Priming mucosal pathogen-agnostic innate immunity with an intranasal TLR2/6 agonist in an aged population

Abstract

Background

Immunosenescence increases the susceptibility to viral respiratory infections, but its effects on nasopharyngeal innate immunity, a critical determinant of response to viral infection, are not well understood. INNA-051 is a Toll-like receptor (TLR)2/6 agonist that activates innate immune pathways associated with antiviral host defence in younger individuals (aged 19–53 years), and in pre-clinical models protects against diverse viruses. Here we assessed INNA-051 in older adults, measuring host defence indicators, and tested its ability to protect aged mice from influenza.

Methods

Clinical safety, tolerability and elicitation of host defence biomarkers by 300 μg of intranasal INNA-051 were assessed in a cohort of 12 healthy older volunteers (aged 66–80 years) included in a recently reported randomised placebo-controlled, dose escalation phase 1 study. Aged mice (64 weeks) received repeated intranasal doses of INNA-051 on days −4 and −1 prior to infection with influenza (H3N2 Udorn, 500 PFU) and response was assessed on day 4 post-infection.

Results

In humans INNA-051 was well tolerated and significantly increased innate immunity host defence pathways within 8 h after each dose. In aged mice, nose-only dosing of INNA-051 stimulated innate nasopharyngeal innate immunity, primed adaptive immunity and effectively reduced lung influenza viral load.

Conclusions

INNA-051 induced innate immune priming in older individuals and attenuated influenza infection in aged mice. TLR2/6 agonist-mediated responses remain functional in older individuals and this study supports further clinical investigation in aged populations known to be susceptible to viral respiratory infections.

Shareable abstract

Activation of TLR2/6 with intranasal INNA-051 safely primed innate mucosal host defence pathways in older adults, and protected aged mice from influenza, suggesting therapeutical utility against respiratory viral disease in the vulnerable elderly https://bit.ly/43hoN9Z

Introduction

Older people endure increasing susceptibility to debilitating, and potentially life-threatening, viral infections as their immunity wanes with advancing immunosenescence [1]. This vulnerability can be further compounded by underlying comorbid conditions, particularly those of the respiratory and cardiocirculatory systems and, as the coronavirus disease 2019 (COVID-19) pandemic tragically underscored, by living conditions [2], particularly living in grouped residential aged care where pathogens with high strike rates can spread with devasting speed [3, 4].

INNA-051 belongs to a potentially transformative new class of medicines called “pathogen-agnostic host-directed therapies”. INNA-051 is a potent agonist of cell-surface Toll-like receptor (TLR)2/6 that is expressed by both human nasal epithelial cells and innate immune cells [5]. It is engineered to have limited systemic bioavailability, through the addition of a short (<30-mer) polyethylene glycol group. In experimental models INNA-051 exerts a rapid modulation of the host innate immune system when administered intranasally that is, at least in part, due to 1) recruiting macrophages to the epithelium and 2) priming host defence genes. This mode of action confers a fundamentally “agnostic” effect independent of the strain, or type, of virus. In pre-clinical animal studies, topical administration of INNA-051, and its close analogues, to the respiratory tract was effective at accelerating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral clearance in ferrets [6], protected mice from lethal and seasonal influenza virus infection [7, 8] and prevented secondary bacterial superinfections [7, 9–11]. In early clinical trials [12], INNA-051 has been shown to be safe, well tolerated and pharmacologically active by inducing local pathways associated with antiviral host defence and accelerating viral clearance after influenza challenge following administration to a young adult population.

However, it is not known if the innate immunity which underlies this type of pathogen-agnostic host-directed response to TLR2/6 is preserved in older people. Here we show that 1) INNA-051 is well tolerated and induces local innate immune stimulation in a cohort of healthy older (aged >65 years) volunteers in a phase 1 clinical trial (registered at https://anzctr.org.au with identifier ACTRN12621000607875p) and 2) in very old mice, repeated intranasal INNA-051 is well tolerated, primes an adaptive immune response and results in reduced lung viral titres following influenza challenge. These data support the concept that intranasal host-directed pathogen-agnostic therapy, exemplified by INNA-051, may be an effective strategy to protect vulnerable older people from viral respiratory illnesses and support further translational research in this highly susceptible population.

Methods

Clinical trial design

This was an aged cohort (aged >65 years) included in a phase 1, single-centre, randomised, double blind, placebo-controlled study to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of multiple ascending, intranasal INNA-051 doses [12]. The trial was conducted in Australia (Scientia Clinical Research Ltd). Repeated doses were administered on days 1, 4, 7 and 10 with either intranasal INNA-051 (300 μg) or placebo. 12 participants were enrolled and randomised by the site in a 3:1 ratio (figure 1a).

FIGURE 1.

Tolerability and safety design. a) Participants received four doses of 300 μg INNA-051 (n=7) or placebo (n=4) at 3-day intervals. Sampling occurred 24 h pre-dose and 6, 12, 24 and 48 h post-dose 1 with a follow-up collection at 6, 24 and 168 h (7 days) post-dose 4. b) Consolidated Standards of Reporting Trials diagram: healthy participants, active or placebo as allocated interventions.

Manufacture

Production of INNA-051 and matching placebo was manufactured and labelled as described previously [12]. Product formulation of INNA-051 was dissolved in 0.9% saline with 0.1% w/w EDTA, administered as an aqueous nasal spray solution via the Aptar cartridge pump system nasal spray pump device (100 µL actuation volume).

Safety assessments

Safety assessments included adverse events, vital signs, clinical laboratory safety tests (haematology, coagulation profile, clinical chemistry and urinalysis), peak nasal inspiratory flow (PNIF), peak expiratory flow (PEF) and visual analogue scale (VAS) scoring of nasopharyngeal symptoms.

Sample collection and processing

Nasosorption FX·i sampling devices (Mucosal Diagnostics, Hunt Developments) were used to absorb mucosal lining fluid and cells from both nostrils at pre- and post-dosing time points [13]. Samples for RNA analysis were collected 24 h pre-dose and 8 h after each dose. Blood for serum isolation was collected pre-dose and 6, 24 and 48 h post-doses 1 and 6; 24 and 168 h (7 days) post-dose 4. RNA extraction and concentration along with analysis with the NanoString nCounter Human PanCancer Immune Profiling Assay were completed as described previously [14–16].

Cytokine analyses

The concentrations of interleukin (IL)-1β (lower limit of quantitation (LLOQ) 1.01 pg·mL⁻¹), macrophage inflammatory protein (MIP)-1α (LLOQ 5.57 pg·mL⁻¹), monocyte chemoattractant protein (MCP)-1 (LLOQ 5.32 pg·mL⁻¹), IL-6 (LLOQ 1.98 pg·mL⁻¹), IL-8 (LLOQ 0.56 pg·mL⁻¹), tumour necrosis factor (TNF)-α (LLOQ 26.61 pg·mL⁻¹), interferon (IFN)-α2a (LLOQ 8.72 pg·mL⁻¹), IFN-γ (LLOQ 6.42 pg·mL⁻¹), IL-10 (LLOQ 0.97 pg·mL⁻¹) and growth-regulated protein-α (LLOQ 0.61 pg·mL⁻¹), in serum and nasal samples were determined using Meso Scale Discovery plates. Samples were analysed on the MS2400imager according to the manufacturer's instructions. Standards and samples were measured in duplicate. LLOQ was determined as the lowest amount of an analyte that could be determined within acceptable precision and accuracy. One participant was included only up to 24 h post-dose 3 due to withdrawal from study.

Animal model

Specific pathogen-free aged and adult male C57Bl/6 mice were obtained from the Animal Resource Centre (Perth, Australia). Mice were aged to ≥64 weeks at the start of the experiment. The animals were housed in sterile passive micro-isolators at a constant 20°C temperature on a 12-h day/night cycle and fed irradiated Barastoc mouse feed with irradiated tap water allowed ad libitum. Mice were anaesthetised with isoflurane and INNA-051 was administered intranasally on days −4 and −1 prior to Udorn infection in a small volume (15 μL), which constrains exposure to the nose only. Mice were then infected at day 0 with an intranasal instillation with 500 PFU of H3N2 mouse-adapted influenza virus A/Udorn/307/72 IAV (Udorn strain) (courtesy of David Jackson, Peter Doherty Institute, the University of Melbourne, Melbourne, Australia), in 10 μL. A/Udorn/307/72 was chosen for its tropism to effectively model the progression viral dissemination from the upper nasal respiratory tract to the lower lung respiratory tract. We have shown previously that this inoculum volume is strictly restricted to the nasal turbinates and does not directly infect the lungs. After dissemination from the nose, viral titres peak in the lung at 3–4 days post-Udorn inoculation [7]. Analyses were performed on day 4 post-Udorn inoculation.

Plaque assay

Infectious viral titres of lung samples were measured by plaque assay on confluent monolayers of Madin Darby canine kidney cells, as described previously [7]. Nasal turbinate and lung sampled from mice were incubated for 3 days at 37°C in 5% carbon dioxide and plaque-forming units, fixed in formaldehyde overnight and stained. Clear circular foci per well were counted. All assays were carried out in duplicate.

Characterisation of cell populations in pre-clinical lung samples

Lung lobes were processed for fluorescence-activated cell sorting by digesting in DMEM/1% fetal calf serum (FCS) containing collagenase D (500 μg·mL⁻¹) and DNase1 (100 g·mL⁻¹) (both Sigma-Aldrich, Merck Group, USA). Cells were suspended in DMEM with 10% FCS and stained. The antibodies used were BV421-F480, PerCP Cy5 .5-GR-1 (Ly6C/6G), FITC-CD45, PECy7-CD11c, PE-Siglec-F, BC711-CD64, APCCy7-Ly6F, BUV395-Cd11b, APC-MertK, Pac Blue-CD4, APC-CD3, PE-CD16/32, BV711-Nk1. 1, AF700-CD8a, PE/Dazzle 594-CD1d, APCCy7-B220 (BD Biosciences and BioLegend, Australia) (supplementary table S2). Cells were passed through a CytoFLEX LX Flow Cytometer (Beckman Coulter, Australia) and analysed in FCS Express (De Novo Software; gating strategy described in supplemental figures S1 and S2). Cell populations were classified as the following: interstitial macrophages, Ly6G⁻F480⁺SiglecF⁻CD64⁺CD11b⁺CD11c⁺; alveolar macrophages, SiglecF⁺CD11c⁺; neutrophils, F480⁻SiglecF⁻CD11b^hiGR-1/Ly6G^hi; cells encompassing dendritic cells and monocytes/macrophages, F480⁻GR-1⁻CD11b^hiCD11c^hi; CD4⁺ T-cells, CD3⁺CD4⁺; CD8⁺ T-cells, CD3⁺CD8⁺; B-cells, CD3⁻B220⁺; natural killer (NK) cells CD3⁻CD1d⁻NK1.1⁺CD16⁺CD32⁺; and NKT-cells, CD3⁺NK1.1⁺CD1d⁺.

Data analyses

VAS data and animal data were analysed in GraphPad Prism v9. Statistical analyses for VAS were performed on baseline subtracted data by mixed-effects model fit using restricted maximum likelihood, with Geisser–Greenhouse correction. Adjusted p-values <0.05 by Sidak's correction for multiple comparisons to placebo were considered statistically significant. For RNA analyses, quality control and normalisation were undertaken utilising nSolver 4.0 (NanoString Technologies, WA, USA). Gene expression data were normalised against controls and filtered to exclude genes expressed below a minimum count of 20. Reference gene normalisation was performed using geNorm. Principal component analysis was performed using DEGreport version 1.32.0 (Pantano 2022). Limma [17] was used to complete differential gene expression, pathway and gene set analysis [14–16] in the R computing environment [18]. Immune pathways scores were estimated as described previously [19]. Differential expression was assessed with a Benjamini–Yekutieli adjusted t-test with a log₂ fold change >1 or <−1 and adjusted p-values <0.05 considered statistically significant. Heatmaps were generated using pheatmap version 1.0.12 [20]. Animal data were tested for normality (Shapiro–Wilk) prior to pairwise comparisons of the treatment groups to placebo controls. Nonparametric datasets were assessed by a two-tailed Mann–Whitney test and parametric data by a two-tailed unpaired t-test, p-values <0.05 were considered statistically significant.

Ethics approval

The phase 1 clinical study was approved (2021-04-454) by the independent ethics review board (Bellberry human research ethics committee) and performed in accordance with International Conference on Harmonization Good Clinical Practice guidelines. All participants gave written informed consent. All animal experimentation was conducted in accordance with institutional regulations and ethics approach (20218, the University of Melbourne animal ethics committee).

Results

Study population, dosing and disposition

For the phase 1 safety, tolerability and pharmacodynamics study, 12 participants aged 66–80 years were enrolled and randomly assigned in a 3:1 ratio to receive repeated doses of either 300 μg INNA-051 (150 μg per nostril) (n=8) or placebo (n=4) (figure 1). One participant withdrew from the study after the third dose due to new-onset atrial fibrillation, which self-reverted within 24 h. It was subsequently disclosed that they had a history of supraventricular tachycardia 5 months earlier, which had self-resolved (case narrative in the supplementary material). Participant disposition and demographics can be found in table 1.

TABLE 1.

Participant disposition and demographics

	Healthy aged volunteers
	Active	Placebo
Randomised	8	4
Completed study	7 (100)	4 (100)
Withdrew from study	1	0
Demographics
Age years	75.2±4.2 (67.8–80.2)	69.9±4.7 (66.0–76.6)
Sex
Female	6 (75.0)	1 (25.0)
Male	2 (25.0)	3 (75.0)
Ethnicity
American Indian or Alaska Native	0 (0)	0 (0)
Asian	0 (0)	0 (0)
White	8 (100)	4 (100)
Other	0 (0)	0 (0)

Data are presented as n, n (%) or mean±sd (range).

Safety assessments

The adverse events profile for INNA-051 in an older population consisted predominantly of findings localised to the nasopharynx, including nasal congestion, nasal discomfort, nasal inflammation and rhinorrhoea, all of which were mild in severity and self-limiting (table 2). There was a trend towards higher incidences of adverse events in the INNA-051 cohorts relative to placebo. A phase 1 trial in adult participants (aged 19–53 years) with multiple dose ranges of INNA-051 showed similar trends of adverse events incidences of INNA-051 cohorts relative to placebo; however, there were no dose relationships identified [12]. No clinically significant laboratory abnormalities were observed during the study. Similarly, no remarkable changes in the PNIF or PEF values relative to baseline were observed. Descriptive statistical analysis found that nasal blockage (figure 2a) and rhinorrhoea (figure 2b) trended notably higher in the INNA-051 cohorts relative to placebo for the first dose administered, with a significant increase in rhinorrhoea 6 h post-dose 1 that resolved in the following 6 h measurement (figure 2b). Of note, with multiple dosing there were no incremental increases, and peak mean VAS scores for nasal blockage and rhinorrhoea were no different to placebo. Portions of the adverse event data have been reported previously in an abstract [21].

TABLE 2.

Treatment-emergent adverse events (TEAEs)^#: 300 μg cohort and pooled placebos

	Aged INNA-051 300 μg		Pooled aged placebo
	Participants	Events	Participants	Events
Participants	8	46	4	16
At least one TEAE	8 (100.0)		3 (75.0)
Blood and lymphatic system disorders
Thrombocytopenia	0	0	0	0
Cardiac disorders
Atrial fibrillation	1¶ (12.5)	1	0	0
Gastrointestinal disorders
Paraesthesia oral	2 (25.0)	2	0	0
General disorders and administration site conditions
Fatigue	0	0	0	0
Hot flush	1 (12.5)	1	0	0
Injury, poisoning and procedural complications
Meniscus injury	0	0	1 (25.0)	1
Nasal injury	0	0	0	0
Phlebitis	0	0	0	0
Musculoskeletal and connective tissue disorders
Back pain	0	0	0	0
Myalgia	1 (12.5)	1	1 (25.0)	1
Nervous system disorders
Dysgeusia	0	0	0	0
Headache	3 (37.5)	4	2 (50.0)	2
Migraine	0	0	1 (25.0)	1
Paraesthesia	0	0	0	0
Pre-syncope	0	0	0	0
Reproductive system and breast disorders
Dysmenorrhoea	0	0	0	0
Respiratory, thoracic and mediastinal disorders
Epistaxis	1 (12.5)	3	0	0
Intranasal paraesthesia	0	0	0	0
Nasal congestion	3 (37.5)	4	0	0
Nasal discomfort	1 (12.5)	1	0	0
Nasal dryness	0	0	0	0
Nasal inflammation	7 (87.5)	11	1 (25.0)	4
Nasal mucosal disorder	5 (62.5)	6	2 (50.0)	2
Nasal pruritus	0	0	0	0
Oropharyngeal pain	1 (12.5)	1	1 (25.0)	1
Rhinorrhoea	5 (62.5)	7	1 (25.0)	1
Sinonasal obstruction	2 (25.0)	2	0	0
Sneezing	0	0	0	0
Throat irritation	0	0	0	0
Skin and subcutaneous tissue disorders
Cheilitis	0	0	1 (25.0)	1
Dermatitis atopic	0	0	0	0
Dermatitis contact	1 (12.5)	1	1 (25.0)	1
Ingrown hair	0	0	0	0
Rash	0	0	0	0
Skin abrasion	1 (12.5)	1	0	0
Vascular disorders
Dizziness	0	0	1 (25.0)	1

Data are presented as n or n (%). Participants who experience multiple events within a category are counted only once in that specific category (n); however, each instance of the event is counted. Percentages are based on the number of participants in the analysis set. Adverse events were coded using Medical Dictionary for Regulatory Activities version 23.1. ^#: defined as adverse events that commence or worsen on or after the first administration of the study drug; ^¶: one participant experienced atrial fibrillation, classified as a moderate adverse event; the participant was withdrawn and commenced apixaban.

FIGURE 2.

Visual analogue scores (VAS) from participants treated with INNA-051 or placebo. Participants received four doses of 300 μg INNA-051 (n=6) or placebo (n=4) at 3-day intervals. VAS scores were taken prior to dosing and at 2, 6, 12, 24 and 48 h and 7 days post-dose. Normalised (baseline subtracted) a) nose blockage and b) rhinorrhoea.

Nasal cytokine assessments

Mechanistic pharmacodynamics biomarkers, MCP-1 and MIP-1α for INNA-051 have previously been identified in nonclinical pharmacology studies [7] as well in a phase 1 healthy adult (aged 18–55 years) clinical trial as potential indicators for biological responses induced by INNA-051 treatment [12]. Multiple intranasal administration of INNA-051 in this older population was also associated with significant local increases in the pharmacodynamics biomarkers MIP-1α and MCP-1 (compared to placebo) at 6 and 12 h after the first dose (supplementary figure S3A and S3B, respectively). Although not statistically significant, we observed that MIP-1α and MCP-1 levels remained elevated in some individuals up to 48 h after dosing. This was also observed in previous clinical studies conducted in younger individuals aged 18–55 years [12] and pre-clinical studies [7]. No drug-related adverse event related to this effect was observed in this study or previous clinical studies [12]. To confirm nonclinical pharmacology studies showing that INNA-051 does not directly stimulate the release of type I interferons [7], concentrations of IFN-α2a were also assessed. IFN-α2a in the nasal samples were below the LLOQ (data not shown).

Serum chemokine and cytokine assessments

Measurements of serum cytokine concentrations were undertaken to determine the effect of intranasal treatment on systemic release of chemokine and cytokines. Of particular interest were IL-6, IFN-γ, TNF-α and IL-1β, which are known markers of cytokine release syndrome [22], and the anti-inflammatory cytokine IL-10, which is produced in response to pro-inflammatory conditions as a negative feedback mediator [23]. There was no evidence of an increased systemic release of storm cytokines (IL-6, IFN-γ, TNF-α or IL-1β) after multiple doses of INNA-051. Serum cytokine levels were analysed at 6, 24 and 48 h after the first dose and fourth dose. This finding is consistent with the inability to detect INNA-051 in plasma (levels below LLOQ; 0.1 ng·mL⁻¹), which suggests a lack of systemic drug exposure. Serum IL-10 and IFN-α2a levels in all samples were at LLOQ or within the ranges seen in the placebo group (data not shown). A low and transient change from baseline of MIP-1α was 6 h post-dose 1 (remaining <90 pg·mL⁻¹) (supplementary figure S3C) as well as a transient increase from baseline of MCP-1, with levels returning to baseline within 24 h (supplementary figure S3D).

Transcriptional immune profiling of nasal mucosa

Principal component analysis indicated that the immune gene expression profiling of the nasal mucosa in pre-dose samples segregated from each of the INNA-051 post-dose 8 h administration time points (figure 3a). Expression increases were observed in key innate immune genes (including IL8, NLRP3 and IL1B), chemokines (CCL2, CCL3 and CCL20) and adhesion molecules (ITGAL, CEACAM1 and ICAM1) (supplementary table S1). An increase in the expression of genes encoding several IFN signal transduction molecules as well as interferon-stimulated genes (ISGs) was observed in the aged cohort, comparable to the pattern in younger adults [12]. Immune cell profiling of nasal mucosa using cell-specific gene signature patterns [24] found that INNA-051 increased the abundance of CD45⁺-expressing immune cells and neutrophils (figure 3b). The pattern of change in immune pathway gene expression and cell abundance scores over the dosing period in older individuals indicates that INNA-051 can elicit a response after each dose on repeated administration. We observed a similar pattern in younger individuals (18–55 years) [12]. Gene set enrichment analysis from pre-treatment to the sequential four 8 h time points post-dose indicated that INNA-051 had an innate immune stimulatory effect, with enrichment of functional pathways [25] associated with innate immunity, in particular the TLR, TNF superfamily, pathogen defence, chemokines, cytokines, macrophage and leukocyte functions (figure 3c). Given the presence of host-response-related pathways among 129 genes differentially expressed from baseline to post-treatment time points, we undertook heatmap visualisation of differentially expressed genes within these pathways. INNA-051 treatment increased expression of 56 genes related to pathways including influenza response (CASP1, ICAM1, HLA-DRA, CCL2, IL1A, NLRP3, TNFRSF1A, IFNGR1, MYD88, PIK3CD, TLR4, IL1B and NFKBIA), pathogen defence (IL1B and IL8) and macrophage function (SYK, CD47, LCP1, SLC11A1 and SBNO2) (figure 4). Notably, the associated enrichment of gene regulatory programmes in the associated pathways was sustained with each repeated dose (figure 4). We further compared the associated enrichment responses to a cohort of adults (aged 19–53 years) [12] and identified that the differentially expressed genes between the two cohorts were highly overlapped. Aged participants who received a 300 μg dose of INNA-051 shared 120 of 129 differentially expressed genes when compared to the adult cohort that received a lower 150 μg dose of INNA-051 (supplementary figure S4). When compared to adults who received an equivalent 300 μg dose of INNA-051, the adult response showed an additional 141 differentially expressed genes in conjunction with 124 of the 129 similarly shared differentially expressed genes between the aged and adult cohorts (supplementary figure S4).

FIGURE 3.

INNA-051 immunomodulates (local) immune cell profiles and enriches innate immunity-associated pathways. a) Principal component (PC) analysis plot of samples from placebo, pre-dose samples and 8 h post-dose. Overt separation of clusters of immune gene expression between the 8 h post-dose relative to the pre-dose and placebo clusters and no cluster separation between each subsequent 8 h post-dose. b) Immune cell profiling using cell-specific gene co-expression patterns showing abundance of CD45⁺ expressing immune cells and neutrophils. c) Immune gene expression analysis was performed using the Nanostring Counter Human Pan Cancer Immune Profiling panel. Associated functional pathway scores (x-axis) were calculated using the Nanostring Pathway Module [19]. TLR: Toll-like receptor; TNF: tumour necrosis factor.

FIGURE 4.

INNA-051 immunomodulates (local) gene regulatory programmes associated with innate immunity and the influenza host-response pathway. Relative gene expression (z-score) in placebo and INNA-051 300 μg aged cohorts at baseline, and at 8 h post-dose for each administration on days 1, 4, 7 and 10. Set of 56 differentially expressed genes with functions that are associated with the enrichments in figure 3c, the influenza Kyoto Encyclopedia of Genes and Genomes pathway, and interferon (IFN) signalling pathway. Pathway membership is indicated in the left-hand panel. TNF: tumour necrosis factor; NK: natural killer; TLR: Toll-like receptor.

Pre-clinical efficacy and tolerability of INNA-051 in aged mice

Aged mice were randomised and received either an intranasal placebo or INNA-051 dose of either a 3 nmol or 10 nmol at −4 days and −1 day prior to Udorn challenge administered by an operator blinded to the treatments (figure 5a). Repeated prophylactic INNA-051 treatment was well tolerated with no overall impact on bodyweight in aged mice (figure 5b). As expected, prophylactic INNA-051 treatment had no effect on preventing initial Udorn infection in the nose (figure 5c). In contrast, Udorn viral titres in the lungs of aged mice showed a trend (p=0.065) to reduced lung viral titres at 3 nmol and significantly reduced lung viral titres at 10 nmol (figure 5d). The 10 nmol, but not 3 nmol (trend) dose significantly increased CD45⁺ immune cells, alveolar and interstitial macrophages, dendritic cells, CD4⁺, CD8⁺ and B-cells, with a trend (p=0.0529) for increased NKT-cells (figure 5e). Because the nasal turbinates were homogenised for viral titre, it was not possible to analyse for cell composition. As INNA-051 was only administered to the nose and is not bioavailable to the lung, we believe cell accumulation in the lung to be a passive process. Doerschuk et al. [26] described that when leukocytes are mobilised from the bone marrow, they marginate in the pulmonary circulation which is the first vascular bed they encounter. These marginated leukocytes may then undergo diapedesis in the interstium and lumen, independent of integrin adherence mechanisms.

FIGURE 5.

Prophylactic repeated administration of INNA-051 to the upper respiratory tract inhibits viral replication in aged lungs and stimulates innate and adaptive immunity. a) Mice (n=8 per group) were administered with sequential INNA-051 at a prophylactic dose of either 3 nmol or 10 nmol prior to challenge with 500 PFU Udorn. b) Change in bodyweight following Udorn infection. c,d) Efficacy of prophylactic treatment was determined by measuring both c) turbinate and d) lung viral titres 4 days post-infection (n=7–8 per group). e) Single-cell lung suspensions stained with fluorochrome-conjugated antibodies of various specificities and analysed by flow cytometry. Statistical analysis for nonparametric datasets was assessed by a two-tailed Mann–Whitney test and parametric data were assessed by a two-tailed unpaired t-test. i.n.: intranasal; NK: natural killer. *: p<0.05, **: p<0.01, ***: p<0.001.

Discussion

Viral respiratory infections particularly afflict older people in whom immunity has waned with advancing age. This vulnerability is thought to be linked to immunosenescence and inflammaging associated with a decline and dysregulation of immune functions [27, 28] and ultimately, the ability to control and eliminate viruses locally before disseminating to the body. As most respiratory viruses first infect the nasopharynx before ramifying to the lungs, the vigour of initial host defence here is a critical determinant of the extent and severity of infection. Accordingly, priming the innate immune system in the nasopharynx is an attractive option, as it confers “pathogen-agnostic” protection against diverse viruses concurrently.

Synthetic agonists of the viral DNA/RNA-recognising TLR molecules, TLR3, TLR7/8 and TLR9, have been shown to boost protective innate immune responses against respiratory viruses. However, their success in the clinic has been limited, mostly due to short duration of benefit or induction of adverse effects related to the systemic release of pro-inflammatory cytokines and activation of the type I IFN pathway, which is associated with flu-like symptoms in humans [29–31]. TLR2 has been shown to recognise a broad variety of commensal and pathogenic microbial molecules [32]. TLR2 typically forms a heterodimer with TLR6 and is highly expressed on primary human nasal epithelial cells [5] and does not directly activate the type I IFN pathway [7, 8], offering an alternative approach.

INNA-051 is a highly potent TLR2/6 agonist with a half-maximal effective concentration of <50 pM against the human TLR2/6 [6], developed to be a self-administered nasal spray to target the predominant site of entry of SARS-CoV-2 and other respiratory viruses [33, 34]. It is engineered to have limited systemic bioavailability through the addition of a polyethylene glycol group which also acts as a solubilising agent. Fast-acting and inducing a durable biological response supporting weekly administration, INNA-051 and close analogues have been shown in nonclinical efficacy and mechanistic studies to mediate upregulation of innate immune defence pathways in airway epithelial cells, defined by early expression of NF-κB-regulated antimicrobial genes, including type III IFN (IFN-λ) that precede immune cell recruitment, such as macrophages, dendritic cells and neutrophils, and support relatively prolonged antiviral defence [7, 8]. This strongly correlates with the observed pre-activated status and virus sensing of epithelial and immune cells of the upper airways in children, which are thought to be responsible for the substantially lower risk of developing COVID-19 in this population [35]. Furthermore, recent studies in natural infection and a SARS-CoV-2 challenge study in adults indicate the importance of nasal mucosa-mediated immunity in the recovery from COVID-19 [36, 37]. Whether this protective effect is preserved with advancing age was the subject of the current study.

This phase 1 study in healthy aged volunteers demonstrated that multiple administration of the synthetic agonist of TLR2/6, INNA-051, was well tolerated, immunomodulates through activation of local innate immunity and rapidly upregulates gene expression profiles associated with innate and influenza host responses within 8 h post-dose over four repeated administrations. INNA-051 treatment in healthy aged volunteers is a localised response with no release of storm or other pro-inflammatory cytokines. Moreover, the sustained activation of innate immune gene regulatory programmes with each sequential dose of INNA-051 in healthy aged volunteers is suggestive that the host-directed priming of local innate immunity with INNA-051 stimulates the waning ageing innate immunity. These are important considerations, especially for aged populations where repeat dosing may reduce vulnerability to diverse respiratory viral infections, including SARS-CoV-2, where nasopharyngeal host defence is critical.

In a recent publication describing influenza-infected participants of a challenge study [12], a dose-related trend was observed showing a reduced duration of influenza symptoms and the associated influenza host-response gene expression profiles. The lower dose (150 μg) in the adult cohort exhibited a similar transient nature of gene regulation in the influenza host-response pathway, as observed in our described healthy aged volunteers. The higher dose (300 μg) in the adult cohort constitutively activated the influenza host-response gene expression profile, and was shown to significantly reduced the duration of infection and viral load [12]. In previous pre-clinical pharmacological studies in young animals, intranasal single and repeated administration of INNA-051 resulted in localised macrophage chemoattractant and reflects the critical role played by macrophages in the innate antiviral mechanism of action of INNA compounds [7]. Here we show that in an aged influenza challenge model, prophylactic intranasal repeated administration of INNA-051 was effective in reducing lung viral titres and stimulated innate immunity and dose-dependently primed an adaptive response with the higher dose producing a log-fold reduction of lung viral load. Considered together, these data suggest that it is likely the dose threshold for prophylaxis INNA-051 will be higher for an aged population to effectively stimulate the innate response to an influenza challenge when compared to a younger adult cohort [12].

Importantly, INNA-051 dosing in healthy aged volunteers was well tolerated and not associated with IFN-α2a production (IFN-β protein was not assessed) at the time points analysed, affirming previous nonclinical pharmacology studies [7], that treatment does not directly stimulate the local or systemic release of type I interferons (both α and β were examined). Mechanistic studies have also shown that the antiviral effect of INNA compounds is primarily associated with early priming for increased expression of type III interferons (IFN-λ), which are known to play an essential role in limiting initial influenza viral replication and dissemination [38]. Ageing negatively affects IFN production and signalling during the early stages of viral infections [39], which is thought to be due to a decline of IFN-producing cells, including macrophages and dendritic cells [40, 41], and impairment in the mechanisms that induce IFN production [42]. Interestingly, while INNA-051 did not directly induce type I IFN (α or β) expression, it did increase the expression of ISGs. ISGs play crucial roles in pathogen defence, encoding factors targeting viral transcription, translation, replication and promoting degradation of viral nucleic acids [39, 43]. Furthermore, recent insights from influenza-infected individuals in participants of a challenge study identified a synergistic effect of INNA-051 prophylaxis with an active influenza infection through increased IFNA7 and IFNL2 expression post-challenge [12]. Selective increased expression of ISGs post-administration of INNA-051 is probably independent of IFN receptor signalling, given that intranasal treatment with a close analogue of INNA-051 protects animals lacking receptors for the type I IFNs (IFN-α and IFN-β) against lethal H1N1 influenza challenge [11]. The ability of INNA-051 to prime ISG expression in healthy aged volunteers independent of waning IFN production may indicate that INNA-051 treatment could augment antiviral defence in elderly and other immunocompromised individuals.

Based on the gene expression data, safety profiles and animal data, INNA-051 is an attractive host defence agent for a new paradigm of prophylactic intervention against respiratory virus infections in older individuals support further evaluation of INNA-051 dose ranges, specifically for aged population groups. Planned studies will assess the efficacy and safety of weekly self- or caregiver-administered intranasal doses INNA-051 in community infections.

Supplementary material

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Supplementary material

01044-2024.SUPPLEMENT

Data availability

The top 20 differentially expressed genes in nasosorption samples are available in supplementary table S1. Other data will be made available on request.