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Journal of Virology, November 2007, p. 12535-12542, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00197-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Recombinant Sendai Virus Is Controlled by CD4⁺ Effector T Cells Responding to a Secreted Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Scott A. Brown,^1,2* Julia L. Hurwitz,^1,2,3 Amy Zirkel,² Sherri Surman,^1,2 Toru Takimoto,^2,5 Irina Alymova,² Chris Coleclough,^1,3,7 Allen Portner,^2,3 Peter C. Doherty,^1,6 and Karen S. Slobod^1,4

Departments of Immunology,¹ Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee,² Departments of Pathology,³ Pediatrics, University of Tennessee, Memphis, Tennessee,⁴ Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 672, Rochester, New York 14642,⁵ Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia,⁶ Silver Bullet Biology, Memphis, Tennessee⁷

Received 29 January 2007/ Accepted 19 June 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The importance of antigen-specific CD4⁺ helper T cells in virus infections is well recognized, but their possible role as direct mediators of virus clearance is less well characterized. Here we describe a recombinant Sendai virus strategy for probing the effector role(s) of CD4⁺ T cells. Mice were vaccinated with DNA and vaccinia virus recombinant vectors encoding a secreted human immunodeficiency virus type 1 (HIV-1) envelope protein and then challenged with a Sendai virus carrying a homologous HIV-1 envelope gene. The primed mice showed (i) prompt homing of numerous envelope-primed CD4⁺ T cell populations to the virus-infected lung, (ii) substantial production of gamma interferon, and interleukin-2 (IL-2), IL-4, and IL-5 in that site, and (iii) significantly reduced pulmonary viral load. The challenge experiments were repeated with immunoglobulin^–/– µMT mice in the presence or absence of CD8⁺ and/or CD4⁺ T cells. These selectively immunodeficient mice were protected by primed CD4⁺ T cells in the absence of antibody or CD8⁺ T cells. Together, these results highlight the role of CD4⁺ T cells as direct effectors in vivo and, because this protocol gives such a potent response, identify an outstanding experimental model for further dissecting CD4⁺ T-cell-mediated immunity in the lung.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antigen-specific CD4⁺ T cells play important but varied roles in experimental models of viral immunity. In every case, CD4⁺ T-cell help is required to promote high-quality antibody production and both B-cell/plasma cell and CD8⁺ T-cell memory. With some pathogens, particularly intracellular bacteria (25) and large DNA viruses like the herpesviruses, gamma interferon (IFN-)-producing CD4⁺ T-cell effectors play a major role in the direct control of the infectious process (11). On the other hand, following respiratory infection with the influenza A viruses, the CD4⁺ T-cell response promotes virus clearance primarily via T-cell help for antibody production (14, 30).

For reasons that are not well understood, many virus-specific CD4⁺ T-cell responses seem to be focused principally on epitopes (major histocompatibility complex class II [MHC-II] protein plus peptide) derived from glycoproteins that are normally expressed both on the surface of the virion and on infected cells. Our previous studies have examined the responses of T cells to human immunodeficiency virus type 1 (HIV-1) envelope proteins (9, 10, 34). We found that vigorous T-cell responses could be elicited in mice by successive immunizations with recombinant DNA and recombinant vaccinia virus vectors, each expressing gp140 envelope proteins. These potent CD4⁺ T-cell activities were generated even though the expressed HIV-1 envelope proteins lacked membrane regions and were therefore not expressed on cell surfaces. Further experimentation showed that dominant epitopes were often located in regions of the gp120 protein that overlapped with targets of neutralizing antibodies. However, analyses with immunoglobulin^–/– (Ig^–/–) µMT mice showed that the specificity profiles were in no way related to any effect on antigen processing mediated by antibody binding (10).

The focused and robust nature of the HIV-1 envelope-specific T-cell response in this system provided an attractive platform for testing CD4⁺ T-cell contributions to protective immunity. As there is no mouse model for HIV-1, we took advantage of the fact that it is possible to engineer the coding sequence for the secreted HIV-1 envelope gp120 protein into Sendai virus. The resultant Sendai virus particle lacks envelope protein (avoiding antibody-mediated clearance) but mediates expression of the soluble protein by virus-infected cells. The basic experimental protocol thus relied on the use of three HIV-1 envelope recombinant vectors; we immunized with the recombinant DNA-vaccinia virus prime-boost regimen to see if this would protect against respiratory challenge with the recombinant Sendai virus. This system was particularly attractive for testing the memory CD4⁺ T cells in that there was no possibility of an ancillary, antibody-related effect resulting from the presence of the antigen on the surface of either the virus or the infected cell. Experimental results showed that the priming regimen elicited envelope-specific CD4⁺ T-cell memory that, on recall following respiratory challenge with the Sendai virus envelope recombinant, mediated rapid control of the infection in the absence of both antibody and CD8⁺ T-cell-mediated effector functions.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice. Female C57BL/6J (B6; H2^b) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and Ig^–/– µMT mice on a B6 background were bred at St. Jude Children's Research Hospital (SJCRH). Both sets of animals were housed under specific pathogen-free conditions in a biosafety level 1, 2, or 3 containment area at the SJCRH animal facility, as specified by the Association for Assessment and Accreditation for Laboratory Animal Care (AAALAC) guidelines. At the time of live virus challenge, mice anesthetized with tribromoethanol (Avertin) were infected intranasally (i.n.) with 10⁶ PFU (for C57BL/6 mice) or 10⁵ PFU (for Ig^–/– µMT mice) of recombinant Sendai virus (see below). All mice were approximately 2 months of age at the initiation of the immunization protocols. The lack of B cells in µMT mice was confirmed by FACScalibur analysis with a B220-specific antibody. Data analyses with BD Cell Quest Pro (Becton Dickinson, Franklin Lakes, NJ) showed that B cells represented <0.5% of the lymphocyte population in these animals.

Immunogens and immunization. Mice were immunized as described previously (9, 10, 34) with a recombinant DNA vector expressing HIV-1 envelope protein from a primary isolate, UG92005 (GenBank accession no. AF338704). The DNA vaccine was prepared by incorporating envelope protein sequence (gp140) into a kanamycin-selectable pVVKan vector containing a cytomegalovirus enhancer/promoter, a cytomegalovirus intron A, a tissue plasminogen activator leader, and a bovine growth hormone poly(A) sequence. The plasmid was purified (EndoFree Plasmid Giga kit; QIAGEN, Valencia, CA) and reconstituted in phosphate-buffered saline (PBS) before injection into mice. Mice were primed and boosted (usually twice) with DNA at 3-week intervals with a 100-µg dose (administered as 50 µg per gastrocnemius muscle). Prior to all challenge experiments, mice were also boosted by intraperitoneal (i.p.) injection with a recombinant vaccinia virus (Western Reserve wild type; bromodeoxyuridine-selected; 10⁷ PFU/mouse) expressing the same UG92005 gp140 envelope protein.

Enzyme-linked immunospot analyses. Overlapping peptides (9- to 15-mer) were produced at the Hartwell Center for Bioinformatics and Biotechnology (9, 10) at SJCRH to represent the entire gp140 envelope protein. Overlapping peptides were generally initiated at every five amino acids. Peptides were tested individually or as pools (pools of 10 peptides each were used for screening purposes). Each peptide was used at a concentration of approximately 10 µg/ml.

At least 1 month after the completion of immunizations, spleens from control and test mice were taken for CD4⁺ T-cell enrichment. Briefly, the splenocytes were treated with rat anti-mouse monoclonal antibodies (MAbs) to MHC-II (TIB120) and CD8 (53-6.72), followed by sheep anti-mouse and sheep anti-rat IgG-coated Dynabeads (Dynal ASA, Oslo, Norway). The samples were then exposed to a magnet to remove the MHC-II⁺, the CD8⁺, and the Ig⁺ populations. Antigen-presenting cells were prepared from naïve mouse spleens by depleting T cells with an anti-mouse Thy1.2 (AT83) and complement (1 part rabbit and 5 parts guinea pig complement [Cedarlane, Ontario, Canada] in Hanks balanced salt solution plus 0.1% bovine serum albumin [BSA]), followed by irradiation with 2,500 rads in a Cs irradiator. Multiscreen HA filtration plates (Millipore, Bedford, MA) were incubated overnight with 10 µg/ml anti-mouse IFN- (clone R4-6A2; BD Biosciences, San Diego, CA) in PBS (100 µl/well) at 4°C. The plates were washed four times with PBS and blocked for at least 1 h at 37°C with complete tumor medium (23, 36), modified Eagle's medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal calf serum, dextrose (500 µg/ml), glutamine (2 mM), 2-mercaptoethanol (3 x 10^–5 M), essential and nonessential amino acids, sodium pyruvate, sodium bicarbonate, and antibiotics. CD4⁺ T cells were plated at 1 x 10⁶ cells/well, and B6 antigen-presenting cells were plated at 5 x 10⁵ cells/well, with and without peptides. Naïve T cells were used as negative controls, while cells stimulated with 4 µg/ml concanavalin A (Sigma, St. Louis, MO) were used as positive controls. The cultures were incubated for 48 h at 37°C in 10% CO₂. Plates were then washed four times with PBS, followed by four washes with PBS wash buffer (PBS plus 0.05% Tween 20). Then, 100 µl of 5 µg/ml biotinylated rat anti-mouse IFN- (clone XMG1.2; BD Biosciences) in PBS containing 0.05% Tween 20 and 1% fetal calf serum (FCS) was aliquoted per well; the plates were incubated at 4°C overnight, and the wells were washed five times with wash buffer. Streptavidin-conjugated alkaline phosphatase (DAKO A/S, Denmark) diluted 1:500 in PBS wash buffer was added (100 µl) to each well, and plates were incubated at room temperature for 1 h. After plates were rinsed five times with wash buffer and four times with water, the IFN- spots were developed with 5-bromo-4-chloro-3-indolyphosphate-nitroblue tetrazolium (BCIP/NBT) alkaline phosphatase substrate (Sigma, St. Louis, MO); the plates were then washed with water to stop the reaction and air dried. Spots were counted using a Zeiss Axioplan 2 microscope and software (München-Hallbergmoos, Germany), and the data were plotted with GraphPad Prism for Windows (version 4.02; San Diego, CA).

Recombinant Sendai virus design and challenge. The UG92005 gp120 envelope gene was first cloned between the Sendai virus P and M genes in the pUC19SV shuttle vector, and then an env gene-containing MluI-NotI fragment from pUC19SV was cloned into pSV(E) to yield a full-length Sendai virus genome with an envelope gene insert (24, 29, 35). To rescue infectious recombinant Sendai virus, Vero cells were grown to confluence and infected with psoralen/UV-treated VVT7-3 (a recombinant vaccinia virus expressing T7 polymerase). Plates were washed, and cells were transfected with the pSV(E) recombinant plasmid and three additional individual plasmids expressing T7 promoter-driven NP, P, and L genes of Sendai virus (24, 29, 35). Fresh medium was added 18 h later, and cells were cultured until cytopathic effects were evident. Cells were then isolated and freeze-thawed to release virus. The cell lysate was then inoculated into the allantoic fluid of embryonated hens' eggs to rescue and expand the recombinant virus, and the virus titer was determined by using a hemagglutination assay. Expression of the HIV-1 envelope by the recombinant was confirmed by Western blotting, developed using heat-inactivated HIV-1 plus serum, followed by an alkaline phosphatase-conjugated anti-human Ig reagent and color reaction. Stock allantoic fluid was diluted in PBS to yield 1 x 10⁶ PFU in 50 µl for the intranasal challenge of C57BL/6J mice and 1 x 10⁵ PFU in 50 µl for the intranasal challenge of Ig^–/– muMT mice. Challenges were conducted at least 4 weeks after vaccination.

In some cases, mice were treated by i.p. injections of the GK1.5 MAb to CD4 or the 2.43.1 MAb to CD8 (20, 31) on days –5, –3, –1, + 1, and + 3 relative to the recombinant Sendai virus challenge. The antibodies were administered as ascites fluid diluted in Dulbecco's PBS. Splenocytes were stained and checked to ensure cell depletion by using flow cytometry with non-cross-reactive MAbs (BD Biosciences Pharmingen, Franklin Lakes, NJ) to CD4 (RM4-4) and CD8ß (53-5.8).

Cytokine measurements. Bronchoalveolar lavage (BAL) was performed with euthanized, virus-infected mice by exposing the trachea, inserting catheters, and washing each lung three times with 1 ml of PBS (3 ml total). Wash samples were centrifuged to remove cellular material, and the supernatants were tested for the presence of five different cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-10, and IFN-), using a Bioplex (Bio-Rad, Hercules, CA). Cytokine samples of known concentrations were used to prepare standard curves. Individual samples were tested in duplicate.

Intracellular staining analyses. Cells from the BAL fluid were collected and incubated on a 60- by 15-mm cell culture dish for 1 h at 37°C in a 10% CO₂ incubator to remove macrophages. Nonadherent cells, removed by gentle washing, were then incubated with immunodominant peptides IVGNIRQAHCNVSKA and GKAMYAPPIAGLIQC (10 µM each) for 5 h (37°C in 10% CO₂) in the presence of brefeldin A (10 µg/ml; Epicentre Biotechnologies, Madison, WI). Following incubation, cells were stained by using rat anti-mouse CD8 PerCP Cy5.5 and anti-mouse CD4 fluorescein isothiocyanate (BD Pharmingen, Franklin Lakes, NJ) and fixed with 1% formaldehyde (Ted Pella, Inc., Redding, CA). Samples were washed and permeabilized using PBS containing 0.5% saponin (Sigma-Aldrich, St. Louis, MO). The cells were then stained using anti-IFN- phycoerythrin stain (BD Pharmingen, Franklin Lakes, NJ). Data were collected with a BD FACScalibur and analyzed using FlowJo software (version 6.4.2).

Virus titers. The lungs were removed sterilely, washed four times in PBS, and homogenized in a total volume of 1 ml of PBS. The suspensions were centrifuged at 2,000 x g for 10 min to clear cellular debris. Virus titers were determined by 50% tissue culture infective dose (TCID₅₀) or by plaque assay. The TCID₅₀ measurements were performed by plating serial 10x dilutions of lung suspension in a final volume of 200 µl with LLC-MK₂ cells in 96-well plates (at least 4 wells per sample dilution) with minimal essential medium containing 0.1% BSA in the presence of 5 µg/ml of acetylated trypsin and 50 µg/ml of gentamicin. Cell supernatants were collected after 4 days of incubation and mixed 1:1 with chicken red blood cells (0.5%) in PBS for hemagglutination detection. TCID₅₀ values were calculated by using the Reed-Muench formula (27).

The plaque assay utilized LLC-MK₂ cells grown to confluence (in six-well plates) in plaquing medium (modified Eagle's medium [Gibco, Grand Island, NY], 0.2% NaHC0₃, 2% GlutaMax [Gibco], and 50 µg/ml gentamicin with 5% FCS). Test and control samples were diluted serially in PBS containing calcium and/or magnesium and plated in duplicate (100 µl/well) onto washed cells. After absorption at room temperature for 1 h with intermittent rocking, cells were overlaid with 4 ml plaquing medium plus 0.15% BSA, supplemental vitamins and amino acids, 5 µg/ml acetylated trypsin (Sigma), and 0.9% agar (Bacto). After the agar was set, plates were inverted and incubated at 37°C in a 5% CO₂ incubator. After 4 days, plates received a second overlay (3 ml), which was similar to the first but with 5% FCS instead of BSA, 0.0035% neutral red, and no trypsin supplement. Plates were incubated for an additional 2 to 3 days, and plaques were counted.

Statistical Analyses. All statistical analyses were performed using GraphPad Prism software version 4.02.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Responses elicited by envelope priming. Our previous work identified a vaccination regimen for the induction of robust HIV-1-envelope-specific CD4⁺ T-cell activities in C57BL/6 mice (10) and mapped respective target peptides within the gp120 molecule. The vaccination regimen of mice consisted of repeat immunizations with recombinant DNA expressing the UG92005 HIV-1 gp140 envelope protein. The peptides targeted by responsive T cells were defined by tests with pools of overlapping peptides covering the entire gp140 molecule and were found to reside predominantly in the V3-C3 and C4 regions of the envelope protein (Fig. 1). Our previous studies also showed that the response could be boosted by immunizations with a vaccinia virus vector expressing the same UG92005 gp140 protein (34). Because these immunizations elicited a dominant CD4⁺ T-cell response, the system provided a unique opportunity to conduct further studies of CD4⁺ T-cell potentials and provided target peptides with which to track T-cell locations and function.

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FIG. 1. Immunodominant T-cell responses in HIV-1 envelope-immunized C57BL/6 mouse CD4+ T cells from C57BL/6 mice vaccinated with recombinant DNA (using a prime-boost regimen) were previously shown to respond predominantly toward the V3-C3 and C4 regions of the U92005 gp120 envelope protein (encompassed by peptide pools G and I, respectively [10] for peptide pool designations). Individual peptides within each positive pool were next tested using IFN- enzyme-linked immunospot assays with splenocytes enriched for CD4+ T cells from vaccinated and control animals. Positive responses are shown in this figure, as well as responses toward peptides flanking the positive peptide targets (data are represented as means ± standard error of the means).

Envelope-recombinant Sendai virus. An established reverse genetics approach was used to create a recombinant Sendai virus carrying the gene for the HIV-1 UG92005 gp120 envelope (Fig. 2A, insertion site). As shown by Western blotting (Fig. 2B), the recombinant virus expressed HIV-1 envelope protein in infected cells. The gp120 recombinant virus (lacking transmembrane and intracellular envelope segments) was selected for use in this system so that envelope protein would not appear on infected cell surfaces or on viral membranes. The envelope-specific antibodies that are known to be induced by the recombinant envelope vaccines (DNA-vaccinia virus [28, 38]) would thus have no target antigen on virus particles or on infected cell surfaces. With the contribution of antibodies removed in this way, the study could focus on CD4⁺ T-cell potential.

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FIG. 2. Design and testing of the envelope recombinant Sendai virus. (A) The gp120 portion of the envelope gene was inserted between the P/C and M genes of the Sendai virus, as described in Materials and Methods. (B) A Western blot was prepared with purified wild-type Sendai virus (wt SeV, lane 1) or 5 µl of allantoic fluid from eggs infected with recombinant Sendai virus (rSeV-UG, lane 2). Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Envelope protein was visualized by incubation with HIV-Ig (heat-inactivated HIV-infected human serum), followed by alkaline phosphatase-conjugated anti-human Ig antibody and a color reaction. gp140 protein from a lysate of transformed recombinant Chinese hamster ovary cells was applied to lane 3 (CHO gp140) as a positive control. Standard molecular masses (in kilodaltons) are indicated.

Envelope-specific CD4⁺ T cells home to the site of infection. C57BL/6 mice were first primed with HIV-1 envelope UG92005 by successive inoculations with DNA (two to three times) and vaccinia virus (once) recombinant vectors (with intervals of at least 3 weeks) and then challenged i.n. at least 1 month later with 10⁶ PFU of the recombinant Sendai virus. The BAL fluid populations were harvested on days 5, 7, and 10 after challenge and pooled for analyses (pools comprised BAL samples from three to five test mice). The results (Fig. 3A) show very clearly there was an influx of CD4⁺ T lymphocytes to the site of recombinant Sendai virus challenge that was greatly enhanced in vaccinated compared to that in unvaccinated animals. The greatest difference in CD4⁺ T-cell counts was seen on day 7, when the numbers were approximately fivefold higher in the primed groups than in the control groups. Intracellular cytokine (IFN-) analysis of this CD4⁺ BAL sample set (Fig. 3B) showed that, by day 5, more than 30% of the population was responsive to the immunodominant peptides IVGNIRQAHCNVSKA and GKAMYAPPIAGLIQC. By day 7, this had increased to more than 60%, giving a much higher prevalence for antigen-specific CD4⁺ T cells than is normally seen with other mouse models of virus-induced respiratory disease.

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FIG. 3. CD4+ T cells home to the site of recombinant Sendai virus challenge in vaccinated animals. (A) BAL fluid from vaccinated (DNA-vaccinia virus prime-boost regimen) and unvaccinated animals were collected on the indicated day following virus challenge (x axis), and cells were isolated. Pooled cells for each group were stained using anti-CD4 antibodies. The average total numbers of BAL fluid CD4+ lymphocytes per mouse are indicated (y axis, four to five animals/group). (B) BAL fluid cells were collected from naïve mice (left panels) and vaccinated mice (right panels) on day 5 (upper panels) and day 7 (lower panels) after recombinant Sendai virus challenge. Pooled BAL fluid cells were tested for responses to immunodominant peptides (IVGNIRQAHCNVSKA and GKAMYAPPIAGLIQC; 10 µg/ml per peptide) by intracellular cytokine staining following 5 h of restimulation in the presence of brefeldin A. Cells were gated on CD4+ T cells prior to flow cytometry analyses. A marker was set to indicate positive responses. Negative control responses among cells that were not stimulated with peptide in vitro were 0.76% and 1.76% for day 5 unvaccinated and vaccinated mice, respectively, and 12.6% and 1.01% for day 7 unvaccinated and vaccinated mice, respectively.

Efforts were also made to detect envelope-specific CD8⁺ T cells in lung-associated tissues. On days 5 and 7, CD8⁺ T cells were present in the BAL fluid, but there was no good evidence of envelope peptide-specific CD8⁺ T-cell activity (data not shown), again suggesting that the envelope-specific T-cell responses were dominated by the CD4⁺ set in this model. Sendai virus-specific responses, however, were apparent in both the CD4⁺ and CD8⁺ T-cell populations, but these de novo responses were not measurable until day 7 (data not shown).

Vaccinated animals show a unique TH1/TH2 cytokine profile in the lung. The cytokine profiles in the BAL fluid recovered from the site of recombinant Sendai virus challenge were analyzed for levels of IL-2, IL-4, IL-5, IL-10, and IFN- (Fig. 4).

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FIG. 4. Both TH1 and TH2 cytokines were detected in the lungs of challenged animals. BAL fluid from mice vaccinated with a DNA-vaccinia virus prime-boost regimen and challenged with the Sendai virus recombinant were examined for IL-2, IFN-, IL-4, IL-5, and IL-10 content (on days 3, 5, 7, and 10 following challenge). Unvaccinated, challenged mice were used as controls. Each symbol represents an average response among animals (three to five animals were tested per group, and data are represented as concentration means ± standard error of the means). *, P < 0.05 using unpaired Student's t test or one sample t test.

Cytokines IL-2, IFN-, IL-4, and IL-5 were detected early (on days 2 to 3) in the vaccinated animals and were never detected in unvaccinated animals in any of the experiments performed at this early time point. Significance was shown for IFN-, IL-2, IL-4, and IL-5 (P < 0.05; unpaired Student's t test in each of two experiments with three to five animals in each group). It is important to emphasize that these cytokines were not detected by in vitro restimulation of T cells but reflected the situation in the lumen of the infected environment. The interleukin concentrations then decreased by days 5 to 7 following the challenge administered to vaccinated mice. In control animals, the interleukin levels were slower to appear and remained high in the second week postchallenge. Interestingly, IL-10 was essentially undetectable in vaccinated/challenged animals but was found in unvaccinated/challenged animals at measurable levels (P < 0.05 in each of two experiments). Overall, the cytokines identified in the BAL wash of vaccinated/challenged animals did not conform to a "typical" TH1 (IFN- with IL-2) or TH2 (IL-4 with IL-5 and IL-10) profile.

Envelope-vaccinated mice show enhanced clearance of challenge virus. Given the prompt influx of responsive cells to the site of recombinant Sendai virus challenge, we asked if the challenge virus was controlled better in vaccinated than in naïve animals. Challenges with Sendai virus in the naïve mouse generally result in peak pulmonary virus loads by the end of week 1 and full clearance by the end of week 2. To examine the effect of immunizations on peak viral loads, lungs were harvested on days 3, 5, and 7 following the virus challenge (10⁶ PFU) of vaccinated and control animals (three to five mice per group per day). After lung homogenization in PBS, the virus titers in clarified supernatants was determined by TCID₅₀ assays with LLC-MK2 cells. As shown by representative data in Fig. 5, the challenge virus titer was substantially lower in vaccinated animals than in controls on days 3, 5, and 7. In repeat experiments, a 1- to 4-log₁₀ reduction of pulmonary challenge virus was found routinely in the vaccinated mice. Results were statistically significant in each of two independent experiments on days 3, 5, and 7 with three to five animals per group (P < 0.05; unpaired Student's t test).

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FIG. 5. Vaccinated animals control virus challenge. Vaccinated (DNA-vaccinia virus prime-boost regimen) and unvaccinated mice were challenged with recombinant Sendai virus (1 x 106 PFU, i.n. administration). On each of days 3, 5, and 7 (x axis), groups of vaccinated and unvaccinated mice were sacrificed (four to five mice per group). Lungs were harvested and challenge virus titers were measured by plaque formation with LLC-MK2 cells, and data are represented as PFU means ± standard error of the means (PFU, y axis). *, P < 0.05 using unpaired Student's t test.

Enhanced clearance is dependent on CD4⁺ T cells but not on B cells or CD8⁺ T cells. As described above, our model was designed to minimize the contribution of antibody to viral clearance, because the expressed envelope protein lacked a transmembrane region necessary for its incorporation into viral or infected cell membranes. However, to ensure that B cells were not required to mediate some indirect mechanism of protection in this model, the vaccination/challenge protocol was repeated with µMT mice (which lack B cells). These immunologically compromised mice are generally more susceptible to respiratory infections, so the challenge dose was reduced 10-fold to 1 x 10⁵ PFU of recombinant Sendai virus. Vaccination induced complete protection by 5 days after virus challenge (Fig. 6), indicating that T cells were able to control the infection in the absence of B cells.

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FIG. 6. Virus clearance in the absence of antibody. Vaccinated (DNA-vaccinia virus prime-boost regimen) and unvaccinated µMT animals were challenged i.n. with 1 x 105 PFU recombinant Sendai virus. Lungs were harvested on day 5 following challenge, and the titers of challenge virus in the lungs were determined by a TCID50 measurement with LLC-MK2 cells. The Reed-Muench formula was used to calculate the TCID50 value. Each symbol represents the TCID50 value of a different animal. *, P < 0.05 using unpaired Student's t test.

Could envelope-specific CD8⁺ T cells, though present at very low levels, contribute to virus clearance in this model? When MAb depletion protocols were used (see Materials and Methods), either CD4⁺ or CD8⁺ T cells were essentially eliminated from the vaccinated µMT mice prior to and during virus challenge. Flow cytometry analysis of BAL fluid populations demonstrated effective depletion of CD4⁺ and CD8⁺ T cells in treated mice (there were <0.2% residual cells in each case) on day 5 after virus challenge. Among µMT mice depleted of CD4⁺ T cells, protection from virus challenge was largely lost (Fig. 7) (two independent experiments gave P values greater the 0.05 by analysis of variance using Dunnett's multiple comparison test). By contrast, eliminating the CD8⁺ T cells from µMT (B cell-deficient) mice did not compromise the early control of this infectious process. In fact, superior protection was observed for vaccinated mice that were depleted of CD8⁺ T cells. Protection within CD8⁺ T-cell-depleted µMT mice was statistically significant in each of two independent experiments (P < 0.01 by analysis of variance using Dunnett's multiple comparison test, with the unvaccinated group as a control). The results thus establish that primed CD4⁺, and not CD8⁺, T cells mediate virus control in this prime/challenge system.

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FIG. 7. Depletion of CD4+ T cells in vaccinated µMT mice eliminates their ability to clear recombinant Sendai virus. µMT mice vaccinated with the DNA-vaccinia virus prime-boost regimen were treated with GK1.5 antibody (to remove CD4+ cells, CD4) and/or 2.43 antibody (to remove CD8+ T cells, CD8) on days –5, –3, –1, +1, and + 3 relative to challenge (see Materials and Methods for details). On day 5 following Sendai virus challenge, lungs were harvested to measure virus infection (by TCID50 measurements with LLC-MK2 cells). The Reed-Muench formula was used to calculate the TCID50 value. Each symbol represents the TCID50 value of a different animal. *, P < 0.05 using unpaired Student's t test.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The general aim of these experiments was to determine whether memory CD4⁺ T cells could control infection by a recombinant Sendai virus expressing the T-cell target antigen. Overall, there is an impression that CD4⁺ T-helper cells often require B-cell or CD8⁺ T-cell function for the control of virus infections (39). Here, we show that envelope-specific CD4⁺ T cells can function without companion lymphocytes to limit the consequences of virus challenge. Primed CD4⁺ T cells can home to the site of infection and reduce viral load. Indeed, isolated CD4⁺ T cells can mediate the control of virus without the involvement of B cells and/or CD8⁺ T cells. Furthermore, as might be expected, T cells primed by vaccines administered at intramuscular and i.p. sites readily home to the lung, and there is no necessity to focus the immunization protocol on draining lymph nodes (5, 6, 8, 26).

A key role for CD4⁺ effector T cells has been described previously in a gammaherpesvirus system. Mice that were chronically infected with virus and depleted of CD8⁺ T cells and B cells were protected from challenge with a gammaherpesvirus variant (4, 11). Unlike the current challenge model, the previous study involved chronic rather than acute viral infection. In many other experimental situations, particularly those involving acute infections, CD4⁺ T cells did not control virus in the absence of other lymphoid effector activities (32, 39). The present system is perhaps uniquely suited for demonstrating CD4⁺ T-cell homing and viral clearance potentials. Because of the robust and focused CD4⁺ T-cell response to HIV-1 envelope, as much as 60% of the CD4⁺ T cells found in the BAL fluid after recombinant Sendai virus challenge were specific for two immunodominant envelope peptides.

The current results raise questions concerning the mechanism by which CD4⁺ T cells function in the absence of B cells and CD8⁺ T cells (2, 3, 13, 18, 19, 33, 37). Likely modes of action are that CD4⁺ T cells secrete cytokines at the site of virus challenge and/or they mediate direct lysis of virus-infected targets (2, 3, 13, 18, 19, 33, 37). Also, CD4⁺ T cells can enhance the activity of "innate" functions such as (i) phagocytosis by macrophages and neutrophils, (ii) activity of natural killer cells, (iii) degranulation of mast cells/basophils or eosinophils, and/or (iv) secretion of cytokines by macrophages. Cytokines, whether produced directly or indirectly by CD4⁺ T cells, may have complex influences on viral clearance mechanisms. Graham et al. (16, 17) have demonstrated that TH1 clones (categorized by IFN- expression) protected mice from influenza virus challenge in the presence of CD8⁺ T cells, while TH2 clones (associated with IL-4 expression) were not protective.

In our studies, the cytokines produced at the site of virus challenge in vaccinated and challenged animals could not be categorized as TH1 or TH2. Rather, a combination of IFN- and IL-2 (typical of a TH1 response) plus IL-4 and IL-5 (typical of a TH2 response) was present immediately postchallenge in protected animals. Future experiments with cytokine knockout animals may help to identify the precise influences of TH1/TH2 combination cytokines on viral clearance in this unique mouse model system (1, 7, 12, 15, 21, 22). In conclusion, the present analysis demonstrates that antigen-specific CD4⁺ T cells primed at distant sites can home to the pneumonic lung and, acting in the absence of other components of the adaptive immune system, terminate an infectious process. The potent responses illustrated by this DNA-vaccinia virus immunization/recombinant virus challenge strategy provide an experimental model that is readily manipulated for further dissection of the nature of CD4⁺ T-cell-mediated effector function.

 
This work was supported in part by NIH NIAID grants P01-AI45142 and R21 AI056974, by NCI Cancer Center Support core grant P30-CA21765, and by the James B. Pendleton Charitable Trust, the Anderson Foundation, and the American Lebanese Syrian Associated Charities (ALSAC).

We thank Tim Lockey, Bart Jones, Brita Brown, Amy Zirkel, Bob Sealy, Ruth Ann Scroggs, and Pam Freiden for assistance with the preparation of reagents. We thank the World Health Organization and James Bradac (AIDS Research and Reference Reagent Repository, Rockville, MD) for virus UG92005, from which a DNA sequence was derived for the preparation of vaccine and challenge virus. The expression cassette, with which the DNA vaccine was made, was kindly provided by James Mullins and Harriet Robinson.

 
* Corresponding author. Mailing address: Department of Immunology, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-2650. Fax: (901) 495-3099. E-mail: address:scott.brown{at}stjude.org

Published ahead of print on 25 July 2007.

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Journal of Virology, November 2007, p. 12535-12542, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00197-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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