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
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 arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chavan, R.
Right arrow Articles by Feinberg, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chavan, R.
Right arrow Articles by Feinberg, M. B.

 Previous Article  |  Next Article 

Journal of Virology, August 2006, p. 7676-7687, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.02748-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Expression of CCL20 and Granulocyte-Macrophage Colony-Stimulating Factor, but Not Flt3-L, from Modified Vaccinia Virus Ankara Enhances Antiviral Cellular and Humoral Immune Responses

R. Chavan,{dagger} K. A. Marfatia,{dagger} I. C. An, D. A. Garber,* and M. B. Feinberg{ddagger}

Emory University Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia 30329

Received 31 December 2005/ Accepted 9 May 2006


arrow
ABSTRACT
 
While modified vaccinia virus Ankara (MVA) is currently in clinical development as a safe vaccine against smallpox and heterologous infectious diseases, its immunogenicity is likely limited due to the inability of the virus to replicate productively in mammalian hosts. In light of recent data demonstrating that vaccinia viruses, including MVA, preferentially infect antigen-presenting cells (APCs) that play crucial roles in generating antiviral immunity, we hypothesized that expression of specific cytokines and chemokines that mediate APC recruitment and activation from recombinant MVA (rMVA) vectors would enhance the immunogenicity of these vectors. To test this hypothesis, we generated rMVAs that express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), human CCL20/human macrophage inflammatory protein 3{alpha} (hCCL20/hMIP-3{alpha}), or human fms-like tyrosine kinase 3 ligand (hFlt3-L), factors predicted to increase levels of dendritic cells (DCs), to recruit DCs to sites of immunization, or to promote maturation of DCs in vivo, respectively. These rMVAs also coexpress the well-characterized, immunodominant lymphocytic choriomeningitis virus nucleoprotein (NP) antigen that enabled sensitive and quantitative assessment of antigen-specific CD8+ T-cell responses following immunization of BALB/c mice. Our results demonstrate that immunization of mice with rMVAs expressing mGM-CSF or hCCL20, but not hFlt3-L, results in two- to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific antibody titers, compared to those responses elicited by nonadjuvanted rMVA. Of note, cytokine augmentation of cellular immune responses occurs when rMVAs are given as primary immunizations but not when they are used as booster immunizations, suggesting that these APC-modulating proteins, when used as poxvirus-encoded adjuvants, are more effective at stimulating naïve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines.


arrow
INTRODUCTION
 
The majority of successful vaccines have been derived from either inactivated organisms or live-attenuated organisms (69). Modified vaccinia virus Ankara (MVA) is an example of a live-attenuated vaccine that is currently in phase I clinical trials as a smallpox vaccine and in development as a vaccine vector for human immunodeficiency virus, malaria, and tuberculosis (2, 5, 23, 50, 53, 54, 59, 62, 86, 94). MVA was originally derived through extensive serial passaging (>500 passages) of vaccinia virus Ankara in chicken embryo fibroblasts, which resulted in multiple deletions and mutations within the MVA genome (3, 49, 56, 81). As a result, MVA exhibits a severely restricted host range phenotype that includes an inability to replicate productively in primary human cells (8, 11, 20, 97). The safety of MVA has been demonstrated through administration of this virus to ~120,000 individuals during the smallpox eradication campaign (48, 81, 95) and, more recently, to immunocompromised hosts including immunosuppressed macaques and immune-deficient mice (82, 98).

MVA has been shown to abortively infect professional antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages (14; unpublished data), cells that play central roles in eliciting antiviral immune responses by mediating effective direct and cross-presentation of microbial antigens to naïve CD4+ and CD8+ T cells in the secondary lymphoid organs to initiate adaptive antiviral immune responses (4, 12, 16, 27, 31, 45, 51, 70). However, the immunogenicity of MVA is likely limited, despite its tropism for APCs, because of an inability to replicate in mammalian hosts that restricts viral gene (antigen) expression to cells infected at the site of immunization. We therefore proposed to enhance the immunogenicity of MVA vectors by generating recombinant viruses that express cytokines or chemokines that have known activities to increase the frequency and/or activation state of APCs, including granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 3{alpha} (MIP-3{alpha}/CCL20), and fms-like tyrosine kinase 3 ligand (Flt3-L).

GM-CSF acts on many immune cells during their early differentiation and plays a major role in the development of immature macrophages from their hematopoeitic precursors (7). Administration of GM-CSF or induction of GM-CSF expression has been reported to induce the differentiation of macrophages and DCs (43, 89). Other studies have demonstrated that GM-CSF, in combination with other cytokines, is capable of driving ex vivo differentiation of DCs from human peripheral blood monocytes (30, 40, 68, 91), whereas in vivo studies have shown that administration of recombinant GM-CSF increases the numbers of myeloid DCs in the spleen, bone marrow, and lymph nodes of naïve mice (18, 72). As a result, GM-CSF has been codelivered as a recombinant protein, or as a gene product encoded by plasmid DNA or poxvirus (avipox and vaccinia virus) vectors, as an adjuvant to enhance the immune responses elicited by vaccines (6, 38, 39, 74, 77, 88, 96).

CCL20 is a chemokine that recruits immature APCs expressing its cognate receptor CCR6 to peripheral sites where they may encounter microbes or infected cells (12, 13, 19, 35). While CCL20 has not been widely studied as a vaccine adjuvant (25), we sought to explore the notion that expressing CCL20 from recombinant MVA (rMVA) would recruit APCs to the site of immunization and result in enhanced antigen presentation and consequent increased vector immunogenicity.

Flt3-L acts to generate DC precursors by modulating hematopoeitic cell differentiation in the bone marrow (17, 36, 37). Several published studies have reported that in vivo administration of recombinant Flt3-L increases the numbers of myeloid and lymphoid DCs in the spleen, bone marrow, and lymph nodes of naïve mice (72). Furthermore, expression of Flt3-L from plasmid DNA can increase the number of DCs in mice and the immunogenicity of target antigens encoded by coadministered plasmids (58, 75, 76). Therefore, we hypothesized that hFlt3-L could also act as an adjuvant when expressed from rMVA vectors.

In this study, we derived rMVAs that express either human (hCCL20 and hFlt3-L) or murine (mGM-CSF) cytokine homologs, in accord with the known functionalities of these homologs in mice (9, 26, 41, 46, 64), to promote APC migration or maturation and, ideally, to increase levels of antigen presentation following immunization of mice with these recombinant vectors. In comparison to immunization with nonadjuvanted MVA, single-dose immunization of mice with rMVAs expressing mGM-CSF or hCCL20 results in durable two- to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific antibody titers. In contrast, expression of hFlt3-L from rMVA did not yield increased T-cell or antibody responses compared to the control vector. Expression of mGM-CSF or hCCL20 from rMVAs enhances the priming of antigen-specific CD8+ T cells but does not enhance the recall of preexisting CD8+ memory responses that had been initially primed via immunization with a DNA vaccine.


arrow
MATERIALS AND METHODS
 
Creation of recombinant MVA viruses. To direct recombination of transgenes into MVA deletion site III, a naturally occurring 3.5-kb deletion within the HindIII A fragment of MVA (85), plasmid pTH5 was constructed by inserting a loxP-flanked gfpzeo gene expression cassette into pG06dH5, which contains bidirectional early (modified H5) viral promoters (D. Garber et al., unpublished data). The lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP gene) (generous gift of Rafi Ahmed) with a 5' Kozak sequence (42) was cloned into the PmeI and AscI sites of pTH5 to create the pTH5NP transfer vector. Genes encoding mGM-CSF, hFlt3-L, and hCCL20 were singly inserted into the SnaBI site of pTH5NP to generate the final MVA{Delta}III transfer vectors pTH5NPmGM-CSF, pTH5NPhFlt3-L, and pTH5NPhCCL20, respectively. The mGM-CSF and hFlt3-L genes were obtained from the National Gene Vector Laboratory (University of Michigan, Ann Arbor, MI). The hCCL20 gene was generated by reverse transcription-PCR using a Titan One Tube reverse transcription-PCR system (Roche, Indianapolis, IN) with mRNA isolated from human peripheral blood mononuclear cells stimulated with lipopolysaccharide for 12 h and the following primers (33): 5'-CTCGAGGCCACCATGTGCTGTACCAAGAGT-3'; 5'-AAGATATCAAAGCCACAGTTTTTACAT-3'. All DNA sequences of amplified cloned genes were confirmed by DNA sequence analysis (Emory University DNA Sequencing Core). For comparison, murine versus human homologs of CCL20, Flt3-L, and GM-CSF exhibit 64% (87), 69%, and 54% (10) amino acid homology, respectively.

Recombinant MVA viruses were generated by homologous recombination in DF-1 fibroblasts (32) (ATCC, Manassas, VA) and isolated by zeocin selection (200 µg/ml) and GFP+ screening as described (Garber et al., unpublished). Recombinant MVAs were plaque purified on DF-1 cell monolayers through five rounds of zeocin selection prior to expansion of viral stocks and purification by sucrose gradient centrifugation. Purified viruses were resuspended in sterile 1x phosphate-buffered saline (PBS) and titers on DF-1 cell monolayers were determined.

Protein expression analyses. In vitro protein expression of hFlt3-L was assessed by immunoblot analysis, mGM-CSF and hCCL20 were measured by enzyme-linked immunosorbent assay (ELISA), and LCMV-NP was detected by flow cytometry (see flow cytometry section below). For all in vitro protein expression experiments, DF-1 cells were infected with MVA, MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or MVA-NP-hFlt3-L at multiplicities of infection of 3 or 5.

Immunoblot analysis. Supernatants were harvested at 24 h postinfection and added to sodium dodecyl sulfate (SDS) running buffer (0.5 M Tris, pH 6.8, glycerol, 20% SDS, bromophenol blue, H2O, ß-mercaptoethanol). Immunoblot analysis was performed as described previously (90) with the exception that proteins were transferred to a nitrocellulose membrane using semidry electroblotting. The hFlt3-L protein was detected with a biotinylated anti-human Flt3-L antibody (0.2 µg/ml) (R&D Systems, Minneapolis, MN) and a neutravidin-horseradish peroxidase (HRP) conjugate (1:10,000 dilution) (Pierce, Rockford, IL) using a Supersignal West Pico system (Pierce, Rockford, IL).

ELISA. Supernatants were harvested at 24 h postinfection and clarified by centrifugation. Protein expression was analyzed using mGM-CSF or hCCL20 Quantikine ELISA kits (R&D Systems, Minneapolis, MN). The in vivo cytokine expression analyses were also performed using mGM-CSF or hCCL20 Quantikine ELISA kits (R&D Systems, Minneapolis, MN), and the assays were performed according to manufacturer's instructions.

Immunization of mice. BALB/c mice (H-2d) (5 to 8 weeks old) were obtained from either the National Cancer Institute (Frederick Cancer Facility, Frederick, MD) or Jackson Laboratories (Bar Harbor, ME) and maintained in the Yerkes National Primate Research Center (Atlanta, GA) vivarium. All procedures pertaining to mouse handling, care, and use were conducted according to the Emory University Institutional Animal Care and Use Committee guidelines.

For all immunization experiments, groups were immunized with a PBS control, MVA, MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or MVA-NP-hFlt3-L intraperitoneally (i.p.) and at a dose of 1 x 106 PFU per immunization. The murine intraperitoneal infection model is a generally accepted model of poxvirus infection and immunity that has been successfully utilized in studies of poxvirus immunogenicity (29), T-cell epitope mapping (92), and rational enhancement of MVA vector immunogenicity (80). Mice were sacrificed at 7 days, 15 days, and 30 days postinfection (dpi), and spleens were removed for the immune response studies. For the in vivo cytokine expression experiments, mice were sacrificed at 1, 6, and 24 h postinfection (hpi), and cardiac blood was collected at each time point.

A similar immunization protocol was followed for the rMVA boost in the DNA prime-MVA boost experiments. Here, mice were boosted with the rMVAs at 30 days following plasmid DNA immunization via gene gun (see below). Mice were sacrificed 7 days after the MVA boost, and spleens were collected for the immune response studies.

Gene gun immunizations. A plasmid expressing the LCMV-NP gene under the control of the cytomegalovirus promoter (pCMVNP) was cross-linked to gold powder as previously described (67). Briefly, 40 mg of gold was added to 0.05 M spermidine and sonicated prior to incubation with the DNA and 1 M CaCl2. The DNA-coated gold was washed with ethanol prior to loading into the completely dry tubing. The tubing was cut into centimeter-long pieces, which contained 1 µg of DNA per 0.5 mg of gold. For the DNA immunizations of the DNA prime-MVA boost experiments, mice were immunized with 2 µg of DNA intradermally on their shaved abdomens.

Serum collection. Cardiac blood was collected in the absence of an anticoagulant and was allowed to rest for 16 to 24 h at room temperature. Serum was collected from the coagulated blood following centrifugation at 8,000 rpm for 15 min.

Intracellular cytokine assay. Mouse spleens collected for immune response analyses were crushed over a 70 µM nylon filter and washed with 1x PBS. Red blood cells were lysed with ACK lysis buffer (BioSource International, Inc., Camarillo, CA) for 1 min at room temperature, and the samples were washed once with 1x PBS. The splenocytes were resuspended in RPMI medium containing 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine at a final concentration of 1.5 x 107 cells/ml.

For the in vitro stimulation experiments, 1.5 x 106 cells were incubated with 5 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) and either RPMI alone, 1 µg/ml LCMV-NP118-126 (containing residues 118 to 126 of NP) peptide, 1.5 x 106 A20 cells (24), 1.5 x 106 MVA-infected A20 cells (16 h infection), or 20 ng/ml phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO) and 500 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO) for 5 h at 37°C. Cells were pelleted and incubated with surface and intracellular protein antibodies, as indicated below.

Flow cytometric analyses. The standard staining protocol used was as follows. Cells were processed according to their purpose and incubated with surface antibodies (1:100) or the LCMV-NP118-126 tetramer for 30 min at 4°C. Cells were washed twice with 0.05% PBS-bovine serum albumin (BSA). Cells stained for only surface proteins or tetramer were fixed with 1% formaldehyde (Fisher Scientific, Fairlawn, NJ). For the intracellular cytokine stains, surface-stained cells were permeabilized with Cytofix/Cytoperm (BD Biosciences, San Jose, CA) for 20 min at 4°C. Cells were then washed twice with 1x Perm/Wash buffer (BD Biosciences, San Jose, CA) and incubated with anti-cytokine antibodies (1:20) for 30 min at 4°C. Cells were washed twice with 1x Perm/Wash buffer and fixed with 1% formaldehyde. All antibodies used were obtained from BD Biosciences (San Jose, CA).

Flow cytometry was performed on a FACSCalibur (BD Biosciences, San Jose, CA) instrument. All flow cytometry data were analyzed using FlowJo software (Treestar, San Carlos, CA), Prism software, and Microsoft Excel (Seattle, WA). Statistical analyses were performed as indicated, and all P values of ≤0.05 are considered significant.

Ex vivo expression of LCMV-NP protein. DF-1 cells were harvested at 24 hpi, and cells were permeabilized with Cytofix/Cytoperm prior to incubation with a purified monoclonal anti-LCMV-NP antibody (1:100) (generous gift from Michael Buchmeier). The LCMV-NP antibody was then incubated with a phycoerythrin-conjugated secondary anti-mouse immunoglobulin G (IgG) antibody (1:400) (Biosource, Camarillo, CA).

NP tetramer staining. Splenocytes were obtained as described for the intracellular cytokine assay. Cells were stained with anti-CD8{alpha} and anti-CD3 antibodies, and an allophycocyanin-conjugated LCMV-NP118-126 tetramer (National Institute of Allergy and Infectious Diseases (NIAID) Tetramer Core, Atlanta, GA).

Intracellular cytokine staining. Stimulated splenocytes were incubated with antibodies against the T-cell markers, CD8{alpha} and either CD4 or CD3. After permeabilization, cells were incubated with an anti-gamma interferon (IFN-{gamma}) antibody.

MVA ELISA. Maxisorb ELISA plates (Nunc, Rochester, NY) were coated with 5 x 105 PFU of MVA per well for 16 to 24 h at 4°C. The plates were washed four times with 1x PBS and blocked at 37°C for 1.5 h with 4% PBS-BSA supplemented with goat serum (1:5). Mouse serum samples that were collected at day 7 (acute phase) and day 30 (memory phase) postinfection were serially diluted (twofold dilutions) in 4% PBS-BSA in 96-well plates. These samples were then added to the blocked ELISA plates and incubated for 1.5 h at 37°C. The plates were washed five times with 1x PBS and incubated with anti-mouse IgG-HRP (1:5,000) (Promega, Madison, WI) for 1.5 h at 37°C. The plates were then washed five times with 1x PBS and incubated with One-Step TMB (3,3',5,5'-tetramethylbenzidine) solution (Pierce, Rockford, IL) for 20 min at room temperature. The reaction was stopped with 4N H2SO4 and read at an absorbance of 450 nm. Antibody titers were calculated as endpoint plasma dilutions with MVA naïve serum as the negative reference value. The significance was calculated using a two-tailed Wilcoxon rank sum test using the R version 1.8.1 (34).


arrow
RESULTS
 
Expression of cytokines from recombinant MVAs. In this study, we sought to enhance the immunogenicity of MVA by generating recombinant MVA viruses that express immunomodulatory proteins (CCL20, GM-CSF, and Flt3-L) that are predicted to positively affect different facets of the antigen presentation process. To facilitate the quantification of antigen-specific CD8+ T-cell immune responses, these viruses also express the well-characterized LCMV-NP that represents an immunodominant antigen in BALB/c mice. Inclusion of the LCMV-NP as a surrogate antigen enables the sensitive and quantitative assessment of LCMV-NP-specific CD8+ T-cell responses by major histocompatibility complex (MHC)-tetramer staining and intracellular cytokine staining (ICS) assays (29, 60, 61).

We initially confirmed the expression of the LCMV-NP, hCCL20, mGM-CSF, and hFlt3-L proteins from recombinant viruses during infection of DF-1 cells in culture. To examine LCMV-NP expression, we infected DF-1 cells with MVA, MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or MVA-NP-hFlt3-L. Infected cells were harvested 24 hpi, and LCMV-NP was detected in those cells with an anti-LCMV-NP antibody by using flow cytometry. LCMV-NP is expressed from all recombinant viruses at similar levels (Fig. 1A, solid and dashed lines), whereas cells infected with the control MVA virus showed no reactivity with the anti-LCMV-NP monoclonal antibody (Fig. 1A, shaded peak).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
  		FIG. 1. hCCL20, mGM-CSF, hFlt3-L, and LCMV-NP are expressed from their respective rMVA vectors in vitro and in vivo. DF-1 cells were infected with MVA, MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or MVA-NP-hFlt3-L and cells (A) or supernatants (B) were harvested at 24 hpi for analysis of LCMV-NP expression (A) or cytokine expression (B). (A) Infected cells were permeabilized and stained with a primary anti-LCMV-NP mouse monoclonal antibody (1:100), followed by incubation with a phycoerythrin-conjugated secondary anti-mouse IgG antibody (1:400), and were analyzed by flow cytometry. The negative controls are the MVA-infected cells, and the positive controls are the MVA-NP-infected cells. (B) Expression of hCCL20 and mGM-CSF in supernatants from cells infected with MVA, MVA-NP, MVA-NP-hCCL20, or MVA-NP-mGM-CSF was measured using cytokine-specific Quantikine ELISA kits. ND, not detected. (C and D) Mice were immunized i.p. with MVA-NP-mGM-CSF (C) or MVA-NP-hCCL20 (D), and sera were analyzed at 1, 6, and 24 h following immunization for mGM-CSF (C) or hCCL20 (D) expression using Quantikine ELISA kits. Data were compiled from at least two independent experiments; symbols represent values determined from individual mice. Neither mGM-CSF nor hCCL20 was detected in sera from mice immunized with control MVA or mock-immunized with PBS (not shown). (E) Supernatants from MVA-NP- or MVA-NP-hFlt3-L-infected cells were incubated with SDS buffer, resolved by SDS-polyacrylamide gel electrophoresis, and prepared for immunoblot analysis. hFlt3-L was detected using a biotinylated anti-Flt3-L antibody (0.2 µg/ml) followed by incubation with neutravidin-HRP conjugate (1:10,000). The positive control is a purified hFlt3-L, and the negative control is the supernatant from cells infected with MVA-NP.

Human CCL20 and mGM-CSF expression was examined using cytokine-specific ELISAs (Fig. 1B). For these experiments, DF-1 cells were infected with MVA, MVA-NP, MVA-NP-hCCL20, or MVA-NP-mGM-CSF. Supernatants were collected from the infected cultures at 24 hpi and analyzed with human CCL20 or mouse GM-CSF Quantikine ELISA kits. Figure 1B demonstrates that expression of each cytokine was specific to its cognate virus and that both hCCL20 and mGM-CSF are expressed at high levels from infected cells in culture. We also examined the level of hCCL20 and mGM-CSF in serum following intraperitoneal immunization of mice with MVA-NP-hCCL20 or MVA-NP-mGM-CSF, respectively, using either hCCL20 or mGM-CSF Quantikine ELISA kits (Fig. 1C and D). Within 6 h following immunization of mice with rMVAs, increased levels of mGM-CSF (Fig. 1C) or hCCL20 (Fig. 1D) were detected. Within their respective immunization groups, mGM-CSF and hCCL20 were detected at similar levels in mouse serum, with peak cytokine levels being observed at 1 h (hCCL20) or 6 h (mGM-CSF) postinfection (Fig. 1C and D). By 24 hpi, neither hCCL20 nor mGM-CSF was detectable in the serum (Fig. 1C and D). Neither mGM-CSF nor hCCL20 was detected at any time in sera from mice immunized with control MVA or mock-immunized with PBS (data not shown).

Expression of hFlt3-L was assayed using immunoblot analysis. DF-1 cells were infected with MVA or MVA-NP-hFlt3-L, and supernatants were collected from these infected cultures at 48 hpi. The hFlt3-L protein was specifically expressed from MVA-NP-hFlt3-L at the correct predicted size of 18 to 20 kDa (Fig. 1E, lane 3). As a positive control for hFlt3-L detection, purified hFlt3-L protein was included on the immunoblot (Fig. 1E, lane 2). We detected an additional band (~24 kDa) in the supernatant from virus-infected cells (Fig. 1E, lane 3) that was not present in the recombinant protein sample (Fig. 1E, lane 2), which we attribute to differential glycosylation. Taken together, our analyses confirm the expression of LCMV-NP, hCCL20, mGM-CSF, and hFlt3-L proteins from their respective recombinant MVA viruses.

Virally expressed mGM-CSF or hCCL20 elicits enhanced frequencies of LCMV-NP118-126-specific CD8+ T-cell responses following single-dose rMVA vaccination in mice. To examine the effects of our cytokine modifications on antigen-specific CD8+ T-cell responses after single-dose vaccination with MVA, mice were immunized i.p. with 1 x 106 PFU of MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, MVA-NP-hFlt3-L, or PBS. Mice were sacrificed at 7 dpi (to examine acute responses) or at 15 dpi or 30 dpi (to examine memory responses), and their spleens were collected. CD8+ T-cell responses that were directed against LCMV-NP were determined in two ways: directly by flow cytometric detection of CD8+ T cells that bound to a MHC class I (MHC-I) tetramer against the immunodominant H-2Dd-restricted LCMV-NP118-126 epitope (1), and functionally by ICS to detect IFN-{gamma} production in CD8+ T-cells following stimulation with the LCMV-NP118-126 peptide.

At 7 dpi, an average of 0.8% of the total CD8+ T cells from mice immunized with MVA-NP were phenotypically (Fig. 2B) and functionally (Fig. 2D) specific for the LCMV-NP118-126 epitope. At this time, the frequencies of LCMV-NP118-126-specific CD8+ T cells in mice that were immunized with either MVA-NP-mGM-CSF or MVA-NP-hCCL20 were at least twice as great as those found in mice that were immunized with the control MVA-NP virus (Fig. 2A and B). These increased frequencies of LCMV-NP118-126-specific CD8+ T cells were highly significant (P < 0.001 for both MVA-NP-mGM-CSF and MVA-NP-hCCL20 versus MVA-NP) as determined by nonparametric analysis of variance (ANOVA; Kruskal-Wallis test) comparison of groups.


Figure 2
View larger version (51K):
[in this window]
[in a new window]
  		FIG. 2. Virally expressed hCCL20 or mGM-CSF enhances LCMV-NP118-126-specific CD8+ T-cell responses following single-dose rMVA vaccination in mice. Groups of mice were immunized i.p. with MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, MVA-NP-hFlt3-L, or PBS, and their spleens were harvested at 7, 15, and 30 days after immunization for LCMV-NP118-126 tetramer (A and B) and IFN-{gamma} ICS (C and D) analyses. (A) Representative LCMV-NP118-126-specific CD8+ T-cell responses at 7, 15, and 30 dpi from individual mice within each experimental group. LCMV-NP118-126-specific CD8+ T cells were detected via flow cytometry following surface staining of splenocytes with an allophycocyanin-conjugated LCMV-NP118-126 tetramer. The frequencies of LCMV-NP118-126-specific CD8+ T cells are expressed as percentages of total CD8{alpha}+ splenocytes. The negative control is the naïve group, and the positive control is the MVA-NP group. (B) Frequencies of LCMV-NP118-126-specific CD8+ splenocytes determined by MHC tetramer analysis compiled from three independent experiments are shown. The symbols denote values determined for individual mice, and the group means are denoted by the horizontal bars. At each time point, a nonparametric ANOVA (Kruskal-Wallis test) was performed to compare all immunization groups. Planned intergroup comparisons (P values shown) were performed via Dunn's multiple comparisons test only when the overall ANOVA was significant (P ≤ 0.05). NS, not significant (i.e., P > 0.05). (C) Representative LCMV-NP118-126-specific CD8+ T-cell responses at 7, 15, and 30 dpi from individual mice within each experimental group. Interferon {gamma}-producing LCMV-NP118-126-specific CD8+ T cells were detected via flow cytometry following stimulation with the LCMV-NP118-126 peptide. The frequencies of LCMV-NP118-126-specific CD8+ T cells are expressed as percentages of total CD8{alpha}+ splenocytes. The negative control is the naïve group, and the positive control is the MVA-NP group. (D) Frequencies of LCMV-NP118-126-specific CD8+ T-cell responses determined by IFN-{gamma} ICS assays compiled from three independent experiments are shown. The symbols denote values determined for individual mice, and the group means are denoted by the horizontal bars. P values were calculated as described above for panel B. NS, not significant.

Consistent with these results, immunization of mice with MVA-NP-mGM-CSF or MVA-NP-hCCL20 resulted in 2.5- to 3-fold increases over MVA-NP-immunized mice in the fraction of CD8+ T cells that produced IFN-{gamma} in response to stimulation with the LCMV-NP118-126 peptide (Fig. 2C and D). These responses were significant (for MVA-NP-mGM-CSF, P < 0.001, and for MVA-NP-hCCL20, P <0.01, versus MVA-NP) as determined by nonparametric ANOVA (Kruskal-Wallis test) comparison of groups. In contrast, no appreciable differences between the percentages of phenotypic (Fig. 2A and B) or functional (Fig. 2C and D) LCMV-NP118-126-specific CD8+ T cells were observed in mice immunized with MVA-NP-hFlt3-L compared to those immunized with MVA-NP.

To determine whether the elevated frequencies of LCMV-NP118-126-specific CD8+ T cells elicited by mGM-CSF- or hCCL20-expressing MVAs at 7 dpi were sustained through the memory phase of infection, tetramer staining and ICS assays were performed at 15 and 30 dpi (Fig. 2). The magnitudes of phenotypic (Fig. 2A and B) and functional (Fig. 2C and D) LCMV-NP118-126 specific CD8+ T-cells at 15 days following immunization of mice with MVA-NP-hCCL20 or MVA-NP-mGM-CSF were, on average, twofold (Fig. 2B) to threefold (Fig. 2D) higher than in mice immunized with the control virus, MVA-NP. These differences are significant (P values of <0.01 to <0.001, as indicated in Fig. 2B and D) as determined by nonparametric ANOVA (Kruskal-Wallis test) comparison of immunization groups. Moreover, the mean frequencies of LCMV-NP118-126-specific CD8+ cells observed at 15 days following immunization of mice with MVA-NP-mGM-CSF or MVA-NP-hCCL20 were higher than the mean levels of LCMV-NP118-126-specific CD8+ responses that were observed at 7 days following immunization of mice with the control virus MVA-NP. In contrast, mice immunized with MVA-NP-hFlt3-L did not exhibit LCMV-NP118-126-specific CD8+ T-cell responses that were different from MVA-NP-immunized mice (Fig. 2B and D).

By 30 dpi, the frequency of LCMV-NP118-126-specific CD8+ T cells within each group of immunized mice was reduced by at least half the amount present during the acute phase (7 dpi) (Fig. 2A to D). Importantly, the day 30 postinfection frequencies of LCMV-NP-specific CD8+ T cells from mice immunized with MVA-NP-mGM-CSF or MVA-NP-hCCL20 remained significantly higher (twofold) than those observed from mice immunized with MVA-NP as determined by NP-tetramer analysis (Fig. 2B) (for MVA-NP-mGM-CSF, P < 0.001, and for MVA-NP-hCCL20, P < 0.05, versus MVA-NP as determined by nonparametric ANOVA comparison of immunization groups) or IFN-{gamma} ICS assay (Fig. 2D) (P < 0.05 for MVA-NP-mGM-CSF versus MVA-NP). In contrast, neither phenotypic (Fig. 2A and B) nor functional (Fig. 2C and D) assessments of day 30 memory CD8+ cell responses in mice immunized with MVA-NP-hFlt3-L were different from those elicited by immunization with MVA-NP. Taken together, these experiments demonstrate that hCCL20 and mGM-CSF expressed from recombinant MVA vectors significantly enhance the magnitude of CD8+ T cells that are directed against the vector-expressed LCMV-NP antigen. Because viral expression of hFlt3-L was not observed to enhance vector immunogenicity, we did not further pursue study of hFlt3-L in the context of a single-dose vaccination regimen.

Virally expressed mGM-CSF or hCCL20 elicits enhanced frequencies of MVA-specific CD8+ T cells following single-dose rMVA vaccination in mice. In addition to its development as a vaccine vector for the expression of heterologous antigens, MVA is currently being evaluated as a potentially safer alternative to the standard Dryvax vaccine for vaccination against smallpox, as well as a "prevaccine" administered prior to Dryvax administration in hopes of ameliorating some of the undesirable adverse consequences of the standard vaccinia virus immunization paradigm (15, 21, 52, 55, 79, 83). Thus, we sought to evaluate whether cytokine expression from our recombinant MVA viruses would also augment cellular and humoral immune responses directed against the vaccinia antigens encoded by the MVA "backbone."

To examine antipoxvirus cellular immune responses, we measured the frequencies of MVA-specific CD8+ and CD4+ splenocytes at 7 days following immunization of mice with 1 x 106 PFU of MVA, MVA-NP-hCCL20, or MVA-NP-mGM-CSF or following mock immunization (PBS). To measure MVA-specific cellular responses, we utilized a coincubation assay, in which MVA-infected syngeneic B cells (A20 cell line [24]) function as APCs to stimulate splenocytes in mixed cultures, and measured the intracellular production of IFN-{gamma} in splenocytes by ICS and flow cytometry (29). Because A20 cells present both MHC-I- and MHC-II-restricted antigens, this assay allows for quantitation of both CD8+ and CD4+ T-cell responses (44).

As demonstrated in Fig. 3, the average frequencies of MVA-specific CD8+ splenocytes in mice immunized with MVA-NP-mGM-CSF (1.12%) or MVA-NP-hCCL20 (0.96%) were two- to threefold greater than those elicited following immunization with the parental control MVA virus (0.38%) (Fig. 3B). These differences are significant (for MVA-NP-mGM-CSF, P < 0.01, and for MVA-NP-hCCL20, P < 0.05 versus MVA-NP) as determined by nonparametric ANOVA (Kruskal-Wallis test) comparison of immunization groups and are of similar magnitude to those observed for LCMV-NP-specific CD8+ responses. We also observed trends toward increased frequencies of MVA-specific CD4+ T cells in mice immunized with either MVA-NP-mGM-CSF or MVA-NP-hCCL20 compared to MVA, but these differences did not achieve significance at a 95% confidence level (Fig. 3B).


Figure 3
View larger version (36K):
[in this window]
[in a new window]
  		FIG. 3. Virally expressed hCCL20 or mGM-CSF enhances MVA-specific CD8+ T-cell and antibody responses following single-dose vaccination in mice. Mice were immunized i.p. with MVA, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or PBS. (A) Representative MVA-specific CD8+ and CD4+ T-cell responses at 7 dpi from individual mice within each experimental group. IFN-{gamma}-producing MVA-specific CD8+ and CD4+ T cells were detected via flow cytometry following stimulation with MVA-infected A20 cells. The frequencies of MVA-specific CD8+ and CD4+ T cells are expressed as percentages of total CD8{alpha}+ or CD4+ splenocytes, respectively. The negative control is the naïve group, and the positive control is the group immunized with MVA. (B) Frequencies of MVA-specific CD8+ and CD4+ T cells (note different scales) were determined at 7 dpi in two independent experiments. The symbols denote values determined for individual mice, and the horizontal bars denote the group means. Nonparametric ANOVAs (Kruskal-Wallis tests) were performed to compare CD8+ or CD4+ T-cell responses between all immunization groups. (C) MVA-specific serum antibody titers were determined by ELISA at 7 days (n = 10 mice per group) and 30 days (n = 5 mice per group) following immunization with the indicated virus. The median titers, range of values, and P values for pairwise comparisons between groups of mice immunized with MVA-NP-mGM-CSF or MVA-NP-hCCL20 versus MVA using a two-tailed Wilcoxon rank sum test are shown.

Virally expressed mGM-CSF or hCCL20 elicits enhanced MVA-specific antibody responses following single-dose rMVA vaccination in mice. Recently, vaccinia virus-specific B-cell responses were reported to be essential for protecting macaques against a lethal monkeypox challenge, thereby demonstrating that the generation of antibody responses against poxvirus antigens is essential for establishing and maintaining protective antipoxvirus immunity (22). To determine whether our rMVAs expressing mGM-CSF or hCCL20, which elicit higher levels of antiviral CD8+ T-cell responses than nonadjuvanted control MVAs, are also capable of eliciting enhanced antibody responses, we measured the titers of MVA-specific antibodies in the serum of mice at various times following single-dose immunization of mice.

To measure MVA-specific antibody titers, mice were immunized i.p. with MVA, MVA-NP-hCCL20, or MVA-NP-mGM-CSF, and serum was collected at 7 and 30 dpi for analysis using an MVA ELISA. MVA-specific antibody titers were calculated as endpoint dilutions and were detected in all serum samples from virus-immunized mice at both time points (Fig. 3C). Median MVA-specific antibody titers at 7 days following immunization of mice with MVA-NP-hCCL20 (titer, 640) or MVA-NP-mGM-CSF (1,024) were 6- to 10-fold greater than that observed in mice immunized with MVA (102) (Fig. 3D). Even larger differences in MVA-specific antibody titers were subsequently observed between immunization groups. At 30 dpi, the median MVA-specific antibody titers in mice immunized with MVA-NP-hCCL20 (2,253) or MVA-NP-mGM-CSF (2,867) were 13- to 17-fold higher than that observed in mice immunized with the control MVA virus (166) (P = 0.012 for both MVA-NP-hCCL20 and MVA-NP-mGM-CSF versus MVA using the two-tailed Wilcoxon rank sum test). Comparison of the rate of change of MVA-specific antibody titers between days 7 and 30 postimmunization within each immunization group demonstrates a higher rate for groups immunized with MVA-NP-hCCL20 (3.5-fold) or MVA-NP-mGM-CSF (2.8-fold) compared to MVA controls (1.6-fold). Thus, expression of either mGM-CSF or hCCL20 from recombinant MVA significantly enhances both the absolute magnitude of MVA-specific antibody titers as well as the rates at which these antibody responses develop in immunized mice.

Cytokine-expressing rMVA vectors do not exhibit enhanced boosting of memory CD8+ T-cell responses. Our results indicate that rMVA vectors expressing mGM-CSF or hCCL20 can be used in single-dose immunization regimens to elicit higher levels of antigen-specific cellular and humoral immune responses. However, the overwhelming majority of MVA-based vaccination strategies currently in development use rMVAs as booster vaccines in heterologous prime-boost vaccination regimens (2, 5, 23, 50, 53, 54, 59, 62, 84, 86). Therefore, we sought to determine whether booster immunization with rMVAs expressing mGM-CSF, hCCL20, or hFlt3-L would elicit an augmented recall of preexisting memory T-cell responses, compared to booster immunization with control rMVA.

To perform these experiments, mice were first immunized (primed) with 2 µg of plasmid pCMVNP, delivered intradermally via gene gun, and boosted 30 days later with 1 x 106 PFU of MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, MVA-NP-hFlt3-L, or PBS. Spleens were harvested at 7 or 30 days postboost, and the frequencies of LCMV-NP-specific CD8+ T cells were measured directly by LCMV-NP118-126 tetramer analysis. At 7 days postboost, all groups boosted with rMVAs exhibited substantial expansions of LCMV-NP-specific CD8+ T cells compared to the mock-boosted group (Fig. 4). However, no differences in the average frequencies of LCMV-NP-specific CD8+ splenocytes were observed between groups of mice boosted with MVA-NP-hCCL20, MVA-NP-mGM-CSF, MVA-NP-hFlt3-L, or MVA-NP control virus. Similarly, no significant differences were observed between any groups of rMVA-boosted mice when LCMV-NP tetramer analyses were determined at a later time (30 days) following booster immunizations (data not shown). Taken together, the data from both the single-dose and DNA prime-rMVA boost vaccination experiments argue that expression of mGM-CSF and hCCL20 from rMVAs enhances the priming of antigen-specific cellular immune responses but not the recall of preexisting memory responses.


Figure 4
View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4. Cytokine-expressing rMVA vectors do not elicit enhanced boosting of memory CD8+ T-cell responses. Mice were immunized with plasmid pCMVNP to prime LCMV-NP-specific CD8+ T-cell responses and were boosted 30 days later with MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, MVA-NP-hFlt3-L, or PBS (Mock). Frequencies of LCMV-NP-specific CD8+ splenocytes were measured at 7 days postboost by MHC-I-tetramer analysis and are expressed as percentages of total CD8{alpha}+ splenocytes. (A) Representative LCMV-NP118-126-specific CD8+ T-cell responses from individual mice within indicated experimental groups. (B) Summary of LCMV-NP118-126-specific CD8+ T cells measured in two independent experiments. Data points represent values derived from individual mice, and the bars denote the group means.


arrow
DISCUSSION
 
MVA is an attenuated orthopoxvirus that is currently in phase I clinical trials as a smallpox vaccine and that is actively being developed as a vector for vaccination against both heterologous infectious diseases including AIDS, malaria, and tuberculosis, as well as human cancers (2, 5, 23, 50, 53, 54, 57, 59, 62, 84, 86, 94). One of MVA's most desirable attributes for use as a vaccine vector is its safety, which is largely due to the fact that MVA does not replicate productively in most mammalian cell types and is, therefore, restricted to undergo less than one complete infectious cycle within a mammalian host (8, 11, 20).

However, this inability to replicate productively in vivo also likely constrains the extent to which current MVA-based vaccine vectors elicit high levels of durable immune responses by restricting de novo synthesis of vector-encoded antigens to that occurring in cells transduced by rMVA vectors at the site of immunization (2, 5, 50, 59, 62, 86). Despite MVA's tropism for DCs, the ensuing abortive infection that prevents cellular maturation and induces apoptosis limits the potential for direct presentation of vector-encoded antigens by infected DCs (14a, 14). Given the central importance of APCs and DCs, in particular, for presentation of antigens encoded by recombinant vaccinia viruses (via direct and/or cross-presentation pathways) (65, 66, 78), strategies designed to recruit additional APCs to the site of immunization, to activate APCs, or to augment antigen presentation and priming of naïve T cells should provide the means to increase the magnitudes of both acute and memory immune responses that are generated against antigens expressed from rMVA vectors.

With this aim of creating MVA recombinants that are more immunogenic than parental vectors, we generated rMVAs that express mGM-CSF, hCCL20, or hFlt3-L. The rationale for expression of human or murine cytokine homologs from MVA recombinants was based on the known functionalities of these molecules in mice. Because mGM-CSF acts in a species-specific manner due to species-specific differences in the subunits that comprise the high-affinity GM-CSF receptor (i.e., the murine GM-CSF receptor does not bind human GM-CSF), the murine homolog was selected for expression from our rMVA vector (41, 64). Similarly, the use of human, rather than murine Flt3-L to mobilize murine DCs in vivo has been extensively characterized (18, 46, 71-73), and several studies have described genetic adjuvant activity associated with both full-length and extracellular forms of human Flt3-L in murine systems (63, 75). Human CCL20 has been shown to effect migration of murine DCs both in vitro and in vivo (26); however, use of CCL20 (human or murine) as an adjuvant (25) has not been previously reported for poxvirus or infectious disease vaccines. Cytokine-expressing viruses were designed to coexpress the LCMV-NP protein because this immunogen elicits strong CD8+ T-cell responses in BALB/c mice (60, 61) and enabled the use of MHC-I tetramers and ICS to facilitate the quantification of total LCMV-NP118-126-specific CD8+ T-cell responses. We examined these viruses in the context of single-dose and heterologous prime/boost vaccination regimens in mice to determine what effects these virally expressed cytokines and chemokine have on priming immune responses versus recalling immune memory responses.

Our results demonstrate that expression of mGM-CSF from a rMVA virus augments the generation of both CD8+ T cell and humoral antiviral immune responses following single-dose immunization of mice with this vaccine vector. As such, our results are consistent with those from studies of the use of GM-CSF as a genetic or recombinant protein adjuvant in alternative model systems (6, 28, 38, 39, 47, 74, 77, 93, 96). For example, immunization of mice with recombinant avipox or vaccinia viruses expressing GM-CSF has been reported to enrich draining lymph nodes with APCs and to effect enhanced T-cell responses that provide protection in a tumor challenge model (38, 39). Alternatively, the approach of administering GM-CSF as a recombinant protein in combination with avipox vectors encoding tumor-specific antigens has advanced into human clinical evaluation (47, 93). Recently, Reali et al. described the use of a recombinant fowlpox virus expressing GM-CSF as an immune adjuvant for codelivered protein, peptide, or poxvirus-encoded gene product antigens (74). Moreover, adjuvant activity of GM-CSF expressed from this recombinant fowlpox vector was at least as potent (and in some cases, more potent) than repeated bolus injections of recombinant GM-CSF, suggesting that the optimal adjuvant activity of GM-CSF resides in its ability to act locally to stimulate the proliferation and maturation of APCs (74). In addition, a number of studies have described the genetic delivery of GM-CSF via plasmid DNA to augment DC recruitment to the site of immunization and to induce immune responses against codelivered antigens (6, 28, 77, 96). The expression of GM-CSF from a vector (particularly one that simultaneously coexpresses antigens of interest, as described in our current study), as opposed to coadministration of recombinant GM-CSF protein with a vaccine vector, promises to simplify experimental immunization regimens and to target GM-CSF to relevant microenvironments to effect optimal adjuvant activity.

The expression of hCCL20 as an adjuvant from poxvirus vaccine vectors has not previously been described. Here, we demonstrate that single-dose immunization of mice with an rMVA vector expressing hCCL20 augments both CD8+ T-cell and humoral antiviral immune responses. Moreover, the magnitudes of hCCL20's adjuvant effects on levels of CD8+ T-cell and antibody responses are similar to those observed following immunization with an rMVA vector that expresses mGM-CSF. While the precise mechanism of this enhancement has not yet been determined, we predict that expression of hCCL20 from rMVA enhances antiviral immune responses via recruitment of immature (CCR6+) APCs to the site of immunization. Because hCCL20 and mGM-CSF may exert their adjuvant effects through different mechanisms, we are evaluating whether coexpression of mGM-CSF and hCCL20 from a single rMVA might result in additive or even synergistic enhancements in the generation of either cellular or humoral antiviral immune responses. Given the observation that neutralizing antibodies constitute a key protective mechanism against pathogenic orthopoxvirus infection (22), our observations that GM-CSF or CCL20 expression from rMVA vectors significantly enhances titers of MVA-specific antibodies in vivo suggest the relevance of expressing these immunomodulatory factors from next generation MVA-based smallpox vaccines.

In our DNA prime-rMVA boost studies, immunization of mice that have an established LCMV-NP118-126-specific CD8+ T-cell memory response with rMVA vectors encoding mGM-CSF, hCCL20, or hFlt3-L was found to boost that response equivalently and to levels similarly achieved by boosting with control rMVA. Taken together with our results from single-dose immunization experiments, we conclude that mGM-CSF and hCCL20, when expressed from rMVA vectors, exert their beneficial adjuvant properties by enhancing the priming of naïve T cells but do not augment the recall of memory CD8+ T-cell responses.

Finally, our results also indicate that viral expression of hFlt3-L had no effect on the generation of antigen-specific immune responses in either single-dose or DNA prime-rMVA boost vaccination regimens. In contrast, delivery of hFlt3-L or hFlt3-L-antigen fusion proteins via repeated immunizations with cytokine-encoding DNA plasmids has been reported to recruit DCs to the site of intramuscular immunization and to result in enhanced levels of CD8+ T-cell responses (75, 76). Importantly, the hFlt3-L gene employed here has been shown to encode a protein that is biologically active and capable of expanding DCs in both murine and nonhuman primate model systems (R. Chavan, unpublished results). The absence of hFlt3-L-associated adjuvant activity in our current study may be attributable to suboptimal temporal or spatial expression of hFlt3-L from rMVA or to expression of an insufficient amount of hFlt3-L than is otherwise necessary to elicit its immunomodulatory effects in vivo.

Overall, we show here that APC maturation and migration factors expressed from rMVA vectors act as adjuvants to enhance the magnitude and duration of induced cellular and humoral immune responses. Practically, the expression of immunomodulatory factors from a single vaccine vector promises to simplify vaccine production compared to vaccination regimens predicated upon the coadministration of a vaccine vector and a recombinant protein or plasmid DNA-encoded adjuvant. Moreover, expression of genetic adjuvants from rMVA vectors provides an opportunity to enhance the immunogenicity of MVA-based vaccines while preserving their inherent safety. Given both the relatively high dose of MVA (≥108 infectious units) that is typically required to elicit appreciable immune responses and the apparent difficulty in achieving large-scale production of MVA-based vaccines, expression of cytokine adjuvants (or other improvements) that increase vaccine immunogenicity and lower the requisite dose to be administered should be of great practical value in the development of MVA-based vaccines. Our results that MVA can be genetically engineered to express APC activation and recruitment proteins to simultaneously enhance both cellular and humoral immune responses directed against MVA and/or heterologous antigens suggest that this approach may be productively exploited to develop equally safe but significantly more immunogenic MVA-based vaccines against smallpox and other infectious diseases, such as malaria or AIDS.


arrow
ACKNOWLEDGMENTS
 
We thank Rafi Ahmed at the Emory University Vaccine Center for providing the LCMV-NP gene, Michael Buchmeier at the Scripps Institute for providing the anti-LCMV-NP monoclonal antibody, and the NIAID Tetramer Core for providing the LCMV-NP118-126 tetramer. We also thank Silvija Staprans, Guido Silvestri, their lab members, and all the members of the Feinberg and Garber labs for helpful discussions.

This work was made possible with support from grant P01 AI 46007 (to M.B.F.), the Southeast Regional Center of Excellence for Emerging Infections and Biodefense (SERCEB)-NIH/NIAID grant U54 AI 057157 (to D.A.G.), and NIH/NIAID grant U19 AI 061728 (to M.B.F.).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Emory Vaccine Center, 954 Gatewood Road NE, Atlanta, GA 30329. Phone: (404) 727-4374. Fax: (404) 727-8199. E-mail: dgarber{at}rmy.emory.edu. Back

{dagger} R.C. and K.A.M. contributed equally to this article. Back

{ddagger} Present address: Merck Vaccine Division, Merck and Co., Inc., West Point, Pa. Back


arrow
REFERENCES
 
    1
  1. Aichele, P., K. Brduscha-Riem, R. M. Zinkernagel, H. Hengartner, and H. Pircher. 1995. T cell priming versus T cell tolerance induced by synthetic peptides. J Exp. Med. 182:261-266.[Abstract/Free Full Text]
    2. 2
  2. Amara, R. R., F. Villinger, J. D. Altman, S. L. Lydy, S. P. O'Neil, S. I. Staprans, D. C. Montefiori, Y. Xu, J. G. Herndon, L. S. Wyatt, M. A. Candido, N. L. Kozyr, P. L. Earl, J. M. Smith, H. L. Ma, B. D. Grimm, M. L. Hulsey, J. Miller, H. M. McClure, J. M. McNicholl, B. Moss, and H. L. Robinson. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292:69-74.[CrossRef][Medline]
    3. 3
  3. Antoine, G., F. Scheiflinger, F. Dorner, and F. G. Falkner. 1998. The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244:365-396.[CrossRef][Medline]
    4. 4
  4. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767-811.[CrossRef][Medline]
    5. 5
  5. Barouch, D. H., S. Santra, M. J. Kuroda, J. E. Schmitz, R. Plishka, A. Buckler-White, A. E. Gaitan, R. Zin, J. H. Nam, L. S. Wyatt, M. A. Lifton, C. E. Nickerson, B. Moss, D. C. Montefiori, V. M. Hirsch, and N. L. Letvin. 2001. Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination. J. Virol. 75:5151-5158.[Abstract/Free Full Text]
    6. 6
  6. Barouch, D. H., S. Santra, K. Tenner-Racz, P. Racz, M. J. Kuroda, J. E. Schmitz, S. S. Jackson, M. A. Lifton, D. C. Freed, H. C. Perry, M. E. Davies, J. W. Shiver, and N. L. Letvin. 2002. Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF. J. Immunol. 168:562-568.[Abstract/Free Full Text]
    7. 7
  7. Barreda, D. R., P. C. Hanington, and M. Belosevic. 2004. Regulation of myeloid development and function by colony stimulating factors. Dev. Comp. Immunol. 28:509-554.[CrossRef][Medline]
    8. 8
  8. Blanchard, T. J., A. Alcami, P. Andrea, and G. L. Smith. 1998. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J Gen. Virol. 79:1159-1167.[Abstract]
    9. 9
  9. Broxmeyer, H. E., L. Lu, S. Cooper, L. Ruggieri, Z. H. Li, and S. D. Lyman. 1995. Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells. Exp. Hematol. 23:1121-1129.[Medline]
    10. 10
  10. Cantrell, M. A., D. Anderson, D. P. Cerretti, V. Price, K. McKereghan, R. J. Tushinski, D. Y. Mochizuki, A. Larsen, K. Grabstein, S. Gillis, et al. 1985. Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor. Proc. Natl. Acad. Sci. USA 82:6250-6254.[Abstract/Free Full Text]
    11. 11
  11. Carroll, M. W., and B. Moss. 1997. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238:198-211.[CrossRef][Medline]
    12. 12
  12. Caux, C., S. Ait-Yahia, K. Chemin, O. de Bouteiller, M. C. Dieu-Nosjean, B. Homey, C. Massacrier, B. Vanbervliet, A. Zlotnik, and A. Vicari. 2000. Dendritic cell biology and regulation of dendritic cell trafficking by chemokines. Springer Semin. Immunopathol. 22:345-369.[CrossRef][Medline]
    13. 13
  13. Caux, C., B. Vanbervliet, C. Massacrier, S. Ait-Yahia, C. Vaure, K. Chemin, M. C. Dieu-Nosjean, and A. Vicari. 2002. Regulation of dendritic cell recruitment by chemokines. Transplantation 73:S7-11.[CrossRef][Medline]
    14. 14
  14. Chahroudi, A., R. Chavan, N. Koyzr, E. K. Waller, G. Silvestri, and M. B. Feinberg. 2005. Vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor. J. Virol. 79:10397-10407.[Abstract/Free Full Text]
    15. 14
  15. Chahroudi, A., D. A. Garber, P. Reeves, L. Liu, D. Kalman, and M. B. Feinberg. J. Virol., in press.
    16. 15
  16. clinicaltrials.gov. 2005 posting date. A database of clinical research conducted in human volunteers. Search term "MVA." U.S. National Institutes of Health. [Online.] Accessed 1 November 2005.
    17. 16
  17. Cutler, C. W., R. Jotwani, and B. Pulendran. 2001. Dendritic cells: immune saviors or Achilles' heel? Infect. Immun. 69:4703-4708.[Free Full Text]
    18. 17
  18. D'Amico, A., and L. Wu. 2003. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. J. Exp. Med. 198:293-303.[Abstract/Free Full Text]
    19. 18
  19. Daro, E., B. Pulendran, K. Brasel, M. Teepe, D. Pettit, D. H. Lynch, D. Vremec, L. Robb, K. Shortman, H. J. McKenna, C. R. Maliszewski, and E. Maraskovsky. 2000. Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murine dendritic cells in vivo: a comparative analysis with Flt3 ligand. J. Immunol. 165:49-58.[Abstract/Free Full Text]
    20. 19
  20. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188:373-386.[Abstract/Free Full Text]
    21. 20
  21. Drexler, I., K. Heller, B. Wahren, V. Erfle, and 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]
    22. 21
  22. Earl, P. L., J. L. Americo, L. S. Wyatt, L. A. Eller, J. C. Whitbeck, G. H. Cohen, R. J. Eisenberg, C. J. Hartmann, D. L. Jackson, D. A. Kulesh, M. J. Martinez, D. M. Miller, E. M. Mucker, J. D. Shamblin, S. H. Zwiers, J. W. Huggins, P. B. Jahrling, and B. Moss. 2004. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature 428:182-185.[CrossRef][Medline]
    23. 22
  23. Edghill-Smith, Y., H. Golding, J. Manischewitz, L. R. King, D. Scott, M. Bray, A. Nalca, J. W. Hooper, C. A. Whitehouse, J. E. Schmitz, K. A. Reimann, and G. Franchini. 2005. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat. Med. 11:740-747.[CrossRef][Medline]
    24. 23
  24. Feng, C. G., T. J. Blanchard, G. L. Smith, A. V. Hill, and W. J. Britton. 2001. Induction of CD8+ T-lymphocyte responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus. Immunol. Cell Biol. 79:569-575.[CrossRef][Medline]
    25. 24
  25. Folsom, V., D. P. Gold, J. White, P. Marrack, J. Kappler, and S. Tonegawa. 1984. Functional and inducible expression of a transfected murine class II major histocompatibility complex gene. Proc. Natl. Acad. Sci. USA 81:2045-2049.[Abstract/Free Full Text]
    26. 25
  26. Furumoto, K., L. Soares, E. G. Engleman, and M. Merad. 2004. Induction of potent antitumor immunity by in situ targeting of intratumoral DCs. J. Clin. Investig. 113:774-783.[CrossRef][Medline]
    27. 26
  27. Fushimi, T., A. Kojima, M. A. Moore, and R. G. Crystal. 2000. Macrophage inflammatory protein 3alpha transgene attracts dendritic cells to established murine tumors and suppresses tumor growth. J. Clin. Investig. 105:1383-1393.[Medline]
    28. 27
  28. Gajewski, T. F., S. R. Schell, G. Nau, and F. W. Fitch. 1989. Regulation of T-cell activation: differences among T-cell subsets. Immunol. Rev. 111:79-110.[CrossRef][Medline]
    29. 28
  29. Haddad, D., J. Ramprakash, M. Sedegah, Y. Charoenvit, R. Baumgartner, S. Kumar, S. L. Hoffman, and W. R. Weiss. 2000. Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cells into injected muscles. J. Immunol. 165:3772-3781.[Abstract/Free Full Text]
    30. 29
  30. Harrington, L. E., R. Most Rv, J. L. Whitton, and 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]
    31. 30
  31. Hausser, G., B. Ludewig, H. R. Gelderblom, Y. Tsunetsugu-Yokota, K. Akagawa, and A. Meyerhans. 1997. Monocyte-derived dendritic cells represent a transient stage of differentiation in the myeloid lineage. Immunobiology. 197:534-542.[Medline]
    32. 31
  32. Hidmark, A. S., G. M. McInerney, E. K. Nordstrom, I. Douagi, K. M. Werner, P. Liljestrom, and G. B. Hedestam. 2005. Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88. J. Virol. 79:10376-10385.[Abstract/Free Full Text]
    33. 32
  33. Himly, M., D. N. Foster, I. Bottoli, J. S. Iacovoni, and P. K. Vogt. 1998. The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology 248:295-304.[CrossRef][Medline]
    34. 33
  34. Hromas, R., P. W. Gray, D. Chantry, R. Godiska, M. Krathwohl, K. Fife, G. I. Bell, J. Takeda, S. Aronica, M. Gordon, S. Cooper, H. E. Broxmeyer, and M. J. Klemsz. 1997. Cloning and characterization of exodus, a novel beta-chemokine. Blood 89:3315-3322.[Abstract/Free Full Text]
    35. 34
  35. Ihaka, R., and R. Gentleman. 1996. R: a language for data analysis and graphics. J. Comput. Graph Stat. 5:299-314.[CrossRef]
    36. 35
  36. Iwasaki, A., and B. L. Kelsall. 2000. Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3{alpha}, MIP-3ß, and secondary lymphoid organ chemokine. J. Exp. Med. 191:1381-1394.[Abstract/Free Full Text]
    37. 36
  37. Karsunky, H., M. Merad, A. Cozzio, I. L. Weissman, and M. G. Manz. 2003. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J. Exp. Med. 198:305-313.[Abstract/Free Full Text]
    38. 37
  38. Karsunky, H., M. Merad, I. Mende, M. G. Manz, E. G. Engleman, and I. L. Weissman. 2005. Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors. Exp. Hematol. 33:173-181.[CrossRef][Medline]
    39. 38
  39. Kass, E., D. L. Panicali, G. Mazzara, J. Schlom, and J. W. Greiner. 2001. Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant. Cancer Res. 61:206-214.[Abstract/Free Full Text]
    40. 39
  40. Kass, E., J. Parker, J. Schlom, and J. W. Greiner. 2000. Comparative studies of the effects of recombinant GM-CSF and GM-CSF administered via a poxvirus to enhance the concentration of antigen-presenting cells in regional lymph nodes. Cytokine 12:960-971.[CrossRef][Medline]
    41. 40
  41. Kiertscher, S. M., and M. D. Roth. 1996. Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4. J. Leukoc. Biol. 59:208-218.[Abstract]
    42. 41
  42. Kitamura, T., K. Hayashida, K. Sakamaki, T. Yokota, K. Arai, and A. Miyajima. 1991. Reconstitution of functional receptors for human granulocyte/macrophage colony-stimulating factor (GM-CSF): evidence that the protein encoded by the AIC2B cDNA is a subunit of the murine GM-CSF receptor. Proc. Natl. Acad. Sci. USA 88:5082-5086.[Abstract/Free Full Text]
    43. 42
  43. Kozak, M. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234:187-208.[CrossRef][Medline]
    44. 43
  44. Krutzik, S. R., B. Tan, H. Li, M. T. Ochoa, P. T. Liu, S. E. Sharfstein, T. G. Graeber, P. A. Sieling, Y. J. Liu, T. H. Rea, B. R. Bloom, and R. L. Modlin. 2005. TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells. Nat. Med. 11:653-660.[CrossRef][Medline]
    45. 44
  45. Lauvau, G., and N. Glaichenhaus. 2004. Mini-review: Presentation of pathogen-derived antigens in vivo. Eur. J. Immunol. 34:913-920.[CrossRef][Medline]
    46. 45
  46. Liu, Y. J. 2005. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 23:275-306.[CrossRef][Medline]
    47. 46
  47. Maraskovsky, E., K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, and H. J. McKenna. 1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184:1953-1962.[Abstract/Free Full Text]
    48. 47
  48. Marshall, J. L., J. L. Gulley, P. M. Arlen, P. K. Beetham, K. Y. Tsang, R. Slack, J. W. Hodge, S. Doren, D. W. Grosenbach, J. Hwang, E. Fox, L. Odogwu, S. Park, D. Panicali, and J. Schlom. 2005. Phase I study of sequential vaccinations with fowlpox-CEA(6D)-TRICOM alone and sequentially with vaccinia-CEA(6D)-TRICOM, with and without granulocyte-macrophage colony-stimulating factor, in patients with carcinoembryonic antigen-expressing carcinomas. J. Clin. Oncol. 23:720-731.[Abstract/Free Full Text]
    49. 48
  49. Mayr, A. 1999. Historical review of smallpox, the eradication of smallpox and the attenuated smallpox MVA vaccine. Berl. Munch. Tierarztl. Wochenschr. 112:322-328. (In German.)[Medline]
    50. 49
  50. Mayr, A. 1976. TC marker of the attenuated vaccinia vaccide strain "MVA" in human cell cultures and protective immunization against orthopox diseases in animals. Zentralbl. Veterinarmed. B 23:417-430. (In German.)[Medline]
    51. 50
  51. McConkey, S. J., W. H. Reece, V. S. Moorthy, D. Webster, S. Dunachie, G. Butcher, J. M. Vuola, T. J. Blanchard, P. Gothard, K. Watkins, C. M. Hannan, S. Everaere, K. Brown, K. E. Kester, J. Cummings, J. Williams, D. G. Heppner, A. Pathan, K. Flanagan, N. Arulanantham, M. T. Roberts, M. Roy, G. L. Smith, J. Schneider, T. Peto, R. E. Sinden, S. C. Gilbert, and A. V. Hill. 2003. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat. Med. 9:729-735.[CrossRef][Medline]
    52. 51
  52. McCullough, K. C., and A. Summerfield. 2005. Basic concepts of immune response and defense development. ILAR J. 46:230-240.[Medline]
    53. 52
  53. McCurdy, L. H., B. D. Larkin, J. E. Martin, and B. S. Graham. 2004. Modified vaccinia Ankara: potential as an alternative smallpox vaccine. Clin. Infect. Dis. 38:1749-1753.[CrossRef][Medline]
    54. 53
  54. McShane, H. 2004. Developing an improved vaccine against tuberculosis. Expert Rev. Vaccines 3:299-306.[CrossRef][Medline]
    55. 54
  55. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, K. Huygen, H. A. Fletcher, and A. V. Hill. 2004. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat. Med. 10:1240-1244.[CrossRef][Medline]
    56. 55
  56. Meseda, C. A., A. D. Garcia, A. Kumar, A. E. Mayer, J. Manischewitz, L. R. King, H. Golding, M. Merchlinsky, and J. P. Weir. 2005. Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus Ankara and licensed smallpox vaccine Dryvax in a mouse model. Virology 339:164-175.[CrossRef][Medline]
    57. 56
  57. Meyer, H., G. Sutter, and A. Mayr. 1991. Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J. Gen. Virol. 72:1031-1038.[Abstract/Free Full Text]
    58. 57
  58. Meyer, R. G., C. M. Britten, U. Siepmann, B. Petzold, T. A. Sagban, H. A. Lehr, B. Weigle, M. Schmitz, L. Mateo, B. Schmidt, H. Bernhard, T. Jakob, R. Hein, G. Schuler, B. Schuler-Thurner, S. N. Wagner, I. Drexler, G. Sutter, N. Arndtz, P. Chaplin, J. Metz, A. Enk, C. Huber, and T. Wolfel. 2005. A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr). Cancer Immunol. Immunother. 54:453-467.[CrossRef][Medline]
    59. 58
  59. Miller, G., V. G. Pillarisetty, A. B. Shah, S. Lahrs, and R. P. DeMatteo. 2003. Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties. J. Immunol. 170:3554-3564.[Abstract/Free Full Text]
    60. 59
  60. Moorthy, V. S., S. McConkey, M. Roberts, P. Gothard, N. Arulanantham, P. Degano, J. Schneider, C. Hannan, M. Roy, S. C. Gilbert, T. E. Peto, and A. V. Hill. 2003. Safety of DNA and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers. Vaccine 21:1995-2002.[CrossRef][Medline]
    61. 60
  61. Murali-Krishna, K., J. D. Altman, M. Suresh, D. Sourdive, A. Zajac, and R. Ahmed. 1998. In vivo dynamics of antiviral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection. Adv. Exp. Med. Biol. 452:123-142.[Medline]
    62. 61
  62. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177-187.[CrossRef][Medline]
    63. 62
  63. Mwau, M., I. Cebere, J. Sutton, P. Chikoti, N. Winstone, E. G. Wee, T. Beattie, Y. H. Chen, L. Dorrell, H. McShane, C. Schmidt, M. Brooks, S. Patel, J. Roberts, C. Conlon, S. L. Rowland-Jones, J. J. Bwayo, A. J. McMichael, and T. Hanke. 2004. A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J. Gen. Virol. 85:911-919.[Abstract/Free Full Text]
    64. 63
  64. Nayak, B. P., G. Sailaja, and A. M. Jabbar. 2006. Augmenting the immunogenicity of DNA vaccines: role of plasmid-encoded Flt-3 ligand, as a molecular adjuvant in genetic vaccination. Virology. 348:277-288.[CrossRef][Medline]
    65. 64
  65. Nishijima, I., T. Nakahata, Y. Hirabayashi, T. Inoue, H. Kurata, A. Miyajima, N. Hayashi, Y. Iwakura, K. Arai, and T. Yokota. 1995. A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. Mol. Biol. Cell 6:497-508.[Abstract]
    66. 65
  66. Norbury, C. C., D. Malide, J. S. Gibbs, J. R. Bennink, and J. W. Yewdell. 2002. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo. Nat. Immunol. 3:265-271.[CrossRef][Medline]
    67. 66
  67. Norbury, C. C., M. F. Princiotta, I. Bacik, R. R. Brutkiewicz, P. Wood, T. Elliott, J. R. Bennink, and J. W. Yewdell. 2001. Multiple antigen-specific processing pathways for activating naive CD8+ T cells in vivo. J. Immunol. 166:4355-4362.[Abstract/Free Full Text]
    68. 67
  68. Pertmer, T. M., M. D. Eisenbraun, D. McCabe, S. K. Prayaga, D. H. Fuller, and J. R. Haynes. 1995. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 13:1427-1430.[CrossRef][Medline]
    69. 68
  69. Pickl, W. F., O. Majdic, P. Kohl, J. Stockl, E. Riedl, C. Scheinecker, C. Bello-Fernandez, and W. Knapp. 1996. Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes. J. Immunol. 157:3850-3859.[Abstract]
    70. 69
  70. Plotkin, S. A., and W. A. Orenstein (ed.). 2004. Vaccines, 4th ed. Saunders, Philadelphia, Pa.
    71. 70
  71. Pulendran, B. 2004. Immune activation: death, danger and dendritic cells. Curr. Biol. 14:R30-R32.[CrossRef][Medline]
    72. 71
  72. Pulendran, B., J. Lingappa, M. K. Kennedy, J. Smith, M. Teepe, A. Rudensky, C. R. Maliszewski, and E. Maraskovsky. 1997. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J. Immunol. 159:2222-2231.[Abstract/Free Full Text]
    73. 72
  73. Pulendran, B., J. L. Smith, G. Caspary, K. Brasel, D. Pettit, E. Maraskovsky, and C. R. Maliszewski. 1999. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc. Natl. Acad. Sci. USA 96:1036-1041.[Abstract/Free Full Text]
    74. 73
  74. Pulendran, B., J. L. Smith, M. Jenkins, M. Schoenborn, E. Maraskovsky, and C. R. Maliszewski. 1998. Prevention of peripheral tolerance by a dendritic cell growth factor: flt3 ligand as an adjuvant. J. Exp. Med. 188:2075-2082.[Abstract/Free Full Text]
    75. 74
  75. Reali, E., D. Canter, H. Zeytin, J. Schlom, and J. W. Greiner. 2005. Comparative studies of avipox-GM-CSF versus recombinant GM-CSF protein as immune adjuvants with different vaccine platforms. Vaccine 23:2909-2921.[CrossRef][Medline]
    76. 75
  76. Sailaja, G., S. Husain, B. P. Nayak, and A. M. Jabbar. 2003. Long-term maintenance of gp120-specific immune responses by genetic vaccination with the HIV-1 envelope genes linked to the gene encoding Flt-3 ligand. J. Immunol. 170:2496-2507.[Abstract/Free Full Text]
    77. 76
  77. Sang, H., V. M. Pisarev, C. Munger, S. Robinson, J. Chavez, L. Hatcher, P. Parajuli, Y. Guo, and J. E. Talmadge. 2003. Regional, but not systemic recruitment/expansion of dendritic cells by a pluronic-formulated Flt3-ligand plasmid with vaccine adjuvant activity. Vaccine 21:3019-3029.[CrossRef][Medline]
    78. 77
  78. Sedegah, M., W. Weiss, J. B. Sacci, Jr., Y. Charoenvit, R. Hedstrom, K. Gowda, V. F. Majam, J. Tine, S. Kumar, P. Hobart, and S. L. Hoffman. 2000. Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus. J. Immunol. 164:5905-5912.[Abstract/Free Full Text]
    79. 78
  79. Serna, A., M. C. Ramirez, A. Soukhanova, and L. J. Sigal. 2003. Cutting edge: efficient MHC class I cross-presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells. J. Immunol. 171:5668-5672.[Abstract/Free Full Text]
    80. 79
  80. Slifka, M. K. 2005. The future of smallpox vaccination: is MVA the key? Med. Immunol. 4:2. [Online.] http://www.medimmunol.com/content/4/1/2.[CrossRef][Medline]
    81. 80
  81. Staib, C., S. Kisling, V. Erfle, and G. Sutter. 2005. Inactivation of the viral interleukin 1beta receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara. J. Gen. Virol. 86:1997-2006.[Abstract/Free Full Text]
    82. 81
  82. Stickl, H., V. Hochstein-Mintzel, A. Mayr, H. C. Huber, H. Schafer, and A. Holzner. 1974. MVA vaccination against smallpox: clinical tests with an attenuated live vaccinia virus strain (MVA). Dtsch. Med. Wochenschr. 99:2386-2392. (In German.)[Medline]
    83. 82
  83. Stittelaar, K. J., T. Kuiken, R. L. de Swart, G. van Amerongen, H. W. Vos, H. G. Niesters, P. van Schalkwijk, T. van der Kwast, L. S. Wyatt, B. Moss, and A. D. Osterhaus. 2001. Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine 19:3700-3709.[CrossRef][Medline]
    84. 83
  84. Stittelaar, K. J., G. van Amerongen, I. Kondova, T. Kuiken, R. F. van Lavieren, F. H. Pistoor, H. G. Niesters, G. van Doornum, B. A. van der Zeijst, L. Mateo, P. J. Chaplin, and A. D. Osterhaus. 2005. Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus. J. Virol. 79:7845-7851.[Abstract/Free Full Text]
    85. 84
  85. Stittelaar, K. J., L. S. Wyatt, R. L. de Swart, H. W. Vos, J. Groen, G. van Amerongen, R. S. van Binnendijk, S. Rozenblatt, B. Moss, and A. D. Osterhaus. 2000. Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies. J. Virol. 74:4236-4243.[Abstract/Free Full Text]
    86. 85
  86. Sutter, G., and B. Moss. 1992. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. USA 89:10847-10851.[Abstract/Free Full Text]
    87. 86
  87. Sutter, G., and C. Staib. 2003. Vaccinia vectors as candidate vaccines: the development of modified vaccinia virus Ankara for antigen delivery. Curr. Drug Targets Infect. Disord. 3:263-271.[CrossRef][Medline]
    88. 87
  88. Tanaka, Y., T. Imai, M. Baba, I. Ishikawa, M. Uehira, H. Nomiyama, and O. Yoshie. 1999. Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans. Eur. J. Immunol. 29:633-642.[CrossRef][Medline]
    89. 88
  89. Tarr, P. E., R. Lin, E. A. Mueller, J. M. Kovarik, M. Guillaume, and T. C. Jones. 1996. Evaluation of tolerability and antibody response after recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and a single dose of recombinant hepatitis B vaccine. Vaccine 14:1199-1204.[CrossRef][Medline]
    90. 89
  90. Tazawa, R., E. Hamano, T. Arai, H. Ohta, O. Ishimoto, K. Uchida, M. Watanabe, J. Saito, M. Takeshita, Y. Hirabayashi, I. Ishige, Y. Eishi, K. Hagiwara, M. Ebina, Y. Inoue, K. Nakata, and T. Nukiwa. 2005. Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis. Am. J. Respir. Crit. Care Med. 171:1142-1149.[Abstract/Free Full Text]
    91. 90
  91. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354.[Abstract/Free Full Text]
    92. 91
  92. Triozzi, P. L., and W. Aldrich. 1997. Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages. J. Leukoc. Biol. 61:600-608.[Abstract]
    93. 92
  93. Tscharke, D. C., G. Karupiah, J. Zhou, T. Palmore, K. R. Irvine, S. M. Haeryfar, S. Williams, J. Sidney, A. Sette, J. R. Bennink, and J. W. Yewdell. 2005. Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines. J. Exp. Med. 201:95-104.[Abstract/Free Full Text]
    94. 93
  94. Ullenhag, G. J., J. E. Frodin, S. Mosolits, S. Kiaii, M. Hassan, M. C. Bonnet, P. Moingeon, H. Mellstedt, and H. Rabbani. 2003. Immunization of colorectal carcinoma patients with a recombinant canarypox virus expressing the tumor antigen Ep-CAM/KSA (ALVAC-KSA) and granulocyte macrophage colony-stimulating factor induced a tumor-specific cellular immune response. Clin. Cancer Res. 9:2447-2456.[Abstract/Free Full Text]
    95. 94
  95. Vollmar, J., N. Arndtz, K. M. Eckl, T. Thomsen, B. Petzold, L. Mateo, B. Schlereth, A. Handley, L. King, V. Hulsemann, M. Tzatzaris, K. Merkl, N. Wulff, and P. Chaplin. 2005. Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine. Vaccine 24:2065-2070.[Medline]
    96. 95
  96. Weber, H. G. 1977. Utilization of the MVA-immunization schedule from the view point of the public health service. Fortschr. Med. 95:1381-1386. (In German.)[Medline]
    97. 96
  97. Weiss, W. R., K. J. Ishii, R. C. Hedstrom, M. Sedegah, M. Ichino, K. Barnhart, D. M. Klinman, and S. L. Hoffman. 1998. A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine. J. Immunol. 161:2325-2332.[Abstract/Free Full Text]
    98. 97
  98. Wyatt, L. S., M. W. Carroll, C. P. Czerny, M. Merchlinsky, J. R. Sisler, and B. Moss. 1998. Marker rescue of the host range restriction defects of modified vaccinia virus Ankara. Virology 251:334-342.[CrossRef][Medline]
    99. 98
  99. Wyatt, L. S., P. L. Earl, L. A. Eller, and B. Moss. 2004. Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. Proc. Natl. Acad. Sci. USA 101:4590-4595.[Abstract/Free Full Text]

Journal of Virology, August 2006, p. 7676-7687, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.02748-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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
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 arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chavan, R.
Right arrow Articles by Feinberg, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chavan, R.
Right arrow Articles by Feinberg, M. B.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»