Communication pathways bridge local and global conformations in an IgG4 antibody

The affinity of an antibody for its antigen is primarily determined by the specific sequence and structural arrangement of the complementarity-determining regions (CDRs). Recent evidence, however, points toward a nontrivial relation between the CDR and distal sites: variations in the binding strengths have been observed upon mutating residues separated from the paratope by several nanometers, thus suggesting the existence of a communication network within antibodies, whose extension and relevance might be deeper than insofar expected. In this work, we test this hypothesis by means of molecular dynamics (MD) simulations of the IgG4 monoclonal antibody pembrolizumab, an approved drug that targets the programmed cell death protein 1 (PD-1). The molecule is simulated in both the apo and holo states, totalling 4 μs of MD trajectory. The analysis of these simulations shows that the bound antibody explores a restricted range of conformations with respect to the apo one, and that the global conformation of the molecule correlates with that of the CDR. These results support the hypothesis that pembrolizumab featues a multi-scale hierarchy of intertwined global and local conformational changes. The analysis pipeline developed in this work is general, and it can help shed further light on the mechanistic aspects of antibody function.

#56: A novel test for diagnosis and surveillance of Wuchereria bancrofti infection

Background Elephantiasis or Lymphatic filariasis (LF) is a parasitic infection that causes significant morbidity and impacts hundreds of millions of people in 73 countries. Most LF is caused by the nematode Wuchereria bancrofti, but Brugia species cause LF in some areas of Southeast Asia. The global program to eliminate LF uses mass drug administration (MDA) of antifilarial drugs in endemic areas to kill the microfilaria (MF) stage of the parasite that is required for ongoing transmission by mosquitos. Better tools are needed for assessing the success of MDA, because of limitations of available diagnostic tests. MF testing is often not feasible, because it requires collection of blood at night in most endemic areas. Existing antibody and antigen tests remain positive long after effective treatment, and their results do not correlate well with current infectivity. The Brugia Rapid test detects antibodies to BmR1, a Brugia protein that is expressed by MF. These antibodies disappear 2–3 years after effective treatment, and that makes the Brugia Rapid test a useful marker for persistent infection in the few countries with brugian filariasis. We set out to develop a novel antibody test for W. bancrofti infection based on a BmR1 homologue in W. bancrofti. Methods We cloned, expressed and purified a Wuchereria bancrofti protein (provisional name WbN1) that is a homologue of the Brugia malayi protein BmR1. Sera from patients infected with Wuchereria bancrofti as well as sera from patients infected with other closely related filarial species were tested for IgG4 antibodies to WbN1 by indirect ELISA. Results The ELISA has a sensitivity of 90.7% for infection with W. bancrofti based on the 80 bancrofti patient samples tested thus far. Specificity was 91.5% with 59 sera samples from patients infected with Onchocerca volvulus or Loa loa, which are filarial parasites that are co-endemic with W. bancrofti in Africa. Specificity was 97.9% with North American control samples. ELISA with sera from a clinical trial in Sri Lanka demonstrated that antibodies to WbN1 decreased significantly faster after treatment than antibodies to the previously described filarial antigen Bm14. Similar declines in antibody to WbN1 occurred after patients in Papua New Guinea received a single dose of triple drug treatment for LF with Ivermectin Diethylcarbamazine and Albendazole, that is effective for clearing MF from the blood without clearing filarial antigenemia. Conclusions While additional studies are needed, this ELISA for IgG4 antibody to the recombinant protein WbN1 could be a promising new surveillance tool for assessing for ongoing transmission of LF following MDA.

Development of Immunochromatographic Test Kit for Rapid Detection of Specific IgG4 Antibody in Whole-Blood Samples for Diagnosis of Human Gnathostomiasis

Human gnathostomiasis is a harmful food-borne zoonosis caused by roundworms of the genus Gnathostoma. The parasite can occasionally migrate to the central nervous system, causing life-threatening disease and death. Here, we report a new point-of-care (POC) test kit, the gnathostomiasis blood immunochromatographic test (GB-ICT) kit. The kit is based on recombinant Gnathostoma spinigerum antigen and detects specific IgG4 antibody in whole-blood samples (WBSs). The GB-ICT kit showed potentially high diagnostic values with simulated WBSs (n = 248), which were obtained by spiking patients’ sera with red blood cells. The accuracy, sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 93.8%, 81.5%, and 100%, respectively. Ten WBSs from clinically suspected gnathostomiasis patients were all positive according to the GB-ICT kit, while 10 WBSs from healthy volunteers were negative. The GB-ICT kit is a simple and convenient POC testing tool using finger-prick blood samples: venous blood sampling and serum separation processes are not required. The GB-ICT kit can support clinical diagnosis in remote areas and field settings without sophisticated equipment facilities.

Development of rSs-NIE-1 and rSs-IR Recombinant Antigen-Based Immunoblot for Detection of Antibody to Strongyloides stercoralis

Strongyloides stercoralis is a soil-transmitted nematode that can cause life-threatening conditions in immunocompromised persons. In the United States, strongyloidiasis should be considered mainly in immigrants, refugees, or travelers. The confirmatory laboratory diagnosis is usually performed by detecting larvae from the stool, duodenal material, and sputum. In persons who are immunocompromised with severe strongyloidiasis, adult worms and eggs can be detected from duodenal material. For serological diagnosis, most assays use crude antigens to detect anti-S. stercoralis IgG. Recently, recombinant proteins such as rSs-NIE-1 and rSs-IR have been used to detect IgG antibodies. We used rSs-NIE-1 and rSs-IR recombinant antigens to develop a biplex Western blot assay to detect the IgG4 antibody in individuals with strongyloidiasis. The sensitivities of rSs-NIE-1 and rSs-IR were 97.4% and 90.8%, respectively, whereas the specificities were 97.6% and 98%, respectively. In conclusion, the biplex rSs-NIE-1 and rSs-IR immunoblot performs well in detecting IgG4 antibody in S. stercoralis-infected persons.

Intestinal Dysbiosis and Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) is a chronic fibro-inflammatory disorder of the pancreas. Recent clinicopathological analysis revealed that most cases of AIP are pancreatic manifestations of systemic IgG4-related disease (IgG4-RD), a newly established disease characterized by enhanced IgG4 antibody responses and the involvement of multiple organs. Although the immuno-pathogenesis of AIP and IgG4-RD has been poorly defined, we recently showed that activation of plasmacytoid dendritic cells (pDCs) with the ability to produce large amounts of IFN-α and IL-33 mediates chronic fibro-inflammatory responses in experimental and human AIP. Moreover, M2 macrophages producing a large amount of IL-33 play pathogenic roles in the development of human IgG4-RD. Interestingly, recent studies including ours provide evidence that compositional alterations of gut microbiota are associated with the development of human AIP and IgG4-RD. In addition, intestinal dysbiosis plays pathological roles in the development of chronic pancreatic inflammation as dysbiosis mediates the activation of pDCs producing IFN-α and IL-33, thereby causing experimental AIP. In this Mini Review, we focus on compositional alterations of gut microbiota in AIP and IgG4-RD to clarify the mechanisms by which intestinal dysbiosis contributes to the development of these disorders.

Hypersensitivity Reactions Secondary to Dupilumab in Two Patients with Moderate to Severe Atopic Dermatitis

Dupilumab is a human monoclonal IgG4 antibody that is widely used to treat atopic dermatitis. Dupilumab was recently FDA approved to treat moderate to severe atopic dermatitis. Here we decribe two cases of patients with moderate to severe atopic dermatitis who developed an acute hypersensitivity reaction within hours of their second dose of dupilumab.