Coadministration of antigen-conjugated and free CpG: effects of in vitro and in vivo interactions in a murine model.

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation.

Generation of gene-engineered chimeric DNA molecules for specific therapy of autoimmune diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis.

Analytical aspects of biosimilarity issues of protein drugs.

The term "biosimilar" is used to designate a follow-on biologic that meets extremely high standards for comparability or similarity to the originator biologic drug that is approved for use in the same indications. Use of biosimilar products has already decreased the cost of treatment in many regions of the world and now a regulatory pathway for approval of these products has been established both in US and in the EU. The analytical tests to demonstrate comparability and similarity of a biosimilar product to a reference drug with respect to protein content, activity, physiochemical integrity, stability, impurities and additives, as well as immunogenicity are discussed.

Modulation of the humoral immune response by targeting CD40 and FcγRII/III; delivery of soluble but not particulate antigen to CD40 enhances antibody responses with a Th1 bias.

Targeted delivery of antigen improves immunogenicity and can obviate the use of adjuvants. In addition to molecular targeting based on affinity interactions, particle-based antigen targeting to myeloid cells is also an efficient means to enhance immune responses. We compared the efficiency of targeting a model antigen, streptavidin, to CD40 and low affinity Fc gamma receptors II and III, either in a soluble or in a particulate form. Single chain fragments targeting these receptors were used to generate soluble tetramers with streptavidin or to decorate streptavidin coated nanobeads, and mice were immunized with the different formulations. Whereas particulate presentation of streptavidin enhanced total IgG1 and IgG2a levels, overall antigen specific antibody production increased in the case of targeted soluble antigen only, as assessed by reverse protein arrays and ELISPOT. In particular, soluble CD40 targeted antigen induced the strongest IgG2a responses, suggesting a Th1 bias compared to FcgammaRII/III targeting. Combined targeting to these receptors did not further increase immunogenicity. Thus, in our model, affinity targeting of soluble antigen to CD40 proved to be superior to particle-mediated delivery both in terms of antibody quantity and quality.

CD16/32-specific biotinylated 2.4G2 single-chain Fv complexed with avidin-FITC enhances FITC-specific humoral immune response in vivo in a CD16-dependent manner.

Fcgamma receptors (FcgammaRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcgammaRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcgammaRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcgammaRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcgammaRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin-FITC, and tested its effect on the FITC-hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11b(high) and CD11c(high) cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC-dextran) or TD (FITC-keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcgammaRIII might be a key mediator of this mechanism.

Modulation of immune response by combined targeting of complement receptors and low-affinity Fcgamma receptors.

Immune complexes (ICs) induce effective pathogen-specific innate and humoral immune response via immunecomplex-binding receptors, such as murine complement receptor type 1 and 2 (mCR1/2) and murine low-affinity Fc receptors for IgG (mFcgammaRII and III). The exact function of mCR1/2 in cooperation with mFcgammaRII/III in modulation of humoral immunity has not yet been adequately clarified. The aim of this study was to target these receptors by specific single-chain fragments of antibody (scFv), either individually or in combination, thus modelling the action of IC. For targeting, we used scFv derived from the well-characterized 7g6 and 2.4g2 monoclonal antibodies recognizing mCR1/2 and mFcgammaRII/III, respectively. These scFvs were monobiotinylated and conjugated to streptavidin or streptavidin-coated microspheres. Such complexes were investigated with respect to target receptor recognition and in vivo localization. Antibody response against the constructs was measured by ELISA and ELISPOT. Results show that targeting streptavidin complexes to mFcgammaRII/III induces stronger IgG1 response than targeting to mCR1/2 yet both strategies enhance the antibody response compared to the control group immunized with non-targeted peptide-streptavidin complexes. Moreover, the immunogenicity of coupled antigens increased using microspheres as carrier, instead of using soluble streptavidin. In summery, our in vivo experiments reveal that mFcgammaRII/III is more potent a target than CR1/2 and show that combined targeting of CR1/2 and FcgammaRII/III receptors does not result in cumulative enhancement of the antigen specific immune response. In addition, microparticle-mediated enhancement of immunization can be further improved by FcgammaRII/III targeting.

Two-dimensional immune profiles improve antigen microarray-based characterization of humoral immunity.

Antigen arrays are becoming widely used tools for the characterization of the complexity of humoral immune responses. Current antibody profiling techniques provide modest and indirect information about the effector functions of the antibodies that bind to particular antigens. Here we introduce an antigen array-based approach for obtaining immune profiles reflecting antibody functionality. This technology relies on the parallel measurement of antibody binding and complement activation by features of the array. By comparing sera from animals immunized against the same antigen under different conditions, we show that identifying the position of an antigen in a 2-D space, derived from antibody binding and complement deposition, permits distinction between immune profiles characterized by diverse antibody isotype distributions. Additionally, the technology provides a biologically interpretable graphical representation of the relationship between antigen and host. Our data suggest that 2-D immune profiling could enrich the data obtained from proteomic scale serum profiling studies.

Physiological up-regulation of inhibitory receptors Fc gamma RII and CR1 on memory B cells is lacking in SLE patients.

Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc gamma Rs and complement receptors. Dysregulation of these mechanisms have been implicated in conditions where IC concentrations reach pathological levels and inflict diseases, like systemic lupus erythematosus (SLE). Our aim was to compare distinct sub-populations of CD19(+) B cells of healthy individuals and SLE patients with regard to their expression of Fc gamma R type II (Fc gamma RII, CD32), complement receptor type 1 (CR1, CD35) and complement receptor type 2 (CR2, CD21) and sIgG/IgM. The following four groups of peripheral CD19(+) B cells were investigated: IgM(+)/CD27(-) naive, IgM(+)/CD27(+) and IgM(-)/CD27(+) memory cells and CD27(high) plasmablasts. We demonstrate that the expression of the inhibitory receptors Fc gamma RII and CR1 is up-regulated on peripheral memory B cells of healthy controls, whereas this up-regulation is considerably impaired on the memory B cells of SLE patients. This reduction affects both the IgM(+) and switched memory B cells. We found a striking difference between the expression of complement receptors CD21 and CD35; namely, no up-regulation of CD21 occurred on the memory B cells of healthy donors, and its decreased expression in SLE patients was characteristic for both the CD27(-) naive and the CD27(+) memory B-cell populations. Our results clearly demonstrate that the previously reported reduced expression of IC-binding receptors is mainly due to the disturbed memory compartment; however, the higher frequency of CD19(+)/CD27(high)/sIg(low) plasmablasts expressing minimal levels of these receptors also contributes to this diminution.

Murine CR1/2 targeted antigenized single-chain antibody fragments induce transient low affinity antibodies and negatively influence an ongoing immune response.

We have generated a single-chain antibody which recognizes murine CR1/2 and carries a genetically fused influenza hemagglutinin derived peptide. Theoretically such a construct is able to crosslink the B cell antigen receptor and CR1/2 on peptide specific B cells. The construct was able to reach its CR1/2 positive target cells, yet intraperitoneal delivery of the construct elicited an IgM response only slightly exceeding that induced by the free peptide. Providing T cell help by the injection of peptide specific lymphocytes did not alter the response in essence, that is anti-peptide IgG was not detectable even after booster immunizations. When used as a booster vaccine following injection of the peptide in adjuvant, the construct even inhibited the development of IgG1 and IgG3 anti-peptide antibodies. These data indicate that although targeting of antigen to CR1/2 on B cells can enhance transient proliferation or differentiation of antigen specific B cells it cannot induce strong, longlasting humoral immune responses. Furthermore, CR1/2 targeting constructs may negatively influence an ongoing immune reaction.

Altered expression of Fcgamma and complement receptors on B cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by B cell hyper-reactivity, autoantibody production, immune complex (IC) deposition, and multiple organ damage. The contribution of IC and B cell-mediated changes in the pathogenesis of SLE is well established, however, the exact role of IC-binding receptors expressed on B cells, Fcgamma receptors, and complement receptors CR1 and CR2 in these pathological processes is unclear. Development of lupus-like symptoms in mice defective for the inhibitory Fc-gammaRIIb and genetic association of certain FcgammaR genes with SLE demonstrate a significant role for these receptors but reports indicating alterations of Fcgamma or complement receptor-mediated B cell functions in human SLE are relatively few. The present review highlights a selected set of data including our own discussing the significance of animal models, genetics, and functional alterations of these IC-binding receptors in the etiopathogenesis of SLE.

On-chip complement activation adds an extra dimension to antigen microarrays.

Antibody profiling on antigen microarrays helps us in understanding the complexity of responses of the adaptive immune system. The technique, however, neglects another, evolutionarily more ancient apparatus, the complement system, which is capable of both recognizing and eliminating antigen and serves to provide innate defense for the organism while cooperating with antibodies on multiple levels. Complement components interact with both foreign substances and self molecules, including antibodies, and initiate a cascade of proteolytic cleavages that lead to the covalent attachment of complement components to molecules in nanometer proximity. By refining the conditions of antibody profiling on antigen arrays we made use of this molecular tagging to identify antigens that activate the complement system. Antigen arrays were incubated with serum under conditions that favor complement activation, and the deposited complement C3 fragments were detected by fluorescently labeled antibodies. We used genetically C3-deficient mice or inhibition of the complement cascade to prove that the technique requires complement activation for the binding of C3 to features of the array. We demonstrate that antigens on the array can initiate complement activation both by antibody-dependent or -independent ways. Using two-color detection, antibody and complement binding to the relevant spots was measured simultaneously. The effect of adjuvants on the quality of the immune response and binding of autoantibodies to DNA with concomitant complement activation in the serum of mice suffering from systemic autoimmune disease was readily measurable by this new method. We propose that measurement of complement deposition on antigen microarrays supplements information from antibody binding measurements and provides an extra, immune function-related fingerprint of the tested serum.