A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2

Ahmed O Hassan, Natasha M Kafai, Igor P Dmitriev, Julie M Fox, Brittany K Smith, Ian B Harvey, Rita E Chen, Emma S Winkler, Alex W Wessel, James Brett Case, Elena Kashentseva, Broc T McCune, Adam L Bailey, Haiyan Zhao, Laura A VanBlargan, Ya-Nan Dai, Meisheng Ma, Lucas J Adams, Swathi Shrihari, Jonathan E Danis, Lisa E Gralinski, Yixuan J Hou, Alexandra Schäfer, Arthur S Kim, Shamus P Keeler, Daniela Weiskopf, Ralph S Baric, Michael J Holtzman, Daved H Fremont, David T Curiel, Michael S Diamond, Ahmed O Hassan, Natasha M Kafai, Igor P Dmitriev, Julie M Fox, Brittany K Smith, Ian B Harvey, Rita E Chen, Emma S Winkler, Alex W Wessel, James Brett Case, Elena Kashentseva, Broc T McCune, Adam L Bailey, Haiyan Zhao, Laura A VanBlargan, Ya-Nan Dai, Meisheng Ma, Lucas J Adams, Swathi Shrihari, Jonathan E Danis, Lisa E Gralinski, Yixuan J Hou, Alexandra Schäfer, Arthur S Kim, Shamus P Keeler, Daniela Weiskopf, Ralph S Baric, Michael J Holtzman, Daved H Fremont, David T Curiel, Michael S Diamond

Abstract

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

Keywords: COVID-19; IgA; SARS-CoV-2; T cells; antibody; intranasal; mucosal immunity; pathogenesis; protection; vaccine.

Conflict of interest statement

Declaration of Interests M.S.D. is a consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals and on the Scientific Advisory Board of Moderna. The Diamond laboratory has received unrelated funding support from Moderna, Vir Biotechnology, and Emergent BioSolutions. M.S.D., D.T.C., A.O.H., and I.P.D. have filed a disclosure with Washington University for possible development of ChAd-SARS-CoV-2. M.J.H. is a member of the DSMB for AstraZeneca and founder of NuPeak Therapeutics. The Baric laboratory has received unrelated funding support from Takeda, Pfizer, and Eli Lilly.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Immunogenicity of ChAd-SARS-CoV-2-S (A) Diagram of transgene cassettes: ChAd-control has no transgene insert; ChAd-SARS-CoV-2-S encodes for SARS-CoV-2 S protein with the two indicated proline mutations. (B) Binding of ChAd-SARS-CoV-2-S transduced 293 cells with anti-S mAbs. (Left) Summary: +, ++, +++, and - indicate 50%, and no binding, respectively. MFI, mean fluorescence intensity. (Right) Representative flow cytometry histograms of two experiments are shown. (C–F) Four-week-old female BALB/c mice were immunized via intramuscular route with ChAd-control or ChAd-SARS-CoV-2-S and boosted 4 weeks later. Antibody responses in sera of immunized mice at day 21 after priming or boosting were evaluated. An ELISA measured anti-S and RBD IgG and IgA levels ([D] and [E]), and an FRNT determined neutralization activity (F). Data are pooled from two experiments (n = 15–30). (G and H) Cell-mediated responses were analyzed at day 7 post-booster immunization after re-stimulation with an S protein peptide pool (Table S1). Splenocytes were assayed for IFNγ and granzyme B expression in CD8+ T cells and granzyme B only in CD4+ T cells by flow cytometry (G). A summary of frequencies and numbers of positive cell populations is shown in (H) (n = 5). Bars indicate median values, and dotted lines are the limit of detection (LOD) of the assays. (I) Spleens were harvested at 7 days post-boost, and SARS-CoV-2 spike-specific IgG+ antibody-secreting cell (ASC) frequency was measured by ELISPOT (n = 13). (J) CD8+ T cells in the lung were assayed for IFNγ and granzyme B expression by flow cytometry after re-stimulation with an S protein peptide pool (n = 4–5). (K) Lung CD8+ T cells also were assayed for expression of CD69 and CD103. (L) Spleens were harvested at 7 days post-boosting, and SARS-CoV-2 spike-specific IgA+ ASC frequency was measured by ELISPOT (n = 4–5). (M and N) SARS-CoV-2 S- (M) and RBD-specific (N) IgG and IgA levels in BAL fluid were determined by ELISA (n = 4–5). Bars and columns show median values, and dotted lines indicate the limit of detection (LOD) of the assays. For (D), (F), (H), (I), and (J): Mann-Whitney test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant. See Figure S1, Figure S2, and S3 and Table S1.
Figure S1
Figure S1
ChAd-SARS-CoV-2-S Vaccine Induces Neutralizing Antibodies as Measured by FRNT, Related to Figure 1 Four-week old female BALB/c mice were primed or primed and boosted with ChAd-control or ChAd-SARS-CoV-2-S via intramuscular route. A-B. Serum samples from ChAd-control or ChAd-SARS-CoV-2 vaccinated mice were collected at day 21 after priming (A) or boosting (B) and assayed for neutralizing activity by FRNT. Serum neutralization curves corresponding to individual mice are shown for the indicated vaccines (n = 15-30 per group). Each point represents the mean of two technical replicates. (C). An ELISA measured anti-SARS-CoV-2 NP IgG responses in paired sera obtained 5 days before and 8 days after SARS-CoV-2 challenge of ChAd-control or ChAd-SARS-CoV-2-S mice vaccinated by an intramuscular route (n = 5: ∗∗p < 0.01; ∗∗∗p < 0.001; paired t test). Dotted lines represent the mean IgG titers from naive sera.
Figure S2
Figure S2
Gating Strategy for Analyzing T Cell Responses, Related to Figure 1 Four-week old female BALB/c mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S and boosted four weeks later. T cell responses were analyzed in splenocytes at day 7 post-boost. Cells were gated for lymphocytes (FSC-A/SSC-A), singlets (SSC-W/SSC-H), live cells (Aqua-), CD45+, CD19- followed by CD4+ or CD8+ cell populations expressing IFNγ or granzyme B.
Figure S3
Figure S3
Impact of Pre-existing ChAd Immunity on Transduction of Mice with Hu-AdV5-hACE2, Related to Figures 1 and 2 Four-week old female BALB/c mice were primed or primed and boosted. Serum samples were collected one day prior to Hu-AdV5-hACE2 transduction. Neutralizing activity of Hu-AdV5-hACE2 in the sera from the indicated vaccine groups was determined by FRNT after prime only (A) or prime and boost (B). Each symbol represents a single animal; each point represents two technical repeats and bars indicate the range. A positive control (anti-Hu-Adv5 serum) is included as a frame of reference.
Figure 2
Figure 2
Protective Efficacy of Intramuscularly Delivered ChAd-SARS-CoV-2-S against SARS-CoV-2 Infection (A) Scheme of vaccination and challenge. Four-week-old BALB/c female mice were immunized ChAd-control or ChAd-SARS-CoV-2-S. Some mice received a booster dose of the homologous vaccine. On day 35 post-immunization, mice were challenged with SARS-CoV-2 as follows: animals were treated with anti-Ifnar1 mAb and transduced with Hu-AdV5-hACE2 via an intranasal route 1 day later. Five days later, mice were challenged with 4 × 105 focus-forming units (FFUs) of SARS-CoV-2 via the intranasal route. (B and C) Tissues were harvested at 4 and 8 dpi for analysis. Infectious virus in the lung was measured by plaque assay (B), and viral RNA levels were measured in the lung, spleen, and heart at 4 and 8 dpi by qRT-PCR (C; n = 3–7). (D) Viral RNA in situ hybridization using SARS-CoV-2 probe (brown color) in the lungs harvested at 4 dpi. Images show low- (top; scale bars, 100 μm) and medium- (middle; scale bars, 100 μm) power magnification with a high-power magnification inset (representative images from n = 3 per group). (E) Fold change in gene expression of indicated cytokines and chemokines from lung homogenates at 4 dpi was determined by qRT-PCR after normalization to Gapdh levels and comparison with naive unvaccinated, unchallenged controls (n = 7). (F and G) Mice that received a prime-boost immunization were challenged on day 35 post-booster immunization. Tissues were collected at 4 dpi for analysis. Infectious virus in the lung was determined by plaque assay (F), and viral RNA was measured in the lung, spleen, and heart using qRT-PCR (G; n = 6–7). (B, C, and E–G) Columns show median values, and dotted lines indicate the LOD of the assays. For (B), (C), (E), (F), and (G): Mann-Whitney test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See Figure S3.
Figure 3
Figure 3
Single-Dose Intramuscular Vaccination with ChAd-SARS-CoV-2-S Protects Mice against SARS-CoV-2-Induced Inflammation in the Lung Four-week-old female BALB/c mice were immunized with ChAd-control and ChAd-SARS-CoV-2-S and challenged following the scheme described in Figure 2. Lungs were harvested at 8 dpi. Sections were stained with hematoxylin and eosin and imaged at 40× (left; scale bar, 250 μm), 200× (middle; scale bar, 50 μm), and 400× (right; scale bar, 25 μm) magnifications. Each image is representative of a group of 3 mice.
Figure 4
Figure 4
Immune Responses after Intranasal Immunization of ChAd-SARS-CoV-2-S (A) Scheme of experiments. 5-week-old BALB/c female mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal route. (B–D) Antibody responses in sera of immunized mice at 1 month after priming were evaluated. An ELISA measured SARS-CoV-2 S- and RBD-specific IgG (B) and IgA levels (C), and a FRNT determined neutralization activity (D). Data are pooled from two experiments with n = 10–25 mice per group. (E–J) Mice that received a booster dose were sacrificed 1 week later to evaluate mucosal and cell-mediated immune responses. SARS-CoV-2 S- and RBD-specific IgG (E) and IgA levels (F) in BAL fluid were determined by ELISA. Neutralizing activity of BAL fluid against SARS-CoV-2 was measured by FRNT (G). CD8+ T cells in the lung were assayed for IFNγ and granzyme B expression by flow cytometry after re-stimulation with an S protein peptide pool (H). CD8+ T cells in the lung also were phenotyped for expression of CD103 and CD69 (I). SARS-CoV-2 spike-specific IgG+ and IgA+ ASC frequency in the spleen harvested 1 week post-boost was measured by ELISPOT (J). Data for mucosal and cell-mediated responses are pooled from two experiments (E–I: n = 7–9 per group; J: n = 5 per group). (B–J) Bars and columns show median values, and dotted lines indicate the LOD of the assays. Mann-Whitney test: ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See Figure S4 and Table S1.
Figure S4
Figure S4
Intranasal Inoculation of ChAd-SARS-CoV-2-S Induces Neutralizing Antibodies as Measured by FRNT and Protects against SARS-CoV-2 Replication, Related to Figures 4 and 5 Five-week old female BALB/c mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal inoculation route. Serum samples collected one month after immunization were assayed for neutralizing activity by FRNT. Mice were boosted at day 30 after priming and were sacrificed one week later to evaluate immune responses. (A) Serum samples from ChAd-control or ChAd-SARS-CoV-2-S vaccinated mice were tested for neutralizing activity with SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 (n = 8-10 per group). (B) Serum samples from ChAd-SARS-CoV-2-S vaccinated mice were tested for neutralization of recombinant luciferase-expressing SARS-CoV-2 viruses (wild-type (left) and D614G variant (middle)). (Right) Paired EC50 values are indicated (n = 5; n.s. not significant, paired t test). (C) BAL fluid was collected from ChAd-control or ChAd-SARS-CoV-2-S vaccinated mice, and neutralization of SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was measured using a FRNT assay (n = 8-10 per group). Each point represents the mean of two technical replicates. (D) On day 35 post-immunization, mice were challenged via intranasal route with 4 × 105 FFU of SARS-CoV-2 five days after Hu-AdV5-hACE2 transduction and anti-Ifnar1 mAb treatment as described in Figure 2. Tissues were collected at 4 dpi for viral burden measurements. Genomic RNA (ORF1a) levels were determined in lungs and nasal turbinates (two experiments, n = 6-9; ∗∗∗∗p < 0.0001; Mann-Whitney test). Columns show median values, and dotted lines indicate the LOD of the assays.
Figure 5
Figure 5
Single-Dose Intranasal Immunization with ChAd-SARS-CoV-2-S Protects against SARS-CoV-2 Infection Five-week-old BALB/c female mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal route. On day 35 post-immunization, mice were challenged as follows: animals were treated with anti-Ifnar1 mAb and transduced with Hu-AdV5-hACE2 via the intranasal route 1 day later. Five days later, mice were challenged intranasally with 4 × 105 FFUs of SARS-CoV-2. (A–C) Tissues and nasal washes were collected at 4 and 8 dpi for analysis. Infectious virus in the lung was measured by plaque assay (A). Viral RNA levels in the lung, spleen, heart, nasal turbinates, and nasal washes were measured at 4 and 8 dpi by qRT-PCR (B). Fold change in gene expression of indicated cytokines and chemokines was determined by qRT-PCR, normalized to Gapdh, and compared to naive controls in lung homogenates at 4 dpi (C; 2 experiments, n = 6–9; median values are shown). Columns show median values, and dotted lines indicate the LOD of the assays. (D) Lungs were harvested at 8 dpi. Sections were stained with hematoxylin and eosin and imaged at 40× (left; scale bar, 250 μm), 200× (middle; scale bar, 50 μm), and 400× (right; scale bar, 25 μm) magnifications. Each image is representative of a group of 3 mice. (E) An ELISA measured anti-SARS-CoV-2 NP IgM (left) and IgG (right) antibody responses in paired sera obtained 5 days before and 8 days after SARS-CoV-2 challenge of ChAd-control or ChAd-SARS-CoV-2-S mice vaccinated by an intranasal route (n = 6). Dotted lines represent the LOD of the assay. Dashed lines indicate the mark for a 4-fold increase of pre-boost IgM and IgG levels. For (A)–(C): Mann-Whitney test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; for (E): ∗∗p < 0.01, ∗∗∗∗p < 0.0001; paired t test. See Figures S4 and S5.
Figure S5
Figure S5
SARS-CoV-2 NP-Specific IgM and IgG Antibody Responses following SARS-CoV-2 Challenge, Related to Figure 5 Five-week-old BALB/c female mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal route. One month later, mice were transduced with Hu-AdV5-ACE2 and challenged with SARS-CoV-2 as described in Figure 5. An ELISA measured anti-SARS-CoV-2 NP IgM and IgG responses in paired sera obtained 5 days before and 8 days after SARS-CoV-2 challenge. Serum ELISA curves corresponding to individual mice are shown for the indicated vaccines or control naive BALB/c mice (n = 6 per group).
Figure 6
Figure 6
Immunogenicity and Protective Efficacy after Intranasal Immunization of ChAd-SARS-CoV-2-S in K18-hACE2 Mice (A) Scheme of experiments. Four-week-old K18-hACE2 female mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal route. (B–D) Antibody responses in sera of immunized mice at 4 weeks after priming were evaluated. An ELISA measured SARS-CoV-2 S-and RBD-specific IgG (B) and IgA levels (C), and a FRNT determined neutralization activity (D; n = 7). (E–G) At 1 month post-immunization, mice were challenged with 103 FFUs of SARS-CoV-2. Tissues and nasal washes were harvested at 4 dpi. Infectious virus in lungs was measured using plaque assay (E). Viral RNA levels in the lung, spleen, heart, nasal turbinates, and nasal washes were measured at 4 dpi by qRT-PCR (F). Fold change in gene expression of indicated cytokines and chemokines was determined by qRT-PCR, normalized to Gapdh, and compared to naive controls in lung homogenates at 4 dpi (G; n = 7). Boxes indicate median values, and dotted lines indicate the LOD of the assays. For (B)–(G): Mann-Whitney test: ∗∗p < 0.01; ∗∗∗p < 0.001. See Figure S6.
Figure S6
Figure S6
Intranasal Inoculation of ChAd-SARS-CoV-2-S Induces Neutralizing Antibodies in K18-hACE2 Mice, Related to Figure 6 Four-week old female K18-hACE2 mice were immunized with ChAd-control or ChAd-SARS-CoV-2-S via an intranasal inoculation route. Serum samples collected four weeks after immunization were assayed for neutralizing activity by FRNT (n = 7 per group). Each point represents the mean of two technical replicates.
Figure 7
Figure 7
Comparison of Immunogenicity of Single-Dose Administration of ChAd-SARS-CoV-2-S by Intramuscular and Intranasal Delivery Routes (Upper panel) After intramuscular inoculation, ChAd-SARS-CoV-2 vaccine transduces antigen-presenting cells (APCs) at the site of injection in muscle tissues. APCs migrate to lymphoid tissues where antigen-specific CD8+T cells become activated, proliferate, and produce IFNγ and granzyme B. Antigen-specific B cells proliferate, some of which become plasmablasts and plasma cells that secrete anti-S IgG. After SARS-CoV-2 challenge, activated CD8+ T cells migrate to the lungs to control infection and anti-S IgG neutralizes virus particles. Intramuscular vaccination protects against lower, but not upper, airway infection and does not efficiently induce mucosal immunity. (Lower panel) After intranasal inoculation, ChAd-SARS-CoV-2 transduces APCs in the upper respiratory tract. APCs then migrate to bronchial- or mucosal-associated lymphoid tissues to present antigens to lymphocytes, including B and T cells. After SARS-CoV-2 challenge, activated CD8+ T cells migrate to the lungs, secrete cytokines, and attack virus-infected cells. Some CD8+ T cells likely adopt a tissue-resident memory phenotype (CD103+ CD69+), enabling them to reside in the lung (or upper airway) and respond more rapidly after re-encountering cognate antigen (SARS-CoV-2 S peptides). The activated B cell becomes responsive after intranasal vaccination produces cells that secrete anti-SARS-CoV-2-S IgG or IgA, the latter of which neutralizes virus within the upper and lower respiratory tracts. The mucosal immunity generated by intranasal inoculation of ChAd-SARS-CoV-2 likely controls infection at the point of initiation in the upper respiratory tract.

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Source: PubMed

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