Impact of human sequences in variable domains of therapeutic antibodies on the location of CD4 T-cell epitopes.
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ABSTRACT:
SUBMITTER: Meunier S
PROVIDER: S-EPMC7264247 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:T cells must play the major role in controlling acute human Lassa virus infection, because patients recover from acute Lassa fever in the absence of a measurable neutralizing antibody response. T cells alone seem to protect animals from a lethal Lassa virus challenge, because after experimental vaccination no neutralizing antibodies are detectable. In order to study human T-cell reactivity to single Lassa virus proteins, the nucleoprotein (NP) of Lassa virus, strain Josiah, was cloned, expressed in Escherichia coli, and affinity purified. Peripheral blood mononuclear cells (PBMC) obtained from 8 of 13 healthy, Lassa virus antibody-positive individuals living in the Republic of Guinea, western Africa, were found to proliferate in response to the recombinant protein (proliferation index >/=10). PBMC obtained from one individual with a particularly high proliferative response were used to generate 50 NP-specific T-cell clones (TCC). For six of these the epitopes were mapped with overlapping synthetic peptides derived from the sequence of the NP. These CD4(+) TCC displayed high specific proliferation and produced mainly gamma interferon upon stimulation with NP. Because variation of up to 15% in the amino acid sequences of the structural proteins of naturally occurring Lassa virus variants has been observed, the reactivity of the TCC with peptides derived from the homologous epitopes of the Nigeria strain of Lassa virus and of the eastern Africa arenavirus Mopeia was tested. With the Nigeria strain of Lassa virus the levels of homology were 100% for two of these epitopes and 85% for three of them, whereas homology with the respective Mopeia epitopes ranged from 92 to 69%. Reactivity of the TCC with peptides derived from the variable epitopes of the Nigeria strain and of Mopeia was reduced or completely abolished. This report shows for the first time that seropositive individuals from areas of endemicity have very strong memory CD4(+) T-cell responses against the NP of Lassa virus, which are partly strain specific and partly cross-reactive with other Lassa virus strains. Our findings may have important implications for the strategy of designing recombinant vaccines against this mainly T-cell-controlled human arenavirus infection.
| S-EPMC111699 | biostudies-literature
Project description:BackgroundThe complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate.MethodsThis review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state.ConclusionRecent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.
| S-EPMC5326600 | biostudies-literature
Project description:CD4 downregulation on infected cells is a highly conserved function of primate lentiviruses. It has been shown to positively impact viral replication by a variety of mechanisms, including enhanced viral release and infectivity, decrease of cell reinfection, and protection from antibody-dependent cellular cytotoxicity (ADCC), which is often mediated by antibodies that require CD4 to change envelope (Env) conformation. Here, we report that incorporation of CD4 into HIV-1 viral particles affects Env conformation resulting in the exposure of occluded epitopes recognized by CD4-induced antibodies. This translates into enhanced neutralization susceptibility by these otherwise nonneutralizing antibodies but is prevented by the HIV-1 Nef accessory protein. Altogether, these findings suggest that another functional consequence of Nef-mediated CD4 downregulation is the protection of viral particles from neutralization by commonly elicited CD4-induced antibodies.IMPORTANCE It has been well established that Env-CD4 complexes expose epitopes recognized by commonly elicited CD4-induced antibodies at the surface of HIV-1-infected cells, rendering them vulnerable to ADCC responses. Here, we show that CD4 incorporation has a profound impact on Env conformation at the surface of viral particles. Incorporated CD4 exposes CD4-induced epitopes on Env, rendering HIV-1 susceptible to neutralization by otherwise nonneutralizing antibodies.
| S-EPMC6819941 | biostudies-literature
Project description:The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.
| S-EPMC3538257 | biostudies-literature
Project description:The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.
| S-EPMC1165436 | biostudies-other
Project description:BackgroundCD4(+)CD25(+) regulatory T cell (Treg)-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used.Methodology/principal findingsSplenic lymphocytes from nonobese diabetic (NOD) mice were stimulated with different glutamic acid decarboxylase (GAD)-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538)-expanded CD4(+)CD25(+) T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528)- or p530 (GAD530-543)-expanded CD4(+)CD25(+) T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes) between the above-mentioned epitopes and MHC class II I-A(g7) were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7), while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7). Furthermore, p524 bound to I-A(g7) more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570), which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+)CD25(+) T cells suppressed the onset of diabetes in NOD mice.Conclusions/significanceThese data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of protective epitopes to expand functional Tregs.
| S-EPMC2736381 | biostudies-literature
Project description:Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.
| S-EPMC6520763 | biostudies-literature
Project description:Antigen binding by antibodies requires precise orientation of the complementarity- determining region (CDR) loops in the variable domain to establish the correct contact surface. Members of the family Camelidae have a modified form of immunoglobulin gamma (IgG) with only heavy chains, called Heavy Chain only Antibodies (HCAb). Antigen binding in HCAbs is mediated by only three CDR loops from the single variable domain (VHH) at the N-terminus of each heavy chain. This feature of the VHH, along with their other important features, e.g., easy expression, small size, thermo-stability and hydrophilicity, made them promising candidates for therapeutics and diagnostics. Thus, to design better VHH domains, it is important to thoroughly understand their sequence and structure characteristics and relationship. In this study, sequence characteristics of VHH domains have been analysed in depth, along with their structural features using innovative approaches, namely a structural alphabet. An elaborate summary of various studies proposing structural models of VHH domains showed diversity in the algorithms used. Finally, a case study to elucidate the differences in structural models from single and multiple templates is presented. In this case study, along with the above-mentioned aspects of VHH, an exciting view of various factors in structure prediction of VHH, like template framework selection, is also discussed.
| S-EPMC7061911 | biostudies-literature
Project description:Constant (C)-region switching of heavy (H) and/or light (L) chains in antibodies (Abs) can affect their affinity and specificity, as demonstrated using mouse, human, and chimeric mouse-human (MH) Abs. However, the consequences of C-region switching between evolutionarily distinct mammalian and avian Abs remain unknown. To explore C-region switching in mouse-chicken (MC) Abs, we investigated antigen-binding parameters and thermal stability of chimeric MC-6C407 and MC-3D8 IgY Abs compared with parental mouse IgGs and chimeric MH Abs (MH-6C407 IgG and MH-3D8 IgG) bearing identical corresponding variable (V) regions. The two MC-IgYs exhibited differences in antigen-binding parameters and thermal stability from their parental mouse Abs. However, changes were similar to or less than those between chimeric MH Abs and their parental mouse Abs. The results demonstrate that mammalian and avian Abs share compatible V-C region interfaces, which may be conducive for the design and utilization of mammalian-avian chimeric Abs.
| S-EPMC6917740 | biostudies-literature
Project description:Antibodies are now indispensable tools for all areas of cell biology and biotechnology as well as for diagnosis and therapy. Antigen-specific single immunoglobulin variable domains that bind to native antigens can be isolated and manipulated using yeast intracellular antibody capture technology but converting these to whole monoclonal antibody requires that complementary variable domains (VH or VL) bind to the same antigenic site. We describe a simple approach (CatcherAb) for specific isolation of such complementary single domains allowing the constitution of functional Fv, forming the basis of antigen-specific whole immunoglobulin and thus antibody production. We illustrate this approach by developing high-affinity Fv from single variable domains binding to RAS and LMO2 oncogenic proteins.
| S-EPMC2655674 | biostudies-literature