Surface Modification of Biodegradable Microparticles with the Novel Host-Derived Immunostimulant CPDI-02 Significantly Increases Short-Term and Long-Term Mucosal and Systemic Antibodies against Encapsulated Protein Antigen in Young Naïve Mice after Respiratory Immunization

Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 μm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 μL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 μm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.


Introduction
The primary requirement for an effective vaccine is the ability to safely generate longterm protective adaptive immune responses against the targeted pathogen above threshold levels that correlate with a significant decrease or elimination of pathogen-related infectious disease [1][2][3][4]. Most licensed vaccines protect against infectious diseases by generating sufficient levels of long-term systemic antibodies after IM or SQ administration [5][6][7] that protect against invasive infections and likely provide backup protection against infections in the lower respiratory tract [8,9]. Administering vaccines by a mucosal route (i.e., oral, nasal, sublingual, buccal, pulmonary, rectal, or vaginal) may provide several advantages over systemic vaccines, including (i.) generating both mucosal and systemic antibodies to protect against initial infection at the portal of entry for most pathogens as well as subsequent invasive infection, (ii.) vaccine immunogenicity regardless of pre-existing systemic immunity, (iii.) the option for frequent boosting, (iv.) easy, pain-free administration that requires little training and increases patient compliance without the risk of spreading blood-borne infections, and (iv.) lower production costs and regulatory burden compared to systemic vaccines [9][10][11][12][13][14].
Currently licensed mucosal vaccines (8 oral and 1 intranasal) are composed of live, live attenuated, or inactivated strains of pathogens that are the most likely to generate the appropriate long-lived protective mucosal and systemic antibodies [9]. These vaccine types, however, are (i.) limited to pathogens that increase protection after natural infection and can be grown in culture, (ii.) are difficult to establish for most bacterial pathogens, (iii.) take a long time to develop, (iv.) are rarely safe and stable, (v.) may not cross-protect against other pathogenic strains, and (vi.), in the case of live/live attenuated vaccines, are not suitable for pregnant women or immunocompromised patients and have the remote possibility of reverting to wild-type virulence [10,[15][16][17].
One approach to potentially overcoming the limitations of currently licensed mucosal vaccines is through mucosal administration of one or more protective antigens (i.e., subunit and recombinant vaccines) [15,18,19] encapsulated in nanoscale biodegradable particles. This can decrease mucosal vaccine degradation and clearance, increase localization to mucosa-associated lymphoid tissues (MALT) (major induction sites of adaptive immune responses), increase the levels and duration of epitope presentation and cross-presentation after internalization by antigen-presenting cells (APC), and increase the magnitudes of short-lived mucosal and systemic adaptive immune responses following mucosal administration [20][21][22][23][24][25][26][27][28][29][30]. Recombinant vaccines can also be designed to generate more potent and broadly protective memory B-cells and T-cells [31]. Encapsulated and unencapsulated subunit and recombinant vaccines, however, require the incorporation of a suitable mucosal immunostimulant to sufficiently activate APC (especially dendritic cells) and generate high levels of long-lived mucosal and systemic adaptive immune responses [32].
Cholera toxin subunit B (CTB) is the only mucosal immunostimulant incorporated as part of a licensed mucosal vaccine (Dukoral: oral, inactivated vaccine) [33,34] but is unsafe for IN administration [35,36] and possibly other routes of mucosal immunization. The most widely developed preclinical immunostimulants are based on pathogen-associated molecular patterns (PAMPs) [37][38][39]. Development and/or incorporation of PAMP-based immunostimulants, however, is extremely challenging due to the large number of PAMP receptors, differences in PAMP receptor activities/cellular distributions, differences in adaptive immune responses and levels of inflammation by individual PAMPs, the complexity and expense of PAMP molecules, and difficulties establishing stable formulations [10,13,40]. Thus, there continues to be a great need for the preclinical development of mucosal immunostimulants that are sufficiently potent, minimally pro-inflammatory, and safe for mass immunization [9,10,12,41].
Lower respiratory infections are the fourth leading cause of death worldwide [9] and develop primarily from initial infections of the upper respiratory tract [53]. As such, developing mucosal vaccines for respiratory immunization that can increase long-term protective mucosal antibodies in the upper and lower respiratory tract in addition to protective systemic antibodies could significantly decrease the incidence of infectious disease.
We recently found that surface modification of biodegradable nanoparticles (~380 nm) with~0.1 wt% CPDI-02 through 2 kDa PEG linkers (i.) increases the activation of immature murine bone marrow-derived DC (BMDC) and (ii.) increases long-lived memory subsets of CD4 + and CD8 + T-cells against an encapsulated model protein immunogen, LPS-free ovalbumin (OVA), in the lungs and spleens of young naïve female C57BL/6 mice after respiratory immunization and subsequent protection against respiratory challenge with OVA-expressing L. monocytogenes [3]. Given that the activation of dendritic cells (DC) is required to increase the generation of both long-lived memory B-cells and T-cells [54], we hypothesized that surface modification of biodegradable microparticles with CPDI-02 will increase the generation of long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory immunization. To test this hypothesis, we encapsulated LPS-free OVA in biodegradable PLGA 50:50 microparticles (MP) (~1 µm diameter) alone or MP surface-modified with inactive scrambled scCPDI-02 (scCPDI-02-MP) or CPDI-02 through 2 kDa PEG linkers (CPDI-02-MP) at~0.4 wt%. We then compared the extent to which intranasal administration of CPDI-02-MP or scCPDI-02-MP increases (i.) magnitudes of short-term OVA-specific antibody-secreting cells (ASCs) in the lungs and spleen 6 days post-immunization, (ii.) titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) OVA-specific antibodies in the nasal cavity, lungs, and sera, and (iii.) long-term signs of inflammation in the lungs 90 days post-immunization compared to vehicle alone.

Diameters and Zeta Potentials of Microparticles
Average hydrodynamic diameters, PDI, and zeta-potentials (mV) ± SD (n = 3 independent samples from at least two batches) were determined using a ZetaSizer Nano ZS90 (Malvern Instruments, Malvern, UK) equipped with a He-Ne laser (λ = 633 nm) as the incident beam. Lyophilized MP were suspended in solution (10 mM NaCl in deionized H 2 O) [0.5 mg/mL] and incubated within the instrument (25 • C, 4 min) before measuring.

Animals
All animal procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Naïve female mice (C57BL/6NCrl~8 weeks old, Charles River Laboratories, Wilmington, MA, USA) were acclimatized in an ABSL-2 facility under pathogen-free conditions at least one week before experiments.

Isolation of Murine Lung Lymphocytes and Splenocytes
Mice were euthanized by isoflurane overdose followed by cervical dislocation at the indicated time point after the final treatment. Following euthanasia, lungs were surgically exposed, perfused by injecting PBS [5 mL] with a 25G needle into the right ventricle of the heart, removed, minced using sterile surgical scissors, and placed directly into a sterile Following incubation, digested lung fragments were homogenized again with the Tissue Dissociator using the same settings. Splenocytes were isolated using sterile forceps, minced with sterile surgical scissors, and then placed in a sterile gentleMACS C tube containing cRPMI [3 mL]. Spleens were then homogenized with a gentleMACS Tissue Dissociator using the "m_spleen_01" setting. Digested spleen or lung cell suspensions were then passed through sterile 30 µm pre-separation filters (Miltenyi) and carefully overlayed onto Lympholyte M (Cedarlane Labs) [5 mL] in a sterile 15-mL centrifuge tube. Cells were centrifuged [1500 RCF, 20 min, r.t., no brakes] and lung lymphocytes or splenocytes were collected from the interphase with a sterile Pasteur pipette and transferred to sterile 15-mL centrifuge tubes. Purified cells were then diluted to 10 mL with cRPMI, pelleted [800 RCF, r.t., 10 min], resuspended in cRPMI [7 mL], pelleted [800 RCF, r.t., 10 min], and resuspended in 0.6 mL (lung lymphocytes) or 1.2 mL (splenocytes) of serum-free CTL-Test Media (Cellular Technology Limited), counted (Cellometer Auto T4 Automated Cell Counter), and diluted in serum-free CTL-Test Media as needed for ELISpot assays (Section 2.11).

IgA, IgG, and IgM ELISpot Assays
Antibody-secreting cells (ASC) were quantitated using Murine Single-Color ELISpot kits (ImmunoSpot) as directed with slight modification. ELISpot plates were coated with OVA [75 µg/mL, 80 µL/well] and incubated at 4 • C overnight the day before lymphocyte/splenocyte isolations. Purified lung lymphocytes or splenocytes (Section 2.10) were diluted as needed in serum-free CTL-Test Media [5 × 10 6 cells/mL], plated in triplicate [0.1 mL/well], and incubated at 37 • C, 5% CO 2 for 18 h. OVA-specific ASC spots were counted with an ImmunoSpot S6 MACRO Plate Analyzer (Cellular Technology Limited) using the "Smart Count Wizard" function (B-cell specific mode; "Small Spots" setting; Spot Separation = 1; Background Balance = 10) and manually adjusting the positive spot gating threshold to a minimum surface area of 0.0015 mm 2 (IgA and IgG) or 0.0035 mm 2 (IgM). Average spot counts per sample were then normalized to 1 × 10 6 plated lymphocytes or splenocytes.

Collection of Serum, BALF, and NLF from Mice
Serum was isolated by collecting whole blood [0.2 to 0.3 mL] into a sterile 0.5-mL centrifuge tube from a submandibular venipuncture (5-mm lancet, MEDIpoint) [60]. Blood was allowed to clot at r.t. for 30 min then centrifuged [2000 RCF, 4 • C, 10 min]. Serum was then transferred to a new sterile 0.5-mL centrifuge tube and stored at −80 • C. Bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF) were collected after isoflurane overdose on Day 14 and Day 90 [61,62]. The tracheas of immunized mice were surgically exposed, and an incision was made below the larynx. A cannula was then inserted and sterile PBS [1.0 mL] was instilled into the lungs and recovered by aspiration. For NLF collection, the cannula was reoriented in the trachea toward the cranium and guided toward the nasopharynx. Sterile PBS [0.6 mL] was then flushed through the nasal cavity and collected in a 6-well plate before being transferred to sterile 0.5-mL centrifuge tubes. BALF or NLF was then centrifuged [400 RCF, 4 • C, 10 min] and supernatants were stored at −80 • C.

OVA-Specific Antibody Titers of Serum, BALF, and NLF
Indirect ELISA was used to determine OVA-specific titers of IgG1, IgG2b, IgG2c, and IgG3 in isolated serum, IgA and total IgG in BALF, and IgA in NLF. Each class-or subclass-specific assay was completed as directed with a few modifications. For all ELISAs, OVA was suspended in ELISA coating buffer (Thermo Scientific) [0.
Negative Net ABS was set to 0. Positive antibody titer cutoff thresholds for individual dilution factors were then calculated statistically at the α = 0.05 level [63] using average NET ABS for mice treated with vehicle only. Net ABS of mice from each treatment group was then compared to positive titer cutoff thresholds by plotting 4-parameter logistic (4PL) curves of NET ABS from individual mice ("bottom" value = 0) and plotting a 4PL curve of positive cutoff thresholds ("bottom" value ≥ 0.01, which is the margin of error of photometric accuracy for the Spectramax iD3 device). Individual titer values were interpolated as the dilution factor at the intersection of the respective 4PL curves for each mouse and the 4PL curve of the positive titer cutoff thresholds [64]. For NLF titers, calculated values were multiplied by a factor of 18.75 to adjust for the volume of the nasal cavity (average nasal cavity volume of mice is~32 µL [65], whereas 600 µL of PBS was used for nasal lavage [600/32 = 18.75]).

Lung Histology
Histologic analyses were completed on lungs of mice on Day 90 as previously described [66]. Lungs were perfused lungs by injecting PBS [5 mL] with a 25G needle through the right ventricle of the heart. Whole lungs were removed and inflated to 10 cm H 2 O pressure with a solution of 10% formalin to preserve anatomical structure. Fixed lungs were embedded in paraffin and sections (4-5 µm) were cut and stained with hematoxylin and eosin by the University of Nebraska Medical Center Tissue Sciences Facility (Omaha, NE, USA). Sample sections were then assessed for signs of inflammation by blinded grading using a scaling system for signs of inflammation where 1 = normal, 2 = mild inflammation, 3 = moderate inflammation, 4 = obvious inflammation, and 5 = severe inflammation.

Statistical Analyses
All statistical analyses were performed using GraphPad Prism version 9.4.0 (San Diego, CA, USA) for Windows, www.graphpad.com (accessed on 26 July 2022). Sample outliers in all experiments were identified by the ROUT method (Q = 1%) and omitted for statistical comparisons. Data from two treatment groups were compared by two-tailed, nonparametric Mann-Whitney U Test (α = 0.05) and data from three or more treatment groups were compared by nonparametric Kruskal-Wallis one-way ANOVA with uncorrected Dunn's multiple comparisons post hoc test (α = 0.05). Additional relevant statistical information is provided in the figure legends.
To next determine if surface modification of biodegradable microparticles with CPDI-02 increases the generation of long-term mucosal antibodies against encapsulated protein antigen, we intranasally administered vehicle alone (50 µL), inactive scCPDI-02-MP in 50 µL IAV, or CPDI-02-MP in 10 µL or 50 µL IAV as before ( Figure 2) and compared titers of OVA-specific IgA in NLF and titers of OVA-specific IgA and IgG in BALF normalized to vehicle alone 90 days post-immunization (104 days post-prime) by ELISA ( Figure 3D-F).

Effect of Surface Modification of~1 µm Biodegradable Microparticles with CPDI-02 and Increased Pulmonary Delivery on Magnitudes of Short-Term Systemic Antibody-Secreting Cells (ASCs) against Encapsulated Protein Antigen in Young, Naïve Mice
Vaccine administration to the respiratory tract, such as other routes of mucosal administration [73], potentially generates both mucosal and systemic antibodies [74]. To provide an initial indication that surface modification of~1 µm biodegradable microparticles with CPDI-02 and increasing delivery to the lungs will likely increase systemic antibodies against encapsulated protein antigen, we intranasally administered vehicle alone (50 µL), inactive scCPDI-02-MP in 50 µL IAV, CPDI-02-MP in 10 µL IAV, or CPDI-02-MP in 50 µL IAV as before but compared magnitudes of OVA-specific IgM and IgG ASCs in the spleen 6 days post-treatment (20 days post-prime) by ELISpot (Figure 4). We focused on systemic IgM given that IgM memory B-cells are most abundant in the early stages of immunization or infection and undergo T helper cell (Th)-dependent class-switching to IgG memory B-cells and on systemic IgG ASCs given that the highest proportion of circulating antibodies are IgG [72].

Surface Modification of~1 µm Biodegradable Microparticles with CPDI-02 and Increased Pulmonary Delivery Greatly Increase Short-Term and Long-Term Systemic IgG Antibody Subclasses against Encapsulated Protein Antigen in the Sera of Young, Naïve Mice
Given that CPDI-02-MP in 50 µL IAV generated higher magnitudes of systemic IgG ASCs against encapsulated LPS-free OVA than in 10 µL IAV but similar magnitudes as inactive scCPDI-02-MP in 50 µL IAV (Figure 4), it remained unclear if both surface modification of~1 µm biodegradable microparticles with CPDI-02 and increased delivery to the lungs are likely to increase systemic antibodies against encapsulated protein antigen as observed for antibodies in the nasal cavities and lungs (Figure 3).

Discussion
Our study provides evidence that surface modification of~1 µm biodegradable microparticles with CPDI-02 significantly increases long-term mucosal and systemic antibodies against encapsulated protein antigen in young naïve mice after respiratory immunization with minimal long-term inflammation in the lungs. We found that IN administration of LPS-free OVA protein encapsulated in~1 µm PLGA 50:50 MP surface-modified with 0.4 wt% CPDI-02 (CPDI-02-MP) through 2 kDa PEG linkers (Table 1) to naïve female C57BL/6 mice in 50 µL of vehicle ("respiratory immunization") greatly increased titers of IgA in NLF and BALF (Figure 2), total IgG in BALF (Figure 2), and serum titers of IgG1, IgG2b, IgG2c, and IgG3 ( Figure 5) against encapsulated LPS-free OVA and showed similar signs of mild inflammation in the lungs 90 days post-immunization (104 days post-prime) ( Figure 6) compared to LPS-free OVA protein encapsulated in MP surface-modified with 0.4 wt% inactive scrambled scCPDI-02.
Our study also provides evidence that increasing delivery of~1 µm microparticles to the lungs increases long-term mucosal and systemic antibodies against encapsulated protein antigen in young naïve mice. We found that IN administration of CPDI-02-MP (Table 1) to naïve female C57BL/6 mice in an IAV of 50 µL expected to deposit~1 µm microparticles primarily in the nasal cavity and lungs of mice ("respiratory immunization") [59] greatly increased titers of OVA-specific IgA in NLF and BALF (Figure 2), total OVA-specific IgG in BALF (Figure 2), and serum titers of OVA-specific IgG1, IgG2b, IgG2c, and IgG3 ( Figure 5) 90 days post-immunization (104 days post-prime) compared to CPDI-02-MP in an IAV of 10 µL expected to deposit microparticles in the nasal cavity alone [59]. Our findings are similar to a previous report that increasing the IAV from 10 µL to 50 µL increases titers of IgA and IgG in the BALF of BALB/c mice against tetanus toxoid encapsulated in~1.8 µm PLA microparticles [57]. This suggests that nasal-associated lymphoid tissue (NALT) in mice is not involved in generating mucosal and systemic antibodies by~1 µm CPDI-02-MP. Considering that the volume of the nasal cavity of~8-week old mice is~32 µL [65], however, it remains possible that IAVs > 30 µL are required to deliver a sufficient amount of CPDI-02-MP to the NALT. Thus, distribution studies with IAVs between 10 µL and 50 µL will be required to determine the extent of NALT involvement with~1 µm CPDI-02-MP in mice.
Surface modification of~1 µm biodegradable microparticles with CPDI-02 may significantly increase long-term mucosal and systemic antibodies against encapsulated protein antigen by initially increasing MP localization to MALT in the nasal cavities (NALT) and lungs (BALT) through increased affinity for C5aR1 receptors expressed on the surface of microfold/membrane cells (M cells) within follicle associated epithelium (FAE) [76]. A similar mechanism has been proposed for M cell-targeted oral vaccines that use ligands to increase affinity for C5aR1 [76,77] or other M cell surface proteins [78]. Whether the nonspecific affinity of MP for M cells already maximizes the rate of M cell transcytosis into MALT or if the potential activation of C5aR1 by surfacCPDI-02 increases the rate of M-cell transcytosis also remains unclear [3]. CPDI-02-MP in the subepithelial compartment of MALT could then (i.) prolong the local release of encapsulated protein antigen for sustained activation of naïve B-cells in the MALT and MALT-draining LN (ii.) activate DC and macrophages in MALT and DC in MALT-draining LN through C5aR1 to create a cytokine microenvironment that supports the generation of long-lived memory B-cells and Th-cells (iii.) localize to MALT-draining LN as observed with 1.1 µm fluorescent polystyrene carboxylate microspheres after IN administration to mice [24] and (iv.) be phagocytosed by CPDI-02-activated DC in the MALT and MALT-draining LN for sustained antigen presentation and activation of naïve Th-cells that, in turn, support the expansion and differentiation of activated naïve B-cells into memory B-cells.
The primary limitation of our study is that it compares titers of long-term mucosal and systemic antibodies against the widely studied model protein antigen OVA but not long-term titers of antibody effector functions (e.g., neutralizing titers, ADCC titers) against a protective antigen. As such, the effect of CPDI-02 surface modification on long-term titers of mucosal and systemic antibody effector functions against encapsulated protective antigens will need to be determined for each pathogen.

Conclusions
In summary, our study indicates that surface modification of~1 µm biodegradable microparticles with CPDI-02 through 2 kDa PEG linkers is likely to greatly increase long-lived mucosal and systemic antibodies against encapsulated protein antigen after respiratory immunization and may be an effective incorporation strategy for other routes of mucosal immunization.