Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

Characterization of gH/gL/pUL128-131 pentameric complex, gH/gL/gO trimeric complex, gB and gM/gN glycoproteins in a human cytomegalovirus using automated capillary western blots.

Human cytomegalovirus (HCMV) is currently a major cause of congenital disease in newborns and organ failure in transplant recipients. Despite decades of efforts, an effective vaccine against HCMV has yet to be developed. However, the discovery of pentameric gH complex on viral surface which contains potent neutralizing epitopes may help enable development of an effective vaccine. In our company ongoing Phase II clinical trial of whole-live virus HCMV vaccine (V160), the pentameric gH complex has been restored on the surface of live attenuated AD169 virus strain. The reconstructed HCMV virus contains a variety of surface glycoproteins including pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, gB glycoprotein, and gM/gN heterodimer complex. To further characterize this virus and enable the monitoring of multiple viral antigens during vaccine process development an effective and efficient analytical strategy was required to detect and quantify several viral surface proteins. In this paper, we present an innovative approach based on capillary western blot technology that allows fast and accurate quantitation of pentameric gH/gL/gUL128-131 complex, trimeric gH/gL/gO complex, and gB glycoprotein. This method is suitable for analyzing target proteins in multiple sample types including supernatants from infected cell culture, purification intermediates, concentration bulk, and the final vaccine product. In addition, the capillary western blot-based technology identified a previously unknown biochemical profile present in some HCMV viruses: triplet gH peaks of viral surface proteins in non-reducing environment, which could potentially present a new strategy for specificity and identity testing.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Use of flow cytometry for characterization of human cytomegalovirus vaccine particles.

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.