Protein-coated nanoparticles are internalized by the epithelial cells of the female reproductive tract and induce systemic and mucosal immune responses.

The female reproductive tract (FRT) includes the oviducts (fallopian tubes), uterus, cervix and vagina. A layer of columnar epithelium separates the endocervix and uterus from the outside environment, while the vagina is lined with stratified squamous epithelium. The mucosa of the FRT is exposed to antigens originating from microflora, and occasionally from infectious microorganisms. Whether epithelial cells (ECs) of the FRT take up (sample) the lumen antigens is not known. To address this question, we examined the uptake of 20-40 nm nanoparticles (NPs) applied vaginally to mice which were not treated with hormones, epithelial disruptors, or adjuvants. We found that 20 and 40 nm NPs are quickly internalized by ECs of the upper FRT and within one hour could be observed in the lymphatic ducts that drain the FRT, as well as in the ileac lymph nodes (ILNs) and the mesenteric lymph nodes (MLNs). Chicken ovalbumin (Ova) conjugated to 20 nm NPs (NP-Ova) when administered vaginally reaches the internal milieu in an immunologically relevant form; thus vaginal immunization of mice with NP-Ova induces systemic IgG to Ova antigen. Most importantly, vaginal immunization primes the intestinal mucosa for secretion of sIgA. Sub-cutaneous (s.c) boosting immunization with Ova in complete Freund's adjuvant (CFA) further elevates the systemic (IgG1 and IgG2c) as well as mucosal (IgG1 and sIgA) antibody titers. These findings suggest that the modes of antigen uptake at mucosal surfaces and pathways of antigen transport are more complex than previously appreciated

Dissemination of Chlamydia from the reproductive tract to the gastro-intestinal tract occurs in stages and relies on Chlamydia transport by host cells.

Chlamydia trachomatis is a Gram-negative bacterial pathogen and a major cause of sexually transmitted disease and preventable blindness. In women, infections with C. trachomatis may lead to pelvic inflammatory disease (PID), ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting the female reproductive tract (FRT), Chlamydia spp. are routinely found in the gastro-intestinal (GI) tract of animals and humans and can be a reservoir for reinfection of the FRT. Whether Chlamydia disseminates from the FRT to the GI tract via internal routes remains unknown. Using mouse-specific C. muridarum as a model pathogen we show that Chlamydia disseminates from the FRT to the GI tract in a stepwise manner, by first infecting the FRT-draining iliac lymph nodes (ILNs), then the spleen, then the GI tract. Tissue CD11c+ DCs mediate the first step: FRT to ILN Chlamydia transport, which relies on CCR7:CCL21/CCL19 signaling. The second step, Chlamydia transport from ILN to the spleen, also relies on cell transport. However, this step is dependent on cell migration mediated by sphingosine 1-phosphate (S1P) signaling. Finally, spleen to GI tract Chlamydia spread is the third critical step, and is significantly hindered in splenectomized mice. Inhibition of Chlamydia dissemination significantly reduces or precludes the induction of Chlamydia-specific serum IgG antibodies, presence of which is correlated with FRT pathology in women. This study reveals important insights in context of Chlamydia spp. pathogenesis and will inform the development of therapeutic targets and vaccines to combat this pathogen

Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally

Nanoparticles (NPs) are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n.) priming followed by i.n. or subcutaneous (s.c.) boosting immunization affects the humoral immune response to chicken ovalbumin (Ova) and Ova conjugated to 20 nm NPs (NP-Ova). We show that i.n. priming with 20 mg of soluble Ova, a dose known to trigger oral tolerance when administered via gastric gavage, induced substantial systemic IgG1 and IgG2c, as well as mucosal antibodies. These responses were further boosted following a s.c. immunization with Ova and complete Freund’s adjuvant (Ova+CFA). In contrast, 100 µg of Ova delivered via NPs induced an IgG1-dominated systemic response, and primed the intestinal mucosa for secretion of IgA. Following a secondary s.c. or i.n. immunization with Ova+CFA or NP-Ova, systemic IgG1 titers significantly increased, and serum IgG2c and intestinal antibodies were induced in mice primed nasally with NP-Ova. Only Ova- and NP-Ova-primed mice that were s.c.-boosted exhibited substantial systemic and mucosal titers for up to 6 months after priming, whereas the antibodies of i.n.-boosted mice declined over time. Our results indicate that although the amount of Ova delivered by NPs was 1000-fold less than Ova delivered in soluble form, the antigen-specific antibody responses, both systemic and mucosal, are essentially identical by 6 months following the initial priming immunization. Additionally, both i.n.- and s.c.-boosting strategies for NP-Ova-primed mice were capable of inducing a polarized Th1/Th2 immune response, as well as intestinal antibodies; however, it is only by using a heterogeneous prime-boost strategy that long-lasting antibody responses were initiated. These results provide valuable insight for future mucosal vaccine development, as well as furthering our understanding of mucosal antibody responses

Vaginally applied NPs reach the lumen of the uterus and are internalized by uterine ECs.

<p>(A) H&E stained tissue section of a mouse uterus; (B) An IFM image of the uterine tissue section 3 h after intra-vaginal administration of 20 nm fluorescent NPs (red). (C, D) Two-color and (E) three color higher magnification IFM image of the uterine epithelium shown in the boxed inset from panel B. (B–E) Tissue sections were stained with actin-binding phalloidin-Alexa350 (blue) and anti-E-cadherin antibodies (green), while fluorescent NPs are shown in red. En-endometrium, My-myometrium, L-lumen, G- mucus glands of the uterus.</p

Intestinal IgA responses in mice primed p.o. with soluble Ova, NP-Ova or PBS.

<p>Mice were administered PBS (control), soluble Ova, or 20 nm NP-Ova p.o. at day 0, 3, 6 and 8, then s.c. boosted with 300 μg Ova+CFA at day 28 (arrow). Fecal extracts were assayed for Ova-specific IgA using ELISA assay. Ova-specific IgA titers are expressed as log₁₀ titer values, with the titer being the highest dilution showing an absorbance value twice that of the background. Data collected from 10 mice per group (2 separate experiments) are expressed as the mean ± SD of the mean. Group means were separated using Tukey’s multiple comparison procedure and were declared significantly different at p<0.05 (*) and p<0.01 (***).</p

NPs instilled vaginally are internalized by vaginal ECs and drain from the FRT via the lymphatics.

<p>(A) A three color IFM image of a vaginal tissue section. (B) An image of the H&E stained vaginal tissue section. (C) A three-color IFM image of the vaginal tissue section depicting red NPs within the vaginal epithelium (Ve) (arrows), and in the sub-epithelial compartment (circled) near CD11c+ DCs (green). (D) A two-color IFM high magnification (630x) image of the vaginal epithelium (blue) harboring internalized NPs (red). (E) A three color IFM image of a vaginal tissue section showing the NPs (red) draining from the vaginal tissue via the lymphatics of the FRT (green). (F) A three-color high magnification image of the boxed inset from panel E showing NPs (red) draining from the FRT. (G, H) Two- and three-color IFM images of lymph ducts draining from the FRT and carrying NPs (red). (A, C–H) Vaginal tissue sections were stained with actin-binding phalloidin-Alexa350 (blue), in combination with (A) E-cadherin (green), (C) CD11c (green), and (E, H) Lyve-1 (green) antibodies, while fluorescent NPs are shown in red. (A–E) Ve-vaginal epithelium, L-vaginal lumen.</p

Antibody titers in sera of mice following priming vaginal immunization with 20 nm NP-Ova and s.c. boosting immunization with Ova in CFA.

<p>Mice were immunized vaginally with 20 nm NP-Ova or PBS (control), then s.c. boosted with 300 µg Ova with CFA at day 28 after priming immunization (arrows). (A) Ova-specific IgG1 and (B) IgG2c antibody titers are expressed as log₁₀ titer values, with the titer being the highest dilution that yielded an OD₄₀₅ absorbance value two times that of the negative control. Values represent the mean ± SD of samples collected from 14 mice per group (3 separate experiments). Group means were separated using Student's t-test and declared significantly different at a P<0.05. Asterisks indicate significant differences between treatment means.</p

NPs distributed within an ILN 5 h after intra-vaginal administration.

<p>(A) A three-color stitched IFM image showing an ILN harboring NPs (red). Two- and three-color higher magnification (630x) IFM images of the top (B, C), middle (D, E) and bottom (F, G) box insets from panel A showing 20 nm NPs (red) within the ILN. Tissue sections were stained with actin-binding phalloidin-Alexa350 (blue) and Lyve-1 antibodies (green), while fluorescent NPs are shown in red.</p

Antibody titers of mice vaginally-primed with 20 nm NP-Ova or PBS (control) and s.c. boosted with 300 µg Ova with CFA at day 28 after priming immunization (arrow).

<p>(A) IgG1 antibody titers in vaginal washings. Antibody titers are expressed as log₁₀ titer values, with the titer being the highest dilution that yielded an OD₄₀₅ absorbance value two times that of the negative control. Values represent the mean ± SD of samples collected from 14 mice per group (3 separate experiments). Group means were separated using Student's t-test and declared significantly different at a P<0.05. Asterisks indicate significantly differences between treatment means. (B) Serum, intestinal, and FRT antibody titers of mice that were fitted with Elizabethan neck collars. Values represent the mean ± SD of samples collected from 6 mice per group.</p

Antibody responses in sera of mice primed p.o. with soluble Ova, NP-Ova or PBS.

<p>Mice were administered PBS (control), soluble Ova, or 20 nm NP-Ova p.o. at day 0, 3, 6 and 8, then s.c. boosted with 300 μg Ova+CFA at day 28 (arrow). Ova-specific serum IgG1 (A) and IgG2c (B) antibody titers are expressed as log₁₀ titer values, with the titer being the highest dilution showing an absorbance value twice that of the background. Data collected from 10 mice per group (2 separate experiments) are expressed as the mean ± SD of the mean. Group means were separated using Tukey’s multiple comparison procedure and were declared significantly different at p<0.05 (*) and p<0.01 (***).</p