Ernesto Oviedo-Orta

Progress in understanding adjuvant immunotoxicity mechanisms

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 Freunds 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.

Gap Junctions and Connexin Hemichannels Underpin Hemostasis and Thrombosis

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.

Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages.

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.

Control of the proliferation of activated CD4+ T cells by connexins

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.

Antigen-induced immunomodulation in the pathogenesis of atherosclerosis

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 (β2GP1), 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.

CD4+ T lymphocyte subsets express connexin 43 and establish gap junction channel communication with macrophages in vitro

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.

Connexin40 regulates platelet function

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 37,43Gap27 on Cx40−/− mouse platelets and of the Cx40 inhibitor 40Gap27 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.

The effect of Chlamydophila pneumoniae Major Outer Membrane Protein (MOMP) on macrophage and T cell-mediated immune responses

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 IFNγ 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 IFNγ 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.

Influenza vaccination promotes stable atherosclerotic plaques in apoE knockout mice.

OBJECTIVE Current evidence suggests a relationship between seasonal Influenza viral infection and cardiovascular disease (CVD). Experimental animals inoculated with Influenza A virus have shown to develop thrombotic complications similar to those seen in humans. Conversely, several epidemiological studies and clinical trials have suggested that Influenza vaccination may have a protective effect on CVD. However, the potential mechanisms behind this protective effect remain unstudied. We aimed to study the effect of Influenza vaccination on atherosclerotic plaque development in apoE(-/-) mice. METHODS AND RESULTS The effect of immunization with increasing doses of Influenza vaccine (0.38, 1.8, 9 and 45 μg/0.5 mL Vaxigrip®, Sanofi-Aventis) on atherogenesis was compared with that of animals immunized with Pneumo23® (pneumococcus vaccine, Sanofi-Aventis) and control group inoculated with phosphate buffered saline (PBS). Animals vaccinated with 45 μg/0.5 mL Vaxigrip®, (the same dose used to immunize humans adults against Influenza) developed smaller atherosclerotic lesions with lower lipid content but richer in smooth muscle cells and collagen when compared with control animals. Concomitantly, they showed lower levels of interferon gamma (IFNγ), interleukin (IL)-2 and tumor necrosis factor alpha (TNFα) but higher levels of IL-4. Furthermore, we found increased levels of anti-Influenza immunoglobulin (Ig) G1 or anti-Pneumo23® IgM specific antibodies in a time and dose dependent fashion in animals immunized with these vaccines. CONCLUSIONS These results indicate that vaccination against Influenza may protect against the development of CVD by promoting smaller and stable atherosclerotic plaques and by inducing atheroprotective immune responses.

Design and evaluation of the immunogenicity and efficacy of a biomimetic particulate formulation of viral antigens

Subunit viral vaccines are typically not as efficient as live attenuated or inactivated vaccines at inducing protective immune responses. This paper describes an alternative ‘biomimetic’ technology; whereby viral antigens were formulated around a polymeric shell in a rationally arranged fashion with a surface glycoprotein coated on to the surface and non-structural antigen and adjuvant encapsulated. We evaluated this model using BVDV E2 and NS3 proteins formulated in poly-(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles adjuvanted with polyinosinic:polycytidylic acid (poly(I:C) as an adjuvant (Vaccine-NP). This Vaccine-NP was compared to ovalbumin and poly(I:C) formulated in a similar manner (Control-NP) and a commercial adjuvanted inactivated BVDV vaccine (IAV), all inoculated subcutaneously and boosted prior to BVDV-1 challenge. Significant virus-neutralizing activity, and E2 and NS3 specific antibodies were observed in both Vaccine-NP and IAV groups following the booster immunisation. IFN-γ responses were observed in ex vivo PBMC stimulated with E2 and NS3 proteins in both vaccinated groups. We observed that the protection afforded by the particulate vaccine was comparable to the licenced IAV formulation. In conclusion, the biomimetic particulates showed a promising immunogenicity and efficacy profile that may be improved by virtue of being a customisable mode of delivery.