Smallpox Vaccines for Biodefense: Need and Feasibility

Andrew W Artenstein; John D Grabenstein

Disclosures

Expert Rev Vaccines. 2008;7(8):1225-1237. 

In This Article

Efficacy of Smallpox Vaccines

The historical premise underlying traditional smallpox vaccines, that of a localized infection with either variola or a cross-reactive orthopoxvirus leading to immune protection, was established long before Jenner published his treatise on vaccination in 1798,[13] and continues to guide the development of newer-generation smallpox vaccines ( Table 1 ). First-generation smallpox vaccines comprising a variety of live vaccinia viruses were used for protection against smallpox yet were hampered by uncommon, potentially life-threatening adverse events that limited their use in the absence of substantial disease risk.[14] This, in concert with concerns regarding the threat of smallpox as a potential agent of bioterrorism has prompted recent efforts toward developing new vaccines with a focus on enhancing safety while maintaining efficacy.

First-generation smallpox vaccines possess a proven track record of clinical effectiveness, highlighted by their success in the global eradication campaign of the 1970s.[3] While immune determinants of protection against smallpox remain incompletely understood, the historical record provides ample data concerning a clinical correlate of protection in humans; observations from the use of variola, cowpox and vaccinia viruses document the direct relationship between a vaccine-associated major cutaneous reaction, or 'take', and protection against smallpox.[3,14,15] The protection appears to be of long duration and to correlate with the presence of neutralizing antibodies.[3] The cellular arm of the immune response is also known to have a significant role in containing vaccinia[12] and, by extrapolation, variola. Smallpox vaccination induces robust vaccinia-specific cytotoxic T lymphocytes (CTLs) and IFN-γ production by T cells in naive recipients, and these may correlate with neutralizing antibody responses.[16]

The original production method of first-generation vaccines involved scarification of calf, sheep or water buffalo skin and viral isolation from skin scrapings containing pus, serum and extruded lymph.[3,17] The resultant liquid suspension of vaccine or 'wet' lymph contained viable bacteria, primarily skin commensals, which were minimized by the use of glycerol and, later, phenol in processing.[3] By the 1950s, liquid vaccine lymph preparations had largely been replaced by lyophilized preparations that enhanced preservation of vaccinia virus viability.[3] Vaccine production by animal scarification was abandoned more than 25 years ago and, because smallpox had been eradicated, essentially no first-generation vaccine has been manufactured since then. This led to the view in 2001 that the stockpiled supply was insufficient to cope with a potential large-scale bioterrorist threat. The stockpile consisted of lymph-derived vaccinia, mainly the last production lots of Dryvax®-brand smallpox vaccine, manufactured by Wyeth Laboratories using the New York City Board of Health (NYCBH) strain of vaccinia. Multiple studies have since demonstrated that existing stockpiles can be expanded by diluting the vaccine; lymph-derived, live vaccinia products retain surrogate clinical efficacy at tenfold dilutions in both vaccinia-naive and vaccinia-experienced subjects.[17,18]

Second-generation smallpox vaccines ( Table 1 ), in which full-strength vaccinia virus is grown in tissue culture rather than in the skin of large mammals, possess theoretical advantages conferred by this modern manufacturing technique: lowered risk of contamination by adventitious agents,[19] viral genetic homogeneity and relative ease of large-scale, consistent production. ACAM1000, a clonal isolate derived from Dryvax and grown in human diploid lung cells (Medical Research Council [MCR]-5), demonstrates similar immunogenicity and cutaneous efficacy at comparable doses to the Dryvax gold standard in animal models, and demonstrates an improved safety profile in preclinical neurovirulence studies in suckling mice and rhesus macaques.[20,21] ACAM2000™, derived from the ACAM1000 master virus by three additional passages in Vero cells,[22] has nearly identical biological characteristics to those of its progenitor in animals.[23]

Randomized Phase II and III clinical trials, in which nearly 1100 vaccinia-naive subjects were vaccinated with ACAM2000, demonstrated its noninferiority compared with Dryvax at similar vaccinia virus inocula, using cutaneous responses (i.e., takes) as an efficacy end point; ACAM2000 did not meet the noninferiority measure using geometric mean neutralizing antibody titers (GMT) on day 30 after vaccination as another efficacy end point.[22,108] In vaccinia-experienced subjects, ACAM2000 only met the noninferiority threshold for the GMT end point but not for cutaneous responses.[108] Nonetheless, in August 2007, ACAM2000 became the initial second-generation smallpox vaccine to be licensed for human use by the US FDA, leading to the delivery of 192.5 million doses to the US government for stockpiling purposes.[109] The vaccine received the following clinical indication: 'active immunization against smallpox disease for persons deemed to be at high risk for smallpox infection'.[108] ACAM2000 is not expected to be commercially distributed in the USA in order to minimize its use and, therefore, its risk.[110] CCSV, another second-generation vaccine grown in MRC-5 cells, compared favorably with Dryvax in a single-center study of 150 vaccinia-naive and 100 vaccinia-experienced subjects.[24] However, this agent was apparently 'deselected' by the manufacturer for further advancement.

Despite the theoretical advantages conferred by second-generation vaccines, they comprise replication-competent, virulent vaccinia viruses and, therefore, possess the potential for a number of uncommon but well-described serious adverse events associated with first-generation smallpox vaccines.[14] Alternative candidates based on attenuated vaccinia strains, third-generation vaccines, may offer more favorable therapeutic ratios.

LC16m8, a replication-competent, highly attenuated vaccinia strain, derives from 53 serial passages of a Lister strain isolate in rabbit kidney cells.[25] LC16m8 appears to be less neurovirulent in animals than unattenuated Lister strain vaccinia[26,27]; its use in more than 100,000 Japanese children in the 1970s demonstrated take rates and neutralizing antibody responses similar to those of lymph-derived smallpox vaccines.[27,28] However, the vaccine was never formally field tested, as smallpox was no longer an epidemic threat in Japan at the time.

Recently, LC16m8 was shown to engender complete protection in both a rabbit model using intradermal rabbitpox challenge and a mouse model using aerosolized ectromelia (i.e., mousepox) virus.[29] In the mouse model, LC16m8-vaccinated animals developed higher vaccinia-specific neutralizing antibody titers, enhanced neutralization of intracellular mature virus (IMV) and comparable capacity to neutralize extracellular enveloped virus (EEV), compared with Dryvax-vaccinated animals.[29] The latter finding is reassuring in that the B5R gene, required for EEV formation, but deleted during the attenuation process in LC16m8, is a neutralizing antibody target. Additional data suggest that LC16m8 may be a safer alternative to unattenuated vaccine strains in immunocompromised hosts. While comparable protection is noted between LC16m8 and Dryvax in a BALB/c mouse vaccinia challenge model, LC16m8 is nonlethal to severe combined immunodeficiency mice.[30,31] Combined data from trials involving nearly 1700 vaccinia-naive subjects demonstrate 95% take rates and neutralizing antibody seroconversions with LC16m8,[32,33] similar to rates reported with first- and second-generation vaccines in naive individuals.[22]

Modified vaccinia Ankara (MVA) strain, a replication-defective, highly attenuated vaccinia virus was initially used as a priming vaccine followed by first-generation smallpox vaccination in more than 120,000 primary vaccinees in Germany in the 1970s.[34] It is attenuated via 570 serial passages in chicken embryo fibroblasts leading to DNA deletions in approximately 15% of its genome, including genes related to host range and immune evasion; thus MVA is generally replication incompetent in mammalian cells.[35] It has been advanced as a third-generation alternative vaccine of potential utility in immunocompromised hosts in whom live vaccinia vaccines are generally contraindicated.[36] Theoretically though, MVA may regain the potential for growth in certain mammalian cell lines owing to reversions at the nucleotide level.[35]

Unlike replication-competent vaccinia, MVA does not result in stereotypical neurovirulence upon intracerebral inoculation of suckling mice and may protect against subsequent intracerebral live vaccinia challenge.[35] Additionally, MVA is not associated with detectable viral replication in irradiated mice and rabbits and protects irradiated mice against live vaccinia challenge.[35] Immunosuppressed cynomolgus macaques demonstrate no significant clinical, hematological or pathological abnormalities following inoculation with high-dose MVA by multiple routes, although vaccinial genomes are detectable by PCR from tissues in the majority of macaques.[37]

Modified vaccinia Ankara strain is immunogenic and protective in both normal and variably immunosuppressed mice.[38,39] However, animals clearly require multiple and higher doses of MVA to achieve comparable antibody titers to those induced by replication-competent vaccinia,[38] and immunosuppressed macaques may fail to develop MVA-specific IgG responses despite high vaccine doses.[37] In comparisons of first-generation vaccinia virus, LC16m8 and MVA, the latter appears to be the least immunogenic, requiring 100-fold more virus to produce similar response levels.[30]

Modified vaccinia Ankara strain protects cynomolgus macaques from lethal intravenous[40] or respiratory[41] monkeypox challenges. Such studies confirm data in mice that high-dose MVA or priming with MVA followed by vaccination with first-generation vaccinia virus is necessary to generate immune responses and protection analogous to those observed with replication-competent vaccinia virus alone.[40,41,42,43] In some cases MVA-immunized animals, while protected against lethal disease, develop pox lesions following viral challenge; thus, this product may not abrogate the transmission potential of orthopoxviruses.

In humans, MVA induces neutralizing antibodies in only 50% of naive subjects receiving a single dose; whereas 80% seroconvert after two doses.[44] The magnitude and duration of humoral immune responses are dose dependent; the proportion of subjects with neutralizing antibodies diminishes by at least half within 3 months following the second dose.[44] Vaccinia-experienced subjects demonstrate more rapid seroconversion or a boosting response and more durable antibody levels after a single dose of MVA.[44] When employed as a priming vaccine in vaccinia-naive subjects, MVA induces a 'modified-take skin reaction' with or without a vesicle upon Dryvax challenge 3 months later, similar to cutaneous responses observed in vaccinia-experienced subjects primed with MVA or administered Dryvax alone.[45] Priming with multiple doses of MVA decreases cutaneous viral shedding after Dryvax challenge in naive subjects. Neutralizing antibody titers are comparable among the vaccinated groups; higher vaccinia-specific CD8+ CTLs are noted in those receiving multiple doses of MVA than in those administered one dose of MVA or Dryvax alone.[45] In summary, MVA modifies the cutaneous reactogenicity of live vaccinia without altering its immunogenicity, and multiple MVA priming doses may enhance immune responses to live vaccinia products.

Other attenuated, replication-defective vaccine candidates may show promise as priming agents in immunocompromised hosts. NYVAC, derived from the Copenhagen vaccine strain of vaccinia and attenuated by the deletion of 18 nonessential open reading frames,[46,47] modulates the effects of Dryvax when used as a priming agent in immunodeficient rhesus macaques,[48] yet fails to protect macaques with AIDS against a lethal, intravenous monkeypox challenge.[49] A replication-defective derivative of the Lister strain of vaccinia, bioengineered by deleting the gene encoding for an essential replication cycle enzyme, uracil-DNA-glycosylase,[50] has similar preclinical characteristics to MVA, but is theoretically unable to revert to virulence because it only grows in permanent cell lines capable of complementing the enzyme deletion.[50,51]

Subunit products are also under investigation as alternative smallpox vaccines. Limited preclinical data support the immunogenicity and protective effect of a vaccinia envelope protein, H3L, in BALB/c mice; passive transfer of H3L-neutralizing antibodies also appears protective.[52] Multiple immunizations with combinations of three outer membrane proteins of IMV (e.g., L1 and A27) and EEV (e.g., A33 and B5) or with combinations of the genes encoding these proteins, are protective in mice and macaque models.[53,54] The latter approach prevents viremia in immunized, challenged monkeys.[54] Animals primed with plasmid DNA encoding the four proteins, then boosted with the analogous proteins, survive lethal monkeypox challenge with significantly milder disease than those immunized with the proteins alone.[55]

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