Niklas Ahlborg

Nickel, cobalt, chromium, palladium and gold induce a mixed Th1- and Th2-type cytokine response in vitro in subjects with contact allergy to the respective metals.

Nickel (Ni), the main cause of contact allergy to metals, induces in vitro production of both Th1‐ and Th2‐type cytokines in peripheral blood mononuclear cells (PBMC) from allergic subjects. Because the knowledge of the cellular immune response to other metals involved in contact allergy has been limited, we investigated the cytokine profile induced by Ni, cobalt (Co), chromium (Cr), palladium (Pd) and gold (Au) in PBMC from patients with patch test reactivity to the respective metals. PBMC from patients with patch test reactivity to Ni, Co, Cr, Au and/or Pd (n = 31) and non‐allergic controls (n = 5) were stimulated in vitro with corresponding metal salts. Th1‐ [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2‐ (IL‐4 and IL‐13) type cytokine responses were measured by enzyme‐linked immunospot (ELISpot) and/or enzyme‐linked immunosorbent assay (ELISA). All metals induced a mixed Th1‐ and Th2‐type cytokine production in PBMC from individual patients with patch test reactivity to the corresponding metal, but not in control PBMC. Significantly higher responses in the patient versus controls were found for Cr (IL‐2 and IL‐13), Pd (IL‐2 and IL‐4), Au (IL‐13 and IFN‐γ) (all P < 0·05) and Ni (all four cytokines; P < 0·01) but not Co. Overall, 71% (37/52) and 89% (81/91) of the positive and negative patch test reactivities to metals, respectively, were matched by the in vitro reactivity. In conclusion, our data suggest that sensitization to Co, Cr, Pd and Au results in a cellular immune response of a character similar to the mixed Th1‐ and Th2‐type cytokine profile shown previously to be induced by Ni.

Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis

Background Patients with suspected allergic contact dermatitis still have to undergo patch testing for a correct diagnosis. As this has several disadvantages there is a need for additional methods, preferentially those that can be performed in vitro.

Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses

B-cell responses after infection or vaccination are often measured as serum titers of antigen-specific antibodies. Since this does not address the aspect of memory B-cell activity, it may not give a complete picture of the B-cell response. Analysis of memory B cells by ELISpot is therefore an important complement to conventional serology. B-cell ELISpot was developed more than 25 years ago and many assay protocols/reagents would benefit from optimization. We therefore aimed at developing an optimized B-cell ELISpot for the analysis of vaccine-induced human IgG-secreting memory B cells. A protocol was developed based on new monoclonal antibodies to human IgG and biotin-avidin amplification to increase the sensitivity. After comparison of various compounds commonly used to in vitro-activate memory B cells for ELISpot analysis, the TLR agonist R848 plus interleukin (IL)-2 was selected as the most efficient activator combination. The new protocol was subsequently compared to an established protocol, previously used in vaccine studies, based on polyclonal antibodies without biotin avidin amplification and activation of memory B-cells using a mix of antigen, CpG, IL-2 and IL-10. The new protocol displayed significantly better detection sensitivity, shortened the incubation time needed for the activation of memory B cells and reduced the amount of antigen required for the assay. The functionality of the new protocol was confirmed by analyzing specific memory B cells to five different antigens, induced in a limited number of subjects vaccinated against tetanus, diphtheria and pertussis. The limited number of subjects did not allow for a direct comparison with other vaccine studies. Optimization of the B-cell ELISpot will facilitate an improved analysis of IgG-secreting B cells in vaccine studies.

Differential induction of immunoglobulin G subclasses by immunization with DNA vectors containing or lacking a signal sequence

The route and method used to immunize mice with antigen-expressing DNA plasmids have an impact on the resulting T-helper cell response and IgG subclass distribution. Previous findings further indicate that the intracellular targeting of expressed antigens influences the differentiation of naive T-cells into either a Th1 or a Th2 type of response. In the present study, we analyzed the levels of IgG1 and IgG2a antibodies, as correlates of Th2 and Th1 responses, respectively, after intramuscular injection of mice with plasmids encoding a chimeric protein containing a Plasmodium falciparum blood stage antigen expressed in two different forms. One plasmid expresses the antigen in a secreted form as it is preceded by a signal sequence while expression from the other plasmid, lacking this sequence, results in cytoplasmic localization of the antigen. Mice immunized with the plasmid encoding secreted antigen responded with predominantly IgG1 antibodies. In contrast, sera from mice immunized with the plasmid expressing cytosolic protein displayed a mixed IgG1/IgG2a profile. In line with previous findings, our results suggest that the intracellular targeting of proteins expressed by DNA plasmids is an important factor for the differentiation of Th cells and the resulting subclass pattern of IgG responses.

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.

Nickel Elicits Concomitant and Correlated in vitro Production of Th1‐, Th2‐Type and Regulatory Cytokines in Subjects with Contact Allergy to Nickel

Nickel (Ni2+) elicits production of functionally distinct cytokines in vitro, but the relation between the cytokine profile and the degree of the allergic reaction in vivo needs to be better defined in order to improve the understanding of the immunological mechanisms involved in contact allergy and to facilitate development of in vitro diagnostics. The aim of the study was to define Th1‐type [interferon‐γ (IFN‐γ)], Th2‐type [interleukin‐4 (IL‐4), IL‐5 and IL‐13] and regulatory (IL‐10) cytokine responses to Ni2+ in peripheral blood mononuclear cells (PBMC) from subjects with varying patch test reactivity to Ni2+. The study included subjects with strong (+3), moderate (+2), weak (+1) or negative (controls) patch test reactivity to Ni2+ (n = 10 per group). All +3 and +2 subjects but only three +1 subjects had a clinical history of contact allergy to Ni2+. Cytokine production of PBMC stimulated with Ni2+ was determined by enzyme‐linked immunospot and/or enzyme‐linked immunosorbent assay. Ni2+ elicited significant production of all cytokines in PBMC from patch‐test‐positive subjects versus controls with a positive correlation between each cytokine and the patch test reactivity as well as with other cytokines. More subjects responded to Ni2+ above cut‐off values with Th2‐type cytokines as compared with IFN‐γ or IL‐10; 100% of +3, 80% of +2, 50% of +1 and 0% of control subjects displayed reactivity to Ni2+ based on IL‐4 and IL‐13 assays. Despite the prevailing view of Ni2+ allergy as a type‐1‐mediated condition, the in vivo reactivity to Ni2+ correlated with a mixed Th1‐type, Th2‐type and regulatory cytokine response to Ni2+in vitro. The results accentuate the importance of type 2 responses in contact allergy and also demonstrate that IL‐4 and IL‐13 are reliable markers for Ni2+ allergy.

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1‐ and Th2‐type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL‐10 is less clear. We therefore investigated the impact of IL‐10 on Ni‐induced Th1‐ (IFN‐γ) and Th2‐type (IL‐4 and IL‐13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni‐PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine‐producing cells and the levels of secreted cytokines were analysed by ELISpot (IL‐4, IL‐13 and IFN‐γ) and ELISA (IL‐10, IL‐13 and IFN‐γ), respectively. The Ni‐induced response was further assessed in the presence of recombinant IL‐10 (rIL‐10) or neutralizing antibody to IL‐10 and the phenotype of the Ni‐specific cytokine‐producing cells regulated by IL‐10 was determined by cell depletion experiments. Ni induced IL‐10 production in Ni‐PBMC (mean, (range); 33·1 pg/ml (0–93·4 pg/ml)) but not control PBMC (2·2 pg/ml (0–14·9 pg/ml)) (P = 0·002). Ni also induced significant production of IL‐4, IL‐13 and IFN‐γ that correlated with the IL‐10 response. Addition of rIL‐10 down‐regulated the Ni‐induced production of all cytokines but with a more pronounced effect on IFN‐γ. However, neutralization of Ni‐induced IL‐10 enhanced the levels of IFN‐γ induced by Ni (P = 0·004) but did not affect the number of IFN‐γ‐producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni‐specific IFN‐γ (and Th2‐type cytokine) producing cells were CD4+ T cells. The impact of IL‐10 on Ni‐induced IFN‐γ responses by CD4+ T cells suggests that an important role of IL‐10 in vivo is to counteract the allergic reactions mediated by Th1‐type cytokines.

ELISpot and ELISA analysis of spontaneous, mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macaques

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.

Immunogens containing sequences from antigen Pf332 induce Plasmodium falciparum-reactive antibodies which inhibit parasite growth but not cytoadherence.

Immunogens based upon sequences from the P. falciparum asexual blood stage antigen Pf332 were assessed for their capacity to induce antibodies inhibiting parasite growth or cytoadherence of infected erythrocytes in vitro. Selection of the Pf332 sequences was based on their reactivity with the human monoclonal antibody (MoAb) 33G2 which inhibits parasite growth as well as cytoadherence in vitro. Octameric multiple antigen peptides (MAP) were assembled based upon either a trimer of the minimal epitope recognized by the MoAb, VTEEI, or a Pf332 sequence including that motif, SVTEE1AEEDK. A dimer of SVTEEIAEEDK was also expressed in Escherichia coli, genetically fused to ZZ, two IgG‐binding domains of staphylococcal protein A. Rabbit antibodies elicited by the immunogens reacted with Pf332 in immunofluorescence and in ELISA with Pf332 peptides which were also recognized by MoAb 33G2. The MAP with branched (VTEEI)3 peptide induced the highest litres of P. falci‐pamm‐reactive antibodies. In contrast to MoAb 33G2, none of the polyclonal Pf332 reactive sera cross‐reacted with repeat sequences of the malaria antigen Pf155jRESA. The polyclonal Pf332‐reactive antibodies inhibited parasite growth efficiently but had no or very low inhibitory effect in a cytoadherence assay. Thus, while Pf332 may be an important target for parasite neutralizing antibodies its involvement in cytoadherence is unclear.

Synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using Fmoc chemistry

Multiple antigen peptides (MAP) consist of lysine residue cores with branching peptide arms and have been demonstrated to be efficient immunogens as well as useful antigens for ELISA. Synthesis of diepitope MAPs with two different branching peptides has previously been described using combined Boc and Fmoc chemistry. Here, the synthesis of a tetrameric diepitope MAP based on Fmoc chemistry is described. A lysine core was synthesized with N alpha- and N epsilon-amino groups othogonally protected by Fmoc and a recently described protection group, Dde, respectively. On the N alpha-amino groups, a sequence from the Plasmodium falciparum antigen Pf332 was synthesized with a capped N-terminus. After removal of Dde, a sequence from the P. falciparum circumsporozoite protein was synthesized on the core. Amino acid analysis of the MAP displayed equimolar amounts of the two peptide sequences, indicating the reliability of the protection group Dde. In ELISA, antibodies specific for either of the two malarial sequences reacted with the MAP. The major advantages of this approach for synthesis of diepitope MAPs are that only a panel of Fmoc-amino acid derivatives is required and that the more complicated cleavage procedure for Boc chemistry can be avoided.