key: cord-0740916-spohl5ey
authors: An, Xingyue; Martinez-Paniagua, Melisa; Rezvan, Ali; Fathi, Mohsen; Singh, Shailbala; Biswas, Sujit; Pourpak, Melissa; Yee, Cassian; Liu, Xinli; Varadarajan, Navin
title: Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2
date: 2020-07-23
journal: bioRxiv
DOI: 10.1101/2020.07.23.212357
sha: 85733c6036a832655e4b68713eac8c5ee0fff1c1
doc_id: 740916
cord_uid: spohl5ey

A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable speed, current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. We report the development of an intranasal subunit vaccine that contains the trimeric or monomeric spike protein and liposomal STING agonist as adjuvant. This vaccine induces systemic neutralizing antibodies, mucosal IgA responses in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA-sequencing confirmed the concomitant activation of T and B cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues (NALT), confirming its role as an inductive site that can lead to long-lasting immunity. The ability to elicit immunity in the respiratory tract has can prevent the initial establishment of infection in individuals and prevent disease transmission across humans.

The COVID19 pandemic has made the development of an efficacious and safe vaccine an urgent priority. Rapid progress in sequencing, protein structure determination, and epitope mapping with cross-reactive antibodies has illustrated that the SARS-CoV-2 spike protein (S protein) binds to human angiotensin-converting enzyme 2 (ACE2) 1, 2 . This, in turn, has made the S protein and the receptor-binding domain (RBD) of the S protein prime candidates for vaccine design to elicit neutralizing antibodies. Indeed, DNA/RNA based vaccine candidates encoded for the S protein have advanced rapidly and are in clinical trials 3, 4 .

Despite this progress, there is considerable uncertainty about the duration of protective immunity elicited by the current vaccine candidates. While neutralizing antibodies are the desired immunological target of the current vaccines, and maybe necessary; it is unclear if serum neutralizing antibodies will be sufficient for sterilizing immunity. Studies have shown that the antibody protection in COVID-19 convalescent patients can be short-lived and that patients can become seronegative in as little as four weeks after exposure [5] [6] [7] . There are also reports of the lack of high levels of neutralizing activity; and in some cohorts, even a lack of detectable neutralizing antibodies in patient convalescent sera 2, 8 .

Adaptive immunity mediated by T cells can complement humoral immunity or can inhibit viral replication independent of humoral immunity. Not surprisingly, there are emerging reports of patients with COVID-19 like symptoms who have detectable T-cell responses without seroconversion 9 . This observation complements other studies that support the existence of robust T cell responses in convalescent patients. Grifoni et al. demonstrated that 100 % of nonhospitalized convalescent patients showed antigen-specific CD4 + T cells, and 70 % of patients showed antigen-specific CD8 + T-cell responses 10 . Weiskopf et al. detected strong Th1 type responses directed towards the S protein in COVID-19 patients admitted to the ICU due to moderate to severe acute respiratory distress syndrome (ARDS), 11 . Similarly, Braun et al. have reported CD4 + T-cell responses targeting the S protein in 83% of COVID19 patients and a third of healthy patients, presumably due to cross-reactivity to other viruses 12 . Taken together, these data highlight that vaccines that target both humoral and cellular responses can deliver lasting protective immunity.

The nose and the upper respiratory tract are the primary routes of entry for inhalation pathogens like SARS-CoV-2. Not surprisingly, the nasal compartment showed particular susceptibility to SARS-CoV-2 infection and can serve as the initial reservoir for subsequent seeding of the virus to the lung 13 . Consequently, pre-existing immunity within the respiratory tract is highly desirable to prevent pathogen invasion. Despite the well-recognized role of mucosal immunity, most vaccines are designed to elicit circulating humoral immunity without necessarily enabling mucosal immunity. Mucosal vaccination can stimulate both systemic and mucosal immunity and has the advantage of being a non-invasive procedure suitable for immunization of large populations. However, mucosal vaccination is hampered by the lack of efficient delivery of the antigen and the need for appropriate adjuvants that can stimulate a robust immune response without toxicity. The identification of the cyclic GMP-AMP Synthase (cGAS) and the stimulator of interferon genes (STING) pathway has enabled the identification and development of STING agonists (STINGa) 14 . STINGa function as novel immunostimulatory adjuvants for mucosal vaccines against respiratory pathogens, including influenza and anthrax, in mice [15] [16] [17] .

In this report, we encapsulated the STINGa, cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP or cGAMP) in liposomes 17 . We used it as the adjuvant for intranasal vaccination with the trimeric or monomeric versions of the S protein. Our results show that the candidate vaccine formulation is safe and elicits systemic immunity (neutralizing antibodies), cellular immunity (spleen and lung), and mucosal immunity (IgA in the nasal compartment and lung, and IgA secreting cells in the spleen). To the best of our knowledge, we report the first COVID-19 vaccine candidate that elicits mucosal immunity and supports further translational studies as an intranasal non-viral candidate that can induce systemic immunity and confers immunity at the primary site of viral entry.

To facilitate efficient priming of the immune system within the respiratory compartment, we encapsulated the STINGa within negatively charged liposomes ( Figure 1A ) 17 . The adjuvant encapsulated liposomes were prepared using a passive drug loading method by hydrating the lipid dry films in buffered solutions containing cGAMP as the STINGa. We removed the free STINGa via ultrafiltration, and the encapsulation efficiency of STINGa was determined to be 35 % by calibration against a standard curve ( Figure S1A ). Dynamic light scattering analysis showed that the mean particle diameter by intensity of STINGa-liposomes was 81 nm, with a polydispersity index of 0.24, while the the size of blank liposomes was 110 nm ( Figure 1B-C) . The mean zeta potential of liposomes was negative both with (-35 mV) and without (-68 mV) encapsulated STINGa ( Figure 1D -E). We tested the stability of the STINGa-liposomes and showed that they were stable for up to two months at 4 °C, as evidenced by the conservation of particle sizes and the absence of aggregates ( Figure S1B ). The surface charge of liposomes was also unaltered (-40 mV) after this period ( Figure S1B ). Collectively, these results established that the negatively charged liposomes had efficiently encapsulated the STINGa and had good stability.

We prepared the vaccine by gently mixing the trimeric S protein (discussed below) with the STINGa-liposomes suspensions at room temperature to allow the adsorption of the protein on the liposomes. The adsorbed Trimer-STINGa-liposomes displayed mean particle diameter of 105 nm and mean zeta potential of -29 mV ( Figure 1F -G), with a polydispersity index of 0.24, slightly bigger and less negative than the STINGa-liposomes (81 nm, -35mV). The results suggested that formulated protein-STINGa-liposomes vaccine exist in a nanoparticulate colloidal form.

We used the recombinant trimeric extracellular domain of the S protein containing mutations to the Furin cleavage site as the immunogen ( Figure 2A ). As expected by extensive glycosylation of the S protein, SDS-PAGE under reducing conditions confirmed that the protein migrated between 180-250kDa ( Figure 2B ). Although previous studies have performed extensive characterization of the lack of toxicity of the adjuvant formulation, we wanted to confirm that the adjuvant does not cause morbidity, weight loss, or other hyper-inflammatory symptoms 17 . Accordingly, we performed an initial pilot experiment with a five BALB/c mice that received a single intranasal dose of the adjuvant without protein and observed no weight loss or gross abnormalities over 14 days ( Figure S2A and S2B). We next immunized two groups of mice by intranasal administration with either a combination of the protein and adjuvant (Trimer-STINGa) or the protein by itself (control). None of the animals showed any clinical symptoms, including loss of weight ( Figure S2C ). Seven days (d7) after immunization, 100 % of the mice that received the Trimer-STINGa seroconverted and robust anti-S IgG levels with mean dilution titers of 1:1,040 were detected ( Figure 2C ). By day 15 (d15), the serum concentration of the anti-S IgG antibodies increased, and mean dilution titers of 1:4,400 were detected ( Figure 2C ). Anti-S IgG was also detected in the bronchoalveolar lavage fluid (BALF) of all three mice tested ( Figure 2D ). We confirmed that the serum anti-S antibodies were neutralizing with a mean 50% inhibitory dose (ID50) of 1:414 as measured by a GFP-reporter based pseudovirus neutralization assay (SARS-CoV-2, Wuhan-Hu-1 pseudotype) [ Figure 2E ].

Emerging data support a role for T-cell responses to contribute to protection independent of antibody responses. We evaluated T-cell responses in the immunized mice using a pool of 15mers that target highly conserved regions of the S-protein ( Figure S3) 18, 19 . At d15, all five animals immunized with the Trimer-STINGa showed robust splenic T cell responses with a mean of 144 IFNγ spots/10 6 cells ( Figure 2F ). Collectively these results show that a single intranasal administration using the Trimer-STINGa elicited robust serum neutralizing antibodies and T-cell responses.

IgA mediated protection is an essential component of mucosal immunity for respiratory pathogens. The Trimer-STINGa improved BALF IgA titers at d15 compared to the control group ( Figure 2G ). We also evaluated the IgA responses in the antibody-secreting cells (ASCs) in the spleen at d15 by ELISPOT assays. The mice immunized with Trimer-STINGa showed an increase in the number of total IgA secreting and S-specific IgA secreting ASCs compared to the control group ( Figure 2H ). Taken together, these results illustrate that intranasal vaccination elicited IgA responses that are an essential component of mucosal immunity.

To investigate if intranasal vaccination can support local inductive responses in the nasal passage, we harvested the NALT from the immunized animals at the time of euthanasia, converted them into single-cell suspensions, and performed scRNA-seq ( Figure 3A and S4) . After filtering, we obtained a total of 1,398 scRNA-seq profiles. By utilizing uniform manifold approximation and projection (UMAP), we identified the myeloid; NK and T; and B cell subpopulations using established lineage markers ( Figure 3B and S5 ). >95 % of the scRNA-seq in both control and Trimer-STINGa groups corresponded to T and B cells, and we performed detailed analyses on these immune cells. Figure  3E ]. These results suggested that successful intranasal vaccination promoted activation of B cells similar to GCs, and we next investigated if T cells within the NALT supported B-cell differentiation. We identified three clusters within the T cells: one CD8 + T cell subpopulation expressing Cd8a, and two CD4 + T cell subpopulations ( Figure 3F and S7) . The CD4 + T cells were classified as naïve T cells (naïve) expressing Cd4 and Npm1; and T follicular helper like (TFH) expressing Cd69, Il6ra, Nr4a1 (Nur77), Tcf7 (TCF1), and Lef1, and also the memory markers Cd27 and Cd28 ( Figure 3G and S7) [22] [23] [24] . The prominent difference in the control and the Trimer-STINGa groups was an increase in the ratio of Tfh/naïve CD4 + T cells ( Figure 3H ). 25 . First, we visualized cell-cell interaction between the different B and T cell clusters in the NALT. We identified that Tfh cells were the dominant interacting cell type and interacted strongly with the GC B cell cluster ( Figure 3I) . At the molecular level, several well-documented receptor-ligand pairs, Cd40-Cd40l, Il21r-Il21, Icosl-Icos, and Baffr (Tnfrsf13c)-Baff (Tnfsf13b) were detected reciprocally on the GC B cells and the Tfh cells within the NALT ( Figure 3J) . These results showed that upon immunization with the Trimer-STINGa, the NALT promoted a GC-like T-cell dependent activation and differentiation of B cells which in turn can lead to long-lasting immunity.

We wanted to evaluate if the monomeric S protein could also elicit a comprehensive immune response. We used a monomeric version of the S protein containing mutations to the Furin binding site and a pair of stabilizing mutations (Lys986Pro and Val987Pro) [ Figure 4A and S7]. SDS-PAGE of the monomeric protein showed a band between 130-180 kDa ( Figure 4B ). We immunized four mice with the monomeric S protein and the adjuvant (Monomer-STINGa) and again confirmed no weight loss in these animals ( Figure S8 ). At d15, 100 % of the animals seroconverted, and the mean serum concentration of the anti-S IgG antibodies was 1:750 ( Figure  4C ). We confirmed that the serum anti-S antibodies were neutralizing with a mean ID50 of 1:188 [ Figure 4D ]. We evaluated T-cell responses with the same pool of immunodominant peptides. At d15, all four animals immunized with the Monomer-STINGa showed robust splenic T cell responses with a mean of 100 IFNγ spots/10 6 cells ( Figure 4E ).

Animal models have shown that T cells in the lung are necesary for protection against pulmonary infection by respiratory pathogens 26 . Accordingly, we evaluated S-protein specific Tcell responses in the lung of the vaccinated animals. T-cell responses in the lung on d15 were detected at a mean of 206 IFNγ spots/10 6 cells ( Figure 4E ). Collectively, these results established that intranasal administration using the Monomer-STINGa also elicited robust serum neutralizing antibodies and T-cell responses in both the spleen and the lung.

We finally investigated the antibody responses in the nasal compartment. We detected total IgA (ELISA) in the nasal wash from two animals. Both these nasal washes also had detectable anti-S IgA antibodies at a mean concentration of 7 ng/ml ( Figure 4F ). Consistent with the ability of the Monomer-STINGa to elicit mucosal immune responses, we also confirmed the detection of S-specific IgA secreting ASCs in the spleen of these mice ( Figure S9 ). These results established that vaccination with the Monomer-STINGa elicits systemic immunity, T-cell responses in the lung and speen, and mucosal IgA responses.

Almost all the vaccine candidates that have advanced in the clinical trials of COVID19 are based on the intramuscular injection of DNA; nanoparticles loaded mRNA or viral vectors 3, 27 . These methods have shown to elicit neutralizing antibodies in the serum of preclinical models and humans [27] [28] [29] . A fundamental limitation of all of these approaches is that they are not designed to elicit mucosal immunity. As prior work with other respiratory pathogens like influenza has shown, sterilizing immunity to virus re-infection requires adaptive immune responses in the respiratory tract and the lung 17, 30, 31 . In the context of COVID19 existing data supports that initial infection in the nasal compartment promotes/facilitates subsequent seeding of the virus to the lung 13 . The ability of vaccines to thus promote immunity at the mucosal sites and specifically within the nasal compartment can prevent seeding of the initial reservoir and control human transmission.

Intranasal vaccination is an attractive platform to elicit systemic and mucosal immunity. The fundamental challenge in intranasal vaccination is the ability to balance safety while ensuring immunogenicity leading to sterilizing immunity. Intranasal administration of live-attenuated vaccines in humans is hampered by concerns of safety 32 and the use of the adenovirus vectored vaccines can be hampered by the presence of pre-existing immunity 33 . Subunit vaccines are attractive candidates that do not suffer from these drawbacks. However, the ability of subunit vaccines to elicit potent and sterilizing immune responses is critically dependent on the choice of appropriate adjuvants.

We demonstrate that STINGa encapsulated in liposomes can function as potent adjuvants. A single intranasal immunization with protein S and the adjuvant can elicit neutralizing antibodies in the serum comparable to other vaccine candidates. We established that intranasal vaccination leads to IgA responses in the lung and directly in the nasal compartment, and we detected B cells secreting IgA in the spleen. We show using scRNA-seq that the NALT functions as an inductive site upon intranasal vaccination, leading to co-ordinated activation/differentiation of B and T cells resembling germinal centers. Moreover, intranasal vaccination also induced S-specific T cell responses in both the spleen and locally in the lung. Collectively, these results illustrate the advantages of optimal intranasal immunization. While we demonstrate cellular and humoral immunity both systemically and locally in the respiratory tract, future studies using appropriate challenge models in non-human primates are required to establish whether we elicit sterilizing immunity and to test the durability of our responses longitudinally. However, we note that due to the severity of the pandemic, most vaccines currently in clinical trials have advanced without testing in preclinical challenge models. Although we have shown the immunity elicited upon a single-dose, we can add a booster dose to increase the durability of the responses, if appropriate.

In summary, our study establishes that intranasal spike subunit vaccines with liposomal STINGa as adjuvants is safe and elicits comprehensive immunity against SARS-CoV-2. In the context of a pandemic, the intranasal vaccine has two compelling advantages. First, the easy access to the nasal cavity makes intranasal administration non-invasive. It is particularly suited for mass vaccinations of large cohorts, including elderly patients and children with minimal clinical infrastructure. Second, from the standpoint of disease control, the ability to control SARS-CoV-2 infection at the first point of entry in the nasal compartment and before spreading to the lung is a desirable option to halt disease progression in individuals and disease transmission across populations. We suggest that our promising vaccine candidate supports human testing.

Lipids and STINGa. We purchased the STINGa, 2′-3'′cyclic guanosine monophosphate adenosine monophosphate (cGAMP) from Chemietek (Indianapolis, IN). 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesterol was obtained from Sigma Aldrich (St. Louis, MO).

Preparation of STINGa loaded liposomes and vaccine formulation. The liposomes were composed of a molar ratio of 10:1:1:1 of DPPC, DPPG, Cholesterol (Chol), and DPPE-PEG2000. To prepare the liposomes, we mixed the lipids in CHCl3 and CH3OH, and the solution was evaporated by a vacuum rotary evaporator for approximately 80 min at 45 °C. We dried the resulting lipid thin film until all organic solvent was evaporated. We hydrated the lipid film by adding a pre-warmed cGAMP solution (0.3 mg/ml in PBS buffer at pH 7.4). The hydrated lipids were mixed at elevated temperature 65 °C for an additional 30 min, then subjected to freeze-thaw cycles. We sonicated the mixture for 60 min using a Brandson Sonicator (40 kHz). The free untrapped cGAMP was removed by Amicon Ultrafiltration units (MW cut off 10kDa). We washed the cGAMP-liposomes three times using PBS buffer. The cGAMP concentration in the filtrates was measured by Take3 Micro-Volume absorbance analyzer of Cytation 5 (BioTek) against a calibration curve of cGAMP at 260 nm. We calculated the final concentration of liposomal encapsulated cGAMP and encapsulation efficiency by subtracting the concentration of free drug in the filtrate.

We mixed the S protein monomer (4 µg) or the trimer (10 µg) with the STINGa-liposomal suspensions at room temperature to allow the adsorption of the protein onto the liposomes. The formulated vaccine was stored at 4 °C and used for up to 2 months. The average particle diameter, polydispersity index, and zeta potential were characterized by Litesizer 500 (Anton Paar) at room temperature.

Mice and immunization. All the animal experiments were reviewed and approved by UH IACUC. Female, 7-9 week-old BALB/c mice were purchased from Charles River Laboratories. Prior to immunization, we anesthetized the mice by intraperitoneal injection of ketamine and xylazine. We immunized the mice intranasally with different formulations: (1) the adjuvant only group was administrated with liposome-STINGa; (2) the control group was administrated with protein only;

(3) the Trimer-STINGa group was administrated with protein and liposome-STINGa; and (4) the Monomer-STINGa group was administrated with protein and liposome-STINGa. The monomeric and trimeric protein was obtained from BEI Resources (VA, USA) and Creative BioMart (NY, USA), respectively.

Body weight monitoring and sample collection. The body weight of the animals was monitored every 2-3 days over two weeks after immunization. Sera were collected seven days and 15 days after post-vaccination for detection of the humoral response. Nasal wash, BALF, NALT, lung, and spleen were harvested and processed 15 days after the administration, essentially as previously described 34, 35 . Sera and other biological fluids (with protease inhibitors) were kept at -80 °C for long-term storage. After dissociation, the splenocytes and lung cells were frozen in FBS+10% DMSO and stored in the liquid nitrogen vapor phase until further use.

following the standard processing workflow in Seurat Package, we acquired the clustering and gene expression data. We removed cells with < 1000 Unique Molecular Identifiers (UMIs) and high mitochondrial gene expression (> 20% of the reads), and we ended up with 1398 single-cell profiles (660 control, 738 treated) with a mean UMI of 1648.

To analyze the cell-cell communication at the molecular level, we used the recovered molecule counts by SAVER in CellPhoneDB analysis tool 25 . First, we transformed the mouse genes to their human orthologous using BiomaRt Package (version 2.38.0) 45 . Then, we categorized all T cells and B cells by their subpopulations and group (control and Trimer-STINGa) into 14 cell types. According to statistical tests calculated CellPhoneDB, we filtered out the ligand-receptor pairs with p values > 0.05 and evaluated the relationship between different cell types with the significant pairs. To generate the network of interactions, we applied igraph Package (version 1.2.5) 46 .

A.

Overall schematic of design of adjuvant and intranasal administration of vaccine. B, C, and F. Distribution of liposomal particle sizes measured by dynamic light scattering (DLS). D, E, and G.

Zeta potential of the liposomes measured by electrophoretic light scattering (ELS).

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This publication was supported by the NIH (U01AI148118) and Owens foundation. XL acknowledges partial funding support from the National Cancer Institute (NIH R15CA182769, P20CA221731, P20CA221696 ) and CPRIT (RP150656). The following reagent was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein (Stabilized) from SARS-Related Coronavirus 2, Wuhan-Hu-1, Recombinant from Baculovirus, NR-52308. Supported by the NIH/NCI under award number P30 CA016672 and used the M.D. Anderson ORION core. We would like to acknowledge Prof. Shaun Zhang for sharing guidance on animal protocols; Drs. Ankita Leekha and Irfan Bandey for assistance with animal experiments; and Prof. Cliona Rooney for sharing ELISPOT plates. We thank BD for the generous loaner of a FACS Melody and Rhapsody; and Intel for the generous loaner of a cluster. 

UH has filed a provisional patent based on the findings in this study.

A. Schematic of trimeric protein used for immunization. B. Denaturing SDS-PAGE gel of the purified trimer protein.C. Humoral immune responses in the serum were evaluated using S-protein based IgG ELISA at day 7 and day 15 after immunization. D. The humoral immune responses in the BALF evaluated using S-protein based IgG ELISA at day 15. E. The ID50 of the serum antibody responses were measured using a pseudovirus neutralization assay F. Cellular immune responses in the spleen were assessed using IFNγ ELISPOT assays. G. IgA levels in the BALF were determined using ELISA. H. Antibody secreting cells (ASCs) secreting IgA and S-protein specific IgA in the spleen were detected using ELISPOT assays. For (C-H) the bar represents the mean, and the error bars represent the standard error. LoD represents limit of detection of assay.