Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

How a different look at latency can help to develop novel diagnostics and vaccines against tuberculosis.

Mycobacterium tuberculosis is one of the most successful human pathogens. It kills every year approximately 1.5 - 2 million people, and at present a third of the human population is estimated to be infected. Fortunately, only a relatively small proportion of the infected individuals will progress to active disease, and most will maintain a latent infection. Although a latent infection is clinically silent and not contagious, it can reactivate to cause highly contagious pulmonary tuberculosis, the most prevalent form of the disease in adults. Therefore, a thorough understanding of latency and reactivation may help to develop novel control strategies against tuberculosis. The most widely held view is that the mycobacteria are imprisoned in granulomatous structures during latency, where they can survive in a non-replicating, dormant form until reactivation occurs. However, there is no hard data to sustain that the reactivating mycobacteria are indeed those that laid dormant within the granulomas. In this review an alternative model, based on evidence from early studies, as well as recent reports is presented, in which the latent mycobacteria reside outside granulomas, within non-macrophage cell types throughout the infected body. Potential implications for new diagnostic and vaccine design are discussed.

Risk stratification of latent tuberculosis defined by combined interferon gamma release assays.

Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA).

Antigen-specific interferon-gamma responses and innate cytokine balance in TB-IRIS.

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune response to TB-antigens. We compared the recovery of IFNγ responses to recall and TB-antigens and explored in vitro innate cytokine production in TB-IRIS patients.

The nature of innate and adaptive interleukin-17A responses in sham or bacterial inoculation

Streptococcus pyogenes is the causative agent of numerous diseases ranging from benign infections (pharyngitis and impetigo) to severe infections associated with high mortality (necrotizing fasciitis and bacterial sepsis). As with other bacterial infections, there is considerable interest in characterizing the contribution of interleukin-17A (IL-17A) responses to protective immunity. We here show significant il17a up-regulation by quantitative real-time PCR in secondary lymphoid organs, correlating with increased protein levels in the serum within a short time of S. pyogenes infection. However, our data offer an important caveat to studies of IL-17A responsiveness following antigen inoculation, because enhanced levels of IL-17A were also detected in the serum of sham-infected mice, indicating that inoculation trauma alone can stimulate the production of this cytokine. This highlights the potency and speed of innate IL-17A immune responses after inoculation and the importance of proper and appropriate controls in comparative analysis of immune responses observed during microbial infection.

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-canies vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, G8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG 2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Selective defect of anti-pneumococcal IgG in a patient with persistent polyclonal B cell lymphocytosis.

BACKGROUND: Persistent polyclonal B cell lymphocytosis (PPBL) is a rare condition characterized by increased IgM and large excess of B cells with an IgD(+) CD27(+) phenotype. In normal individuals, these cells play a central role in the defense against pneumococcal infection. So far, few studies have characterized humoral immune responses in PPBL patients. We therefore measured IgG directed against S. pneumoniae antigens in a 51 yr-old woman with PPBL before and after vaccination with a pneumococcal 23-valent polysaccharide vaccine. METHODS: Antibodies against pneumococcal antigens were measured first with an overall immunoassay using microplates coated with the 23-valent pneumococcal vaccine. A serotype-specific test was also performed according to the WHO consensus protocol. RESULTS: Despite a large number of IgD(+) CD27(+) cells, our patient had low baseline titers of IgG directed against pneumococcal antigens and did not significantly respond to a 23-valent polysaccharide vaccine against S. pneumoniae. On the contrary, she had good titers of IgG directed against tetanus toxoid. CONCLUSION: IgM(+) IgD(+) CD27(+) cells which accumulate in this patient with typical PPBL patient failed to perform IgG isotype switch after a polysaccharide vaccine. The potential mechanisms and relationships with the main features of PPBL are discussed. Further studies on a larger number of similar patients are needed.

Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses.

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.

Modulation of the infant immune responses by the first pertussis vaccine administrations.

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis-tetanus-diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-gamma in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

Monomerization of dimeric IgG of intravenous immunoglobulin (IVIg) increases the antibody reactivity against intracellular antigens

Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens.