A multicomponent intranasal adjuvant drives durable humoral, cellular, and mucosal immune responses to SARS-CoV-2 in young and aged mice

Multiple FDA-approved SARS-CoV-2 vaccines provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a marked decrease in immunogenicity with age. Robust antigen-specific IFNγ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

immunity in both young and aged populations [5][6][7][8] . 70 There has been much interest in developing mucosal vaccines which induce both systemic and 71 respiratory mucosal immunity against SARS-CoV-2. This is especially pertinent given the immunity is not achieved, mucosal immunity in the upper respiratory tract provides the major 77 advantage of blocking viral dissemination into the lower respiratory tract, and memory T cells 78 within the respiratory tract can provide more effective protection [10][11][12][13][14] . 79 Here we expand on our previous work on a rationally designed combination intranasal (IN) 80 adjuvant composed of a nanoemulsion-based adjuvant (NE) and an RNA-based agonist of RIG-I 81 (IVT DI) 15,16 . NE is an oil-in-water emulsion consisting of soybean oil, a nonionic (Tween80) 82 and cationic (cetylpyridinium chloride) surfactant, and ethanol 17,18 . This adjuvant has established 83 Phase I clinical safety as an IN adjuvant in two different human trials [19][20][21] . NE induction of 84 mucosal and systemic immune responses is mediated, at least in part, through TLR2 and 4 85 activation and through NLRP3 activation via induction of immunogenic apoptosis 22,23 . IVT DI is 86 a highly selective RIG-I agonist, derived from the full-length (546nt) copy-back defective   Finally, we demonstrate that the humoral and cellular immune responses induced by NE/IVT are 112 both durable and long-lived. 115 and elicits mucosal antibody responses in both young and aged mice 116 The ability of NE and NE/IVT to induce immune responses in the context of aging was receptor binding domain (RBD) was selected as antigen to better differentiate responses, as it has 120 lower immunogenicity than either the full-length spike (S) protein or the S1 subunit previously 121 examined with the NE/IVT adjuvant 15 . The RBD contains the region of the S protein necessary 122 for binding to the human ACE2 receptor (hACE2) and for viral entry, and thus contains most 123 epitopes targeted by neutralizing antibodies (nAbs) as well as multiple T cell epitopes [26][27][28] . 124 Mice were immunized IN with three doses of 10 or 20 g of RBD with either PBS, 20% NE, or 125 young mice, no RBD-specific IgG was detectable for groups given RBD 10 only or IM Advx/10 138 RBD. However, low but detectable RBD-specific IgG was detectable in some of the young mice 139 in the NE/10 RBD (2/5), and NE/IVT/10 RBD (1/5) treatment groups at this early time point. For 140 animals given the higher dose of RBD, no RBD-specific IgG was detected in the RBD alone, or 141 NE/20 RBD groups, whereas low but significant IgG titers were induced in most mice in the 142 combined adjuvant NE/IVT/20 RBD group (4/5), demonstrating the advantage of the combined 143 NE/IVT adjuvant in both young and aged animals.

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After the second immunization (prime/boost), IgG titers increased in all adjuvanted groups in 145 both age cohorts ( Figure 1B) that are maintained in the context of aging. After the third immunization, (prime/boost/boost) 160 RBD-specific IgG titers further increased for all adjuvanted groups in young and aged mice 161 ( Figure 1C). Similar high IgG titers were induced in young mice given NE/10 RBD, NE/IVT/10 162 RBD, and IM Advx/10 RBD (1.2x10 4 , 1.6x10 4 , 1.2x10 4 , respectively). While comparable titers 163 were induced after the final boost in young and aged mice given NE/IVT/10 RBD, titers for aged 164 mice immunized with NE/10 RBD and IM Advx/10 RBD did not increase as much as those for 165 the corresponding young groups, supporting the advantage of the combined NE/IVT adjuvant. In 166 young mice given 20 g RBD, similar RBD-specific IgG titers were observed for the NE and 167 NE/IVT groups, which were comparable to those in young mice immunized with 10 g RBD 168 with the same adjuvants. However, for the NE only groups, the spread in induced IgG titers was 169 reduced at the higher RBD dose, suggesting a more optimal response with the higher antigen 170 dose for the single adjuvant. As was observed after the second immunization, similar or higher 171 titers were seen in the aged mice given NE/IVT/20 RBD as compared to the young animals after 172 the third immunization. While no difference in RBD-specific IgG was observed in young mice 173 when comparing the NE/IVT adjuvanted 10 and 20 g RBD groups, a half-log increase was 174 observed in aged mice with the higher antigen dose.

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RBD-specific IgG subclass distributions for IgG1, IgG2b and IgG2c were analyzed at the 10 wk 176 time point (Figure 1D-F). Subclass analysis indicated a balanced TH1/TH2 profile for the NE and 177 NE/IVT adjuvants, consistent with previous studies 15, 16,29,30 . IgG1 titers followed the same 178 6 relative pattern in each treatment group across age cohorts as observed for total IgG. NE/IVT 179 induced higher IgG1 titers than NE alone in both young and aged mice, and both adjuvants 180 induced similar IgG1 titers in young and aged groups ( Figure 1D). Robust IgG2b titers were also 181 induced by the NE and NE/IVT in young and aged mice. As in previous studies, inclusion of 182 IVT in the NE/IVT enhanced IgG2b and 2c titers relative to NE alone, consistent with the greater 183 TH1-bias of NE/IVT. Interestingly, NE/10 RBD and NE/IVT/10 RBD induced higher IgG2b 184 titers (half log higher) in aged as compared to young mice. In contrast to the IN adjuvants, IM 185 Advx/10 RBD induced notably less IgG2b relative to IgG1, particularly in aged mice, consistent 186 with the more TH2-polarizing properties of this adjuvant. IgG2c titers induced by NE and 187 NE/IVT were lower than IgG2b, appearing to require a higher antigen dose for optimal 188 induction. However, NE/IVT also appeared to induce higher IgG2c in aged mice as compared to 189 young, as particularly evident at the higher RBD dose (NE/IVT/20 RBD) ( Figure 1F).   Table S1).   Table S1). To further assess the protection provided by antibodies from young and aged mice immunized 294 with NE and NE/IVT in the absence of cellular immunity, sera from immunized mice were 295 transferred into 8-week old naïve recipient mice. Sera from mice in each immunization group 296 were pooled after two immunizations at wk 6 (2 wks after the last immunization), and 50 L of  consistent with the relative neutralization titers observed in these immunization groups.

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Achieving sterilizing immunity will likely be promoted by a combination of both B and T cell 334 responses. The latter may be particularly the case for the RBD antigen, since it has been shown 335 to be less protective as a soluble protein (unconjugated to nanoparticles) than the S1 subunit or 336 full-length S protein, particularly against heterologous viral variants 32 .  The importance of robust T cell responses in protection against SARS-CoV-2 has been clearly 364 established especially for protection against severe disease. T cell responses are responsible for 365 maintaining immunity when nAbs wane and for imparting immunity against divergent variants 366 that nAbs fail to effectively neutralize given that T cell epitopes are typically more highly 367 13 conserved across different variants of concern. Accordingly, we evaluated T cell antigen recall  (Figures 4A, 5A). Surprisingly, showed significantly reduced levels of IL-2 in aged animals (Figures 4B, 5B). NE/IVT similarly 391 enhanced IP-10 levels relative to NE alone in aged mice, however differences were small within 392 the spleen and more clearly observable in the cLN (Figures 4C, 5C). Thus, for these TH1 393 cytokines, NE/IVT/10 RBD induced similar or higher levels of cytokine secretion in aged, 394 immunized mice as in young immunized mice, which was in contrast to the consistent reduction 395 observed with IM Advx/10 RBD in aged mice. To further examine the contribution from 396 antigen-specific CD8 + T cells, splenocytes from aged mice immunized with NE, NE/IVT, or 397 Advx with 10 g RBD were compared for IFN- production by ELISpot in response to 398 stimulation with the H-2K b class-I restricted peptide (VVLSFELL, the only identified class I 399 mouse epitope within the RBD) (Fig. S2). ELISpot analysis revealed a robust antigen-specific 400 CD8 + T cell response with the NE/IVT which was enhanced compared to the NE alone.  Relatively low levels of IL-13 were induced in the spleen or cLN across all of the young 426 treatment groups (Figures 4G, 5G). However, IL-13 was elevated for young mice immunized 427 with Advx/10 RBD, consistent with its TH2 bias. In light of concerns over TH2 associated  (Figures 4H, 5H). Interestingly IL-6 levels were also increased in the spleen and cLN of aged 436 mice for all adjuvanted groups as compared to the corresponding young groups. However, 437 minimal induction of IL-6 was observed in the cLN for NE/10 RBD and IM Advx/10 RBD in 438 either age group. immunoprotective mechanisms, it has also been associated with pathology in certain contexts 35 . 455 However, it has been shown to be protective and non-pathogenic in the context of IL-10 co-456 production. Indeed, significant IL-10 was elicited in the spleen by NE/IVT/10 RBD in both 457 young and aged groups which was further enhanced at the higher RBD dose (Figures 4J, 5J). 458 Interestingly, IM Advx/10 RBD elicited higher levels of IL10 in the spleen than NE or NE/IVT 459 at the same antigen dose in young mice but not in aged mice. In contrast, marked enhancement   Table S1).  Table S1).  Table S1).  Figure 7E). Interestingly, while IL-5 appeared modestly increased at wk 33 in the spleen for 536 NE and NE/IVT groups, levels remained low <70 pg/mL, and were markedly reduced in both 537 groups in the cLN at wk 33 relative to wk 10 ( Figure 7F). Similarly, IL-13 remained low in the 538 spleen and were reduced to undetectable levels in the cLN at wk 33 ( Figure 7G). Thus, while 539 TH1-associated cytokines were maintained or enhanced over six months, TH2-associated 540 cytokines were maintained in the spleen but were consistently reduced in the cLN. In contrast, 541 IL-6 levels showed a similar pattern as the TH1 cytokines, with IL-6 in the spleen increasing after 542 six months in both NE and NE/IVT groups, while cLN levels were unchanged ( Figure 7H). 543 Finally, comparison of IL-17A levels at wk10 and wk33 showed maintenance of the high levels  Induction of potent antibody responses at mucosal surfaces, the port of entry for respiratory 680 viruses, by vaccination is required to provide optimal protection and prevent transmission.  Other groups have also demonstrated promise with IN immunization against SARS-CoV-2.

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Much interest has focused on adenovirus vectored IN vaccines, for example a chimp-adenovirus 716 vectored S1/nucleocapsid/RdRp construct and an adenovirus-type 5 vectored RBD. Both 717 constructs were highly effective at inducing mucosal IgA after a single dose with induction of 718 airway memory T cells. However, both showed minimal or no induction of antigen-specific T 719 cell responses in the spleen, which were short-lived (<14 days). RBD has shown potential as a 720 vaccine antigen in different animal models and has been in clinical trials as a vaccine candidate, 721 albeit as a dimer rather than a monomer, most likely because of the poor intrinsic 722 immunogenicity of the RBD 42, 43 . An IN adjuvanted recombinant RBD vaccine has been tested in 723 preclinical animal models, however, the adjuvant was polyethyleneimine, a cationic lipid.

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Polycationic lipids are an interesting avenue that is being explored for mucosal adjuvants, but 725 need to be modified and carefully tested to reduce reactogenicity at mucosal sites particularly 726 where there is possibility for unintended delivery to the lung, due to interactions with 727 phospholipid cellular membranes. This is expected to be an important field of future research 728 given their inclusion in lipid nanoparticles for mRNA vaccines as well.

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Adjuvants and antigen 743 NE was produced by emulsifying cetylpyridinium chloride (CPC) and Tween 80 at a 1:6 (w/w) 744 ratio, with ethanol (200 proof), super refined soybean oil (Croda) and molecular grade water 745 using a high-speed homogenizer as previously described 18 . The emulsion was homogenized to a 746 uniform particle size (d=450-550 nm) and charge (zeta potential=50-55mV). The sequence and 747 synthesis of IVT DI RNA has previously been described in detail 15, 24 . Briefly, SeV DI RNA was 748 amplified using a 5' primer with the T7 promoter and a 3' primer with the hepatitis delta virus 749 genomic ribozyme site followed by the T7 terminator and cloned into a pUC19 plasmid. IVT DI 750 was in vitro transcribed using a HiScribe T7 High Yield RNA synthesis kit (New England