Protection from cytomegalovirus infection by antibodies
Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals and congenitally infected infants. HCMV infections or reactivations represent a major clinical complication in transplant patients resulting in increased morbidity and mortality. There are no licensed vaccines and improved understanding of the mechanisms of protection is urgently needed to guide novel interventions. The major goal of current HCMV vaccine concepts is the induction of neutralising antibodies (mAbs) (Nelson et al., 2018a). However, wild-type HCMV strains may escape the neutralising mAb response by cell-to-cell spread (Falk et al., 2018). Thus, other, non-neutralising mAb-dependent effector functions may contribute to protection against HCMV and should therefore be included in concepts of vaccination (Nelson et al., 2018b; Baraniak et al., 2018). Recent in vitro data demonstrated that antibody effector functions like antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) correlate with protection against HCMV (Vietzen et al., 2020; Vlahava et al., 2021; Semmes et al., 2022). Importantly, these in vitro assays have not identified immunodominant antigens – expressed on the surface of infected cells - which have to be targeted for efficiently preventing virus dissemination in vivo.
We have been studying the protective antibody response against CMV both, in the human (HCMV) and the murine (MCMV) system. For HCMV, we have extensively characterised the antibody response against the structural proteins gB and gH (Pötzsch et al., 2011; Spindler et al., 2014; Wiegers et al., 2014; Thomas et al., 2021). Using MCMV as a model system, we demonstrated that prophylactic or therapeutic administration of both, neutralising and non-neutralising anti-gB mAbs protected MCMV-infected immunodeficient RAG-/- animals (Bootz et al., 2017). We currently investigate the mechanism of protection of mAbs against the structural glycoprotein B and establish assays for the in vitro quantification of Fc-effector functions.
We propose to use MCMV as a model system to identify antigen specificity and mode of action of antibodies directed against viral membrane proteins presented on infected cells. To this end, it is planned to generate an extensive set of mAbs from MCMV-infected animals. Ideally, this set will include neutralising and non-neutralising mAbs and mirror the human response to the corresponding HCMV antigens. First, antibodies against viral antigens will be identified by using an existing library of MCMV membrane proteins and screening for binding to MCMV-GFP-infected cells. Second, the variable mAb sequences will be obtained by using the 10x Genomics platform and transferred into existing expression vectors before mAbs are purified from HEK293 cells according to the established protocols. Third, the recombinant mAbs will be characterised in vitro with respect to target specificity, ADCC, ADCP and inhibition of cell-to-cell spread. The mAbs will be further optimised for target-binding based on bioinformatics modelling and improved in their Fc-effector function. In addition, the derived mAbs provide the basis for the generation of novel CARs and BiTEs with further and/or improved specificities. Fourth, in order to test for their in vivo protective capacity, mAbs with different binding specificities will be administered therapeutically or prophylactically into MCMV-infected RAG-/- mice. Once target antigens of protective mAbs have been identified, we will finally attempt to induce the respective mAbs by immunisation. Here, it is planned to use different antigen formats such as soluble proteins, DNA or adenoviral and orthoreoviral vectors for in vivo antigen expression. Using structural information of the antibody targets, further optimisation of the vaccine antigens is intended. In summary, the proposed project aims at determining the contribution of the humoral immune response against viral membranes proteins for the in vivo protection from CMV, which is instrumental for a rational vaccine design against human CMV.
