Human cytomegalovirus (HCMV) is an important, ubiquitously occurring, human pathogen in immunocompromized hosts. The virus can cause severe disease in transplant recipients. In large parts of the world HCMV is also the most common viral infection acquired in utero. In the USA and Europe an estimated 0.2% -1.2% of all live born infants are infected with HCMV and 5-15% of these babies develop long term sequalae resulting from congenital infection. As a consequence of the importance of congenital HCMV infection for public health, the Institute of Medicine at the National Institutes of Health (NIH), USA, has ranked the development of a HCMV vaccine as a top priority. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective responses that should be induced by the vaccine. HCMV is a complex virus containing numerous antigens within the viral envelope that could be targets for protective antibodies. The fusogen glycoprotein B (gB) and/or the heterodimeric gH/gL which are components of the receptor binding trimeric (gH/gL/gO) or pentameric (gH/gL/UL128,130,131A) complexes are important targets for neutralizing antibodies and hence interesting molecules for intervention strategies such as vaccination or passive immunotherapy.
The main research areas of our lab are:
- Uncovering novel neutralizing epitopes on cytomegaloviral glycoproteins
- Characterizing the mechanisms of protection conferred by monoclonal antibodies in vivo.
- Identification of antibody signatures as predictors for HCMV disease and transmission
- Molecular insights into the fusion process of HCMV-gB and SARS-CoV-2 spike protein
To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (mabs) were generated following immunization of mice with HCMV. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent mabs, 6E3, 3C11, and 2B10 (Figure 1). Mabs 6E3 and 3C11 blocked infection of all viral strains that were tested, while mab 2B10 neutralized only 50% of the HCMV strains analyzed. The strain-specific determinant for neutralization by mab 2B10 was identified as a single Met/Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation.
We have used the murine model system of CMV (MCMV) to explore the potential of gB-specific monoclonal antibodies (mabs) in immunotherapy or prophylaxis. The mabs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB (see Figure 2). When these mabs were used in immunodeficient RAG-/- mice to limit an ongoing infection we observed a reduction in viral load both with mabs having potent neutralizing capacity in vitro as well as mabs classified as non-neutralizing (Figure 2). In a therapeutic setting, nt mabs more potently reduced the viral burden compared to nnt mabs. Efficacy was correlated with sustained concentration of virus neutralizing mabs in vivo rather than their nt capacity in vitro. Combinations of nt mabs further augmented the antiviral effect and were as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mabs before infection was also protective and both nt and nnt mabs equally prevented immune-deficient mice from the lethal course of infection. In summary, our data argue that therapeutic application of potently neutralizing mabs against gB represents a strategy to block CMV infection in immunodeficient hosts. When present before infection, both nt and nnt anti-gB mabs were equally protective.
HCMV primary infection, reactivation, or reinfection can hardly be discriminated and predictions for the risk of HCMV transmission and likelihood to progress to disease cannot be made. Previous studies of our working group demonstrated that following primary HCMV infection, antibodies against the glycoproteins gB or gH were formed with a delay compared to tegument proteins such as pp65 (Schoppel et al., 1997). Via a test for antibodies against a strain-specific gH-epitope, the reinfection in a CMV-seropositive pregnant woman could be diagnosed, which correlated with intrauterine transmission followed by symptomatic neonatal infection (Boppana et al., 2001). In vivo, both neutralizing as well as non-neutralizing antibodies showed significant protection with neutralizing antibodies being superior (Bootz et al., 2017). In addition, it is known that antibodies of different IgG-subclasses may have different functional activities according to the distinct affinities for Fc-receptors (Vidarsson et al., 2014). This project aims to identify HCMV antibody signatures induced by various HCMV antigens that might serve as predictors for HCMV disease and transmission. As an alternative to inactivated virus particles, recombinantly produced viral proteins are increasingly being used as antigens for serological test methods. The production of viral glycoproteins and their coupling to surfaces require complex development and validation work. A variable that is difficult to control is the spatial orientation and presentation of the proteins immobilized on the surfaces used. Specific hetero-dimerizing peptide tags, so-called coiled-coils (Bind & Bite technology), offer a clear improvement (Lindhout et al. 2004). For this, heterodimers of two peptides with K-coil or E-coil are formed and can be stabilized by covalent coupling. Various structural and non-structural HCMV-antigens were cloned for prokaryotic or eukaryotic expression in-frame to a C-terminal K-coil-His-tag (Master thesis Zhiyu Xiao, 2022). Via cooperation with our industry partner Virion\Serion (Würzburg, Germany) we were able to purify recombinant HCMV proteins via Bind & Bite technology with E-coil-coupled paramagnetic beads from crude cell lysates. As a proof-of-principle, antigen-loaded magnetic beads were incubated with HCMV-positive-sera from the diagnostic department and stained with a PE-labeled secondary antibody to determine the IgG/IgM-specific antibody responses. As these pilot experiments successfully reconfirmed the seropositive individuals, the set of analyzed HCMV-antigens is currently extended, as it will be for the IgG-subclass-specific antibody responses and FcR-binding capacity of the respective sera.
HCMV as well as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficiently spread cell-to-cell through mechanisms facilitated by their membrane glycoproteins gB or spike, respectively. In order to dissect the cell membrane fusion activity of gB and spike in further detail, we established a dual split protein (DSP) assay based on complementation of GFP and luciferase, to quantify the fusogenic activity of the viral fusion proteins (see Figure 4). The DSP assay allows screening for inhibitors or antibodies that interfere with the fusion process and may therefore contribute to both rational vaccine design and development of novel treatment options against the human pathogenic viruses HCMV or SARS-CoV-2.