Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Nanomedicine. 2014 Mar 4;10(6):1311–1321. doi: 10.1016/j.nano.2014.02.009

Poly (lactic acid)-poly (ethylene glycol) nanoparticles provide sustained delivery of a Chlamydia trachomatis recombinant MOMP peptide and potentiate systemic adaptive immune responses in mice

Saurabh Dixit a, Shree R Singh a, Abebayehu N Yilma a,1, Ronald D Agee II a,2, Murtada Taha a,3, Vida A Dennis a,*
PMCID: PMC4119847  NIHMSID: NIHMS572505  PMID: 24602605

Abstract

PLA-PEG [poly (lactic acid)-poly (ethylene glycol)], a biodegradable copolymer, is underexploited for vaccine delivery although it exhibits enhanced biocompatibility and slow release immune-potentiating properties. We document here successful encapsulation of M278, a Chlamydia trachomatis MOMP (major outer membrane protein) peptide, within PLA-PEG nanoparticles by size (~73–100 nm), zeta potential (−16 mV), smooth morphology, encapsulation efficiency (~60%), slow release pattern, and non-toxicity to macrophages. Immunization of mice with encapsulated-M278 elicited higher M278-specific T-cell cytokines [Th1 (IFN-γ, IL-2), Th17 (IL-17)] and antibodies [Th1 (IgG2a), Th2 (IgG1, IgG2b)] compared to bare M278. Encapsulated-M278 mouse serum inhibited Chlamydia infectivity of macrophages, with a concomitant transcriptional down-regulation of MOMP, its cognate TLR2 and CD80 co-stimulatory molecule. Collectively, encapsulated-M278 potentiated crucial adaptive immune responses, which are required by a vaccine candidate for protective immunity against Chlamydia. Our data highlights PLA-PEG’s potential for vaccines, which resides in its slow release and potentiating effects to bolster immune responses.

Keywords: Chlamydia trachomatis, bacteria, PLA-PEG nanoparticles, antibody, cytokines

Background

Biodegradable polymers typically employed for fabrication of nanoparticles provide controlled release, low toxicity, high encapsulation efficiency, sub-cellular size and bio-distribution in vivo1. They are also excellent delivery systems for bioactive macromolecules, including peptides and proteins2 and can be fabricated to evoke immune responses, either by direct stimulation of antigen presenting cells (APCs) or delivering antigens to specific cellular compartments3. Reportedly, PLA-PEG [poly (lactic acid)-poly (ethylene glycol), a biodegradable copolymer, can control release of proteins over several weeks4, which is ideally attractive for vaccine delivery5. PLA-PEG sustained release, sub-cellular size, and enhanced biocompatibility facilitate uptake of antigens by APCs and their influx to the injection site6. Encapsulation within PLA-PEG has tremendous benefits in vivo including protecting the integrity and activity of biomolecules while augmenting their immune-potentiating effects. This copolymer apparently enhances the therapeutic activity of encapsulated drugs5,7 and consequently is more commonly used for drug delivery. Conversely, it is not widely exploited for vaccine delivery8, in spite of its aforementioned inherent properties, which are desirable for vaccines.

Chlamydia trachomatis, the most frequently reported bacterial sexually transmitted infection worldwide, causes considerable morbidity and socioeconomic burdens9. Currently, there is no approved vaccine against Chlamydia, perhaps due to ineffective delivery systems or formulations that do not bolster immune responses to achieve long-lasting protective immunity against this intracellular pathogen. The major outer membrane protein (MOMP) of C. trachomatis is highly immunogenic and hence frequently employed with conventional adjuvants for vaccine development. Nevertheless, these vaccine formulations have only afforded partial protection against Chlamydia infections10–12. Adjuvants although commonly used for vaccine studies suffer from side effects, ineffectiveness to certain antigens, poor ability to elicit cell-mediated immune responses13, inconsistencies in producing humoral immunity, and instability to freezing and drying, which may restrict their applicability against intracellular pathogens14. Thus, there is a necessity to explore alternative nonconventional delivery systems for MOMP, such as biodegradable nanoparticles, which can stimulate long-lasting protective immune responses.

In the present study, we employed PLA-PEG as a delivery system for M27815, a C. trachomatis recombinant MOMP peptide, and hypothesized that encapsulation of M278 will provide for its sustained slow release to potentiate and bolster immune responses in mice. First, by in vitro studies we determined the physiochemical characteristics of encapsulated-M278 (size, zeta potential, morphology, absorbance and chemical compositions), encapsulation efficiency, release pattern, and toxicity to macrophages. Next, we compared adaptive immune response outcomes triggered by encapsulated-M278 with that of bare M278 in immunized mice by quantifying M278-specific cellular (Th1, Th2 and Th17 cytokines, and chemokines) and serum antibodies (Th1 and Th2). Finally, we determined immune serum-mediated inhibition of Chlamydia infectivity of macrophages, and the ensuing effect on the mRNA transcriptional expression of MOMP, its cognate TLR2, and the CD80 co-stimulatory molecule. Herein, we present and discuss our findings.

Methods

Fabrication of nanoparticles

Recombinant MOMP-278 (M278) was cloned as previously reported15 and encapsulated in PLA-PEG nanoparticles using a modified water/oil/water double emulsion evaporation technique16,17 and then lyophilized in the presence of 5% trehalose (as a stabilizer) to obtain encapsulated-M278 (PLA-PEG-M278). Phosphate buffered saline (PBS) was similarly encapsulated in PLA-PEG to serve as a negative control (PLA-PEG-PBS). All lyophilized nanoparticles were stored at −80°C in a sealed container until used.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

The morphology of nanoparticles was assessed using SEM (Zeiss EVO 50 VPSEM) and TEM (Zeiss EM10 TEM) as recently published16–18. For SEM, nanoparticles were mounted on metal pegs using conductive double-sided tape and sputter coated with a gold layer prior to examination. For TEM, nanoparticles were dispensed in distilled water and added to formvar/carbon grids prior to microscopy analysis.

Zetasizer and zeta potential measurements

The mean sizes and zeta potentials of nanoparticles were measured using a zetasizer Nano-ZS instrument (Malvern Instruments, UK)16–18. Each nanoparticle sample was measured in triplicates.

Fourier transform-infrared (FT-IR) spectrometry

Chemical analyses of the functional groups present in nanoparticles were determined using FT-IR16,18. The spectra were obtained with 64 scans per sample ranging from 4000 to 500 cm-1 and a resolution of 4 cm-1.

Ultraviolet visualization (UV-Vis)

UV-Vis was employed to ascertain M278 encapsulation within PLA-PEG as recently described17,18. Nanoparticles and bare M278 were each diluted in deionized water and their absorbance and spectral wavelengths were assessed using the DU 800UV/Vis spectrophotometer (Beckman Coulter, Fullerton, CA).

Encapsulation efficiency

The encapsulation efficiency (EE) was extrapolated from measurements of the total M278 encapsulated within PLA-PEG as described previously16,17 and calculated as: EE=A-B/A × 100 %, where A is the total M278 amount, B is the free M278 amount. These measurements were performed three times.

In vitro release of encapsulated-M278

Release of encapsulated-M278 was determined as reported16, 17. Briefly, nanoparticles (100 mg each) were suspended in PBS containing 0.01% sodium azide and incubated at 37°C. At predetermined time-intervals (up to 20 days), tubes were centrifuged, and the supernatants were removed followed by replenishment of fresh PBS to nanoparticles. The Micro BCA protein assay was used to quantify M278 in collected supernatants with absorbance readings at 562 nm using a microplate reader.

Cell viability assay

The effect of nanoparticles on mouse J774 macrophages viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay and the Cell-Titer 96 Cell Proliferation Assay kit (Promega) as described19. Absorbance at 570 nm was measured using a microplate reader.

Mice immunizations

Female 6–8 weeks old BALB/c mice (Charles River Laboratory, Raleigh, NC) were used for this study. The animal studies were performed following a protocol approved by the Alabama State University Institutional Animal Care and Use Committee (IACUC). Mice were housed under standard pathogen-free environmental conditions at ambient temperatures of 25°C, and provided with sterile food and water ad libitum. Four groups of mice (n=6) received three subcutaneous immunizations at two-week intervals with PBS, M278 and nanoparticles (PLA-PEG-PBS or PLA-PEG-M278) (Figure 1). The PLA-PEG-M278 mice each received 50 µg/100 µL of encapsulated-M278 in PBS; those in the PLA-PEG-PBS group received an equivalent weight of nanoparticles in 100 µL of PBS. Mice in the M278 group received 50 µg/100 µL of purified M278; those in the PBS group received 100 µL of PBS only. Two weeks following the last immunization (day 42), all mice were sacrificed to collect spleen and serum samples for cellular and antibody analyses, respectively.

Figure 1.

Figure 1

Open in a new tab

Schematic of immunization experiments in mice.

T-cells purification and stimulation

Spleens were collected from groups of immunized mice and pooled per group in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. Single spleen cell suspensions were obtained using gentleMACS™ Dissociator (Miltenyi Biotech, Auburn, CA), filtered through 40-µm nylon mesh strainer (BD Biosciences), and washed prior to red blood cells lyses using ACK lysing solution (Gibco). Cells were incubated with anti-CD 90.2-conjugated magnetic beads and total T cells were isolated by positive selection over MACS columns (Miltenyi Biotec). Purity of T cells was greater than 95% as assessed by flow cytometry employing PE-conjugated anti-CD 90.2 antibodies (data not shown). Naïve single spleen cell suspensions were treated with Mitomycin-C (25 µg/mL) for 30 min at 37°C in a 5% CO2 humidified atmosphere to serve as antigen presenting cells (APCs).

Purified T-cells and APCs at 2 × 106/mL (1 × 106 T cells and 1 × 106 APCs) co-cultures were stimulated with purified M278 (10 µg/mL) in 24-well plates and incubated at 37°C in a 5% CO2 humidified atmosphere. The selected 10 µg/mL of M278 was based on previous titration studies in our laboratory. Cell free-culture supernatants were collected by centrifugation after 48 hr and stored at −80°C until used.

Quantification of cytokines and chemokines

Cytokines and chemokines were quantified in cell free-culture supernatants using multiplex ELISA as described17,19–21. Only those cytokines and chemokines that were statistically significant (P ≤ 0.05) above the detection limit (3.2 pg/mL) are reported in this study. The multiplex ELISA was repeated twice and each sample was run in triplicates.

Quantification of serum antibodies

Sera collected from groups of mice were pooled (per group) and used to detect M278-specific antibodies as previously described11,17 except that 1 µg/mL of purified M278 was used to coat plates in the present study.

Serum inhibition assay

Serum inhibition assay was used to investigate the functional role of immune serum on C. trachomatis infectivity of (or multiplication in) mouse J774 macrophages. Macrophages were plated at 106/mL/well in 24-well plates and infected with live C. trachomatis (105 inclusion forming unit, IFU/well) in antibiotic-free DMEM medium in the presence or absence of pooled immune serum (1:100 dilution). The plates were incubated at 37°C in a 5% CO2 humidified atmosphere, and after 48 hr supernatants were removed by centrifugation, and cells were washed three times with PBS prior to extraction of total RNA using Qiagen RNeasy kit.

Quantitative real-time PCR (qRT-PCR)

TaqMan qRT-PCR was employed as described20 to assess the mRNA gene transcripts of MOMP (as a marker of C. trachomatis infectivity of macrophages), TLR2 (C. trachomatis cognate receptor) and the CD80 co-stimulatory molecule. TaqMan qRT-PCR was conducted using TaqMan RNA-to-Ct 1-step kit and TaqMan gene expression assay (for TLR2, CD80 and GAPDH) according to the manufacturer’s instructions (Life Technologies). MOMP primers and probe were designed using primer express software (Life Technologies). All values were normalized with respect to the GAPDH “housekeeping” gene mRNA levels. Results are presented as the fold increase over control (unstimulated cells) using the ΔΔCT method20.

Immunofluorescence staining (IFA)

We conducted IFA to confirm C. trachomatis infectivity of mouse J774 macrophages. Macrophages (2.5 × 104 cells/well) were seeded in 8-well chamber slides and infected with live C. trachomatis (2.5 × 103 IFU/well) in the absence or presence of pooled immune serum (1:100 dilution). After 48 hr, macrophages were washed, fixed with 2% paraformaldehyde and blocked with 10% normal goat serum. Macrophages were stained with FITC-conjugated anti-C. trachomatis polyclonal antibody (1:2000 dilution) as reported22 and PE-conjugated macrophage surface marker ER-MP58 (1:100 dilution) for 1 hr, washed, and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) combined with an anti-fade mounting solution (Life Technologies). Chlamydia was visualized in macrophages using a Nikon Eclipse Ti Confocal Microscope (Nikon Instrument, Melville, NY).

Statistical analysis

Data were analyzed by one- or two-way analysis of variance (ANOVA) followed by Tukeys post-hoc test or the two-tailed Mann Whitney test using GraphPad Prism 5 Software. Significance was established at P < 0.001 = ***, P < 0.01 = ** and P < 0.05 = *.

Results

Physiochemical properties of nanoparticles

SEM and TEM microscopy techniques were employed to assess the morphology and size of nanoparticles. By SEM analysis, PLA-PEG-PBS appeared to be coagulated particles and in the conformation of what is referred to as PEGylated “brush” with rough outer surface23 (Figure 2, A). Magnification of this brush structure within PLA-PEG-M278 revealed a web-like matrix that contained grape-like structures dispersed throughout (Figure 2, B). Additionally, TEM images of PLA-PEG-PBS (Figure 2, C) and PLA-PEG-M278 (Figure 2, D) revealed their appearances to be spherical and smooth in shape with nanosizes < 100 nm.

Figure 2.

Figure 2

Open in a new tab

Physiostructural characterization of nanoparticles. The size and morphology of nanoparticles were determined using SEM (A and B) and TEM (C and D) where one drop of the nanoparticles was deposited on a formvar grid or metal stub, respectively. Size (E and F) and zeta potential (G and H) of nanoparticles were evaluated by the Zetasizer.

As particle size plays an important role in determining the level of cellular and tissue uptake24, we employed zetasizing, which revealed PLA-PEG-PBS and PLA-PEG-M278 were 83 and 73 nm, respectively (Figure 2, E, F), thereby corroborating the TEM results. Next, we measured the zeta potential, which is one of the most essential particle characteristics affecting particle stability. The zeta potentials of PLA-PEG-PBS (Figure 2, G) and PLA-PEG (Figure 2, H) were −10 mV and −16 mV, respectively. These findings demonstrate that encapsulation of M278 did not adversely change the property and size of the nanoparticle, which are essential for maintaining its integrity and stability.

FT-IR and UV-Vis analyses

FT-IR further identified variations in chemical functional groups within and between PLA-PEG nanoparticles. Appearances of typical characteristic peaks for -C-H stretch (at 2883 cm-1), C-O stretch (at 1452 cm-1) -C(O)-O-C-(ester C-O) group formation (at 1342 cm-1) and the presence of the ester carbonyl (CO) group (at 1751 cm-1) were observed within the PLA-PEG-PBS nanoparticles (Figure 3, A). After encapsulation, the positions of some characteristic peaks shifted towards lower frequency for –C-H stretch (to 2880 cm-1) and towards higher frequency for the -C(O)-O-C-(ester C-O) group (to 1352 cm-1). This shift in the spectra after encapsulation may indicate a chemical interaction between M278 and PLA-PEG. The presence of M278 within PLA-PEG-M278 is evident with the –NH peak (protein amide band/amines) frequency (at 1650 cm-1) (Figure 3, B) that is absent from within PLA-PEG-PBS. This finding provides additional evidence of successful encapsulation of M278 within PLA-PEG nanoparticles.

Figure 3.

Figure 3

Open in a new tab

Physiochemical characterization, release and cytotoxicity studies. FT-IR (A and B) and UV-Vis spectra (C) confirmed successful encapsulation of M278 within PLA-PEG. In vitro release of encapsulated-M278 over a 20-day period (D). Cytotoxicity of nanoparticles to mouse J774 macrophages over a 72 hr time-period (E and F).

To rule out the possibility of a burst effect, which can result from adsorption of protein to the outer surface of nanoparticles during fabrication, we next compared their absorbance spectra with that of bare M278. As shown in Figure 3, C, UV-Vis of bare M278 showed protein absorption at a wavelength of ~ 285 nm. In contrast, minimal absorption was seen on the outer surface of nanoparticles, further validating the successful encapsulation of M278.

Encapsulation efficiency and release profile of M278 from PLA-PEG

A modified water/oil/water double emulsion method was employed for fabrication of encapsulated-M278, resulting in ~60 % encapsulation efficiency. The cumulative release of encapsulated-M278 was one of a biphasic release pattern (Figure 3, D) whereby phase one was rapid and lasted up to day 1 (18–21%), followed by a gradual release over a 20-day period. Only 32% of M278 was released the entire time-period, thus confirming its sustained slow release profile.

Cytotoxicity effect of PLA-PEG nanoparticles on mouse macrophages

Toxicity is of major concerns when using nanoparticles in biomedical applications, including even biodegradable polymers. Given the significance of macrophages in innate immunity and Chlamydia replication in macrophages, we tested the toxicity of nanoparticles against mouse J774 macrophages. Both dose and time-dependent toxicity studies were conducted by exposing macrophages to concentrations of nanoparticles ranging from 2 to 0.1 mg/mL over a 24, 48 and 72 hr time-period. At all examined time-points, all concentrations of nanoparticles do not affect the viability of macrophages (Figure 3, E and F), which indicate they are non-toxic to macrophages, and substantiate their safety and biocompatibility for in vivo studies.

Cellular adaptive immune responses

Next, we compared potentiating of T cell-specific cellular immune responses by PLA-PEG-M278 with that of bare M278 in mice (on day 42 post-immunization). Co-cultures of purified T cells and APCs were re-stimulated with M278 to quantify cytokines and chemokines by multiplex ELISA. Compared to bare M278, immunization with PLA-PEG-M278 significantly augmented (P < 0.001) M278-specific T-cell responses including Th1 (IFN-γ, IL-2), Th17 (IL-17), IL-3, GM-CSF, M-CSF and LIF cytokines (Figure 4, A-G) along with the CXCL10 chemokine (Figure 4, H). IL-4 (Th2 cytokine) was also secreted, albeit of lower level and significance (P < 0.05) (Figure 4, I). In contrast, T-cells from bare M278-immunized mice produced significantly (P < 0.001) higher IL-5 (Th2 cytokine) as compared to PLA-PEG-M278 immunized mice (Figure 4, J). Control groups of mice (PBS and PLA-PEG-PBS) did not produce T cell-specific responses (Figure 4, A-J). Our results show that encapsulated-M278 potentiates adaptive T cell-specific immune responses in mice.

Figure 4.

Figure 4

Open in a new tab

Immunization with encapsulated-M278 potentiates specific cellular immune responses in mice. Responses were assessed on day 42 post-immunization where purified T cells (1 × 106/mL) were co-cultured with antigen presenting cells (APCs, 1× 106/mL) and re-stimulated with M278 (10 µg/mL) for 48 hr. Cytokines (A-G, I-J), and chemokines (H) in cell free-culture supernatants were quantified by multiplex ELISA. Significance between groups was established at P < 0.001 = ***, P < 0.01 = ** and P < 0.05 = *.

Antibody adaptive immune responses

We also investigated the capacity of encapsulated-M278 to potentiate antibody immune responses in mice by quantifying M278-specific serum antibodies on day 42 post-immunization by ELISA. Immunization with PLA-PEG-M278 elicited significantly (P < 0.001) higher IgG, IgG2a (Th1) as well as IgG1 and IgG2b (Th2) antibodies compared to bare M278 immunized mice (Figure 5, A). Moreover, the antibody response was predominantly Th2-driven with IgG1/IgG2a and IgG2b/IgG2a ratios of 6.7 and 14, respectively.

Figure 5.

Figure 5

Open in a new tab

Immunization with encapsulated-M278 potentiates specific antibody immune responses in mice. Serum was collected from mice on day 42 post-immunization and used to determine IgG, IgG1, IgG2a and IgG2b antibodies by ELISA (A). To determine antibody titers (B), two-fold serial dilutions of serum were made and the endpoint titer was considered the last dilution with readings higher than the mean + 3 standard deviations of negative controls. Significance between groups was established at P < 0.001 = ***.

We further determined isotype-specific antibody titers by performing serial 2-fold dilutions of sera and observed that PLA-PEG-M278 induced higher isotype-specific antibody titers than did bare M278 with fold differences of 4, 4, 16 and 64, respectively for IgG, IgG1, IgG2a and IgG2b (Figure 5, B), again suggesting more of a Th2-driven response. Control mice (PBS and PLA-PEG-PBS) did not produce serum-specific antibodies (Figure 5, A and B). These findings confirm the encapsulated-M278 potentiating effect also on antibody adaptive immune responses in mice.

In vitro serum inhibition studies

Because of the high levels of serum antibodies triggered by PLA-PEG-M278 in mice, we hypothesized, that immune serum might function to inhibit Chlamydia infectivity and/or replication in macrophages. Therefore, we infected mouse J774 macrophages with C. trachomatis in the absence or presence of immune serum followed by RNA extraction to quantify MOMP mRNA gene transcript, as a marker of infectivity. Expression of MOMP was significantly reduced (P < 0.001) in macrophages exposed to serum from PLA-PEG-M278 immunized mice (Figure 6, A), suggesting, perhaps, less C. trachomatis infectivity. In contrast, MOMP expression was significantly enhanced (P < 0.001) in macrophages exposed to serum from other immunized groups (PLA-PEG-PBS, PBS and M278; Figure 6, A), which may suggest more C. trachomatis infectivity of macrophages.

Figure 6.

Figure 6

Open in a new tab

Serum-mediated inhibition of Chlamydia infectivity of macrophages. Mouse J774 macrophages (106/mL) were infected with C. trachomatis (CT; 105 IFU, Inclusion Forming Units) and incubated for 48 hr in the absence and presence of mouse immune serum (1:100 dilution). qRT-PCR was used to quantify mRNA expression of MOMP (A), TLR2 (B) and CD80 (C). Immunofluorescence microscopy visualization of Chlamydia in macrophages (D-H). Columns 1, 2, 3 and 4, respectively reflect staining for nuclear (blue fluorescence), macrophage surface (red fluorescence), anti-Chlamydia antibody (green fluorescence) and their overlay images (yellow fluorescence).

Given that TLR2 is critical for Chlamydia-mediated host cell activation and MOMP signaling, we assessed in parallel the ensuing impact of reduced MOMP expression on TLR2 mRNA gene transcripts in macrophages. TLR2 expression was reduced (P < 0.001) in macrophages exposed to PLA-PEG-M278 immune serum as compared to other groups (PLA-PEG-PBS, PBS and M278) of mice (Figure 6, B). Moreover, we similarly observed significant reduction (P < 0.05) in the expression of the CD80 co-stimulatory molecule (Figure 6, C).

To confirm infectivity, IFA was used for direct visualization of C. trachomatis within macrophages. As shown in Figure 6, columns 1, 2, 3 and 4, respectively reflect staining for nuclear (blue fluorescence), macrophage surface (red fluorescence), anti-Chlamydia antibody (green fluorescence) and their overlay images (yellow fluorescence). Our results demonstrate that cells alone did not contain Chlamydia (Figure 6, D). However, marked infectivity of cells was seen in the presence of PLA-PEG-PBS immune serum (Figure 6, E), and less infectivity when cells were exposed to PLA-PEG-M278 immune serum (Figure 6, F). Cells exposed to M278 immune serum (Figure 6, G) and PBS (Figure 6, H) were also markedly infected with Chlamydia, thus underscoring the inhibition of Chlamydia infectivity of macrophages by encapsulated-M278 mouse immune serum.

Discussion

Problems associated with conventional adjuvants for vaccines are protein denaturation and low bioavailability in vivo25. PLA-PEG offers an attractive alternative to conventional adjuvants as a vaccine-delivery system because it can augment the immunogenicity of proteins by providing their controlled slow release to the immune system1,2,26. Moreover, PLA-PEG can enhance the protein loading capacity, reduce its burst effect, prevent degradation, and increase the circulation time and bioavailability of proteins without altering their spatial configuration27,28. In the present study, we exploited PLA-PEG immune-potentiating and controlled slow release properties as a vaccine-delivery system for M278, a C. trachomatis MOMP peptide. We show (i) successful M278 encapsulation, its in vitro physiochemical characterization and release profile (ii) that encapsulated-M278 triggered higher specific cellular and antibody adaptive immune responses in mice, as compared to bare M278, and (iii) that encapsulated-M278 immune serum markedly inhibited Chlamydia infectivity of macrophages, with concomitant transcriptional down-regulation of MOMP, its cognate TLR2, and the CD80 co-stimulatory molecule.

Encapsulating proteins in biodegradable polymers has become of major concerns because heat generated during sonication and organic solvents may adversely affect their biological functions. Here we employed ethyl acetate, an organic solvent of PLA, which in the presence of PEG acts as a surfactant in an ethyl acetate-water system29 and PVA as the emulsifying agent for fabricating stable PLA-PEG nanoparticles. PLA-PEG-M278 nanoparticles had a −16 mV zeta potential, 73–100 nm size and ~60% encapsulation efficiency, thus corroborating the > −10 mV zeta potential and > 50% encapsulation efficiency attained for other PLA-PEG nanoparticles fabricated with ethyl acetate28–30. The inability of PLA-PEG-M278 to affect macrophages viability may have resulted from their non-toxic properties or alternatively to their rapid uptake and processing by macrophages, henceforth greater viability. Indeed, subcellular sizes of fabricated PLA-PEG, as reported herein, can facilitate uptake of antigens by APCs6. In addition, the PEGylated brush structures visualized via SEM, most likely contributed to augmenting PLA-PEG-M278 stability by forming a “sheath” like system due to the hydrophobic and hydrophilic domains of the nanoparticle having separate dispositions following lyophilization. Reportedly, this brush structure can prevent adsorption of biological components that trigger the uptake of particulates by macrophages, and aid in the stability of proteins 30,31.

An initial burst release of encapsulated-M278 followed by a gradual slow release of 30–32% over a 20-day period was observed and is consistent with previous reports whereby hepatitis antigen encapsulated within PLA-PEG exhibited a 40% cumulative release over a 42-day period8. We did not investigate the mechanism(s) of encapsulated-M278 release but it may have resulted from degradation of the nanomatrix and diffusion and/or collaborative processes27. Most studies attribute an initial burst release to outer protein adsorption32, however our UV-Vis data supports M278 encapsulation, and further suggests that its initial burst and sustained release may result from the overall degradation kinetics of PLA-PEG. Further support of our data comes from an elegant pharmacokinetics study conducted in mice, which documented that PLA-PEG prolonged the half-life of the antiretroviral drug zidovudine (AZT) delivery over time by increasing its Tmax 33. These investigators also reported that, due to the slow release of AZT from within PLA-PEG, its metabolic breakdown was slower resulting in an increase in both its main half-life and bioavailability33. Performing pharmacokinetics studies of PLA-PEG-M278 in mice are of future investigations in our lab.

Immunization with encapsulated-M278 potentiated M278-specific adaptive immune responses when compared with bare M278. Specifically our observation of heightened levels of Th1 cytokines including IL-2 and especially IFN-γ, is congruent with marked production of IFN-γ by PLA-PEG26. Increased levels of IFN-γ and IL-2 are of significance as they are associated with protection against Chlamydia and other intracellular pathogens including Leishmania major and Mycobacterium tuberculosis34–35. IFN-γ activates Th1 CD4 and CD8 effector T cells that are required for a successful Chlamydia vaccine13 as well as for antigen presentation and lysosome activity in macrophages36. Additionally, IFN-γ has several anti-chlamydial actions in clearing infections by inducing indoleamine-2, 3-dioxgenase (IDO), which lowers the intracellular tryptophan pool necessary for chlamydial metabolism37 and limits chlamydial growth by depleting its intracellular iron37. The impact of IFN-γ for Chlamydia resistance is underscored by lack of protective immunity in IFN-γ receptor-deficient mice17, and for resistance to Chlamydia infections in humans38.

IL-17 production by encapsulated-M278 immune T-cells adds credence to our findings given that it can synergize with IFN-γ for inhibition of Chlamydia lung infections39. Similarly, a Chlamydia vaccine formulated with MOMP, PmpG, PmpE/F-2 and the DDA/TDB adjuvant conferred protection against chlamydial infections, with heightened IFN-γ, IL-17 and TNF-α production by CD4+ T cells35. Noteworthy of mention is triggering of CXCL10 and GM-CSF by encapsulated-M278, which are essential for stimulating cell growth, differentiation of precursors needed by immune cells40, and for their contribution to clearance of Chlamydia infections in mice41–43. Elevated levels of CXCL10 and CCL5 have been noted in the genital tract of Chlamydia-infected mice41, and mice deficient in their putative CXCR3 and CCR5 receptors, respectively are unable to clear their Chlamydia genital tract infections41. Studies also show that topical application of GM-CSF followed by transcutaneous immunization with MOMP protected mice against genital and respiratory tract chlamydial infections, and protection was afforded by IFN-γ and serum antibody42. Likewise, GM-CSF combined with UV-inactivated EBs protected mice against Chlamydia infection43. Overall, our findings support that encapsulated-M278 potentiates T cell-mediated immune responses in mice, especially those that are prerequisites for protection against C. trachomatis.

Encapsulated-M278 triggered high M278-specific Th1 (IgG2a) and Th2 (IgG1 and IgG2b) antibodies in mice, which are congruent with other studies of PLA-PEG as a delivery system for tetanus toxoid and hepatitis B antigens8,32. Seemingly, IgG1 and IgG2b are associated with protection against Chlamydia44,45 although the precise contribution of antibody to Chlamydia protection is still not completely understood. Chief amongst our findings was encapsulated-M278 serum-mediated inhibition of Chlamydia infectivity and/or multiplication in macrophages. We did not explore the definitive role of antibodies in the serum-mediated inhibition of Chlamydia infectivity of macrophages but nonetheless, strong evidence suggests that antibodies could be involved in resistance to reinfection46 or in blocking attachment of C. trachomatis to cells47. In support of this notion is that C. trachomatis pre-coated with IgG2b antibodies and administered intra-vaginally to mice afforded them reduced infection rates48. In the present study, the reduction in MOMP and TLR2 expression in infected macrophages exposed to encapsulated-M278 serum are indicative of less Chlamydia infectivity of macrophages. Conversely, as TLR2, a cognate C. trachomatis receptor is critical for Chlamydia-mediated host cell-activation and MOMP signaling49; the presence of more Chlamydia in macrophages would correlate with higher TLR2 and MOMP expressions as observed in macrophages exposed to bare M278 or control sera. The low level of CD80 expression, that is critical for T cell-activation, also agrees with results obtained in CD80/CD86 knockout mice, which are resistant to a primary C. trachomatis infection50. Therefore, encapsulated-M278 immunization elicited functional immune serum due to the transcriptional down-regulation of MOMP and TLR2, as markers of C. trachomatis infectivity.

In summary, this is the first report, to our knowledge, to employ PLA-PEG as a vaccine-delivery system for C. trachomatis MOMP or its peptide derivative. We demonstrate that immunization with encapsulated-M278 promotes enhanced adaptive immune responses and serum-mediated inhibition of Chlamydia infectivity in macrophages, which are all desirable for protection against this pathogen. Our data further provides compelling evidence that PLA-PEG holds strong promise as a vaccine-delivery system for not only C. trachomatis but also other intracellular pathogens. The potential of PLA-PEG for vaccine delivery undeniably resides in its inherent potentiating and controlled slow release effects to bolster immune responses.

Supplementary Material

01

Acknowledgments

Special thanks go to Yvonne Williams, Lashaundria Lucas, Juwana Smith Henderson and Maiya Moore for their excellent administrative assistance; Eva Dennis for the graphical abstract, and Michael Miller, Auburn University, for assistance with SEM and TEM imaging.

Financial support: This research was supported by National Science Foundation-CREST (HRD-1241701), NSF-HBCU-UP (HRD-1135863) and National Institutes of Health-MBRS-RISE (1R25GM106995-01) grants.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

All authors declare no conflict of interest related to the submitted manuscript.

An abstract of this manuscript was presented at the TechConnect World Conference, May 11–16, 2013, Washington, DC, and at the 113th American Society for Microbiology General Meeting, May 18–21, 2013, Denver, CO.

References

  • 1.Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces. 2010;75:1–18. doi: 10.1016/j.colsurfb.2009.09.001. [DOI] [PubMed] [Google Scholar]
  • 2.Csaba N, Garcia-Fuentes M, Alonso MJ. Nanoparticles for nasal vaccination. Adv Drug Deliv Rev. 2009;61:140–157. doi: 10.1016/j.addr.2008.09.005. [DOI] [PubMed] [Google Scholar]
  • 3.Kalkanidis M, Pietersz GA, Xiang SD, Mottram PL, Crimeen-Irwin B, Ardipradja K, Plebanski M. Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity. Methods. 2006;40:20–29. doi: 10.1016/j.ymeth.2006.05.018. [DOI] [PubMed] [Google Scholar]
  • 4.Irache JM, Esparza I, Gamazo C, Agüeros M, Espuelas S. Nanomedicine: novel approaches in human and veterinary therapeutics. Vet Parasitol. 2011;180:47–71. doi: 10.1016/j.vetpar.2011.05.028. [DOI] [PubMed] [Google Scholar]
  • 5.Ishihara T, Takahashi M, Higaki M, Mizushima Y, Mizushima T. Preparation and characterization of a nanoparticulate formulation composed of PEG-PLA and PLA as anti-inflammatory agents. Int J Pharm. 2010;385:170–175. doi: 10.1016/j.ijpharm.2009.10.025. [DOI] [PubMed] [Google Scholar]
  • 6.De Souza Rebouças J, Esparza I, Ferrer M, Sanz ML, Irache JM, Gamazo C. Nanoparticulate adjuvants and delivery systems for allergen immunotherapy. J Biomed Biotechnol. 2012;2012:474605. doi: 10.1155/2012/474605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Essa S, Louhichi F, Raymond M, Hildgen P. Improved antifungal activity of itraconazole-loaded PEG/PLA nanoparticles. J Microencapsul. 2013;30:205–217. doi: 10.3109/02652048.2012.714410. [DOI] [PubMed] [Google Scholar]
  • 8.Jain AK, Goyal AK, Gupta PN, Khatri K, Mishra N, Mehta A, Mangal S, Vyas SP. Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B. J Control Release. 2009;136:161–169. doi: 10.1016/j.jconrel.2009.02.010. [DOI] [PubMed] [Google Scholar]
  • 9.Sheringham J, Mann S, Simms I, Stafford M, Hart GJ, Raine R. It matters what you measure: a systematic literature review examining whether young people in poorer socioeconomic circumstances are more at risk of chlamydia. Sex Transm Infect. 2013;89:175–180. doi: 10.1136/sextrans-2011-050223. [DOI] [PubMed] [Google Scholar]
  • 10.Hafner L, Beagley K, Timms P. Chlamydia trachomatis infection: host immune responses and potential vaccines. Mucosal Immunol. 2008;1:116–130. doi: 10.1038/mi.2007.19. [DOI] [PubMed] [Google Scholar]
  • 11.Singh SR, Hulett K, Pillai SR, Dennis VA, Oh MK, Scissum-Gunn K. Mucosal immunization with recombinant MOMP genetically linked with modified cholera toxin confers protection against Chlamydia trachomatis infection. Vaccine. 2006;24:1213–1224. doi: 10.1016/j.vaccine.2005.08.097. [DOI] [PubMed] [Google Scholar]
  • 12.Champion CI, Kickhoefer VA, Liu G, Moniz RJ, Freed AS, Bergmann LL, Vaccari D, Raval-Fernandes S, Chan AM, Rome LH, Kelly KA. A vault nanoparticle vaccine induces protective mucosal immunity. PLoS One. 2009;4:e5409. doi: 10.1371/journal.pone.0005409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliv Rev. 2008;60:915–928. doi: 10.1016/j.addr.2007.05.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sandhya M, Srinivas P and Sadanandam M. Nanoparticulate systems and other novel approaches in the diagnosis, prevention and treatment of hiv/aids. Int. J. Pharm. Sci. 2010;2:738–749. [Google Scholar]
  • 15.Taha MA, Singh SR, Hulett K, Pillai SR, Agee R, Dennis VA. A peptide containing T-Cell epitopes of Chlamydia trachomatis recombinant MOMP induces systemic and mucosal antibody responses in mice. W. J. V. 2011;1:138–147. [Google Scholar]
  • 16.Taha MA, Singh SR, Dennis VA. Biodegradable PLGA 85/15 nanoparticles as a delivery vehicle for Chlamydia trachomatis recombinant MOMP-187 peptide. Nanotechnology. 2012;23:325101. doi: 10.1088/0957-4484/23/32/325101. [DOI] [PubMed] [Google Scholar]
  • 17.Fairley SJ, Singh SR, Yilma AN, Waffo AB, Subbarayan P, Dixit S, Taha MA, Cambridge CD, Dennis VA. Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper-1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine. Int J Nanomedicine. 2013;8:2085–2099. doi: 10.2147/IJN.S44155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Cambridge CD, Singh SR, Waffo AB, Fairley SJ, Dennis VA. Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles. Int J Nanomedicine. 2013;8:1759–1771. doi: 10.2147/IJN.S42723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yilma AN, Singh SR, Dixit S, Dennis VA. Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis. Int J Nanomedicine. 2013;8:2421–2432. doi: 10.2147/IJN.S44090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Gautam A, Dixit S, Embers M, Gautam R, Philipp MT, Singh SR, Morici L, Dennis VA. Different patterns of expression and of IL-10 modulation of inflammatory mediators from macrophages of Lyme disease-resistant and -susceptible mice. PLoS One. 2012;7:e43860. doi: 10.1371/journal.pone.0043860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yilma AN, Singh SR, Morici L, Dennis VA. Flavonoid Naringenin: A Potential Immunomodulator for Chlamydia trachomatis Inflammation. Mediators Inflamm. 2013;2013:102457. doi: 10.1155/2013/102457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yilma AN, Singh SR, Fairley SJ, Taha MA, Dennis VA. The anti-inflammatory cytokine, interleukin-10, inhibits inflammatory mediators in human epithelial cells and mouse macrophages exposed to live and UV-inactivated Chlamydia trachomatis. Mediators Inflamm. 2012;2012:520174. doi: 10.1155/2012/520174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered controlled release vesicles. J Control Release. 2004;96:37–53. doi: 10.1016/j.jconrel.2003.12.021. [DOI] [PubMed] [Google Scholar]
  • 24.Waeckerle-Men Y, Gander B, Groettrup M. Delivery of tumor antigens to dendritic cells using biodegradable microspheres. Methods Mol Med. 2005;109:35–46. doi: 10.1385/1-59259-862-5:035. [DOI] [PubMed] [Google Scholar]
  • 25.Wu F, Jin T. Polymer-based sustained-release dosage forms for protein drugs, challenges, and recent advances. AAPS PharmSciTech. 2008;9:1218–1229. doi: 10.1208/s12249-008-9148-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Gutierro I, Hernández RM, Igartua M, Gascón AR, Pedraz JL. Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres. Vaccine. 2002;21:67–77. doi: 10.1016/s0264-410x(02)00435-8. [DOI] [PubMed] [Google Scholar]
  • 27.Xiao RZ, Zeng ZW, Zhou GL, Wang JJ, Li FZ, Wang AM. Recent advances in PEGPLA block copolymer nanoparticles. Int J Nanomedicine. 2010;5:1057–1065. doi: 10.2147/IJN.S14912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wang B, Jiang W, Yan H, Zhang X, Yang L, Deng L, Singh GK, Pan J. Novel PEG-graft-PLA nanoparticles with the potential for encapsulation and controlled release of hydrophobic and hydrophilic medications in aqueous medium. Int J Nanomedicine. 2011;6:1443–1451. doi: 10.2147/IJN.S19765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Makoto M, Ken H, Tsutomu O. PEG–PLA nanoparticles prepared by emulsion solvent diffusion using oil-soluble and water-soluble PEG–PLA. Materials Letters. 2010;64:969–971. [Google Scholar]
  • 30.Vila A, Sánchez A, Tobío M, Calvo P, Alonso MJ. Design of biodegradable particles for protein delivery. J Control Release. 2002;78:15–24. doi: 10.1016/s0168-3659(01)00486-2. [DOI] [PubMed] [Google Scholar]
  • 31.Vonarbourg A, Passirani C, Saulnier P, Benoit JP. Parameters influencing the stealthiness of colloidal drug delivery systems. Biomaterials. 2006;27:4356–4373. doi: 10.1016/j.biomaterials.2006.03.039. [DOI] [PubMed] [Google Scholar]
  • 32.Alonso MJ, Gupta RK, Min C, Siber GR, Langer R. Biodegradable microspheres as controlled-release tetanus toxoid delivery systems. Vaccine. 1994;12:299–306. doi: 10.1016/0264-410x(94)90092-2. [DOI] [PubMed] [Google Scholar]
  • 33.Mainardes RM, Khalil NM, Gremião MP. Intranasal delivery of zidovudine by PLA and PLA-PEG blend nanoparticles. Int J Pharm. 2010;395:266–271. doi: 10.1016/j.ijpharm.2010.05.020. [DOI] [PubMed] [Google Scholar]
  • 34.Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ, Hoff ST, Andersen P, Reed SG, Morris SL, Roederer M, Seder RA. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med. 2007;13:843–850. doi: 10.1038/nm1592. [DOI] [PubMed] [Google Scholar]
  • 35.Forbes EK, Sander C, Ronan EO, McShane H, Hill AV, Beverley PC, Tchilian EZ. Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice. J Immunol. 2008;181:4955–4964. doi: 10.4049/jimmunol.181.7.4955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Morré SA, Karimi O, Ouburg S. Chlamydia trachomatis: identification of susceptibility markers for ocular and sexually transmitted infection by immunogenetics. FEMS Immunol Med Microbiol. 2009;55:140–153. doi: 10.1111/j.1574-695X.2009.00536.x. [DOI] [PubMed] [Google Scholar]
  • 37.Hu VH, Holland MJ, Burton MJ. Trachoma: protective and pathogenic ocular immune responses to Chlamydia trachomatis. PLoS Negl Trop Dis. 2013;7:e2020. doi: 10.1371/journal.pntd.0002020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Karunakaran KP, Yu H, Foster LJ, Brunham RC. Development of a Chlamydia trachomatis T-cell Vaccine. Hum Vaccin. 2010;6:676–680. doi: 10.4161/hv.6.8.12299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhang Y, Wang H, Ren J, Tang X, Jing Y, Xing D, Zhao G, Yao Z, Yang X, Bai H. IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth. PLoS One. 2012;7:e39214. doi: 10.1371/journal.pone.0039214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science. 2010;327:291–295. doi: 10.1126/science.1183021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Olive AJ, Gondek DC, Starnbach MN. CXCR3 and CCR5 are both required for T-cell-mediated protection against Chlamydia trachomatis infection in the murine genital mucosa. Mucosal Immunol. 2011;4:208–216. doi: 10.1038/mi.2010.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Hickey DK, Bao S, Ikeda LT, Carey AJ, Beagley KW. Induction of anti-chlamydial mucosal immunity by transcutaneous immunization is enhanced by topical application of GM-CSF. Curr Mol Med. 2005;5:599–605. doi: 10.2174/1566524054863906. [DOI] [PubMed] [Google Scholar]
  • 43.Lu H, Xing Z, Brunham RC. GM-CSF transgene-based adjuvant allows the establishment of protective mucosal immunity following vaccination with inactivated Chlamydia trachomatis. J Immunol. 2002;169:6324–6331. doi: 10.4049/jimmunol.169.11.6324. [DOI] [PubMed] [Google Scholar]
  • 44.Hansen J, Jensen KT, Follmann F, Agger EM, Theisen M, Andersen P. Liposome delivery of Chlamydia muridarum major outer membrane protein primes a Th1 response that protects against genital chlamydial infection in a mouse model. J Infect Dis. 2008;198:758–767. doi: 10.1086/590670. [DOI] [PubMed] [Google Scholar]
  • 45.Morrison SG, Morrison RP. A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection. J Immunol. 2005;175:7536–7542. doi: 10.4049/jimmunol.175.11.7536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Igietseme JU, Eko FO, He Q, Black CM. Antibody regulation of T-cell immunity: implications for vaccine strategies against intracellular pathogens. Expert Rev Vaccines. 2004;3:23–34. doi: 10.1586/14760584.3.1.23. [DOI] [PubMed] [Google Scholar]
  • 47.Pal S, Theodor I, Peterson EM, de la Maza LM. Monoclonal immunoglobulin A antibody to the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar protects mice against a chlamydial genital challenge. Vaccine. 1997;15:575–582. doi: 10.1016/s0264-410x(97)00206-5. [DOI] [PubMed] [Google Scholar]
  • 48.Peterson EM, Cheng X, Motin VL, de la Maza LM. Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection. Infect Immun. 1997;65:2693–2699. doi: 10.1128/iai.65.7.2693-2699.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Massari P, Toussi DN, Tifrea DF, de la Maza LM. Toll-like receptor 2-dependent activity of native major outer membrane protein proteosomes of. Chlamydia trachomatis. Infect Immun. 2013;81:303–310. doi: 10.1128/IAI.01062-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Chen L, Cheng W, Shivshankar P, Lei L, Zhang X, Wu Y, Yeh IT, Zhong G. Distinct roles of CD28- and CD40 ligand-mediated costimulation in the development of protective immunity and pathology during Chlamydia muridarum urogenital infection in mice. Infect Immun. 2009;77:3080–3089. doi: 10.1128/IAI.00611-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS572505-supplement-01.docx (19.5KB, docx)

RESOURCES