Introduction: Mucosal vaccines have become increasingly sought owing to their needle-free delivery, lower cost, and ability to raise strong immune responses at the site of application, notably production of IgA and tissue-resident T-cells. We have developed a self-assembling nanofiber system known as Coil29 that is amenable to lengthwise control and addition of sequences of interest to its N-terminus. Here, Coil29 with C5a epitopes from the complement system are employed to tackle chronic inflammatory reactions through intranasal mucosal delivery. However, it has been previously observed that Coil29 causes Th1 bias and is proinflammatory when administered with our mucosal adjuvant cyclic-di-AMP, contrary to our desired goals. Moreover, this adjuvant may traffic to the brain by crossing the blood-brain barrier due to its small molecular size and the proximity of application to the brain in intranasal administration. Therefore, we employed a “Prime and Spike” approach to evaluate an adjuvant-free method for obtaining protective mucosal immunity with unadjuvanted peptide nanofibers. The method consists of priming the subject subcutaneously and then boosting intranasally, both with unadjuvanted formulations. Originally applied to a SARS-CoV-2 vaccine, we show here that prime and spike is able to elicit strong systemic and mucosal responses when using unadjuvanted peptide nanofibers.
Materials and
Methods: Coil29-C5a peptides were synthesized with Liberty Blue solid-phase peptide synthesizer and purified via HPLC. The molecular weight of synthesized peptides was verified through MALDI-TOF mass spectrometry. Peptides were assembled into nanofibers using previously described methods, and their morphology was assessed through atomic force microscopy (AFM). In a murine model, nanofibers were administered in three different modalities, in which each mouse received three immunizations in total: 1) intranasal prime/intranasal boost/intranasal boost; 2) subcutaneous prime/subcutaneous boost/subcutaneous boost; and 3) subcutaneous prime/intranasal boost/intranasal boost. Nanofiber solution delivery consisted either of 40 μL intranasally or 100 μL subcutaneously during each immunization. Priming occurred in week 0 and boosts were applied on a biweekly basis in week 2 and 4. Blood was collected on week 0, 1, 3, and 5. Feces were collected on week 0 and 5 and processed to isolate antibodies. Then, blood serum and feces were subjected to ELISAs to measure the presence of IgA and IgG. At endpoint in week 5, splenocytes were collected and subjected to an ELISpot to quantify cytokine secretion and T-cell bias. Additionally, nasal-associated lymphoid tissue (NALT) and Peyer’s patches were cultured and antibody titers from these tissues were quantified via ELISA. Finally, data was analyzed through one-way or two-way ANOVA in GraphPad Prism.
Results, Conclusions, and Discussions: To evaluate C5a-Coil29 immunogenicity, we first monitored IgG levels in the serum to determine systemic immunity. In week 5, prime & spike achieved equally high anti-C5a titers as the subcutaneous group, while intranasal showed less consistent responses with only about 60% of the mice responding. We then analyzed IgG and IgA titers in the nasal-associated lymphoid tissue (NALT), Peyer’s patches, and feces, in addition to serum IgA. NALT collected on week 5 also showed consistently high IgG levels for prime & spike. Conversely, intranasal immunization showed consistently low NALT IgG titers and subcutaneous immunization only produced titers as high as prime and spike in 60% of mice. Additionally, week 5 fecal IgG was significantly higher for prime & spike when compared to the other methods. Results from Peyer’s patches further emphasize the consistency of IgG production through the prime & spike method using the C5a-Coil29 platform. Serum IgA increased steadily over the course of the experiment for mice in the prime & spike group, achieving comparable results as intranasal immunization. NALT, Peyer’s Patches, and fecal samples have also been observed to show similar IgA responses. Despite low responses, the steady increase in serum IgA for prime & spike coupled with similar trends as intranasal immunization might suggest the possibility of mucosal immunity induction via this novel method while offering higher IgG responses. In searching for an unadjuvanted method of C5a-Coil29 delivery, we have demonstrated a more cost-efficient and effective approach that elicits equally high IgG titers as intranasal and conventional subcutaneous immunization. Prime and spike elicits more robust and consistent systemic immunity and comparable mucosal immunity. Moreover, prime and spike cleared the need for an adjuvant while also requiring 40% less nanofibers to be delivered when compared to subcutaneous immunization. In clinical application, this could lead to more patients being vaccinated as well as a reduction in supply-chain costs. Further, avoidance of adjuvant improves the safety profile of synthetic vaccines and streamlines clinical development.
