Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities Tod J. Merkela,1, Pin-Yu Pererab,1, Vanessa K. Kellya, Anita Vermaa, Zara N. Llewellync, Thomas A. Waldmannd,2, Joseph D. Moscae, and Liyanage P. Pererad,2 aCenter for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; bVeterans Affairs Medical Center, Washington, DC 20422; cSouthern Research Institute, Birmingham, AL 35255; dMetabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and eJDM Technologies, Ellicott City, MD 21042 Contributed by Thomas A. Waldmann, September 2, 2010 (sent for review May 26, 2010) Bioterrorism poses a daunting challenge to global security and smallpox vaccinations in the civilian population ceased over 30 y public health in the 21st century. Variola major virus, the etiological ago, and we are now faced with an ever-increasing naive pop- agent of smallpox, and Bacillus anthracis, the bacterial pathogen ulation, along with declining immunity in the older generation responsible for anthrax, remain at the apex of potential pathogens who were vaccinated in their childhood. Although the currently that could be used in a bioterror attack to inflict mass casualties. available licensed vaccine [ACAM 2000 (Acambis, Inc.), a cell- Although licensed vaccines are available for both smallpox and an- culture-grown derivative (7) of the earlier Dryvax vaccine used in thrax, because of inadequacies associated with each of these vac- the smallpox eradication campaigns] is highly effective, it can cines, serious concerns remain as to the deployability of these vac- cause serious complications in some vaccinated individuals, in- cines, especially in the aftermath of a bioterror attack involving cluding encephalitis, progressive vaccinia, myocarditis, or myo- pericarditis. Additionally, its use is contraindicated in patients these pathogens. We have developed a single vaccine (Wyeth/IL- with atopic skin diseases and immune deficiencies and during 15/PA) using the licensed Wyeth smallpox vaccine strain that is ef- – ≈ fi pregnancy. Unlike 3 4 decades ago, 25% of the individuals in cacious against both smallpox and anthrax due to the integration contemporary populations are estimated to represent “at-risk of immune-enhancing cytokine IL-15 and the protective antigen (PA) ” B. anthracis groups for whom live ACAM 2000 vaccine is contraindicated (7). of into the Wyeth vaccinia virus. Integration of IL-15 Thus, a safer vaccine that can match the efficacy of Dryvax/ fi renders Wyeth vaccinia avirulent in immunode cient mice and en- ACAM 2000 vaccine but that is devoid of its residual virulence is hances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred urgently needed for contemporary populations. sterile protection against a lethal challenge of B. anthracis Ames Anthrax caused by the bacterial pathogen B. anthracis clinically strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected manifests itself in cutaneous, gastrointestinal, and inhalational 33% of the vaccinated A/J mice against a lethal spore challenge 72 h forms, all of which can be fatal, although the inhalational form later whereas a single dose of licensed anthrax vaccine protected causes the highest mortality (8). A number of antibiotics such as only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequa- ciprofloxacin and doxycycline are very effective in eliminating cies associated with the licensed vaccines, and the inherent ability vegetative bacilli, but the ability of the antibiotic-resistant spores to of Wyeth vaccinia virus to be lyophilized without loss of potency remain dormant in the tissues (9) and subsequently reactivate to makes it cold-chain independent, thus simplifying the logistics of cause fatal disease increases the difficulty of treating this disease storage, stockpiling, and field delivery in the event of a bioterror with antibiotics alone. In fact, even with antibiotic coverage, attack involving smallpox or anthrax. mortality could be as high as 60–70% especially with the inhalation form. The pathogenesis of anthrax is driven by plasmid-borne γ biodefense | IL-15 | bioterrorism virulence factors that include the poly- -D-glutamic acid capsule and the tripartite exotoxin that consists of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA plays a central role he 2001 terror attacks in the United States have heightened in virulence by mediating the entry of LF and EF into cells. Col- Tthe potential threat of bioterrorism in the United States and lectively, these virulence factors promote the overwhelming sys- around the globe. Because of the purported past use of Variola temic pathophysiology associated with B. anthracis infection, often major and Bacillus anthracis in biowarfare and the reported pos- leading to a fatal outcome. The available evidence supports the sible existence of military programs for weaponization of these notion that induction of antibodies solely against PA can be pro- agents by some nations and rogue regimes, smallpox and anthrax tective against anthrax (10). PA is also the major component of represent the most deadly bioterror entities (1–3) that are a threat the anthrax vaccine licensed for human use in the United States as MEDICAL SCIENCES to our national security and public health. Therefore, the de- an aluminum hydroxide-adsorbed cell-free filtrate of an acapsular velopment of medical countermeasures against their potential strain (V770-NP1-R) of B. anthracis [Biothrax or anthrax vaccine use remains a national priority (4). adsorbed (AVA)]. In addition to concerns regarding adverse Smallpox is a highly communicable, often fatal disease with effects associated with this vaccine, its undefined composition, lot- mortality rates approaching 30% in susceptible individuals (5). to-lot variation, and the requirement for multiple doses over a Currently there is no approved treatment for smallpox although protracted period to achieve adequate levels of protective immu- vaccination within the first few days of exposure may provide some nity make this vaccine less than optimal for use in response to level of protection against the disease (6) (http://www.bt.cdc.gov/ agent/smallpox/vaccination/facts.asp). Variola virus, the etiologi- cal agent of smallpox, is a member of the Orthopoxvirus subfamily Author contributions: T.J.M., P.-Y.P., T.A.W., J.D.M., and L.P.P. designed research; T.J.M., of viruses, which also includes the vaccinia virus that induces P.-Y.P., V.K.K., A.V., Z.N.L., and L.P.P. performed research; Z.N.L. supervised rabbit immu- potent cross-protective immunity against Variola virus. The medi- nization and challenge studies under contract; T.J.M., P.-Y.P., A.V., T.A.W., J.D.M., and cal triumph of eradicating smallpox as a natural disease in 1977 L.P.P. analyzed data; and L.P.P. wrote the paper. was achieved with the use of a calf-lymph–derived live vaccinia The authors declare no conflict of interest. virus vaccine (Dryvax containing the Wyeth strain in the United 1T.J.M. and P-Y.P. contributed equally to this work. fi 2 States), thus attesting to the ef cacy and immunogenicity of To whom correspondence may be addressed. E-mail: [email protected] or tawald@ vaccinia viruses against Variola virus. In the United States, helix.nih.gov. www.pnas.org/cgi/doi/10.1073/pnas.1013083107 PNAS | October 19, 2010 | vol. 107 | no. 42 | 18091–18096 Downloaded by guest on October 2, 2021 a bioterrorism incident. Furthermore, the limited shelf life of ≈4y of Biothrax/AVA results in the need for periodic replenishment of vaccine in the strategic national stockpile. Therefore, a compelling need exists for a better vaccine against B. anthracis that can confer rapid immunity with an abbreviated immunization schedule that can be stored long term and deployed quickly in the event of a bioterror event. We recently reported that the integration of IL-15, a cytokine with pleiotropic immune modulatory activities, into the Wyeth strain of vaccina virus derived from the licensed smallpox vaccine resulted in the development of a smallpox vaccine candidate (Wyeth/IL-15) with superior efficacy and immunogenicity. This smallpox vaccine candidate out-performed the parental vaccine in Fig. 1. The expression of glycosylated B. anthracis PA by recombinant two animal models that sufficiently recapitulate the pathophysi- vaccinia viruses with an integrated IL-15 gene. BHK-21 cells were infected ology of smallpox in humans; mice were protected when lethally with Wyeth/IL-15/PA and MVA/IL-15/PA for 48 h, and the infected BHK-21 challenged with a neurotropic strain of vaccinia virus intranasally cell lysates were subjected to SDS/PAGE on a 10% acrylamide gel. Western and cynomolgus monkeys were protected from a lethal i.v. chal- blot analyses were performed using a polyclonal goat PA-specific antibody lenge of monkeypox virus (11, 12). Moreover, unlike the parental (List Biological Laboratories). The glycosylation of vaccinia virus-expressed virus, our Wyeth/IL-15 is nonlethal in T-cell–deficient athymic PA polypeptide in BHK21 cells was assessed by the treatment of vaccinia- nude mice at a dose of 107 plaque-forming units (pfu) and un- infected cell lysates with PNGase F enzyme (New England Biolabs) before dergoes enhanced clearance in these mice, attesting to its safety SDS/PAGE. The positions of molecular weight markers are on shown on left. even in immunocompromised hosts (12). Having demonstrated the superiority in immunogenicity and attenuation of virulence of fi – Immunogenicity and Ef cacy of Wyeth/IL-15/PA in Rabbits. To assess IL-15 integrated Wyeth vaccinia, we exploited our Wyeth/IL-15 the immunogenicity and efficacy of Wyeth/IL-15/PA in compar- platform to generate a dual vaccine effective against both smallpox ison with the licensed Biothrax/AVA, groups of female New and anthrax by integrating the PA gene of B.