ACAM1000; Vaccinia virus, live, MRC-5 cell cultured
Status: not approved; no longer manufactured; controlled/government distribution
Organizations involved:
Joint Vaccine Acquisition Program (JVAP), DOD – R&D; Tech.; USA mark.
U.S. Department of Defense (DOD) – Parent org.
DVC LLC – Manuf.; R&D
DynPort Vaccine Company LLC – Former
Acambis plc – Manuf.; R&D; Tech.
BioReliance, Inc. – Manuf.; R&D.
Invitrogen Corp. – Former
OraVax, Inc. – Former
Salk Institute, The (TSI) – R&D; Tech.
Cross ref.: See the entries for Smallpox Vaccine Products (#535) and other smallpox vaccines.
Description: A new variola virus infection (smallpox) vaccine involving live lyophilized (freeze-dried) vaccinia virus cultured in the human diploid MRC-5 cell line has been manufactured under U.S. Department of Defense (DOD) contracts. This new cell cultured vaccinia virus vaccine is a formulation of live “Connaught” or Conn-Master 17633 (New York City Board of Health; NYCBH) strain of vaccinia virus cultured in the MRC-5 diploid human cell line and subjected to modern purification and quality control methods.
The new vaccine will be administered by the classic bifurcated needle scarification method. ACAM1000 is packaged in 100 dose vials, like Dryvax and ACAM2000 (see entry below), for reconstitution, with a dating period of 5 years stored at 2-8˚C (refrigerated).
ACAM1000 was originally primary slated for use by the U.S. military, with a later government contract granted for manufacture and maintenance of a stockpile of million doses. With ACAM1000 and ACAM2000 essentially being the same vaccine (same virus) cultured in different human host cells and with ACAM2000 further along in development and manufacture, ACAM1000 was “deselected,” and the government has consolidated its large-scale smallpox vaccine stockpiling efforts and switched to manufacture of ACAM2000 (see entry below), with ACAM1000 no longer being manufactured and existing stockpiles held in reserve in the U.S. biodefense stockpile.
Nomenclature: Smallpox vaccine/MRC-5 [BIO]; ACAM1000 [TR]; vaccinia virus, live [SY]; Cell Cultured Smallpox Vaccine [SY]; CCSV [SY]
Companies.: The Department of Defense (DOD) has used DynPort Vaccine Co, LLC, now DVC LLC, as its prime contractor for smallpox vaccine research, development and manufacture, as part of a 10-year, $322 million contract with DynPort for bioterrorism/biological warfare defense vaccine R&D and procurement granted by the Joint Vaccine Acquisition Program (JVAP), DOD, in fall 2000. Dyn-Port was originally a joint venture of DynCorp, a defense contractor, which was merged into Computer Sciences Corp., and Porton International Ltd., a subsidiary of Beaufour Ipsen S.A., now Ipsen S.A. In Aug. 2004, CSC acquired the 49% of DynPort owned by Porton/Ipsen, with the company now a fully-owned subsidiary of CSC and renamed DVC LLC.
In May 2003, the federal government consolidated its ACAM1000 and ACAM2000 R&D, manufacturing and stockpiling contracts into a single contract with Acambis plc, and a later phased out ACAM1000. manufacture of ACAM1000 was subcontracted to BioReliance Corp. (formerly Microbiological Associates, later part of Invitrogen Corp.). Invitrogen Corp. acquired Bio-Reliance in Dec. 2003, and later Acambis acquired BioReliance’s vaccine manufacturing facility in Rockville, MD. BioReliance was originally expected to produce 300,000 doses of ACAM1000 for DOD.
ACAM2000, not ACAM1000, is now the primary vaccine being manufactured in large-scale (over 200 million doses) for U.S. government use, including military and civilian use. ACAM1000 was manufactured in much smaller quantities for backup, in case it is needed (e.g., ACAM-1000 fails in trials), and is no longer being manufactured. The CDC cancelled its orders for undelivered ACAM1000, and instead ordered 54 million more doses of ACAM2000 to be delivered over the next 12 months, in addition to the 209 million doses of ACAM2000 already contracted for. This ACAM2000 contract will also be used to supply long-term, annual “warm-base’ production capability (“surge capacity”) for ACAM1000, i.e., the capabilities of rapidly scaling-up manufacture of ACAM2000 will be maintained. Presumably, JVAP and DOD will maintain small stockpiles of ACAM1000 for use, if needed.
History: ACAM1000 is based on a vaccine originally developed by the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID; Fort Detrick, MD). This was an injectable vaccine using cell cultured vaccinia virus manufactured by The Salk Institute (TSI). The product “was transitioned to advanced development” by the Army in Dec. 1993 with the filing of an IND (BB-IND 4984). The vaccine has shown safety in laboratory animal studies and multiple small clinical trials. A one-dose regimen has shown efficacy against aerosol challenge in non-human primates using the monkeypox model for variola virus infection. Phase I clinical studies in 1994 demonstrated safety. A Phase IIA trial conducted in 1999 sponsored by JVAP indicated this vaccine administered by the traditional skin scarification method had a take rate of over 90%.
Manufacture: The vaccinia virus NYCBH strain used for manufacture of ACAM1000 was derived from Dryvax (see #536). Samples of Dryvax from three lots were pooled, plaque purified, and ACAM1000 Master Seed virus was extracted from passage number seven (i.e, seventh cycle of culture in MRC-5 cells). This clonal virus provides a pure, consistent source for manufacture, unlike Dryvax vaccine, which has been shown to contain a diversity of vaccinia virus strains, including some with increased infectivity.
ACAM1000 was apparently manufactured using two 10-plate Nunc cell 500 L bioreactors. Comparisons of Nunc cell factory and perfusion parallel-plate (PPB) bioreactors for ACAM1000 manufacture were reported in “Production of Cell Cultured Smallpox vaccine (CCSV) in a Parallel-Plate Bioreactor (PPB),” M. Valle, et al., BioReliance Corp. and Dynport Vaccine Co., Abst. 229, Div. of Biochem. Tech., Amer. Chem. Soc. Nat. Meeting, March 28-April 1, 2004. The authors concluded the PBB method was “feasible and desirable over the current method consisting of Nunc cell factories,” but other authoritative sources report Nunc cell bioreactors are being used for manufacture of ACAM1000. Further information was obtained from,”Development of a Manufacturing Process for a Cell Culture-Derived Smallpox Vaccine,” presented at the same meeting (provided by BioReliance). Information about ACAM1000 downstream processing was disclosed in “Improved Vaccinia Virus Recovery for Cell-Cultures Smallpox Vaccine (CCSV),” Feldman, S., et al., BioReli-ance Corp. and Dynport Vaccine Co., Abst. 289, Div. of Biochem. Tech., Amer. Chem. Soc. Nat. Meeting, March 28-April 1, 2004. [Note, much of the following manufacturing information has been assembled and surmised from multiple sources).
The manufacturing process involves five unit operations - seeding, growth, infection, production and harvest. Seeding is accomplished over 2 days (one side of the modules/day) by introducing MRC-5 cells expanded in T-flasks and roller bottles into each module and allowing 3 hours for the cells to attach. The cell are then cultured at 37˚C. The culture medium used is apparently Dulbecco’s Modified Eagles Medium (DMEM) optimized for seeding density, culture volume, feeding frequency, and fetal bovine serum concentration (20%). About 1 week after seeding, culture is stopped, and infection of the cells with virus is achieved by introducing virus, e.g., at a multiplicity of infection (MOI) of 0.1 pfu/cell. About three days later, virus-infected cells are harvested, disrupted by homogenization (e.g., 4 cycles of sonication) and concentrated (e.g., sucrose cushion) for downstream processing. Titers of disrupted cells provide a yield of about 2.7-6.4 x 106 pfu/cm2.
Downstream processing includes membrane filtration for clarification of the cell homogenate; Benzonase treatment for enzymatic removal for DNA; and tangential flow filtration (UF/DF) for concentration and buffer exchange, resulting in 28-fold reduction in total protein and 60% recovery of virus. Benzonase is a recombinant Serratia marcescens endonuclease enzyme expressed in E. coli strain W3110 (strain of K12) which attacks and degrades all forms of DNA and RNA (single stranded, double stranded, linear and circular), resulting in complete digestion of nucleic acids. During development, filtration was identified as a unit operation resulting in major loss of product (>60% loss). Polypropylene filters with a pore size in the range of 1.2-5 µm provided significant improvement. The storage temperature of the infected cell harvest, cell density prior to disruption and homogenization, protein load of the feed stream prior to filtration, and cell density prior to disruption and homogenization, in this respective order of significance, were shown to impact product recovery (along with selection of filter material). Process improvements during development resulted in ~100-fold improvement in production, including an ~15-fold increase in virus production.
BioReliance’s Rockville facilities include 58,000 sq. ft. of manufacturing space; bioreactors expandable to 3,000 liter scale; sterile automated filling capacity of up to 40,000 units/lot; and a 110 sq. ft. Telstar lyophilizer able to handle ~30,000 vials/batch. A dry vacuum pump heats exhausted air to 170˚C to eliminate discharge of live microorganisms outside the plant.
FDA class: Biologic BLA
Status: Development and testing of ACAM1000 was initiated in Oct. 2000. FDA Master File BB-MF #10227 and IND BB-IND #10253 were submitted (and allowed) in Jan. 2002. Large scale manufacturing in bioreactors began in early 2003, but may have been halted prior to or soon after start. Several Phase I and II trials had been completed (see Trials section below), and a Phase III trial is in progress.
The DOD contracts included clinical development plans with the objective of obtaining FDA approval for this new and untested vaccine. Approval would be based on surrogate markers of efficacy, particularly evidence of “take” or induction of localized skin infection at the inoculation site (associated with efficacy using the older calf--derived vaccines), induction of antibody and cellular immune responses effective in vitro and in animal models against variola virus, and comparison with Dryvax vaccination. Distribution of the vaccine, particularly prior to FDA approval, is controlled by DOD.
This vaccine has been criticized by right-to-life and anti-abortion activists, because the MRC-5 cell line was derived from embryonic tissue from an aborted fetus.
Tech. transfer: The NYCBH strain of vaccinia virus has been in use for over a century and has long passed into the public domain (i.e., any patents have long expired). BioReliance and/or DynPort/DVC may have applied for manufacturing methods-related patents.
Trials: Initial Phase I clinical trial results with ACAM1000 were reported in late 2002. The trial compared the safety, tolerability, and immunogenicity of ACAM1000 and Dryvax (see related entry), the vaccine used in the U.S. until smallpox vaccination was halted in the early 1980s. Sixty subjects in this randomized double-blind trial received either the standard dose of Dryvax or an equivalent dose (in terms of viable vaccinia virus) of ACAM1000. A “take” or localized infection pustules, the primary surrogate marker of vaccine efficacy, was observed in 100% of ACAM1000 recipients (n = 30) and 97% of Dryvax recipients (n = 30). Lesion size and appearance were identical in the two groups. No serious or unexpected adverse events were reported with ACAM1000.
In April 2003, DynPort reported results from a double-blind Phase I trial comparing ACAM2000 and Dryvax in 350 vaccinia (Dryvax)-naive and vaccinia-experienced (having received Dryvax) health volunteers. Those receiving ACAM2000 has at least 8% lower incidence of vaccine-related fatigue, application site rash, fever, headache, lymphangitis, and nausea, compared to Dryvax recipients.
Index Terms:
Companies involvement:
Full monograph
539 Smallpox Vaccine/MRC-5
Nomenclature:
Smallpox vaccine/MRC-5 [BIO]
ACAM2000 [TR]
CCSV [SY]
Cell Cultured Smallpox Vaccine [SY]
Vaccinia virus, live, MRC-5 [SY]
variola virus vaccine [SY]
FDA Class: Biologic BLA
biopharmaceutical products
live microorganisms (as active agent)
vaccines, live
vaccines, viral
benzethonium chloride
bioreactors, 10,000 Liter
bioreactors, 10,000 Liter
cells, human
conjugates
fibroblasts, human
mammalian cell culture
MRC-5 human diploid fibroblast cells
neutropenia
Nunc cell factories
parainfluenza virus type 1, murine
perfusion bioreactors
Sepharose, Erythrina trypsin inhibitor (ETI)-
vaccinia virus vaccine
virus culture
benzethonium chloride
lyophilized (freeze-dried)
BHK-21 (C-13)
conjugates
North American coral snake
EU000 Not yet/Never filed with EU
UM100 Controlled/Gov't Distribution in US
US666 Biodefense stockpile
EM999 Not Available/Not Marketed in EU
Copyright© 2020, Biotechnology Information Institute