New Approaches to the Development and Evaluation of Human Cytomegalovirus (HCMV) Vaccines
Haruhiko Murata, MD, PhD
Office of Vaccines Research and Review
Division of Viral Products
Laboratory of DNA Viruses
Biosketch
Dr. Haruhiko Murata obtained his MD and PhD degrees from Washington University School of Medicine in St. Louis, MO. He was then accepted into the Cancer Prevention Fellowship Program sponsored by the National Cancer Institute, during which time he completed the Master of Public Health program with a concentration in epidemiology and biostatistics at the Johns Hopkins University Bloomberg School of Public Health. Dr. Murata pursued his fellowship research on virology and vaccinology in the Office of Vaccines Research and Review (OVRR), CBER, FDA. He is currently a Principal Investigator in the Division of Viral Products, OVRR, CBER, FDA.
General Overview
Infection with human cytomegalovirus (HCMV) is very common, but obvious health problems are usually not observed in infected individuals with normal immunity. However, this virus is a major cause of serious infection in the fetus of a pregnant woman. Babies born with HCMV infection can suffer a range of permanent disabilities, including hearing loss and mental retardation. Each year, approximately 7000 babies are born in the US with HCMV disease, more than other, more familiar diseases and conditions, such as Down syndrome. HCMV is also an important cause of disease in immunocompromised individuals. For these reasons, developing a vaccine to prevent HCMV disease is recognized as a high public health priority, a position endorsed by the US National Academy of Medicine.
We recently developed a test that measures the ability of components in blood samples to inhibit (neutralize) viral infection of cells grown in the laboratory. The test format is "high-throughput," that is, simple enough to be able to process many blood samples at once. Also, our approach can be applied to a broad range of viral targets. We plan to use our new test as one approach to further investigate the how antibodies in blood, or chemical compounds, can inhibit viruses such as HCMV. We also intend to develop other high-throughput tests to measure immune responses to infection and vaccination for HCMV and other viruses.
Neutralizing antibodies target viral proteins on the surface (envelope) of the virus. These viral envelope proteins are often involved in important steps in the virus life cycle, such as infecting susceptible cells. By studying how neutralizing antibodies target key viral envelope proteins, we hope to understand viral entry mechanisms. Several HCMV vaccine candidates in development are deliberately designed to induce antibodies that target recently identified viral envelope protein complexes. Thus, our work might help to identify factors necessary to improve vaccine quality and efficacy. Our work might also enable us to offer technical guidance to manufacturers of CBER-regulated products.
Finally, we are assessing potential safety issues associated with the use of tumorigenic cells (cells capable of forming tumors when injected into experimental animals) for manufacturing vaccines, including investigational HCMV vaccines. Our results might lead to changes in regulatory testing requirements for vaccine cell substrates (cells used for vaccine manufacture). Addressing perceived safety issues associated tumorigenicity might also expand the repertoire of available vaccine cell substrates.
Scientific Overview
We recently developed high-throughput qPCR-based neutralization assays for several viruses (SV40, influenza virus, and RSV), an approach with numerous advantages. The sensitivity of qPCR allows assessment of virus neutralization after a short duration, thereby saving time and reducing assay variability. The dynamic range of qPCR allows the assay to be performed over a wide range of virus input, thereby contributing to assay robustness. Other benefits include adaptability to automation and ease of inter-laboratory standardization through distribution of frozen RNA/DNA reagents that can serve as calibration standards.
For RNA targets, we use RT-qPCR-ready crude cell lysates. Sample preparation includes 1) washing infected cells in a 96-well format; 2) applying the cell-lysis reagent; 3) collecting samples after a brief incubation. The resulting cell-lysate samples, which contain target RNA, can be stored frozen or used as input templates for downstream one-step RT-qPCR. This eliminates the need for sample nucleic acid purification, a rate-limiting step for conventional PCR workflows. This approach also avoids the variability in RT-qPCR data due to non-quantitative RNA recovery that occurs using traditional extraction procedures.
We recently applied our approach to the study of HCMV. Prior studies suggested that the neutralization behavior of HCMV can depend on the host cell type. We therefore established our assay using both human fibroblasts (MRC-5) and human retinal epithelial cells (ARPE-19). We designed PCR primers to the conserved and abundantly expressed immediate early gene IE1. When we assessed seropositive human serum samples using our neutralization assay, there were substantially higher titers in epithelial cells compared with fibroblasts. This supports the hypothesis that there are both qualitative and quantitative differences in neutralization according to the cell type used in the assay. A manuscript describing our assay was recently published.
We intend to develop other high-throughput assays to measure immune responses to infection and vaccination for HCMV, as well as other viral targets. A major focus is on key envelope glycoprotein complexes that are both determinants of cell entry and targets for neutralizing antibodies; consequently, these complexes are also key antigens for investigational vaccines.
Finally, a portion of our effort is directed at assessing potential safety issues associated with the use of tumorigenic cells for the manufacture of vaccines, including investigational HCMV vaccines. Historical concerns have limited the use of tumorigenic cells for vaccine manufacture. Our work might alter, or perhaps eliminate, current tumorigenicity testing requirements for vaccine cell substrates.
Publications
- Vaccine: X 2019 Apr 11;(1):100004
Responsiveness to basement membrane extract as a possible trait for tumorigenicity characterization.
Murata H, Omeir R, Tu W, Lanning L, Phy K, Foseh G, Lewis AM Jr, Peden K - Vaccine 2018 Sep 5;36(37):5580-90
Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response.
Cui X, Cao Z, Wang S, Lee RB, Wang X, Murata H, Adler SP, McVoy MA, Snapper CM - Biologicals 2017 Nov;50:35-41
Binding and neutralizing anti-cytomegalovirus activities in immune globulin products.
Wang X, Xu Y, Scott DE, Murata H, Struble EB - Data Brief 2016 Dec;9:417-21
Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis.
Wang X, Teferedegne B, Shatzkes K, Tu W, Murata H - Anal Biochem 2016 Nov 15;513:21-7
Endogenous RNase inhibitor contributes to stability of RNA in crude cell lysates: Applicability to RT-qPCR.
Wang X, Teferedegne B, Shatzkes K, Tu W, Murata H - Vaccine 2015 Dec 16;33(51):7254-61
RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.
Wang X, Peden K, Murata H