A synthetic lymph node containing inactivated Treponema pallidum cells elicits strong, antigen-specific humoral and cellular immune responses in mice

Authors

  • Lola V. Stamm,

    Corresponding author
    1. The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
    • Correspondence

      Lola V. Stamm, Infectious Diseases Program, Department of Epidemiology, Gillings School of Global Public Health, Michael Hooker Research Building, S. Columbia St., The University of North Carolina at Chapel Hill, NC 27599-7435, USA.

      Tel.: +919 966 3809

      fax: +919 966 0584

      e-mail: lstamm@email.unc.edu

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  • Rebecca L. Drapp

    1. The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
    Current affiliation:
    1. Rebecca L. Drapp, North Carolina State University College of Veterinary Medicine, Raleigh, NC, USA
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  • In this interesting study by Stamm and Drapp the authors demonstrate that synthetic lymph nodes implanted into mice that carried Treponema pallidum antigen induce both strong humoral and cellular immune responses. Animal models for studying syphilis are difficult to work with and are limited. This study suggests that the mouse model has the potential to be employed as a tool for investigating immune responses to Treponema pallidum.

Abstract

The goal of this study was to investigate the use of a synthetic lymph node (SLN) for delivery of Treponema pallidum (Tp) antigens. Immune responses of C57BL/6 mice were analyzed at 4, 8, and 12 weeks after SLN implantation. Group 1 mice received SLN with no antigen; Group 2, SLN with formalin-inactivated Tp (f-Tp); and Group 3, SLN with f-Tp plus a CpG oligodeoxynucleotide. When tested by ELISA, sera from Group 2 and Group 3 mice showed stronger IgG antibody reactivity than sera from Group 1 mice to sonicates of f-Tp or untreated Tp, but not to sonicate of normal rabbit testicular extract at all times. The IgG1 level was higher than IgG2c level for Group 2 mice at all times and for Group 3 mice at 4 and 8 weeks. IgG1 and IgG2c levels were nearly equivalent for Group 3 mice at 12 weeks. Immunoblotting showed that IgG from Group 2 and Group 3 mice recognized several Tp proteins at all times. Supernatants of splenocytes from Group 2 and Group 3 mice contained significantly more IFNγ than those from Group 1 mice after stimulation with f-Tp at all times. A significant level of IL-4 was not detected in any supernatants. These data show that strong humoral and cellular immune responses to Tp can be elicited via a SLN.

Introduction

Syphilis is a chronic, multistage disease caused by infection with Treponema pallidum subsp. pallidum (Tp) (Ho & Lukehart, 2011; Stamm & Mudrak, 2013). Syphilis is usually transmitted through contact with active lesions of a sexual partner or from an infected pregnant woman to her fetus. Although syphilis has remained endemic in sub-Saharan Africa and South-East Asia, it has recently re-emerged in several developed countries in the form of small, sporadic outbreaks and large, widespread epidemics (Stamm & Mudrak, 2013). Due to the lack of a vaccine, the control of syphilis is mainly dependent upon the identification and treatment of infected individuals and their contacts. While intramuscular penicillin is effective for early syphilis, Tp strains with high-level resistance to oral macrolide antibiotics (e.g. erythromycin, azithromycin) are now present in several countries (Stamm, 2010; Stamm & Mudrak, 2013).

The resurgence of syphilis and the emergence and rapid spread of macrolide-resistant Tp are a clear indication of the need to develop new strategies to prevent infection. Progress along these lines has been hindered due to the unique biology of Tp. This spirochete cannot be cultivated in vitro for sustained periods, it is genetically intractable, and the fragility of its outer membrane has impeded the unambiguous identification of surface-exposed outer membrane proteins (OMPs) that are putative targets for vaccine development. Thus far, only one immunization study has demonstrated solid protection against experimental infection with Tp (Miller, 1973). This study used γ-irradiated Tp cells given intravenously to rabbits over several months. When challenged intradermally with virulent Tp, the immunized rabbits did not develop lesions, and transfer of their tissues to naïve rabbits did not produce infection. Unfortunately, the identity of the Tp antigens that elicited the protective response has remained elusive, although presumably they are rare, poorly immunogenic, cell-surface-exposed OMPs (Radolf et al., 1989; Walker et al., 1989; Lewinski et al., 1999). Subsequent studies that employed immunization with heat-inactivated Tp, fractionated Tp, or recombinant-produced Tp antigens achieved only partial protection in the rabbit model (Cullen & Cameron, 2006; Ho & Lukehart, 2011). Most of these studies used an intramuscular route for the delivery of Tp antigens, often in combination with adjuvants that promoted a T-helper type 2 (Th2) response. However, experimental data suggest that a Th1 response, which is associated with clearance of Tp during early infection and characterized by production of opsonizing IgG antibody and activated macrophages, may be more relevant to protection (Baker-Zander & Lukehart, 1992; Arroll et al., 1999; Podwinska et al., 2001; Leader et al., 2007). It is likely that the development of a protective Th1 response would require that Tp antigens (i.e. cell-surface-exposed OMPs) be administered in their native conformation and delivered by a strategy that engages both the innate and adaptive responses to elicit long-term memory (Cullen & Cameron, 2006).

Although outbred rabbits are the most widely used animal model for syphilis research, several investigators have shown that mice can be chronically infected with Tp (Gueft & Rosahn, 1948; Magnuson et al., 1949; Ohta, 1972; Klein et al., 1980; Folds et al., 1983; Saunders & Folds, 1985). Infected mice develop humoral and cellular responses to Tp, but unlike rabbits, they do not exhibit pathology. However, inbred mice offer a well-defined genetic and immunologic background, and reagents for studying their immune response are readily available. Thus, mice are a practicable tool for screening new approaches to modulate the immune response to Tp. Here, we report the results of a novel strategy for the delivery of Tp antigens that employed subcutaneous implantation of a ‘synthetic lymph node’ (SLN) in mice. We hypothesized that this approach would promote the development of a strong immune response to Tp. The SLN is composed of a porous matrix within a perforated, but otherwise impermeable membrane (Garin et al., 2007). It acts by retaining antigen at the implantation site and by attracting immune cells [e.g. antigen presenting cells (APCs) and lymphocytes], which proliferate and secrete cytokines and chemokines after antigen exposure. Because antigens, cytokines, and chemokines move only by diffusion, they are retained largely within the SLN. In contrast, the activated immune cells can migrate out of the SLN to initiate a systemic immune response. Our data show that strong humoral and cellular immune responses specific to Tp can be elicited by the delivery of inactivated Tp cells via a SLN.

Materials and methods

Research animals

Eight-week-old, male C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Adult male New Zealand white rabbits were purchased from Robinson Services, Inc. (Mocksville, NC). Animals were housed in a temperature-controlled facility under specific pathogen-free conditions with antibiotic-free food and water provided ad libitum. All animal procedures were reviewed and approved by the University of North Carolina at Chapel Hill (UNC-CH) Institutional Animal Care and Use Committee.

Cultivation of Tp

Rabbits were infected intratesticularly with Tp Nichols strain and treated as previously described (Stamm & Bassford, 1985). At peak orchitis, the rabbits were sacrificed, and testicular tissue was removed aseptically and minced in a laminar flow hood. Treponemes were extracted in phosphate-buffered saline (PBS) containing 10% heat-inactivated normal rabbit serum (Sigma-Aldrich, St. Louis, MO). Extracts were centrifuged at low speed to remove contaminating rabbit cells. The supernatants were pooled and centrifuged, and the pelleted Tp were washed twice with PBS, quantitated by dark-field microscopy with a Petroff–Hausser counting chamber (Hausser Scientific, Horsham, PA), and frozen. For formalin treatment, freshly extracted, washed treponemes were incubated overnight at room temperature in PBS with neutral-buffered formalin (Sigma-Aldrich) added to a final concentration of 0.2%. The formalin-inactivated Tp (f-Tp) were pelleted, washed twice with PBS, suspended in PBS, and stored at 4 °C. Normal rabbit testicular extract (NRTE) was prepared by extracting testicular tissue from uninfected rabbits as described above.

SLN construction and implantation

SLNs were prepared using a 1.25-cm length of 0.15-cm-interior-diameter SILASTIC silicone tubing (Dow Corning Corp., Midland, MI) with twenty 20-gauge needle holes evenly spaced across the tubing (Garin et al., 2007). The SLNs were fitted with a 1.25-cm length of hydroxylated polyvinyl acetate wound dressing sponge (MEROCEL; Medtronic Inc., Minneapolis, MN) and sterilized by autoclaving. Anesthetized mice were randomly divided into three groups. Group 1 (six mice) received SLN loaded with PBS (empty SLN; no antigen control), Group 2 (nine mice) received SLN loaded with c. 2 × 107 f-Tp cells (Tp SLN), and Group 3 (nine mice) received SLN loaded with c. 2 × 107 f-Tp cells plus 9.5 μg CpG oligodeoxynucleotide 1826 (InvivoGen, San Diego, CA), a TLR9 agonist that promotes a TH1 response (Tp+CpG SLN) (Hanagata, 2012). Each mouse received only one SLN in its shaved, right dorsal flank. Two mice from Group 1 and three mice each from Group 2 and Group 3 were bled at 4, 8, and 12 weeks after SLN implantation to obtain serum and then sacrificed. Spleens were collected and pooled for each group of mice.

ELISA for detection of IgG antibody to Tp

Serum from individual mice or pooled sera from each group of mice were tested by ELISA to detect mouse IgG antibody to Tp using a modification of the protocol of Folds et al. (1983). Briefly, untreated Tp (u-Tp) cells, f-Tp cells, or NRTE were sonicated on ice with a Virsonic 50 (The Virtis Co. Inc., Gardiner, NY) and centrifuged to remove insoluble material, and the supernatant was frozen at −80 °C. Thawed sonicate was diluted 1 : 10 in 0.05 M carbonate buffer (Sigma-Aldrich). Aliquots representative of c. 2 × 106 Tp or an equivalent volume of NRTE were added to microtiter plate wells (Costar 9018, Corning, NY), and the plates were incubated overnight at 4 °C. Before use, the plates were washed with PBS-0.05% Tween 20 and blocked with PBS–0.05% Tween 20 containing 1% BSA. Serum samples diluted in blocking solution were added in duplicate to the washed wells, and the plates were incubated overnight at 4 °C. After washing, HRP-conjugated goat anti-mouse IgG (R & D Systems, Inc., Minneapolis, MN) or HRP-conjugated goat anti-mouse IgG1 or IgG2c (Southern Biotech, Birmingham, AL) was diluted in blocking solution as recommended by the manufacturer and added to the wells. Plates were incubated at room temperature for 60 min. Enzyme substrate (R & D Systems, Inc.) was added after the wells were washed, and the color was allowed to develop. The reaction was stopped by addition of 2N H2SO4. Plates were read at 450 nm with a 570-nm reference using a Benchmark microplate reader with Microplate Manager III data analysis software (Bio-Rad, Richmond, CA). Mean optical density (OD) values of Group 2 (Tp SLN) and Group 3 (Tp+CpG SLN) sera that were at least two standard deviations (SD) above the mean OD of the Group 1 (empty SLN) sera were considered positive for an IgG antibody response to Tp.

Western blot

Solubilized Tp proteins were separated by electrophoresis on 15% acrylamide slab gels as previously described (Stamm & Bassford, 1985) and electrophoretically transferred to nitrocellulose sheets (0.45-μm pore size; Schleicher & Schuell, Inc., Keene, NH) (Saunders & Folds, 1985), which were then cut into strips. The strips were blocked with 5% nonfat dry milk in PBS–0.05% Tween 20 and incubated with pooled mouse sera diluted 1 : 100 in blocking solution at 4 °C overnight. After washing with PBS–0.05% Tween 20, the strips were incubated for 60 min in HRP-conjugated goat anti-mouse IgG (R & D Systems, Inc.) diluted 1 : 1000 in blocking solution, washed, incubated for 1 min in chemiluminescence developing reagents (Thermo Scientific, Rockford, IL), and exposed to X-ray film.

Splenocyte preparation and cytokine assays

Pooled spleens from mice that were sacrificed at 4, 8, and 12 weeks after SLN implantation were processed by a modification of the protocol of Klein et al. (1980). Briefly, spleens were mechanically dissociated in RPMI medium 1640 (Life Technologies Corp., Grand Island, NY), passed through a 40-μm nylon strainer, centrifuged, and resuspended in lysis buffer to eliminate red blood cells. Splenocytes were washed in cold RPMI medium and resuspended in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 10 mM Hepes, 100 units mL−1 penicillin, and 100 μg mL−1 streptomycin. Cell viability was assessed by trypan blue exclusion, and cell numbers were quantitated with a hemocytometer. Duplicate sets of splenocytes were seeded in triplicate at 1 × 106 cells per well in 48-well tissue culture plates with medium only (negative control) or with medium containing c. 1 × 107 f-Tp cells and incubated for 48 h at 37 °C in 5% CO2. Supernatants were collected and stored at −80 °C. IFNγ and IL-4 levels in the supernatants were determined by ELISA as recommended by the manufacturer (R & D Systems, Inc.). Student's t-test was performed to assess any significant differences (< 0.05) between the groups using prism 5.0c (GraphPad Software Inc., San Diego, CA).

Results

IgG antibody response of mice to Tp after SLN implantation

The serum IgG antibody response of the SLN-implanted mice to Tp was assessed with an ELISA that used sonicated f-Tp as antigen. Sera from two mice (M1 and M2) from Group 1 (empty SLN) that received SLN containing PBS showed only minimal reactivity to f-Tp sonicate at all time points after SLN implantation (Fig. 1). In contrast, serum from each of three mice (M1-M3) from Group 2 (Tp SLN) and Group 3 (Tp+CpG SLN) that received SLN containing f-Tp cells showed strong reactivity to f-Tp sonicate at each time point after SLN implantation (Fig. 1). Although the serum reactivity to f-Tp sonicate varied between the mice in Group 2 or Group 3 at each time point, it was consistently higher than that of the Group 1 mice at all time points.

Figure 1.

IgG antibody response of mice to Tp after SLN implantation. Serum samples from Group 1 (empty SLN), Group 2 (Tp SLN), and Group 3 (Tp+CpG SLN) mice that were sacrificed at 4, 8, and 12 weeks after SLN implantation were tested at a 1 : 40 dilution by ELISA using sonicate prepared with formalin-inactivated Tp. For Group 1, pooled sera from two mice (M1 & M2) were tested. For Group 2 and Group 3, serum from each of three mice (M1–M3) was tested. Values presented are the mean (± SD) of samples tested in duplicate. *Samples with a mean OD value at least two SD above the mean OD value of Group 1 are considered positive for an IgG antibody response to Tp.

The ELISA was repeated with sonicate prepared from Tp cells that had not been formalin-inactivated to determine whether sera from the Group 2 and Group 3 mice react with u-Tp. Pooled sera from the Group 2 and Group 3 mice reacted strongly to both f-Tp and u-Tp sonicates at all time points, confirming that these sera recognize native Tp antigens (Fig. 2). Because Tp was derived from testicular tissue of experimentally infected rabbits, the pooled mouse sera were tested by ELISA with sonicate of NRTE. Group 2 and Group 3 sera did not exhibit significant reactivity over that of Group 1 sera to NRTE at any time point, indicating that the IgG antibody response of the Group 2 and Group 3 mice is specifically directed against Tp rather than contaminating rabbit cells (Fig. 2).

Figure 2.

Serum IgG antibody reactivity of mice after SLN implantation to sonicates of f-Tp, untreated Tp (u-Tp), and NRTE. Samples of pooled sera from Group 1 (empty SLN), Group 2 (Tp SLN), and Group 3 (Tp+CpG SLN) mice that were sacrificed at 4, 8, and 12 weeks after SLN implantation were tested at a 1 : 40 dilution by ELISA using sonicates prepared with f-Tp, u-Tp, or NRTE. Values presented are the mean (± SD) of samples tested in duplicate. *, Samples with a mean OD value at least two SD above the mean OD value of Group 1 are considered positive for an IgG antibody response to Tp.

IgG isotyping indicated that both IgG1 and IgG2c antibodies to Tp were present in pooled sera from the Group 2 and Group 3 mice (see Supporting Information, Fig. S1). The IgG1 level was higher than the IgG2c level for the Group 2 mice at all time points and for the Group 3 mice at 4 and 8 weeks after SLN implantation. However, IgG1 and IgG2c levels were nearly equivalent for the Group 3 mice at 12 weeks after SLN implantation. Western blot analysis with pooled sera from the three groups of mice showed that only the Group 2 and Group 3 mice exhibited strong reactivity to Tp (Fig. 3). IgG antibodies in the sera of these mice recognized several Tp proteins, most of which were in the range of c. 55–31 kDa, at all time points after SLN implantation. The latter results are very similar to those of Saunders & Folds (1985) for Western blots performed with sera of mice that had been infected intradermally with virulent Tp for 12–18 weeks. Interestingly, these investigators reported that sera from mice that were given a single intradermal injection of 1 × 107 heat-inactivated Tp cells did not show any reactivity to Tp when tested by Western blot.

Figure 3.

Western blot analysis of Tp proteins recognized by serum IgG antibodies of mice after SLN implantation. Tp proteins were separated by gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane. Nitrocellulose strips were probed with a 1 : 100 dilution of pooled sera from Group 1 (empty SLN), Group 2 (Tp SLN), and Group 3 (Tp+CpG SLN) mice sacrificed at 4, 8, and 12 weeks after SLN implantation. Reactivity was detected with HRP-labeled goat anti-mouse IgG. Lane 1, pooled Group 1 sera for 4, 8, and 12 weeks, respectively; Lanes 2–4, pooled Group 2 sera for 4, 8, and 12 weeks, respectively; Lanes 5–7, pooled Group 3 sera for 4, 8, and 12 weeks, respectively. Only the relevant portion of the blot is shown. Numbers on the left (× 103) refer to the molecular mass of protein standards run on the same gel.

Splenocyte cytokine response of mice to Tp after SLN implantation

Secretion of IFNγ (Th1 cytokine) by pooled splenocytes from the three groups of mice was examined at 4, 8, and 12 weeks after SLN implantation. Incubation of splenocytes with medium only resulted in an undetectable level of IFNγ (i.e. below the assay detection limit of 31.25 pg mL−1) in the supernatants for all groups of mice at all time points (data not shown). In contrast, stimulation of splenocytes with f-Tp cells resulted in secretion of a significantly higher level of IFNγ for Group 2 (Tp SLN) and Group 3 (Tp+CpG SLN) mice compared with Group 1 (empty SLN) mice at all time points (P < 0.001; Fig. 4). Interestingly, stimulated splenocytes from Group 2 mice secreted a level of IFNγ comparable to that of stimulated splenocytes from Group 3 mice at 4 weeks and at 8 weeks, respectively, whereas stimulated splenocytes from Group 2 mice secreted significantly more IFNγ at 12 weeks than stimulated splenocytes from Group 3 mice (P < 0.001).

Figure 4.

IFNγ response of Tp-stimulated pooled splenocytes from mice after SLN implantation. Duplicate samples of Group 1 (empty SLN), Group 2 (Tp SLN), and Group 3 (Tp+CpG SLN) splenocytes prepared from mice sacrificed at 4, 8, and 12 weeks after SLN implantation were plated in triplicate and incubated for 48 h with medium only or 1 × 107 f-Tp cells. Supernatants were tested at a 1 : 20 dilution for IFNγ by ELISA. Values are the mean (± SD). IFNγ levels of splenocyte supernatants incubated with medium alone are not shown because they were below the assay detection limit (31.25 pg mL−1) for all groups of mice at all time points. **Significantly different (P < 0.001) by t-test compared with the Group 1 value at the corresponding time point.

For comparative purposes, pooled splenocytes from naïve, male C57BL/6 mice were stimulated with f-Tp cells. Stimulated splenocytes from naïve mice that were age-matched to Group 1 mice at 4 weeks after SLN implantation secreted about one-third of the level of IFNγ detected in the supernatants of the stimulated splenocytes from Group 1 mice. Stimulated splenocytes from naïve mice that were age-matched to Group 1 mice at 12 weeks after SLN implantation secreted a low level of IFNγ, similar to that of the stimulated splenocytes from Group 1 mice (see Fig. S2). The higher level of IFNγ secreted by the stimulated splenocytes from Group 1 mice at 4 weeks after SLN implantation was presumably due to an early, but transitory inflammatory response to the empty SLN that waned over time.

Secretion of IL-4 (Th2 cytokine) by pooled splenocytes from the three groups of mice was also examined at 4, 8, and 12 weeks after SLN implantation. An IL-4 level significantly higher than that of the medium control was not detected in any of the supernatants at any time point (data not shown).

Discussion

The goal of this study was to investigate the use of a SLN as a novel strategy to elicit humoral and cellular immune responses to Tp. We found that the Group 2 (Tp SLN) and Group 3 (Tp+CpG SLN) mice, but not the Group 1 mice (empty SLN), produced serum IgG antibodies that reacted strongly and specifically to sonicates of f-Tp and u-Tp at 4, 8, and 12 weeks after SLN implantation. Both IgG1 and IgG2c antibodies to Tp were present in the sera of the Group 2 and Group 3 mice. The IgG1 level was higher than the IgG2c level at all time points for the Group 2 mice and at 4 and 8 weeks for the Group 3 mice, whereas the IgG1 and IgG2c levels were nearly equivalent for the Group 3 mice at 12 weeks. Western blot analysis showed that IgG antibodies in sera from the Group 2 and Group 3 mice recognized several Tp proteins at all time points after SLN implantation. Furthermore, splenocytes from the Group 2 and Group 3 mice secreted a significantly higher level of IFNγ after stimulation with f-Tp cells than splenocytes from the Group 1 mice at all time points. A significant level of IL-4 was not detected in supernatants of f-Tp-stimulated splenocytes from any of the mice. Collectively, these data are consistent with the development of a mixed Th1-/Th2-mediated immune response to f-Tp cells by the Group 2 mice at all time points and by the Group 3 mice at 4 and 8 weeks after SLN implantation. Addition of CpG oligodeoxynucleotide, a TLR9 agonist that promotes a Th1 immune response (Hanagata, 2012), did not appear to increase the serum IgG2c level (Th1 response) compared with the IgG1 level (Th2 response) of the Group 3 mice at 4 and 8 weeks after SLN implantation. However, IgG1 and IgG2c levels were virtually identical in the Group 3 mouse sera at 12 weeks, suggesting that the addition of CpG oligodeoxynucleotide had a delayed effect and that the immune response of these mice to f-Tp was more Th1-biased.

Earlier studies by other investigators showed that mice respond slowly and poorly to inactivated Tp cells delivered without adjuvant by various routes (McLeod & Magnuson, 1951; Ohta, 1972; Folds et al., 1983; Saunders & Folds, 1985). Although these studies assessed the humoral response of mice to Tp using serological assays (e.g. fluorescent antibody staining, microhemagglutination, ELISA, and immunoblotting), they did not examine the cellular response of the mice. We cannot compare our results directly with those of the other studies because they used different methods for inactivation of Tp (i.e. heat or antibiotic treatment), different strains of mice, and different routes of immunization. Nonetheless, we have clearly demonstrated that a single, relatively low dose of f-Tp cells, delivered subcutaneously via a SLN, elicits strong humoral and cellular responses specific to Tp at least as early as 4 weeks after SLN implantation and that these immune responses are sustained for at least a 12-week period. We postulate that two key features of the SLN were critical to promoting the strong immune response to f-Tp. First, the SLN retained antigen at the implantation site (i.e. depot effect), which likely allowed for more efficient capture of Tp antigen by APCs that were attracted to the site due to the transient, host inflammatory response to minor tissue injury resulting from implantation of the SLN. Second, the SLN retained secreted cytokines and chemokines at higher concentrations, which presumably enhanced the stimulation of immune cells that were essential to development of the adaptive response.

To our knowledge, this is the first report of the use of the SLN for delivery of bacterial antigens. However, data obtained using the SLN with other types of microbial antigens have shown that this approach can elicit the production of robust humoral and cellular immune responses in mice and rabbits that can protect them against certain infections (S. Meshnick, pers. commun.). Although evaluation of the status of the Group 2 and Group 3 mice to challenge infection with Tp was beyond the scope of this study, we believe that our findings encourage further investigation of the SLN for eliciting an immune response to Tp and possibly the expansion of this novel strategy to the outbred rabbit model of syphilis, for which protection to experimental challenge infection is more easily assessable.

Acknowledgements

This work was supported by a research grant from the UNC-CH University Research Council to L.V.S. We thank Carla Hand and Steve Meshnick for providing the SLNs and Charlene Santos and the UNC-CH Animal Studies Core Facility for SLN implantation.

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