Antibody-mediated protection against cytomegalovirus

Human cytomegalovirus (HCMV) is a globally prevalent pathogen that presents significant clinical challenges, particularly for immunosuppressed individuals and infants infected in utero. Among transplant recipients, HCMV reactivation and infection are major contributors to increased morbidity and mortality. In congenital infections, HCMV is the leading cause of sensorineural hearing loss. Current therapeutic options are constrained by the emergence of escape mutations and drug-related toxicities. Furthermore, the specificities and protective mechanisms of antibody responses following infection remain poorly understood, contributing to the modest efficacy observed in vaccine trials. This thesis investigates the mechanisms of antibody-mediated protection targeting CMV glycoproteins through in vitro characterizations and in vivo mouse models. To produce anti-CMV monoclonal antibodies (mAbs), the well-characterized Hybridoma-derived anti-gB M11 antibody was cloned into expression plasmids for recombinant production. The original IgG2b subclass of M11 was switched, and Fc effector function-modulating mutations were introduced. During production, notable batch variability in expression levels was observed across subclasses and antibody variants. Size Exclusion Chromatography revealed aggregation and fragmentation in certain mAbs, leading to reduced functionality. Aggregated mAbs failed to bind their respective antigens, underscoring the critical impact of aggregation on antibody activity. Notably, storage conditions and buffer composition significantly influenced antibody stability. Under optimized conditions re-produced M11 mAbs exhibited consistent antigen-binding and neutralization capacities across IgG subclass variants compared to the original Hybridoma-derived antibody. The Fc mutations IgG2b DANG, IgG2c N297A and IgG2c LALA-PG decreased Fcγ receptor and complement binding, whereas IgG2c SAEAKA and IgG2c NA mutations enhanced these interactions. Analysis of the Hybridoma-derived mAb 32.22 confirmed specific binding to gN within the gM/gN complex, recognizing only one of the three known gN splicing variants. To date, it is the only reported anti-gN mouse antibody in CMV. Binding studies demonstrated that gN expression on the plasma membrane requires gM co-expression. Two additional mAbs, BK1 and BK2, were characterized as anti-gH mAbs. BK1 exhibited potent neutralizing activity, while BK2 lacked neutralizing ability even at high concentrations. Serological analysis of MCMV-infected mice showed a high prevalence against gM/gN (> 80 %) and gH (100 %), highlighting these complexes as critical CMV vaccine targets. Structural predictions using AlphaFold revealed homology between murine CMV glycoproteins and those of human beta-herpesviruses and other animal CMVs, further underscoring their importance. The in vivo efficacy of Hybridoma-derived mAbs M11, BK1 and BK2 was assessed in MCMV-infected immunodeficient RAG-/- mice. Therapeutic administration was more effective on day 3 than on day 1 post infection, as indicated by bioluminescence imaging, organ viral loads, serum neutralization assays and IgG titres. Among the mAbs, M11 provided the highest protection, while BK1 unexpectedly failed to significantly reduce viral loads, in contrast to the non-neutralizing mAb BK2. Collectively, this study advances our understanding of antibody-mediated protection against CMV, particularly the roles of glycoproteins gB, gM, gN and gH. These findings provide valuable insights for the development of improved vaccines and passive immunization strategies.