Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters

Coronavirus disease 2019 (COVID-19) in humans is often a clinically mild illness, but some individuals develop severe pneumonia, respiratory failure and death1–4. Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in hamsters5–7 and nonhuman primates8–10 have generally reported mild clinical disease, and preclinical SARS-CoV-2 vaccine studies have demonstrated reduction of viral replication in the upper and lower respiratory tracts in nonhuman primates11–13. Here we show that high-dose intranasal SARS-CoV-2 infection in hamsters results in severe clinical disease, including high levels of virus replication in tissues, extensive pneumonia, weight loss and mortality in a subset of animals. A single immunization with an adenovirus serotype 26 vector-based vaccine expressing a stabilized SARS-CoV-2 spike protein elicited binding and neutralizing antibody responses and protected against SARS-CoV-2-induced weight loss, pneumonia and mortality. These data demonstrate vaccine protection against SARS-CoV-2 clinical disease. This model should prove useful for preclinical studies of SARS-CoV-2 vaccines, therapeutics and pathogenesis.

vector-based vaccine 19 encoding a stabilized SARS-CoV-2 spike (S) in this stringent model.
We inoculated 20 Syrian golden hamsters (10-12 weeks old) with 5 × 10 4 50% tissue culture infective dose (TCID 50 ) (n = 4; low-dose) or 5 × 10 5 TCID 50 (n = 16; high-dose) SARS-CoV-2 by the intranasal route. In the high-dose group, four animals were necropsied on day 2, four animals were necropsied on day 4 for tissue viral loads and histopathology and the remaining eight animals were followed longitudinally. All remaining animals were necropsied on day 14. In the low-dose group, hamsters lost a median of 14.7% of body weight by day 6 but fully recovered by day 14 (Fig. 1a,b), consistent with previous studies [5][6][7] . In the high-dose group, hamsters lost a median of 19.9% of body weight by day 6. Of the eight animals in this group that were followed longitudinally, four met Institutional Animal Care and Use Committee humane euthanasia criteria of more than 20% weight loss and respiratory distress on day 6, and two additional animals met these criteria on day 7. The remaining two animals recovered by day 14. These data demonstrate that high-dose SARS-CoV-2 infection in hamsters led to severe weight loss and partial mortality.
Tissue viral loads were assessed in the four animals that received high-dose SARS-CoV-2 and were necropsied on day 2, the four animals that were necropsied on day 4 and five of six of the animals that met euthanasia criteria on days 6-7 (Fig. 1c). High median tissue viral loads on day 2 of 10 12 RNA copies per gram in lung tissue and 10 8 -10 9 RNA copies per gram in nares and trachea were observed, with a median of 10 5 -10 8 RNA copies per gram in heart, gastrointestinal tract, brain, spleen, liver and kidney, indicative of disseminated infection. By days 6-7, tissue viral loads were approximately 2 logs lower, despite continued weight loss.

NATuRE MEDICINE
Hamsters infected with high-dose SARS-CoV-2 were assessed by histopathology on days 2 (n = 4), 4 (n = 4), 6-7 (n = 6) and 14 (n = 2). Infection was associated with marked inflammatory infiltrates and multifocal epithelial necrosis of the nasal turbinate ( Fig. 2a) and bronchiolar epithelium, resulting in degenerative neutrophils and cellular debris in the lumen (Fig. 2b). The endothelium of nearby vessels was reactive with adherence of mononuclear cells to the endothelium and transmigrating within vessel walls, indicative of endothelialitis (Fig. 2b). There was moderate to severe multifocal interstitial pneumonia characterized by pulmonary consolidation affecting 30-60% of the lung parenchyma as early as day 2 after SARS-CoV-2 infection (Fig. 2c). Inflammatory infiltrates consisted of massive numbers of macrophages and neutrophils with fewer lymphocytes. The nasal turbinate epithelium (Fig. 2d) and bronchiolar epithelial cells (Fig. 2e) were strongly positive for SARS nucleocapsid protein (SARS-CoV-N) by immunohistochemistry (IHC) in regions of inflammation and necrosis. SARS-CoV-N IHC also showed locally extensive staining of the alveolar septa and interstitial mononuclear cells morphologically consistent with macrophages ( Fig. 2f). Similarly, substantial SARS-CoV-2 viral RNA (vRNA) was observed in the bronchiolar epithelium and the pulmonary interstitium in regions of inflammation (Fig. 2g,h).
Levels of both SARS-CoV-2 vRNA and SARS-CoV-N protein expression in lung were highest on day 2 and diminished by day 4, with minimal vRNA and SARS-CoV-N protein detected by day 7 (Extended Data Fig. 1). The pneumonia was characterized by large inflammatory infiltrates of ionized calcium-binding adaptor protein 1 (Iba-1) + macrophages in the lung interstitium as well as CD3 + T lymphocytes (Fig. 2i,j). Many viable and degenerative neutrophils were detected throughout the lung, especially in regions of necrosis, with high expression of neutrophil myeloperoxidase (MPO) throughout the lung (Fig. 2k). Diffuse expression of the interferon inducible gene product MX1 was also detected in the lung (Fig. 2l). In contrast with the kinetics of SARS-CoV-2 vRNA and SARS-CoV-N detection, which peaked on day 2, these markers of inflammation peaked on day 7 (Extended Data Fig. 1), coincident with maximal weight loss and mortality (Fig. 1a,b)  Syrian golden hamsters (10-12 weeks old; male and female; n = 20) were infected with 5 × 10 4 TCID 50 (low-dose; n = 4) or 5 × 10 5 TCID 50 (high-dose; n = 16) of SARS-CoV-2 by the intranasal route. a, Median percent weight change after challenge. The numbers reflect the number of animals at each time point. In the high-dose group, four animals were necropsied on day 2, four animals were necropsied on day 4, four animals met euthanization criteria on day 6 and two animals met euthanization criteria on day 7. b, Percent weight change after challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Gray lines indicate animals with scheduled necropsies on day 2 and day 4. c, Tissue viral loads as measured by log 10 RNA copies per gram of tissue (limit of quantification, 100 copies per gram) in the scheduled necropsies at day 2 and day 4 and in 2-5 of 6 animals that met euthanization criteria on days 6-7. extended tissues were not harvested on day 6.

NATuRE MEDICINE
anti-sense vRNA (Extended Data Fig. 2a-e) but also positive-sense vRNA (Extended Data Fig. 2f-j), which overlapped in location and pattern, from day 2 to day 7 after challenge. SARS-CoV-2 vRNA expression (both anti-sense and sense) was present in lung with robust ACE2 receptor expression (Extended Data Fig. 2k-o).
Systemic vRNA was also detected in distal tissues, including the brain stem, gastrointestinal tract and myocardium (Extended Data Fig. 3a-f). Prominent endothelialitis and perivascular inflammation with macrophages and lymphocytes was observed in these tissues, despite minimal SARS-CoV-N staining (Extended Data Fig. 3g-j). Focal lymphocytic myocarditis was noted in one animal and corresponded to the presence of vRNA (Extended Data Fig. 3k-l). Other sites of virus detection included peripheral blood mononuclear cells in thrombi in lung (Extended Data Fig. 4a-c) and bone marrow of the nasal turbinate (Extended Data Fig. 4d-f).
We produced recombinant, replication-incompetent Ad26 vectors encoding 1) SARS-CoV-2 S with deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain with a foldon trimerization domain (S.dTM.PP) or 2) full-length S (S.PP), both with mutation of the furin cleavage site and two proline stabilizing mutations 20 (Fig. 3a). We recently reported the immunogenicity and protective efficacy of these vaccines against SARS-CoV-2 challenge in rhesus macaques 13 .
We immunized 50 Syrian golden hamsters with 10 10 or 10 9 viral particles (vp) of Ad26 vectors encoding S.dTM.PP or S.PP (n = 10 per group) or sham controls (n = 10). Animals received a single vaccination by the intramuscular route at week 0. We observed receptor-binding domain (RBD)-specific binding antibodies by enzyme-linked immunosorbent assay (ELISA) 10,11 (Fig. 3b) and neutralizing antibodies (NAbs) by a pseudovirus neutralization  We further characterized S-specific and RBD-specific antibody responses in the vaccinated animals at week 4 by systems serology 22 . IgG, IgG2a, IgG3, IgM, Fc-receptors FcRγ2, FcRγ3 and FcRγ4 and antibody-dependent complement deposition (ADCD) responses were assessed ( Fig. 3d-f). Higher and more consistent responses were observed with Ad26-S.PP than with Ad26.S.dTM. PP (Fig. 3d,f), and a principal component analysis (PCA) of these antibody features confirmed that these two vaccines had distinct profiles ( Fig. 3e).
At week 4, all animals were challenged with 5 × 10 5 TCID 50 SARS-CoV-2 by the intranasal route. Three animals in each group were necropsied on day 4 for tissue viral loads and histopathology, and the remaining seven animals in each group were followed until day 14. In the sham controls, hamsters lost a median of 19.6% of body weight by day 7, and 43% (3/7) of the animals that were followed longitudinally met euthanasia criteria on days 6-7 ( Fig. 4a,b). The Ad26-S.dTM.PP vaccinated animals lost a median of 8.7% body weight, and the Ad26-S.PP vaccinated animals lost a median of 4.0% body weight (Fig. 4a,b). Maximum percent weight loss was markedly lower in both vaccinated groups compared to sham controls (P < 0.0001, two-sided Mann-Whitney tests; Fig. 4c), and animals that received Ad26-S.PP showed less weight loss than animals that received Ad26.S.dTM.PP (P < 0.0001, two-sided Mann-Whitney tests; Fig. 4c). Both vaccines protected against mortality, defined as meeting humane euthanization criteria, as compared to sham controls (P = 0.02, two-sided Fisher's exact tests; Extended Data Fig. 5b). A combined analysis of the two hamster experiments confirmed that both vaccines effectively protected against mortality (P = 0.007, two-sided Fisher's exact tests; Extended Data Fig. 5c). ELISA responses at week 2 (R = −0.8992, P < 0.0001) and week 4 (R = −0.9344, P < 0.0001) correlated inversely with maximum percent weight loss (Extended Data Fig. 6a). NAb responses at week 2 (R = −0.7380, P < 0.0001) and week 4 (R = −0.8075, P < 0.0001) also correlated inversely with maximum percent weight loss (Extended Data Fig. 6b).
Tissue viral loads were assessed in the subset of animals necropsied on day 4 and in the remaining surviving animals on day 14. On day 4 after high-dose SARS-CoV-2 challenge, virus was detected in tissues in all animals by subgenomic RNA reverse trancriptionpolymerase chain reaction (RT-PCR), which is thought to measure replicating virus 10,23 (Extended Data Fig. 7a). Median viral loads in lung tissue were approximately 10 12 RNA copies per gram in the sham controls compared to 10 8 RNA copies per gram in the Ad26-S. dTM.PP vaccinated animals and 10 6 RNA copies per gram in the Ad26-S.PP vaccinated animals. Reduced TCID 50 infectious virus titers per gram of lung tissue were also observed for the Ad26-S. dTM.PP and Ad26.S.PP vaccinated animals compared to sham controls (P = 0.02 and P = 0.01, respectively, two-sided Mann-Whitney tests; Extended Data Fig. 7b). By day 14, virus was still detected in lung and nares of the surviving sham controls but was observed in only a minority of Ad26-S.dTM.PP vaccinated animals and in none of the Ad26-S.PP vaccinated animals (Extended Data Fig. 7c).
The surviving sham controls developed potent binding and neutralizing antibody responses by day 14 after challenge (Extended Data Fig. 9b). Vaccinated animals also demonstrated higher ELISA and NAb responses after challenge (Extended Data Fig. 9b), consistent with tissue viral loads showing low and transient levels of virus replication in these animals after high-dose SARS-CoV-2 challenge.
Vaccinated animals also demonstrated diminished pathology compared to sham controls on day 4 after challenge (Extended Data Fig. 10). Ad26-S.PP vaccinated animals demonstrated minimal to no evidence of viral interstitial pneumonia, disruption of the bronchiolar epithelium or peribronchiolar aggregates of CD3 + T lymphocytes and macrophages. Histiocytic and neutrophilic inflammatory infiltrates were markedly reduced in all lung lobes, and significantly reduced SARS-CoV-2 vRNA was observed in Ad26-S.dTM.PP and Ad26-S.PP vaccinated hamsters compared to sham controls (P = 0.004 and P = 0.004, respectively, two-sided Mann-Whitney tests; Fig. 4d).
In this study, we demonstrated that a single immunization of an Ad26 vector encoding a full-length prefusion stabilized S immunogen (S.PP) protected against severe clinical disease after high-dose SARS-CoV-2 challenge in hamsters. Sham controls demonstrated marked weight loss, severe pneumonia and partial mortality. In contrast, vaccinated animals showed minimal weight loss and pneumonia and no mortality. Vaccine-elicited binding and neutralizing antibody responses correlated with protection against clinical disease as well as reduced virus replication in the upper and lower respiratory tract.
The severity of clinical disease in this model contrasts with previous studies involving SARS-CoV-2 infection in hamsters [5][6][7] and other species [8][9][10][14][15][16][17][18] . Hamsters are a permissive model for SARS-CoV-2 as a result of their homology to the human ACE2 Humoral immune responses were assessed at weeks 0, 2 and 4 by b) RBD-specific binding antibody eLISA and c) pseudovirus neutralization assays. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation. d, S-and RBD-specific IgG subclass, FcγR and ADCD responses at week 4 are shown as radar plots. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (antibody subclass, red; FcγR binding, blue; ADCD, green). e, PCA plot showing the multivariate antibody profiles across vaccination groups. each dot represents an animal; the color of the dot denotes the group; and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution. f, The heat map shows the differences in the means of z-scored features between vaccine groups S.PP and S.dTM.PP. The two groups were compared by two-sided Mann-Whitney tests, and stars indicate the Benjamini-Hochberg-corrected q values (*q < 0.05, **q < 0.01 and ***q < 0.001).

NATuRE MEDICINE
receptor 5 , and transmission among hamsters has been reported 6 . The high challenge dose resulted in extensive clinical disease in the present study, although biologic factors that remain to be fully defined might also affect clinical disease, such as animal age, animal origin and viral challenge stock.
In summary, our data demonstrate that a single immunization of Ad26-S.PP provides robust protection against severe clinical disease after high-dose SARS-CoV-2 infection in hamsters. To our knowledge, vaccine protection against severe SARS-CoV-2 pneumonia and mortality has not previously been reported. Ad26-S.PP, which is also termed Ad26.COV2.S, is currently being evaluated in clinical trials. This hamster severe disease model should prove useful for testing of SARS-CoV-2 vaccines, therapeutics and other countermeasures.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41591-020-1070-6.

Methods
Animals and study design. Seventy male and female Syrian golden hamsters (Envigo), 10-12 weeks old, were randomly allocated to groups. All animals were housed at Bioqual. Animals received Ad26 vectors expressing S.dTM.PP or S.PP or sham controls (n = 10 per group). Animals received a single immunization of 10 10 or 10 9 vp Ad26 vectors by the intramuscular route without adjuvant at week 0. At week 4, all animals were challenged with 5.0 × 10 5 TCID 50 (6 × 10 8 vp, 5.5 × 10 4 plaque-forming units (PFU)) or 5.0 × 10 4 TCID 50 (6 × 10 7 vp, 5.5 × 10 3 PFU) SARS-CoV-2, which was derived with one passage from USA-WA1/2020 (NR-52281, BEI Resources) 10 . Virus was administered as 100 μl by the intranasal route (50 μl in each nare). Body weights were assessed daily. All immunologic and virologic assays were performed blinded. On day 4, a subset of animals was euthanized for tissue viral loads and pathology. All animal studies were conducted in compliance with all relevant local, state and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee. RNAscope in situ hybridization. RNAscope in situ hybridization was performed as previously described 10 using SARS-CoV2 anti-sense specific probe v-nCoV2019-S (ACD, cat. no. 848561) targeting the positive-sense viral RNA and SARS-CoV2 sense specific probe v-nCoV2019-orf1ab-sense (ACD, cat. no. 859151) targeting the negative-sense genomic vRNA. In brief, after slides were deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water, retrieval was performed for 30 min in ACD P2 retrieval buffer (ACD, cat. no. 322000) at 95-98 °C , followed by treatment with protease III (ACD, cat. no. 322337) diluted 1:10 in PBS for 20 min at 40 °C. Slides were then incubated with 3% H 2 O 2 in PBS for 10 min at room temperature. Before hybridization, probe stocks were centrifuged at 13,000 r.p.m. using a microcentrifuge for 10 min and then diluted 1:2 in probe diluent (ACD, cat. no. 300041) to reduce probe aggregation tissue artifacts. Slides were developed using the RNAscope 2.5 HD Detection Reagents-RED (ACD, cat. no. 322360).
Quantitative image analysis. Quantitative image analysis was performed using HALO software (v2.3.2089.27 or v3.0.311.405; Indica Labs) on at least one lung lobe cross-section from each animal. In cases where more than one cross-section was available, each lung lobe was quantified as an individual data point. For SARS-CoV-N, the Multiplex IHC v2.3.4 algorithm was used with an exclusion screen for acid hematin to determine the percentage of SAR-N protein positive cells as a proportion of the total number of cells. For Iba-1, the Multiplex IHC v2.3.4 algorithm was used for quantitation. For SARS-CoV-2 RNAscope ISH and Mx1 quantification, the Area Quantification v2.1.3 module was used to determine the percentage of total SARS-CoV-2 anti-sense or sense probe or Mx1 protein as a proportion of the total tissue area. For MPO (neutrophil) and CD3 + cell quantification, slides were annotated to exclude blood vessels (>5 mm 2 ), bronchi, bronchioles, cartilage and connective tissue; subsequently, the Cytonuclear v1.6 module was used to detect MPO + or CD3 + cells and frequency was calculated as a proportion of total alveolar tissue (polymorphonuclear leukocytes per mm 2 ), determined by running the Area Quantification v2.1.3 module. In all instances, manual inspection of all images was performed on each sample to ensure that the annotations were accurate.
Subgenomic mRNA assay. SARS-CoV-2 E gene subgenomic mRNA (sgmRNA) was assessed by RT-PCR using primers and probes as previously described 10,11,23 . Briefly, total RNA was extracted from tissue homogenates from several anatomical sites using a QIAcube HT (Qiagen) and RNeasy 96 QIAcube HT Kit (Qiagen). A standard curve was generated using the SARS-CoV-2 E gene sgmRNA by cloning into a pcDNA3.1 expression plasmid; this insert was transcribed using an AmpliCap-Max T7 High Yield Message Maker Kit (Cellscript). Before RT-PCR, samples collected from challenged animals or standards were reverse transcribed using Superscript III VILO (Invitrogen) according to the manufacturer's instructions. A Taqman custom gene expression assay (Thermo Fisher Scientific) was designed using the sequences targeting the E gene sgmRNA. Reactions were carried out on QuantStudio 6 and 7 Flex Real-Time PCR Systems (Applied Biosystems) according to the manufacturer's specifications. Standard curves were used to calculate sgmRNA copies per gram tissue; the quantitative assay sensitivity was 100 copies.
ELISA. RBD-specific binding antibodies were assessed by ELISA essentially as described 10,11 . Briefly, 96-well plates were coated with 1 µg ml ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance two-fold above background.
To determine the neutralization activity of the antisera from vaccinated animals, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 × 10 4 cells per well overnight. Three-fold serial dilutions of heat-inactivated serum samples were prepared and mixed with 50 µl of pseudovirus. The mixture was incubated at 37 °C for 1 h before adding to HEK293T-hACE2 cells. Forty-eight hours after infection, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer's instructions. SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in relative light units was observed relative to the average of the virus control wells.

Luminex.
To detect relative quantity of antigen-specific antibody titers, a customized Luminex assay was performed as previously described 25   Extended Data Fig. 5 | Correlation of antibody titers and survival curves. a, Correlations of binding eLISA titers and pseudovirus NAb titers at week 2 and week 4. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests. b, Survival curve for the vaccine study. P values indicate two-sided Fisher's exact tests. N denotes number of animals in each group. c, Combined analysis of the two hamster studies involving all animals that received the 5x10 5 TCID 50 challenge dose and were followed longitudinally. P values indicate two-sided Fisher's exact tests. N denotes number of animals in each group. . N reflects all animals that were followed for weight loss or that were necropsied for lung or nasal turbinate viral loads. Significant correlations are indicated by stars after multiple testing correction using the Benjamini-Hochberg procedure (*q < 0.05, ** q < 0.01, *** q < 0.001). b, eLISA and NAb responses in surviving hamsters on day 14 following SARS-CoV-2 challenge.