Computational Construction of a Single-Chain Bi-Paratopic Antibody Allosterically Inhibiting TCR-Staphylococcal Enterotoxin B Binding

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRβ complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRβ chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions.

H2-M and H2-O as Targeting Vehicles for the MHC Class II Processing Compartment Promote Antigen-Specific CD4+ T Cell Activation

CD8 and CD4 T cell activation are both required for a strong and long-lasting T cell immune response. Endogenously expressed proteins are readily processed by the MHC class I antigen presentation pathway, enabling activation of CD8+ T cells. However, the MHC class II antigen presentation pathway, necessary for CD4+ T cell activation, is generally not sufficiently accessible to endogenously expressed proteins, limiting the efficiency of mRNA- or DNA-based vaccines. In the current study, we have evaluated the feasibility of using antigen sequences fused to sequences derived from the H2-M and H2-O proteins, two complexes known to participate in MHC class II antigen processing, for the enhancement of CD4 T-cell activation. We analyzed T cell activation after genetic immunization with mRNA-encoding fusion proteins with the model antigen ovalbumin and sequences derived from H2-M or H2-O. Our results show that H2-M- or H2-O-derived sequences robustly improve antigen-specific CD4 T-cell activation when fused to the antigen of interest and suggest that the approach could be used to improve the efficiency of mRNA- or DNA-based vaccines.

Repurposing Saquinavir for Host-Directed Therapy to Control Mycobacterium Tuberculosis Infection

Despite the available antibiotics, tuberculosis (TB) has made its return since the 90’s of the last century as a global threat mostly due to co-infection with HIV, to the emergence of drug resistant strains and the lack of an effective vaccine. Host-directed strategies could be exploited to improve treatment efficacy, contain drug-resistant strains, improve immune responses and reduce disease severity. Macrophages in the lungs are often found infected with Mycobacterium tuberculosis (Mtb) and/or with HIV. The long-term survival of lung macrophages infected with Mtb or with HIV, together with their ability to produce viral particles, especially during TB, makes these niches major contributors to the pathogenicity of the infection. Among the available drugs to control HIV infection, protease inhibitors (PIs), acting at post-integrational stages of virus replication cycle, are the only drugs able to interfere with virus production and release from macrophages during chronic infection. For Mtb we recently found that the pathogen induces a general down-regulation of lysosomal proteases, helping bacteria to establish an intracellular niche in macrophages. Here we found that the PI saquinavir, contrary to ritonavir, is able to induce an increase of endolysosomal proteases activity especially of cathepsin S in Mtb infected macrophages and during co-infection with HIV. Our results indicate that saquinavir treatment of infected macrophages led not only to a significant intracellular killing of Mtb but also: (i) to an improved expression of the HLA class II antigen presentation machinery at the cell surface; (ii) to increased T-lymphocyte priming and proliferation; and (iii) to increased secretion of IFN-γ. All together the results indicate saquinavir as a potential host directed therapy for tuberculosis.

Generation of Unexpected Allele-Specific Anti-HLA Antibodies after the Transplantation of a Fully-Matched Kidney Allograft and the Diagnostic Approaches Required for Excluding Harmful Effects after Subsequent Renal Regrafts

The specification of anti-human leukocyte antigen (HLA) antibodies is an important task for patients awaiting kidney allografts. Especially the patients immunized in previous transplantations, transfusions, or pregnancies must be carefully observed, since grafting patients with HLA antigens/phenotypes recognized by their pre-formed antibodies are the main cause of harmful hyperacute and acute rejection. The complement-dependent lymphocytotoxicity-based de facto (physical) crossmatching (CDC-CM) has thus been implemented as the last diagnostic obstacle before kidney allografting. Here, an assay is performed by incubating the donors’ lymphocytes with the sera of the prospective recipients, and a negative outcome was desired for eligibility of the underlying organ allocation. Furthermore, valid antibody specification has to be performed at least quarterly for each patient on the kidney waiting list, as defined by certain guidelines, for example, the Eurotransplant guidelines. Based on the exclusion of these specificities, also referred to as virtual crossmatching, certain donors are a priori listed as unacceptable for these recipients. In this case report, we showed that defining unacceptable antigens may be difficult if the recipients’ antibodies are allele-specific after being generated in the patient who is expressing the HLA-class II antigen DQ6 and also developing antibodies against this antigen. Low resolution (two-digit) typing is used before kidney allografting. Thus, these antibodies are generally not definable, as donors and recipients share the same antigen (allelic group). Here, we demonstrate the diagnostic approaches required to exclude inadequate kidney donors for a patient exhibiting antibodies only against the HLA-DQB1*06:04 allelic variant and not against the common phenotype HLA-DQ6. In practice, the patient’s HLA-class II high resolution (four-digit) typing, as well as his antibody specification at the highest (single antigen) resolution, are included. Furthermore, we critically discuss, according to the Eurotransplant guidelines, the missing possibility to declare own HLA-antigens unacceptable, which may be very helpful for recipients who exhibit allele-specific antibodies.

Innovation in Nucleotide-Binding Oligomerization-Like Receptor and Toll-Like Receptor Sensing Drives the Major Histocompatibility Complex-II Free Atlantic Cod Immune System

The absence of MHC class II antigen presentation and multiple pathogen recognition receptors in the Atlantic cod has not impaired its immune response however how underlying mechanisms have adapted remains largely unknown. In this study, ex vivo cod macrophages were challenged with various bacterial and viral microbe-associated molecular patterns (MAMP) to identify major response pathways. Cytosolic MAMP-PRR pathways based upon the NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) were identified as the critical response pathways. Our analyses suggest that internalization of exogenous ligands through scavenger receptors drives both pathways activating transcription factors like NF-kB (Nuclear factor-kappa B) and interferon regulatory factors (IRFs). Further, ligand-dependent differential expression of a unique TLR25 isoform and multiple NLR paralogues suggests (sub)neofunctionalization toward specific immune defensive strategies. Our results further demonstrate that the unique immune system of the Atlantic cod provides an unprecedented opportunity to explore the evolutionary history of PRR-based signaling in vertebrate immunity.