339 research outputs found
Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens
Background
Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assays are not widely available.
Methods
We developed six quantitative suspension array assays to measure IgG1, IgG2, IgG3, IgG4, IgM and IgE specific to multiple P. falciparum antigens. Secondary and tertiary antibodies, as well as human purified antibodies for standard curves, were tested among several commercially available sources. Positive and negative controls included plasmas from malaria hyper-immune African adults and from malaria-naïve European adults, respectively. Reagents were selected and optimal antibody and test sample dilutions established according to sensitivity, specificity and performance of the standard curves. The variability between replicates and plates was assessed with 30 test samples and controls.
Results
Assays were able to detect P. falciparum antigen-specific antibodies for all isotypes and subclasses in samples from malaria-exposed individuals, with low background signal in blank wells. Levels detected in malaria-naïve individuals were overall low except for IgM. For the IgG2 and IgE assays, a triple sandwich was required for sensitivity. Standard curves with 5-parameter logistic fit were successfully obtained in all assays. The coefficients of variation for measurements performed in different days were all <30%, and <5% when comparing duplicates from the same plate.
Conclusion
The isotype/subclass assays developed here were sensitive, specific, reproducible and of adequate quantification dynamic range. They allow performing detailed immuno-profiling to large panels of P. falciparum antigens to address naturally- and vaccine-induced Ig responses and elucidate correlates of malaria protection, and could also be applied to other antigenic panels
Chronic Exposure to Malaria Is Associated with Inhibitory and Activation Markers on Atypical Memory B Cells and Marginal Zone-Like B Cells
In persistent infections that are accompanied by chronic immune activation, such as human immunodeficiency virus, hepatitis C virus, and malaria, there is an increased frequency of a phenotypically distinct subset of memory B cells lacking the classic memory marker CD27 and showing a reduced capacity to produce antibodies. However, critical knowledge gaps remain on specific B cell changes and immune adaptation in chronic infections. We hypothesized that expansion of atypical memory B cells (aMBCs) and reduction of activated peripheral marginal zone (MZ)-like B cells in constantly exposed individuals might be accompanied by phenotypic changes that would confer a tolerogenic profile, helping to establish tolerance to infections. To better understand malaria-associated phenotypic abnormalities on B cells, we analyzed peripheral blood mononuclear cells from 55 pregnant women living in a malaria-endemic area of Papua Nueva Guinea and 9 Spanish malaria-naïve individuals using four 11-color flow cytometry panels. We assessed the expression of markers of B cell specificity (IgG and IgM), activation (CD40, CD80, CD86, b220, TACI, and CD150), inhibition (PD1, CD95, and CD71), and migration (CCR3, CXCR3, and CD62l). We found higher frequencies of active and resting aMBC and marked reduction of MZ-like B cells, although changes in absolute cell counts could not be assessed. Highly exposed women had higher PD1+-, CD95+-, CD40+-, CD71+-, and CD80+-activated aMBC frequencies than non-exposed subjects. Malaria exposure increased frequencies of b220 and proapoptotic markers PD1 and CD95, and decreased expression of the activation marker TACI on MZ-like B cells. The increased frequencies of inhibitory and apoptotic markers on activated aMBCs and MZ-like B cells in malaria-exposed adults suggest an immune-homeostatic mechanism for maintaining B cell development and function while simultaneously downregulating hyperreactive B cells. This mechanism would keep the B cell activation threshold high enough to control infection but impaired enough to tolerate it, preventing systemic inflammation
Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne’s Disease using MAP Protein Microarrays
Considerable effort has been directed toward controlling Johne’s disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n = 30); negative high exposure (NH, n = 30); fecal- positive, ELISA-negative (F + E−, n = 60); and both fecal- and ELISA-positive (F + E+, n = 60). The analyses identified a total of 49 candidate antigens in the NH, F + E−, and F + E+ with reactivity compared with the NL group (p \u3c 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E− (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E−, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E− groups, have potential utility for the early sero-diagnosis of MAP infection
Early detection of Mycobacterium avium subsp. paratuberculosis infection in cattle with multiplex-bead based immunoassays
Johne’s Disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), results in significant economic loss to livestock production. The early detection of MAP infection in animals with extant serological assays has remained challenging due to the low sensitivity of commercially available ELISA tests, a fact that has hampered the development of effective JD control programs. Our recent protein microarray-based studies identified several promising candidate antigens that are immunogenic during different stages of MAP infection. To evaluate these antigens for use in diagnostic assays and reliably identify animals with MAP infection, a multiplex (Luminex®) assay was developed using color-coded flourescent beads coupled to 6 MAP recombinant proteins and applied to screen 180 serum and 90 milk samples from cows at different stages of MAP infection including negative (NL), fecal test positive/ELISA negative (F+E-), and fecal positive/ELISA positive (F+E+). The results show that while serum antibody reactivities to each of the 6 anti-gens were highest in F+E+ group, antibody reactivity to three of the six antigens were identified in the F+E- group, suggesting that these three antigens are expressed and provoke antibody responses during the early infection stages with MAP. Further, antibodies against all six antigens were elevated in milk samples from both the F+E- and F+E+ groups in comparison to the NL group (
Thermal Emission of WASP-14b Revealed with Three Spitzer Eclipses
Exoplanet WASP-14b is a highly irradiated, transiting hot Jupiter. Joshi et
al. calculate an equilibrium temperature Teq of 1866 K for zero albedo and
reemission from the entire planet, a mass of 7.3 +/- 0.5 Jupiter masses and a
radius of 1.28 +/- 0.08 Jupiter radii. Its mean density of 4.6 g/cm3 is one of
the highest known for planets with periods less than 3 days. We obtained three
secondary eclipse light curves with the Spitzer Space Telescope. The eclipse
depths from the best jointly fit model are +/- at 4.5
{\mu}m and +/- at 8.0 {\mu}m. The corresponding brightness
temperatures are 2212 +/- 94 K and 1590 +/- 116 K. A slight ambiguity between
systematic models suggests a conservative 3.6 {\mu}m eclipse depth of
+/- and brightness temperature of 2242 +/- 55 K. Although extremely
irradiated, WASP-14b does not show any distinct evidence of a thermal
inversion. In addition, the present data nominally favor models with day night
energy redistribution less than . The current data are generally
consistent with oxygen-rich as well as carbon-rich compositions, although an
oxygen-rich composition provides a marginally better fit. We confirm a
significant eccentricity of e = 0.087 +/- 0.002 and refine other orbital
parameters.Comment: 16 pages, 16 figure
Identification of Novel Seroreactive Antigens in Johne’s Disease Cattle by Using the Mycobacterium tuberculosis Protein Array
Johne’s disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. aviumsubsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing 75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis- infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having 70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne’s disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne’s disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne’s disease diagnostic assays
Genomic and panproteomic analysis of the development of infant immune responses to antigenically-diverse pneumococci
Streptococcus pneumoniae (pneumococcus) is a nasopharyngeal commensal and respiratory pathogen. This study characterises the immunoglobulin G (IgG) repertoire recognising pneumococci from birth to 24 months old (mo) in a prospectively-sampled cohort of 63 children using a panproteome array. IgG levels are highest at birth, due to transplacental transmission of maternal antibodies. The subsequent emergence of responses to individual antigens exhibit distinct kinetics across the cohort. Stable differences in the strength of individuals’ responses, correlating with maternal IgG concentrations, are established by 6 mo. By 12 mo, children develop unique antibody profiles that are boosted by re-exposure. However, some proteins only stimulate substantial responses in adults. Integrating genomic data on nasopharyngeal colonisation demonstrates rare pneumococcal antigens can elicit strong IgG levels post-exposure. Quantifying such responses to the diverse core loci (DCL) proteins is complicated by cross-immunity between variants. In particular, the conserved N terminus of DCL protein zinc metalloprotease B provokes the strongest early IgG responses. DCL proteins’ ability to inhibit mucosal immunity likely explains continued pneumococcal carriage despite hosts’ polyvalent antibody repertoire. Yet higher IgG levels are associated with reduced incidence, and severity, of pneumonia, demonstrating the importance of the heterogeneity in response strength and kinetics across antigens and individuals
Changing plasma cytokine, chemokine and growth factor profiles upon differing malaria transmission intensities
Background: Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence
of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions
and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or
tolerance that are afected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune
responses are responsible for the symptoms and pathological alterations during disease and are expected to change
rapidly upon malaria exposure or cessation.
Methods: The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from
a malaria endemic area of southern Mozambique were compared between 2 years of diferent MTI: 2010 (lower,
n=234) and 2013 (higher, n=143). The efect of the year on the correlations between cytokines, chemokines and
growth factors and IgGs to Plasmodium falciparum (markers of exposure) was explored. The efects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed.
Results: An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of
correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors,
regardless of their immune function, had higher concentrations in 2010 compared with 2013 in P. falciparum-infected
and uninfected subjects. Age and neighbourhood showed an efect on analyte concentrations.
Conclusions: The results show a diferent regulation of the cellular immune response in 2010 vs 2013 which could
be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune
mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance
Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity
Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes
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