly represents one of the main hurdles in the pathway to produce effective vaccines for seniors. This article reviews in a succinct form the current state of the art on the development of vaccines for the elderly and critically assesses the past and current literature on this topic, while also proposing new avenues for future studies.
Nature communications, 2013
The presence of multiple connexins was recently demonstrated in platelets, with notable expressio... more The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor (37,43)Gap27 on Cx40(-/-) mouse platelets and of the Cx40 inhibitor (40)Gap27 on Cx37(-/-) mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.
Infection and immunity, May 28, 2013
Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. diff... more Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.
PloS one, Apr 2013
Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively s... more Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE(-/-) mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.
Expert review of vaccines, Mar 13, 2013
This Special Focus Issue on ‘Therapeutic Vaccines and Immunotherapies: Current Challenges and New... more This Special Focus Issue on ‘Therapeutic Vaccines and Immunotherapies: Current Challenges and New Frontiers’ covers a broad range of topics, including: cancer immunotherapy, with particular reference to prostate cancer, non-small-cell lung cancer, HPV therapeutic vaccines and dendritic cells in cancer immunotherapy; immunotherapy for autoimmune diseases and vaccines against atherosclerosis; and vaccines against substance abuse and tobacco addiction. This issue will provide a critical overview of the state of the art of therapeutic vaccines and immunotherapies highlighting both their strengths and weaknesses, and should stimulate reflection on what has been achieved and on the key hurdles ahead.
BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric h... more BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair.
METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function.
CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.
Vaccines and antibiotics have significantly contributed to improve health and also to increase th... more Vaccines and antibiotics have significantly contributed to improve health and also to increase the longevity of human beings. The fast-acting effect of antibiotics makes them indispensable to treat infected patients. Likewise, when the causative agent of the infection is unknown and in cases of superinfections with different species of bacteria, antibiotics appear to be the only therapeutic option. On the contrary, vaccines are usually not efficacious in people already infected and their action is generally limited to a much narrowed range of pathogens. However, vaccines have contributed to the eradication of some of the most deadly infectious agents worldwide, can generate immunity to infections lasting for several years or life-long, and are able to induce herd immunity. Nonetheless, infectious diseases are still among the leading causes of morbidity and mortality worldwide. This is mainly owing to the emergence of bacterial resistance to antibiotics and the lack of efficacious medications to treat several other infectious diseases. Development of new vaccines appears to be a promising solution to these issues. Indeed, with the advent of new discovery approaches and adjuvants, today is possible to make vaccines virtually against every pathogen. In addition, while vaccine-resistant bacteria have never been reported, accumulating literature is providing evidence that vaccination can reduce the raise of antibiotic resistant strains by decreasing their use.
Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparatio... more Peptides derived from apolipoprotein B (apoB)-100 have been previously used in vaccine preparations to treat atherosclerosis. Such vaccines have been shown to reduce atherosclerotic plaque development by 50 % in experimental animals, and this effect is associated with induction of T helper (Th)2 immune responses. In this study we immunised apolipoprotein E-deficient (apoE(-/-)) mice with apoB-100-derived peptides P2, P45 and P210. Animals received BSA-conjugated peptides or peptide-loaded bone marrow-derived dendritic cells (DCs). We used enzyme-linked immunosorbent assays to assess the synthesis of anti-peptide-specific IgG1 and IgG2a as well as the levels of interleukin (IL-)10 and interferon gamma (IFN-γ) in plasma of immunised animals. We also measured the effect of immunisation on the number of spleen-derived CD4(+) and CD8(+) regulatory T cells (Tregs) in these animals. Peptide and peptide-loaded DC immunisation significantly increased the levels of peptide-specific immunoglobulins and the number of Tregs in apoE(-/-) mice. This was accompanied by a significant increase in the secretion of IL-10 with no effect on IFN-γ levels. The results also show that the peptides can modulate the homing properties of DCs. Altogether, this study provides novel evidence for the immune mechanisms excerpted by apoB-100-derived peptides and their effect on Tregs and DCs relevant to their use in vaccine preparations.
Over the last twenty years research has provided an important insight into the mechanisms respons... more Over the last twenty years research has provided an important insight into the mechanisms responsible for the immunotoxicity of both local and systemic adverse reactions following the use of immunostimulating drugs and adjuvants. In this article we provide an update of the present knowledge relating to the various parameters and reactants of the immune system at the cellular as well as molecular level that are believed to play a key role in reactogenicity. We discuss evidence obtained from observations in vitro, in vivo in animal models and from clinical applications, including adjuvants used in large scale vaccination today. The data discussed are mainly taken from animal models following hyperstimulation of the immune system; either by the use of very powerful adjuvants, like Freund's that are too toxic for use in practical vaccination, by deliberate high dose application of adjuvants or by the in vivo application of cytokines. Although such hyperstimulating regimens are unlikely to find their way into practical vaccination of humans, this information is of great value as it may facilitate the understanding of the toxicity mechanisms, aid the design of standardised models for the assessment of adjuvant safety and the possible application of new adjuvants in vaccines for humans.
The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins... more The Major Outer Membrane Protein (MOMP) belongs to the membrane complex of cysteine-rich proteins of Chlamydophila pneumoniae. Although MOMP can elicit strong immune responses it fails to confer long-term protection against infection in animal models. This effect has been attributed, at least in part, to an inadequate induction of protective Th1-mediated immune responses. In an effort to understand the cellular mechanisms associated to the immunomodulatory properties of MOMP we studied the effect of this protein on mouse macrophages and naïve T-lymphocytes. We found that incubation of mouse macrophages with recombinant MOMP (rMOMP) results in an increased secretion of MMP-9 and a down-regulation of MHC class II, CD86 and CD40. This was accompanied by an increase in IL-10 and IFNgamma but not in IL-12 secretion. rMOMP induced a down-regulation of the expression of CD69 and CD154 markers by activated CD4(+) T cells, and enhanced the secretion of IL-2 and IL-10 by these cells. Conversely, rMOMP-treated macrophages up-regulated the expression of CD69 but not CD154, inhibited the synthesis of IL-10 and up-regulated the production of IFNgamma by activated CD8(+) T cells. Immunization of mice with MOMP induced the synthesis only of MOMP-specific IgG1 but no differences in cytokine profile were observed compared to controls. Our results provide new evidence on the role of MOMP in modulating T cell-mediated immune responses.
As expression of Cxs in cells of the immune system increases upon cellular activation, we investi... more As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.
Toxicology letters, Jan 1, 2009
There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydroca... more There is experimental and epidemiological evidence demonstrating that polycyclic aromatic hydrocarbons (PAHs) are involved in the pathogenesis of cardiovascular diseases. However, heterocyclic amines (HAs), a class of carcinogenic compounds present in food, which share many biochemical features with PAHs, have not received much attention. Previous reports have shown that the heterocyclic amine 2-amino-9H-pyrido[2,3-b]indole (AalphaC) binds and metabolically affects endothelial cells in animal models suggesting a potential role in vascular remodeling. The present study investigates the effect of exposure to HAs on atherosclerotic plaque development in the apoE(-/-) mice. We observed that animals treated with AalphaC developed atherosclerotic lesions characterized by lower lipid content but richer in inflammatory cells and collagen content when compared with control animals. Moreover, atherosclerotic plaques from AalphaC-treated apoE(-/-) mice were also smaller with a marked reduction in the tunica media thickness. Furthermore, total cholesterol levels were significantly reduced in AalphaC-treated apoE(-/-) mice. In contrast to what has been previously reported for PAHs, we provide for the first time evidence that HAs may protect against cardiovascular disease by inducing stable atherosclerotic plaques and reducing circulating cholesterol levels. These results open new avenues to further investigate the role of these food-borne carcinogens in cardiovascular physiology and pathology.
Journal of …, Jan 1, 2007
Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary ly... more Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4+ Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.
…, Jan 1, 2008
Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalli... more Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti-inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte-conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40-CD40 ligand (CD40L) interactions were involved in T-cell-stimulated MMP secretion from macrophages. Under baseline conditions, MMP-2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP-9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP-2 and MMP-9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP-9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP-2 and MMP-9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40-CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.
Clinical and …, Jan 1, 2008
Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of monocytes... more Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of monocytes/macrophages, smooth muscle cells, and lymphocytes within the arterial wall in response to the release of proinflammatory molecules. Such accumulation results in the formation of the atherosclerotic plaque, which would eventually evolve to complications such as total artery occlusion, rupture, calcification, or aneurysm. Although the molecular mechanism responsible for the development of atherosclerosis is not completely understood, it is clear that the immune system plays a key role in the development of the atherosclerotic plaque and in its complications. There are multiple antigenic stimuli that have been associated with the pathogenesis of atherosclerosis. Most of these stimuli come from modified self-molecules such as oxidised low-density lipoproteins (oxLDLs), beta2glycoprotein1 (beta2GP1), lipoprotein a (LP(a)), heat shock proteins (HSPs), and protein components of the extracellular matrix such as collagen and fibrinogen in the form of advanced glycation-end (AGE) products. In addition, several foreign antigens including bacteria such as Porphyromonas gingivalis and Chlamydia pneumoniae and viruses such as enterovirus and cytomegalovirus have been associated with atherosclerosis as potentially causative or bystander participants, adding another level of complexity to the analysis of the pathophysiology of atherosclerosis. The present review summarises the most important scientific findings published within the last two decades on the importance of antigens, antigen stimulation, and adaptive immune responses in the development of atherosclerotic plaques.