BackgroundFor most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases.MethodsIn this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses.ResultsOf the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent.ConclusionsThis antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.

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A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG 1 , IgG 2a , and IgG 2b switch variants of murine monoclonal antibody (mAb) 3E5 IgG 3 with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.Antibodies are heterodimeric proteins composed of two heavy (H) 2 and two light (L) chains. Each H and L chain contains a V domain that when combined defines the antigen (Ag) binding site. In the mammalian genome, there are five major classes of heavy constant (C H ) domains that determine Ab isotype. These are usually assumed to have effector functions without directly affecting the Ag binding affinity and/or specificity. However, the C H domains are known to contribute to the apparent affinity through avidity effects that result from the polyvalent nature of certain immunoglobulins (Ig) classes. Hence, the classical view of Ig function is that the V domains are solely responsible for Ab affinity and specificity, whereas the C region is responsible for the biological properties such as complement activation, Fc receptor binding, avidity, and serum half-life (1). This concept posits that when B cells switch from one C H region to another, the avidity and effector functions of an Ab change without altering the specificity for the antigen (2). However, V region-identical Abs have been reported to manifest differences in the magnitude of binding to Ag, fine specificity, and idiotypic (Id) recognition (3-7), thus challenging this central immunological dogma.A critical condition for Ab-Ag binding is the formation of a specific complex between the Ab and the Ag. Understanding the interaction of these two biological macromolecules requires detailed knowledge of the structure and functional characteristics of the complex. The structure of the Ab-Ag complex can be described using x-ray crystallography and computer-generated structural models. The functional activity can be described by the kinetic rate constants (k on/off ), equilibrium constants (K), and thermodynamic parameters of the binding complex. The equilibrium constant is related...

Ab specificity is determined by V region sequence. The murine Mab 18B7 (IgG1) binds to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan and produces annular immunofluorescence (IF) on yeast cells. The heavy and light V regions of 18B7 were expressed with the human C regions μ, γ1, γ2, γ3, γ4, and α1, and the specificity and binding properties of these mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2, chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3 produced punctate IF, despite identical V region sequences. Competition experiments with murine Abs that competed with mAb 18B7 and binding assays to peptide mimetics of glucuronoxylomannan provided additional evidence for altered specificity in some of the ch Abs. Expression of 18B7 heavy V region with murine μ C region produced IgM with a punctate IF, indicating that a change in fine specificity also accompanied the change from murine IgG1 to IgM. Our results show that Ab fine specificity can be a function of isotype. This phenomenon may be most apparent for Abs that bind to Ag with repeating epitopes, such as polysaccharides, where the quarternary structure of the Ag-Ab complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc interactions, Ab size, and solvent accessibility to exposed surfaces. Alterations in Ab fine specificity following isotype change could have important implications for current concepts on the generation of secondary Ab responses to certain Ags and for the isotype preference observed in Abs to polysaccharides.

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.

SUMMARYGerm-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T-or B-celldepleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-g , IL-12, TNF, IL-10 and TGF-b production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-g , IL-12 and TNF than did non-TG MLN cells . In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-b . T cell depletion abolished IFN-g and decreased IL-12 production, but did not affect IL-10 and TGF-b production. Conversely, neither IL-10 nor TGF-b was produced in cultures of B cell-depleted MLN. In addition, CD4 + T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-g when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-b , but not IFN-g , IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-g -producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.

The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9 -2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.

The present results suggest that the antimicrobial efficacy of RIT involves killing through promotion of fungal cell apoptosis-like death, reduction in yeast capsule size, cooperation with macrophages, and modulation of the inflammatory response.