PLoS One. 2013 Oct 9;8(10):e75574.

A Vaccine made of two viruses: Combined Poxvirus/Alphavirus vaccine vectors

(original title: A Vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles)

Juana M. Sánchez-Puig, María M. Lorenzo and Rafael Blasco

Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Madrid, Spain



Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well established vaccine vectors having large insertion capacity, excellent stability and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.



Live recombinant viruses constitute outstanding tools for the prevention or treatment of many diseases, including infectious diseases and cancer. Among the available vectors with excellent immunization power, Poxviruses and Alphaviruses are prominent. Representative members of those virus families, Vaccinia Virus and Semliki Forest Virus are well characterized, can be easily modified by genomic engineering, and have been shown to mediate expression of foreing genes in a wide variety of cell lines and animals. Vaccinia virus, used for over 200 years as the Smallpox vaccine, has a large DNA genome that provides a hefty insertion capacity, allowing the expression of large or multiple genes. In contrast, Alphavirus-based vectors are expression systems with small genome size and limited insertion capacity, but constitute attractive vaccine candidates shown to induce strong immune responses. For reviews on Pox and Alphavirus vectors see (1-7).

Despite their potential, some drawbacks for the use of those vectors exist. For instance, although Vaccinia Virus was used extensively during the Smallpox erradication campaigns, “classical” vaccine strains retain some pathogenicity, leading to a small number of clinical complications. The problem of vaccine pathogenicity has been addressed by developing new vaccine strains with growth restricted characteristics or harboring severely attenuating mutations. While those changes improve the safety profile of the vaccine vectors, they usually lead to a decreased immune response against the desired antigen. Thus, achieving an optimal balance of attenuation and immunogenic potential is of prime importance for Poxvirus vaccine development. In contrast to Vaccinia virus, Alphavirus vectors are a relatively new addition to the palette of available live virus vaccine vectors, and therefore the safety record is still limited. Alphavirus replicons are usually delivered as replicon-containing infectious single cycle particles, that are difficult and expensive to produce and purify.

In this work we have demonstrated the concept of combining two viral vectors with the aim of using a large DNA virus (Vaccinia virus) as a delivery device for a small RNA virus vector (Semliki Forest virus) and to exploit the benefits of each of the individual vectors involved. We have shown that, by inserting an Alphavirus replicon in the genome of vaccinia virus, and simultaneously providing the constituents of the Alphavirus capsid, we can generate (in vaccinia virus-infected cells) Alphavirus particles containing the replicon (Fig 1). This strategy results in a single Vaccinia virus recombinant, eliminating the need to produce in vitro recombinant Alphavirus particles. After constructing the combined VV/SFV virus, the release of Alphavirus particles capable of infecting cells and express the foreign antigen was evident in cell culture monolayers and by induced changes in the virus plaque phenotype (Fig 2). Also, by direct inspection of cells infected with the combined vector by electron microscopy, we detected typical Alphavirus particles assembling and budding from the plasma membrane of infected cells or accumulating in the extracellular space (Fig 3).

Our approach opens the prospect of using combined virus vectors directly as immunizing agents in vivo, by using a large virus recombinant as a shuttle for a smaller vector inside the injected tissues. The two-virus expression cascade also provides a potential means to enhance the efficacy of either Alphavirus- or Poxvirus- based vaccines, because of enhanced antigen expression. Besides, the system may facilitate improvements in the safety of classical vaccine strains, since it could be easily applied to replication-defective Poxvirus vectors that do not expand within the tissues in mammalian hosts. In that situation, the combined vector would produce Alphavirus single-cycle particles even in the absence of VV production, and may therefore lead to increased antigen expression without complete viral replication.

One interesting question is whether antigen expression from the combined vectors may influence the immunological response to the relevant antigen. Of note, foreign antigens inserted in standard VV recombinants are expressed as regular VV genes, and are therefore detected by the immune system in a context of hundreds of vector-specific antigens. During the last decades, different approaches have been used to try to single-out the response to the relevant antigen. For instance, a well studied strategy relies on priming the immune response to the foreign antigen by DNA immunization before Poxvirus injection. It is tempting to speculate that the combined vector system described here, that results in the expression of the antigen in two different cellular contexts (the VV-infected cell and the Alphavirus infected cell) might have consequences with respect to the immune responses to the antigen. Furthermore, our system could be used in combination with prime-boost immunization regimens, immune modulators or additional mutations to further increase the responses. All those aspects need futher experimentation.



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Corresponding author:

Rafael Blasco

Departamento de Biotecnología

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.)

Madrid, Spain

Tlf:       34-91-347 39 13

Fax:    34-91-357 22 93



FIGURE LEGENDS Figure 1Fig. 1.  Rationale for the combined VV/SFV vector. A vaccinia virus infecting a cell mediates the expression of the SFV replicon and the SFV genes encoding the structural genes, including capsid and envelope genes. After transcription by the Vaccinia RNA polymerase, the SFV replicon directs its own replication and the expression of the foreign gene, in this case GFP (green box). The vaccinia infected cell produces single-cycle alphavirus particles that, in turn, infect aditional cells, where the replicon RNA is replicated autonomously and the foreign gene expressed. Note that in this cell, structural genes of the Alphavirus are not present, and therefore no virus particles can be produced.


figure_2Fig. 2. Plaque formation by the combined VV/SFV virus. Left, virus plaques formed on BHK-21 cell monolayers under the microscope. GFP expression (green) was detected by fluorescence, and Vaccinia virus infection was revealed by staining with anti-Vaccinia antiserum (red). Note the comet tail made of GFP positive cells that is caused by the release of single-cycle Alphavirus particles from VV-SFV combined virus-infected cells (VV-SFV combined virus). Vaccinia viruses expressing GFP (VV-GFP) or expressing the replicon but not structural proteins (VV-Replicon) are shown for comparison. Right, virus plaque morphology. Plaques of the combined vector (VV-SFV combined virus) showing a comet shape, which is indicative of the release of single-cycle Alphavirus particles. The plaques formed by a VV recombinant containing the SFV replicon alone (no Alphavirus structural proteins) are shown for comparison (VV-Replicon).


figure_3Fig. 3. Electron microscopy of cells infected with the combined VV/SFV vector. Vaccinia virus (oval particles, approximately 250 nm, arrows) and smaller, SFV-like particles (spherical, approximately 50nm diameter, arrowheads) are shown. Alphavirus particles budding from the plasma membrane or accumulating in the extracellular space are seen.

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