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Vaccinia Viral Vectors

Vaccinia virus (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome of approximately 190 kb in length, which encodes for around 250 genes. The genome is surrounded by a lipoprotein core membrane. The poxviruses are the largest known DNA viruses and are distinguished from other viruses by their ability to replicate entirely in the cytoplasm of the host cell, outside of the nucleus.

The dimensions of the virion are roughly 360 × 270 × 250 nm (see figure 1). Vaccinia virus is well-known for its role as a vaccine that eradicated the smallpox disease, making it the first human disease to be successfully eradicated by mankind. This endeavour was carried out by the World Health Organization (WHO) under the Smallpox Eradication Program. Post eradication of smallpox, scientists study Vaccinia virus to use as a tool for delivering genes into biological tissues (gene therapy and genetic engineering).

The natural host of Vaccinia virus is unknown, but the virus can replicate in cows and humans. During its replication cycle, Vaccinia virus 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.

Vaccinia virus can accept as much as 25 kb of foreign DNA, making it useful for expressing large genes. Foreign genes are integrated stably into the viral genome, resulting in efficient gene expression. Vaccinia viruses re-engineered to express foreign genes are robust vectors for production of recombinant proteins. Vaccinia viruses have been engineered to express immunizing antigens of herpesvirus, hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses.

Figure 1. An electron micrograph of a Vaccinia virus.

Concerns about the safety of the vaccinia virus have been addressed by the development of vectors based on attenuated Vaccinia viruses. Recombinant vaccinia viruses have been created from different strains of Vaccinia virus. Certain highly attenuated, host-restricted, non- or poorly replicating poxvirus strains have been developed for use as substrates in recombinant vaccine development. These strains include the Orthopoxviruses, Modified Vaccinia Ankara (MVA) and NYVAC (derived from the Copenhagen vaccinia strain), and the Avipoxviruses, ALVAC and TROVAC (derived from canarypox and fowlpox viruses, respectively).

The attenuated strains of Vaccinia virus have been reported to be avirulent and safe among humans. Because of the biological properties and accumulated attenuation data for MVA, NYVAC, ALVAC, and TROVAC, the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health (NIH) reduced the biosafety level for these viruses to lowest biosafety level. The Occupational Safety and Health Board of NIH no longer requires vaccinia (smallpox) vaccination for personnel manipulating MVA or NYVAC in a laboratory where no other vaccinia viruses are being manipulated.

The Modified Vaccinia Ankara (MVA) virus, is a highly attenuated strain of vaccinia virus that was developed towards the end of the campaign for the eradication of smallpox in the seventies of the previous century. MVA was derived from Vaccinia strain Ankara by over 570 passages in chicken embryo fibroblast cells (CEF). This resulted in six major deletions corresponding to the lost of about 10% of the vaccinia genome. The complete genomic sequence is known and has a length of 178 kp corresponding to 177 genes. The numerous mutations explain the attenuated phenotype of MVA and its inability to replicate in mammalian cells.

MVA is widely considered as the Vaccinia virus strain of choice for clinical investigation because of its high safety profile. MVA has been administered to numerous animal species including monkeys, mice, swine, sheep, cattle, horses, and elephants, with no local or systemic adverse effects. Over 120,000 humans have been safely and successfully vaccinated against smallpox with MVA by intradermal, subcutaneous, or intramuscular injections. Studies in mice and nonhuman primates have further demonstrated the safety of MVA under conditions of immune suppression. Compared to replicating vaccinia viruses, MVA provides similar or higher levels of recombinant gene expression even in non-permissive cells. In animal models, recombinant MVA vaccines have been found immunogenic and to protect against various infectious agents including influenza, parainfluenza, measles virus, flaviviruses, and plasmodium parasites. The combination of a very good safety profile and the ability to deliver antigens in a highly immunogenic way makes MVA suitable as a vaccine vector.

The attenuated NYVAC vector has been derived from a plaque-cloned isolate of the Copenhagen vaccinia vaccine strain by the deletion of 18 open reading frames (ORFs) from the viral genome. The deleted genes encode for virulence factors and human host range replication proteins. The result is a highly attenuated Vaccinia virus that retains the capacity to express foreign genes and produce an immune response in a immunized host. Due to the deletions of both the C7L ad K1L host range genes, NYVAC cannot replicate in human cells. Just like MVA, NYVAC is being extensively studied in clinical vaccination trials against numerous infection diseases, including HIV.

ALVAC is an attenuated canarypox virus vector that was a plaque-cloned derivative of Kanapox, a licensed canarypox vaccine. The ALVAC attenuated poxvirus vector replicates only in avian species. ALVAC is a ubiquitous vector with high biosafety and is not known to replicate in mammalian cells. This feature provides an important safety barrier for human use.

On human cell cultures, ALVAC replication is aborted early in the viral replication cycle prior to viral DNA synthesis. The canarypox vaccine vector technology has been used to develop several vaccines for animals, horses as well as humans. A number of clinical trials in humans are ongoing using for instance ALVAC HIV-1 vaccines.

TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus, which is licensed for vaccination of one day old chicks. Just like ALVAC, these viruses do not grow in mammalian cells and, therefore, do not produce clinical infections among humans.