Encapsulating protein subunit vaccines in biodegradable microparticles (MP) can increase induction of long-term protective mucosal and systemic antibodies after respiratory administration but requires incorporation of a mucosal immunostimulant. We previously found that incorporating complement peptide-derived immunostimulant-02 (CPDI-02) with MP-encapsulated LPS-free OVA through surface modification of the same ~1 µm PLGA 50:50 MP greatly increases mucosal and systemic OVA-specific antibodies in young, naïve female C57BL/6 mice at 14 and 90 days post-intranasal administration. Here, we directly compared effects of incorporating CPDI-02 or inactive scCPDI-02 with MP-encapsulated LPS-free OVA by separate encapsulation (SE), co-encapsulation (CE), or surface modification (SM) on induction of OVA-specific IgA, IgM, and IgG antibody secreting cells (ASCs) in the lungs and spleen, IgA antibodies in nasal lavage fluid, IgA and IgG antibodies in bronchoalveolar lavage fluid, and IgG subclasses in the serum at 14 days post-intranasal administration. We found that SE incorporation induced greater or similar levels of mucosal and systemic OVA-specific ASCs and mucosal IgA and IgG titers but greater titers of systemic IgG subclasses than CE or SM incorporation versus inactive scCPDI-02. Thus, given that surface modification with CPDI-02 induces high titers of OVA-specific systemic and mucosal antibodies at 14 and 90 days post-intranasal immunization under the same experimental conditions, separate encapsulation of CPDI-02 is expected to more broadly induce long-term mucosal and systemic antibodies against MP-encapsulated protein vaccines than co-encapsulation and surface modification after intranasal and possibly other routes of mucosal immunization.

Background/Objectives: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) greatly complicates the treatment of skin and soft tissue infections (SSTI). It was previously found that subcutaneous (SQ) treatment with the mononuclear phagocyte (MP)-selective activator complements peptide-derived immunostimulant-02 (CPDI-02; formerly EP67) and increases prophylaxis of outbred CD-1 mice against SQ infection with CA-MRSA. Here, we determined if treatment with CPDI-02 also increases curative protection. Methods: Female CD-1 mice were challenged SQ with CA-MRSA USA300 LAC, then CPDI-02 or inactive scCPDI-02 was administered by a topical, SQ, IM, or IV route at 6 or 24 h post-challenge. Abscess sizes were compared over 10 days and CA-MRSA burden, neutrophils, MP, and pro-inflammatory cytokines were compared in subcutaneous abscesses. CPDI-02 PK and distribution in female CD-1 mice were compared after IM or IV dosing and CPDI-02 toxicity in male and female CD-1 mice was determined by IM dose escalation and repeat IM dosing. Results: Repeat IM treatment starting at 6 h post-challenge decreased maximum abscess surface area, CA-MRSA burden, and time to resolution, whereas repeat treatment by a topical, SQ, or IV route had no effect. Repeat treatment starting at 24 h post-challenge was ineffective by the current routes. Single IM treatment starting at 6 h post-challenge was as effective as repeat IM treatment, increased systemic exposure to CPDI-02, and, in subcutaneous abscesses, initially decreased IL-1β and increased MP. CPDI-02 was tolerated between 130 and 170 mg/kg after IM dose escalation and between 65 and 130 mg/kg after repeat IM dosing with males being more tolerant. Conclusions: Single early-stage IM treatment with CPDI-02 may increase curative protection against SSTI caused by CA-MRSA and/or other pathogens controlled by activated MP.

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.

EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.

An EGFR-targeting peptide with Ala-Glu-Tyr-Leu-Arg sequence was assembled directly onto the surface of spray-dried chitosan microparticles using solid-phase peptide synthesis with Fmoc chemistry. Both targeted and scrambled peptides were cleaved from chitosan with enzymatic digestion, isolated using reversed-phase HPLC, then identified with LC/MS/MS and amino acid analysis. Particles with conjugated peptides and fluorescent-labeled were characterized for binding with A549 (cancer) and WI-26 VA4 (normal) lung cells using flow cytometry and confocal microscopy. The purity of peptide synthesis was in the range of 74–77%. The cell binding studies revealed that particles modified with the peptide bind to lung cancer cells 8.3 times higher than the normal lung cells. The binding potential of surface-modified targeted particles to the tumor cells was compared with scrambled and unmodified ones and was found to be significantly different. The binding was 7.3–7.5-fold higher than the scrambled and unmodified particles, respectively. Modification of chitosan particles with direct assembly of Ala-Glu-Tyr-Leu-Arg peptide on their surface enhanced their targeting potential to lung cancer cells and could be used as a potential carrier for delivery and therapy. This approach can further be utilized for assembly of other short peptide ligands on micro- or nanoparticulate systems for tumor targeting.

Encapsulation of protein vaccines in biodegradable nanoparticles (NP) increases T-cell expansion after mucosal immunization but requires incorporating a suitable immunostimulant to increase long-lived memory T-cells. EP67 is a clinically viable, host-derived peptide agonist of the C5a receptor that selectively activates antigen presenting cells over neutrophils. We previously found that encapsulating EP67-conjugated CTL peptide vaccines in NP increases long-lived memory subsets of CTL after respiratory immunization. Thus, we hypothesized that alternatively conjugating EP67 to the NP surface can increase long-lived mucosal and systemic memory T-cells generated by encapsulated protein vaccines. We found that respiratory immunization of naïve female C57BL/6 mice with LPS-free ovalbumin (OVA) encapsulated in PLGA 50:50 NP (∼380 nm diameter) surface-conjugated with ∼0.1 wt% EP67 through 2 kDa PEG linkers (i) increased T-cell expansion and long-lived memory subsets of OVA323-339-specific CD4+ and OVA257-264-specific CD8a+ T-cells in the lungs (CD44HI/CD127/KLRG1) and spleen (CD44HI/CD127/KLRG1/CD62L) and (ii) decreased peak CFU of OVA-expressing L. monocytogenes (LM-OVA) in the lungs, liver, and spleen after respiratory challenge vs. encapsulation in unmodified NP. Thus, conjugating EP67 to the NP surface is one approach to increase the generation of long-lived mucosal and systemic memory T-cells by encapsulated protein vaccines after respiratory immunization.