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J Virol. 2010 November; 84(22): 11602–11613.
Published online 2010 September 8. doi:  10.1128/JVI.00677-10
PMCID: PMC2977905

Impact of ETIF Deletion on Safety and Immunogenicity of Equine Herpesvirus Type 1-Vectored Vaccines [down-pointing small open triangle]

Abstract

Heterologous gene transfer by viral vector systems is often limited by factors such as preexisting immunity, toxicity, low packaging capacity, or weak immunogenic potential. A novel viral vector system derived from equine herpesvirus type 1 (EHV-1) not only overcomes some of these obstacles but also promotes the robust expression of a delivered transgene and the induction of antigen-specific immune responses. Regarding an enhanced safety profile, we assessed the impact of the gene encoding the sole essential tegument protein, ETIF, on the replication and immunogenicity of recombinant EHVs. The deletion of ETIF severely attenuates replication in permissive RK13 cells and a human lung epithelial cell line but without influencing transgene expression. Whereas the intranasal administration of a recombinant luciferase EHV in BALB/c mice resulted in transgene expression in nasal cavities and lungs for 5 to 6 days, the ETIF deletion limited expression to 2 days and resulted in 30-fold-less luminescence. Attenuated replication was accompanied by a decreased capacity to induce CD8+ T cells against a delivered HIV Gag transgene in BALB/c mice following repeated intranasal application. However, a single subcutaneous immunization with a gag DNA vaccine primed specific T cells for substantial expansion by two subsequent intranasal booster immunizations with either the gag recombinant ETIF mutant or the parental virus. In addition to inducing Gag-specific serum antibodies, this prime-boost strategy clearly outperformed three sequential immunizations with the parental or EHV-ΔETIF virus or repeated DNA vaccination by inducing substantial specific secretory IgA (sIgA) titers.

Viruses are evolutionarily predestined to transfer genetic material into eukaryotic cells and use the cellular machinery to express virally harbored genes. Hence, intensive research has focused on the potential of various viruses to deliver heterologous genes (for a review, see references 8 and 44). Depending on the type of viral vector system used, it is possible to induce efficient transgene-specific T- and B-cell immune responses (15, 34, 36, 43) as well as mucosal immune responses (16, 51), which can be enhanced by applying homologous or heterologous prime-boost strategies (4, 25, 56).

Unfortunately, the use of common viral vector systems for heterologous gene transfer is often limited by factors such as neurovirulence (e.g., rabies virus) (12, 28), weak immunogenic potential (e.g., avian poxviruses) (44), or low packaging capacity (e.g., adeno-associated viruses and first- and second-generation adenoviral vectors) (21, 44). However, the most important obstacle in vector development is preexisting immunity. Due to high infection rates (e.g., adenoviruses, adeno-associated viruses, and human herpesviruses) and systematic vaccination programs (e.g., poxviruses and measles virus) (14, 45), a large percentage of the human population has already acquired strong immune responses to currently used vector systems. Although it has been assumed that preexisting immunity to the administered recombinant virus would simply weaken the induction of transgene-specific immune responses, a recent clinical anti-HIV vaccination study revealed that the risk of infection with HIV might even be increased with inept vector systems used as delivery vehicles. Male subjects who had received a recombinant adenoviral vaccine harboring the HIV-1 Gag, Pol, and Nef genes showed an increased risk of HIV infection compared to a relevant control group (30). Thus, the further development of existing vector systems as well as the establishment of novel ones are necessary for successful heterologous gene delivery.

We previously showed that equine herpesvirus type 1 (EHV-1) strain RacH is a promising candidate as a novel viral vector system. The virus has been attenuated by 256 passages on porcine embryonic kidney cells (37) and lacks both copies of the IR6 gene, previously described as one of the main virulence factors of EHV-1 (41). The expression of all viral genes depends on one single immediate-early gene (20) that is trans-activated by the essential equine trans-inducing factor (ETIF), the EHV-1 homologue of HSV-1 VP16, also known as α-trans-inducing factor (α-TIF) (32, 42). ETIF is a major tegument protein encoded in the unique long region and is expressed at late times during viral replication. Besides transactivation of the immediate-early gene, it is also involved in the secondary envelopment of virions (33, 53).

All currently available data indicate that EHV-1 strain RacH is apathogenic in humans, with severely impaired replication in human cells. Nevertheless, the virus effectively infects a broad spectrum of different mammalian cells and can be grown to high titers in vitro. In addition, due to the absence of the virus in humans, it can be assumed that the problem of preexisting immunity should be of minor importance. Furthermore, it is known that neutralizing antibodies directed against human herpesviruses show no cross-reactivity to EHV-1. We could also show that recombinant EHV-1 is able to elicit considerable transgene-specific cellular immune responses in BALB/c mice via various routes of application (50). This is most likely due to the virus's natural tropism for peripheral blood mononuclear cells (PBMCs), which play an important role in inducing innate and adapted immune responses (3, 54). Above all, due to the virus's ability to infect epithelial cells, EHV-1 is characterized by inducing strong mucosal immune responses (9). Considering that distinct mucosal immune responses have been defined as one of the correlates of protective immunity against HIV (10, 29), this represents an important feature for a vector system, not only for a potential HIV vaccine but also for diseases transmitted by the mucosal route in general.

Therefore, we aimed to extend previous findings and characterize transgene expression and viral replication of a recombinant EHV-1 with the ETIF open reading frame (ORF) (UL48) deleted for purposes of attenuation. In addition, we studied the immunogenic potential of ETIF-deleted EHV-1. More specifically, we examined the quality and magnitude of transgene-specific immune responses upon the application of homologous or heterologous prime-boost strategies, including recombinant virus or plasmid DNA.

MATERIALS AND METHODS

Plasmids, BACs, and bacteria.

All plasmids were constructed and maintained in Escherichia coli strain DH5α cells (24) grown at 37°C in Luria-Bertani (LB) medium. The maintenance and mutagenesis of the EHV-1 bacterial artificial chromosome (BAC) pRacH (50) was performed with E. coli strain EL250, encoding in its genome the λ phage recombination enzymes Exo, Beta, and Gam under the control of a heat-inducible promoter (31).

Generation of recombinant virus.

Recombinant EHV-1 was generated by using two-step “en passant” mutagenesis (49) to introduce an expression cassette for the green fluorescent protein (GFP) (EHV-gfp), the HIV-1 group-specific antigen (EHV-syngag), or the firefly luciferase (EHV-luc) into the miniF plasmid sequence of the EHV-1 BAC pRacH.

For the insertion of transgene sequences, a linear transfer construct containing the transgene open reading frame (ORF) under the control of a cytomegalovirus (CMV) promoter and the bovine growth hormone (BGH) poly(A) site was generated. Furthermore, the transfer construct included a kanamycin resistance cassette (aphAI) framed by two I-SceI sites and flanked by pRacH homologous sequences (5′-flanking sequence 5′-AACCGGGCTGCATCCGATGCAAGTGTGTCGCTGTCGAGTTTAAACA-3′ and 3′-flanking sequence 5′-ATGGCCGCATAACTTCGTATAGCATACATTATACGAAGTTATCTAGCAGATC-3′) for the sequence-oriented insertion of the transfer cassette in the infectious clone. Transfer constructs were based on the pcDNA 3.1 plasmid (Invitrogen), which contains either the ORF encoding green fluorescent protein (pc-gfp) or the HIV-1 precursor Gag protein (pc-syngag) (13, 19). The gfp ORF and part of the CMV promoter region (MluI/BamHI) or the syngag ORF (KpnI/BamHI) was excised and cloned into transfer plasmid pEPkan-S (49), which resulted in the transfer construct pEPgfp or pEPsyngag, respectively. The luciferase gene from the vector pGL4,13 (catalog number E6681; Promega) was amplified by PCR using primers 5′-GGGATCCGTAAAGCCACCATGGAAGATG-3′ and 5′-GCGATATCTTACACGGCGATCTTGCC-3′ and cloned into pEPkan-S via BamHI and EcoRV, resulting in the transfer construct pEPluc.

The cleavage of the particular pEP constructs with I-CeuI resulted in the linear transfer construct used for subsequent recombination.

In an attempt to enhance vector safety, EHV-1 unique long open reading frame 48 (UL48), coding for the essential tegument protein ETIF (equine α-trans-inducing factor), was deleted from the BAC. For this, a second round of “en passant” mutagenesis was applied to the EHV BAC, resulting in the viral constructs EHV-gfpΔETIF, EHV-syngagΔETIF, and EHV-lucΔETIF.

To delete the UL48 ORF from pRacH, a kanamycin resistance cassette (aphAI) framed by two I-SceI sites encoded on plasmid pEPkan-S was amplified by PCR. Primers Ep_D48_for (5′-GCTGGTATTTTGACGCGCGCCCGGCAGCTTCAATAGTTTAATACAATAAAGTATGTTAGGGATAACAGGGTAATCGATTT-3′) and Ep_D48_rev (5′-CCAACATGTTAAGTCTGGAAACATACTTTATTGTATTAAACTATTGAAGCTGCCGGGGCCAGTGTTACAACCAATTAACC-3′) contain 56- to 57-nucleotide homology arms flanking ETIF and 23 to 24 nucleotides (boldface type) for the amplification of the aphAI cassette from plasmid pLAY2.

One hundred nanograms of the applicable transfer construct was electroporated into recombination-competent EL250 cells harboring pRacH and helper plasmid pBAD-I-SceI. Two-step Red-mediated recombination was performed as described previously (31, 49).

Infectious virus was reconstituted by transfecting recombinant BAC DNA into RK13 or RK-ETIF cells using calcium phosphate precipitation (47). A schematic overview of viral constructs is presented in Fig. Fig.11.

FIG. 1.
Schematic overview of EHV-1 viral constructs. (A) Genomic organization of the EHV-1 RacH genome. The genome can be divided into a unique long region (UL) and a unique short region (Us), which is flanked by an internal repeat region (IR) and a terminal ...

Viruses and cells.

EHV-1 strain RacH expressing enhanced green fluorescent protein (EHV-gfp) (50) and its derivatives were propagated on rabbit kidney cells (RK13) or complementing rabbit kidney cells coding for EHV-1 ETIF (RK-ETIF) (53). Analysis of recombinant EHV-1 was carried out with RK13 cells, RK-ETIF cells, or a human lung epithelial cell line (A549) (ATCC CCL-185). All cells were cultured at 37°C with 5% CO2 in Dulbecco minimal essential medium (Invitrogen) containing 10% fetal calf serum (FCS), 100 IU of penicillin, and 100 μg of streptomycin per ml (PAN Biotech). In addition, medium for RK-ETIF was supplemented with 500 μg Geneticin (Invitrogen) per ml. Primary splenocytes from immunized BALB/c mice were cultured in RPMI 1640 medium (PAN Biotech) completed with 5% FCS, 100 IU of penicillin, and 100 μg of streptomycin per ml (PAN Biotech) as well as 50 μM β-mercaptoethanol.

PCR analysis of viral DNA.

Viral DNA was isolated from eukaryotic cells by using the QIAamp DNA blood minikit (Qiagen). Purified viral DNA was analyzed by PCR and sequencing using primers P1 (5′-CTGGACCCAACGGTTACGAT-3′) and P2 (5′-GGCCAACATGTTAAGTCTGGAA-3′) to verify the successful deletion of gene 12 from the EHV-1 genome.

Western blot analysis.

Total cell lysates were prepared at 48 h posttransfection or 16 to 24 h postinfection (p.i.) by using a triple-detergent buffer system (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% [wt/vol] sodium dodecyl sulfate [SDS], 1% [wt/vol] Nonidet P-40, 0.5% [wt/vol] Na-deoxycholate) supplemented with a cocktail of protease inhibitors (Complete minikit; Roche). Protein concentrations were determined by Bradford analysis (Bio-Rad protein assay; Bio-Rad Laboratories) and adjusted to equal protein levels. The indicated amounts of total protein were separated by SDS-12.5% polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane (0.45 μm; Schleicher & Schuell) by semidry blotting (Bio-Rad).

Free binding sites on the membrane were blocked with Tris-buffered saline (TBS) buffer (20 mM Tris-HCl [pH 7.5], 500 mM NaCl) containing 5% skim-milk powder. Membranes were then probed with either a polyclonal rabbit anti-GFP antibody (Takara/Clontech), a monoclonal mouse anti-Gag antibody (13-5) (55), or a monoclonal mouse anti-β-actin antibody (AC-15) (Sigma), followed by incubation with an appropriate alkaline phosphatase (AP)-labeled anti-mouse (Bio-Rad) or anti-rabbit (Dako) secondary antibody. Antigen-antibody complexes were then visualized by using NBT-BCIP solution (0.3% 4-nitroblue tetrazolium chloride [NBT], 0.3% 5-bromo-4-chloro-3-indolylphosphate [BCIP], 100 mM Tris, 100 mM NaCl, 50 mM MgCl2 [pH 9.5]; Roche). For the detection of luciferase, membranes were incubated with an anti-luciferase antibody (Acris), followed by an anti-goat horseradish peroxidase (HRP) antibody (Dako). The antigen-antibody complexes were incubated with SuperSignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's instructions and visualized by using a ChemilusPro instrument (Intas).

Growth kinetics.

A total of 4 × 105 RK13 or RK-ETIF cells were infected with 4 × 102 to 106 PFU of EHV-gfp or EHV-syngag, resulting in a multiplicity of infection (MOI) of 0.001 to 10. Infected cells were incubated at 37°C to allow the penetration of virus. At 2 h p.i., cells were washed with phosphate-buffered saline (PBS) (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl) thoroughly to remove residual input virus, and fresh medium was added.

The amount of GFP- or Gag-positive cells was determined by fluorescence-activated cell sorter (FACS) analysis. Infected cells were harvested, washed with PBS, and resuspended in FACS buffer (PBS with 1% FCS and 1 mg/ml NaN3). While GFP was detected directly, Gag was visualized by intracellular staining with a Gag-specific phycoerythrin (PE)-labeled antibody (KC57-RD1; Beckman Coulter). Staining was performed according to the manufacturer's instructions.

Determination of virus titers.

The amount of progeny virus in supernatants of infected cells was determined. Three days after plating of serially diluted cell culture supernatants on RK-ETIF cells and incubation at 37°C under a 0.6% methylcellulose overlay, cells were fixed with 5% formaldehyde and stained with crystal violet, and plaques were counted.

In vivo imaging.

For biodistribution analysis, 1 × 105 PFU of EHV-luc, 106 PFU of EHV-lucΔETIF, or 106 PFU EHV was administered intranasally in a volume of 25 μl to 8- to 12-week-old BALB/c mice. Before in vivo imaging, 100 μl (60 mg/ml) of d-luciferin (Promega) was injected intraperitoneally. Images were obtained with an IVIS charge-coupled-device (CCD) camera (Xenogen) and analyzed with Living Image 2.60.1 software (Xenogen) 5 min after d-luciferin administration. Mice were anesthetized with 2% isoflurane during the procedure.

Vaccination of mice.

Female BALB/c mice (Charles River) aged 8 to 12 weeks were allocated into groups of three to six mice each and were immunized according to the indicated prime-boost regimen. Immunizations were performed either (i) subcutaneously by saline injection of 200 μl containing 80 μg pc-syngag or (ii) intranasally by applying a total volume of 25 μl PBS, including the indicated amount of EHV, into the nostrils by using a micropipette. For intranasal immunizations, animals were under light isoflurane anesthesia. All experiments were conducted in accordance with the legal requirements of local and national authorities.

Synthetic peptides.

The p24(CA)-derived 9-mer peptide A9I (AMQMLKETI), representing a defined Dd-restricted cytotoxic T-lymphocyte (CTL) epitope in BALB/c mice, as well as the V3-IIIB-specific epitope (SERIQRGPGRAFVTIGKI) used as a negative control were purchased from Biosyntan (Berlin, Germany).

Evaluation of antibody responses.

Mice were bled at the indicated time points, and serum was collected and analyzed for Gag-specific total immunoglobulin. Bronchoalveolar lavage (BAL) was performed by infusing 1 ml of PBS into the trachea. After two washes, at least 0.7 to 0.9 ml could be recovered by gentle aspiration. The fluid was centrifuged at 20,000 × g for 10 min. Supernatants were collected and tested for Gag-specific secretory immunoglobulin A (sIgA).

Anti-Gag total immunoglobulin and sIgA were quantified by an endpoint dilution enzyme-linked immunosorbent assay (ELISA) with serum or BAL fluid of individual animals. In brief, flat-bottomed 96-well Maxisorb microtiter plates (Nunc) were coated with purified HIV-1 clade IIIB Pr55gag precursor protein (50 ng in 100 mM NaHCO3 [pH 9.5]/well) and incubated overnight at 4°C. Plates were then washed with washing buffer (PBS with 0.05% Tween 20) and blocked with 200 μl/well blocking buffer (PBS containing 2% FCS) for 1 h at 37°C. Serum or BAL fluid was serially diluted in dilution buffer (PBS containing 2% fetal calf serum and 3% Tween 20) (2-fold dilutions from a 1:100 dilution [serum] or a 1:10 dilution [BAL fluid], respectively) and applied onto the blocked plates. After 2 h of incubation at 37°C, the plates were washed and subsequently incubated with 100 μl/well HRP-conjugated anti-mouse immunoglobulin (Dako) (1:2,000 in PBS with 2% FCS) or 100 μl/well of biotin-conjugated anti-mouse IgA (BD Biosciences) (1:1,000 in PBS with 2% FCS) for 1 h at 37°C. For the IgA ELISA, an additional incubation step at 37°C with 100 μl/well HRP substrate (Roche) (1:2,000 in PBS with 2% FCS) was performed for 1 h. After a final washing, 100 μl HRP substrate (TMB substrate kit; Pierce Perbio) was added to each well. The reaction was stopped by the addition of 100 μl/well 2 M H2SO4 to the mixture, and the optical density (OD) was measured at 450 nm. The reported titers were determined as the reciprocal of the highest serum dilution showing a 2-fold-higher OD value than the corresponding dilution of preimmune serum or of BAL fluid from untreated mice.

Intracellular IFN-γ staining and FACS analysis.

Single-cell suspensions were prepared aseptically from spleens of mice at the indicated time points. Gamma interferon (IFN-γ) expression of CD8+ T cells was detected by intracellular staining followed by FACS analysis. Splenocytes were stimulated for 6 h with 10 μM p24(CA)-derived nonameric peptide A9I (AMQMLKETI), representing a defined Dd-restricted CTL epitope in BALB/c mice (Biosyntan, Germany); an irrelevant V3 HIV-1 clade IIIB-specific 18-mer peptide (SERIQRGPGRAFVTIGKI) (Biosyntan, Germany), or growth medium in the presence of brefeldin A (5 mg/ml). Medium and the V3 peptide served as negative controls. Intracellular staining was performed as described previously (6). In total, 3 × 104 CD8+ lymphocytes were analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD).

Statistical analysis.

The statistical methods used for data analysis in this paper are 2-fold. Differences between cellular immune responses based on FACS analysis were shown by using t tests. To detect differences between IgA and IgG results, a nonparametric Mann-Whitney U test (35) was used. To ensure a better approximation of normality, the original FACS data were transformed by using a Box-Cox power transformation (7). t tests were employed for detecting differences between the means of treatments.

RESULTS

Biochemical characterization of recombinant EHV after deletion of gene 12 (ETIF).

Recombinant EHV-1 derivatives were generated based on RacH strains encoding GFP, firefly luciferase, or the HIV-1 Gag protein. To determine the impact of ETIF on the replication and immunogenicity of EHV-1, the ETIF coding sequence was deleted from the genomes of these viruses (Fig. (Fig.1).1). Recombinant virus was analyzed by PCR and Western blots. As expected, PCR of the ETIF region from cells infected with the deletion virus yielded a shortened PCR product (278 bp) compared to the parental sequence (1,603 bp) (data not shown).

To confirm correct transgene expression, permissive RK13 cells were infected with recombinant EHV at an MOI of 1, and cell lysates were screened for the expression of Gag, GFP, or luciferase by Western blotting. We showed that both the ETIF-deleted and parental virus were able to induce the expression of the specific transgenes in infected cells, and the size of specifically detected GFP, Gag, or Luc proteins correlated with their calculated molecular weights. In addition, infection of RK13 cells with ETIF-deleted or parental virus at an MOI of 1 yielded comparable amounts of Gag and GFP, indicating that neither gene delivery nor transgene expression is affected by the deletion of the ETIF gene (Fig. (Fig.22).

FIG. 2.
Protein expression from recombinant viruses. (A) RK13 cells were infected with an MOI of 1 of the indicated viruses or transfected with the indicated plasmids (pc-syngag or pc-gfp). Infected cells were harvested 24 h postinfection, and transfected cells ...

ETIF deletion self-limits virus replication in permissive cells in vitro.

Next, we addressed the issue of vector safety. It was reported previously that the ETIF protein is essential for the replication of EHV since it is essential for viral tegumentation and transactivation of the sole immediate-early gene of EHV-1 (53). We therefore determined the kinetics of viral replication to compare the dynamics of ETIF-deleted virus to those of the parental virus. We infected RK13, RK-ETIF, or human A549 cells with different doses of ETIF-deleted and parental viruses (MOI of 0.001 to 1) and monitored the numbers of infected cells (Fig. (Fig.3)3) and viral loads in supernatants (Fig. (Fig.4)4) over a period of 5 days.

FIG. 3.
Determination of GFP-positive RK13 and RK-ETIF cells after infection with EHV-gfp, EHV-gfpΔETIF, and mock controls by FACS analysis. A total of 4 × 105 cells were infected with the indicated amounts of virus, and the expression of GFP ...
FIG. 4.
Determination of virus titers in cell supernatants from RK13 and RK-ETIF cells infected with EHV-gfp, EHV-gfpΔETIF, and mock controls by plaque assays. Supernatants were harvested at the indicated time points and titrated on RK-ETIF cells. Shown ...

RK13 cells are productively infected by EHV-gfp, and viral doses even as low as an MOI of 0.001 were sufficient to infect the whole population of cells within 72 h postinfection. However, when RK13 cells were infected with different amounts of ETIF-deleted virus (MOI of 0.001 to 1), the maximum percentage of infected cells increased in a dose-dependent manner. While 2% of the cells were infected at an MOI of 0.001, 11% were infected at an MOI of 0.01, 54% were infected at an MOI of 0.1, and 90% were infected at an MOI of 1, as detected by GFP expression. The relatively high percentage of infected cells at MOIs of <1 reflects the fact that the ETIF-deleted virus is propagated in RK-ETIF cells that trans-complement the ETIF protein. Thus, the complemented virions transport functional ETIF into infected RK13 cells, resulting in a “recycling” of the ETIF protein for at least another round of virion assembly. However, the incorporated ETIF protein is diluted out from one round of infection in RK13 cells to the next, until the infection limits itself. Indeed, a time-dependent decrease in numbers of infected RK13 cells was observed at 96 h after infection with ETIF-deleted virus, whereas the percentage of EHV-gfp-infected cells remained stable (Fig. (Fig.3,3, white symbols). Similar results were obtained by using EHV-syngag or EHV-syngagΔETIF instead of the GFP-encoding viruses (data not shown).

The reduction in infected cells after infection with ETIF-deleted virus coincided with the finding that virus amounts in the supernatants of EHV-gfpΔETIF-infected RK13 cells decreased 72 h after infection. In fact, 120 h after infection with the lowest viral dose (MOI of 0.001), infectious particles could no longer be detected in the supernatants (Fig. (Fig.4,4, white circles).

The number of infected cells and viral loads in the supernatants could be restored when RK-ETIF cells were used for infection experiments instead of RK13 cells (Fig. (Fig.33 and and4,4, black symbols). These results provide good evidence that the observed effects can be specifically attributed to the ETIF defect.

In conclusion, our findings support previous data showing that infection of an EHV-1-permissive cell line with an ETIF deletion virus is self-limiting unless complemented with constitutively expressed ETIF in trans (53). In terms of a viral vector system for therapeutic applications, this mechanism of self-control promises a strongly enhanced safety profile.

No increase of ETIF-deleted virus after infection of human cells.

Taking into account that the therapeutic use of a vector system generally aims at applications for humans, we addressed the question of whether the ETIF deletion would affect infection and transgene expression in human cells. We infected a human lung epithelial cell line (A549) with different doses of EHV-syngag or EHV-syngagΔETIF and observed the rates of infected cells and viral loads in supernatants by FACS analysis and plaque assays over a period of 5 days (Fig. (Fig.55).

FIG. 5.
Determination of Gag expression (A) and virus titers (B) in cell supernatants of a human lung epithelial cell line (A549) after infection with EHV-syngag and EHV-syngagΔETIF compared to noninfected cells serving as negative controls. (A) A total ...

It was shown previously that recombinant EHV-1 is able to infect a wide variety of human cell lines and primary cells at low infection doses (50). Nevertheless, EHV infected A549 cells less efficiently than RK13 or RK-ETIF cells, which could be overcome by using higher MOIs. In these experiments almost all A549 cells expressed the Gag transgene within 24 h after infection with EHV-syngag at an MOI of 1 or 10. Although the percentage of transgene-expressing cells started to decline from 24 h after infection, our results confirm that recombinant EHV successfully enters human cells and drives transgene delivery and expression. This was also true, although to a much lesser extent at an MOI of 1, using EHV-syngagΔETIF for infection instead of EHV-syngag. However, since early in infection (6 h p.i.), the same number of Gag-positive cells was found in A549 populations infected with either the ETIF-deleted virus or the parental virus, the ETIF deletion does not seem to have a negative impact on transgene delivery and expression in human cells (Fig. (Fig.55).

While infection kinetics at an MOI of 10 were similar for the two virus derivatives, the number of cells expressing the transgene after infection with the ETIF-deleted virus at an MOI of 1 fell far short of that with the parental virus, with just under 50% of A549 cells being positive for Gag expression. Consistent with this, we detected clear differences between the amounts of virus in supernatants from A549 cells infected with EHV-syngag or EHV-syngagΔETIF (Fig. (Fig.5).5). The replication of recombinant EHV-syngag in A549 cells was severely impaired but not completely disabled. Between 12 and 24 h after infection there was a 10-fold increase in viral loads in the supernatants of infected A549 cells, pointing to further infection, replication, and virus release. For the ETIF-deleted virus, however, the amount of virus in supernatants declined between 6 and 12 h after infection, indicating continued uptake but little virus replication in A549 cells but, however, explaining the subsequent increase in numbers of Gag-positive cells during EHV-syngagΔETIF infection.

In conclusion, these results suggest that the residual replication of EHV-1 in human cells might be fully blocked by the deletion of the essential tegument protein ETIF without affecting transgene delivery and expression.

In vivo distribution.

To determine the potential impact of absent ETIF on the biodistribution and attenuation of the recombinant viruses in a permissive in vivo model, 1 × 105 or 1 × 106 PFU of EHV, EHV-luc, or EHV-lucΔETIF expressing firefly luciferase was administered intranasally to BALB/c mice. In all cases, and independently from the administered dose, bioluminescence analysis with the luciferase reporter revealed transgene expression within the borders of sensitivity to be restricted exclusively to the nasal cavity and lungs of treated mice (Fig. (Fig.6).6). The luciferase signal was detectable as soon as 6 h p.i. and remained stable for 4 to 5 days. Bioluminescence peaked at 2 days p.i. in both the nasal cavity and lungs. Although substantial luminescence was monitored after the administration of the lower dose of EHV-luc (105 PFU), a 10-fold dose escalation resulted in a further increase in signal intensity (data not shown). In comparison, the deletion of the ETIF gene from the EHV-1 genome and the administration of 105 PFU EHV-lucΔETIF resulted in background levels of luciferase expression. Increasing the dose of EHV-lucΔETIF by 1 order of magnitude to 106 PFU led to luciferase transgene expression in the lungs that was limited to less than 2 days and resulted in 30-fold-reduced lung-associated bioluminescence even if compared to a 10-fold-lower dose (105 PFU) of the parental virus. Taken together, these findings clearly suggest that the deletion of ETIF substantially attenuates virus replication in vivo and may thus add to increasing the safety of EHV-based vector systems.

FIG. 6.
In vivo analysis of luciferase expression after intranasal administration of EHV, EHV-luc, and EHV-lucΔETIF to BALB/c mice. The indicated doses (PFU) of viruses were administered intranasally and imaged with a CCD camera at the indicated time ...

Single immunization with EHV-syngagΔETIF results in decreased transgene-specific cellular immune responses in vivo.

We previously showed that the intranasal application of recombinant EHV (EHV-syngag) expressing a viral antigen (HIV-1 group-specific antigen [Gag]) is capable of inducing transgene-specific cellular immune responses in BALB/c mice (50). To evaluate if the ETIF deletion has any impact on the induction of transgene-specific immune responses, we performed a viral dose titration with BALB/c mice. Mice were immunized once with different intranasal doses (4 × 105, 4 × 106, and 4 × 107 PFU) of EHV-syngag or EHV-syngagΔETIF. Animals receiving a single subcutaneous application of a Gag expression plasmid (pc-syngag) served as a positive control.

Ten days after the administration of virus or DNA, spleen cells of immunized mice were isolated, and Gag-specific cellular immune responses were quantified by FACS analysis to determine the number of IFN-γ-producing CD8+ T cells upon stimulation with Gag-specific nonameric peptide A9I or an irrelevant HIV-1 envelope-derived peptide specific for V3 (Fig. (Fig.7).7). As expected, a single subcutaneous application of plasmid DNA (pc-syngag) resulted in strong Gag-specific immune responses. A single intranasal inoculation with EHV-syngag was sufficient to trigger a distinct Gag-specific cellular immune response in a dose-dependent manner. In fact, the highest viral dose, 4 × 107 PFU, even reached the level of CD8+ T-cell responses detected after a single administration of plasmid DNA. In contrast, EHV-syngagΔETIF induced substantially lower numbers of Gag-specific CD8+ T cells, which was not enhanced by increasing the immunization dose. Even a viral dose as high as 4 × 107 PFU did not reach the level of Gag-specific CD8+ T cells induced by inoculation with 4 × 105 PFU of EHV-syngag.

FIG. 7.
Dose titration of EHV-syngag and EHV-syngagΔETIF in BALB/c mice. Shown are data from determinations of Gag-specific CD8+ cells in BALB/c mice (three per group) immunized once intranasally (i.n.) with the indicated amount of virus or subcutaneously ...

Hence, our data clearly indicate that the ETIF deletion and the concomitant reduction of in vivo replication mitigate the induction of transgene-specific immune responses after a single administration to BALB/c mice. To compensate for this reduced activity in subsequent experiments, viral doses were set at 1 × 105 PFU for the parental virus and 1 × 106 PFU for the ETIF-deleted virus.

Magnitude of transgene-specific cellular and humoral immune responses induced by ETIF-deleted and parental virus are substantially increased by application of heterologous prime-boost regimens.

To investigate the influence of homologous and heterologous prime-boost strategies on the induction of transgene-specific immune responses, BALB/c mice received a total of three immunizations comprising either an intranasally applied dose of virus (EHV-syngag or EHV-syngagΔETIF) or a subcutaneously administered Gag expression plasmid (pc-syngag). Initial priming was followed by two booster immunizations after 5 and 9 weeks. Animals receiving three subcutaneous applications of a Gag expression plasmid (pc-syngag) served as positive controls. Ten days after the last immunization, we terminated the experiment and determined Gag-specific cellular immune responses (Fig. (Fig.8)8) as well as levels of Gag-specific total immunoglobulin and secretory IgA (Fig. (Fig.99).

FIG. 8.
Determination of Gag-specific cellular immune responses by FACS analysis. Female BALB/c mice (six per group) were immunized three times at 4-week and 5-week intervals with the indicated virus or DNA; 10 days after the last immunization splenocytes from ...
FIG. 9.
Determination of Gag-specific IgG and IgA levels in serum and bronchoalveolar lavage (BAL) fluid from immunized BALB/c mice. Mice were immunized three times at 4-week and 5-week intervals with the indicated virus or DNA. Each symbol ([filled lozenge]) represents ...

Whereas DNA priming followed by two DNA booster immunizations significantly enhanced Gag-specific T-cell responses compared to a single pc-syngag DNA administration (group 2) (compare Fig. Fig.77 and and8),8), repeated immunization with recombinant EHV-syngag or EHV-syngagΔETIF (groups 3 and 4) (Fig. (Fig.8)8) did not expand the level of antigen-specific T-cell responses seen after a single immunization (Fig. (Fig.7).7). However, a single subcutaneous DNA immunization provided sufficient priming to substantially increase the number of antigen-specific T cells induced by two subsequent administrations of either EHV-syngag or EHV-syngagΔETIF (groups 5, 6, and 7) (Fig. (Fig.8).8). Moreover, the numbers of Gag-specific CD8+ T cells induced by the heterologous prime-boost regimen were in the same range as those evoked in the three consecutive DNA immunization controls (group 2).

In conclusion, our data show that the subcutaneous application of a Gag expression plasmid followed by two booster immunizations with recombinant EHV-syngag, EHV-syngagΔETIF, or a combination of both resulted in a potent transgene-specific cellular immune response in BALB/c mice. Furthermore, although a single administration of ETIF-deleted EHV did have a negative impact on the induction of transgene-specific immune responses compared to that of the parental virus, no significant difference was observed between mice that received EHV-syngagΔETIF and those that received EHV-syngag as a booster immunization.

Recombinant EHV-1 induces transgene-specific humoral immune responses, including the secretion of secretory IgA.

These findings were also confirmed at the level of Gag-specific humoral immune responses. Gag-specific total immunoglobulin levels in serum were determined by endpoint dilution ELISA (Fig. (Fig.9A).9A). Statistical analysis of the results showed that animals immunized according to a heterologous prime-boost regimen (groups 5, 6, and 7) developed significantly higher levels of Gag-specific total immunoglobulin than animals that had received 3 doses of either virus or plasmid DNA. Again, there was no difference whether EHV-syngagΔETIF, EHV-syngag, or a combination of both was used for booster immunizations.

A further criterion for the eligibility of EHV-1 as a vector system was whether recombinant EHV-syngag is also capable of eliciting transgene-specific mucosal immune responses. To investigate this, Gag-specific sIgA levels in bronchoalveolar lavage fluid from immunized mice were determined by using endpoint dilution ELISA (Fig. (Fig.9B).9B). The sIgA ELISA data revealed that three-time DNA immunization did not induce the formation of Gag-specific sIgA (group 2). However, substantial amounts of Gag-specific secretory IgA were induced in animals receiving pc-syngag priming followed by two viral booster immunizations with EHV-syngag, EHV-syngagΔETIF, or a combination of both viruses. Differences between immunization groups 5, 6, and 7 suggesting a trend toward higher levels of sIgA where EHV-syngagΔETIF was used as a booster vaccine were not statistically significant and may have been impacted by the fact that 10-fold-higher doses of the attenuated EHV-syngagΔETIF (106 PFU) were used than the replication-competent EHV-syngag (105 PFU). Although we cannot exclude that increasing the dose of the parental virus might lead to higher titers of Gag-specific sIgA responses, these data clearly show that the highly attenuated phenotype of the deletion mutant can be compensated for by increasing the dose by 1 order of magnitude.

In summary, our data reveal that a heterologous prime-boost regimen comprising DNA priming and two viral boosts was capable of inducing substantial amounts of sIgA in BALB/c mice. Furthermore, although a Gag expression plasmid applied systemically could not induce Gag-specific secretory IgA in immunized mice on its own, it was able to prime for a transgene-specific mucosal immune response.

DISCUSSION

The natural purpose of viruses to infect cells and deliver their genetic information has been exploited successfully in the past, resulting in a variety of different viral vector systems (for a review, see reference 44). Despite the availability of these vectors, it is broadly accepted that additional viral vectors are needed to both support novel gene therapy strategies and contribute to developing new vaccines targeting current epidemics, including but not limited to HIV, malaria, and tuberculosis.

Herpesviral vectors used for vaccination or gene therapy approaches so far are comprised primarily of herpes simplex virus type 1 (HSV-1) but also include Epstein-Barr virus (EBV) and human CMV (11, 18, 22, 23, 44). The main advantage of these replicating or nonreplicating vectors is the targeting of mucosal surfaces in order to induce mucosal immune responses. However, many vaccination strategies suffer from preexisting immunity, and the use of HSV, EBV, or CMV in humans is highly limited due to the high prevalence of their parental viruses.

It was shown previously that antibodies directed against human herpesviruses show no cross-reactivity to EHV-1 (50). Since the virus is absent in humans (40), it can be assumed that the problem of preexisting immunity should be of minor importance. Furthermore, EHV-1 infects a broad spectrum of different eukaryotic cells (50), and due to its ability to infect epithelial cells, EHV-1 challenge is characterized by strong mucosal immune responses (9). Due to these properties, EHV-1 strain RacH was selected recently as a novel vector for delivering vaccine genes into human cells (50, 53). We recently showed that recombinant EHV-1 RacH efficiently transduces primary human antigen-presenting cells, allowing the robust expression of a delivered transgene. In addition, strong transgene-specific cellular immune responses could be elicited in BALB/c mice after inoculation with recombinant virus via various routes (50).

Providing additional evidence that recombinant EHV replicates poorly in human cells, we demonstrate here that the deletion of ETIF, the transactivator of the sole immediate-early gene of EHV-1, abrogates the residual replication of EHV in a human lung epithelial cell line (A549). Similarly, in a permissive cell line (RK13), the deletion of ETIF from the EHV-1 genome severely attenuated EHV replication without influencing the expression of the GFP reporter. This phenotype can be reverted by stably expressing ETIF in trans (RK-ETIF) (53). Furthermore, our in vivo analyses with BALB/c mice demonstrated that EHV infection is restricted to the nasal cavities and the lungs and is self-limiting after ~6 days postinoculation. Moreover, in accordance with our ex vivo analyses, the deletion of ETIF reduced the time of luciferase expression to 2 days as well as luminescence intensity by 30-fold compared those of the parental virus, demonstrating an additional attenuated phenotype in the RacH vaccine strain.

A single intranasal administration of various doses of the ETIF-deleted mutant in BALB/c mice clearly revealed that the attenuation results in reduced CD8+ cellular immune responses to the Gag transgene compared to the parental virus. This finding suggests that the replicative capacity monitored during the bioimaging analyses correlates with the level of transgene-specific T-cell responses. Vector immunity after more than one application of the EHV vector may limit the effect of booster immunizations (Fig. (Fig.77 and and8).8). Indeed, BALB/c mice that had been primed with EHV-gfp or EHV-gfpΔETIF followed by two booster immunizations with EHV-syngag hardly developed any Gag-specific immune response (data not shown).

It is noteworthy, as shown in Fig. Fig.8,8, that a single subcutaneous immunization with gag DNA primed specific T cells for substantial expansion by two subsequent intranasal booster immunizations using either the gag recombinant ETIF deletion mutant or the parental virus. The level of antigen-specific T cells induced by this prime-boost regimen reached the T-cell responses measured after three DNA immunizations and clearly exceeded those responses measured after a single DNA immunization (Fig. (Fig.7)7) or a homologous prime-boost strategy of 3 intranasal doses of virus (Fig. (Fig.8).8). This finding correlates with previous results from our group and others showing that the strengths of immune responses induced by different vector systems are often not simply additive but actually synergistic (4, 25, 56). Moreover, in terms of inducing systemic Gag-specific serum antibody levels, the DNA prime-EHV boost strategy was superior to the repeated administration of the DNA vaccine or either EHV vector.

Secretory IgA levels are generally very low, rarely exceeding values above 1,000 (38, 39, 48), and often can be determined only qualitatively (2, 26, 27, 52). Immune responses against EHV-1 in horses, however, are characterized by high levels of EHV-specific IgA, identified as the predominant isotype upon natural EHV-1 infection (9). In agreement with these data we found that heterologous prime-boost regimens comprising subcutaneous priming with plasmid DNA and two nasal boosts with recombinant EHV-1 or EHV-ΔETIF induced high levels of transgene-specific sIgA in BALB/c mice, as assessed with bronchoalveolar lavage (BAL) fluid. Such sIgA levels were not reached either by three subsequent immunizations with the parental or EHV-ΔETIF virus or by repeated DNA vaccination. Interestingly, although plasmid DNA applied subcutaneously could not induce a mucosal immune response on its own, it could still prime the induction of a transgene-specific mucosal immune response when boosted with one of the two EHV-1 derivatives. In this setting, compared to parental EHV-1, the further attenuation of EHV-1 by deleting ETIF had no negative impact on the stimulation of sIgA-producing B cells.

The strength of the mucosal immune response was measured in bronchoalveolar lavage fluid from immunized mice, which is the primary sIgA induction site after the intranasal application of a vaccine. Although this provides no evidence that a transgene-specific mucosal immune response is also elicited in distant mucosal compartments, it has been reported repeatedly that intranasal immunizations are capable of inducing mucosal immune responses at secondary effector sites such as the vagina or oral mucosa (5, 57). In fact, it has even been shown that the intranasal application of a vaccine elicits stronger mucosal immune responses in vaginal mucosa than does a corresponding vaginal application (17). For HIV (10, 29), and also other sexually or mucosally transmitted diseases in general, protective immunity at the site of pathogen entry plays an important role in controlling and preventing disease transmission.

In conclusion, EHV-based vectors seem to be well suited to induce transgene-specific cellular, humoral, and mucosal immune responses. ETIF deletion self-limits EHV replication in permissive cells in vitro and in vivo and may thus contribute to vector safety. DNA-EHV prime-boost regimens, irrespective of whether the parental or the deletion mutant was used in this regimen, were superior to the administration of either component alone. Highly attenuated replication resulting from the ETIF deletion can be compensated for by a 1-log dose increase of the administered deletion virus in a DNA-EHV prime-boost setting. In sum, EHV-1's safety profiles combined with its potential to induce transgene-specific cellular, humoral, and mucosal immune responses recommend this animal herpesvirus as a potent and promising new candidate for heterologous gene delivery. It certainly deserves further investigation.

Acknowledgments

This work was supported by grants M29 and M45.2 from the Hector Foundation, Germany.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 September 2010.

The authors have paid a fee to allow immediate free access to this article.

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