key: cord-1048595-e74xs35u
authors: Jeyanathan, M.; Fritz, D. K.; Afkhami, S.; Aguirre, E.; Howie, K. J.; Zganiacz, A.; Dvorkin-Gheva, A.; Thompson, M. R.; Silver, R. F.; Cusack, R. P.; Lichty, B. D.; O'Byrne, P. M.; Kolb, M.; Medina, M. F. C.; Dolovich, M. B.; Satia, I.; Gauvreau, G. M.; Xing, Z.; Smaill, F.
title: Safety and immunopotency of an adenovirus-vectored tuberculosis vaccine delivered via inhaled aerosol to healthy humans: a dose and route comparison phase 1b study
date: 2021-09-14
journal: nan
DOI: 10.1101/2021.09.09.21263339
sha: c602863c5f42a9ee753df247ebc6db8450fc64d9
doc_id: 1048595
cord_uid: e74xs35u

Background: Adenoviral (Ad)-vectored vaccines are typically administered via intramuscular injection to humans, but this route of delivery is unable to induce respiratory mucosal immunity which requires respiratory mucosal route of vaccination. However, inhaled aerosol delivery of Ad-vectored vaccines has remained poorly characterized and its ability to induce respiratory mucosal immunity in humans is still unknown. The goal of our study was to evaluate and compare the safety and immunogenicity of a human serotype 5 Ad-based tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to healthy humans via inhaled aerosol or intramuscular injection. Methods: In this open-labeled phase 1b trial, 31 healthy adults between 18 and 55 years of age with a history of BCG vaccination were enrolled at McMaster University Medical Centre, Hamilton, Ontario, Canada. AdHu5Ag85A was administered by a single-dose aerosol using the Aeroneb Solo Vibrating Mesh Nebulizer or by intramuscular (IM) injection; 11 in the low dose (LD, 1x10e6 PFU) aerosol group, 11 in the high dose (HD, 2x10e6 PFU) aerosol group and 9 in the IM (1x10e8 PFU) group. The primary outcome was safety of a single administration of vaccine delivered to the respiratory tract by aerosol or by IM injection. The vaccine-related local and systemic adverse events were collected from participants from a self-completed diary for 14 days after vaccination and at scheduled follow-up visits. Routine laboratory biochemical and haematological tests were measured at 2, 4 and 12 weeks after vaccination and lung function was measured at 2, 4, 8 and 12 weeks after vaccination. The secondary outcome was comparison of immunogenicity among the different routes and aerosol dose groups. Immunogenicity to aerosol or IM vaccination was measured both in the peripheral blood and bronchoalveolar lavage samples by Luminex, and cell surface and intracellular cytokine immunostaining. Anti-AdHu5 antibodies and neutralization titers were determined before and after vaccination using ELISA and bioassay, respectively. This trial is registered with ClinicalTrial.gov, NCT02337270. Results: The aerosol droplets generated by Aeroneb Solo Nebulizer were mostly <5.39 micrometer in size, suitable for efficient Ad-vectored vaccine deposition to major human airways. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and the intramuscular injection were safe and well-tolerated. Respiratory adverse events were infrequent, mild, transient and similar among groups. IM injection was associated with a mild local injection site reaction in two participants. Systemic adverse events were also infrequent, mild, transient and similar among all groups. There were no grade 3 or 4 adverse events reported nor any serious adverse events. Both aerosol doses, particularly LD, but not IM, vaccination markedly induced Ag85A-specific airway tissue-resident memory CD4 and CD8 T cells of polyfunctionality. While as expected, IM vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages indicative of trained innate immunity. Interpretation: Inhaled aerosol delivery of Ad-vectored vaccine is a safe, economical and superior way to elicit respiratory mucosal immunity. The results of this study encourage further development of aerosol vaccine strategies against not only TB but also other respiratory pathogens including COVID-19.

Pulmonary tuberculosis (TB) continues to be a major global health issue, accounting for

In the present phase 1b study, we have characterized the property of AdHu5Ag85A aerosol 148 droplets generated by the AeroNeb Solo nebulizer. We evaluated the two aerosol doses and 149 compared the safety and immunogenicity of the vaccine delivered via the respiratory mucosal route 150 or intramuscular route in previously BCG-vaccinated healthy adults. Our study is the first to safely 151 deliver an adenovirus-vectored vaccine via inhaled aerosol to humans and to demonstrate its 152 superiority in inducing respiratory mucosal immunity over intramuscular injection. a randomization list was generated containing sequential codes linked to route of study vaccine 184 assignment, either IM injection or aerosol (Fig 1) . Following a review of the data after the 185 enrolment of the second cohort, the protocol was amended, and an additional 3 participants were 186 enrolled in the high dose aerosol group and an additional 3 participants were enrolled in the low 187 dose group. The study was not blinded. Each participant served as their own control (before and 188 after vaccination) and there was no placebo group. For safety reasons, 2 participants were first 189 vaccinated for each aerosol dose, and followed for 2 weeks after vaccine administration before 190 immunizing the rest of the participants in the dose group. Reports detailing AEs and SAEs for 4 191 weeks post-dose were reviewed by the safety monitoring committee before moving to the higher 192 dose. All participants were followed for a total of 24 weeks after vaccine administration. aerosol droplet size characteristics using the NGI Cascade Impactor operated at 15Lpm were 199 measured. Salbutamol sulphate served as the tracer for saline droplets containing vaccine particles. 200 Regional deposition of vaccine droplets in the lung was estimated from the particle size metrics of 201 the carrier (salbutamol) aerosol. An indication of the available vaccine dose at the mouth was 202 predicted from ED. The amount of aerosol-containing vaccine estimated to be deposited into the 203 lung was calculated using ED in combination with particle size statistics. Viability of the 204 aerosolized vaccine was determined by plaque forming assay.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Immunology Research Center. The first aspirate was discarded after obtaining the cell count. 2 nd , 224 3 rd and 4 th BALF was saved separately and stored at -80°C for future analysis. Cells were then 225 pooled and counted.

Interferon gamma release assay was performed using QuantiFERON TB Gold in tube or 227 QuantiFERON TB Gold Plus (Qiagen) according to manufacturer's instruction to determine latent 228 TB status 8 . Collected serum and concentrated BALF samples (20-fold concentrated from 10 ml 229 of combined aspirates 2 and 3 using Centriprep 3kDa Cut-off centrifugal filter unit-Millipore-230 Sigma) were measured for anti-AdHu5 IgG antibodies as previously described 8 . AdHu5-231 neutralizing antibody (nAb) levels in serum and concentrated BALF were assessed as a function 232 of GFP-expressing AdHu5 infection using A549 cells as previously described 18 . For both total 233 IgG and nAb titers, serum samples at a 1:10 and BALF at a 1:5 dilution were used.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint We counted the total cells in the BALF and calculated the number of cells per ml of BALF.

Differential cell count was performed on the cells from BALF using cytospin. We quantified the 236 Ag85A-and M.tb antigen-specific T cell responses in the peripheral blood and in the airways 237 represented by the BALF using ELISA and/or intracellular cytokine staining (ICS) assay as For determination of CD4 and CD8 T cell responses to vaccine using the whole blood 249 culture 10 , 1ml of blood was stimulated with each of the antigens mentioned above in the presence 250 of 1 μg/mL αCD28, 1 μg/mL αCD49d (BD Biosciences). Samples were incubated at 37°C in 5% 251 CO2 for 6h with Ag85A p. pool or PHA stimulation or for 12h with MtbCF-rAg85A stimulation.

Brefeldin-A was added for last 5h (Ag85A p. pool or PHA) or last 6h (MtbCF-rAg85A or 253 unstimulated whole blood). At the end of incubation, red blood cells were lysed, and samples were 254 frozen in liquid nitrogen until FACS analysis. To determine the CD4 and CD8 T cell responses in 255 the airways, 0.5-1x10 6 BALF cells were plated and stimulated as for the whole blood. At the end 256 of incubation, cells were immediately stained for ICS and FACS analysis.

Surface immunostaining and ICS was done as previously described 8 . Briefly, frozen cells 258 from whole blood were thawed and permeabilized before staining with a cocktail of fluorochrome-259 conjugated monoclonal antibodies; CD3 (FITC), CD4 (PB), CD8 (PECy7), IFNγ (PE), IL-2 (APC), 260 and TNFα (PerCP-Cy5.5). BALF cells were surface-immunostained for viability (Molecular 261 Probes LIVE/DEAD fixable stain (Aqua), Invitrogen) followed by CD4 (AF700), CD14 (V450), 262 CD19 (V450). Cells were then permeabilized and stained for intracellular cytokines IFNγ (PE), 263 IL-2 (APC), and TNFα (FITC). Ag85A p. pool-stimulated whole blood and BALF cells were also 264 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint stained for surface markers CD103 (APC), CD69 (PerCP-Cy5.5) and CD49d (PE-dazzle) to 265 evaluate the surface expression of tissue-resident memory T cell-or T cell lung trafficking-266 associated molecules. Cells were analyzed with LSRII flow cytometer and assessed with Flowjo 267 version 9.9.6 (Tree Star Inc.). 268 We evaluated the induction of trained innate immunity in airway macrophages in the LD 269 dose group. After reviving the frozen BALF cells for 6h, one million viable cells were seeded on Ontario, Canada. 4 participants were excluded (two withdrew consent and two were withdrawn 315 before vaccination because they were unable to comply with the study visit requirements) and one 316 did not complete any follow-up visits after vaccination because of COVID restrictions. 31 317 participants completed the study, 11 in the low dose aerosol group, 11 in the high dose aerosol 318 group and 9 in the intramuscular group (Fig 1) . The demographic and baseline characteristics of 319 the study participants were similar among study groups (Table 1) .

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The copyright holder for this this version posted September 14, 2021. device 323 The Aeroneb® Solo Micropump was selected to be part of the device set up for aerosol 324 generation and delivery in our study (Fig S1) . A fill volume of 0.5 ml in the nebulizer was 325 determined to be optimal for vaccine delivery in saline. Subjects completed inhalation of this 326 volume containing the vaccine via tidal breathing in approximately 2.5 minutes ( Table 2 ). The 327 emitted dose of vaccine available at the mouth was found to be approximately 50% of the loaded 328 dose in the nebulizer ( Table 2 ). The majority of aerosol droplets were <5.39 µm (85%) or between 329 2.08 and 5.39 µm in diameter conducive to vaccine deposition in major airways. Thus, the amount 335 Both low and high doses of AdHu5Ag85A administered by aerosol inhalation or the 336 intramuscular injection were safe and well-tolerated. Respiratory adverse events were infrequent, 337 mild, transient and similar among groups (Table 3) . Intramuscular injection was associated with a 338 mild local injection site reaction in two participants. Systemic adverse events were also infrequent, 339 mild, transient and similar among groups (Table 3) Bronchoscopy and bronchoalveolar lavage were generally well tolerated in all participants.

As expected, in some participants the procedures were associated with mild cough, sore throat, Ag-specific CD4 T cells in the airways (Fig 2A) . Compared with CD4 T cells, although the levels 374 of airway CD8 T cell responses were much smaller, they were significantly increased at both 2 375 and 8wk, particularly following LD aerosol vaccination ( Fig 2D) . Of interest, there was also a 376 small but increased number of CD8 T cells at 2wk after IM vaccination.

Analysis of polyfunctionality of vaccine-activated CD4 and CD8 T cells reactive to Ag85A 378 in the airways revealed that LD aerosol vaccination led to induction of higher magnitude of CD4 379 T cells that co-expressed IFNγ, TNFα and IL-2 (3+) and any of two cytokines (2+) than those 380 producing single cytokine (1+) (Fig 2E/F) . Importantly, polyfunctional CD4 T cells remained 381 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint we next determined whether antigen-specific T cells induced by aerosol AdHu5Ag85A 412 vaccination were of tissue-resident memory phenotype and compared them to those by IM 413 vaccination. BALF cells obtained before and at select timepoints post-vaccination were stimulated 414 with Ag85A p. pool and immunostained for co-expression of two key TRM surface markers CD69 415 and CD103 by antigen-specific IFNγ-producing CD4 or CD8 T cells (Fig 3A) . Marked increases 416 in Ag85A-specific IFN-γ+ CD4 and CD8 T cells co-expressing CD69 and CD103 were seen only 417 in the airway of LD and HD aerosol vaccine groups, but not in IM group (Fig 3B/C) . Although 418 TRM increases at 8wk post-aerosol vaccination compared to the baseline were only marginally 419 statistically significant (95% CI) probably due to small sample sizes, remarkable proportions of 420 Ag85A-specific CD4 T cells (~20%) and CD8 T cells (~54%) present in the airways of aerosol 421 vaccine groups were TRM ( Fig 3D) . As expected, there was no detectable antigen-specific TRM in 422 the peripheral blood before and after vaccination. 423 We also studied T cell surface expression of α4β1 integrin (VLA-4; or CD49d for α4), known from each other (Fig S4-A) . We then identified the differentially expressed genes (DEGs) by 445 comparing wk0-and wk8-stimulated AM with respective unstimulated AM. A total of 2,726 genes 446 were differentially expressed upon stimulation in pairwise analysis of which 1667 genes (61%) 447 were shared between the baseline (wk0) Group 3/1 and aerosol vaccine (wk8) Group 4/2 (Fig S4- Ag85A-specific CD8 T cells up to 16wk (Fig 4F) . By comparison, aerosol vaccination minimally 491 induced such CD8 T cells in the circulation (Fig 4F) . Compared to circulating CD4 T cells, similar 492 to the overall kinetics of total cytokine+ CD8 T cells (Fig 4F) , circulating Ag85A-specific 493 polyfunctional CD8 T cells peaked behind the peak CD4 T cell responses in all vaccine groups 494 (Fig 4G) .

The kinetics of polyfunctional profiles of circulating CD4 T cells were further examined 496 in greater detail with a focus on LD aerosol vaccine group and its comparison with IM group.

There existed considerable differences in the polyfunctional profile of circulating Ag85A-specific 498 CD4 T cells between LD aerosol and IM groups (Fig S5-A) . In LD aerosol group, the proportion 499 of IFNγ+TNFα+IL-2+ progressively shrank and at 16wk, ~ 75% of the population were TNFα+IL-500 2+ and IFNγ+TNF-α+ together with single TNFα+ CD4 T cells. In comparison, in IM group the 501 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint proportion of IFNγ+TNFα+IL-2+ progressively expanded, constituting ~ 75% of the population 502 at 16wk (Fig S5-A) . 503 Upon examination of circulating BCG-specific CD4 T cells (reactive to M.tbCF+rAg85A 504 stimulation), we found that they were not strikingly increased in aerosol and IM vaccine groups 505 although the trend was higher in IM group (Fig S5-B/C) and AUC values did not differ 506 significantly between aerosol and intramuscular groups (LD aerosol, p=0.0870; HD aerosol, 507 p=0.2666 compared to IM). However, LD and HD aerosol vaccination had a significant enhancing 508 effect on the polyfunctionality of pre-existing circulating BCG-specific CD4 T cells (Fig S5-C) .

Similar to BCG-specific circulating CD4 T cells (Fig S5-B) , BCG-specific circulating CD8 T cells 510 were not significantly increased by either aerosol or IM vaccination (Fig S5-D) .

These data together indicate that besides markedly induced mucosal T cell immunity ( AdHu5-specific total IgG in most of the trial participants (10 4 -10 5 ), and the levels were comparable 525 between the groups (using Kruskal-Wallis test p=0.2048) ( Table 4 ). These titres significantly 526 increased after HD aerosol or IM AdHu5Ag85A vaccination but not after LD aerosol vaccination.

In comparison, pre-existing levels of anti-AdHu5 total IgG in the airways were 1-1.5 log less than 528 the levels in the circulation and were comparable between groups (using Kruskal-Wallis test 529 p=0.2048). Of interest, LD and HD aerosol as well as IM vaccination did not alter the pre-existing 530 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint anti-AdHu5 total IgG levels in the airways (Table 4 ) but the data from HD aerosol and IM groups 531 should be interpreted with caution due to the small sample size at 8wk.

Because the total anti-AdHu5 Ab titres may not always correlate with AdHu5-neutralizing 533 capacity in the circulation 8 , we further assessed the AdHu5-neutralizing Ab (nAb) titres before 534 and after vaccination in the circulation and airways by using a bioassay. The pre-existing AdHu5-535 nAb titres in the circulation were comparable between groups (using Kruskal-Wallis test p=0.3588) 536 with 27%, 54% and 66% of participants in LD, HD and IM groups having >10 2 AdHu5-nAb titres, (Table 4 ). On the other hand, similar to total anti-AdHu5 IgG levels, pre-existing AdHu5-540 nAb titers in the airways were ~1 log less than those in the circulation (Table 4 ). Of importance, 541 63%, 36% and 33% of participants in LD, HD and IM groups, respectively, had no detectable 542 baseline AdHu5-nAb titers in their airways which remained unaltered following vaccination 543 (Table 4) . We further found a significant positive correlation between AdHu5-nAb and total 544 AdHu5 IgG titres both in the circulation and airways (Fig S6-A/B ).

Given that many of the trial participants had moderate to significant levels of AdHu5-nAb 546 titers in the circulation and ~50% of them also had a small but detectable level of pre-existing 547 AdHu5-nAb titres in the airways, we next examined whether such nAbs present in the airways and 548 blood may have negatively impacted the immunopotency of LD aerosol and IM vaccination, 549 respectively. To this end, % airways or blood total cytokine+ Ag85A-specific CD4 T cells at the 550 peak response time (2 weeks post-vaccination) for individual participants was plotted against 551 corresponding pre-existing AdHu5-nAb titre and Spearman rank correlation test was performed.

There was no significant correlation between pre-existing airways AdHu5-nAb titers and the 553 magnitude of vaccine-induced CD4 (Fig S6-C) and CD8 (Fig S6-D) T cell responses in the airways 554 following LD aerosol vaccination. Of note, one participant who hardly responded to aerosol 555 vaccine did have the highest neutralization titers in the cohort (Fig S6-C) . On the other hand, 556 consistent with our previous observation 8 , there was no significant correlation between pre-557 existing circulating AdHu5-nAb titers and the magnitude of antigen-specific CD4 (Fig S6-E) and 558 CD8 (Fig S6-F ) T cell responses in the blood following IM vaccination. The above data together 559 suggest that while there is high prevalence of pre-existing circulating anti-AdHu5 nAb in humans 560 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This represents the first clinical study to have fully characterized the method to deliver 567 aerosolized adenoviral-vectored vaccine to human lungs and to demonstrate its superiority in 568 inducing respiratory mucosal immunity over the intramuscular injection.

Both low and high aerosol doses of AdHu5Ag85A were safe and well-tolerated. has met with threats from emerging variants of concern, dwindling vaccine-induced immunity, 639 and increasing break-through infections 16 . Of note, several approved first-generation COVID-19 640 vaccines are adenoviral-vectored but they are all administered via intramuscular injection 15 .

Although a recent study has shown that it is safe and well-tolerated to deliver via inhaled aerosol 

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The copyright holder for this this version posted September 14, 2021. ;

Induction of multifunctional T cells in the airways following aerosol or intramuscular vaccination . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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Induction of airway tissue-resident memory T cells (TRM) following aerosol or intramuscular vaccination . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint Figure S3 Induction of multifunctional T cells specific for Ag85A or a cocktail of mycobacterial antigens in the airways following aerosol or intramuscular vaccination . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint Figure S4 Transcriptomic analysis of alveolar macrophages (AM) following LD aerosol vaccination . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint Figure S5 Induction of multifunctional T cells specific for Ag85A or a cocktail of mycobacterial antigens in the peripheral blood following aerosol or intramuscular vaccination . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 14, 2021. ; https://doi.org/10.1101/2021.09.09.21263339 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint Local pain n/a n/a n/a n/a 0 0 Redness n/a n/a n/a n/a 1 11.1 Swelling n/a n/a n/a n/a 1 11. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

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