JOURNAL OF VIROLOGY, June 2005, p. 7135–7145 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.11.7135–7145.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 11
Characterization of Human Immunodeficiency Virus Gag-Specific Gamma Interferon-Expressing Cells following Protective Mucosal Immunization with Alphavirus Replicon Particles Soumi Gupta,1 Ramesh Janani,2 Qian Bin,1 Paul Luciw,1 Catherine Greer,2 Silvia Perri,2 Harold Legg,2 John Donnelly,2 Susan Barnett,2 Derek O’Hagan,2 John M. Polo,2 and Michael Vajdy2* Department of Pathology and Center for Comparative Medicine, University of California, Davis,1 and Chiron Vaccines, 4560 Horton Street, Emeryville,2 California Received 10 November 2004/Accepted 23 January 2005
A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8ⴙ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed ␣47 or ␣E7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed ␣47 or CD62L than expressed ␣E7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues. can induce the best immune responses in the vaginal mucosa, the draining lymph nodes, and systemic tissues as well as providing protection in a vaginal challenge model. Traditionally, vaccines against bacterial or viral pathogens have been designed to produce neutralizing antibodies against the envelope proteins of pathogens. However, it has become increasingly evident that CD4 T-helper responses, and particularly CD8 T cells with cytotoxic activity, play a major role in conferring protection against many infectious diseases. Compared to available mucosal adjuvants and delivery systems used for gene-based vaccine candidates, mucosally applicable replication-defective viral delivery vectors that can also induce cellmediated protective responses in mucosal and systemic lymphoid tissues are attractive gene-based vaccine candidates. Alphaviruses, including Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE), are enveloped RNA viruses that have been developed into replication-defective “suicide” vectors (42, 45). Alphavirus replicon RNA vectors maintain the nonstructural protein gene and cis replication sequences required to drive abundant expression of heterologous antigens from the viral subgenomic 26S promoter but are devoid of any alphaviral structural protein genes required for propagation and spread. These vectors also offer the prospect of natural adjuvanticity and stimulation of the innate immune response, in addition to the antigen-specific adaptive response arising from the cytoplasmic amplification of these vectors through double-stranded RNA intermediates (29).
The vaginal mucosa serves as the portal of entry for human immunodeficiency virus (HIV) and several other clinically important pathogens. Vaginal and systemic cytotoxic T-lymphocyte (CTL) responses specific for simian immunodeficiency virus (SIV) or HIV have been demonstrated for nonhuman primates and humans, respectively (25, 30, 59). Thus, mucosal and systemic anti-HIV CTL may play an important role in the elimination of HIV-infected cells. However, a practical mucosal route of immunization that can confer protection in the genitourinary tract needs to be explored. Intranasal (i.n.) immunization induces local immunity, not only in the nasally associated lymphoid tissue and the lung but also in the female genital tract, in rodents (1, 14, 31, 53, 61) as well as humans and nonhuman primates (3, 20, 34, 44). Similarly, intravaginal (IVAG) and intrarectal immunization of rodents also induces local immunity in the genital tract. However, i.n. vaccination, which is more practical than vaginal vaccination, may induce sufficient anti-HIV protective immune responses in the genital tract to make it the immunization route of choice. Importantly, if HIV virions or HIV-infected cells that enter through the vaginal mucosa are not eliminated in situ, they may spread systemically through the draining lymphatics (32, 37, 54). It is therefore important to delineate which route of immunization * Corresponding author. Mailing address: Chiron Vaccines, 4560 Horton Street, M/S 4.3, Emeryville, CA 94608. Phone: (510) 923-8595. Fax: (510) 923-2586. E-mail:
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Replicon vectors have been widely evaluated as vaccine immunogens, both as plasmid DNA replicon vaccines and as viruslike replicon particles (46). In a previous study, we found that vaginal and rectal immunizations with Sindbis virus-based replicon particles encoding HIV Gag conferred protection against a vaginal challenge with a vaccinia virus expressing the same Gag antigen (VV-Gag). In this model, i.n. immunizations conferred partial protection, whereas intramuscular (i.m.) immunizations conferred no protection against the vaginal challenge (55). More recently, we found that VEE-derived and chimeric replicon particles, with VEE-derived replicon RNA and SIN-derived surface glycoproteins (VEE/SIN), expressing the HIV type 1 (HIV-1) Gag antigen induced significantly stronger systemic cell-mediated responses than previously described SIN replicon particles following systemic (i.m.) immunization with the vectors (39). The study reported here included the SIN-Gag, VEE-Gag, and VEE/SIN-Gag replicon particles and was designed to investigate four issues with regards to the immunogenicity of alphavirus delivery systems. First, the contribution of the alphavirus envelope glycoproteins, which dictate host cell tropism, to immunogenicity was evaluated. Second, the homing and chemokine receptors on intracellular gamma interferonexpressing cells (iIFNEC) were analyzed in mucosal and systemic lymphoid tissues. Third, a correlation was sought between the number of cells that expressed the encoded HIV Gag antigen following a mucosal or systemic immunization with each type of replicon particle and the ensuing T-cell responses after immunizations and a vaginal challenge. Finally, the protective efficacies of the different alphavirus-based replicon particles delivered through various mucosal and systemic immunization routes were determined. MATERIALS AND METHODS Preparation of alphavirus replicon particles. The VEE-Gag, VEE/SIN-Gag, and SIN-Gag replicon particles were prepared as described previously (39). Briefly, replicon particles were generated by cotransfection of in vitro-transcribed RNA species corresponding to a replicon and two defective helpers, with one helper expressing the capsid protein and the other expressing the envelope glycoproteins E2 and E1 (41). Titers of replicon particles on BHK-21 cells were determined as previously described (38). Replicon particles expressing HIV-1 p55gag were harvested as culture supernatants at 20 and 30 h posttransfection, clarified by filtration, and purified by cation-exchange chromatography. Replicon particle titers, reported in infectious units (IU) per ml, were determined by intracellular staining of the expressed Gag after an overnight infection of BHK-21 cells with serial dilutions of particles. Infected cells were permeabilized, fixed by use of a Cytofix/Cytoperm kit (Pharmingen), and then stained with fluorescein isothiocyanate (FITC)-conjugated antibodies to the HIV-1 core antigen (Coulter). Using flow cytometry analysis, we determined the percentages of Gag-positive cells and used them to calculate titers. The absence of contaminating replication-competent viruses was determined by five consecutive infections of naive BHK-21 cell monolayers and confirmation of the absence of PFU. Endotoxin levels were measured for all replicon particle samples and were shown to be ⬍0.5 endotoxin unit/ml. Immunizations and vaccinia virus challenge. Groups of five female BALB/c mice that were 6 to 8 weeks old at the onset of immunization were used for each vaccine or immunization route, and the tissues were pooled upon sacrifice. Immunizations were performed three times at 2-week intervals. The vaginal challenge was performed 6 weeks after the final immunization. i.m. immunizations were performed on mice under anesthesia with 2.5 ⫻ 106 IU of SIN-Gag, VEE/SIN-Gag, or VEE-Gag particles in 50 l of phosphate-buffered saline (PBS) injected into the right and left anterior tibialis muscles. i.n. immunizations were performed without anesthesia with 2.5 ⫻ 106 IU of SIN-Gag, VEE/SINGag, or VEE-Gag particles in 25 l of PBS. IVAG immunizations were performed with 107 IU of SIN-Gag, VEE/SIN-Gag, or VEE-Gag, and the IVAG
J. VIROL. challenge was performed with 108 PFU of VV-Gag. The replicon particles and the VV-Gag virus were applied IVAG to anesthetized mice in a volume of 12.5 l, after which the mice were kept in dorsal recumbency for 20 min. The mice were sacrificed 1 week following the final immunization or 5 days following VV-Gag challenge. A standard PFU assay was performed on pairs of ovaries, collected 5 days following the VV-Gag challenge, from a total of 10 mice per group. Student’s t test on paired samples for means was performed by using Microsoft Excel’s statistical analysis software program. Tissue collection and preparation of single-cell suspensions. Spleens (SP), iliac lymph nodes (ILN), and vaginal/uterine mucosae (VUM) were harvested and pooled from five mice per group. Single-cell suspensions were used for fluorescence-activated cell sorting (FACS) analysis. One week following the final immunization or 5 days following the IVAG challenge, SP, ILN, and VUM from groups of five immunized mice each were harvested and pooled. SP and ILN tissues were teased through a nylon mesh with a pore diameter of 250 m, washed three times in medium (ELISPOT assay medium, composed of RPMI containing 10% fetal calf serum, antibiotics, HEPES, and L-glutamine [complete RPMI]), counted, and seeded into wells. Single-cell suspensions were prepared from VUM by removing the entire vagina, uterus, and uterine horns from five mice per group as previously described (55). Uterine horns were cut longitudinally, and together with vaginal and uterine tissues, were diced into 5-mm pieces. The tissue pieces were washed three times in Hanks buffered salt solution (HBSS) without Ca2⫹ and Mg2⫹ containing 10% fetal calf serum and 5 mM HEPES (complete HBSS) and treated enzymatically with agitation at 37°C once with 1 mg/ml collagenase/dispase (Sigma) plus 0.5 mg/ml DNase (Roche Molecular Biochemicals, Indianapolis, Ind.) in 20 ml of complete HBSS for 30 min and twice with 800 U/ml collagenase (type XI; Sigma) plus 0.5 mg/ml DNase in 20 ml of complete RPMI for 45 min. Following each enzymatic treatment, the released cells were recovered and washed twice with complete RPMI. The cell suspensions from each enzymatic treatment were pooled and counted. This method routinely resulted in the recovery of a minimum of 107 mononuclear cells per five mice at a viability level of ⬎90%. Flow cytometric analysis. Seven days after the final immunization with the replicon particles or 5 days following the IVAG VV-Gag challenge, ILN, VUM, and SP were harvested from pools of five mice, and single-cell suspensions were prepared as described above. ILN and SP cells (1 ⫻ 106 to 2 ⫻ 106) or VUM cells (0.5 ⫻ 106 to 1 ⫻ 106) were cultured at 37°C in the presence or absence of 10 g of p7g peptide/ml or with monoclonal antibodies directed against CD3 and CD28 (both from Pharmingen, San Diego, Calif.) as positive controls. The HIV Gag p24-derived peptide p7g, used for the in vitro restimulation of cells from various tissues, has previously been shown to be the most immunodominant peptide from this protein (10). This peptide is nine amino acids long, with the sequence AMQMLKETI, and has been shown to specifically recognize major histocompatibility complex class I (MHC I)-restricted CD8⫹, but not CD4⫹, T cells (10, 55). BD GolgiPlug (Pharmingen, San Diego, Calif.) was added to block cytokine secretion. After 5 h, the cells were washed, incubated with Fc Block (anti-CD16/ 32; Pharmingen) to block Fc␥ receptors, and stained for cell surface antigens with a BD Cytofix/Cytoperm kit (Pharmingen) per the manufacturer’s instructions. The cells were then fixed in 2% (wt/vol) paraformaldehyde and stored overnight at 4°C. The following day, the cells were permeabilized and then stained for intracellular gamma interferon (IFN-␥) with a phycoerythrin (Pharmingen)-conjugated mouse anti-IFN-␥ monoclonal antibody in the presence of 0.1% (wt/vol) saponin. The cells were then washed and analyzed on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems). Table 1 lists the antibodies used for phenotypic and functional analysis of the cells. We tested the p7g peptide on cells from naı¨ve, VV-Gag-challenged mice, which had a response rate of 0.3% CD8⫹ iIFNEC (Fig. 1). Unstimulated splenic CD8⫹ iIFNEC from mice immunized i.m. with VEE/SIN-Gag and challenged IVAG with VV-Gag had a background level response of 0.03%, while p7g-stimulated spleen cells from the same mice responded with 2.31% CD8⫹ iIFNEC (Fig. 1). A response of a twofold increase above the background response of 0.3% was considered positive for CD8⫹ iIFNEC. For all flow cytometric analyses, the data are presented as means of two pools of five mice each plus standard deviations (SD). Data from one representative experiment of two with similar results are shown. Twofold or larger differences between the mean values of the groups were considered significant. Immunohistological staining. Mice were immunized once through the i.m., i.n., or IVAG route with SIN-Gag or VEE/SIN-Gag as described above. Sixteen hours later, the mice were sacrificed and ILN were carefully excised, embedded in OCT compound (Miles Inc., Elkhart, Ind.), and snap-frozen in 2-methylbutane (J. T. Baker, Phillipsburg, N.J.) that had been chilled on dry ice. Thin sections (7 m) were cut with a cryostat, transferred onto glass slides, and kept at ⫺80°C until staining. Before staining, the sections were fixed in ice-cold
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TABLE 1. Reagents used for flow cytometric analysis Antibody target
b
CD3 IFN-␥ ␣47 TCR CD103/␣E7 CD62L CD11b CD11c Gag SAv-FITC SAv-PE SAv-PerCP Avidin-APC a b
Fluorochrome
a
FITC, PerCp PE PE FITC, CYC Biotinylated Biotinylated, PE FITC, PE, APC PE, APC RD1 (PE or FITC) FITC PE PerCP APC
Clone
Vendor
Cat. No.
145-2C11 XMG1.2 DATK32 DATK32 H57-597 2E7 MEL-14 M1/70 HL3 KC57
Pharmingen Pharmingen Southern Biotechnology Associates Pharmingen Pharmingen eBioscience Pharmingen Pharmingen Pharmingen Coulter Pharmingen Pharmingen Pharmingen Pharmingen
553062 554412 1870-08 553811 553171 13-1031-85 553149 557396 557401 6604667 554060 554061 554064 554067
PerCP, peridinin chlorophyll protein; PE, phycoerythrin; APC, allophycocyanin. SAv, streptavidin.
acetone for 10 min. After extensive washes in PBS and blocking in blocking buffer (PBS–2% bovine serum albumin–5% rat serum) (Dako, Calif.) for 1 h, the sections were washed in PBS and stained with a FITC-conjugated anti-HIV-core (Gag) antibody (Table 1) at a 1:200 dilution in blocking buffer for 1 h in the dark at room temperature. The slides were then washed six to eight times with PBS. The stained sections on slides were then mounted in Fluorsave (Calbiochem, Calif.) and photographed with a digital camera linked to a fluorescent microscope with FITC and phycoerythrin filters. The immunostaining results shown are representative of at least two staining experiments using two serial sections from one ILN per mouse, with three mice per group.
RESULTS IFN-␥ responses by CD8ⴙ cells in mucosal inductive and effector sites and a systemic tissue. To compare the immunogenicities of the SIN-Gag, VEE-Gag, and VEE/SIN-Gag alphavirus-based replicon particles, we immunized mice i.n., i.m., or IVAG at 2-week intervals and then challenged them IVAG with VV-Gag 6 weeks later. Five days after the IVAG challenge, single-cell suspensions were prepared from VUM, ILN, and SP and restimulated with the Gag peptide p7g. The cells were stained for intracellular IFN-␥ and surface CD8 expression and analyzed by flow cytometry. Notably, in preliminary studies, i.m. or i.n. immunization with SIN-Gag, VEE/ SIN-Gag, or VEE-Gag, without a subsequent VV-Gag challenge, induced IFN-␥ responses in SP with no detectable responses in ILN or VUM, and VEE/SIN-Gag induced stronger responses than did SIN-Gag (data not shown). Moreover, IVAG immunizations without a VV-Gag challenge induced no detectable responses in any tissues (data not shown). In general, following i.m. or i.n. immunization with VEE/SIN-Gag and an IVAG challenge with VV-Gag, a larger number of Gag-specific CD8⫹ iIFNEC were detected in ILN, which drain the VUM and SP, than were observed for immunizations with SIN-Gag. i.m. immunizations with VEE/SIN-Gag or VEE-Gag followed by an IVAG challenge with VV-Gag induced a higher percentage of CD8⫹ iIFNEC in SP and ILN than did i.m. immunizations with SIN-Gag followed by an IVAG challenge with VV-Gag (Fig. 2A). For i.n. immunizations followed by an IVAG challenge with VV-Gag, responses were generally lower overall than for i.m. immunizations, but VEE/SIN-Gag appeared to induce a modestly higher percentage of CD8⫹ iIFNEC per tissue in ILN than that induced by i.n. immuniza-
tions with VEE-Gag or SIN-Gag followed by IVAG challenge with VV-Gag (Fig. 2B). IVAG immunizations with VEE/SINGag, VEE-Gag, or SIN-Gag followed by an IVAG challenge with VV-Gag also induced generally lower numbers of CD8⫹ iIFNEC than did i.m. immunizations, and the responses were similar among the groups, except for an increase in the ILN of the VEE-immunized groups (Fig. 2C). Notably, the vaginal challenge of naı¨ve mice with VV-Gag induced only background-level responses (Fig. 1B). These data suggest that, particularly for i.m. and i.n. immunizations, the alphavirus-based replicon particles containing the VEE replicon RNA, i.e., VEE/SIN-Gag and VEE-Gag, induced larger numbers of CD8⫹ iIFNEC than did similar immunizations with SIN-Gag in local and/or systemic lymphoid tissues. Surface expression of homing receptors by iIFNEC. Because lymphocyte trafficking to and from mucosal tissues plays an important role in the generation of mucosal and systemic immune responses, we determined the surface expression of the mucosal lamina propria homing receptor, ␣47, the peripheral homing receptor, CD62L, and the intraepithelial retention receptor, ␣E7, on the iIFNEC after various routes of immunization with each replicon particle type followed by an IVAG challenge with VV-Gag. The phenotypes of the cells were similar regardless of the replicon particle type, and therefore only the data generated following immunizations with VEE/ SIN-Gag are presented. In VUM, regardless of the route of immunization, a relatively high percentage of iIFNEC expressed ␣47, followed by ␣E7, with a relatively very low percentage expressing CD62L (Fig. 3). In contrast, in SP, regardless of the route of immunization, the highest percentage of iIFNEC expressed ␣47, followed by CD62L, with a relatively low percentage of iIFNEC expressing ␣E7 (Fig. 3). In ILN, regardless of the route of immunization, relatively high and similar percentages of all three homing receptors were expressed by the iIFNEC (Fig. 3). Thus, in general, while the expression patterns of the homing receptors differed among the tissues analyzed, the expression patterns did not appear to vary based on the route of immunization or the type of replicon particle. However, relatively higher percentages of IFNEC expressing ␣E7 were detected in ILN following i.n. or IVAG immunizations with VEE/SIN than were seen after i.n. or
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FIG. 1. CD8⫹ IFN-␥-secreting cells in SP. Groups of five female BALB/c mice were immunized with VEE/SIN or PBS three times through the i.m. route and then were challenged IVAG with VV-Gag. Single-cell suspensions from the various SP were pooled from five immunized/challenged mice restimulated with medium alone (A) or from naı¨ve mice challenged IVAG with VV-Gag and restimulated with p7g (B) or the HIV Gag peptide p7g, representing a single MHC I-restricted epitope (C). The cells were analyzed by intracellular staining for IFN-␥ and surface staining for CD8. The data are presented as mean total percentages of CD8⫹ IFN-␥⫹ cells from one pool of five mice. The data are representative of four similar results from two independent experiments and from various tissues.
IVAG immunizations with SIN (Fig. 4). Because CD62L has been shown to be proteolytically cleaved on antigen-activated cells, Fig. 5 demonstrates representative FACS plots for various tissues differentially expressing CD62L compared to ␣E7
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FIG. 2. CD8⫹ intracellular IFN-␥-expressing cells (iIFNEC) in VUM, ILN, and SP. Groups of five female BALB/c mice were immunized with VEE, VEE/SIN, or SIN three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-␥ and surface staining for CD8. The data are presented as mean total percentages of CD8⫹ IFN-␥⫹ cells ⫾ SD of four pools of five mice each from two independent experiments.
or ␣47. Thus, while iIFNEC from spleens and ILN express CD62L, VUM cells had very low expression of this marker. Collectively, these data show that following mucosal or systemic routes of immunization and an IVAG challenge with VV-Gag, similar percentages of iIFNEC in the mucosal effector site (VUM), the mucosal inductive site (ILN), and the distant systemic lymphoid tissue (SP) express the mucosal homing receptor ␣47. Surface expression of the chemokine receptor CCR5 by iIFNEC. Chemokines have been shown to induce an influx of cells to sites of infection (4, 6–8, 18, 51, 58). Therefore, the expression of the chemokine receptor CCR5 on iIFNEC was examined. Regardless of the route of immunization, a rela-
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FIG. 3. Expression of mucosal and peripheral homing receptors of IFN-␥⫹ cells in VUM, ILN, and SP following immunizations with VEE/SIN-Gag. Groups of five female BALB/c mice were immunized with VEE/SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing an MHC I-restricted single epitope, and analyzed by intracellular staining for IFN-␥ and surface staining for ␣47, ␣E7, or CD62L. The data are presented as mean total percentages of iIFNEC cells coexpressing ␣47, ␣E7, or CD62L ⫾ SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.
FIG. 4. Surface expression of ␣E7 by iIFNEC in VUM, ILN, and SP following immunizations with VEE-Gag, VEE/SIN-Gag, or SINGag. Groups of five female BALB/c mice were immunized with VEEGag, VEE/SIN-Gag, or SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-␥ and surface staining for ␣E7. The data are presented as mean total percentages of iIFNEC cells per tissue coexpressing ␣E7 ⫾ SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.
tively high percentage of CCR5⫹ iIFNEC was detected in ILN, whereas a relatively low percentage of CCR5⫹ iIFNEC was detected in SP (Fig. 6). An intermediate percentage level of CCR5⫹ iIFNEC was detected in VUM. This observation was possibly due to the differential percentages of total CCR5⫹ cells in these tissues, which were 50% lower for the SP than for the VUM or ILN (data not shown). Thus, differential expression of CCR5 by iIFNEC was observed in ILN depending on the route of immunization and the replicon particle type administered, with VEE/SIN-Gag inducing the highest percentages of iIFNEC-expressed CCR5 following i.n. immunizations (Fig. 6).
Gag-expressing cells in ILN following a single immunization with VEE/SIN-Gag compared to those after immunization with SIN-Gag. The immune response by CD8⫹ T cells is initiated by antigen-presenting cells (APC) taking up the antigen and presenting it to T cells. Therefore, comparisons were made of the ability of VEE/SIN-Gag and SIN-Gag replicon particles to enter local APC and express the Gag antigen. To this end, mice were immunized with a single i.m., i.n., or IVAG dose of VEE/SIN-Gag or SIN-Gag, and 1 day later the ILN were removed, freeze-embedded, and sectioned for immunohistological staining. VEE/SIN-Gag given i.n. or IVAG induced
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FIG. 5. Flow cytometric plots demonstrating expression of mucosal and peripheral homing receptors of IFN-␥⫹ cells in VUM, ILN, and SP following immunizations with VEE/SIN-Gag. Groups of five female BALB/c mice were immunized with VEE/SIN-Gag three times through the i.n. route and then challenged IVAG with VV-Gag. Single-cell suspensions from the VUM (A, D, and G), ILN (B, E, and H), and SP (C, F, and I) were pooled from five mice per group, restimulated with an HIV Gag peptide representing an MHC I-restricted single epitope, and analyzed by intracellular staining for IFN-␥ and surface staining for CD62L (A, B, and C), ␣E7 (D, E, and F), and ␣47 (G, H, and I). The data are presented as mean total percentages of iIFNEC cells per tissue coexpressing ␣47, ␣E7, or CD62L ⫾ SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.
severalfold larger numbers of Gag-expressing cells in the ILN than did SIN-Gag (Fig. 7 and 8). In contrast, i.m. immunization with VEE/SIN-Gag or SIN-Gag induced relatively small and similar numbers of Gag-expressing cells in ILN (Fig. 7 and 8). Flow cytometric analyses of CD11b⫹ and CD11c⫹ cells revealed that the ratio of CD11b⫹ to CD11c⫹ cells in both VUM and ILN was approximately 3:1 (Fig. 9). It was particularly noteworthy that the percentages of CD11b⫹ and CD11c⫹ cells were four and three times higher, respectively, in VUM than in
ILN (Fig. 9). These data show that the vaginal mucosa contains large numbers of dendritic cells or macrophages with the potential to take up and deliver the replicon particles to ILN. Furthermore, the data showed larger numbers of Gag-expressing cells in ILN following a mucosal immunization with VEE/ SIN-Gag than after immunization with SIN-Gag. Protective responses against vaginal challenge with vaccinia virus. The protective efficacies of the replicon particles administered through various routes of immunization were deter-
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FIG. 7. Gag-expressing cells in ILN following immunization with VEE/SIN-Gag or SIN-Gag. Groups of three female BALB/c mice were immunized i.m., i.n., or IVAG with a single dose of VEE/SINGag or SIN-Gag. After 16 h, ILN were removed and snap-frozen. Immunohistological staining was then performed on 7-m thin sections with a FITC-conjugated anti-HIV Gag antigen. The data are presented as mean numbers of Gag-expressing cells per high-power field ⫾ SD for three mice per group.
Gag and VEE/SIN-Gag induced the best protective responses. These results show that VEE/SIN-Gag induced stronger protective responses than SIN-Gag through any route of immunization (significantly stronger for i.m. immunization) and that mucosal immunizations induced better protection against vaginal challenge than systemic immunizations. DISCUSSION
FIG. 6. Surface expression of CCR5 by iIFNEC in VUM, ILN, and SP following immunizations with VEE-Gag, VEE/SIN-Gag, or SINGag. Groups of five female BALB/c mice were immunized with VEEGag, VEE/SIN-Gag, or SIN-Gag three times through the i.m. (A), i.n. (B), or IVAG (C) route and then challenged IVAG with VV-Gag. Single-cell suspensions from the various tissues were pooled from five mice per group, restimulated with an HIV Gag peptide representing a single MHC I-restricted epitope, and analyzed by intracellular staining for IFN-␥ and surface staining for CCR5. The data are presented as mean total percentages of iIFNEC cells coexpressing CCR5 ⫾ SD of two pools of five mice each from one experiment. Data from one representative experiment of two with similar results are shown.
mined. Mice were immunized three times through the i.n., i.m., or IVAG route and challenged IVAG with VV-Gag 6 weeks later. Five days following the challenge, the ovaries were removed for use in a PFU assay. Statistically significant differences were observed between the various immunization routes and the type of replicon particle used. In general, immunizations with SIN-Gag induced the least protective responses compared to VEE-Gag and VEE/SIN-Gag through any route of immunization. Moreover, i.m. versus i.n. or IVAG immunizations with SIN-Gag induced the least protective responses (Fig. 10). In contrast, IVAG and i.n. immunizations with VEE-
This study addressed several important questions about the mechanisms of vaccine-induced immunity in response to alphavirus-based replicon particles. We first determined whether the surface envelope glycoproteins, which dictate the receptor tropism of the particles, rather than properties of the replicon RNA expression vector packaged within the particles, played a role in the stimulation of IFN-␥ immune responses following mucosal versus systemic immunizations. We recently reported that SIN- and VEE-derived replicon particles and chimeras between the two induced different responses following systemic (i.m.) immunizations, in that the replicon particles containing VEE replicon RNA induced stronger cell-mediated responses to an expressed HIV-1 Gag antigen regardless of whether the envelope glycoproteins used for packaging were derived from VEE or SIN (39). However, the question of whether a similar observation would hold true for mucosal immunizations remained unanswered. With the current study, we confirmed the previously published data for i.m. immunizations. Furthermore, we found that mucosal (i.n. or IVAG) immunizations alone with all three replicon particles induced weak cellular responses that were significantly boosted after IVAG challenge with a vaccinia virus vector expressing the same antigen. Under these conditions, the largest difference in immunogenicity among the three replicon particles was detected after i.m. immunizations, whereas after i.n., and particularly, IVAG immunizations, this difference was not pronounced. Notably, the percentages of CD8⫹ IFNEC in Fig. 1 represent the percentages of IFNEC that expressed CD8 per tissue (e.g., for i.m. immunization with VEE/SIN, the percent-
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FIG. 9. Relative percentages of CD11b⫹ and CD11c⫹ monocytelineage cells in VUM and ILN. Single-cell suspensions from the various tissues were pooled from five female BALB/c mice per group and then analyzed for surface expression of CD11c or CD11b by threecolor flow cytometry. The data are presented as mean relative percentages of CD11b⫹ or CD11c⫹ cells ⫾ SD for two experiments.
data presentation format, the differences between the groups and tissues remained similar. The envelope glycoprotein structure dictates the affinity and tropism of microorganisms for host cell receptors. In this study, we demonstrated that the origin of the viral envelope glycoproteins in the replicon particles studied was less important for immunogenicity and protection than the nature of the replicon RNA following mucosal and systemic immunizations. This result may be due to several factors. We previously showed that higher HIV Gag expression resulted from replicon particles
FIG. 8. Representative photomicrographs of Gag-expressing cells in ILN following immunization with VEE/SIN-Gag or SIN-Gag. Groups of three female BALB/c mice were immunized i.n. with a single dose of VEE/SIN-Gag (A) or SIN-Gag (B). After 16 h, ILN were removed and snap-frozen. Immunohistological staining was then performed on 7-m thin sections with a FITC-conjugated anti-HIV Gag antigen or with a control antibody from the same species but with a different specificity (C).
age of IFNEC in the SP was 1.9%). The percentages of CD8⫹ IFNEC relevant to total CD8⫹ cells per tissue were severalfold higher (e.g., for i.m. immunization with VEE/SIN, the percentage of IFNEC in the SP was 14%). However, regardless of the
FIG. 10. Protective efficacy of alphavirus-based replicon particles against IVAG challenge with VV-Gag. Two groups of five female BALB/c mice were immunized with VEE, VEE/SIN, or SIN three times through the i.m., i.n., or IVAG route and then challenged IVAG with VV-Gag. Five days after the challenge, the ovaries were removed and a PFU assay was performed. The data are presented as PFU from pairs of ovaries from individual mice, with horizontal bars representing mean PFU per pair of ovaries from 5 (naı¨ve group) or 10 (all other groups) mice per group. The numbers on the x axis show the numbers of mice with no PFU, i.e., protected mice. The statistical significance between groups is shown by the P values on dotted lines between the groups.
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containing VEE replicon RNA and that this expression difference was not due to a higher RNA replication rate or transcription, but possibly to factors playing a role at the level of translation (39). Another possibility may be the sensitivity of replicon RNA to IFN-␣/ (reviewed in reference 24). Our previous results suggested that replicon particles containing SIN-derived RNA are more sensitive to IFN-␣/ than particles containing VEE-derived RNA, and the origin of envelope glycoproteins did not significantly affect the sensitivities of these vectors (39). Another important aim for this study was to characterize the antigen-specific IFN-␥-secreting cells induced in mucosal and systemic lymphoid tissues, particularly with regards to their homing receptors. Although several studies have reported the characterization of CD4⫹ T cells in the murine female genital mucosa, reports on the characterization of CD8⫹ T cells at this site are more limited (12, 17, 26, 35, 36, 40, 48). ␣47 has been implicated in CD8⫹ T cell homing to the intestinal mucosa in protection against rotavirus (43), and CD8⫹ cells have been shown to increase in size in the vagina following infection with herpes simplex virus type 2 (HSV-2), particularly after immunization (36). The phenotypic characterization of the CD8⫹ IFNEC in the mucosal effector and inductive sites as well as in a distant systemic lymphoid tissue in our study revealed interesting information about the homing patterns. Our data showed that, regardless of the route of immunization, after IVAG challenge with VV-Gag relatively few antigen-specific CD62L⫹ iIFNEC were induced in VUM, although such cells were readily found in ILN and SP, suggesting that they may not originate in VUM. However, although we have no functional data demonstrating a direct involvement of ␣47 in homing from VUM to ILN or SP, ␣47⫹ iIFNEC were found in relatively large and comparable numbers. Although evidence for the transient expression of MadCAM-1 (the ligand for ␣47) in Chlamydia-infected vaginal mucosa (26) has been shown, most evidence points to a lack of expression of MadCAM-1 but a positive expression of E-cadherin (the ligand for ␣E7) in the vaginal uterine mucosa (22, 28, 47). Thus, whether ␣47 is the homing receptor involved in trafficking of cells to and from the vaginal uterine mucosa needs more direct evidence, such as the use of ␣47 gene knockout mice. Our data also showed that the frequency of iIFNEC expressing ␣E7 in ILN correlated with the better immunogenicity of VEE/SIN or VEE than that of SIN following i.n. and IVAG, but not i.m., immunizations. Notably, the percentages of IFNEC expressing various homing receptors shown in Fig. 2 represent the percentages of IFNEC that expressed a given homing receptor per tissue (e.g., for i.m. immunization, the percentage of IFNEC expressing ␣47 in the SP was 2%). The percentage of IFNEC expressing a given homing receptor relevant to total IFNEC cells per tissue was severalfold higher (e.g., for i.m. immunization, the percentage of IFNEC expressing ␣47 in the SP was 49%). However, regardless of the data representation format, the differences between the groups and tissues remained similar. Our data are consistent with other reports of the recruitment of 7⫹ CD4⫹ T cells to the vaginal mucosa following vaginal infection with the intracellular bacterium Chlamydia trachomatis (17, 26), although another report argues that ␣L2 and ␣41 are important homing receptors expressed on T cells in the vaginal
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mucosae of mice infected with this pathogen (40). Importantly, the low CD62L and high ␣E7 expression levels on vaginal T cells have also been reported for nonhuman primates (50). The majority of the antigen-specific iIFNEC expressed Tcell receptor ␣ (TCR␣) (data not shown). This was expected because the Gag-derived peptide used for the restimulation of the cell cultures is an MHC I-restricted peptide that has been shown to only stimulate CD8⫹ T cells (10). It is also well known that TCR␣ is expressed on T cells during the presence of antigen that occurs during the restimulation of cell cultures (27). T cells with uncommon phenotypes have been reported as present in the genitourinary tract (21, 23). Thus, TCR␥␦⫹ CD8⫹ or CD8⫺ T cells are found in the mucosal effector sites of the murine genitourinary tract (23). In this regard, protection against genital herpes simplex virus type 2 (HSV-2) in mice has been shown to be independent of TCR␥␦ cells (33). However, because TCR␥␦ cells are known to have a restricted response to antigenic epitopes, it is unlikely that they would have participated in the adaptive, antigen-specific phase of the response in our study. This surmise does not preclude the possibility that the TCR␥␦ T cells participate in the innate phase of the immune response, following either the immunizations with replicon particles or the IVAG challenge with VV-Gag. The finding that the iIFNEC in the mucosal effector and inductive sites were CCR5⫹ suggests that such cells move toward a chemokine gradient that is generated locally at these sites. Our data are supported by a report in which coexpression of CCR5 and interleukin-2 (IL-2) was found in human genital but not systemic (blood) T cells (19). Moreover, nonhuman primates have larger numbers of vaginal CD4⫹ T cells expressing CCR5 than does peripheral blood (57). The presence of RANTES, which binds CCR5⫹ cells, has been demonstrated in mice that were treated with CpG and protected against a vaginal HSV-2 challenge (16). Moreover, an increased secretion of macrophage inflammatory protein 1␣, which also binds CCR5, in the murine genital tract following infection with Chlamydia trachomatis has been associated with increased TH1 activity (9). We also sought to find a correlation between the number of cells that expressed the gene of interest, that for HIV Gag, following a mucosal or systemic immunization with each of the replicon particle types and the ensuing T-cell responses and protection. It has become increasingly evident that antigenpresenting cells, and in particular dendritic cells (DC), play an important role in the initiation of adaptive cellular responses (5, 15, 49, 52). Therefore, for the present study, the major APC populations of the VUM and ILN were characterized. Although several studies have reported the presence of various populations of DC in the vaginal mucosa or in ILN, to our knowledge there has been no report of a comparison of the relative percentages of the various DC and macrophage populations in VUM and ILN. It is important to note that our procedure for the preparation of single-cell populations from VUM yielded both intraepithelial and submucosal (lamina propria) cells. We found severalfold larger numbers of CD11b⫹ and CD11c⫹ cells in VUM than in ILN, suggesting that the vaginal mucosal portal of virus entry has large numbers of potential antigen-presenting cells. In this regard, it has been shown that protection against the genital tract pathogen
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Chlamydia trachomatis is associated with the recruitment of CD45⫹ CD86⫹ CD40⫹ MHC II⫹ antigen-presenting cells into the uterine tissue (48). In the current study, VEE/SIN-Gag given i.n. or IVAG, but not i.m., induced severalfold larger numbers of Gag-expressing cells in ILN than did SIN-Gag. This correlated with the larger number of iIFNEC expressing ␣E7 in ILN following i.n. or IVAG, but not i.m., immunization with VEE/SIN-Gag than that after immunization with SIN-Gag. This may be due to the possibility that while both SIN-Gag and VEE/SIN-Gag have the same envelope glycoproteins and most likely enter APC in similar numbers, the VEE replicon RNA in VEE/SIN-Gag is better able to express the encoded antigen. This appears to be due to factors at the stage of translation, since a previous study demonstrated similar mRNA expression levels in cells infected with VEE/SIN or SIN replicon particles (39). The protective immune responses supported the cellular responses in that there was a statistically significant difference in the protective responses induced by VEE/SIN-Gag compared to those induced by SIN-Gag. The overall PFU titers were lower than what is expected following an intraperitoneal challenge with vaccinia virus. It is important that because we were interested in characterizing the antigen-specific T cells, we did not subject the mice to hormonal (progesterone) treatment to regulate their estrous cycle, because such treatments alter the cellular composition of various organs and also interfere with the induction of protective mucosal responses (11–13, 60). Previously, we reported that SIN replicon particles encoding HIV Gag given intrarectally or IVAG, but not i.n. or i.m., were protective following a vaginal or rectal challenge with VV-Gag. Similar to the results of the previous study, the IVAG and i.n. routes of immunization were more effective than the i.m. route of immunization with SIN-Gag for inducing protective responses against vaginal challenge with VV-Gag, while the VEE/SIN-Gag chimera afforded even greater protection by these routes. Thus, regardless of the type of replicon particle used, the i.n. and IVAG routes of immunization were more protective than the i.m. route of immunization against a vaginal challenge with the virus. Protection against a rectal challenge with vaccinia virus expressing an HIV Env peptide has been shown to be mediated by antigen-specific CD8⫹ IFN-␥secreting cells (2). However, in the current, as well as our previous, study (55), we did not find a correlation between the number of peptide-specific IFN-␥-secreting cells and protection from a vaginal challenge with VV-Gag. Interestingly, though, it was shown for IFN-␥-deficient mice that protection from intraperitoneal infection with vaccinia virus appeared not to be dependent on IFN-␥ (56). The higher frequency of Gag⫹ cells in ILN 24 h after a mucosal immunization than that after an i.m. immunization did not correlate with higher iIFNEC numbers but did correlate with better protection following mucosal immunization. The i.m. route of immunization induced small numbers of Gag⫹ cells in ILN but large numbers of iIFNEC and was less effective at protection against vaginal challenge. Because we performed immunohistological analysis 1 day after a mucosal or an i.m. immunization, we did not address the length of time it takes for the replicon particles to reach the ILN after one or three i.m. immunizations, as was done for the immunogenicity and protection studies. As for the discrepancy between immu-
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nogenicity and protection, we reason that factors other than IFN-␥ may play a role in protection. Thus, while the aim of this study was not to analyze the correlates of protection against VV-Gag in mice, an elucidation of the correlates of protection against vaccinia virus will need further clarification. ACKNOWLEDGMENTS This study was supported by NIH R21 grant 1 R21 AI50430-01, by NIH IPCAVD grant 1 U19 AI51596, and by the Chiron Corporation. We thank Gillis Otten for a critical reading of the paper and Nelle Cronen for expert assistance with the illustrations. REFERENCES 1. Asanuma, H., C. Aizawa, T. Kurata, and S. Tamura. 1998. IgA antibodyforming cell responses in the nasal-associated lymphoid tissue of mice vaccinated by intranasal, intravenous and/or subcutaneous administration. Vaccine 16:1257–1262. 2. Belyakov, I. M., J. D. Ahlers, B. Y. Brandwein, P. Earl, B. L. Kelsall, B. Moss, W. Strober, and J. A. Berzofsky. 1998. The importance of local mucosal HIV-specific CD8(⫹) cytotoxic T lymphocytes for resistance to mucosal viral transmission in mice and enhancement of resistance by local administration of IL-12. J. Clin. Investig. 102:2072–2081. 3. Bergquist, C., E. L. Johansson, T. Lagergard, J. Holmgren, and A. Rudin. 1997. Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina. Infect. Immun. 65:2676–2684. 4. Campbell, D. J., G. F. Debes, B. Johnston, E. Wilson, and E. C. Butcher. 2003. Targeting T cell responses by selective chemokine receptor expression. Semin. Immunol. 15:277–286. 5. Castellano, G., A. M. Woltman, F. P. Schena, A. Roos, M. R. Daha, and C. van Kooten. 2004. Dendritic cells and complement: at the cross road of innate and adaptive immunity. Mol. Immunol. 41:133–140. 6. Cyster, J. G. 1999. Chemokines and cell migration in secondary lymphoid organs. Science 286:2098–2102. 7. Cyster, J. G. 2003. Lymphoid organ development and cell migration. Immunol. Rev. 195:5–14. 8. D’Ambrosio, D., P. Panina-Bordignon, and F. Sinigaglia. 2003. Chemokine receptors in inflammation: an overview. J. Immunol. Methods 273:3–13. 9. Darville, T., C. W. Andrews, Jr., J. D. Sikes, P. L. Fraley, L. Braswell, and R. G. Rank. 2001. Mouse strain-dependent chemokine regulation of the genital tract T helper cell type 1 immune response. Infect. Immun. 69:7419– 7424. 10. Doe, B., and C. M. Walker. 1996. HIV-1 p24 Gag-specific cytotoxic Tlymphocyte responses in mice. AIDS 10:793–794. 11. Gallichan, W. S., and K. L. Rosenthal. 1996. Effects of the estrous cycle on local humoral immune responses and protection of intranasally immunized female mice against herpes simplex virus type 2 infection in the genital tract. Virology 224:487–497. 12. Ghaleb, M., M. Hamad, and K. H. Abu-Elteen. 2003. Vaginal T lymphocyte population kinetics during experimental vaginal candidosis: evidence for a possible role of CD8⫹ T cells in protection against vaginal candidosis. Clin. Exp. Immunol. 131:26–33. 13. Gillgrass, A. E., A. A. Ashkar, K. L. Rosenthal, and C. Kaushic. 2003. Prolonged exposure to progesterone prevents induction of protective mucosal responses following intravaginal immunization with attenuated herpes simplex virus type 2. J. Virol. 77:9845–9851. 14. Giuliani, M. M., G. Del Giudice, V. Giannelli, G. Dougan, G. Douce, R. Rappuoli, and M. Pizza. 1998. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. J. Exp. Med. 187:1123– 1132. 15. Granucci, F., I. Zanoni, S. Feau, G. Capuano, and P. Ricciardi-Castagnoli. 2004. The regulatory role of dendritic cells in the immune response. Int. Arch. Allergy Immunol. 134:179–185. 16. Harandi, A. M., K. Eriksson, and J. Holmgren. 2003. A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection. J. Virol. 77:953–962. 17. Hawkins, R. A., R. G. Rank, and K. A. Kelly. 2000. Expression of mucosal homing receptor alpha4beta7 is associated with enhanced migration to the Chlamydia-infected murine genital mucosa in vivo. Infect. Immun. 68:5587– 5594. 18. Heeney, J. L., V. J. Teeuwsen, M. van Gils, W. M. Bogers, C. De Giuli Morghen, A. Radaelli, S. Barnett, B. Morein, L. Akerblom, Y. Wang, T. Lehner, and D. Davis. 1998. Beta-chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques. Proc. Natl. Acad. Sci. USA 95:10803–10808. 19. Hladik, F., G. Lentz, E. Delpit, A. McElroy, and M. J. McElrath. 1999. Coexpression of CCR5 and IL-2 in human genital but not blood T cells:
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