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

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.

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.

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-γ (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 × 10⁻⁵ M), essential and nonessential amino acids, sodium pyruvate, sodium bicarbonate, and antibiotics. CD4⁺ T cells were plated at 1 × 10⁶ cells/well, and B6 antigen-presenting cells were plated at 5 × 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-γ (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-γ 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).

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 × 10⁶ PFU in 50 μl for the intranasal challenge of C57BL/6J mice and 1 × 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 CD8α (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 CD8β (53-5.8).

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-γ), using a Bioplex (Bio-Rad, Hercules, CA). Cytokine samples of known concentrations were used to prepare standard curves. Individual samples were tested in duplicate.

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-γ phycoerythrin stain (BD Pharmingen, Franklin Lakes, NJ). Data were collected with a BD FACScalibur and analyzed using FlowJo software (version 6.4.2).

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 × 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 10× 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.

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

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.

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.

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-γ) 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.

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).

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-γ (Fig. 4).

Cytokines IL-2, IFN-γ, 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-γ, 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-γ with IL-2) or TH2 (IL-4 with IL-5 and IL-10) profile.

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).

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 × 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.

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.