Smallpox vaccines: circling the wagons.

Poxviruses are the largest and most complex viruses that infect humans. They belong to the genus Orthopoxvirus, family Poxviridae, which includes the viral agents vaccinia, monkeypox, cowpox, camelpox and ectromelia [1]. Pox-viruses are made of a single molecule of double-stranded DNA and have the ability to replicate in the cytoplasm rather than the nucleus of susceptible cells [2]. The linear genome contains approximately 200 genes, ranging in size from 130 to 260 kb, with those in the central region encoding proteins involved in virus uncoating, genome replication or virion structure. This large number of viral antigens which can serve as immune targets is an important issue for smallpox vaccine development and is likely to be at the core of why members of the pox-viral family generate crossreactive immunity that is broadly protective. The original vaccine, developed by Jenner in the late 1700s, was a cowpox virus derived directly from infected animals – 160 years later, the field tested and licensed a smallpox vaccine that was a lyophilized preparation of live vaccinia virus (VACV), made by using strain New York City calf lymph (NYC_CL), obtained from infecting cows through scarification. In 1979, following a worldwide vaccination campaign, eradication of naturally occurring smallpox disease was reached – perhaps the greatest success in the history of interventional medicine. No new vaccine was developed and the process for making the vaccine was laid to rest. It was not until the tragedy of 9/11 and the subsequent release of anthrax in the USA that the apparent risks of a deliberate release of smallpox on a vaccine-naive population became clear. Accordingly, a strategy to produce a new generation of smallpox vaccine was developed [3,4].

The eradication of smallpox was not achieved without a cost. This live virus vaccine was associated with reports of several adverse complications including dermatologic and CNS disorders, vaccinia necrosum, generalized vaccinia, pericarditis, arthritis, malignant tumors at vaccination scars and eczema vaccinatum [4-16]. Dermatologic and CNS disorders were the most frequently recognized adverse events, which included case fatalities that occurred almost exclusively in people with cellular immunodeficiency [12]. Such a risk rate is likely to be unacceptable today. Furthermore, times have changed and the number of people currently at risk for serious adverse events with the original vaccine is estimated to have grown dramatically. There is a large number of otherwise healthy individuals who are at risk of adverse events associated with the current VACV vaccine, including the elderly, pregnant women, non-VACV immunized people due to inadvertent spread, and individuals with certain skin conditions including eczema. Of concern is that a large number of individuals who are immunocompromised, including HIV-infected individuals, intravenous drug users, transplant recipients and individuals on immunosuppressive drugs living in North America in the case of a deliberate bioterrorism release of smallpox, would be potential vaccine recipients.

Recently, efforts have focused on developing a tissue culture preparation of the original VACV preparation [17]. In theory, it was reasoned that such a passaged preparation would be more attenuated and therefore safer than the original vaccine. Accordingly, Weltzin and colleagues at the biotechnology company Acambis adapted the existing DryVax® vaccine, which is derived from the crossprotective vaccinia virus, to a human cell line for production in tissue culture. These investigators selected a viral stock that appeared to have some attenuation benefit regarding decreased pathogenesis in vitro and decreased neurovirulence in an animal model. In a small clinical study in humans, 100% of subjects vaccinated with the candidate vaccine exhibited the hallmark of vaccine take, a significant cutaneous reaction at the site of inoculation resulting in scarification, compared with 97% in DryVax-vaccinated subjects. Furthermore, the viral strain appeared to be immunogenic in animals and humans, inducing both antibody and cytotoxic T-lymphocyte responses. Accordingly, large field trials for the assessment of immunogenicty were undertaken. Unfortunately, field trials of this tissue culture-adapted vaccine were halted due to a high adverse event rate; in particular, three cases of myocarditis were observed. A more detailed look at the results of these studies are highly informative. All three cases of myocardial adverse events were in vaccine-naive individuals. A side-by-side study of the vaccine in 1752 individuals who had previously received the older smallpox vaccine did not show the same adverse events. This study is highly important as it demonstrates that prior vaccination will further attenuate vaccine-induced side effects and that more intense efforts will be needed to generate less virulent replacement vaccines for the primary immunization phase of a smallpox vaccine effort to limit adverse events.

These studies allow several conclusions to be drawn. The simplest is that the present Acambis vaccine is likely to be a good approach for previously vaccinated populations. Reboosting of these populations is likely to provide increased benefit in the event of a smallpox release. More complex conclusions regarding the immunization of pox vaccine-naive subjects can also be drawn. One is that immunization of vaccine-naive individuals with ACAM2000 should only be considered in the event of a smallpox outbreak, due to the possibility of vaccine-induced complications and imminent danger to the population. A second is that the lack of a current strategy for testing a completely new smallpox vaccine prototype in the field suggests that abandoning the currently effective vaccine is also not an easy option. Importantly, studies aimed at determining potential immune correlates for novel vaccine development are critical. Alternatively, the development of a more attenuated vaccine strategy to be used as a prime for the Acambis vaccine is likely to be a highly promising concept that would take advantage of the best of both strategies. The more mild attenuated approach would not necessarily be guaranteed to be as effective in protection against virulent smallpox. However, it is likely to provide a good immune prime which would then limit adverse events associated with the vaccine-naive status. Recently, an attenuated MVA was developed that appears to be promising in nonhuman primate models [18]. Earl and colleagues compared a highly attenuated MVA with the licensed DryVax vaccine in a monkey challenge model. After two doses of MVA or one dose of MVA followed by DryVax, antibody binding and neutralizing titers as well as T-cell responses were equivalent or higher than those induced by DryVax alone. Furthermore, challenge of vaccinated animals with monkeypox virus demonstrated that nonimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic. These studies are very encouraging. Recently, the description of a DNA vaccine approach, effective at limiting poxviral pathogenesis in a nonhuman primate model, was also reported [19]. Hooper and colleagues tested the efficacy of a four poxviral antigen cocktail for their immunogenicity and protective efficacy in a rhesus macaque model system. Plasmids encoding four vaccinia virus antigens (L1R, A27L, A33R and B5R) were developed and administered as DNA by gene-gun immunization in rhesus macaques. This vaccination strategy was able to induce significant immunogenicity and protection from severe disease following challenge with monkeypox virus [19]. The authors selected these four immunogens due to their role as targets of neutralizing or otherwise protective antibody responses.

These, and other more attenuated approaches, may represent reasonable primers for such a two-step immunization protocol. It will be important to assess the effects of the primers on the resulting immunogenicity induced following the boost with the Acambis replication-competent vaccine. In theory, this boost should broaden the immune response, but due to the priming, does not result in the previously observed significant adverse events. One concern is that if the prime approach induces a strong neutralizing antibody response, then the Acambis vaccine will not provide a significant boost to the cellular immune response. In this regard, studies in another model of the efficacy of recombinant adeno-viral vaccines have demonstrated that prior neutralizing antibody responses will completely prevent the ability of these recombinant viruses to induce a cellular immune response in humans. These experimental systems suggest that developing a strong cellular antipoxviral immune response with the priming strategy will be the most useful in such a prime–boost protocol. A strong cellular response, induced by a priming vaccination in the absence of a neutralizing antibody response, will not prevent infection and boosting by the live vector. However, this cellular response should be sufficient to limit pathogenesis of the live vector boost. Studies identifying potential candidate protective cellular antigens will be important to test such a hypothesis. These strategies bring us closer to a full protection option against smallpox for non-immunocompromised populations. However, more creative approaches, such as the development of self-adjuvanting vaccine approaches providing their own immune help, may be required in the most severe at-risk populations. Work in these areas should be encouraged.