The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 7158 -7166
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Day, S. L.
Right arrow Articles by Ranasinghe, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Day, S. L.
Right arrow Articles by Ranasinghe, C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

Differential Effects of the Type I Interferons {alpha}4, β, and {epsilon} on Antiviral Activity and Vaccine Efficacy1

Stephanie L. Day2,*, Ian A. Ramshaw*, Alistair J. Ramsay{dagger} and Charani Ranasinghe*

* Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia; and {dagger} Gene Therapy Program, Clinical Sciences Building, Louisiana State University Health Sciences Center, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The type I IFNs exert a range of activities that include antiviral, antiproliferative, and immunomodulatory effects. To study this further, we have constructed recombinant vaccinia viruses expressing HIV or hemagglutinin (HA) Ags along with murine type I IFNs, IFN-{alpha}4 (HA-VV-IFN-{alpha}4), IFN-β (HA-VV-IFN-β), or IFN-{epsilon} (HIV-VV-IFN-{epsilon}), a recently discovered member of this family. Our aims were to characterize IFN-{epsilon} functionality as a type I IFN and also to study the biological properties of these factors toward the development of safer and more effective vector-based vaccines. HIV-VV-IFN-{epsilon} and HA-VV-IFN-β grew to lower titers than did their parental controls in murine cell lines. In vivo, however, HIV-VV-IFN-{epsilon} growth was not attenuated, while IFN-β demonstrated potent local antiviral activity with no replication of HA-VV-IFN-β detected. Flow cytofluorometric analysis of B lymphocytes incubated with virally encoded IFN-{epsilon} showed up-regulation of activation markers CD69 and CD86, while RT-PCR of IFN-{epsilon}-treated cells revealed that gene expression levels of antiviral proteins were elevated, indicating the induction of an antiviral state. The use of these constructs in a poxvirus prime-boost immunization regime led to robust humoral and cellular immune responses against the encoded Ags, despite the lack of replication in the case of HA-VV-IFN-β. Thus, coexpression of these factors may be beneficial in the design of safer vector-based vaccines. Our data also indicate that while IFN-{epsilon} exhibits certain biological traits similar to other type I IFNs, it may also have a specific role in mucosal immune regulation that is quite distinct.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interferons are a group of cytokines first discovered based on their antiviral activity against influenza (1). Two families of IFNs, type I and type II, can be distinguished. Type I comprises a family of cytokines, while type II consists of a single cytokine, IFN-{gamma}. The type I IFN family includes multiple IFN-{alpha} subsets, a single IFN-β and IFN-{omega} gene, and also the recently discovered IFN-{epsilon} (2). All members of the type I IFN family bind the same cell surface-specific receptor, IFN-IR, and all share sequence homology, with all type I IFN genes located in a single gene cluster both in humans and in mice (2, 3, 4).

In a viral infection, induction of the production of IFN-{alpha} and IFN-β occurs via two pathways. TLR signaling triggers production of IFN from APCs upon recognition of viral nucleic acids endocytosed from infected cells (5, 6). A second more widespread system recognizes viral infection within the cytosol of the infected cell and triggers production of IFN by signaling via RNA helicases, retinoic acid inducible gene-1 (RIG-1), and melanoma differentiation-associated gene 5 (MDA5) (7, 8). The IFN response to infection is rapid, and these cytokines serve as a frontline of defense against many pathogens and diseases. Previous studies have shown that IFN-{alpha}/β play a major role in the generation and survival of effector and memory T cells in response to viral infection (9, 10, 11). Additionally, type I IFNs demonstrate a range of activities on innate cells of the immune system, such as the activation and survival of NK cells during viral infection (12).

IFN-{epsilon} is a newly discovered member of the type I IFN family. Its expression has been localized to reproductive as well as to brain tissues (2). However, no significant work has been performed to elucidate any immunomodulatory or regulatory functions of IFN-{epsilon} in these tissues. In the human cervix following exposure to seminal fluid, increased expression of IFN-{epsilon} has also been reported (13). Seminal fluid delivers a range of signaling molecules that prime the environment to receive and protect an embryo and to provide a barrier against infection. Thus, IFN-{epsilon} may be involved in the recruitment, activation, and function of local leukocytes and APCs, and it may have a role in mucosal immunity.

Vaccinia virus (VV)3 is a member of the genus orthopoxvirus, which includes variola virus, the causative agent of smallpox. These viruses are very closely related and, as such, infection or vaccination from one member of the genus can confer a level of cross-protection against other members. Consequently, VV has been effectively used in the worldwide eradication of smallpox (14) and has also been extensively studied as an expression vector for foreign genes in a number of experimental systems (15, 16, 17). Using VV as a live recombinant vaccine, many groups have demonstrated that potent cell-mediated and humoral immunity can be generated against an encoded vaccine Ag (18, 19). The capacity to insert large pieces of foreign DNA into VV or other poxvirus vectors has enabled the construction of recombinant vaccinia viruses (rVV) expressing multiple proteins, such as vaccine Ags and costimulatory molecules (20, 21), that can enhance cell-mediated and humoral immunity to the encoded Ag or the vaccine vector (22, 23, 24). In particular, cytokines known to have antiviral activity such as IFN-{gamma}, or those that influence the differentiation or activation of cell subsets involved in the immune response such as IL-4, have been found to significantly modulate the immune response (24, 25).

Due to the type I IFNs pleiotropic effects on immune cells both of the innate and adaptive immune systems, as well as their promising use as therapeutic agents, we have constructed rVV coexpressing the murine type I IFNs, IFN-{alpha}4, IFN-β, and IFN-{epsilon}, along with a model Ag, hemagglutinin (HA) from the influenza virus, or HIV gag/pol genes. We have demonstrated that the expression of IFN-{alpha}4 or IFN-β causes a marked attenuation of viral virulence both in immunocompetent and immunosuppressed mouse models. Furthermore, we elucidate that viral attenuation by the expression of IFN-{alpha}4 or IFN-β occurs without compromising the effectiveness of the immune response to HA or to the viral vector. Results indicate that expression of IFN-{alpha}/β could increase the safety and efficacy of rVVs as vaccine vectors. In contrast, IFN-{epsilon} expression causes a reduction in the viral growth in vitro in murine cells but does not cause viral attenuation in vivo. Additionally, expression of IFN-{epsilon} does not alter the immunogenicity of the vaccine vector. Thus, the functionality of IFN-{epsilon} as a member of the type I IFN family remains to be fully characterized.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female athymic BALB/c nude mice (4–6 wk old) were obtained from the Animal Resource Centre, Perth, Western Australia. Female BALB/c (H-2d) mice, type I IFN receptor knockout (IFN-IR–/–), and wild-type 129 mice (4–6 wk old) were obtained from the Animal Breeding Facility at the John Curtin School of Medical Research. Female type II IFN receptor knockout (IFN-IIR–/–) mice (8–11 wk old) were kindly provided by Sue Fordham at The Canberra Hospital. All mice were bred and maintained under specific pathogen-free conditions and used in accordance with animal ethics guidelines.

Cells

African green monkey kidney CV-1 cells, human osteosarcoma 143B cells, and murine fibrosarcoma L929 cells were grown in MEM (Invitrogen) supplemented with 5% FCS, 1 mM L-glutamine, 10 mM HEPES, and antibiotics at 37°C in 5% CO2. CV-1, 143B, and L929 cells were used for VV growth.

Generation of recombinant vaccinia and fowlpox viruses

RNA was extracted from BALB/c ovarian tissue and cDNA was reverse transcribed. For cloning purposes, SalI restriction enzyme sites were added to the primers. IFN-{epsilon} cDNA was PCR amplified using Elongase enzyme mix (Invitrogen) according to the manufacturer’s instructions and using the following primers; IFN-{epsilon} forward: 5'-CCC GAA TTC TGA TCA AGA TCC CTG TGT GCC CTC CAC CAT-3'; IFN-{epsilon} reverse: 5'-CCC GAA TTC GTC GAC TCC TCC CAC TCC GCC ACC AA-3'. PCR conditions were 94°C for 2 min and then 35 cycles of 94°C for 30 s, 60°C for 40 s, and 72°C for 1 min. The IFN-{epsilon} gene was cloned into pTK7.5 digested with SalI, which was then transfected into cells infected with the parental HIV-VV thymidine kinase-negative (TK–) virus, which contained HIV B clade gag/pol genes. Recombinants were selected and IFN-{epsilon} expression was confirmed by PCR. Fowlpox virus recombinants (rFPV) expressing HA and HIV gag/pol were constructed as described previously (26, 27). rVVs expressing IFN-{alpha}/β were derived from the Western reserve (WR) strain of the virus as described previously (18, 28). They express the HA gene from the A/PR/8/34 strain of influenza virus as well as genes encoding murine IFN-{alpha}4 (mIFN-{alpha}4) or murine IFN-β (mIFN-β; we thank Dr. M. Rentrop (German Cancer Research Center, Heidleberg, Germany) for plasmid containing mIFN-β). Expression of the IFNs was confirmed by virus cytopathic effect assay with respect to international reference standards provided by the National Institutes of Health (see Ref. (29)).

Peptide

The H-2Kd restricted, well-defined HA epitope (533IYSTVASSL541) from the PR8 influenza virus (30) was synthesized using the 9-fluorenylmethyl-oxycarbonyl (FMOC) method on a Rainen Symphony/Multiplex peptide synthesizer and supplied by the Australian Cancer Research Foundation Biomolecular Resource Facility at the John Curtin School of Medical Research, Australian National University. An HIV overlapping 15mer Gag peptide pool was obtained from the AIDS Research and Reference Reagent Program (National Institutes of Health, Bethesda, MD).

Virus growth curves

Monolayers of CV-1 cells or L929 cells were infected with each rVV (HA-VV-IFN-{alpha}4, HA-VV-IFN-β, HA-VV, HIV-VV-IFN-{epsilon}, HIV-VV) at a multiplicity of infection (MOI) of 0.01 for 1 h at 37°C in 6-well plates. MEM (2 ml) supplemented with 5% FCS was added to each well and the cells were cultured at 37°C for 72 h. At each time point both the intracellular and extracellular fractions were collected as described previously (31). The fractions were frozen, thawed, sonicated (2 x 15 s), and trypsinized. Viral titers were then determined by titration onto 143B cell monolayers in 6-well plates by plating out dilutions of 1/10 to 1/106 and staining plaques with crystal violet after 48 h incubation at 37°C.

Virus growth in immunocompetent mice

Groups of BALB/c mice (n = 4) were immunized i.v. with 107 PFU for each rVV (HA-VV-IFN-{alpha}4, HA-VV-IFN-β, HA-VV, HIV-VV-IFN-{epsilon}, HIV-VV) in 200 µl sterile PBS. Mice were sacrificed after 72 h by CO2 asphyxiation after which ovaries were harvested, homogenized, and trypsinized, and dilutions were plated onto monolayers of 143B cells to determine viral titers.

Vaccinia virulence in nude mice

Viral pathogenicity was measured in groups of BALB/c nude mice (n = 4–5). Mice were immunized i.v. with doses of the different rVVs ranging from 106 to 108 PFU/ml in 200 µl sterile PBS. Mice were monitored daily and were sacrificed when they showed signs of severe weight loss and reduced responsiveness. Spleen, ovaries, lung, and brain were harvested, frozen immediately, and stored at –20°C. Viral titers in the organs were enumerated by plaque assays, by plating dilutions of homogenized organs onto monolayers of 143B cells in 6-well plates.

ELISPOT assay

Spleen cells were assayed by IFN-{gamma} ELISPOT for HA- or HIV Gag-specific T cells as described elsewhere (32). Briefly, MultiScreen HTS filter plates (Millipore) were coated overnight with 5 µg/ml purified rat anti-mouse IFN-{gamma} capture Ab (BD Pharmingen). Splenocytes/well (2 x 105) were incubated for 20 h at 37°C in the presence of 1 µg/ml 9mer HA peptide (533IYSTVASSL541), 0.5 µg/ml 15mer Gag peptide pool, without peptide, or 5 µg/ml Con A as a positive control. For vaccinia-specific IFN-{gamma} responses, naive spleen cells were infected with a wild-type vaccinia WR strain (VV-WR) at a MOI of 10 for 1 h at 37°C, and then washed cells were added to primed cells at a ratio of 1:4 and incubated as described above. Following incubation, the plates were washed and incubated with biotinylated rat anti-mouse IFN-{gamma} Ab (BD Pharmingen) and further incubated with streptavidin alkaline phosphatase (Amersham Life Science). Spot forming units (SFU) were developed using BCIP/NBT alkaline phosphatase substrate (Moss) and counted using an ELISPOT Bioreader-4000 PRO-X (Bio-Sys). The background counts from unstimulated cells were subtracted for the purpose of analysis. Results are expressed as SFU per 1 x 106 cells and represent duplicate or triplicate experiments.

ELISA

HA-specific serum Abs were detected by ELISA. Ninety-six-well ELISA plates (Thermo LabSystems) were coated with PR8 influenza virus diluted in borate buffer (BupH borate buffer, Pierce Biotechnology) and incubated overnight at 4°C. The plates were washed with PBS 0.05% Tween and then blocked with PBS containing 5% skim milk for 2 h at 37°C. The mouse serum diluted in PBS was added to the plates at appropriate dilutions and incubated for 90 min at 37°C, and then plates were incubated with 0.5 µg/ml biotinylated goat anti-mouse IgG1 and IgG2a Abs (Southern Biotechnology Associates) for 2 h at 37°C. Finally, plates were incubated with streptavidin alkaline phosphatase diluted in PBS containing 1% BSA for a further 90 min at 37°C. After each step plates were washed 4x with PBS 0.05% Tween. The serum Ab was detected by developing with alkaline phosphate substrate solution (Sigma-Aldrich) for 20 min, and the absorbances were read at 405 nm using a ThermoMax plate reader and SoftMax software (MDS Analytical Technologies).

Properties of IFN-{epsilon} protein

For biological IFN-{epsilon} assays, L6 plate monolayers of CV-1 cells were infected with 0.01 MOI HIV-VV-IFN-{epsilon} or HIV-VV and incubated for 24 h at 37°C. The medium was collected and live virus removed by double filtration through 0.22-µm filters. Supernatant was incubated with primary splenocytes for 16 h at 37°C, after which cells were washed, incubated with Abs to cell surface markers, and analyzed by flow cytometry to assess cellular activation states. CD8-allophycocyanin, B220-FITC, CD69-PE, and CD86-PE Abs were purchased from BD Pharmingen. Recombinant mouse IFN-{alpha} was used as a positive control (Chemicon International).

Expression of antiviral proteins was assessed at the mRNA level by RT-PCR. L929 cells were incubated as above with inactivated supernatant, and then RNA was isolated as described previously (33). cDNA was synthesized and PCR was performed using Scientifix Taq polymerase and the following primers: protein kinase R (PKR) forward: 5'-ATG CAC GGA GTA GCC ATT ACG-3'; PKR reverse: 5'-GTT TTC GGC GGG CTC TTT AAC-3'; 2'5'oligoadenylate synthetase (2'5'OAS) forward: 5'-ATG GAG CAC GGA CTC AGG A-3'; 2'5'OAS reverse: 5'-TGC TTC AGG AAG TTC CGC TG-3'; L32 forward: 5'-TTA AGC GAA ACT GGC GGA AAC-3'; L32 reverse: 5'-CGT TGG GAT TGG TGA CTC TGA TGG-3'. PCR conditions were 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 58°C for 40 s, and 72°C for 40 s. PCR products were separated on a 1.5% agarose gel.

Real-time PCR

To detect and quantify antiviral gene expression, real-time PCR was performed using Applied Biosystems SYBR Green PCR master mix. Reactions were performed in Eppendorf twin-tec 96-well PCR plates in 20 µl reaction volumes, which contained 10 µl master mix, 1.5 pmol each forward and reverse primers, and 1–10 ng cDNA. All primers were designed to have the midpoint of thermal denaturation (Tm) of 60°C using the PrimerBank facility (34). Primers used were 2'5'OAS forward: as above; 2'5'OAS reverse: 5'-TCACACACGACATTGACGGC-3'; PKR forward: as above; PKR reverse: 5'-TGACAATCCACCTTGTTTTCGT-3'; L32 forward and reverse: as above. Reactions were run using the following cycling conditions: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Samples were run on an ABI PRISM 7700 sequence detection system (Applied Biosystems) and analyzed using SDS 1.9.1 software. Fold increases in mRNA were calculated as described previously (35).

Ectromelia challenge studies

Groups of BALB/c mice (n = 4) were immunized i.m. with 1 x 107 PFU of rVV diluted in 100 µl sterile PBS, or with PBS only. At 21 days postimmunization mice were challenged with 1 x 103 PFU of ectromelia virus (ECTV) Moscow strain by footpad injection. Mice were monitored daily and were sacrificed when showing symptoms of acute mousepox. These symptoms included lethargy, eye inflammation, ruffled fur, and a haunched posture.

Statistics

p values were calculated using a two-tailed, two-sample, equal variance Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of type I IFN expression on the growth of rVV in vitro

Coexpression of cytokines and other immune-modulating genes by poxvirus vaccine vectors has proven to be a useful tool in optimizing the vaccine response. rVV expressing IFN-{alpha}4, IFN-β, and IFN-{epsilon} were constructed from parental viruses expressing HA gene from the influenza virus A/PR/8/34 or HIV gag/pol genes, as described in Materials and Methods.

Confirmation of IFN-{alpha}/β cytokine expression by the rVVs was assayed by the cytopathic effect inhibition bioassay using influenza virus and L929 cells. At 24 h postinfection (PI), we observed a 10-fold increase in the bioactivity of virally expressed IFN-β compared with IFN-{alpha}4, with 6000 IU/ml IFN-{alpha}4 and 63,000 IU/ml IFN-β detected. To establish whether the increase in antiviral activity of IFN-β was due to differing viral expression levels, IFN-{alpha}4 and IFN-β expression in rVV-infected CV-1 cells was assayed by real-time PCR. Results show no difference in the viral mRNA expression levels of the two cytokines at 6 h following 10-fold MOI (Fig. 1a). Viral mRNA expression of IFN-{epsilon} was assayed by RT-PCR and indicates a time- and virus dose-dependent level of gene expression (Fig. 1b).


Figure 1
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 1. IFN expression by rVV. CV-1 cells were infected with rVV at the indicated MOIs. a, At 6 h PI, IFN-{alpha}4 and IFN-β mRNA levels were assayed by real-time PCR. mRNA levels were denoted as fold increase above uninfected cells and were normalized against L32 expression. b, At 6 and 24 h, IFN-{epsilon} mRNA levels were evaluated by PCR.

 
To study the growth characteristics of the rVVs, low MOI (0.01) growth curves were conducted using murine L929 cells (responsive to murine type I IFNs) and nonmurine CV-1 cells (nonresponsive to murine type I IFNs). We observed no difference in the replication of HA-VV-IFN-{alpha}4 or HA-VV-IFN-β as compared with the HA-VV parental in the CV-1 cells (Fig. 2, a and c). In contrast, addition of the IFN-β greatly attenuated the levels of viral replication in mouse L929 cells, while IFN-{alpha}4 expression had no observable effect (Fig. 2, b and d). Likewise, viral expression of IFN-{epsilon} resulted in a reduction in virus growth in murine L929 cells but not in nonmurine CV-1 cells compared with the HIV-VV parental (Fig. 3), validating functional protein expression of IFN-{epsilon}.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 2. HA-VV-IFN-β replicates to lower levels in murine L929 cells. Monolayers of CV-1 (a and c) or L929 (b and d) cells were infected at an MOI of 0.01 with HA-VV, HA-VV-IFN-{alpha}4, or HA-VV-IFN-β. Intracellular (a and b) and extracellular (c and d) fractions were collected over 72 h and virus titers determined by plating onto monolayers of 143B cells. Data are representative of two experiments performed in duplicate.

 

Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 3. HIV-VV-IFN-{epsilon} replicates to lower levels in murine L929 cells. Monolayers of CV-1 (a and c) or L929 (b and d) cells were infected at an MOI of 0.01 with HIV-VV or HIV-VV-IFN-{epsilon}. Intracellular (a and b) and extracellular (c and d) fractions were collected during 72 h and virus titers determined by plating onto monolayers of 143B cells. Data are representative of two experiments performed in duplicate.

 
Up-regulation of antiviral genes by IFN-{epsilon}

The reduction in HA-VV-IFN-β and HIV-VV-IFN-{epsilon} growth in a cell-specific manner suggests that vaccinia expression of the murine IFN-β/{epsilon} may be activating an antiviral state in the murine cells, inhibiting the ability of the virus to replicate. IFN-β has been well characterized as to its antiviral properties (36, 37); however, little or no work has been performed to determine the properties of IFN-{epsilon}. To assess the ability of virally expressed IFN-{epsilon} to up-regulate antiviral molecules on lymphocytes, inactivated supernatant from HIV-VV-IFN-{epsilon} infected CV-1 cells was incubated with L929 cells, and up-regulation of antiviral genes PKR and 2'5'OAS were determined by RT-PCR. As shown in Fig. 4, components of the viral infection alone can affect gene expression, as supernatant from HIV-VV-infected cells up-regulated expression of PKR and 2'5'OAS (Fig. 4a). However, the addition of IFN-{epsilon} further increased the magnitude of PKR and 2'5'OAS mRNA expression 4- and 10-fold, respectively, above HIV-VV supernatant (Fig. 4b). In addition to providing antiviral activities during a viral infection, the type I IFNs also have immunoregulatory features. Thus, to investigate the regulatory effects of IFN-{epsilon} on lymphocyte activation, primary splenocytes from wild-type 129 or IFN-IR–/– mice were incubated with the inactivated supernatants as above. As shown in Fig. 5, elevated CD69 and CD86 expression on B cells incubated with HIV-VV-IFN-{epsilon} (Fig. 5, a and b) compared with HIV-VV (Fig. 5, c and d) or medium only were observed. Although it appeared that some component of the viral infection present in the supernatant was causing an increase in CD86 expression compared with medium alone, the data suggest that this is independent of IFN-IR signaling, as both wild-type 129 and IFN-IR–/– B cells showed elevated CD86 expression levels when incubated with HIV-VV supernatant (Fig. 5d). The presence of IFN-{epsilon} elevated both CD69 and CD86 levels compared with HIV-VV supernatant in a IFN-IR-dependent manner, as an increase in expression levels was observed in wild-type mice and not in IFN-IR–/– mice (Fig. 5, a and b). Positive controls of cells incubated with 100 U rIFN-{alpha} are shown in Fig. 5, e and f.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 4. Induction of antiviral molecules by IFN-{epsilon}. Virus-inactivated supernatant from rVV-infected CV-1 cells was incubated with L929 cells for 24 h. a, mRNA was isolated and levels of antiviral gene expression were analyzed by PCR. b, Quantitative real-time PCR of L929 cells after 24 h incubation with HIV-VV-IFN-{epsilon} supernatant compared with HIV-VV supernatant. The graph represents the fold change in mRNA, normalized against L32 expression.

 

Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 5. IFN-IR dependent B lymphocyte activation by IFN-{epsilon}. Virus-inactivated supernatant from rVV (HIV-VV, HIV-VV-IFN-{epsilon}) infected CV-1 cells was incubated with splenocytes from IFN-IR–/– and wild type mice. Expression of CD69 and CD86 on B220+ B cells was measured by FACS. Medium gray line indicates wild type; light gray line, IFN-IR–/–; solid black line, resting cells. Incubation with HIV-VV-IFN-{epsilon} supernatant (a and b), HIV-VV supernatant (c and d), and 100 U rIFN-{alpha} (e and f).

 
Type I IFN expression attenuates vaccinia virulence in BALB/c mice

To assess whether the observed in vitro antiviral properties of IFN-β/{epsilon} coexpression conferred an inhibition of replication of the rVVs in vivo, groups of 4 female BALB/c mice were injected i.v. with 107 PFU of HA-VV-IFN-{alpha}4, HA-VV-IFN-β, HA-VV, HIV-VV-IFN-{epsilon}, or HIV-VV, and the viral titers in the ovaries were measured after 72 h as described in Materials and Methods. Results indicated a significant reduction (i.e., >1 log) of viral titers of HA-VV-IFN-{alpha}4 in comparison to HA-VV (p < 0.05), for which titers were as high as 4.8 x 107 in the ovaries (Fig. 6a). Additionally, the coexpression of IFN-β appeared to aid the rapid clearance of HA-VV-IFN-β following infection, or suppressed viral dissemination, as virus could not be detected in the ovaries at this time to the limits of the plaque assay (102 PFU). In contrast, coexpression of IFN-{epsilon} resulted in a moderate (<1 log) but insignificant reduction in the growth of HIV-VV-IFN-{epsilon} compared with HIV-VV (Fig. 6b).


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 6. rVV expressing type I IFNs are attenuated in immunocompetent and immunodeficient mice. a, BALB/c mice were infected i.v. with 1 x 107 PFU HA-VV-IFN-{alpha}4, HA-VV-IFN-β, HA-VV, or (b) HIV-VV-IFN-{epsilon} or HIV-VV, and at 3 days PI viral titers in the ovaries were enumerated by titration onto 143B cell monolayers. */**, p < 0.05. c and d, Immunodeficient BALB/c nude mice (n = 4–5) were infected i.v. with 1 x 106 PFU of either virus and monitored twice daily for disease and mortality. Data are pooled from two experiments.

 
Coexpression of type I IFNs attenuates rVV in nude mice

To assess the contribution of T lymphocytes in the reduction of in vivo viral titers, we compared the ability of the rVVs to replicate in athymic BALB/c nude mice, which lack functional T cells. Nude mice were infected i.v. with 106 PFU of HA-VV-IFN-{alpha}4, HA-VV-IFN-β, HA-VV, HIV-VV-IFN-{epsilon}, or HIV-VV, and their survival was monitored. Nude mice infected with HA-VV all developed pox lesions and displayed severe weight loss before succumbing to disease at an average of 16 days PI. In contrast, nude mice infected with HA-VV-IFN-{alpha}4 showed a longer average survival period of up to 30 days. Furthermore, nude mice infected with HA-VV-IFN-β did not develop the weight loss or pox lesions typical of pox infection in immunodeficient mice and survived the infection during the period of observation (90 days) (Fig. 6c). Coexpression of IFN-{epsilon} did not have such a marked effect on the survival of nude mice as did IFN-{alpha}/β, as mice survived no longer (17 days PI) than did mice given the control virus HIV-VV (15 days PI) (Fig. 6d).

At the time of sacrifice, ovaries, spleen, and brain were taken and titrated for rVV (data not shown). Surprisingly, no virus was detected at the time of death in the brain, ovaries, or spleen of nude mice infected with HA-VV-IFN-{alpha}4, despite a disease progression similar to pox disease observed ~5 days leading up to death. However, virus replication was observed in the ovaries and brains of nude mice infected with 106 PFU of the control virus (HA-VV), with titers of up to 107 PFU detected in the ovaries. In concurrence with the observation that nude mice infected with HA-VV-IFN-β remained healthy throughout the experimental period, no evidence of virus replication in the organs at the time of sacrifice was observed. Furthermore, nude mice infected with 5 x 106 and 5 x 107 PFU HA-VV-IFN-β also remained healthy for the duration of the observation period (90 days), and no virus was detected in the organs at the time of sacrifice. Organs from nude mice infected with HIV-VV-IFN-{epsilon} yielded similar recoverable virus levels to mice infected with HIV-VV, suggesting that IFN-{epsilon} expression was unable to curb virus growth in the T cell-deficient model, thus indicating that T cells may be a target for IFN-{epsilon} activity (data not shown).

The significant attenuation of HA-VV-IFN-β observed in both immunocompetent and immunodeficient mice suggests a local antiviral state induced by expression of IFN-β, which is independent of T lymphocytes. Studies investigating antiviral therapies have suggested that a highly antiviral state can be induced by coactivation of IFN-{alpha}/β and IFN-{gamma} receptor pathways (38). To evaluate whether endogenous IFN-{gamma} can act in synergy with the expressed IFN-β, IFN-{gamma} receptor knockout mice (IFN-IIR–/–), which have defective resistance to virus infection and increased susceptibility to VV (39), were used. However, IFN-IIR–/– mice given 107 PFU of HA-VV-IFN-β did not exhibit any signs of disease and remained healthy throughout the duration of the observation (37 days PI), nor was any virus detected in the ovaries of these mice at sacrifice (data not shown).

Coexpression of the type I IFNs in a prime-boost vaccination strategy generates high levels of cellular and humoral immunity

The data suggest that coexpression of different type I IFNs by rVV can have varying effects on the virus replication both in vitro and in vivo, leading to different levels of viral attenuation in both immunocompetent and immunocompromised mice. Coexpression of IFN-β/{epsilon} by VV reduces rVV replication in murine cells in vitro; however, IFN-β expression also reduced viral virulence in both nude and normal mice. To study the immunomodulatory effects of cytokine coexpression on the vaccine-generated T cell responses to the coexpressed and viral Ags, BALB/c mice were prime-boost immunized i.m. with 1 x 107 PFU rVV followed by 1 x 107 PFU rFPV i.m. at a 2-wk intervals. Results indicate that mice immunized with HA-VV-IFN-β generated a robust anti-HA CD8+ T cell response (357 ± 169 SFU/106 cells) when splenocytes were restimulated with the MHC class I-restricted Kd HA533–541 peptide. However, these CD8+ IFN-{gamma} responses were unchanged compared with the control vaccine group (514 ± 151 SFU/106 cells) (Fig. 7a). Likewise, when immunized via the i.m. route, HA-VV-IFN-{alpha}4 followed by HA-FPV generated similar numbers of IFN-{gamma}-secreting CD8+ T cells (338 ± 214 SFU/106 cells) compared with mice primed with HA-VV.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7. Coexpression of the type I IFNs in the prime of a prime-boost vaccination does not affect anti-HIV T cell responses or anti-HA T cell or humoral responses. BALB/c mice were primed with 1 x 107 PFU HIV-VV-IFN-{epsilon}, HIV-VV, HA-VV-IFN-{alpha}4, HA-VV-IFN-β, or HA-VV i.m. and boosted with 1 x 107 PFU HIV-FPV or HA-FPV at 2-wk intervals. At 14 days after boost, splenocytes were stimulated either with the overlapping 15mer gag peptide pool (b) or with the HA 533IYSTVASSL541 peptide (a). HIV- and HA-specific IFN-{gamma}-secreting T cells were detected by ELISPOT. Anti-HA- (c) IgG1 and (d) IgG2a levels in serum were evaluated by ELISA. Data are representative of three experiments of n = 4.

 
Similarly, BALB/c mice immunized with HIV-VV-IFN-{epsilon} i.m. followed by a HIV-FPV booster immunization i.m. elicited elevated numbers of HIV Gag-specific T cells (1380 ± 170 SFU/106 cells), with similar counts being found in mice immunized by HIV-VV followed by HIV-FPV (826 ± 420 SFU/106 cells) (Fig. 7b).

ELISAs were conducted on serum from vaccinated mice at sacrifice to assess the humoral immune response induced by vaccination with HA-VV-IFN-{alpha}4 and HA-VV-IFN-β as described above. No significant differences were observed in the HA-specific IgG1 or endpoint titers between mice that were primed with HA-VV, HA-VV-IFN-{alpha}4, or HA-VV-IFN-β (Fig. 7, c and d). Priming with HA-VV-IFN-{alpha}4 generated a significantly higher HA-specific IgG2a endpoint titer than did priming with HA-VV-IFN-β (p = 0.04); however, this was not significantly higher than that generated by priming with HA-VV (p = 0.075).

High levels of antivaccinia cellular immunity generated by prime-boost vaccination with rVV-IFNs

Our data demonstrate that coexpression of murine IFN-{alpha}4 or IFN-β attenuates replication of the viruses both in immunocompetent and immunodeficient mice; indeed, rVV coexpressing IFN-β could not be detected in vivo. Both viruses also induced cellular and humoral immune responses against HA in a vaccine regime to similar levels as induced by the control virus. Presently, the smallpox vaccination carries the risk of severe reactions and adverse effects due to the local dissemination and shedding of live virulent VV, which is introduced under the skin to cause local infection and a cellular immunity and Ab response. Therefore, we assessed whether the attenuated rVV, HA-VV-IFN-{alpha}4 and HA-VV-IFN-β, could be used to reduce risk yet induce a high vaccinia-specific cellular response. Using the poxvirus prime-boost regime as described previously, and using vaccinia-infected splenocytes as stimulator cells at a stimulator-to-responder cell ratio of 1:4, we found no statistical difference in the high levels of vaccinia-specific IFN-{gamma}-secreting T cells generated by priming with either HA-VV-IFN-{alpha}4 (1012 ± 280 SFU/106 cells), HA-VV-IFN-β (860 ± 145 SFU/106 cells), or HA-VV (945 ± 167 SFU/106 cells) (Fig. 8).


Figure 8
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 8. Coexpression of the type I IFNs in a prime-boost regime does not affect antivaccinia T cell responses. BALB/c mice were primed with 1 x 107 PFU HA-VV-IFN-{alpha}4, HA-VV-IFN-β, or HA-VV i.m. and boosted with 1 x 107 PFU HA-FPV. At 14 days after boost, splenocytes were incubated at a ratio of 1:4 with splenocytes infected with VV-WR at an MOI of 10. VV-specific responses were measured by IFN-{gamma} ELISPOT.

 
Protection against the ECTV

Our data show that coexpression of IFN-β limits viral replication in vivo, yet is able to induce high numbers of vaccinia-specific T cells. To investigate whether the cellular immunity generated by HA-VV-IFN-β is protective, a challenge model with the highly virulent mousepox virus, ECTV, was used as described in Materials and Methods. Both VV and ECTV are members of the gene family orthopoxvirus, which are closely related, and infection or immunization with one such virus is known to confer a level of cross-protection. C57BL/6 mice immunized with HA-VV-IFN-β or the control virus (HA-VV) were protected against challenge by otherwise lethal doses of the Moscow strain of ECTV.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cytokines play an important role in the immune response as the initiators and regulators of both the innate and adaptive immune networks. The type I IFNs play an integral role in the antiviral immune response, promoting an antiviral state and contributing to the CD8+ T cell clonal expansion and memory cell survival. In the present study, we used rVV vectors to deliver the local release of the murine type I IFNs, IFN-{alpha}4, IFN-β, and the newly discovered IFN-{epsilon}, to study their effects on the immunogenicity and safety of the virus vectors, as well as to determine their ability to modulate the immune response to the vaccine Ags in a poxvirus prime-boost strategy.

It is well established that the type I IFNs can induce an antiviral state in infected cells by signaling through type I IFN receptor-linked JAK-STAT pathways to induce expression of IFN-stimulated genes (37, 40). The present in vitro studies suggest that the local expression of IFN-β and IFN-{epsilon} elicit a similar antiviral state, resulting in an impaired ability of these viruses to replicate in murine L929 cells (compared with the control viruses; Figs. 2 and 3). This indicates an induction of antiviral effects by IFN-stimulated genes rather than a defect in the pathology of the viruses themselves. Additionally, we have shown that IFN-{epsilon}, similar to other type I IFNs, can up-regulate antiviral proteins such as PKR and 2'5'OAS (Fig. 4). However, IFN-{alpha}4 did not reduce viral growth in vitro as was observed with IFN-β and IFN-{epsilon}. Although all characterized type I IFNs bind the same receptor (IFN-IR), which is composed of two subunits, IFNAR-1 and IFNAR-2, binding to this receptor can occur with different affinities, leading to multiple signaling cascades, which can lead to distinct biologic outcomes such as conferring antiviral resistance to different virus species (41, 42). Using IFN-β-deficient mice, Deonarain and colleagues (43) demonstrated that IFN-β is essential for protection against VV, and that many of the IFN-{alpha} subtypes, including IFN-{alpha}4, are dependent on IFN-β for induction. We postulate that the observed impaired ability of HA-VV-IFN-β to replicate in murine cells in vivo may be due to rVV-encoded IFN-β causing a considerable antiviral response to the VV. In contrast, signaling induced by the virally encoded IFN-{alpha}4 may not have as high an antiviral response against VV.

The induction of IFN-inducible proteins PKR and 2'5'OAS by IFN-{epsilon} suggests that it, too, signals via the type I IFN receptor. Early viral infection causes the activation of lymphocytes, which can be measured by the up-regulation of cell surface molecules, and this is thought to be a direct consequence of type I IFN production (44, 45). Indeed, our findings that IFN-{epsilon} can up-regulate CD69 and CD86 expression on B cells from wild-type mice (Fig. 5) but not in mice lacking IFN-IR indicates signaling via the common receptor and demonstrates that this highly conserved signaling mechanism is used by IFN-{epsilon}. Similarly, CD8 T cells cultured with IFN-{epsilon} were found to up-regulate CD69 expression in an IFN-IR-dependent manner (data not shown), although this increase was not as high as that observed on B cells.

Results indicate that both HA-VV-IFN-β and HIV-VV-IFN-{epsilon} demonstrate reduced replication in murine cells in culture; however, the extent of these antiviral effects of the cytokines in vivo could be different. Indeed, only the virally encoded IFN-β was able to attenuate the virus highly in a systemic infection model. BALB/c mice infected with HA-VV-IFN-{alpha}4 or HIV-VV-IFN-{epsilon} had ~1 log reduction in virus titers detectable in their ovaries at 3 days PI compared with the controls, HA-VV, and HIV-VV, respectively. Interestingly, mice infected with HA-VV-IFN-β had no detectable virus in the ovaries, suggesting significant virus attenuation by IFN-β expression, which may be beneficial for improved safety of the vector for use as a vaccine or immunotherapeutic treatment. In many cases the use of live viral vaccines is contraindicated for immunocompromised patients, due to the risk of vaccine-related complications, and thus improvements in vaccine attenuation is of great potential value.

Our data demonstrate that local IFN-β expression appeared to greatly enhance the safety of vaccinia for use as a vaccine, as immunocompromised nude mice that lack functional T lymphocytes (important effector cells in the primary response against vaccinia infection) were nonetheless able to resist or overcome infection by HA-VV-IFN-β. We found that systemic infection induced by a dose of 106 PFU of HA-VV resulted in rapid weight loss and pox lesion formation, and the mice succumbed to pox infection 17.8 ± 1.3 days PI, which was confirmed by high viral titers recovered from their ovaries at the time of death. However, systemic infection with up to 107 PFU of HA-VV-IFN-β did not cause any signs of disease or infection for up to 9 mo monitored. In contrast, the mice infected with the HA-VV-IFN-{alpha}4 died at 22.7 ± 5 days PI. Interestingly, even though these mice displayed classic symptoms of pox infection such as pox lesions and high weight loss, at the time of death no virus was detectable in any organs. It is possible that this pathology is being caused by IFN produced by virus that is present below levels detectable in our assay system (100 PFU).

Our data highlight clear differences in the ability of IFN-{epsilon}, IFN-{alpha}4, and IFN-β to induce antiviral states; whereas HIV-VV-IFN-{epsilon} demonstrated a similar reduction in growth in vitro to HA-VV-IFN-β, in the nude and BALB/c mouse models the effect of IFN-{epsilon} on viral growth was minimal.

Several studies have shown that the replication of some viruses such as HSV and human CMV can be blocked by the coactivation of the IFN-IR and IFN-IIR signaling pathways (38, 46). To investigate whether the lack of detectable replication of HA-VV-IFN-β in vivo is caused by a synergistic effect of virally encoded IFN-β and endogenous IFN-{gamma}, we looked at an in vivo model of infection in the absence of IFN-{gamma} responsiveness by infecting IFN-IIR–/– mice with 1 x 107 PFU HA-VV-IFN-β or HA-VV and monitoring them for signs of vaccinial disease. These mice were defective in their ability to clear VV infection, as 1 x 106 PFU VV-WR proved fatal to most mice in 3–4 days (39). Interestingly, neither mice infected with HA-VV or HA-VV-IFN-β showed any sign of infection for >30 days (data not shown). VV-WR is known to be a virulent strain of VV, which would explain the differences in susceptibility observed.

We have shown that the coexpression of the type I IFNs, especially IFN-β, can improve the safety of the vector, as demonstrated in both immunocompetent and immunodeficient mice. Introduction of the HA-VV to nude mice induced a progressive fatal vaccinia infection, which could be circumvented by the coexpression of IFN-β. To determine the efficacy of the rVVs as vaccine vectors, it was necessary to demonstrate that the reduction in replication did not occur at a cost to the immunogenicity of the viruses.

Previous work has demonstrated that a poxvirus prime-boost regime can generate better immune responses to coexpressed Ags than a single vaccination or using a DNA/poxvirus combination prime-boost regime (32, 47). Likewise, coexpression of immunomodulatory cytokines and molecules by the vaccine vectors has shown to enhance or modulate the vaccine-induced immune response (48, 49, 50). Thus, we used the poxvirus prime-boost strategy to examine the ability of the type I IFNs to modulate the anti-HA or anti-HIV immune response. In this model, IFN-{epsilon} coexpression was unable to enhance the levels of HIV-specific T cells generated by immunization. Little is known regarding the activities of IFN-{epsilon} and immune regulation. The data to date indicate that IFN-{epsilon} expression is restricted to reproductive tissues (2) and that IFN-{epsilon} expression is up-regulated with exposure to seminal fluid (13). This suggests that it may have important immunomodulatory or protective roles in these regions, and thus systemic immunization may not be an effective measure of elucidating a role of IFN-{epsilon}. We postulate that IFN-{epsilon} may have an antiviral, protective, or inhibitory role in the mucosa, as the cervix is both a site of entry for many sexually transmitted pathogens, but conversely, it is also subject to exposure to seminal fluid to which no immunity should be raised.

Our data show that despite the greatly reduced replicative capacity of HA-VV-IFN-β, a considerable level of transcription and translation of the viral proteins could be occurring in the mice, as the use of this vector in the prime-boost immunization did not significantly alter the HA-specific or vaccinia-specific T cell responses as detected by IFN-{gamma} ELISPOT (Fig. 7a and 8). Similarly, no significant changes in the HA-specific IgG1 and IgG2a (Fig. 7, c and d) levels in the serum of vaccinated mice were observed. The viral attenuation by expression of IFN-β without cost to vaccine immunogenicity suggests that such vaccines can be used as alternatives to the currently available smallpox vaccine. Several studies have been conducted to test the ability of using rVV such as modified VV Ankara as a safer smallpox vaccine that can induce an effective immune response without replication of the VV (51, 52). Similar to our characterization of HA-VV-IFN-β, modified VV Ankara is highly attenuated and yet it induces a robust immune response upon vaccination that can protect against challenge by virulent poxviruses. However, as modified VV Ankara has an extremely narrow host cell range in which it can replicate (53), the large-scale synthesis of the vaccine could prove difficult. In contrast, HA-VV-IFN-β appears to have reduced replication only in murine cells due to the specific immunomodulatory actions of the murine cytokine, and thus it has a better potential as a live attenuated vaccine.

To assess whether the antivaccinia immunity generated by vaccination with the rVVs is cross-protective, BALB/c mice were immunized i.m. with 1 x 107 PFU HA-VV, HA-VV-IFN-β, or HA-VV-IFN-{alpha}4 and challenged at 21 days PI with 1 x 103 ECTV Moscow strain. Although BALB/c mice are susceptible to ECTV growth, which results in fatal disease within 7 days (54), we found that all rVVs were able to protect against infection from the ECTV Moscow strain (data not shown), indicating that a single immunization is sufficient to provide cross-protection against an otherwise lethal dose of ECTV.

We have demonstrated that coexpression of the type I IFNs in rVV causes varying degrees of attenuation of the different rVVs. Local IFN-{epsilon} expression imparts a low level of antiviral activity to the rVV, as seen by a reduction in viral growth in vitro and also up-regulation of antiviral genes; however, this does not translate to an enhancement of the immune response generated to the viral vector. Tissue-restricted expression of IFN-{epsilon} suggests a specialized role that may be quite distinct (i.e., mucosally related) to the antiviral activities of IFN-{alpha}/β. Reports examining the expression of IFN-{epsilon} in various tissues have found low levels of gene expression in the lung (2). We have found that in an allergic BALB/c model, IFN-{epsilon} appeared to be up-regulated in the lung when compared with the control BALB/c lung. Quantitative real-time PCR showed a 5-fold increase in message RNA levels of IFN-{epsilon} in allergic lung compared with the control. However, levels of IFN-{alpha}/β increased 29- and 17-fold, respectively. Nonetheless, constitutive expression levels of IFN-{epsilon} were higher than the other IFNs. Additionally, unlike IFN-{epsilon}, neither IFN-{alpha} nor IFN-β was up-regulated when cervical epithelial cells were exposed to seminal fluid (13). We thus propose a role for IFN-{epsilon} in mucosal immune regulation that may be quite distinct from the other type I IFNs, and we are currently investigating this possibility in other models of mucosal immunity and disease.


   Acknowledgments
 
We thank Jill Medveczky and Sandra Beaton for their invaluable technical assistance. Thanks to Prof. Paul Hertzog (Centre for Functional Genomics and Human Disease at the Monash Institute of Medical Research) for assaying viral IFN-{alpha}/β production by cytopathic effect inhibition assay. Also thanks to Dr. Dianne Webb at the John Curtin School of Medical Research for providing cDNA from allergic BALB/c mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by National Health and Medical Research Council Program Grant 299907. Back

2 Address correspondence and reprint requests to Dr. Stephanie Day, Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia. E-mail address: stephanie.day{at}anu.edu.au Back

3 Abbreviations used in this paper: VV, vaccinia virus; ECTV, ectromelia virus; FPV, fowlpox virus; HA, hemagglutinin; MOI, multiplicity of infection; 2'5'OAS, 2'5'oligoadenylate synthetase; PI, postinfection; PKR, protein kinase R; SFU, spot forming units; WR, Western reserve. Back

Received for publication November 14, 2007. Accepted for publication March 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Isaacs, A., J. Lindenmann. 1957. Virus interference. I. The interferon. Proc. R. Soc. London Ser. B 147: 258-267. [Medline]
  2. Hardy, M. P., C. M. Owczarek, L. S. Jermiin, M. Ejdeback, P. J. Hertzog. 2004. Characterization of the type I interferon locus and identification of novel genes. Genomics 84: 331-345. [Medline]
  3. Trent, J. M., S. Olson, R. M. Lawn. 1982. Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of in situ hybridization. Proc. Natl. Acad. Sci. USA 79: 7809-7813. [Abstract/Free Full Text]
  4. Dandoy, F., K. A. Kelley, J. DeMaeyer-Guignard, E. DeMaeyer, P. M. Pitha. 1984. Linkage analysis of the murine interferon-alpha locus on chromosome 4. J. Exp. Med. 160: 294-302. [Abstract/Free Full Text]
  5. Lund, J., A. Sato, S. Akira, R. Medzhitov, A. Iwasaki. 2003. Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198: 513-520. [Abstract/Free Full Text]
  6. Lund, J. M., L. Alexopoulou, A. Sato, M. Karow, N. C. Adams, N. W. Gale, A. Iwasaki, R. A. Flavell. 2004. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101: 5598-5603. [Abstract/Free Full Text]
  7. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730-737. [Medline]
  8. Yoneyama, M., M. Kikuchi, K. Matsumoto, T. Imaizumi, M. Miyagishi, K. Taira, E. Foy, Y. M. Loo, M. Gale, Jr, S. Akira, et al 2005. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J. Immunol. 175: 2851-2858. [Abstract/Free Full Text]
  9. Kolumam, G. A., S. Thomas, L. J. Thompson, J. Sprent, K. Murali-Krishna. 2005. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J. Exp. Med. 202: 637-650. [Abstract/Free Full Text]
  10. Cousens, L. P., R. Peterson, S. Hsu, A. Dorner, J. D. Altman, R. Ahmed, C. A. Biron. 1999. Two roads diverged: interferon {alpha}/β and interleukin 12-mediated pathways in promoting T cell interferon {gamma} responses during viral infection. J. Exp. Med. 189: 1315-1328. [Abstract/Free Full Text]
  11. Brinkmann, V., T. Geiger, S. Alkan, C. H. Heusser. 1993. Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells. J. Exp. Med. 178: 1655-1663. [Abstract/Free Full Text]
  12. Nguyen, K. B., T. P. Salazar-Mather, M. Y. Dalod, J. B. Van Deusen, X. Q. Wei, F. Y. Liew, M. A. Caligiuri, J. E. Durbin, C. A. Biron. 2002. Coordinated and distinct roles for IFN-{alpha}β, IL-12, and IL-15 regulation of NK cell responses to viral infection. J. Immunol. 169: 4279-4287. [Abstract/Free Full Text]
  13. Sharkey, D. J., A. M. Macpherson, K. P. Tremellen, S. A. Robertson. 2007. Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells. Mol. Hum. Reprod. 13: 491-501. [Abstract/Free Full Text]
  14. Fenner, F., D. Henderson, I. Arita, Z. Jezek, I. Ladnyi. 1988. Smallpox and Its Eradication World Health Organization, Geneva.
  15. Ramshaw, I. A., M. E. Andrew, S. M. Phillips, D. B. Boyle, B. E. Coupar. 1987. Recovery of immunodeficient mice from a vaccinia virus/IL-2 recombinant infection. Nature 329: 545-546. [Medline]
  16. Wang, L. Y., R. S. Fujinami. 1997. Enhancement of EAE and induction of autoantibodies to T-cell epitopes in mice infected with a recombinant vaccinia virus encoding myelin proteolipid protein. J. Neuroimmunol. 75: 75-83. [Medline]
  17. Johnson, T. R., J. E. Fischer, B. S. Graham. 2001. Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine. J. Gen. Virol. 82: 2107-2116. [Abstract/Free Full Text]
  18. Andrew, M. E., B. E. Coupar, G. L. Ada, D. B. Boyle. 1986. Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants. Microb. Pathog. 1: 443-452. [Medline]
  19. De Rose, R., C. J. Batten, M. Z. Smith, C. S. Fernandez, V. Peut, S. Thomson, I. A. Ramshaw, B. E. Coupar, D. B. Boyle, V. Venturi, et al 2007. Comparative efficacy of subtype AE simian-human immunodeficiency virus priming and boosting vaccines in pigtail macaques. J. Virol. 81: 292-300. [Abstract/Free Full Text]
  20. Abaitua, F., J. R. Rodriguez, A. Garzon, D. Rodriguez, M. Esteban. 2006. Improving recombinant MVA immune responses: potentiation of the immune responses to HIV-1 with MVA and DNA vectors expressing Env and the cytokines IL-12 and IFN-{gamma}. Virus Res. 116: 11-20. [Medline]
  21. Tsang, K. Y., C. Palena, J. Yokokawa, P. M. Arlen, J. L. Gulley, G. P. Mazzara, L. Gritz, A. G. Yafal, S. Ogueta, P. Greenhalgh, et al 2005. Analyses of recombinant vaccinia and fowlpox vaccine vectors expressing transgenes for two human tumor antigens and three human costimulatory molecules. Clin. Cancer Res. 11: 1597-1607. [Abstract/Free Full Text]
  22. Karupiah, G., A. J. Ramsay, I. A. Ramshaw, R. V. Blanden. 1992. Recombinant vaccine vector-induced protection of athymic, nude mice from influenza A virus infection: analysis of protective mechanisms. Scand. J. Immunol. 36: 99-105. [Medline]
  23. Harrington, L. E., R. Most Rv, J. L. Whitton, R. Ahmed. 2002. Recombinant vaccinia virus-induced T-cell immunity: quantitation of the response to the virus vector and the foreign epitope. J. Virol. 76: 3329-3337. [Abstract/Free Full Text]
  24. Legrand, F. A., P. H. Verardi, K. S. Chan, Y. Peng, L. A. Jones, T. D. Yilma. 2005. Vaccinia viruses with a serpin gene deletion and expressing IFN-{gamma} induce potent immune responses without detectable replication in vivo. Proc. Natl. Acad. Sci. USA 102: 2940-2945. [Abstract/Free Full Text]
  25. Jackson, R. J., A. J. Ramsay, C. D. Christensen, S. Beaton, D. F. Hall, I. A. Ramshaw. 2001. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. J. Virol. 75: 1205-1210. [Abstract/Free Full Text]
  26. Boyle, D. B., B. E. Coupar. 1988. Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 10: 343-356. [Medline]
  27. Boyle, D. B., M. A. Anderson, R. Amos, R. Voysey, B. E. Coupar. 2004. Construction of recombinant fowlpox viruses carrying multiple vaccine antigens and immunomodulatory molecules. BioTechniques 37: 104-106, 108–111. [Medline]
  28. Boyle, D. B., B. E. Coupar, G. W. Both. 1985. Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses. Gene 35: 169-177. [Medline]
  29. Pestka, S.. 1986. Interferon standards and general abbreviations. Methods Enzymol. 119: 14-23. [Medline]
  30. Tamura, M., K. Kuwano, I. Kurane, F. A. Ennis. 1998. Definition of amino acid residues on the epitope responsible for recognition by influenza A virus H1-specific, H2-specific, and H1- and H2-cross-reactive murine cytotoxic T-lymphocyte clones. J. Virol. 72: 9404-9406. [Abstract/Free Full Text]
  31. Verardi, P. H., L. A. Jones, F. H. Aziz, S. Ahmad, T. D. Yilma. 2001. Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene (B8R) are attenuated in vivo without a concomitant reduction in immunogenicity. J. Virol. 75: 11-18. [Abstract/Free Full Text]
  32. Ranasinghe, C., J. C. Medveczky, D. Woltring, K. Gao, S. Thomson, B. E. Coupar, D. B. Boyle, A. J. Ramsay, I. A. Ramshaw. 2006. Evaluation of fowlpox-vaccinia virus prime-boost vaccine strategies for high-level mucosal and systemic immunity against HIV-1. Vaccine 24: 5881-5895. [Medline]
  33. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. [Medline]
  34. Wang, X., B. Seed. 2003. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 31: E154[Medline]
  35. Ranasinghe, C., S. J. Turner, C. McArthur, D. B. Sutherland, J. H. Kim, P. C. Doherty, I. A. Ramshaw. 2007. Mucosal HIV-1 pox virus prime-boost immunization induces high-avidity CD8+ T cells with regime-dependent cytokine/granzyme B profiles. J. Immunol. 178: 2370-2379. [Abstract/Free Full Text]
  36. Muller, U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264: 1918-1921. [Abstract/Free Full Text]
  37. Samuel, C. E.. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14: 778-809. [Abstract/Free Full Text]
  38. Pierce, A. T., J. DeSalvo, T. P. Foster, A. Kosinski, S. K. Weller, W. P. Halford. 2005. Beta interferon and gamma interferon synergize to block viral DNA and virion synthesis in herpes simplex virus-infected cells. J. Gen. Virol. 86: 2421-2432. [Abstract/Free Full Text]
  39. Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R. M. Zinkernagel, M. Aguet. 1993. Immune response in mice that lack the interferon-gamma receptor. Science 259: 1742-1745. [Abstract/Free Full Text]
  40. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264. [Medline]
  41. James, C. M., M. Y. Abdad, J. P. Mansfield, H. K. Jacobsen, A. R. Vind, P. A. Stumbles, E. J. Bartlett. 2007. Differential activities of alpha/beta IFN subtypes against influenza virus in vivo and enhancement of specific immune responses in DNA vaccinated mice expressing haemagglutinin and nucleoprotein. Vaccine 25: 1856-1867. [Medline]
  42. Harle, P., V. Cull, M. P. Agbaga, R. Silverman, B. R. Williams, C. James, D. J. Carr. 2002. Differential effect of murine alpha/beta interferon transgenes on antagonization of herpes simplex virus type 1 replication. J. Virol. 76: 6558-6567. [Abstract/Free Full Text]
  43. Deonarain, R., A. Alcami, M. Alexiou, M. J. Dallman, D. R. Gewert, A. C. Porter. 2000. Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon. J. Virol. 74: 3404-3409. [Abstract/Free Full Text]
  44. Alsharifi, M., M. Lobigs, M. Regner, E. Lee, A. Koskinen, A. Mullbacher. 2005. Type I interferons trigger systemic, partial lymphocyte activation in response to viral infection. J. Immunol. 175: 4635-4640. [Abstract/Free Full Text]
  45. Braun, D., I. Caramalho, J. Demengeot. 2002. IFN-{alpha}/β enhances BCR-dependent B cell responses. Int. Immunol. 14: 411-419. [Abstract/Free Full Text]
  46. Sainz, B., Jr, H. L. LaMarca, R. F. Garry, C. A. Morris. 2005. Synergistic inhibition of human cytomegalovirus replication by interferon-alpha/beta and interferon-gamma. Virol. J. 2: 14[Medline]
  47. Hodge, J. W., J. P. McLaughlin, J. A. Kantor, J. Schlom. 1997. Diversified prime and boost protocols using recombinant vaccinia virus and recombinant non-replicating avian pox virus to enhance T-cell immunity and antitumor responses. Vaccine 15: 759-768. [Medline]
  48. Leong, K. H., A. J. Ramsay, D. B. Boyle, I. A. Ramshaw. 1994. Selective induction of immune responses by cytokines coexpressed in recombinant fowlpox virus. J. Virol. 68: 8125-8130. [Abstract/Free Full Text]
  49. Harrison, J. M., E. M. Bertram, D. B. Boyle, B. E. Coupar, C. Ranasinghe, I. A. Ramshaw. 2006. 4-1BBL coexpression enhances HIV-specific CD8 T cell memory in a poxvirus prime-boost vaccine. Vaccine 24: 6867-6874. [Medline]
  50. Iwasaki, A., B. J. Stiernholm, A. K. Chan, N. L. Berinstein, B. H. Barber. 1997. Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J. Immunol. 158: 4591-4601. [Abstract]
  51. Phelps, A. L., A. J. Gates, M. Hillier, L. Eastaugh, D. O. Ulaeto. 2006. Comparative efficacy of modified vaccinia Ankara (MVA) as a potential replacement smallpox vaccine. Vaccine 25: 34-42. [Medline]
  52. Ober, B. T., P. Bruhl, M. Schmidt, V. Wieser, W. Gritschenberger, S. Coulibaly, H. Savidis-Dacho, M. Gerencer, F. G. Falkner. 2002. Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara. J. Virol. 76: 7713-7723. [Abstract/Free Full Text]
  53. Drexler, I., K. Heller, B. Wahren, V. Erfle, G. Sutter. 1998. Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells. J. Gen. Virol. 79: 347-352. [Abstract]
  54. Chaudhri, G., V. Panchanathan, R. M. Buller, A. J. van den Eertwegh, E. Claassen, J. Zhou, R. de Chazal, J. D. Laman, G. Karupiah. 2004. Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox. Proc. Natl. Acad. Sci. USA 101: 9057-9062. [Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Day, S. L.
Right arrow Articles by Ranasinghe, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Day, S. L.
Right arrow Articles by Ranasinghe, C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS