Improved Cutaneous Genetic Immunization by Microneedle Array Delivery of an Adjuvanted Adenovirus Vaccine.

To the Editor

Genetic immunization based on recombinant DNA technology is an attractive approach for induction of robust antiviral or antitumor immunity (Condon et al., 1996, He et al., 2006). Specifically, adenoviral vaccines encoding target antigen transgenes are the subject of extensive preclinical studies owing to their established capacity for generating immune responses (Kim et al., 2016, Zaric et al., 2019). The skin is an ideal vaccination site containing an innate immune network that is exquisitely responsive to environmental stimuli and capable of inducing a proinflammatory microenvironment favoring the generation of strong and long-lasting adaptive immunity (Kabashima et al., 2019, Kashem et al., 2017). To exploit the readily accessible cutaneous immune network, vaccine delivery technologies, such as microneedle arrays (MNAs), have been developed to precisely and reproducibly target immunologically active cargos to skin microenvironments (Kim et al., 2012, Sullivan et al., 2010).

Though often effective in inducing antibody responses, traditional vaccines frequently fail to generate robust cytotoxic T-cell responses that are essential to prevent or treat many cancers or infectious diseases. Currently, induction of antigen-specific cellular immunity is a point of emphasis in the vaccine field, as evidenced by recent efforts to generate universal vaccines for mutable infectious agents (e.g., influenza, HIV, and coronaviruses). These vaccines target infected cells expressing functionally essential viral antigens, instead of, or in addition to, more traditional viral surface proteins that are targeted by antibodies but are highly mutable (Herold and Sander, 2020). Successful integration of adenovector vaccines onto coated or into dissolvable MNAs has been shown to induce efficacious and durable antigen-specific responses in preclinical studies (Bachy et al., 2013, DeMuth et al., 2013, Vrdoljak et al., 2012). Despite these promising results, clinical translation of adenovector vaccines has been hampered by limited efficacy, thereby defining an unmet need to enhance the immune responses induced by adenovirus vaccines. Here, we generated a three-dimensional multicomponent skin-targeted vaccine platform, combining an adenovirus-encoded antigen with an adjuvant to induce stronger cellular immune responses. Specifically, we developed dissolving MNAs to simultaneously co-deliver adenovectors encoding the transgene for the model antigen ovalbumin (OVA) together with polyinosinic acid:polycytidylic acid (Poly[I:C]), a toll-like receptor (TLR) 3‒triggering double-stranded RNA molecule, to the cutaneous microenvironment, with the primary goal of enhancing antigen-specific cellular immune responses. Dissolvable MNAs are designed to mechanically penetrate the superficial cutaneous layers, rapidly dissolve upon insertion into the skin, and deliver uniform quantities of biocargo to a defined three-dimensional space within the skin (Korkmaz et al., 2015). They enable localized delivery of low amounts of drugs or vaccines to achieve high concentrations in a specific skin microenvironment.

Innate cell-signaling pathways (e.g., downstream of TLRs) are well-studied targets for the rational design of vaccine adjuvants for protein subunit vaccines (Schijns and O’Hagan, 2006); however, they have yet to be comprehensively evaluated in the context of recombinant viral vectored vaccines because of significant mechanistic differences, including differences in the kinetics and amount of antigen expression. Among TLR family members, TLR3 signaling imparts unique responses, such as secretion of immunostimulatory IFN-β and CXCL10, owing to its distinct downstream pathways. Although other TLRs signal through adaptor protein MyD88, TLR3 uses the TRIF adapter protein, with subsequent activation of IFN regulatory transcription factor 3 and IFN regulatory transcription factor 7 (Boehme and Compton, 2004, Schijns and O’Hagan, 2006). Here, using in vivo mouse models, we demonstrate that MNA delivery of the TLR3 agonist Poly(I:C) with antigen-encoding adenovectors results in proinflammatory changes in the targeted skin microenvironment that correlate with robust antigen-specific cellular and humoral adaptive immune responses. For these studies, mice were used at 8–10 weeks of age, and all experiments were conducted in accordance with Institutional Animal Care and Use Committee-approved protocols and guidelines.

Dissolvable MNAs incorporating adenovirus vaccines (Ad5.OVA) with or without Poly(I:C) were manufactured using a composition of two generally-regarded-as-safe water-soluble biomaterials, carboxymethyl cellulose and trehalose. Optical microscopy images of MNAs before and after in vivo application to skin demonstrated high-quality micron-scale needles after fabrication and effective dissolution upon skin insertion, respectively (Figure 1 a and b). Importantly, the multicomponent MNAs effectively delivered adenovirus (Figure 1c) and Poly(I:C) (Figure 1d) to the skin microenvironment in vivo (Figure 1e), resulting in transgene (OVA) expression (Figure 1f). Interestingly, compared with MNA delivery of adenovector alone, inclusion of Poly(I:C) was associated with significantly increased expression of OVA mRNA that was sustained through 48 hours (P < 0.0001).

Figure 1

MNAs effectively penetrate the skin and deliver live adenovector vaccines and Poly(I:C) to the same cutaneous microenvironment, driving robust antigen transgene expression. Dissolvable MNAs incorporating Ad.OVA ± Poly(I:C) were fabricated using a spin-casting method, applied to the mouse skin for 10 minutes, and then removed. Images of MNAs (a) before and (b) after the application were obtained using optical stereomicroscopy. Bar = 500 μm. In vivo multicomponent vaccine delivery performance of MNAs was evaluated by fluorescent live animal imaging following application of MNAs incorporating Alexa488-labeled Poly(I:C) and Alexa555-labeled Ad.OVA to the right ears of mice. Mice were imaged using the IVIS 200 system with filters corresponding to (c) Alexa488 and (d) Alexa555 to demonstrate simultaneous co-delivery of Ad.OVA and Poly(I:C). (e) MNA-treated mouse skin was excised and imaged by epifluorescent microscopy and bright-field microscopy to show the intercutaneous delivery of multicomponent vaccines in vivo. Bar = 100 μm. (f) To quantify transgene (OVA) expression in the skin, mouse skin that was treated with Ad.OVA ± Poly(I:C) MNAs was recovered after 24, 48, and 72 hours, and OVA mRNA expression in the skin was quantified by RT-qPCR. Data are presented as mean ± SD. Significance was determined by two-way ANOVA followed by Sidak multiple comparison test. ∗∗P < 0.01 and ∗∗∗∗P < 0.0001. MNA, microneedle array; OVA, ovalbumin.

Intercutaneous vaccination with MNAs generated robust antigen-specific cytotoxic and humoral-immune responses. Remarkably, multicomponent MNA vaccine platforms incorporating both antigen-encoding adenovector and Poly(I:C) augmented OVA-specific lytic immunity by approximately two-fold compared with MNA delivery of the same adenovector alone (Figure 2 a). In addition to cell-mediated immunity, MNA-adenovirus vaccine platforms with or without the addition of Poly(I:C) elicited strong and robust antigen-specific antibody responses (IgG1 and IgG2c) (Figure 2b and c). Thus, adding Poly(I:C) to this MNA-delivered adenovirus vaccine significantly improved antigen-specific cellular immunity while maintaining strong antibody responses. Notably, multicomponent MNAs integrating both Poly(I:C) and adenovirus retained their immunogenicity after 1 month of storage at 4 °C, as indicated by no statistically significant loss in cell-mediated or antibody responses (Figure 2a–c).

Figure 2

Intercutaneous immunization with multicomponent MNA vaccine platforms incorporating adenovector-encoded OVA and Poly(I:C) adjuvants more effectively engineers a proinflammatory skin microenvironment in vivo Mice were immunized with Ad.OVA ± Poly(I:C) MNAs or blank MNAs (control). Antigen-specific cell-mediated and humoral immune responses were determined at the indicated time points using established lytic and ELISA assays, respectively. To assess the stability of multicomponent MNAs, intercutaneous immunization experiments were repeated with Ad.OVA+Poly(I:C) MNAs stored at 4 °C for 1 month. (a) Quantification of OVA-specific lytic responses. (b, c) Quantification of serum concentrations of OVA-specific IgG1 and IgG2c antibodies, respectively. Data are presented as mean ± SD and analyzed by one-way ANOVA, followed by Tukey’s post-hoc test. ns > 0.05, ∗P < 0.05, ∗∗P< 0.01, ∗∗∗∗P < 0.0001. (d–g) To investigate key immune mediators in the skin microenvironment induced by immunization, MNAs with the indicated components or blank MNAs were applied as described above, and expression of (d)IFNB1,(e)CXCL10,(f)IL1B, and (g)IL6 mRNA levels was quantified by RT-qPCR at the indicated time points. Data are presented as mean ± SD and analyzed by two-way ANOVA, followed by Tukey’s multiple comparisons test. Significant differences between treatment groups at each time point are indicated by ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. MNA, microneedle array; ns, nonsignificant; OVA, ovalbumin.

Mechanistically, simultaneous co-delivery of Poly(I:C) with adenovector vaccines impacted the proinflammatory microenvironment at the immunization site (Figure 2d–g). In particular, statistical analyses showed that the addition of Poly(I:C) significantly increased IFNB1 (Figure 2d) and CXCL10 (Figure 2e) expression at 6 hours with respect to blank (empty) MNAs or MNA-adenovirus vaccine alone, which suggests that Poly(I:C) plays a distinctive role during early skin immunomodulation. Furthermore, the inclusion of Poly(I:C) continued to significantly enhance the expression of CXCL10 (Figure 2e) at later time points (24 hours and 48 hours) compared with the groups with blank MNA and MNA-adenovirus vaccine alone, consistent with a sustained chemoattractant effect of Poly(I:C). Importantly, these proinflammatory effects of Poly(I:C) correlate with enhanced systemic cytotoxic T-cell responses. Expression of the proinflammatory cytokines IL 1 B and IL 6, which can be induced by a broad range of pathogen-associated molecular patterns and danger-associated molecular patterns, was elevated in MNA-immunized skin microenvironments at early time points (6 hours) regardless of vaccine components (Figure 2f and g) likely as a result of the mechanical stress of microneedle application. Interestingly, at later time points (48 hours and 72 hours), after the resolution of the transient mechanical stress generated by microneedles, both adenovirus and/or Poly(I:C) evoked significant increases in IL 1B expression in the skin microenvironment, and the combination of Ad.OVA and Poly(I:C) sustained elevated levels of IL 6 through 48 hours.

Collectively, our results demonstrate improved immunogenicity of skin-targeted adenovector vaccines by simultaneous co-delivery of the TLR3 ligand Poly(I:C) and support further development of pathogen-associated molecular pattern and/or danger-associated molecular pattern ligand integration in MNA-delivered viral vector vaccines. Specifically, our results demonstrate that Poly(I:C)-adjuvanted MNA-adenovirus vaccines elicit significantly improved cytotoxic T-cell responses compared with adenovirus alone while generating antibody responses at least as good as adenovirus alone. MNA-delivered vaccines have the potential to offer advantages of ease of fabrication, application, and storage compared with other vaccine delivery platforms. Our results suggest that by uniquely enabling delivery of both adjuvant and antigen-encoding viral vectors to the same skin microenvironment, multicomponent MNA vaccine platforms result in improved immunogenicity, including cellular immune responses, thereby contributing to the efforts to develop universal vaccines and improve global immunization capabilities.

Data availability statement

Data related to this article are available on request.

ORCIDs

Geza Erdos: http://orcid.org/0000-0001-7530-7371

Stephen C. Balmert: http://orcid.org/0000-0002-4938-0329

Cara Donahue Carey: http://orcid.org/0000-0002-3602-099X

Gabriel D. Falo: http://orcid.org/0000-0002-1669-8701

Nikita A. Patel: http://orcid.org/0000-0002-7162-9135

Jiying Zhang: http://orcid.org/0000-0002-4344-9794

Andrea Gambotto: http://orcid.org/0000-0001-8154-7419

Emrullah Korkmaz: http://orcid.org/0000-0002-8808-5445

Louis D. Falo Jr: http://orcid.org/0000-0001-9813-0433

Conflict of Interest

GE and LDF are inventors of related intellectual property.