Enhanced immune responses to vaccine antigens in the corneal stroma

Abstract

To examine the widely accepted dogma that the eye is an immune-privileged organ that can suppress antigen immunogenicity, we explored systemic immune responses to a model vaccine antigen (tetanus toxoid) delivered to six compartments of the rodent eye (ocular surface, corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, vitreous body). We discovered that antigens delivered to corneal stroma induced enhanced, rather than suppressed, antigen-specific immune responses, which were 18- to 30-fold greater than conventional intramuscular injection and comparable to intramuscular vaccination with alum adjuvant. Systemic immune responses to antigen delivered to the other ocular compartments were much weaker. The enhanced systemic immune responses after intrastromal injection were related to a sequence of events involving the formation of an antigen “depot” in the avascular stroma, infiltration of antigen-presenting cells, up-regulation of MHC class II and costimulatory molecules CD80/CD86, and induction of lymphangiogenesis in the corneal stroma facilitating sustained presentation of antigen to the lymphatic system. These enhanced immune responses in corneal stroma suggest new approaches to medical interventions for ocular immune diseases and vaccination methods.

1. Introduction

It is widely accepted that the eye has developed immune privilege as a protective mechanism in which immune responses to endogenous and exogenous antigens are suppressed and sometimes completely inhibited [1, 2]. Ocular immune privilege protects the eye from generating severe immune response to pathogens and deleterious ocular inflammation, which can distort vision. This immune privilege is believed to be related to a lack of direct blood and lymphatic drainage from the interior of the eye, immunological ignorance, active immunosuppressive mechanisms by immunomodulatory molecules, and the antigen-specific down-regulation of delayed-type hypersensitivity response to antigens introduced into the eye (termed anterior chamber-associated immune deviation (ACAID)) [3, 4].

Despite the ocular immune privilege, autoimmune and immune-mediated ocular diseases occur with high frequency, including autoimmune keratitis, inflammatory uveitis, dry eye disease, age-related macular degeneration, and autoimmune retinopathy [5, 6]. These diseases are likely related to the aberrant reaction of the immune system to self-antigens or foreign antigens localized to the ocular surface and associated tissues [5]. However, the unique ocular immune responses to antigens and their related mechanisms are still poorly understood. The understanding of ocular immunology and the potential role of immune mechanisms in pathology are reflected in our evolving understanding of the molecular and cellular processes underpinning immunological responses, and can facilitate the development of new therapeutics for the treatment of ocular diseases [7, 8].

Prior studies suggest that immune responses may differ in different compartments of the eye. For example, antigen injected into the anterior chamber can lead to immune tolerance, by a mechanism believed to involve antigen presentation in a tolerogenic fashion in the spleen [9]. This finding was further demonstrated when injection of S antigen (a retinal soluble antigen) into the anterior chamber of rats markedly reduced the incidence of experimental autoimmune uveitis due to ACAID induction by the S antigen [10]. Even though, in another study, tumor antigen in the anterior chamber was found to drain exclusive to submandibular lymph nodes, leading to activation of tumor-specific cytotoxic T cell reaction, with no antigen drainage found in the spleen [11]. While the vitreous cavity is known as an immunologically privileged site [12], foreign proteins introduced into the vitreous body of the rabbit generated an inflammatory response extending to various ocular tissues and stimulating antigen-specific antibody formation [13]. Gene therapy administered to the eye via intravitreal, subretinal, and suprachoroidal routes has a risk of serious ocular inflammation and local or systemic immune responses to the delivery vectors [8]. Conjunctival immunization with TT alone or with various adjuvants induced TT-specific local and systemic immune responses [14].

The cornea’s immune privilege has enabled corneal transplants to be the most widely used and most successful form of solid organ transplantation [15]. This “immune privilege” is not only due to the physical barrier function of the mucosal lining but also actively maintained through a variety of immunoregulatory mechanisms that prevent the disruption of immune homeostasis [16]. The absence of lymphatic-drainage pathways in the cornea is important for shielding ocular antigens from the immune system and keeping effector cells from reaching the graft [17]. The ACAID-related tolerance, the role of regulatory T (T_reg) cells, immunological ignorance, and active immunosuppressive mechanism promote corneal immune privilege [3, 18].

However, the immune privilege of the cornea recently has been challenged by the discovery of unique immune cells in the cornea, such as resident memory T cells [19] and antigen-presenting cells [20]. The immune privilege and immunosuppressive microenvironment of the cornea can be compromised when challenged with inflammation, infection, or injury [21]. For example, corneal grafts in a host cornea with neovascularization due to inflammation have a much higher incidence of rejection, creating a high-risk setting [22]. Another example is herpes keratitis caused by infection of herpes simplex virus (HSV) in the cornea, which can lead to serum antibodies against HSV that can persist even after the virus has been cleared, as well as T-cell-mediated inflammation in the corneal stroma [23]. Compromised ocular immune privilege in the cornea can also be seen by the formation of “Wessely rings” of immune infiltrate in the cornea after corneal infections as well as intracorneal injection of serum albumin [24, 25].

Given the above findings, we hypothesized that the nature of the immune response to antigens in the eye depends on the site of antigen administration, type of antigen (e.g., involving toll-like receptor (TLR) activation), and route of antigen presentation. Because immune responses to antigens administered to different compartments of the eye have not been extensively compared before, we sought to systematically study the immune response to foreign antigens delivered to six different parts of the eye: topical application to the ocular surface, as well as injection into the corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space and vitreous body (Fig. S1 and Fig.S2). We conducted this study using model vaccine antigens (i.e., TT and subunit influenza vaccines) and compared systemic immune responses after ocular delivery to intramuscular delivery, which is the conventional route of administration for these vaccines. We are not aware of any prior study that has directly compared systemic immune responses among a collection of different sites in the eye, and for some of these ocular compartments, immune responses have not been studied before at all.

2. Experimental section

2.1. Animals

All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines of the Georgia Institute of Technology. The adult male Wistar rats (250–300 g) were supplied by Charles River Laboratories (Wilmington, MA). The animals were kept in a 12 h/12 h light/dark cycle at the animal care facility, given free access to diet and water, and acclimatized for at least 7 days before the experiments.

2.2. Reagents and Antibodies

Tetanus toxoid monobulk (MW≈150kDa) without aluminum adjuvant was kindly provided by Serum Institute of India (Pune, India). This monobulk contained the same vaccine antigen as found in the tetanus toxoid vaccine sold by Serum Institute of India. Influenza vaccine antigen (B/Brisbane/60/08) was kindly provided by Seqirus (Maidenhead, UK). This monobulk contained the same vaccine antigen as found in the inactivated influenza virus vaccine sold by Seqirus (Fluvirin). The liquid monobulk B/Brisbane/60/08 was concentrated by 100-fold and buffer-exchanged with phosphate-buffered saline using ultrafiltration (Vivaspin^® 20, 30,000 MWCO, Sartorius, Germany) before use. Bovine serum albumin (BSA), DNase I and collagenase D were purchased from Sigma-Aldrich (St. Louis, MO). Vectashield antifade mounting medium with DAPI was obtained from Vector Laboratories (CA, USA). CD45R (B220) antibody (Alexa Fluor 647 label) and anti-rat CD4 antibody (FITC label and 10% neutral buffered formalin were purchased from BioLegend (San Diego, CA). The rat IgG, IgG1, IgG2a, IgG2c, and corresponding HRP-linked mouse anti-rat IgG, IgG1, IgG2a, and IgG2c were purchased from Southern Biotech (Birmingham, AL). Alexa Fluor 750 NHS ester, Alexa Fluor 555 NHS ester, normal goat serum (10%), EDTA (0.5 M), anti-rat CD80 (PE label), anti-rat CD86 (FITC label), anti-rat MHC class II (FITC label), Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) recombinant rabbit anti-rat monoclonal antibody (JF0979), goat anti-mouse IgGs (Alexa Fluor 635 and 488 labeled), and goat anti-rabbit IgGs (Alexa Fluor 405 and 647 labeled) were purchased from ThermoFisher Scientific (eBioscience, Waltham, MA).

2.3. Vaccine administration and tissue collection

The injection procedures were optimized before vaccine administration (Fig. S1). At week 0, each group of rats (6 rats per group) were anesthetized with isoflurane and then given 4 Lf (i.e., 10 μg) TT vaccine by injections to the corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, or vitreous body under stereomicroscope-guided imaging (M80, Leica, Germany). The injections to the corneal stroma, anterior chamber, subconjunctival space, and vitreous body were performed using a sub-microliter injection system (Nanofil, Sarasota, FL) and a 36 G, 35 G, or 34 G, beveled needle (Nanofil) while viewing under a surgical microscope. Suprachoroidal space injection was performed with a custom-made 100 μm-length glass microneedle which was guided across the sclera to reach the suprachoroidal space using a 3D-printed micro-positioner to create a microscopic puncture. Another group was given the same dose by topical eye drops (10 μL). The same dose of TT vaccine with or without alum adjuvant administered by intramuscular injection in 10 μL to the biceps femoris was used as control. On week 4, a booster dose was administered by the same route of administration to each animal following the same procedures. A naïve group that received no injection was used as a negative control. Because the immune response to TT is antigen-specific, we did not think a saline injection group was necessary in addition to a naïve control. The procedures for all the injections are detailed in the Supplementary Information. The timeline for TT vaccine administration to the rat eye and blood sample collection is shown in Fig. S2.

Blood samples were collected on weeks 3, 5, and 8. The serum was separated by centrifugation at 8160 x g for 5 min for antibody analysis. On week 12, the rats were euthanized by carbon dioxide inhalation, after which the spleen, and the right, and left ipsilateral mandibular lymph nodes were collected.

To study whether local immunological reactions in corneal stroma induced by injection of an antigen improved immune responses to TT vaccine, the cornea was primed by influenza vaccine delivery to induce a local immunological reaction before injection of TT vaccine antigen. In another experiment, the rats were divided into 3 groups (6 rats per group). At week 0, two groups were primed with influenza vaccine (10 μg) by intrastromal injection or intramuscular injection, a third group was untreated. On week 1, all the three groups were given 4 Lf TT vaccine by intrastromal injection (i.e., to the same eye for the group that received intrastromal influenza vaccination). Blood samples were collected at week 3, from which serum was separated for antibody analysis.

2.4. Anti-TT specific antibody analysis

The anti-TT-specific antibody levels were quantified by ELISA. Briefly, to capture TT-specific antibodies, Nunc Maxisorb 96-well plates (ThermoFisher Scientific, Waltham, MA) were incubated with 100 μL pH 9.6 carbonate buffer solution containing 4 μg/mL TT at 4°C overnight, then blocked with 5% BSA in carbonate buffer solution. Rat Anti-TT IgG, IgG1, IgG2a, and IgG2c isotypes were quantified from the standard curves generated with corresponding purified rat immunoglobulin isotypes and isotype-specific HRP-labeled anti-rat antibodies. HRP-labeled anti-rat antibodies were diluted with PBS-T (phosphate-buffered saline with 0.1% w/v Tween-20) solution at suitable times to obtain standard curves with a good regression coefficient (R²>0.999) and reproducibility. The serum was diluted for suitable times (Table S1) with PBS-T to make the absorption in an appropriate range of the standard curve. The color was developed by commercial 3,3',5,5'-tetramethylbenzidine (TMB) substrate and stopped by a commercial stop solution (BD Biosciences, San Jose, CA). The absorption of each well was read at 620 nm by iMark microplate absorbance reader (Bio-Rad, Hercules, CA). The concentration of the anti-TT antibodies was calculated based on the corresponding standard curves.

2.5. Splenocyte restimulation and cytokine analysis

Rat spleens collected on week 12 after vaccination were diced and treated with collagenase D for 30 min, then passed through a 40-μm cell strainer (Sigma-Aldrich) to prepare a single-cell suspension with a density of 1×10⁶ cells in 150 μL RPMI media (Invitrogen) per well containing 10% characterized fetal bovine serum (HyClone, Logan, UT), 1% penicillin-streptomycin, 2 mM glutamine, 1x beta-mercaptoethanol, 1 mM sodium pyruvate. Fifty microliters of 20 μg/mL TT vaccine were added to each well. After incubation for 72 h, the supernatant was collected and IFNg, IL-4, and TNF-α were measured by Luminex.

2.6. Elimination of TT vaccine from eye after administration

TT vaccine was tagged with Alexa Fluor 750 following the standard protocol of the manufacturer. Unconjugated dye was removed by ultrafiltration. Four Lf of Alexa Fluor 750-tagged TT was administered to rats following the same procedures as the immunization study. The fluorescence intensity at specified time points was determined by an in vivo imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA). In an in vitro study, we did not find photobleaching of Alexa Fluor 750 over the course of 17 days (Fig. S5). Because the closure of the eyelid could affect measured fluorescence intensity, the eyelids were fully opened before making fluorescence intensity measurements. The percentage of TT vaccine remaining in the eye was calculated by dividing the fluorescence intensity at different time points (t) by the fluorescence intensity at time 0 (i.e., within 5 min) after vaccine administration. The curves for TT vaccine elimination were modeled by Excel using a simple exponential decay model, that is, P(t) = P₀e^−kt, where P(t) and P₀ are the percentages of TT vaccine remaining in the tissue at time t and time 0; k is the decay rate constant.

2.7. Immunofluorescence staining

Alexa Fluor 555-tagged TT vaccine (labeled according to the manufacturer’s protocol) was injected into the rat cornea under anesthesia. The rats were euthanized 7 days post injection. The eyeballs were enucleated and fixed in 10% formalin neutral buffer overnight before staining. Before immunofluorescence staining of the cornea, the corneas were treated with 0.2 mL 0.05% Trypsin for 10 min at 37°C, then transferred into 1 mL methanol for 30 min incubation, followed by incubation in 0.5 mL PBS containing 0.2% Triton-X-100 and 2% BSA for 20 min at room temperature (20 – 25°C). The corneas were then blocked by 0.2 mL 10% goat serum at 4°C overnight.

The immunofluorescence staining of the cornea was performed according to the reference with minor modification [26]. The primary antibodies and secondary antibodies were diluted 100× by 10% serum before use. The corneas were incubated with 0.15 mL primary antibody solutions (mouse anti-rat MHC Class II antibody or rabbit anti-rat LYVE1 antibody) at 4°C overnight, then thoroughly washed by three changes of 1 mL PBS-T shaking 30 min at room temperature. Corneas were blocked again, then incubated with 0.15 mL secondary antibody solutions (goat anti-mouse IgG Alexa Fluor 488 or goat anti-rabbit IgG Alexa Fluor 405) in a black box on a rocking board at 37°C for 3 h. The corneas were washed with 1 mL PBS, shaking at 4°C overnight, then pLaced for additional two times with 1 mL PBS each on a shaking board for 30 min. The corneas were secured with a pair of forceps in one hand, and made 8 slits of symmetric slides around the cornea with a scalpel blade to flatten out the cornea on a glass slide, and then added a drop of Vectashield antifade mounting medium with or without DAPI. The corneal tissue was covered with a glass coverslip and sealed to the slide with colorless nail polish. Tissues were imaged by laser scanning confocal microscope (LSM 700, Carl Zeiss, Germany).

Ipsilateral mandibular lymph nodes were harvested at day 7 post intrastromal injection of Alexa Fluor 555-tagged TT vaccine. The lymph nodes were immersed in a 30% sucrose/ 70% fixative solution (formalin buffer) overnight, and then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, CA) and stored at −20° C until ready for sectioning. Lymph nodes were cut to 20 μm thickness by Cryostat (Leica CM1950, Leica Biosystems, Germany) and mounted onto gelatin or poly-L-lysine coated slides. The sections were washed twice with PBS-T containing 1% goat serum and then blocked by incubating the tissue sections in PBS-T containing 5% goat serum for 30 min at room temperature. Two drops of 5% goat serum containing 10 μg/mL Alexa Fluor 647 anti-rat CD45R (B220) antibody and FITC anti-rat CD4 antibody were added to the tissue sections and then incubated in a wet dish at 4°C overnight. The sections were washed twice with 5 mL 1% goat serum in PBS-T for 10 min each time. A drop of Vectashield antifade mounting medium was added. The sections were covered and sealed to the slide with colorless nail polish, and then imaged by LSM.

2.8. Flow cytometry analysis

The corneas of two groups of rats (3 rats with 6 corneas in each group) were injected with 4 Lf TT vaccine on day 0 or day 5. Another two groups of rats were injected with saline (0.9% NaCl) on day 0 or day 5. A naïve group of 3 rats that received no injection was used as a negative control. On day 7, the rats in all groups were euthanized, and the eyeballs were enucleated. The fresh corneas were harvested immediately. The corneal limbus was removed by a blade, and the sclera was cut away as much as possible without damaging the cornea. The iris was peeled from the back of the cornea under a microscope. Corneas were then cut into small pieces (~2 mm²) using scissors and then digested by DNase I (2 mg) and collagenase D (4 mg) in 1 mL of RPMI-1640 for 60 min at 37°C. Fetal bovine serum (FBS) (200 μL) was added to each tube to stop the digestion. The resulting single-cell suspension was filtered by passing through a 40-μm strainer.

After centrifugation (500 g, 5 min, 4°C), the supernatant was poured off and 2 mL flow cytometry buffer (FACS buffer, PBS containing 0.5-1% BSA) was added. The cell suspension was transferred to a FACS tube (Fisher Scientific, Hampton, NH) and the final cell density was adjusted to approximately 10⁶ cells/mL by adding more FACS buffer. The cell suspension was divided into two suspensions with an equal volume of 1 mL each. After centrifugation, both suspensions were blocked by Fc receptor-blocking antibody, then, one suspension was added with 50 μL of a solution containing 0.5 μg anti-rat CD80 (PE) and anti-rat CD86 (FITC). Another suspension was added with 0.5 μg anti-rat MHC class II (FITC). All the samples were kept at 4°C for 30 min. The unbonded antibodies were removed by centrifugation.

The stained cell suspensions were fixed by CytoFix (Biolegend Fixation Buffer or 4% PFA in PBS) for 10 min at room temperature, and the fixation buffer was then removed by centrifugation (500 g, 5 min, 4°C). The fixed cells were resuspended in 300 μL FACS buffer and analyzed by flow cytometry (BD Fortessa) within one week. The naïve corneas and corneas injected with 0.9% NaCl were used as the controls and processed following the same procedures as the vaccine-injected corneas.

2.9. Clinical observation and histological analysis of the eye

We examined the rat cornea after intrastromal injection of TT vaccine and saline (0.9% NaCl) by stereomicroscope and slit lamp biomicroscope for at least 14 days. At week 12, rats were euthanized. Eyes that had received TT vaccine were enucleated and fixed in 10% formalin neutral buffer. The eyes were then dehydrated by an automatic tissue dehydration system, followed by embedding in paraffin, sectioned at 5 μm thickness by rotary microtome (Leica RM2155, Leica Biosystems, Germany), and stained by hematoxylin and eosin with Leica Autostainer XL following the manufacturer’s protocol (Leica Biosystems, Germany). Three slides per eye (each slide having 8 sections) were stained. All tissue sections were observed and imaged by an inverted microscope (IX73, Olympus Life Science, Tokyo, Japan). We evaluated the cornea, iris, lens, ciliary body, retina, choroid, and optic nerve for inflammation, hemorrhage, and necrosis, and graded each tissue as grade 0 (normal), 1, 2, and 3. A score of 1 was up to 1/3 of the tissue involved, 2 was 1/3-2/3 and 3 was greater than 2/3.

2.10. Statistical analysis

All data presented in this study represent mean ± standard deviation. Statistical analysis was performed using a two-sided Student’s t-test or ANOVA, with the software GraphPad Prism 8.0 (GraphPad, San Diego, CA). A value of p < 0.05 was considered significant.

3. Results

3.1. Injection of a model vaccine antigens into the corneal stroma induces enhanced adaptive immune responses

Our first objective was to study systemic immune responses to the TT vaccine as a model antigen administered to different sites in the eye. We measured serum anti-TT-specific IgG, IgG1, IgG2a, and IgG2c titers for 8 weeks after vaccination. Considering total IgG, antibody responses to vaccination in the eye via anterior chamber, subconjunctival, suprachoroidal space and vitreous body all yielded similar responses that were not significantly different from IM vaccination, which was used as a positive control (p>0.05, Fig. 1, Table S2).

Fig. 1. Humoral immune responses in rats immunized with TT vaccines via different routes.

(A) Rats were immunized with tetanus toxoid vaccine at weeks 0 and 4, and blood samples were withdrawn at weeks 3, 5, and 8 (Fig. S2). Anti-tetanus toxoid-specific IgG (A), IgG1 (B), IgG2a (C), and IgG2c (D) were determined by ELISA. TP: topical drop; IS: intrastromal injection; IC: intracameral injection; SJ: subconjunctival space injection; SCS: suprachoroidal space injection; InVitr: intravitreal injection; IM: intramuscular injection; IM+adj: intramuscular injection antigen with adjuvant. (***p<0.001). Data shown mean ± standard deviation from n=6 independent replicates from separate animals.

Surprisingly, vaccination in the corneal stroma generated anti-TT IgG titers that were significantly higher than all of the other ocular vaccination routes (p<0.001, Fig. 1, Table S2). Vaccination in the corneal stroma was also higher than IM vaccination, measuring 18- to 30-fold higher antibody responses (p<0.001, Fig. 1, Table S2). We also included IM vaccination with TT combined with alum adjuvant, which increased anti-TT IgG titers by 12- to 40-fold compared to unadjuvanted IM vaccination (p<0.001, Fig. 1, Table S2). Remarkably, intrastromal vaccination without adjuvant produced IgG responses as strong as IM vaccination with alum adjuvant (p>0.05, Fig. 1, Table S2). Intrastromal vaccination also generated stronger systemic immune responses as measured by IgG subclasses compared to IM vaccination and the other ocular routes. The IgG1 subclass titers, which accounted for the majority of IgG responses, were 26- to 28-fold higher after intrastromal vaccination compared to the IM route (p<0.001, Fig. 1, Table S3) and were not significantly different compared to adjuvanted IM vaccine (p>0.05, Fig. 1, Table S3). Similarly, intrastromal vaccination generated antibody subclasses IgG2a and IgG2c that were 11- to 26-fold (p<0.001) and 6- to 17-fold (p<0.05) higher than IM vaccination, respectively (Fig. 1, Tables S4 and S5). IgG titers of all three subclasses were similar after administration of unadjuvanted intrastromal vaccine compared to adjuvanted IM vaccine (p>0.05).

To assess antibody classes and their relative abundance, we calculated the IgG2a-to-IgG1 ratio, and found that topical surface antigen administration was inclined to produce IgG2a compared with other administration routes which produced IgG1 predominant subclass (p<0.05, Fig. S3). There was no significant difference in the ratios among other immunization routes. In addition, the adjuvanted IM delivery shifted from IgG2a to IgG1 predominant subclass starting from week 5 (p < 0.05, Fig. S3). Considering the other ocular administration sites, TT vaccination by topical drops produced significantly lower anti-TT IgG titers than intravitreal and IM injections (p<0.05, Fig. 1, Table S2). There was no significant difference in anti-TT IgG, IgG1, and IgG2c titers between intracameral, subconjunctival, suprachoroidal, and IM injections (p>0.05, Fig. 1, Tables S2, S3 and S5). However, intracameral injection produced a significantly lower anti-IgG2a titer than IM delivery (p<0.05, Fig. 1, Table S4).

We next determined if the enhanced immune responses in the corneal stroma can be generalized to other vaccines by injecting an influenza vaccine into the corneal stroma. This analysis similarly found that intrastromal injection produced 21- to 24-fold higher anti-influenza IgG, IgG1, and IgG2a titers compared with IM vaccination, which was measured at week 3 (p<0.001, Fig. 2), demonstrating that the enhanced systemic immune response can be seen with multiple antigens.

Fig. 2. Humoral immune responses in rats immunized with influenza vaccine by intrastromal injection.

Anti-influenza-specific IgG (A), IgG1 (B), IgG2a (C), and IgG2c (D) were determined by ELISA. Data shown mean ± standard deviation from n=6 independent replicates from separate animals. (***p<0.001).

In addition to humoral responses, we also studied whether the route of antigen administration affects cellular immune responses, as determined by levels of cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α (secreted by T helper type 1 (Th1) cells) [27] and interleukin (IL)-4 cytokine (secreted by T helper type 2 (Th2) cells) [28] in the culture supernatants of splenocytes from vaccinated animals after stimulation with TT vaccine. There were significantly higher levels of IFN-γ and TNF-α after intrastromal vaccination than other sites of administration (p<0.05, Fig. 3), while no significant difference was found in IL-4 levels among the ocular and IM vaccination sites (p>0.05, Fig. 3). These findings indicate that intrastromal injection preferably enhanced Th1 cellular immune responses compared with other sites of administration.

Fig. 3. Cytokine levels (IFN-γ, TNF-α, IL-4) secreted by splenocytes after 72 h stimulation with TT vaccine.

Rats were administered the TT vaccine at weeks 0 and 4. The splenocytes were harvested at week 12 (Fig. S2) (* 0.01<p<0.05, ** 0.001<p<0.01, *** p<0.001). Data shown mean ± standard deviation from n=4 independent replicates from separate animals.

3.2. Elimination of TT vaccine antigen from the sites of administration

To understand why immune responses varied among different sites of vaccination, we studied the kinetics of TT clearance from different sites in the eye. At all sites, a vaccine depot was formed at the site of injection immediately after administration, as demonstrated by strong fluorescence from fluorescently tagged TT at the injection site (Fig. 4A, Fig. S4). However, the elimination rate of TT vaccine from the injection sites varied dramatically among the sites of administration.

Fig. 4. Clearance of TT vaccine from the eye after administration to the eye via different routes.

The fluorescence intensity of Alexa Fluor 750-tagged TT was determined by IVIS. (A) Representative IVIS images of eye showing TT distribution in the eye at 0 h (highest fluorescence intensity), 50% TT clearance and 90% TT clearance from the site of administration for intrastromal injection, subconjunctival injection and topical drop administration in rats. The corresponding times after TT administration is shown (* 90% cleared; ^# 97.7% cleared). Additional IVIS imaging of TT clearance after injection by all routes of administration is shown in Fig. S4. (B). The percentage of TT that remained in the eye was calculated by normalizing the fluorescence intensity at different times to the intensity at 0 h to obtain the kinetic curves for the clearance of TT after administration to the eye via different routes. A kinetic curve of TT clearance after IM injection is shown in Fig. S6. Data shown mean ± standard deviation from n=4 independent replicates from separate animals.

The fastest elimination of TT occurred after topical administration, where 90% of TT was eliminated within 0.5 h (Fig. 4B, Table S6). This rapid clearance is consistent with known rapid tear fluid turnover via drainage through nasolacrimal ducts to the nasal cavity [29]. In our study, fluorescence signal (Alexa Fluor 750 tagged TT vaccine) was detected in the nasal cavity (2 out of 4 rats) (Fig. 4A), suggesting that the observed systemic immune responses could be due to a combination of antigen delivery to the ocular surface and the nasal mucosa.

TT elimination from the non-topical ocular sites occurred more slowly. Intracameral clearance occurred by first-order exponential elimination kinetics, with a half-life (t_1/2) of 2.2 h, as determined by regression analysis (Fig. 4B, Table S6). The anterior chamber of the eye is actively cleared by aqueous humor flow primarily via the trabecular meshwork [30], which may explain the relatively rapid clearance.

Elimination of TT from the subconjunctival space followed two-phase elimination kinetics, with fast elimination accounting for 64% of TT clearance with t_1/2 of 2.1 h in 3 h, and slow elimination of the rest with t_1/2 of 38.5 h (Fig. 4B, Table S6). Suprachoroidal space clearance also exhibited two-phase elimination kinetics, with 44% of the antigen eliminated with t_1/2 of 5.0 h in 4 h, and the rest with t_1/2 of 63.0 h. Both of these tissue sites are highly vascularized, which may explain the relatively rapid TT elimination from these sites, which may be accelerated during clearance of the liquid injection and is then more slowly cleared from the residual TT depot [31].

After intravitreal injection, TT was cleared by first-order elimination with t_1/2 of 69.3 h in the whole process (Fig. 4B, Table S6). Lacking vasculature in the vitreous body, compounds are eliminated from the eye by diffusion anteriorly where they are cleared by aqueous humor or in other directions to vasculature in other ocular tissues [32].

Intrastromal injection resulted in a still different elimination profile, where essentially no clearance was seen within 96 h after injection (p>0.5, Fig. 4B, Table S6). After that, TT was eliminated from the cornea by first-order kinetics with t_1/2 of 63 h. This slow elimination rate may be due to the avascular nature of the cornea, and the delayed clearance may be associated with the onset of new lymphatic vessel formation in the cornea, as discussed below.

Finally, TT elimination after IM injection also exhibited two-phase clearance kinetics and t_1/2 of 12.8 h before 3 days, and 86.6 h after 3 days (Fig. S6, Table S6). More detailed discussion of TT elimination kinetics and pathways via the various routes of administration can be found in the Supplementary Information.

3.3. Up-regulation of MHC class II and costimulatory molecules (CD80, CD86) in the cornea

To better understand the nature of the systemic immune response generated after intrastromal vaccination, we identified MHC II⁺ cells in the cornea by confocal microscopy after immune staining. The number of the MHC II⁺ cells was increased at the injection site 7 days after intrastromal injection of TT, and showed good colocalization of MHC II⁺ cells with TT, as demonstrated by both confocal microscopy imaging and flow cytometry analysis (Fig. 5). More specifically, TT could be found inside the MHC II⁺ cells, suggesting acquisition of TT by the cells (Fig. 5A). The MHC II⁺ cells accounted for 13.2 ± 2.9% of cornea cells in the naïve eye and increased to 30.7 ± 2.8% two days after intrastromal injection of TT (p<0.001) (Fig. 6A). Intrastromal injection of isotonic saline also increased MHC II⁺ cells of the cornea (23.0 ± 1.1%) compared with naïve eye probably due to inflammation caused by the injection alone [33], but this increase in MHC II⁺ cells due to saline injection was still smaller than after intrastromal injection of TT (p<0.001).

Fig. 5. Uptake of TT vaccine by MHC II⁺ cells.

Alexa Fluor 555-tagged TT (red) was injected into the corneal stroma of rats. (A) The corneas were harvested on day 7 and immunolabelled by antibodies against MHC II (green). Cell nuclei were stained by Hoechst 33342 (blue). Representative images of the cornea were then obtained by confocal microscopy. The MHC II⁺ cells were identified at the injection site and showed good colocalization with the TT (arrows). (B) Single-cell suspensions of the cornea were prepared and immunolabelled by antibody against MHC II. The fluorescence intensity of the cell population was analyzed by flow cytometry. Representative flow cytometry dot plots of cornea MHC II⁺ cell population of different days after intrastromal injection of TT show the changes of cell population and colocalization of TT with MHC II⁺ cells.

Fig. 6. Infiltration of MHC II⁺ cells and expression of costimulatory molecules CD80 and CD86.

Single-cell suspensions of the cornea were prepared and immunolabelled by antibody against (A) MHC II, (B) CD80 and (C) CD86 using naïve corneas, as well as corneas 2 and 7 days after intrastromal TT vaccination or injection of normal saline (0.9% NaCl). Flow cytometry analysis identified the percent cells positive for the indicated marker (* 0.01<p<0.05, **0.01<p< 0.001, *** p<0.001). Data shown mean ± standard deviation from n = 4~6 independent replicates from separate eyes.

MHC class II molecules are normally found only on professional antigen presenting cells (APCs) such as dendritic cells (DCs), and are essential for T-cell priming and generating an effective and specific immune response to foreign antigens [34]. Corneal DCs can rapidly up-regulate surface MHC class II in response to alteration in their microenvironment induced by activation by foreign antigens and tissue injury [35]. The costimulatory molecules CD80/CD86 are also critical for efficient antigen presentation and priming of antigen-specification naïve T cells [36]. Upregulation of CD80 and CD86 was also observed in the cornea two days after intrastromal injection of TT (p<0.001) (Fig. 6B, 6C), which further confirmed the activation of APCs in the cornea.

3.4. Injection of vaccine antigen into the corneal stroma induced isolated lymphangiogenesis

We hypothesized that the lymphangiogenesis in the normally avascular cornea may have played a role in the systemic immune response to intrastromal vaccination. While naïve eyes did not show evidence of lymphatic vessels in the cornea, the lymphatic endothelial marker LYVE1 was seen at the injection site 7 days after intrastromal TT injection, demonstrating lymphangiogenesis in the cornea induced by the antigen injection (Fig. 7). Imaging showed that TT was not limited to the injection site, and was also seen diffusing across the cornea to the limbus and conjunctiva, where abundant lymphatic vessels exist. In this way, intrastromal delivery may form an antigen depot for slow release in part to the limbus and conjunctiva. (Fig. 7).

Fig. 7. Lymphangiogenesis in the avascular cornea after intrastromal injection of TT.

Corneas and neighboring tissue were harvested 7 days after injection of Alexa Fluor 555-tagged TT (red). The lymphatics were immuno-stained by LYVE1 antibody (blue). A representative photograph (left) shows a rat eye 7 days after TT injection and identifies the sites of microscopic imaging. Representative confocal microscopy imaging of the cornea and neighboring tissues shows lymphatic vessels in the naïve eye limbus, but not in the cornea. In eyes injected with TT, cornea, limbus and conjunctiva all show the presence of TT as well as lymphatic vessels (n=6 eyes).

The cornea is circumferentially surrounded by lymphatic vessels located at the limbus. These vessels connect to the conjunctival lymphatic network, but, under homeostatic conditions, do not enter the cornea [37]. However, under inflammatory conditions, such as injury or infection, these lymphatic vessels can give rise to new lymphatics, which may extend into the cornea as part of a non-specific inflammatory response driven by the innate immune system [26, 38]. This response may be the source of corneal lymphangiogenesis seen in this study too.

3.5. Antigen presentation to the lymphatic system

Seven days after intrastromal injection, we identified TT vaccine (with a red fluorescent tag) in the ipsilateral mandibular lymph nodes, which are known sites for lymphatic drainage from conjunctiva [39, 40] (Fig. 8A). Within the lymph node, TT vaccine was observed in the lymphatic capillaries (Fig. 8A-1) and mostly located at the B-cell zone (Fig. 8A-2) and subcapsular sinus (Fig. 8A). No evidence of red fluorescence was seen in the lymph node of naïve rats (Fig. S7). Subcapsular sinus macrophages are typically positioned at the lymph-tissue interface in the lymph node to trap and present antigens to B cells [41, 42]. Extended TT vaccine colocalization with lymphatic vessels in the cornea, limbus and conjunctiva suggests that the TT vaccine might be transported by lymphatic vessels to secondary lymphoid tissue to activate the systemic immune response [43].

Fig. 8. TT vaccine in the ipsilateral mandibular lymph nodes 7 days after intrastromal injection.

(A) Representative confocal microscopy image of a mandibular lymph node from a rat 7 days after intrastromal injection of Alexa Fluor 555-tagged TT. Cells are identified by staining by DAPI (blue). The red fluorescence signal demonstrates the presence of Alexa Fluor 555-tagged TT vaccine in the capillary lymphatic vessels (A-1) and B-cell zone (A-2) of the mandibular lymph node; yellow arrows indicate the subcapsular sinus. (B) Representative confocal microscopy image shows germinal center formation in the lymph node, where T-lymphocytes (CD4, green) and B-lymphocytes (B220, purple) are present in large numbers, and some of the immune cells were colocalized with the vaccine (appearing white).

The germinal center formation was also observed in the lymph node, where T- and B-lymphocytes were present in large numbers, and some of the immune cells were colocalized with the TT vaccine, indicating antigen presentation and initiation of systemic immune response to the antigens (Fig. 8B).

3.6. Priming the cornea with influenza vaccine enhanced humoral immune response of TT vaccine

We hypothesized that corneal lymphangiogenesis and other local immunological reactions in corneal stroma associated with injection of foreign antigen should generally improve immune responses to injected antigen. Consistent with this hypothesis, we found that humoral immune response of TT vaccine was boosted by priming the cornea with influenza vaccine a week before administrating TT vaccine to the corneal stroma (p<0.05, Fig. 9). In contrast, priming the rats with influenza vaccine administered IM did not affect the systemic immune response to TT vaccine administered intrastromally (p>0.05), suggesting that local microenvironmental activation in the cornea boosted the immune response to TT vaccine.

Fig. 9. Humoral immune response of TT vaccine after priming the cornea with influenza vaccine.

(A) A prime dose of influenza vaccine was administered to the cornea by intrastromal (IS) injection, or a prime dose of influenza vaccine was administered to the muscle by intramuscular (IM) injection or no prime dose was given at all to rats at week 0. One week later, TT vaccine was administered to the cornea by IS injection to all rats (i.e., in the same eye for those with IS priming). (B) Serum TT-specific IgG, IgG1, IgG2a, and IgG2c antibody titers at 3 weeks after TT vaccination were determined. (* 0.01<p<0.05; ** 0.001<p<0.01; *** p<0.001; ns: no significant difference). Data shown mean ± standard deviation from n=6 independent replicates in 6 separate animals.

This type of tissue priming is fundamentally different from conventional vaccination involving two doses of the same vaccine, where the first vaccination stimulates the immune system to generate a response involving immunological memory that is antigen-specific and boosts the antigen-specific immune response upon later exposure to the same antigen [44]. In contrast, priming the corneal stroma with a first dose of antigen in this study appears to modify tissue immune environment in a way that non-specifically potentiates immune responses to subsequent exposure to antigen, whether the same or different from the first antigen.

3.7. Clinical observation and histological analysis

We examined the rat cornea after intrastromal injection by stereomicroscope and slit lamp biomicroscope for 14 days, and determined that the injection procedure was generally well tolerated. Immediately after injection, an opaque region measuring approximately 2 mm in diameter was seen in the cornea at the site of injection, presumably representing the liquid depot of vaccine or saline (i.e., whichever was injected) (Fig. S8. By day 2, the opaque region and any evidence of the site of injection in the cornea could no longer be seen. Other than this transient, localized opacity, the corneas were optically clear without evidence of surface irregularities, and no evidence of cell and flare or other significant findings were observed by slit lamp exam in all 12 eyes studied (i.e., 6 with TT injection and 6 with saline injection) over the 14 days of study (Fig. S8 and S9).

As a further assessment of tissue response to ocular vaccination, histological sections of ocular tissues were evaluated by an ophthalmic pathologist 12 weeks after injection. We evaluated the cornea, iris, lens, ciliary body, retina, choroid, and optic nerve for inflammation, hemorrhage, and necrosis by grading as 0 (normal), 1, 2, and 3 (severe). We did not see evidence of hemorrhage or necrosis in any of the tissues. We also saw no abnormal findings in any of the tissues other than the cornea. For intrastromal injection, localized grade 1 corneal scarring was observed in one out of 6 vaccinated eyes (Fig. S10); corneal neovascularization (i.e., with blood vessels) and grade 2 inflammation were also observed in one out of the 6 vaccinated eyes (Fig. S11), while all other eyes for intrastromal injection were normal (Fig. S10).

4. Discussion

This study showed that delivery of TT vaccine to the corneal stroma elicited an enhanced systemic antibody immune response with anti-TT antibody titers 18- to 30-fold greater than after IM injection and comparable to IM injection with alum adjuvant. Other ocular delivery routes, such as intracameral, subconjunctival, intravitreal and SCS injections, induced humoral immune responses comparable to IM injection and inferior to intrastromal delivery. The systemic immune response elicited by intrastromal injection related to a sequence of events involving sustained presentation of antigen to the lymphatic system via an antigen “depot” effect, infiltration of APCs, up-regulation of MHC class II and costimulatory molecules CD80/CD86, and induction of corneal lymphangiogenesis in the avascular cornea stroma.

These findings raise interesting further questions, such as (i) why is the systemic immune response after intrastromal injection of antigen into an avascular and immune-privileged tissue much more efficient compared to the IM and other ocular routes (and how can this be used therapeutically)? and (ii) why are immune responses to injected vaccines at other ocular sites with immune privilege (e.g., anterior chamber) not weaker compared to the IM injection route?

4.1. Depot effect and antigen kinetics

A vaccine depot with slow clearance of vaccine for more than a week was created in the corneal stroma after intrastromal injection. This slow clearance of antigen appeared to occur by a combination of diffusion, new lymphatic vessels in the cornea, and existing blood and lymphatic vessels in the limbal conjunctiva. This slow clearance of antigen enabled antigen presentation to the lymphatic system and extended stimulation to the immune system, which may have played an important role in amplifying systemic immune responses. For example, “depot” formation is a known component of how alum-based adjuvants enhance vaccine immune response by prolonging exposure to the immune system and enhancing antigen uptake by APCs [45]. Antigen presentation kinetics have been reported to have profound effects on the strength of vaccine immune responses [46, 47] and the quality of the germinal center response [48]. Extended antigen dosing profiles lead to better antigen capture in the germinal center, eliciting an enhanced and long-term humoral immunity [49]. However, in a traditional bolus immunization, the half-life of antigen present in lymph nodes is shorter than the time scale of germinal center formation, which produces poorer IgG antibodies [49].

Antigen clearance from corneal stroma can be compared to prior findings regarding antigen clearance from other ocular tissues. For example, antigen in the anterior chamber is rapidly eliminated following the aqueous humor flow pathway, and can travel to secondary lymphoid organs via conjunctival lymphatics and vascular routes [39]. Potential APCs in the iris and ciliary body are capable of internalizing intracameral antigen [50]. Both factors could induce immune response to antigens.

The conjunctiva is well vascularized with blood and lymphatic vessels, both of which significantly eliminate macromolecules injected in the subconjunctival space [51]. Antigens delivered to the subconjunctival space can be presented to the lymphatic system and are known to induce systemic immune responses [52].

Molecules delivered to the suprachoroidal space can be transported into adjacent sclera, choroid and retina, which all contain blood vessels [31]. Sclera and retina are free of lymphatic vessels, and it is controversial for choroid [53, 54]. Antigen delivered to the suprachoroidal space is mainly cleared by blood flow in the neighboring choroid, and this process depends in part on molecular size, where larger molecules persist in the suprachoroidal space for a longer time [55].

Compounds in the vitreous body are known to be eliminated either via the anterior route or posterior route [32]. The anterior route is based on molecular diffusion through the vitreous humor towards the posterior chamber where it enters the aqueous humor. The posterior route is associated with crossing the blood-ocular barriers, including vascular endothelia and epithelia in the ciliary body and iris, and the blood-retina barrier. The anterior route has the potential to travel to secondary lymphoid organs, which is similar to that of the delivery to anterior chamber. Humoral immune response after intravitreal injection was observed before [56]. Further discussion of antigen clearance kinetics in this study is provided in Supplementary Information.

4.2. Local immune responses induced by antigen injection in the cornea

We hypothesize that the potent systemic immune responses to intrastromal vaccination derive in part from an inflammatory response created in the cornea by intrastromal injection of antigen. It is well-known that inflammatory responses are essential for the induction of adaptive immunity after vaccination [57]. We found that the humoral immune response to TT vaccine was enhanced by priming the cornea with influenza vaccine a week before TT vaccination (p<0.05), while priming the rats with influenza vaccine by IM injection did not affect the immune response to intrastromal TT vaccine (p>0.05).

Influenza vaccine probably induced a local immunological reaction in the cornea, which facilitated increased immunogenicity of subsequent TT injection. We expect that priming the cornea with other immunostimulatory molecules (e.g., ovalbumin, which is commonly used as a model antigen in animals [58]) would similarly potentiate immune responses to subsequent vaccination with a different antigen. This local immunological reaction in the cornea is also evidenced by an immune ring (or Wessely ring) which forms in the cornea after cornea keratitis infection [59] and intracorneal injection of high-dose serum albumin [60]. The reason for this phenomenon is likely due to the formation of antigen-antibody complexes in the cornea and the infiltration of inflammatory cells.

APCs play a crucial role in the immune response to antigens. The central cornea contains immature/precursor-type DCs, and the corneal periphery contains mature and immature resident DCs [61]. Such immature APCs are thought to maintain tolerance to auto- and exo-antigens in the absence of inflammatory mediators associated with APC maturation [62]. However, when immature APCs acquire antigens within an inflammatory environment, a maturation process involving up-regulation of MHC class II and B7 (CD80/CD86) costimulatory molecules (i.e., as seen in this study) renders them highly efficient at immunogenic presentation of antigens to naïve T cells, and facilitates a strong systemic immune response [34, 63]. Professional APCs, such as DCs and macrophages were found in the cornea stroma [61, 64]. These cells are recognized as essential for regulating the initiation of immune responses.

Intrastromal injection of saline also increased MHC class II and B7 costimulatory molecules of the cornea compared with naïve eye. Intrastromal injection alone could lead to corneal epithelial abrasion, which could induce an acute inflammatory response that resulted in the maturation of dendritic cells in response to the injury or inflammation through the over-expression of MHC-II and B7 costimulatory molecules [65, 66].

4.3. Lymphangiogenesis in the cornea

We also hypothesize that lymphangiogenesis in the cornea facilitated antigen presentation to the lymphatic system. Lymphatic vessels are crucial to the host’s ability to respond to infectious agents and antigens, as they facilitate the trafficking of antigens and APCs to draining lymph nodes, where they present antigen to naïve T cells leading to an adaptive immune response [67]. Prior studies have reported that lymphangiogenesis is often a component of the local inflammatory immune response in the cornea, and is essential for fluid homeostasis as part of the acute inflammatory response during wound healing [68, 69]. When the delicate balance between pro- and anti-(lymph)angiogenic factors shifts [70], the avascular cornea can be invaded by lymphatic vessels [71]. In these processes, MHC II⁺ cells and especially macrophages are important for maintaining lymphatic vessels associated with the secretion of inflammatory cytokines [72]. LYVE-1 is widely accepted as the most reliable lymphatic marker [53]. Here, we observed endothelial markers LYVE1 and lymphatic structures at the injection sites of all 6 eyes one week after intrastromal injection of TT. Lymphangiogenesis is also known to mediate corneal transplant rejection and regulate corneal edema, dry eye disease, and allergic responses in the eye [38, 68, 73].

It is possible that lymphangiogenesis could be due at least in part to inflammation caused by microneedle insertion and injection in the cornea (i.e., irrespective of the introduction of antigen or other immunostimulatory compounds). Concerning this point, we previously examined eyes treated by insertion of microneedles into rat cornea without delivery of any compounds, and found no significant increase of macrophages or vascularization in the cornea, suggesting that insertion of microneedles into the cornea is minimally invasive and thereby has little or no effect on inflammatory or vascularization responses in the eye [74]. Another study also showed that injection of saline using a 31-gauge microneedle did not induce adverse effects or ocular inflammation after injection. The intraocular pressure and corneal thickness returned to near baseline within 24 h and normalized in all eyes by 5 days after injection [75].

In our study, corneal lymphangiogenesis was not accompanied by angiogenesis (i.e., by blood vessels) after intrastromal vaccine injection. We did not observe angiogenesis in the cornea two weeks after injection (i.e., in the presence of significant lymphangiogenesis), and only one out of six eyes exhibited corneal neovascularization on week 12. In other contexts, lymphangiogenesis and angiogenesis usually occur in concert, but do not always accompany each other [53, 76]. For example, in dry eye disease, which is a low-grade corneal inflammatory disorder, lymphangiogenesis often occurs without angiogenesis [77]. In addition, it has recently been shown that acute changes in corneal stromal fluid balance in the murine model of acute keratoconus can also lead to temporarily isolated ingrowth of lymphatic vessels [68]. The rapid intrastromal fluid injection during intrastromal vaccination in our model here mimics that scenario.

4.4. Immune privilege of the eye

The enhanced systemic immune response to the vaccine in the cornea observed in this study may at first appear inconsistent with the well-known immune privilege of the eye, as demonstrated by the low immunogenicity of antigens placed within or arising from the eye and the immunosuppressive microenvironment reflected by extended survival of corneal transplants and foreign tissues placed in the anterior chamber, vitreous cavity and subretinal space [12, 78]. For example, prior work showed that antigens in the anterior chamber can induce a systemic suppression of humoral responses and cell-mediated responses to the antigen due to ACAID [79].

Nonetheless, it is well established that corneal immune privilege and angiogenic privilege can be lost, such as in infectious diseases that pose a danger to the whole organism. For example, in herpetic keratitis, corneal immune privilege is terminated to prevent systemic danger [80]. It is thought that activation of innate immune cells, such as via TLRs, leads to termination of immune privilege and, for example, in the case of herpetic keratitis resulting in stromal scarring [81]. Similarly, corneal neovascularization leads to loss of ACAID, thus rendering corneal grafts placed into pathologically pre-vascularized recipients at high risk for rejection [82].

The intrastromal vaccination setting described here has many similarities to the scenario of high-risk corneal transplantation. Whereas corneal transplants placed into avascular, “normal-risk” settings enjoy an extended graft survival without immunosuppression, grafts placed into “high-risk” beds are swiftly rejected. Similar to the vaccination setting, a high-risk bed is characterized by infiltration of APCs into the corneal stroma, up-regulation of MHC class II and costimulatory molecules CD80/CD86, induction of corneal lymphangiogenesis and sustained presentation of antigen (i.e., from the transplanted cornea) to the lymphatic system [17, 83]. This suggests that the strong systemic immune response to intrastromal vaccination is indeed not unique, but similar to high-risk corneal transplantation settings [3]. In both scenarios, immune privilege at the site is lost due to the ingrowth of predominantly lymphatic vessels, which seem to be key mediators of immune responses in the cornea [82, 84].

In our study, total antigen-specific IgG production was similar among the intracameral, subconjunctival, SCS, and intravitreal sites of administration and IM injection possibly due to antigen elimination from the eye by ocular blood flow producing a similar extent of antigen presentation to the regional lymphatic system as IM delivery (even though IgG2a titers for intracameral, subconjunctival, and SCS injections were significantly lower than IM delivery (p<0.05, Table S4). The antibody levels after intracameral injection are consistent with a study showing that total IgG production was unaffected after administration of soluble antigens, such as OVA, in the anterior chamber, while the IgG isotypes including IgG2a, IgG2b, and IgG3, were inhibited in some strains of ACAID mice, suggesting inhibition of Th1 function of CD4+ T cells in the form of tolerance to foreign antigens [85]. We cannot exclude the possibility that responses to the trauma from the injection procedure itself played a role in the temporary loss of immune privilege at normally protected sites.

4.5. Enhance vaccination and immunotherapy for ocular diseases

These new mechanisms for regulating adaptive immunity might have future clinical value using corneal stroma’s strong immune response to enhance vaccination responses. In this study, we evaluated systemic immunity and found that vaccination in the cornea can offer significant immunological advantages over intramuscular vaccination. However, intrastromal injection can have risks, such as causing lamellar separation of corneal stroma, which could possibly lead to corneal layer separation, corneal fibrosis or scar formation [86, 87]. The cornea with induced immune response may also suffer from increased risk of graft rejection after a future corneal transplantation [88]. Therefore, these immunological benefits need to be balanced against possible risks to the patient and provider acceptance of administering intrastromal injections, possible (transient) effects on vision and other possible risks to the eye, However, we note, that intrastromal injections in this study were generally well tolerated, with only mild abnormalities observed through 3 months of observation following vaccination in a rat model.

This risk-benefit comparison may be more favorable for vaccination and immunotherapy for local ocular diseases. For example, studies have shown that a local periocular vaccination was more effective than systemic vaccination against herpes simplex virus type 1-induced corneal diseases including conjunctivitis, iritis, epithelial keratitis, and corneal clouding [89]. Other studies showed that local periocular vaccination (conjunctival immunization) induced Salmonella typhimurium bacterial ghost-specific local and systemic immune responses and a Th1-biased response that is critical for the control of Salmonella species [52].

4.6. Limitations and Expectations

Chemical composition of antigens and cytokines produced by lymphocytes and APCs affect antibody class switching during the immune response to antigens [90]. Bacterial toxin protein antigens, such as TT, usually trigger class switching to IgG1 or IgG3; viral polysaccharide antigens, such as influenza vaccines, usually induce class switching to IgG2 [91]. Different subclasses of antibodies have different effector functions. The effects of antigen composition and cytokines produced after antigen delivery via various ocular routes on the antibody responses of different subclasses should be further studied. In addition, characterizing the proportional change of CD4⁺ T cells and CD8⁺ T cells after immunization via intrastromal injection and other routes may generate interesting results that can help to further understand ocular immunology, and may be valuable for immunotherapy of ocular diseases. A more in-depth and comprehensive study on these topics is needed in the future to better understand the possible immunological and other advantages of intrastromal vaccination and immunotherapy for ocular diseases, as well as the safety, tolerability and acceptability of this approach.

5. Conclusions

This study found that injection of TT antigen into six different compartments of the eye could produce strong humoral and cellular antigen-specific immune responses, in many cases similar to intramuscular injection, despite known mechanisms of ocular immune privilege. Notably, delivering vaccine antigens to the corneal stroma elicited a surprisingly strong immune response, which was 18- to 30-fold stronger than IM vaccination and comparable to IM vaccination with alum adjuvant. This enhanced systemic immune response to intrastromal injection was related to a sequence of events involving infiltration of APCs into the corneal stroma, up-regulation of MHC class II and costimulatory molecules CD80/CD86, induction of corneal lymphangiogenesis and sustained presentation of antigen to the lymphatic system via an antigen “depot” effect and induction of an efficient germinal center reaction. This work demonstrates the highly immune-responsive nature of cornea stromal to injected vaccine antigen, which may offer new approaches to promote immune responses to vaccination and immunotherapy, as well as suppress immune responses to other corneal interventions.

Highlights.

1. We explored immune responses to vaccine antigens delivered to six compartments of the rodent eye, including ocular surface, corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, and vitreous body.

2. Antigens delivered to corneal stroma induced enhanced, rather than suppressed, antigen-specific immune responses.

3. The mechanism for enhanced immune responses to antigens delivered to corneal stroma was investigated.

Abbreviations

ACAID

anterior chamber-associated immune deviation

TLR

toll-like receptor

TT

tetanus toxoid

SCS

suprachoroidal space injection

MHC

major histocompatibility complex molecules

HSV

herpes simplex virus

BSA

Bovine serum albumin

FBS

Fetal bovine serum

FACS

fluorescence-activated single cell sorting

FITC

Fluorescein isothiocyanate

DC

dendritic cells

APC

antigen presenting cell

LYVE-1

Lymphatic vessel endothelial hyaluronan receptor 1

OVA

Ovalbumin