Assessing antibody decline after chemotherapy of early chronic Chagas disease patients

Background Chagas disease remains a significant public health problem in Latin America. There are only two chemotherapy drugs, nifurtimox and benznidazole, and both may have severe side effects. After complete chemotherapy of acute cases, seropositive diagnosis may revert to negative. However, there are no definitive parasitological or serological biomarkers of cure. Methods Following a pilot study with seven Bolivian migrants to Spain, we tested 71 serum samples from chronic patients (mean age 12.6 years) inhabiting the Argentine Chaco region. Benznidazole chemotherapy (5–8 mg/kg day, twice daily for 60 days) was administered during 2011–2016. Subsequently, pre-and post-chemotherapy serum samples were analysed in pairs by IgG1 and IgG ELISA using two different antigens and Chagas Sero K-SeT rapid diagnostic tests (RDT). Molecular diagnosis by kDNA-PCR was applied to post-treatment samples. Results Pilot data demonstrated IgG1 antibody decline in three of seven patients from Bolivia 1 year post-treatment. All Argentine patients in 2017 (averaging 5 years post-treatment), except one, were positive by conventional serology. All were kDNA-PCR-negative. Most (91.5%) pre-treatment samples were positive by the Chagas Sero K-SeT RDT, confirming the predominance of TcII/V/VI. IgG1 and IgG of Argentine patients showed significant decline in antibody titres post-chemotherapy, with either lysate (IgG, P = 0.0001, IgG1, P = 0.0001) or TcII/V/VI peptide antigen (IgG, P = 0.0001, IgG1, P = 0.0001). IgG1 decline was more discriminative than IgG. Antibody decline after treatment was also detected by the RDT. Incomplete treatment was associated with high IgG1 post-treatment titres against lysate (P = 0.013), as were IgG post-treatment titres to TcII/V/VI peptide (P = 0.0001). High pre-treatment IgG1 with lysate was associated with Qom ethnicity (P = 0.045). No associations were found between gender, age, body mass index and pre- or post-treatment antibody titres. Conclusions We show that following chemotherapy of early chronic Chagas disease, significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure. Graphical abstract We show that following chemotherapy of early chronic Chagas disease, a significant decline in IgG1 antibody suggests cure, whereas sustained or increased IgG1 is a potential indicator of treatment failure. Due to restricted sensitivity, IgG1 should not be used as a diagnostic marker but has promise, with further development, as a biomarker of cure.

Molecular Dynamics Simulations Reveal Interactions of an IgG1 Antibody With Selected Fc Receptors

In a survey of novel interactions between an IgG1 antibody and different Fcγ receptors (FcγR), molecular dynamics simulations were performed of interactions of monoclonal antibody involved complexes with FcγRs. Free energy simulations were also performed of isolated wild-type and substituted Fc regions bound to FcγRs with the aim of assessing their relative binding affinities. Two different free energy calculation methods, Molecular Mechanical/Generalized Born Molecular Volume (MM/GBMV) and Bennett Acceptance Ratio (BAR), were used to evaluate the known effector substitution G236A that is known to selectively increase antibody dependent cellular phagocytosis. The obtained results for the MM/GBMV binding affinity between different FcγRs are in good agreement with previous experiments, and those obtained using the BAR method for the complete antibody and the Fc-FcγR simulations show increased affinity across all FcγRs when binding to the substituted antibody. The FcγRIIa, a key determinant of antibody agonistic efficacy, shows a 10-fold increase in binding affinity, which is also consistent with the published experimental results. Novel interactions between the Fab region of the antibody and the FcγRs were discovered with this in silico approach, and provide insights into the antibody-FcγR binding mechanism and show promise for future improvements of therapeutic antibodies for preclinical studies of biological drugs.

PD-1 checkpoint blockade disrupts CD4 T cell regulated adaptive B cell tolerance to foreign antigens

Adaptive B cell immunity to environmental antigens must be regulated by multiple CD4 T cell dependent tolerance mechanisms. Using integrated single cell strategies, we demonstrate that acute PD-1 blockade induces extensive and selective local anti-inflammatory IgG1 plasma cell (PC) differentiation. Expansion of pre-existing IgG1 germinal center (GC) B cell and enhanced GC programming without memory B cell involvement reveals an isotype-specific GC checkpoint that blocks steady-state IgG1 antibody maturation. While there was no adjuvant impact on immunization, acute PD-1 checkpoint blockade exaggerates anti-commensal IgG1 antibody production, alters microbiome composition and exerts its action in a CD4 T cell dependent manner. These findings reveal a PD-1 controlled adaptive B cell tolerance checkpoint that selectively constrains maturation of pre-existing anti-inflammatory antibodies to prevent over-reaction to steady-state foreign antigens.

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 109/L, respectively, to ≈13 days (both HR and RR) at 350 × 109/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.