Immunity to smallpox and vaccinia: the future of smallpox vaccines

The last case of smallpox occurred in 1977 [1]. Despite the absence of cases, recent decades have brought major advances in our understanding of the structure, function, genetics and immunology of smallpox and other orthopoxviruses. Orthopoxviruses are the largest and most complex DNA viruses that infect humans [2]. A number of strains of smallpox and vaccinia have been completely sequenced and have remarkably similar and stable genomes. Vaccinia, the most readily accessible member of the family, has linear double-stranded DNA containing at least 200 genes, with approximately one half involved with the construction of the virions, and about half devoted to interactions with the host immune system. These genes orchestrate a very complex replication process, involving multiple forms of the virus, inside and outside human cells and elaboration of several immunomodulatory proteins [2–5].

The immune response to infection with vaccinia (and presumably smallpox) is correspondingly complex. Initial infection involves natural killer cells, interferon, the complement system, inflammatory cells and, if the host has had previous experience with an orthopoxvirus, neutralizing antibodies and sensitized cytotoxic lymphocytes. Viral construction takes place in the cytoplasm and switches the cell from host to viral protein synthesis. The viral genes cause the infected cells to secrete a number of proteins that are immunosubversive and modify the host’s response. Some three dozen structural proteins are produced, as well as approximately two dozen viral enzymes. These create a rich soup to which the host’s immune system must respond. This response is, in turn, complex.

Our understanding of the mechanism by which the immune system protects us against smallpox remains imperfect. Hemaglutination-inhibiting antibodies and complement-fixing antibodies do not seem to be major protective elements. Complement-fixing antibodies do not appear in all vaccinees and are usually gone 2 years after vaccination [1,6]. Hemaglutination-inhibiting antibody titers drop to very low levels within 5 years of vaccination and frequently do not have an anamnestic response after revaccination, while clinical protection persists much longer [1,6]. Neutralizing antibodies appear to be a better candidate. They persist for many years and get a vigorous boost on revaccination [6–9]. Unfortunately, there is considerable variation from one laboratory to another in methods of measuring titers of neutralizing antibodies. In one animal model using monkeys infected with monkeypox (monkeys are not the normal ecological host of monkeypox virus, but when given monkeypox, their clinical disease has many similarities to human smallpox), circulating neutralizing antibodies appear to be both necessary and sufficient to limit infection to a nonlethal exanthem [10]. However, there is no specific titer known to correlate well with clinical protection of humans against smallpox and the few trials done using intramuscular injections of neutralizing antibodies (in the form of vaccinia immune globulin) failed to provide complete protection [11]. Efforts are underway to make high concentration monoclonal human antibodies to smallpox itself (as opposed to vaccinia) by recombinant techniques. This will start with antibodies taken from patients who have recovered from smallpox. Such a preparation should be more protective than vaccinia immune globulin, but there are no data to prove so.

Work with other orthopoxviruses suggests that while both cellular and humoral immunity are important, cytotoxic T cells appear to provide good protection [12–14]. There are insufficient data to permit us to make a dogmatic statement about the relative importance of cell-mediated and humoral antibody in vaccinia-induced protection from smallpox. Historically, the simplest indicator of cellular immunity, and a good indicator of clinical protection, was a severely modified dermal reaction to challenge with vaccinia virus. Unfortunately, this is nonquantitative and difficult to use to compare individuals. Patients who lack competent T cells develop progressive vaccinia when vaccinated, even if they had immunity before developing their T-cell defect [15,16]. Patients who have defects in antibody production but have a good cell-mediated immune response usually develop normal primary vaccinations [1]. In mice, CD8⁺ T cells seem to protect against disease, but CD4⁺ T-cell-dependent antibody production is important in clearing the virus during acute infection [12,13].

These and many other studies of humoral and cell-mediated immunity show a complex pattern of immunity, which may vary from one animal species to another and from one orthopoxvirus to another. Perhaps this should not be surprising given the large number of genetically mediated mechanisms that these viruses have, and their complex life cycle.

There is a consensus that we need an improved smallpox vaccine. The current licensed product is Dryvax^®, a New York City Board of Health strain vaccinia grown on the skin of live calves. It protects against smallpox but is associated with many well-known adverse events, particularly in patients with compromized immune status [15,16]. The 2002–2003 vaccination efforts brought to light the occurrence of vaccine-associated myopericarditis, probably the result of circulating immune complexes [17,18]. Dryvax is considered a first-generation vaccine. An improved second-generation vaccine will probably be licensed shortly. It is a single clone isolated from Dryvax, grown in cell culture so it is free of adventitial viruses and bovine antigens [19]. Several third-generation vaccines are being investigated. These are either attenuated by traditional means (serial passage on eggs or tissue culture, such as Modified Vaccinia Ankara and LC16m8) or by conscious deletion of genes believed to be partly responsible for pathogenicity (NYVAC) [20–22].

Postexposure vaccination with a first-generation vaccine can prevent smallpox if given within approximately 3 days of exposure [23]. Presumably, the same would be true of a second-generation vaccine, since it is made with the same virus. Perhaps pre-exposure vaccination with one of the third-generation vaccines can enhance that effect and make it safer. If people with contraindications to vaccination acquire some basic immunity from immunization with a third-generation vaccine, then they would have an anamnestic response to revaccination with a full strength first- or second-generation vaccine. They would be protected from smallpox more rapidly than primary (first-time) vaccinees, and would (probably) be protected from major adverse events. Vaccinia strains are good vaccines against themselves, as well as against smallpox.

How can we decide whether these third-generation vaccines are protective? If we cannot prove their efficacy perfectly, how will we use them? Obviously we cannot challenge vaccinees with smallpox. Animal models are improving, but currently there is no animal model that fully mimics human smallpox [24]. Since we have no data set from the era of intense research on using actual smallpox patients (approximately 1950–1975), which established a level of humoral antibodies, or of any given measure of cellular immunity, which was protective, we have no surrogate index of efficacy of vaccines. The US FDA has suggested an animal rule by which the candidate vaccine must be equal to or superior to first-generation vaccinia in two different animal models.

The US federal government has taken steps to stockpile large quantities of second-generation vaccine, in addition to the limited supply of first-generation vaccines. The government has also stated that it will purchase third-generation vaccines for the stockpile, even if they are not licensed in the traditional manner. However, the government has not enunciated a policy for the use of these vaccines. We will have the vaccines but, in the horrible event of a bioterrorist attack, we will have very little time to debate how to use them. I personally would not want to use a vaccine that has not been proven to protect against smallpox for patients who have been exposed to the disease. I am unsure whom to vaccinate at present, in the absence of a clear threat of a bioterrorist attack. I would suggest that we should start discussing this issue now.