Abstract
Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.
Highlights
herpes simplex virus 2 (HSV-2) is the primary cause of infectious ulcerative genital disease worldwide [1,2,3,4]
We describe a comprehensive purification procedure with reasonable yields, which is comprised of eluting the candidate vaccine ACAM529 from infected complementing Vero cell (AV529-19) culture using dextran sulfate and further processing the dextran sulfate-released virus to sufficiently high purity for testing in vivo using a combination of endonuclease treatment, depth filtration, anion exchange chromatography and hollow fiber tangential flow filtration (TFF)
ACAM529 prepared by the methods described here is 200-fold more pure with respect to host cell proteins and 2 orders of magnitude more pure with respect to the process stream contaminant dextran sulfate
Summary
HSV-2 is the primary cause of infectious ulcerative genital disease worldwide [1,2,3,4]. HSV-2 infects epithelial cells at the mucosal surface. It establishes latency by retrograde transport along nerve axons to the dorsal root ganglia [5]. The virus travels by anterograde transport to the skin surface where it can cause outward disease symptoms (genital ulcers, nonspecific symptoms or lesions and/or prodrome such as tingling, itching and pain) or may be asymptomatic. Worldwide there are an estimated 23 million new HSV-2 infections per annum [6], which underscores the clinical importance of this viral pathogen. As further evidence to the burden of this disease to US healthcare, Fisman et al estimate that the cost to treat HSV-2 infection could top $2.5 billion per year by 2015 [7] with care centered around oral antiviral treatment and in some cases prevention of transmission with valacyclovir
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