Development of Smallpox Virus Vaccines

Virus structure and life cycle

Smallpox was an infectious disease caused by variola virus (often called smallpox virus) which belongs to the genus orthopoxvirus, poxvirus family. They are unique among DNA viruses because they replicate only in the cytoplasm of the host cell, outside of the nucleus. Therefore, the large genome is required for encoding various enzymes and proteins involved in viral DNA replication and gene transcription. During its replication cycle, VV produces four infectious forms which differ in their outer membranes: intracellular mature virion (IMV), the intracellular enveloped virion (IEV), the cell-associated enveloped virion (CEV) and the extracellular enveloped virion (EEV). Although the issue remains contentious, the prevailing view is that the IMV consists of a single lipoprotein membrane, while the CEV and EEV are both surrounded by two membrane layers and the IEV has three envelopes. The IMV is the most abundant infectious form and is thought to be responsible for spread between hosts. On the other hand, the CEV is believed to play a role in cell-to-cell spread and the EEV is thought to be important for long range dissemination within the host organism ^[2-4].

The genome of orthopoxviruses is a double-stranded DNA with covalently closed ends, varying in length from 186 to 230 kbp. The central genomic region, with a length of about 100 kbp, containing 102 genes, is conserved for all of the species belonging to this genus. The species-specific distinctions of the orthopoxvirus genome are most pronounced in their terminal variable regions. The very first comparisons of the DNA nucleotide sequences of various VARV strains demonstrated a high conservation of their genomes and revealed specific differences between the South American and the studied Asian, Central African, and East African VARV strains. VARV has the smallest genome (186 kbp) among the orthopoxviruses and presumably evolved from an orthopoxvirus with a larger genome and a wide host range similar to cowpox virus via deletions and mutations in a number of genes localized to the terminal variable genomic regions ^[5,6].

First-generation VACV vaccines and global smallpox eradication

Because all orthopoxviruses are antigenically related, immunization with nearly any orthopoxvirus can protect against challenge with other orthopoxviruses. In the late 1700s, Edward Jenner discovered that cowpox virus could be used successfully to protect humans against smallpox. Since vaccination with cowpox was much safer than variolation, it quickly became the primary method of conferring protection. Over time, VACV replaced cowpox virus as the agent used for vaccination. An intensified global smallpox eradication program was begun in 1966. The basic strategy for this program involved mass vaccination campaigns in each country and the development of surveillance systems to detect and contain outbreaks. Typically following vaccination, a vesicular or pustular skin lesion at the site of inoculation is indicative of a successful vaccination or "take" ^[7].

Third-generation vaccines: attenuation through sequential passage

One commonly used technique to attenuate VACVs involves multiple passaging of the wt viruses in tissue culture cells from alternative hosts, which has been shown to alter properties such as viral host range, virulence, and genome composition.

VACV LC16m8, DIs and MVA have all been obtained by passage on alternative hosts. Nonetheless, while there are some similarities among the three viruses, there are numerous differences as well. DIs and MVA were both obtained by passage in chicken cells, and both have large deletions in the host range determining region, which makes both viruses replication defective in most mammalian cells. These additional mutations in innate immune evasion genes in MVA may be responsible for the unique ability of this virus, among non-genetically engineered orthopoxviruses, to induce proinflammatory signal transduction, proinflammatory gene expression, and to lead to an increased migration of immune cells to the site of immunization. The ability of MVA to induce proinflammatory cytokines may have a profound influence on its ability to induce adaptive immunity. LC16m8 was obtained by passage through rabbit kidney cells, and rather than having large deletions it has one single base pair deletion in B5R, and likely has other small mutations that alter gene function to give the temperature restriction and low pathogenicity of this virus. LC16m8 has an intact host range determining region, and appears to be replication competent in most mammalian cells. As a replication competent virus LC16m8 may have properties closer to a wt-VACV than DIs or MVA. However, the failure to make EV may at least potentially affect the efficacy of LC16m8 as a replacement smallpox vaccine ^[9].

Summary

The eradication of smallpox combined with the advent of genetically engineered recombinant poxvirus technology demonstrated the feasibility of using VACVs to protect against specific pathogens. With the relative ease of generating recombinant VACVs to express heterologous genes, widespread attention has been given to the idea of using these vectors as a vehicle for an antigen delivery system against different diseases. Replication-competent recombinant VACV-based vaccines have received increasing attention as potential vaccine vectors for many infectious diseases, since they are presumably able to elicit potent humoral and cell mediated immune responses, and confer lasting protection. Different approaches have been taken to lower the virulence and enhance the immune response in an effort to generate a safe vaccine platform. We are confident that continued efforts to genetically modify poxvirus vectors will not only improve our understanding of these viruses but also allow preservation of safety while taking advantage of the immunogenic benefits of replication competence.