key: cord-0958334-tf5a48i2
authors: Nouailles, Geraldine; Adler, Julia M.; Pennitz, Peter; Peidli, Stefan; Alves, Gustavo Teixeira; Baumgart, Morris; Bushe, Judith; Voss, Anne; Langenhagen, Alina; Pott, Fabian; Kazmierski, Julia; Goekeri, Cengiz; Simmons, Szandor; Xing, Na; Langner, Christine; Vidal, Ricardo Martin; Abdelgawad, Azza; Herwig, Susanne; Cichon, Günter; Niemeyer, Daniela; Drosten, Christian; Goffinet, Christine; Landthaler, Markus; Blüthgen, Nils; Wu, Haibo; Witzenrath, Martin; Gruber, Achim D.; Praktiknjo, Samantha D.; Osterrieder, Nikolaus; Wyler, Emanuel; Kunec, Dusan; Trimpert, Jakob
title: A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants
date: 2022-05-16
journal: bioRxiv
DOI: 10.1101/2022.05.16.492138
sha: 73ec014bf00c31d5720d9ab086212bd52afe650d
doc_id: 958334
cord_uid: tf5a48i2

Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.

As of early 2022, ten COVID-19 vaccines fulfill the quality, safety and efficacy requirements that 14 allow emergency use listing (EUL) by the WHO (1). Currently authorized vaccines employ 15 classical approaches such as inactivated virus and subunit vaccines, as well as more recent 16 technology, including the use of adenoviral vectors and the novel nucleoside-modified mRNA 17 vaccines (2). Despite initially high vaccine efficacy and long-lasting protection from severe illness, 18 waning of protection from infection and symptomatic disease is now evident, particularly 19

following the emergence and spread of the omicron variant (3, 4). Moreover, global disparity in 20 vaccine access remains alarmingly high: By 6 May 2022, only 15.8% of people in low-income 21 countries had received at least one dose of vaccine despite the surplus possessed by wealthy nations 22

(5). This situation, together with the continued evolution of SARS-CoV-2, warrants an even more 23 robust vaccine and immunization strategy. Optimal COVID-19 vaccines would not just protect 24 from severe disease, but also provide protection from infection with a broad spectrum of virus 25 variants, while, at the same time, they would prevent or significantly limit SARS-CoV-2 26 transmission. Although live attenuated vaccines (LAV) are used very successfully to tackle many 27 virus infections such as measles, mumps and rubella (MMR) (6), studies investigating the efficacy, 28 effectiveness and cross-comparison of promising intranasal LAVs with other routes of delivery 29 remain limited (7, 8) . Notably, LAVs do not depend on adjuvants (7) and can be administered 30 locally, for example intranasally, as is the case for influenza LAVs (9). Comprised of replication-31 competent viruses, intranasal LAVs mimic the natural course of infection and antigen production, 32 which distinguishes them from locally administered, replication-incompetent vector-or antigen- To perform RNA bulk sequencing, RNA was isolated from lung tissue using Trizol reagent 271 according to the manufacturer's instructions (ambion, life technologies). Briefly, 1 ml Trizol was 272 added to the homogenized organ sample and vortexed thoroughly. After an incubation time of 273 20 min, 200 µL of chloroform were added. The samples were vortexed again and incubated for 10 274 min at room temperature. Subsequently, tubes were centrifuged at 12,000 × g for 15 min at 4°C 275 and 500 µL of the aqueous phase were transferred into a new tube containing 10 µg GlycoBlue. 276

Isopropanol (500 µL) was added followed by vortexing, incubating and centrifuging the samples 277 as described above. Thereafter, isopropanol was removed and 1 mL of ethanol (75 %) was applied. 278

The tubes were inverted shortly and centrifuged at 8.000× g for 10 min. After freeing the pellet 279 from ethanol, RNA was resuspended in 30 µL of RNAase-free water and stored at -80°C. 280 281 Cell isolation from blood and lungs 282

White blood cells were isolated from EDTA-blood as previously described, steps included erylysis 283 and cell filtration prior counting. Lung cells (caudal lobe) were isolated as previously described 284 (32), steps included enzymatic digestion, mechanical dissociation and filtration prior counting in 285 trypan blue. Buffers contained 2 µg/mL actinomycin D to prevent de novo transcription during the 286 procedures. 287 288 Cell isolation from nasal cavities 289

To obtain single cell suspensions from the nasal mucosa of SARS-CoV-2 challenged hamsters, the 290 skull of each animal was split slightly paramedian, such a way that the nasal septum remained 291 intact on the left side of the nose. The right side of the nose was carefully removed from the 292 cranium and stored in ice-cold 1× PBS with 1 % BSA and 2 µg/mL actinomycin D until further 293 use. Nose parts were transferred into 5 mL Corning® Dispase solution supplemented with 750 294 U/mL Collagenase CLS II, and 1 mg/mL DNase and incubated at 37°C for 15 min. For preparation 295 of cells from the nasal mucosa, the conchae were carefully removed from the nasal cavity and re-296 incubated in digestion medium for 20 min at 37°C. Conchae tissue was dissociated by pipetting 297 and pressing through a 70-µm filter with a plunger. Ice-cold PBS with 1 % BSA and 2 µg/mL 298 actinomycinD was added to stop the enzymatic digestion. Cell suspension was centrifuged at 4°C 299 for 15 min at 400× g and supernatant discarded. The pelleted nasal cells were resuspended in 5 mL 300 red blood cell lysis buffer and incubated at RT for 2 min. Lysis reaction was stopped with 1× PBS 12 with 0.04 % BSA and cells centrifuged at 4°C for 10 min at 400× g. Pelleted Sequencing reads were initially processed using the multi command of the 10× Genomics Cell 337 Ranger 6.0.2 software. For the cellplex demultiplexing, the assignment thresholds were partially 338 adjusted (see the github page accompanying this manuscript, https://github.com/Berlin-Hamster-339

Single-Cell-Consortium/Live-attenuated-vaccine-strategy-confers-superior-mucosal-and-340 systemic-immunity-to-SARS-CoV-2, for details). Further processing was done using the Seurat R 341 package (76). In the next step, cells were filtered by a loose quality threshold (minimum 250 342 detected genes per cell) and clustered. Cell types were then annotated per cluster, and filtered using 343 cell type specific thresholds (cells below the median or in the lowest quartile within a cell type 344 were removed). The remaining cells were processed using the SCT/integrate workflow (77), and 345 cell types again annotated on the resulting Seurat object. All code for downstream analysis is 346 available on github, https://github.com/Berlin-Hamster-Single-Cell-Consortium/Live-attenuated-347 vaccine-strategy-confers-superior-mucosal-and-systemic-immunity-to-SARS-CoV-2. 348 349

Utilizing the widely employed mRNA vaccine BNT162b2 and two original vaccine candidates -351 an adenoviral vector vaccine carrying the spike glycoprotein of SARS-CoV-2 (28) and a live 352 replicating virus levels below the detection, indicating sterilizing immunity following a booster 382 vaccination with sCPD9 regardless of heterologous (mRNA) or homologous (sCPD9) priming 383 ( Figure 1H ). On a virus transcript level, these results were confirmed by sequencing of bulk RNA 384 extracted from the lungs ( Figure S1B ). 385 386 LAV is superior in preventing inflammatory damage to the lung 387

To determine the degree of protection from virus-induced tissue damage and inflammation, 388 infected hamster lungs were examined by histopathology. We found that after single vaccination, 389 sCPD9 prevented inflammation and pneumonia more efficiently than other vaccines. sCPD9 390 vaccinated animals showed less consolidated lung areas ( Figure 1M Humoral immunity against SARS-CoV-2 is most potent when the LAV is included in the 435 vaccination regimen 436

To determine the potency of humoral responses, we quantified the ability of hamster sera collected 437 before challenge (day 0) and on days 2 and 5 after challenge to neutralize the ancestral SARS- (e.g., Pax5 is downregulated in plasmablasts, but upregulated here), they could be interpreted as 497 an early "memory recall gene expression signature" visible in the scRNA-seq data at 2 dpc. 498

Notably, expression of this signature in blood B cells was strongest in hamsters that had received 499 homologous or heterologous prime-post vaccination with sCPD9, which likewise generated 500 highest antibody titers ( Figure 3E -H) . In line with these findings, the numbers and frequencies 501 amongst blood cells of pre-PB (cluster 3) and mem->pre-PB (cluster 6) cells were significantly 502 higher in sCPD9+sCPD9 vaccinated hamsters ( Figure 3M ). 503 504 19 LAV enhances T cell proliferation in response to challenge with SARS-CoV-2 505

To investigate the occurrence of T cell memory recall responses, we proceeded with subclustering 506 T and NK cells. To this end, we assayed CD4+, CD8+, and proliferating T cells in blood ( Figure  507 4A, B; S10, S11). As expected, CD4+ and CD8+ T cells recapitulate the pattern generally observed 508 for T cells ( Figure 4A, 3J) . Analyses of gene expression indicative of proliferation (Mki67, Top2a), 509 naïve or central memory status (Sell, Ccr7, Lef1, Il7r) and activation of T cells (Cd69, Cd44, 510

Klrg1, Tnfsf9, Icos, Cd40lg) revealed that most T cells in the blood displayed either naïve or 511 central memory phenotypes (cluster 0 -4, Figure S10B -E). In blood taken at 2 dpc, type 1-512 immunity effector genes (Tbx21, Gzma, Gzmb, Faslg, Ifn) were only expressed by NK cells 513 (cluster 5, Figure S11 ). The proliferating T cell population consisted of activated T cells expressing 514 memory markers, such as Il7r (cluster 6, Figure S11 ). Of note, the concentration and frequencies 515 of proliferating T cells, albeit generally small, were significantly increased after heterologous 516 vaccination, when compared to unvaccinated hamsters ( Figure 4B ). Analysis of gene expression 517 in cluster 6 revealed that the fraction of cells expressing proliferation markers as well as the 518 expression level of proliferation associated genes was higher when animals were vaccinated, and 519 highest when sCPD9 was included in the vaccination regimen ( Figure 4C ). 520 Next, we examined whether different prime-boost vaccination strategies differed in the ability 521 to re-activate tissue-resident memory T cells (Trm) in lungs of Syrian hamsters (39, 40). To 522 characterize pulmonary T cell subsets, we subclustered the initial T and NK cell clusters into 10 523 subclusters ( Figure S12A ). In analogy to the blood T cell readouts, we identified clusters 3, 7 and 524 9 as NK cells based on gene expression (Nkg7-true, Cd3-false), cluster 4 as CD8+ T cells (Cd3e,  525 Cd8a), clusters 0, 1, 2, 6 as CD4+ T cells (Cd3e, Cd4) and clusters 8 and 5 as proliferating T cells 526 (Cd3e, Mki67, Top2a) ( Figure S12B , C). In line with NK cell gene expression pattern in the blood, 527 NK cells located in the lungs likewise presented the most pronounced type 1-immunity effector 528 gene expression (Tbx21, Faslg, Gzma, Gzmb) as well as Klrg1 ( Figure S12D , E). Cluster 2 was 529 dominated by CD4+ T cells and displayed a mixed phenotype of effector, activation and memory 530 gene markers ( Figure S12B -E, S13A, B) . At 2 dpc of prime-boost vaccinated hamsters, most 531 identified CD8+ T cells (cluster 4) were primarily of naïve or central memory type, whereas few 532 CD8+ T cells scattered into the CD4+ T cell dominated clusters 5 and 8 of proliferating T cells. 533 CD4+ T cells in clusters 0 and 1 were reminiscent of naïve or central memory type (Sell, Ccr7, 534 Lef1, Il7r, Tcf7, S1pr1) ( Figure S13A ). In cluster 6, we did not find genes associated with naïve 535 20 or central memory-associated signatures ( Figure S13A ) but combined and strong expression of T 536 cell-homing and tissue retention genes (Cxcr6, Rgs1, Prdm1 (Blimp-1), Znf683 (Hobit), Itga1 537 (CD49a) and Itgae (CD103)), a signature indicative of Trm status ( Figure S13B , C, S14). At 2 dpc, 538

Trm (cluster 6), activated (cluster 2) and proliferating (cluster 5 and 8) T cell populations were 539 small and represented less than 2 % of all lung cells in each case ( Figure 4D -E) . Trm (cluster 6) 540 and activated T cells (cluster 2) trended towards higher frequencies and numbers in the lungs of 541 sCPD9-vaccinated hamsters ( Figure 4D, E) . Gene expression analysis of Trm (cluster 6) showed 542 that the gene expression level and cell fraction expressing Cxcr6, a prominent tissue homing 543 receptor, was highest in sCPD9-vaccinated groups while lymph node retention receptor S1pr1 was 544 least detected here ( Figure S15A ). Across activated T cells (cluster 2), gene expression of 545 activation and effector genes was more uniform between groups and, thus, likely independent of 546 prior vaccination ( Figure S15A ). Notably, like in blood, at 2 dpc numbers and frequencies of 547 proliferating T cells were significantly higher in vaccinated groups and highest when animals had 548 received sCPD9 as part of their vaccination regimen ( Figure 4F ). However, contrary to 549 proliferating T cells in the blood, their lung counterparts expressed higher levels of effector genes 550 such as Ifng and Gzma ( Figure 4G ). Moreover, when we scored the seurat clusters for a published 551 human Trm gene set (41), we observed a subset of cells in cluster 5 (proliferating T cells) with a 552 high Trm signature score ( Figure 4H ). At 2 dpc the Trm signature score in proliferating T cells 553 (cluster 5) was remarkably higher when sCPD9 was part of the vaccination strategy ( Figure 4I, J) . 554

In cluster 8 (proliferating T cells), overall cell numbers were too low to generate interpretable 555 scores, and no cells from unvaccinated animals were identified ( Figure S15B ). To evaluate the 556 extent to which the clusters are connected, we used a partition-based graph abstraction (PAGA) 557 approach (42), which indicates particularly strong cellular connectivity between clusters 2, 8 and 558 5 and clusters 2 and 6 as well as a possible connection between cluster 6 and 8 ( Figure 4K) . 559

Ordering cells according to global expression similarity by diffusion pseudotime (43) and plotting 560 this rank against the Trm signature further corroborates a path between clusters 2/6 and clusters 8 561 and 5, which are accompanied by variable Trm-like gene expression ( Figure 4L ; S15C, D). 562

Overall, these findings suggest that a subset of proliferating T cells is Trm recall-derived and 563 activated in response to SARS-CoV-2 challenge infection in sCPD9-boosted hamsters. 564 565 21 LAV induces superior mucosal immunity against SARS-CoV-2 566

In addition to potent T cell memory and humoral immunity, induction of protective mucosal 567 immunity is a distinguishing property of LAVs that are administered locally at the natural site of 568 virus replication (44). However, in case of SARS-CoV-2, induction of limited mucosal antibody 569 responses following mRNA vaccination is reported (45, 46) . To assess induction of protective 570 mucosal immune responses in the upper respiratory tract of hamsters that received different 571 vaccine regimens, we measured SARS-CoV-2 spike-specific IgA levels after prime-only 572 vaccinations. We found that sCPD9 vaccinated animals harbored considerably larger quantities of 573 IgA in nasal washes prior challenge and at all tested time points post challenge ( Figure 5A 

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We thank V.M. Corman, Charité, for his help in study design and prolific discussions on 872 results and conclusions and S. Reiche, Friedrich-Loeffler-Institut, for providing anti-SARS-CoV-873 2 nucleocapsid antibody. We thank C. Thöne-Reineke for support in animal welfare and 874 husbandry. GN (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE200596), along with bulk RNA-seq 922 read count tables, and h5 matrices and Seurat objects for the single-cell RNA-seq data. Code is 923 available through github at https://github.com/Berlin-Hamster-Single-Cell-Consortium/Live-924 attenuated-vaccine-strategy-confers-superior-mucosal-and-systemic-immunity-to-SARS-CoV-2. 925