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Introduction: Malaria remains a significant burden, and a fully protective vaccine against Plasmodium falciparum is critical for reducing morbidity and mortality. Antibody responses against the blood-stage antigen Merozoite Surface Protein 2 (MSP2) are associated with protection from P. falciparum malaria, but its extensive polymorphism is a barrier to its development as a vaccine candidate. New tools, such as long-read sequencing and accurate protein structure modelling allow us to study the genetic diversity and immune responses towards antigens from clinical isolates with unprecedented detail. This study sought to better understand naturally acquired MSP2-specific antibody responses. Methods: IgG responses against recombinantly expressed full-length, central polymorphic regions, and peptides derived from the conserved termini of MSP2 variants sequenced from patient isolates, were tested in plasma from travelers with recent, acute malaria and from individuals living in an endemic area of Tanzania. Results: IgG responses towards full MSP2 and truncated MSP2 antigens were variant specific. IgG antibodies in the plasma of first-time infected or previously exposed travelers did not recognize the conserved termini of expressed MSP2 variants by ELISA, but they bound 13-amino acid long linear epitopes from the termini in a custom-made peptide array. Alphafold3 modelling suggests extensive structural heterogeneity in the conserved termini upon antigen oligomerization. IgG from individuals living in an endemic region, many who were asymptomatically infected, did not recognize the conserved termini by ELISA. Discussion: Our results suggest that responses to the variable regions are critical for the development of naturally acquired immunity towards MSP2. [ABSTRACT FROM AUTHOR]

Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles. Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13. Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations. Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified. [ABSTRACT FROM AUTHOR]

Identifying the mechanism of naturally acquired immunity against Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens (MSP-119, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria. [ABSTRACT FROM AUTHOR]

A new expression system involving Escherichia coli-Staphylococcus shuttle vector pSPPmABPXM help target heterologous recombinant peptides on Staphylococcus carnosus cell surfaces. A Staphylococcus hyicus lipase polypeptide enables the release of gene fusion products from the cell surface. An LPXTGX motif-containing receptor region, which also contains a charged tail and an hydrophobic C-terminus, and a charged repetitive region X are essential for cell surface anchoring.

Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four Plasmodium falciparum blood-stage antigens, MSP-119, MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria. [ABSTRACT FROM AUTHOR]

Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5mAbs in each were defined; 2mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. [ABSTRACT FROM AUTHOR]

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of doublesecreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease. [ABSTRACT FROM AUTHOR]

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8–87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases. [ABSTRACT FROM AUTHOR]

Background: Atopic eczema is a chronic inflammatory skin disease in which several subgroups of cases can be identified. Atopy patch testing (APT) reveals allergen sensitization also in atopic eczema patients devoid of detectable allergen-specific IgE, suggesting the importance of factors other than IgE in the reaction. Here we investigate the relationship between APT reactions and allergen-specific peripheral IgE and T cell reactivity in atopic eczema patients. Methods: Adult patients with atopic eczema (n = 64) and healthy controls (n = 24) were analyzed for reactivity to Malassezia sympodialis extract by APT, measurement of specific plasma IgE and in vitro determination of the frequency of allergen-reactive peripheral blood mononuclear cells producing interleukin-4 and interleukin-5 using the ELISpot method. Results: When combining the results of the APT, IgE measurements and the ELISpot analyses, reactivity to M. sympodialis was found in a majority of the atopic eczema patients (69%), whereas the healthy controls were negative throughout. T cell reactivity to M. sympodialis, manifested by production of both interleukins 4 and 5, was highly predictive for a positive APT reaction and displayed a strongly positive correlation with the APT score. In contrast, the allergen-specific IgE levels did not predict the APT outcome, and no correlation could be found between the IgE levels and the APT score. Conclusion: Peripheral allergen-specific T helper 2 cell-mediated reactivity appears to be required for a positive APT reaction to M. sympodialis. The diagnostic potential of measuring peripheral allergen-specific T cell responses should be considered in atopic eczema. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Detection of cytokines secreted by ex vivo antigen-stimulated peripheral blood mononuclear cells (PBMC) by ELISA is hampered by low frequencies of specific T cells and cellular receptor consumption. We investigated if ELISpot, measuring cytokine production at the single cell level, facilitated a better detection of the Th2 cytokines IL-4 and IL-5. PBMC from nickel-allergic (n = 31) and non-allergic subjects (n = 10) were stimulated with nickel or tetanus toxoid (TT) and cytokine production assessed by ELISpot and ELISA. By IL-4 ELISpot, 74% of the allergic and 0% control subjects responded to nickel and 56% of all subjects to TT. ELISA detected IL-4 after nickel stimulation only in 13% of the allergic subjects. Also detection of TT-induced IL-5 was improved by ELISpot with 54% subjects responding versus 24% in ELISA. In contrast, detection of nickel-induced IL-5 was more comparable between methods, most likely due to the 7-fold higher IL-5 production per cell in response to nickel versus TT. The low IL-5 response to TT was associated with a higher induction of the down-regulatory cytokine IL-10 by TT as compared to nickel (p < 0.001). Overall, ELISpot displayed a better detection of IL-4 as well as low intensity IL-5 responses thus emphasizing the importance of selecting suitable methods for the measurement of cytokine production ex vivo. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family. [Copyright &y& Elsevier]