Use of flow cytometry for characterization of human cytomegalovirus vaccine particles

Abstract

Despite a 40-year effort, an effective vaccine against human cytomegalovirus (HCMV) remains an unmet medical need. The discovery of potent neutralizing epitopes on the pentameric gH complex (gH/gL/UL128/130/131) has reenergized HCMV vaccine development. Our whole-virus vaccine candidate, currently in a Phase I clinical trial, is based on the attenuated AD169 strain with restored expression of the pentameric gH complex. Given the complexity of a whole-virus vaccine, improved analytical methods have been developed to better characterize heterogeneous viral particles released from infected cells during vaccine production. Here we show the utility of a commercial flow cytometer for the detection of individual HCMV particles, either via light scattering or using fluorescence after labeling of specific antigens. Rapid measurements requiring minimal material provide near real-time information on particle concentration, distributions of different particle types, and product purity. Additionally, utilizing immunoreagents has allowed us to characterize the distribution of key antigens across individual particles and particle types.

Introduction

Congenital human cytomegalovirus (HCMV) infection is the leading viral cause of congenital neurological defects and HCMV infections in immunosuppressed individuals can lead to serious complications [1]. Development of an effective vaccine to prevent congenital HCMV is widely recognized as a significant unmet medical need. Early clinical trials with attenuated fibroblast-adapted HCMV strains, Towne and AD169, yielded disappointing results, showing only marginal immune response compared to natural infection (reviewed in [1], [2]). More recently, a vaccine based on recombinant gB protein in MF59 adjuvant was evaluated in a Phase II efficacy trial in HCMV seronegative women and was shown to be 50% effective in preventing primary HCMV infection [3]. The same gB-based vaccine also showed an ability to reduce viremia in a Phase II trial with patients receiving solid organ transplants [4].

Over the past decade, the importance of the pentameric gH complex (gH/gL/UL128/130/131) for humoral immunity has become widely recognized. This glycoprotein complex is important for the infection of epithelial and endothelial cells [5] and is the predominant target of the neutralizing activity in human sera with regard to these important cell types [6], [7]. In an effort to improve upon immune responses observed following vaccination with either fibroblast-adapted attenuated strains (Towne and AD169) or recombinant gB, we restored the expression of the pentameric gH complex in the attenuated AD169 strain by repairing the frameshift mutation in the UL131-128 locus [8]. This whole-virus vaccine candidate has possible advantages over other vaccine approaches by having the potential to present the full array of native HCMV glycoproteins to drive a humoral response capable of protecting multiple cell types, and by having the potential to elicit a more robust CD8 response than subunit vaccines due to the de novo expression of viral antigens. The new vaccine candidate strain is currently being evaluated in Phase I clinical trials.

Despite the potential advantages of an attenuated, whole-virus vaccine for eliciting broad humoral and cellular immunity, developing such a complex vaccine against HCMV presents many analytical challenges. HCMV is a complex virus containing dozens of structural proteins [9], [10] and producing multiple particle types [11], [12]. Infectious virions compose only a small percentage of the total viral particles released from infected cells. These virions are approximately 200 nm in diameter [13] and contain an encapsidated viral genome enclosed by a multi-protein tegument layer surrounded by a viral envelope containing a large number of glycoprotein complexes. In addition to infectious virions, in vitro cultures also produce large numbers of dense bodies (DB) – spherical particles of uniform electron density that consist predominantly of the main tegument protein pp65 surrounded by a viral envelope. Dense bodies are non-infectious particles lacking both capsid and viral genome [11] and can be variable in size. Infected cultures also produce a third particle type called non-infectious enveloped particles (NIEPs), which are structurally similar to virions except they lack DNA in the capsid [12]. These three viral particle types (infectious virions, NIEPs and DB) can be separated by density gradient centrifugation [14].

Detection of viruses by flow cytometry was demonstrated over 30 years ago [15]. This technology, however, has not gained wide-spread use for routine characterization of viral particles due to the lack of sensitivity of most conventional flow cytometers and has been limited to custom-made instruments [15], [16], [17]. One instrument that has been used in virology labs is the Virus Counter made by Virocyt (Boulder, CO) that uses proprietary dyes that bind to either proteins or nucleic acid. Potentially infectious viruses are differentiated from other particles because they have both protein and DNA signal. Specific antigens, however, cannot be labeled with the Virus Counter thereby limiting its usefulness for characterizing specific antigen distribution across different particle types. Flow cytometers have had more widespread use in characterizing extracellular vesicles and several reviews have been published [18], [19], [20]. The power of flow cytometry for characterization of viral particles was demonstrated in a recent work by Gaudin and Barteneva [17]. Using a customized flow cytometer the authors were able to show a link between virus infectivity and the concentration of viral glycoproteins on the surface of the virus.

Here we demonstrated the utility of an inexpensive, commercially available flow cytometer from Apogee Flow Systems (A50 Micro) to support vaccine development by providing information on multiple attributes of a virus-containing sample. The unique optical design of the Apogee flow cytometer allows detection of particles down to approximately 100–150 nm in diameter by light scattering (depending on the refractive index) and provides exceptional resolution of populations of small particles [20]. Using only light scattering, the A50 Micro was capable of providing near real-time characterization data that accurately predicted a variety of quality attributes including infectious titer, total antigen concentration, and purity. Such data were invaluable for monitoring infected cultures in lieu of labor or time-intensive methods that are not practical for real-time process monitoring. Furthermore, combining immunochemistry with the A50 Micro flow cytometer allowed for characterization of the distribution of two key antigens (pentameric gH complex and gB) across the various particle types independent of labor-intensive separation techniques.