Open Access Research

L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines

Sole Maria Pacchioni1, Massimiliano Bissa1, Carlo Zanotto1, Carlo De Giuli Morghen12, Elena Illiano3 and Antonia Radaelli234*

Author Affiliations

1 Department of Medical Biotechnologies and Translational Medicine, University of Milan, via Vanvitelli 32, 20129 Milan, Italy

2 Cellular and Molecular Pharmacology Section, CNR Institute of Neurosciences, University of Milan, via Vanvitelli 32, 20129 Milan, Italy

3 Department of Medical Biotechnologies and Translational Medicine, University of Milan, via Vanvitelli, 32, 20129 Milan, Italy

4 Laboratory of Molecular Virology and Recombinant Vaccine Development, Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy

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Journal of Translational Medicine 2013, 11:95  doi:10.1186/1479-5876-11-95


The electronic version of this article is the complete one and can be found online at: http://www.translational-medicine.com/content/11/1/95


Received:21 December 2012
Accepted:4 April 2013
Published:11 April 2013

© 2013 Pacchioni et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity.

Methods

Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence.

Results and conclusions

Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non–cross-reactive with vaccinia virus. These recombinants might therefore represent safer and more promising immunogens that can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans.

Keywords:
Smallpox vaccine; Orthopoxvirus infections; Fowlpox recombinants; Transgene expression

Background

Preventive vaccination still remains the most effective control against orthopoxvirus (OPXV) infections, as it can elicit neutralising antibodies against an incoming virus. The traditional smallpox vaccine was administered by scarification, but its use was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, attenuated strains of vaccinia virus (VV) have been produced and tested in humans in attempts to develop strains with lower reactogenicity and fewer side effects. Indeed, in spite of the discontinuation of the smallpox vaccination programmes, the threat of deliberate release of variola virus for bioterrorism and the need for protection from new zoonotic infections still remain [1-4]. Also, the monkeypox virus (MPXV) resembles smallpox in the severity of its symptoms, and it might be a potential bioweapon if cowpox and MPXV can adapt to grow and spread in humans [5].

VV recombinants that express different transgenes have already been used in clinical studies for the prevention and immunotherapy of different infectious diseases. However, although extremely effective, VV raises safety concerns due to its high reactogenicity, its ability to spread to non-vaccinated subjects, and its moderate to severe side effects [6], especially in immune-compromised individuals [7]. Such side effects are not acceptable in a post-endemic smallpox era [6,8]. Clinical trials have also been performed with the attenuated VV-derived Lister clone LC16m8 and the modified vaccinia Ankara (MVA). LC16m8, which replicates in humans, showed protective efficacy in animal models, and was safely used for over 50,000 children in Japan in 1974 [9]. MVA has an extensive history of safety in humans, it is immunogenic and efficacious in both mice and non-human primates, where it can also protect against MPXV, and it is a leading candidate for an alternative smallpox vaccine [10-13]. However, as MVA replication in mammals is only partially abortive [14], and induces lower immunogenicity than the traditional smallpox vaccine [15], the search for new alternatives is still ongoing [16,17]. Failure of protection with MVA has been demonstrated in animals with CD4/CD8 combined immunodeficiency [6] and in Rhesus macaques infected with simian immunodeficiency virus with a very low cell count of the immune repertoire [18].

The VV antigens that protect against smallpox are not completely known. However, analysis of the immune responses after immunisation with traditional vaccines has shown that the neutralising antibodies are mainly directed against the surface proteins of the two infectious forms of OPXVs: the intracellular mature virions (MVs) and the extracellular virions (EVs). MVs are released after cell lysis, and they are responsible for the host-to-host spread because of their stability in the extracellular environment. In contrast, EVs are wrapped by an additional envelope, and they have an important role in cell-to-cell spread; i.e., in the dissemination within the host [19,20]. In particular, the L1 and A27 proteins on MVs and the A33 and B5 proteins on EVs are involved in the attachment, fusion and penetration of the virus into target cells [20,21]. Combined DNA-based vaccines expressing these four proteins are more protective than vaccines carrying individual immunogens [22,23], possibly because of the induction of synergistic antibodies that can act at the different infection phases, both at the initial exposure, and during viral dissemination [24,25].

Although other immunogens have been used to induce protective immunity, when these four genes have been delivered as DNA expression plasmids [22] or as virus-like replicons [17], they have already been shown to induce functional antibodies and to protect mice against VV parenteral/ intranasal challenge, and monkeys against MPXV intravenous challenge [26,27].

In the present study, four novel recombinants were constructed based on the fowlpox (FP) genetic background that express the VV L1R, A27L, A33R and B5R genes independently. Their correct expression was determined by RT-PCR, Western blotting and immunofluorescence, both in replication-permissive chick embryo fibroblasts (CEFs) and in non-permissive mammalian Vero and MRC-5 cells. The ability of the proteins expressed by the four novel recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated by immunoprecipitation followed by Western blotting (IP/WB) using hyperimmune mouse serum. The recognition by VV-specific antibodies of a mixture of the four FP recombinants (4FPmix) was tested using a plaque-reduction neutralisation assay.

FP vectors are replication-restricted to avian species [28], but they are permissive for entry and transgene expression in mammalian cells, while being immunologically non–cross-reactive with VV. They therefore represent safer immunogens [29], as they can circumvent neutralisation by vector-generated immunity in smallpox-vaccine-experienced humans. The advanced replication cycle, long transgene expression, and balanced Th1/Th2 cytokine induction of FP-based recombinants [28] might elicit a more effective immune response.

Methods

Cells

Specific-pathogen-free primary CEFs were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated calf serum (Gibco Life Technologies, Grand Island, NY, USA), 5% Tryptose Phosphate Broth (Difco Laboratories, Detroit, MI, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. Green monkey kidney (Vero) cells, and normal human lung fibroblasts (MRC-5 cells) were grown in DMEM supplemented with 10% heat-inactivated calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin.

Construction of the recombinants

Four FP-based recombinants expressing the VV MV L1 and A27 proteins and the EV A33 and B5 proteins were obtained by in-vitro homologous recombination [30-32], with minor modifications. FPwt was obtained by J. Taylor (Wadsworth Center, NY State Dept. of Health, Albany, NY) [29]. Molecular cloning of pFPA33R and preparation of the FPA33R recombinant have already been described [33]. For FPL1R, FPA27L and FPB5R, these genes were PCR-amplified from the Lancy strain of VV DNA (Berna Biotech, courtesy of M. R. Capobianchi, L. Spallanzani National Institute for Infectious Diseases, Rome, Italy). They were separately inserted into the pcDNA3 and pBSII plasmids (L1R), the pBSII plasmid (A27L), or the pcDNA3 and pAFTd plasmids (B5R). Insertion into the pFPMCS recombinant plasmid was performed downstream of the VV H6 early/ late promoter [34], inside the 3-β-hydroxysteroid dehydrogenase 5-delta 4 isomerase gene interrupted by a multiple cloning site. The amplification of L1R was carried out using the forward V182 (5’ GGG AAG CTT TTA AAT GGG TGC CGC AGC AAG CAT ACA 3’) and reverse V183 (5’ GGG CTC GAG ATT TTC AGT TTT GCA TAT CCG TGG TAG 3’) primers. For A27L, the forward V354 (5’ CCC GGG AAG CTT AAT GGA CGG AAC TCT TTT C 3’) and reverse V353 (5’ TTT TGG TAC CAT AAA AAT TAC TCA TAT GGG CGC CG 3’) primers were used. For B5R, the forward V184 (5’ GGG AAG CTT AAA AAT GAA AAC GAT TTC CGT TGT TAC 3’) and reverse V185 (5’ GGG CTC GAG ATA TTT ACG GTA GCA ATT TAT GGA ACT 3’) primers were used. Amplifications were performed as described previously [35], using 2.5 mM MgCl2 and 2 mM MgSO4, with annealing at 61°C for 30 s (L1R and A27L), or at 57°C for 30 s (B5R) and extension at 72°C for 45 s. The β-actin reference gene was amplified using the forward V84 (5’ CTG ACT ACC TCA TGA AGA TCC T 3’) and reverse V85 (5’ GCT GAT CCA CAT CTG CTG GAA 3’) primers, with 1 mM MgSO4 and with annealing at 60°C for 30 s. The plasmid DNAs were purified and the genes were sequenced (Genenco, MMedical, Milan, Italy), to exclude any mutations arising from the PCR amplification. The plasmids are designated as pFPL1R (8,992 bp), pFPA27L (8,568 bp), pFPA33R (8,764 bp), and pFPB5R (9,173 bp). Recombinants were obtained by in-vitro homologous recombination in CEFs, using FPwt and the different pFP recombinants, with minor modifications. Recombinant plaques were identified by autoradiography after hybridisation with [32P]-labelled specific probes, and then subjected to multiple cycles of plaque purification. One clone was selected for correct and high expression of each gene by Western blotting, using specific antibodies. The recombinant viruses were amplified in CEFs, purified on discontinuous sucrose density gradients, and titrated essentially as already described [36]. Briefly, the cells were harvested, ultracentrifuged at 30,000× g for 2 h at 4°C, and the pellets resuspended in 1 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4. The pellet then had 0.06% trypsin added, and was incubated for 5 min at 37°C, and the virus was released from the cells by sonication. The supernatant was overlaid onto a discontinuous 30% to 45% (w/w) sucrose gradient, in the same buffer. After ultracentrifugation at 38,000× g for 1 h, the viral band at the interface was recovered, diluted with 1 mM Tris-HCl, pH 9, and pelletted at 67,000× g for 1 h. The purified virus was resuspended in Ca++- and Mg++-free phosphate-buffered saline (PBS-), disaggregated by sonication, aliquoted, and frozen at -80°C until use.

RT-PCR

The expression of L1R, A27L, A33R and B5R was investigated by RT-PCR. The cells were infected with 1 plaque-forming unit (PFU)/cell of the FPL1R, FPA27L, FPA33R or FPB5R recombinants, and the mRNAs were extracted 24 h post-infection (p.i.) from CEFs and Vero and MRC-5 cells, as described previously [28]. Briefly, 50 ng RNA from each sample was used in a final volume of 10 μl, in the presence of 1 μM of each primer, 200 mM of each dNTP, 0.1 U/μl Thermus flavus DNA polymerase, and 0.1 U/μl avian myeloblastosis virus reverse transcriptase, using the V182/V183 primers for L1R (779 bp), V354/V353 for A27L (363 bp), V186/V187 for A33R (584 bp), V184/V185 for B5R (980 bp), and V84/85 (518 bp) for β-actin detection under the conditions described above. RNA from FPwt-infected cells was used as a negative control. The RT-PCR products were quantified using the ImageJ software [37].

Western blotting

To determine whether the L1, A27, A33, and B5 proteins were expressed by the recombinants at the same levels in the different cell lines, CEFs and Vero and MRC-5 cells were infected (10 PFU/cell) and examined by Western blotting, as already described [38]. The blotted nitrocellulose membranes were incubated overnight at 4°C with 1:100 dilutions of the primary antibody. Alternatively, two different specific antibodies (Beiresources, Manassas, VA, USA) were used for each gene: a mouse monoclonal antibody followed by goat anti-mouse horseradish-peroxidase-conjugated serum, and a rabbit polyclonal antibody followed by goat anti-rabbit horseradish-peroxidase-conjugated serum (1:2,000 dilution; DakoCytomation, Carpinteria, CA, USA). After a 1-h incubation and 2 h of washes, the proteins were revealed using the ECL system (EuroClone, Pero, Milan, Italy). Cells infected with FPwt were used as the negative control.

Immunoprecipitation/ Western blotting analysis

Conventional Western blotting was also performed after immunoprecipitation of cell lysates using specific antibodies (IP/WB). Vero cells were infected as for Western blotting, and immunoprecipitation was performed essentially as already described [35], with minor modifications. Sixteen hours p.i., the cells were harvested by resuspension in 1 ml lysis buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 0.2 mg/ml PMSF, 1% NP40, 0.01% sodium azide) per Petri dish, and 0.6 TIU aprotinin (Sigma, St Louis, MO, USA). The lysate was clarified by centrifugation at 9,000× g for 20 min at 4°C, and immunoprecipitation was performed with 10 μl anti-IHD-J mouse hyperimmune serum, from our laboratory. The proteins were resolved using 15% SDS-PAGE, identified using a polyclonal antibody (Beiresources), and revealed using the ECL system.

Immunofluorescence

Protein expression by the recombinant viruses was also examined by immunofluorescence, which was carried out in CEFs and Vero and MRC-5 cells, essentially as described previously [35]. In particular, after infection at 37°C for 1 h with 1 or 3 PFU/cell (depending on the different cytopathogenicity of the recombinants), the cells were grown for 6 or 15-18 h before immunofluorescence. The cells were fixed either with only fresh 2% paraformaldehyde in PBS- for 10 min, for membrane immunofluorescence, or with paraformaldehyde followed by 100% cold acetone for 5 min at -20°C, for cytoplasmic immunofluorescence. The samples were incubated for 1 h with monoclonal or polyclonal antibodies (Beiresources) (Table 1). FITC 1:100-diluted anti-mouse or anti-rabbit secondary antibodies (Cappel, MP Biomedicals, Inc., Aurora, OH, USA) were used. Cells infected with FPwt were the negative controls. The samples were viewed under a Zeiss Axioskop epifluorescence microscope.

Table 1. Antibody dilutions for IF

IHD-J and preparation of the four FP recombinants mixture

The IHD-J strain of VV was kindly obtained from S. Dales (University of Western Ontario, London, Canada) [39] grown in Vero cells, and used to repeatedly infect Balb/c mice (1 × 105 PFU/mouse) via the airways, to obtain VV-specific hyperimmune serum. A mixture of the four FP recombinants (4FPmix) was also prepared in CEFs, by co-infection with the four FP viruses (3 PFU/cell/each recombinant virus). The IHD-J and the 4FPmix viruses were amplified in Vero cells and CEFs, respectively, purified on discontinuous sucrose density gradients, and titrated as already described.

Virus neutralisation assays

Recognition of the 4FPmix by VV-specific mouse antibodies was tested using the virus neutralisation assay. VV IHD-J hyperimmune mouse serum was used to determine the inhibition of the infectivity of the 4FPmix. Neutralisation assays were performed by pre-incubating the 4FPmix with an equal volume of heat-inactivated hyperimmune serum, for 1 h at 37°C. Pre-immune serum was used as a negative control. The IHD-J virus was used in a parallel test with both pre-immune and hyperimmune serum. The sera were diluted starting from a 1:20 dilution in DMEM. The viral inoculum was adjusted to give approximately 102 PFU/Petri dish. Infection was allowed to proceed for 1 h at 37°C. The cells then had 5 ml DMEM with agarose LE 0.7% (SeaKem, FMC BioProducts, Rockland, ME, USA) added. The plaque numbers were counted on day 4 p.i., after adding an agarose layer containing 1.5% neutral red (Gibco). The neutralisation is expressed as the percentage of reduction of plaque numbers versus the control, where the viral inoculum was incubated with no serum.

Results

Transcript expression by the FP recombinants is similar in the different cell lines

After RNA isolation from the infected CEFs and Vero and MRC-5 cells, the transcripts were detected by RT-PCR after an overnight incubation, as 779-bp, 363-bp, 584-bp and 980-bp fragments in all of the cell lines infected by the FPL1R, FPA27L, FPA33R and FPB5R recombinants, respectively (Figure 1, lanes A). As determined by densitometric analysis, similar levels of expression were observed in the different cells for each recombinant, except for FPB5R, that expressed the B5R mRNA 2.1 times more in human MRC-5 cells than in CEF and Vero cells. The amplification of human β-actin RNA is also shown as a 518-bp band (Figure 1, lanes A). As expected, the FPwt-infected cells used as a negative control did not show any specific band (Figure 1, lanes B).

thumbnailFigure 1. Expression of the FPL1R, FPA27L, FPA33Rand FPB5Rtranscripts by RT-PCR in replication-permissive and non-permissive cells. Following infection of CEFs and Vero and MRC-5 cells with the four recombinants, transcript expression was detected after overnight infection, as 779-bp (panel a), 363-bp (panel b), 584-bp (panel c) and 980-bp (panel d) fragments. Expression of human β-actin is shown as a 518-bp band (panels a-d). No specific band is seen for cells infected with FPwt, which was used as a negative control (lanes B).

The recombinant proteins are expressed by all of the FP recombinants in all of the different cell lines, although at different levels

The FP recombinants expression was verified by Western blotting on lysates of both replication-permissive CEFs and non-permissive Vero and MRC-5 cells, which were infected separately with FPL1R, FPA27L, FPA33R and FPB5R, or with FPwt. The polyclonal primary antibodies recognised specific 27-kDa, 12.7-kDa, 21-kDa and 37-kDa bands, which corresponded to the L1, A27, A33 and B5 proteins, respectively, in the infected CEFs and Vero and MRC-5 cells (Figure 2, lanes B). As determined by densitometric analysis, the expression levels for FPL1R were 4.2-fold and 3.9-fold higher in CEFs and Vero cells than in MRC-5 cells, those for FPA27L were 2.9-fold and 2.5-fold higher in CEFs and Vero cells than in MRC-5 cells, those for FPA33R were 2.6-fold and 3-fold higher in CEFs and MRC-5 cells than in Vero cells, and those for FPB5R were 2.1-fold and 3.1-fold higher in Vero and MRC-5 cells than in CEFs (Figure 2; lanes B). No specific bands were recognised when the cells were infected with FPwt (Figure 2; lanes A). The corresponding monoclonal antibodies showed similar specificity, but lower binding activity (data not shown).

thumbnailFigure 2. Transcript expression by Western blotting in replication-permissive and non-permissive cell lines. All of the different cell lines, the CEFs and Vero and MRC-5 cells, were infected with 10 PFU/cell of FPL1R, FPA27L, FPA33R or FPB5R, and harvested 15-18 h p.i.. Rabbit polyclonal primary antibodies recognised the corresponding proteins, in all of the cell lines (lanes B). The expression levels were quantified by densitometric determinations. No specific bands were seen when the cells were infected with FPwt (lanes A).

Vaccinia hyperimmune mouse serum recognises FP-expressed heterologous proteins

IP/WB was used to test whether the hyperimmune mouse sera could also recognise the FP-expressed proteins. After infection of the Vero cells with the FPL1R, FPA27L, FPA33R and FPB5R recombinants, the specific proteins were immunoprecipitated from cell lysates using sera from mice that had been repeatedly immunised with VV IDH-J (Figure 3, lanes B). No specific bands were seen for cells infected with FPwt (Figure 3, lanes A).

thumbnailFigure 3. Immunoprecipitation/Western blotting. To verify the recognition of the FP-expressed proteins also by VV-specific hyperimmune mouse sera, immunoprecipitation was performed, followed by Western blotting. After infection with FPL1R, FPA27L, FPA33R or FPB5R, the specific proteins were immunoprecipitated by the hyperimmune sera (lanes B). No specific bands were seen in cells infected with FPwt (lanes A).

Different localisation of the foreign proteins is detected by immunofluorescence

To identify differences in the heterologous protein expression and subcellular localisation in the different cell lines, immunofluorescence was performed at 15-18 h p.i. in CEFs and Vero and MRC-5 cells infected with the FPL1R, FPA27L, FPA33R and FPB5R recombinants, or with FPwt (Figure 4). The cells infected with FPL1R and FPA27L showed diffuse cytoplasmic fluorescence (Figure 4A, 1a-c, 2a-c), although the specific staining with FPL1R was only slightly greater than the negative control, in all of the cell lines. Conversely, there was specific, greater, granular perinuclear and cytoplasmic localisation in the cells infected with FPA33R and FPB5R (Figure 4A, 3a-c, 4a-c), which was particularly evident in the MRC-5 cells infected with FPA33R. FPwt-infected cells were always negative (Figure 4A, 5a-c). Immunofluorescence performed at 6 h p.i. gave similar results (data not shown). Membrane immunofluorescence was present only after infection with the FPA33R recombinant in MRC-5 cells (Figure 4B, 1b vs. 1a). Polyclonal and monoclonal antibodies were used at different dilutions for the different recombinants (Table 1).

thumbnailFigure 4. Heterologous protein localisation by immunofluorescence. (A) Intracellular immunofluorescence was detected in all of the cell lines, with differences between the proteins ascribed to MV or EV particles. In cells infected with FPL1R or FPA27L the cytoplasmic immunofluorescence was diffuse (panels 1a-c, 2a-c), and in FPL1R-infected cells, the difference with the negative control was very low. Conversely, specific, granular perinuclear localisation was shown by the cells infected with FPA33R or FPB5R (panels 3a-c, 4a-c), which was particularly evident in MRC-5 cells infected with FPA33R. FPwt-infected cells were negative (panels 5a-c). (B) MRC-5 cells showed membrane immunofluorescence only after infection with the FPA33R recombinant (panel 1bvs. 1a).

The 4FPmix is not neutralised by VV hyperimmune serum

To determine whether the FP recombinant virions produced by the CEFs were carrying the structural products of the four transgenes on their surface, the 4FPmix, containing a mixture of all of the FP recombinants, was incubated with VV IHD-J hyperimmune serum. No reduction in plaque numbers was detected, as compared to samples where pre-immune serum was incubated with the IHD-J virus. High neutralising activity was seen in samples where IHD-J was pre-incubated with IHD-J hyperimmune serum (Figure 5).

thumbnailFigure 5. Virus neutralisation assay. A mixture of all of the four FP recombinants (4FPmix) was incubated with IHD-J hyperimmune serum to determine whether the proteins expressed by the recombinants were localised on the surface of the new viral progeny. High neutralising activity was detected only in the samples where IHD-J was pre-incubated with IHD-J-hyperimmune mouse serum.

Discussion

Development of alternative replication-deficient vaccine candidates against smallpox and other zoonotic OPXV infections is still needed as a defence against new poxvirus outbreaks as well as to reduce the adverse reactions of traditional vaccines and the limited immune responses in VV-experienced individuals. Along with discontinuation of the smallpox vaccination campaign and the decline in herd immunity in most countries, an increase in MPXV infections has been described [40,41], which has mostly occurred in children, young adults, and immune-compromised patients [42]. MPXV is widely distributed in a variety of African rodents, and particularly in squirrels, which might be the reservoir [43], and it has the potential to be used as a bio-weapon [41,44,45]. Outbreaks of human MPXV infections were also reported in the Unites States in 2003, with 69 people infected following the importation of MPXV-infected rodents from West Africa. Also, in the Republic of Congo in 1996-1997, there were 92 cases and 3 deaths [46], which might have been related to the overall vanishing immunity against poxviruses throughout the world community.

The available smallpox vaccines based on VV show relatively high rates of adverse side effects, and new strategies should therefore be devised to improve the safety of traditional smallpox vaccines. Replication-deficient vaccines should reduce the risks for animal handlers that arise from accidental exposure to cowpoxvirus and other OPXV, limit the OPXV-infection of domestic animals, and should be useful for protective immunization in the event of intentional or accidental release of variola virus.

In the present study, we have described the characterisation of four new FP recombinants, FPL1R, FPA27L, FPA33R and FPB5R, that express the VV L1R, A27L, A33R and B5R genes. We demonstrated that: (i) all of these recombinants can express the proteins correctly, although at different levels in different cell lines, as revealed by RT-PCR and Western blotting; (ii) MV and EV proteins show different subcellular localisations; (iii) recombinant FP virions (4FPmix) are resistant to neutralisation by VV-specific serum; and (iv) the four FP recombinants express functional foreign proteins that are immunoprecipitated by hyperimmune serum from IHD-J-immunised mice.

Using Western blotting, heterologous VV-specific proteins were detected in cells infected by the four recombinants. Their expression in human and non-human primate cells was often higher than in CEFs, where the virus replicates. This can be ascribed to the cytocidal effect of the FP recombinants in avian cells, although it is not clear why this occurs only for the expression of specific genes. In particular, the higher transgene expression by FPB5R in human MRC-5 cells confirms the results already shown by RT-PCR. IP/WB was used to overcome the difficulty of revealing cell-expressed antigens by conventional Western blotting, especially when using mouse or human hyperimmune sera. This assay, which better avoids nonspecific binding to nitrocellulose and makes the native proteins detectable, showed that these proteins can be recognised by VV-specific hyperimmune mouse serum.

By immunofluorescence, the heterologous proteins were mainly localised at the cytoplasmic level for all of the recombinants, and also at the membrane surface when MRC-5 cells were infected with FPA33R. Although no specific differences were observed among the cells lines, the expression of the proteins belonging to MV particles was always lower than the expression of the proteins belonging to EV particles. In particular, the intracellular expression of A33 and B5 was granular, perinuclear and cytoplasmic, which confirms previously reported data, and demonstrates their high expression in the juxtanuclear area and Golgi region [47]. Membrane fluorescence was only detectable after infection with FPA33R. This also confirms previous data showing the spontaneous translocation of the A33 protein to the plasma membrane, which, using a FPB5R recombinant, was only observed in very low amounts by immunoelectron microscopy, but not by immunfluorescence [47]. The very low fluorescence detected after FPL1R infection can either be ascribed to limited protein expression, which was not confirmed by the Western blotting, or to the low avidity of the antibody. This is also confirmed by other studies, where low expression was overcome by the use of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) [48].

The absence of neutralising activity of IHD-J-specific hyperimmune mice sera against the virus particles released by CEFs infected with the four FP recombinants suggests that the L1, A27, A33 and B5 gene products may not be inserted into the envelope of the new viral progeny. This enhances the importance of these FP recombinants for their use in individuals who have already been vaccinated against smallpox, where these can be administered as a recall. As human peripheral blood monocytic cells and macrophages are permissive for penetration of the recombinants and expression of foreign proteins [28], antigen cross-presentation should occur, which should result in complete humoral and cellular immune responses.

Conclusions

To avoid lytic infection, ulceration and scab formation after dermal scarification with VV for smallpox prophylaxis, FP recombinants represent alternative safer immunogens. This arises from their natural host-range restricted replication to avian species [49], their correct transgene expression in mammalian cells, and their ability to elicit a complete immune response in vaccinated hosts [50]. Although these L1R, A27L, A33R and B5R VV-derived genes have already been successfully expressed using recombinant DNA [22,48], the use of avipox recombinants expressing these genes might provide better control against zoonotic OPXV infections, will offer advances in the fight against the threat of bioterrorism, and can also be used in VV-experienced individuals.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SP performed the production and purification of the FP recombinants, the Western blotting, and the IP/WB. MB performed the production and purification of the FP recombinants, and the immunofluorescence assays. CZ performed the molecular cloning, prepared the primary cell cultures, prepared the Figures, and analysed the data and the study results. CDGM conceptualised and supervised the whole study. EI performed the RT-PCR and animal immunisations. AR designed the study, performed the neutralisation assays, assisted in the animal immunisations, analysed the data, interpreted the study results, and prepared the manuscript. All of the authors have read and approved the present version of the manuscript.

Acknowledgements

The following reagents were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: the monoclonal anti-VV (WR) L1 protein, residues 1 to 185 (similar to VMC-2; produced in vitro), NR-417; the polyclonal anti-VV (WR) L1 protein (antiserum, rabbit), NR-631; the monoclonal anti-VV (WR) A27 protein, residues 1 to 110 (similar to VMC-38; produced in vitro), NR-567; the polyclonal anti-VV (WR) A27 protein (antiserum, rabbit), NR-627; the monoclonal anti-VV (WR) A33 protein, residues 58 to 185, NR-565; the polyclonal anti-VV (WR) A33 protein, NR-628; the monoclonal anti-VV (WR) B5 protein, residues 20 to 275 (ectodomain; similar to VMC-20; produced in vitro), NR-551; and the polyclonal anti-VV (WR) B5 protein (antiserum, rabbit), NR-629. We also thank Dr. Christopher Berrie for editorial assistance with the manuscript.

References

  1. Whitley RJ: Smallpox: a potential agent of bioterrorism.

    Antiviral Res 2003, 57:7-12. PubMed Abstract | Publisher Full Text OpenURL

  2. Vogel S, Sardy M, Glos KKHC, Ruzicka T, Wollenberg A: The Munich outbreak of cutaneous cowpox infection: transmission by infected pet rats.

    Acta Derm Venereol 2012, 92:126-131. PubMed Abstract | Publisher Full Text OpenURL

  3. Cardeti G, Brozzi A, Eleni C, Polici N, D'Alterio G, Carletti F, Scicluna MT, Castilletti C, Capobianchi M, Di Caro A, Autorino GL, Amaddeo D: Cowpox virus in llama, Italy.

    Emerg Infect Dis 2011, 17:1513-1515. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  4. Megid J, Borges IA, Trindade GS, Appolinário CM, Ribeiro MG, Allendorf SD, Antunes JM, Silva-Fernandes AT, Kroon EG: Vaccinia virus zoonotic infection, São Paulo State, Brazil.

    Emerg Infect Dis 2012, 18:189-191. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  5. Lewis-Jones S: Zoonotic poxvirus infections in humans.

    Curr Opin Infect Dis 2004, 17:81-89. PubMed Abstract | Publisher Full Text OpenURL

  6. Wiser I, Balicer RD, Cohen D: An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies.

    Vaccine 2007, 25:976-984. PubMed Abstract | Publisher Full Text OpenURL

  7. Bray M: Pathogenesis and potential antiviral therapy of complications of smallpox vaccination.

    Antiviral Res 2003, 58:101-114. PubMed Abstract | Publisher Full Text OpenURL

  8. Enserink M: Smallpox Vaccines: Looking Beyond the Next Generation.

    Science 2004, 304:809. PubMed Abstract | Publisher Full Text OpenURL

  9. Kenner J, Cameron F, Empig C, Jobes DV, Gurwith M: LC16m8: an attenuated smallpox vaccine.

    Vaccine 2006, 24:7009-7022. PubMed Abstract | Publisher Full Text OpenURL

  10. Earl PL, Americo JL, Wyatt LS, Eller LA, Whitbeck JC, Cohen GH, Eisenberg RJ, Hartmann CJ, Jackson D, Kulesh DA, Martinez MJ, Miller DM, Mucker EM, Shamblin JD, Zwiers SH, Huggins JW, Jahrling PB, Moss B: Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox.

    Nature 2004, 428:182-185. PubMed Abstract | Publisher Full Text OpenURL

  11. Stittelaar KJ, van Amerongen G, Kondova I, Kuiken T, van Lavieren RF, Pistoor FH, Niesters HG, van Doornum G, van der Zeijst BA, Mateo L, Chaplin PJ, Osterhaus ADME: Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus.

    J Virol 2005, 79:7845-7851. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  12. Wyatt LS, Earl PL, Eller LA, Moss B: Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge.

    Proc Natl Acad Sci USA 2004, 101:4590-4595. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  13. Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD, Franchini G, Moss B, Berzofsky JA: Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses.

    Proc Natl Acad Sci USA 2003, 100:9458-9463. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  14. Blanchard TJ, Alcami A, Andrea P, Smith GL: Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine.

    J Gen Virol 1998, 79:1159-1167. PubMed Abstract | Publisher Full Text OpenURL

  15. Ferrier-Rembert A, Drillien R, Tournier JN, Garin D, Crance JM: Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine.

    Vaccine 2008, 26:1794-1804. PubMed Abstract | Publisher Full Text OpenURL

  16. Kennedy RB, Ovsyannikova I, Poland GA: Smallpox vaccines for biodefense.

    Vaccine 2009, 27:D73-D79. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Hooper JW, Ferro AM, Golden JW, Silvera P, Dudek J, Alterson K, Custer DM, Rivers B, Morris J, Owens G, Smith JF, Kamrud KI: Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates.

    Vaccine 2010, 28:494-511. OpenURL

  18. Edghill-Smith Y, Bray M, Whitehouse CA, Miller D, Mucker E, Manischewitz J, King LR, Robert-Guroff M, Hryniewicz A, Venzon D, Meseda C, Weir J, Nalca A, Livingston V, Wells J, Lewis MG, Huggins J, Zwiers SH, Golding H, Franchini G: Smallpox vaccine does not protect macaques with AIDS from a lethal monkeypox virus challenge.

    J Infect Dis 2005, 191:372-381. PubMed Abstract | Publisher Full Text OpenURL

  19. Smith GL, Vanderplasschen A, Law M: The formation and function of extracellular enveloped vaccinia virus.

    J Gen Virol 2002, 83:2915-2931. PubMed Abstract | Publisher Full Text OpenURL

  20. Roberts KL, Smith GL: Vaccinia virus morphogenesis and dissemination.

    Trends Microbiol 2008, 16:472-479. PubMed Abstract | Publisher Full Text OpenURL

  21. Moss B: Smallpox vaccines: targets of protective immunity.

    Immunol Rev 2011, 239:8-26. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Hooper JW, Custer DM, Thompson E: Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicitis appropriate antibody responses in nonhuman primates.

    Virology 2002, 306:181-195. OpenURL

  23. Fogg C, Lustig S, Whitbeck JC, Eisenberg RJ, Cohen GH, Moss B: Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions.

    J Virol 2004, 78:10230-10237. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. Edghill-Smith Y, Golding H, Manischewitz J, King LR, Scott D, Bray M, Nalca A, Hooper JW, Whitehouse CA, Schmitz JE, Franchini G: Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus.

    Nat Med 2005, 11:740-747. PubMed Abstract | Publisher Full Text OpenURL

  25. Panchanathan V, Chaudhri G, Karupiah G: Antiviral protection following immunization correlates with humoral but not cell-mediated immunity.

    Immunol Cell Biol 2010, 88:461-467. PubMed Abstract | Publisher Full Text OpenURL

  26. Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, Schmaljohn CS, Schmaljohn AL, Jahrling PB: Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox.

    J Virol 2004, 78:4433-4443. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  27. Golden JW, Josleyn MD, Hooper JW: Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies.

    Vaccine 2008, 26:3507-3515. PubMed Abstract | Publisher Full Text OpenURL

  28. Zanotto C, Pozzi E, Pacchioni S, Volonté L, De Giuli Morghen C, Radaelli A: Canarypox and fowlpox viruses as recombinant vaccine vectors: a biological and immunological comparison.

    Antiviral Res 2010, 88:53-63. PubMed Abstract | Publisher Full Text OpenURL

  29. Taylor J, Weinberg R, Languet B, Desmettre P, Paoletti E: Recombinant fowlpox virus inducing protective immunity in nonavian species.

    Vaccine 1988, 6:497-503. PubMed Abstract | Publisher Full Text OpenURL

  30. Radaelli A, De Giuli MC: Expression of HIV-1 envelope gene by recombinant avipoxvirus.

    Vaccine 1994, 12:1101-1109. PubMed Abstract | Publisher Full Text OpenURL

  31. Parks RJ, Krell PJ, Derbyshire JB, Nagy E: Studies of fowlpox virus recombination in the generation of recombinant vaccines.

    Virus Res 1994, 32:283-297. PubMed Abstract | Publisher Full Text OpenURL

  32. Pozzi E, Basavecchia V, Zanotto C, Pacchioni S, De Giuli Morghen C, Radaelli A: Construction and characterization of recombinant fowlpox viruses expressing human papilloma virus E6 and E7 oncoproteins.

    J Virol Methods 2009, 158:184-189. PubMed Abstract | Publisher Full Text OpenURL

  33. Bissa M, Pacchioni S, Zanotto C, De Giuli Morghen C, Radaelli A: GFP co-expression reduces the A33R gene expression driven by a fowlpox vector in replication permissive and non-permissive cell lines.

    J Virol Methods 2013, 187:172-176. PubMed Abstract | Publisher Full Text OpenURL

  34. Rosel JL, Earl PL, Weir J, Moss B: Conserved TAAATG Sequence at the Transcriptional and Translational Initiation Sites of Vaccinia Virus Late Genes Deduced by Structural and Functional Analysis of the Hindlll H Genome Fragment.

    J Virol 1986, 60:436-449. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  35. Zanotto C, Pozzi E, Pacchioni S, Bissa M, De Giuli Morghen C, Radaelli A: Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein.

    J Transl Med 2011, 9:190-200. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  36. Pacchioni S, Volonté L, Zanotto C, Pozzi E, De Giuli Morghen C, Radaelli A: Canarypox and fowlpox viruses as recombinant vaccine vectors: an ultrastructural comparative analysis.

    Arch Virol 2010, 155:915-924. PubMed Abstract | Publisher Full Text OpenURL

  37. Rasband WS: U.S. National Institutes of Health. Bethesda, Maryland, USA;

    http://rsb.info.nih.gov/ij/ webcite

    OpenURL

  38. Radaelli A, De Giuli Morghen C, Zanotto C, Pacchioni S, Bissa M, Franconi R, Massa S, Paolini F, Muller A, Venuti A: A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers.

    Virus Res 2012, 170:44-52. PubMed Abstract | Publisher Full Text OpenURL

  39. Wilton S, Gordon J, Dale S: Identification of antigenic determinants by polyclonal and hybridoma antibodies induced during the course of infection by vaccinia virus.

    Virology 1986, 148:84-96. PubMed Abstract | Publisher Full Text OpenURL

  40. Hutin YJ, Williams RJ, Malfait P, Pebody R, Loparev VN, Ropp SL, Rodriguez M, Knight JC, Tshioko FK, Khan AS, Szczeniowski MV, Esposito JJ: Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997.

    Emerg Infect Dis 2012, 7:434-438. OpenURL

  41. Reed KD, Melski JW, Graham MB, Regnery RL, Sotir MJ, Wegner MV, Kazmierczak JJ, Stratman EJ, Li Y, Fairley JA, Swain GR, Olson VA, Sargent EK, Kehl SC, Frace MA, Kline R, Foldy SL, Davis JP, Damon IK: The detection of monkeypox in humans in the Western Hemisphere.

    New Engl J Med 2004, 350:342-350. PubMed Abstract | Publisher Full Text OpenURL

  42. Schulze C, Alex M, Schirrmeier H, Hlinak A, Engelhardt A, Koschinski B, Beyreiss B, Hoffmann M, Czerny CP: Generalized fatal Cowpox virus infection in a cat with transmission to a human contact case.

    Zoonoses Public Health 2007, 54:31-37. PubMed Abstract | Publisher Full Text OpenURL

  43. Reynolds MG, Carroll DS, Olson VA, Hughes C, Galley J, Likos A, Montgomery JM, Suu-Ire R, Kwasi MO, Jeffrey Root J, Braden Z, Abel J, Clemmons C, Regnery R, Karem K, Damon IK: A silent enzootic of an orthopoxvirus in Ghana, West Africa: evidence for multi-species involvement in the absence of widespread human disease.

    Am J Trop Med Hyg 2010, 82:746-754. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  44. Rimoin AW, Mulembakani PM, Johnston SC, Lloyd Smith JO, Kisalu NK, Kinkela TL, Blumberg S, Thomassen HA, Pike BL, Wolfe ND, Shongo RL, Graham BS, Formenty P, Okitolonda E, Hensley LE, Meyer H, Wright LL, Muyembe JJ: Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.

    Proc Natl Acad Sci USA 2010, 107:16262-16267. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  45. Parker S, Nuara A, Buller RML, Schultz DA: Human monkeypox: an emerging zoonotic disease.

    Future Microbiol 2007, 2:17-34. PubMed Abstract | Publisher Full Text OpenURL

  46. Cohen J: Is an old virus up to new tricks?

    Science 1997, 277:312-313. PubMed Abstract | Publisher Full Text OpenURL

  47. Lorenzo M, Galindo I, Griffiths G, Blasco R: Intracellular localization of vaccinia virus extracellular enveloped virus envelope proteins individually expressed using a Semliki Forest virus replicon.

    J Virol 2000, 74:10535-10550. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  48. Hooper JW, Golden JW, Ferro AM, King AD: Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge.

    Vaccine 2007, 25:1814-1823. PubMed Abstract | Publisher Full Text OpenURL

  49. Baxby D, Paoletti E: Potential use of nonreplicating vectors as recombinant vaccines.

    Vaccine 1992, 10:8-9. PubMed Abstract | Publisher Full Text OpenURL

  50. Skinner MA, Laidlaw SM, Eldaghayes I, Kaiser P, Cottingham MG: Fowlpox virus as a recombinant vaccine vector for use in mammals and poultry.

    Expert Rev Vaccines 2005, 4:63-76. PubMed Abstract | Publisher Full Text OpenURL