Final Report Summary - MEGALOVAX (In search of new cytomegalovirus vaccine antigens)
MEGALOVAX is aimed to search for novel human cytomegalovirus (HCMV) antigens involved in viral entry into the host cell as well as comprehensive profiling of the human antibody repertoire against HCMV. The project consists of four objectives:
(1) immunological screening of selected HCMV sequences;
(2) establishment of an in vitro neutralisation assay system and execution of the assays;
(3) in vitro expression of selected genes of HCMV and assessment of their subcellular localisation;
(4) profiling of the antibody repertoire in HCMV seropositive individuals.
The final outcomes of these objectives are described hereafter.
(1) Immunological screening of selected HCMV sequences
Dr Kessler was able to subclone all gene sequences that are predicted to encode surface exposed HCMV proteins, which were subsequently used to immunise mice using alphavirus replicon particles.
(2) Establishment of an in vitro neutralisation assay system and execution of the assays
The protocol of HCMV cell culture has been successfully established within the proposed timeline. Neutralisation assays were performed on HCMV with the mouse sera obtained from O1. None of the selected proteins was able to raise a neutralising antibody response in mice. Dr Kessler also obtained the technology of bacterial artificial chromosome (BAC). This technology allows us to modify the genome of HCMV, thus allowing the characterisation of individual HCMV gene products.
(3) In vitro expression of selected genes of HCMV and assessment of their subcellular localisation
All HCMV antigens selected in O1 used for the immunisation of mice were expressed in vitro by transfection of cell lines. The expression of each HCMV gene was verified by immunoblotting using antibodies against an attached tag, thus preselecting proteins for further analyses and determining their subcellular localisation. Fluorescence activated cell sorting (FACS) analysis of the transfectants using sera from immunised mice was impaired by high background for some gene products.
(4) Profiling of the antibody repertoire in HCMV seropositive individuals
Discussion with the NVD protein microarray group took place to evaluate the technical feasibility of the approach. Material availability (production of recombinant HCMV proteins) for serum profiling was confirmed by a Doctor of Philosophy (PhD) student under supervision of Dr Kessler. Subsequently, pilot experiments were performed and the results were evaluated with the protein microarray team. Based on the results, we considered serum profiling of a larger number of subjects using the protein microarray system at NVD to be feasible. We contacted several universities / hospitals for access to a larger number of human plasma / serum samples. Unfortunately we could not establish the collaboration because of contractual disagreement. As a consequence, we could only perform antibody repertoire profiling on a smaller set of commercially available samples.
Overall, Dr Kessler pursued the MEGALOVAX project very well. Because we were unable to fulfil only the last part of the milestones for non-scientific, non-technical reasons; we continue to seek additional financial support to complete the serum profiling of HCMV infected individuals. The expected results may provide an opportunity to identify new vaccine candidates against HCMV infection, for which currently no licensed vaccine is available. With further studies the outcome of MEGALOVAX would have a large socio-economic impact.
(1) immunological screening of selected HCMV sequences;
(2) establishment of an in vitro neutralisation assay system and execution of the assays;
(3) in vitro expression of selected genes of HCMV and assessment of their subcellular localisation;
(4) profiling of the antibody repertoire in HCMV seropositive individuals.
The final outcomes of these objectives are described hereafter.
(1) Immunological screening of selected HCMV sequences
Dr Kessler was able to subclone all gene sequences that are predicted to encode surface exposed HCMV proteins, which were subsequently used to immunise mice using alphavirus replicon particles.
(2) Establishment of an in vitro neutralisation assay system and execution of the assays
The protocol of HCMV cell culture has been successfully established within the proposed timeline. Neutralisation assays were performed on HCMV with the mouse sera obtained from O1. None of the selected proteins was able to raise a neutralising antibody response in mice. Dr Kessler also obtained the technology of bacterial artificial chromosome (BAC). This technology allows us to modify the genome of HCMV, thus allowing the characterisation of individual HCMV gene products.
(3) In vitro expression of selected genes of HCMV and assessment of their subcellular localisation
All HCMV antigens selected in O1 used for the immunisation of mice were expressed in vitro by transfection of cell lines. The expression of each HCMV gene was verified by immunoblotting using antibodies against an attached tag, thus preselecting proteins for further analyses and determining their subcellular localisation. Fluorescence activated cell sorting (FACS) analysis of the transfectants using sera from immunised mice was impaired by high background for some gene products.
(4) Profiling of the antibody repertoire in HCMV seropositive individuals
Discussion with the NVD protein microarray group took place to evaluate the technical feasibility of the approach. Material availability (production of recombinant HCMV proteins) for serum profiling was confirmed by a Doctor of Philosophy (PhD) student under supervision of Dr Kessler. Subsequently, pilot experiments were performed and the results were evaluated with the protein microarray team. Based on the results, we considered serum profiling of a larger number of subjects using the protein microarray system at NVD to be feasible. We contacted several universities / hospitals for access to a larger number of human plasma / serum samples. Unfortunately we could not establish the collaboration because of contractual disagreement. As a consequence, we could only perform antibody repertoire profiling on a smaller set of commercially available samples.
Overall, Dr Kessler pursued the MEGALOVAX project very well. Because we were unable to fulfil only the last part of the milestones for non-scientific, non-technical reasons; we continue to seek additional financial support to complete the serum profiling of HCMV infected individuals. The expected results may provide an opportunity to identify new vaccine candidates against HCMV infection, for which currently no licensed vaccine is available. With further studies the outcome of MEGALOVAX would have a large socio-economic impact.