key: cord-0322499-kynw1cc3 authors: Lapuente, Dennis; Fuchs, Jana; Willar, Jonas; Antão, Ana V; Eberlein, Valentina; Uhlig, Nadja; Issmail, Leila; Schmidt, Anna; Oltmanns, Friederike; Peter, Antonia Sophia; Mueller-Schmucker, Sandra; Irrgang, Pascal; Fraedrich, Kirsten; Cara, Andrea; Hoffmann, Markus; Pöhlmann, Stefan; Ensser, Armin; Pertl, Cordula; Willert, Torsten; Thirion, Christian; Grunwald, Thomas; Überla, Klaus; Tenbusch, Matthias title: Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization date: 2021-08-03 journal: bioRxiv DOI: 10.1101/2021.08.03.454858 sha: 154d66aeed8fd0b86c5d7b30846e274da7864a15 doc_id: 322499 cord_uid: kynw1cc3 Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and caused 39 a worldwide pandemic accounting for over 190 million infections and 4 million deaths at the time of 40 this report 1 . In an unprecedented speed, academic institutions and biotech companies developed, 41 evaluated, and licensed several SARS-CoV-2 vaccines. Beside traditional approaches like protein 42 subunit or inactivated virus vaccines, gene-based vaccines were at the forefront of the developmental 43 process and the first to become licensed 2 . 44 Vaccines based on messenger RNA (mRNA) or adenoviral vectors (Ad) demonstrated efficacy against 45 SARS-CoV-2 infections and, most importantly, against severe coronavirus disease 2019 (COVID-19) and 46 death 3-6 . Humoral as well as cellular immune responses against the spike (S) surface protein were 47 4 feature of TRM is the direct localization at barrier tissues, which makes a time-consuming migration into 75 the inflamed lung redundant. A second remarkable characteristic is the ability to exert innate and 76 adaptive functions within a few hours after secondary infection 36,37 , in part due to the storage of ready-77 made mRNAs encoding cytokines like IFNγ at steady state 38, 39 . Altogether, these unique features of 78 mucosal immune responses enable an immediate and effective countermeasure against pulmonary 79 infections as described for flu 40,41 , respiratory syncytial virus (RSV) 42 , and Mycobacterium tuberculosis 80 43, 44 . The great majority of these finding were generated in animal models, partly due to the invasive 81 nature of bronchoalveolar lavages (BAL) and biopsy sampling. However, small experimental human 82 challenge studies started to look precisely at the role of mucosal immunity against respiratory viruses 83 45, 46 . 84 A few preclinical studies investigated intranasal SARS-CoV-2 vaccines so far. In a series of publications, 85 one group reported protective efficacy of a one shot vaccination with an chimpanzee adenoviral vector 86 (ChAd) vaccine encoding for the spike protein in mice, hamsters, and rhesus macaques [47] [48] [49] . 87 Importantly, van Doremalen et al. have shown that intramuscular ChAd vaccination prevents 88 pneumonia in macaques but allow for virus replication in the upper respiratory tract 50 . However, 89 administered intranasally, the vaccine attenuated nasal virus replication more efficiently 51 . It is 90 important to investigate intranasal vaccine candidates not only as standalone modality but also in the 91 context of pre-existing immunity induced by a previous vaccination. On one hand, this is important 92 due to the broad employment SARS-CoV-2 vaccines in recent vaccination campaigns. On the other 93 hand, first clinical data point towards suboptimal immunogenicity of solely intranasal vaccinations 94 against SARS-CoV-2 in humans without pre-existing immunity, but also provides evidence for robust 95 immunity after heterologous prime-boost vaccinations 52, 53 . 96 Here we demonstrate that a systemic DNA or mRNA prime followed by an intranasal boost with an 97 adenoviral serotype 5 vector (Ad5) enables a comprehensive systemic and local T cell immunity as well 98 as substantial mucosal neutralization of SARS-CoV-2 VOCs. Concomitantly, the mucosal boost 99 9 The analysis of spike-specific, cytokine producing CD8 + T cells showed a similar compartmentalization. 200 Although the overall numbers of CD107a + , IFNγ + , and TNFα + CD8 + T cells were highest in the lungs of 201 the 2x RNA group, these cells were almost exclusively found in the vascular compartment (iv-labelled, 202 Fig. 8 A-C) . The same is true for the homologous immunization with Ad5, albeit reaching much lower 203 percentages of reactive cells. In line with the phenotypic analyses, RNA-Ad5 induced both systemic 204 and local T cell responses, whereas DNA-Ad5 provoked mainly TRM. The trends observed for CD8 + T cell 205 responses in the iv-labelled lung population were largely mirrored by the splenic responses ( Fig. 8 D) , 206 further underlining that the former population reflects circulating T cells present in the lung 207 vasculature at the time of sampling. Spike-specific, tissue-resident CD4 + T cell responses were also 208 effectively established by the mucosal boost strategies ( Fig. 9 A and B ) and systemic CD4 + T cells in the 209 spleen were induced by all vaccine schedules with two RNA shots being the most effective strategy 210 ( Fig. 9 D) . 211 In conclusion, only intranasal vaccinations schedules were able to induce profound mucosal immunity 212 in the respiratory tract consisting of neutralizing IgG, IgA, and lung TRM. Compared to DNA-Ad5, the 213 RNA-Ad5 strategy provoked a more efficient neutralization of VOCs and established a comprehensive 214 T cell immunity consisting of both TRM and circulatory T cells. 215 In order to assess the protective efficacy of the vaccination strategies, human ACE2 transgenic mice 217 (K18-hACE2) were immunized as described before and challenged four weeks after the boost 218 immunization with 9x10 3 FFU of the SARS-CoV-2 strain BavPat1 as previously described 56 . Since the 2x 219 Ad5 immunization was less immunogenic than the 2x RNA immunization, this group was replaced by 220 another 2x Ad vaccination regime consisting of an intramuscular Ad19a prime followed by the 221 established intranasal Ad5 boost (Fig. 10 A) . Seven out of eight unvaccinated control animals reached 222 humane endpoints at day five indicating a severe and lethal course of the disease (Fig. 10 B) . They 223 presented weight loss starting at day four post-infection with a concomitant increase of clinical signs 224 ( Fig. 10 C and D) . In contrast, all vaccinated groups were largely protected from weight loss, clinical 225 signs of disease, and mortality ( Fig. 10 B-D) . High levels of viral RNA in lung homogenates and BAL fluids 226 were only detected in unvaccinated animals indicating efficient viral replication, while from the 227 vaccinated animals only two of the 2x RNA group had viral RNA copy numbers in the lung above the 228 detection limit (Fig. 10 E) . Similarly, infectious virus was retrieved from the lungs of unvaccinated 229 animals but not from the immunized groups (Fig. 10 F) . Due to the nature of this challenge model, high 230 viral RNA copy numbers were also detected in the brains of naïve animals (Fig. S7) . Although viral RNA 231 was still detectable in the majority of the vaccinated animals, the copy numbers were reduced by 4-5 232 logs, and no significant differences among the vaccine groups could be seen. 233 Taken together, the mucosal boost strategies were able to fully prevent mortality and symptomatic 234 disease upon experimental SARS-CoV-2 infection. The protective efficacy was equal to the current 235 approved vaccination regimen consisting of two intramuscular injections of Comirnaty®. 236 The SARS-CoV-2 pandemic had and still has a deep impact on social, economic, and healthcare aspects 238 of the world community. As a reaction, academic institutions, biotech companies, and regulatory 239 agencies released safe and effective vaccines in an unprecedented speed. While early in the pandemic 240 the vaccine efficacies of the respective vaccine schedules were in focus, the interest now shifts towards 241 investigating immunogenicity and efficacy of mixed modality vaccinations, the maintenance of long-242 term immunity, and the protection against emerging variants. So far, the heterologous combination of 243 different vaccine modalities is mostly connected to a superior immunogenicity in preclinical 57,58 and 244 clinical studies 59-62 . As now most countries with progressed vaccination campaigns discuss the 245 employment of booster vaccinations, a possible next step might be the implementation of mucosal 246 immunizations in order to harness the full potential of mucosal immunity at the entry port of SARS-247 CoV-2 infections. It is tempting to speculate that anti-vector immunity induced by the primary immunization might have 290 dampened the effect of the homologous booster. This mechanism is also discussed in the context of 291 the lower vaccine efficacy in humans reported with two standard doses Vaxzevria® (ChAdOx1) 292 compared to the low dose-standard dose schedule 5 . 293 Mucosal antibody levels were higher in the groups having received a mucosal boost compared to the 294 repeated systemic vaccination regimens. In regard to the levels of mucosal IgG, this trend was less 295 pronounced as for mucosal IgA levels, presumably because serum IgG can be transudated into the 296 respiratory lumen, whereas IgA is more stringently connected to a local immune reaction. Most 297 importantly, the increased antibody responses in the mucosa also translated into more efficient virus 298 neutralization by BAL samples. Only BAL samples from groups with mucosal vaccinations displayed 299 13 effective neutralization of all tested VOCs. Although definitive evidence is currently missing, mucosal 300 virus neutralization might be key to supress initial infections with SARS-CoV-2 and therefore minimize 301 the risk of transmission to and by vaccinees. Interestingly, we observed distinct neutralization profiles 302 between the DNA-Ad5 and RNA-Ad5 schemes probably originating from the use of different spike 303 antigens. Thus, it is important to investigate the role of the prefusion conformation stabilization 70 304 regarding neutralization profiles in more detail. 305 An important advantage of intranasally administered genetic vaccines is the induction of TRM in the 306 respiratory tract. In the present study, tissue-resident memory was exclusively established by mucosal 307 vaccinations. This is congruent with published research showing that local antigen expression is 308 essential for the development of respiratory TRM 30,32,40 . Moreover, in combination with a mucosal 309 boost, a priming immunization with RNA provoked a broader cellular immunity compared to a DNA 310 prime consisting of not only TRM in the lung but also of significant numbers of circulating memory T 311 cells. We speculate that such comprehensive T cell immunity is more effective against breakthrough The gating strategy is shown in Fig. S3 . BALs or lung homogenates were applied to the cells for 3 hours. After replacing the supernatant with 475 overlay medium (DMEM with 1 % methyl cellulose, 2 % FBS and 1% penicillin/streptomycin), cells were 476 incubated for further 27 hours. SARS-CoV-2 infected cells were visualized using SARS-CoV-2 S-protein 477 specific immunochemistry staining with anti-SARS-CoV-2 spike glycoprotein S1 antibody (Abcam) 90 . 478 Results are shown as mean ± SEM or as median ± interquartile range except it is described differently. ) 642 2. WHO, W. H. O. COVID-19 vaccine tracker and landscape Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against -2: an interim analysis of four randomised controlled trials in Brazil, South Africa, 651 and the UK Safety and efficacy of an rAd26 and rAd5 vector-based heterologous 653 prime-boost COVID-19 vaccine: an interim analysis of a randomised An mRNA Vaccine against SARS-CoV-2 -Preliminary Report Safety, Immunogenicity, and Efficacy of the BNT162b2 Covid-19 Vaccine in 658 BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in 660 humans Safety and immunogenicity of an rAd26 and rAd5 vector-based 662 heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised 663 phase 1/2 studies from Russia Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against 665 Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern 668 in BNT162b2-mRNA-vaccinated individuals Vaccine against the B.1.1.7 and B.1.351 Variants Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 674 variant of concern 202012/01 (B.1.1.7): an exploratory analysis of a randomised controlled 675 trial Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B Vaccine Breakthrough Infections with SARS-CoV-2 Variants Transmission event of SARS-CoV-2 Delta variant reveals multiple vaccine 681 breakthrough infections BNT162b2 vaccine breakthrough: clinical characteristics of 152 fully 684 vaccinated hospitalized COVID-19 patients in Israel Initial report of decreased SARS-CoV-2 viral load after inoculation 687 with the BNT162b2 vaccine Effect of Vaccination on Household Transmission of SARS-CoV-2 in England Secretory IgA antibodies provide cross-protection against infection with 691 different strains of influenza B virus Role of IgA versus IgG in the Control 693 of Influenza Viral Infection in the Murine Respiratory Tract Potential Role of Nonneutralizing IgA Antibodies in Cross-Protective Immunity 696 against Influenza A Viruses of Multiple Hemagglutinin Subtypes IgA polymerization contributes to efficient virus neutralization on human 698 upper respiratory mucosa after intranasal inactivated influenza vaccine administration The role of nasal IgA in children vaccinated 701 with live attenuated influenza vaccine Nasal IgA Provides Protection against Human Influenza Challenge in 703 Human immune responses elicited by an intranasal inactivated H5 influenza 705 vaccine Intranasal vaccination with a replication-deficient 707 influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans Intranasal vaccination with an inactivated whole influenza virus vaccine induces 710 strong antibody responses in serum and nasal mucus of healthy adults IL-1β as mucosal vaccine adjuvant: the specific induction of tissue-resident 713 memory T cells improves the heterosubtypic immunity against influenza A viruses Comparative evaluation of two severe acute respiratory syndrome (SARS) 716 vaccine candidates in mice challenged with SARS coronavirus Superior immune responses induced by intranasal 719 immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein 720 of Middle East respiratory syndrome coronavirus Optimal Generation of Tissue-Resident but Not Circulating Memory T Cells 722 during Viral Infection Requires Crosspriming by DNGR-1+ Dendritic Cells Cutting Edge: Tissue-Retentive Lung Memory CD4 T Cells Mediate Optimal 725 Protection to Respiratory Virus Infection Specific niches for lung-resident memory CD8 + T cells at the site of tissue 727 regeneration enable CD69-independent maintenance Local proliferation maintains a stable pool of tissue-resident memory T cells 729 after antiviral recall responses Sensing and alarm function of resident 731 memory CD8(+) T cells Hobit and Blimp1 instruct a universal transcriptional program of tissue 733 residency in lymphocytes. Science (80-. ) The Molecular Signature of Tissue Resident Memory CD8 T Cells Isolated 735 from the Brain Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-737 protection against pulmonary virus infection Vaccine-generated lung tissue-resident memory T cells 739 provide heterosubtypic protection to influenza infection Cells in the Lungs Protect against Acute Respiratory Syncytial Virus Infection Prevention of tuberculosis in macaques after intravenous BCG 744 immunization Cutting Edge: Control of Mycobacterium tuberculosis Infection by a Subset of 746 Lung Parenchyma-Homing CD4 T Cells RSV-specific airway resident memory CD8+ T cells and differential disease 748 severity after experimental human infection Epitope-specific airway-resident CD4+ T cell dynamics during experimental 750 human RSV infection A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower 752 Respiratory Tracts against SARS-CoV-2 A single intranasal dose of chimpanzee adenovirus-vectored vaccine 754 protects against SARS-CoV-2 infection in rhesus macaques A single intranasal or intramuscular immunization with chimpanzee 757 adenovirus-vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces shedding 762 of SARS-CoV-2 D614G in rhesus macaques Altimmune Announces Update On AdCOVID TM Phase 1 Clinical Trial Safety, tolerability, and immunogenicity of an aerosolised adenovirus type-5 Ad5-nCoV) in adults: preliminary report of an open-label and 769 randomised phase 1 clinical trial Rapid response flow cytometric assay for the detection of antibody 772 responses to SARS-CoV-2 Cutting Edge: Intravascular Staining Redefines Lung CD8 T Cell 775 A pair of non-competing neutralizing human monoclonal antibodies 777 protecting from disease in a SARS-CoV-2 infection model Heterologous vaccination regimens with self-amplifying RNA and 780 adenoviral COVID vaccines induce robust immune responses in mice Heterologous prime-boost: breaking the protective immune response bottleneck 783 of COVID-19 vaccine candidates Immune responses against SARS-CoV-2 variants after heterologous 785 and homologous ChAdOx1 nCoV-19/BNT162b2 vaccination Immunogenicity and reactogenicity of BNT162b2 booster in ChAdOx1-S-788 primed participants (CombiVacS): a multicentre, open-label, randomised, controlled, phase 2 789 trial Heterologous ChAdOx1 nCoV-19 and mRNA-1273 Vaccination /NEJMc2110716 792 62. Tenbusch, M. et al. Heterologous prime-boost vaccination with ChAdOx1 Evaluation of adenovirus 19a as a novel 795 vector for mucosal vaccination against influenza A viruses A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of 797 Circulating and Genital-Resident Intraepithelial CD8 + Memory T Cells ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-800 CoV-2 shedding in ferrets Isolation of a common receptor for Coxsackie B viruses and 802 adenoviruses 2 and 5 Adenoviruses with A549 Cellular Receptors: Sialic Acid versus alpha v Integrins Adenovirus type 5 pseudotyped with 807 adenovirus type 37 fiber uses sialic acid as a cellular receptor Membrane Cofactor Protein Is a Receptor for Adenoviruses Associated with 809 Epidemic Keratoconjunctivitis Structure-based design of prefusion-stabilized SARS-CoV-2 spikes A mouse-adapted model of SARS-CoV-2 to test COVID-19 813 countermeasures The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese 815 and aged mice and is neutralized by convalescent and post-vaccination human sera SARS-CoV-2 infection aggravates chronic comorbidities of cardiovascular diseases 818 and diabetes in mice Preliminary Assessment of the Efficacy of a T-Cell-Based Influenza Vaccine Efficacy of FLU-v, a broad-spectrum influenza vaccine, in a randomized 822 phase IIb human influenza challenge study Neutralizing antibody levels are highly predictive of immune protection 824 from symptomatic SARS-CoV-2 infection Evidence for antibody as a protective correlate for COVID-19 vaccines Longitudinal analyses reveal immunological misfiring in severe COVID-19 Safety and immunogenicity of adenovirus-vectored nasal and 830 epicutaneous influenza vaccines in humans Safety and Immunogenicity of a Novel Intranasal Influenza Vaccine A Phase 2 Randomized SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their 834 Requirements for Receptor Expression and Proteolytic Activation Engineering adenovirus genome by bacterial artificial 836 chromosome (BAC) technology BNT162b vaccines protect rhesus macaques from SARS-CoV-2 Innate signalling molecules as genetic adjuvants do not alter the efficacy of 840 a DNA-based influenza A vaccine Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 842 is persistent and critical for survival SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies SARS-CoV-2 variant B.1.617 is resistant to bamlanivimab and evades 846 antibodies induced by infection and vaccination Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by 849 42 infection and BNT162b2 vaccination Detection of SARS-CoV-2 in human breastmilk Growth, detection, 853 quantification, and inactivation of SARS-CoV-2