key: cord-0706661-quogvgl4 authors: Bewley, Kevin R.; Gooch, Karen; Thomas, Kelly M.; Longet, Stephanie; Wiblin, Nathan; Hunter, Laura; Chan, Kin; Brown, Phillip; Russell, Rebecca A.; Ho, Catherine; Slack, Gillian; Humphries, Holly E.; Alden, Leonie; Allen, Lauren; Aram, Marilyn; Baker, Natalie; Brunt, Emily; Cobb, Rebecca; Fotheringham, Susan; Harris, Debbie; Kennard, Chelsea; Leung, Stephanie; Ryan, Kathryn; Tolley, Howard; Wand, Nadina; White, Andrew; Sibley, Laura; Sarfas, Charlotte; Pearson, Geoff; Rayner, Emma; Xue, Xiaochao; Lambe, Teresa; Charlton, Sue; Gilbert, Sarah; Sattentau, Quentin J.; Gleeson, Fergus; Hall, Yper; Funnell, Simon; Sharpe, Sally; Salguero, Francisco J.; Gorringe, Andrew; Carroll, Miles title: Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques date: 2020-12-21 journal: bioRxiv DOI: 10.1101/2020.12.21.423746 sha: 9759bb6f2c61b2a62366000a61264210300ef7cb doc_id: 706661 cord_uid: quogvgl4 There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity. Laemmli buffer (Sigma, S3401) and heated at 90°C for 5 min and loaded onto a 10- 140 well NuPAGE 4-12% Bis-Tris gel, 1.0mm (ThermoFisher). 5µL SeeBlue Plus2 141 7 (ThermoFisher) ladder was loaded as a marker and gels were stained with SimplyBlue 142 SafeStain (ThermoFisher). Western Blot: Samples were processed as described for 143 SDS PAGE and transferred to PVDF membrane with iBlot2 (ThermoFisher). After 144 transfer, membranes were washed with tris-buffered saline 0.1% Tween20 (TBST) for 145 5 min at room temperature, followed by 1 h in blocking buffer (TBST, 5% skimmed 146 milk powder). Membranes were washed three times for 5 min with TBST. MERS The relative density of this band (20.7%) permitted estimation of the proportion of the 157 FIV total protein that was Coronavirus-specific (178µg/mL). 10mg/kg). SARS-CoV-2 Victoria/01/2020 26 was prepared as described previously 24 . It 202 was delivered to ferrets by intranasal instillation (1.0mL total, 0.5mL per nostril) diluted 203 in PBS. A single dose of virus (5x10 6 pfu/ferret) was delivered to Ad-GFP-(n=4) and 204 FIV-(n=6) vaccinated ferrets. Macaques were challenged with 5 x 10 6 delivered by the 205 intratracheal route (2ml) and intranasal instillation (1ml total, 0.5ml per nostril). The 206 schedule of euthanisation and sampling is shown in Table 1 . 207 Nasal washes were obtained by flushing the nasal cavity with 2mL PBS. Throat swabs 208 were collected using a standard swab (Sigma Virocult ® ) gently stroked across the back 209 of the pharynx in the tonsillar area. Throat swabs were processed, and aliquots stored 210 in viral transport media (VTM) and AVL at ≤ -60°C until assay. Clinical signs of disease 211 were monitored as described previously 24 25 . The necropsy procedures were also as CoV-2 N ORF (accession number NC_045512.2) with quantification between 1 x 10 1 229 and 1 x 10 6 copies/µL. Positive samples detected below the limit of quantification were 230 assigned the value of 5 copies/µL, whilst undetected samples were assigned the value 231 of 2.3 copies/µL, equivalent to the assay's lower limit of detection. The severity of the histopathological lesions was scored as: 0=none (within normal 320 limits), 1=minimal, 2=mild, 3=moderate, and 4=severe. The following samples from each rhesus macaque were fixed, processed, cut and 338 stained as described above: left cranial and caudal lung lobes, trachea, larynx, 339 mediastinal lymph node, tonsil, spleen, liver, kidney, duodenum and colon. Fig. 2) . inflammatory foci within the parenchyma (Fig. 3B) . Moreover, perivascular cuffing 494 was observed frequently (Fig. 3C) , with the infiltrates being mostly mononuclear 495 cells, including CD3 + T lymphocytes identified by immunohistochemistry (IHC) 496 staining (Fig. 3D) . Occasionally, neutrophils and eosinophils were also present ( Fig. 497 3C, insert). The cuffing also affected numerous airways (Fig. 3C) . Due to the small 498 numbers of animals, the differences in scores observed between FIV and Ad-GFP-499 vaccinated groups did not reach significance. In contrast, at 13-15 days pc, the lesions observed were minimal to mild with no 501 obvious differences between groups (Fig. 3A) . 502 RNAScope ISH technique was used to detect viral RNA in lung and nasal cavity tissue immune responses before SARS-CoV-2 challenge (Fig 4) . geometric mean viral RNA copies per ml (p=0.168) were measured in macaques that 539 received FIV than no vaccine controls (Fig 4C) . No significant changes in body 540 temperature (Supplementary Fig. 2C) where observed. Slight weight loss was 541 observed in both groups (Supplementary Fig. 2D ), but no adverse clinical signs were 542 recorded for any macaque despite frequent monitoring during the study period. in the total CT score in the FIV group compared to the scores attributed to macaques 559 in the unvaccinated group (Fig. 6D) . Similarly, the FIV vaccine reduced both the 560 amount of abnormalities induced (pattern score) and distribution of disease (zone 561 score) (Supplementary Fig. 4 H and I) . macaques. The total pathology score for the no vaccine group was greater than the 569 FIV group (Fig. 6B, p=0.013 ). RNAScope analysis of the percentage of area positively 570 stained for SARS-CoV-2 RNA showed a greater lung area infected in the no vaccine 571 group than FIV-vaccinated macaques (Fig. 6C, p = 0.0238) . Immune responses to FIV in rhesus macaques. Serum from control macaques 573 obtained on the day of challenge did not show any N, RBD or S-specific IgG but rises 574 in RBD and S-specific IgG were detected in serum from the FIV-vaccinated macaques 575 (Fig. 7) . FIV-vaccinated animals also showed a rise in neutralising antibody titre on the 576 day of challenge (p=0.0287). Both groups showed a rise in neutralising antibody titre 577 25 7 days following challenge (Fig. 7) . Similarly, on the day of challenge, a higher 578 frequency of spike-specific IFNγ-secreting cells was measured by ELISPOT assay in 579 the FIV group compared to that determined in the unvaccinated group suggesting the 580 induction of a modest but significant (p = 0.0433) SARS-CoV-2-specific cellular 581 response (Supplementary. Figure 3B) . The trend reversed six to eight days after 582 challenge when frequencies were assessed at the end of the study, with higher Binding curves revealed that ligands binding to formaldehyde-treated S protein gave 610 substantially lower maximum binding than that to the untreated S counterpart (Fig. 611 8A) . Area under the curve (AUC) analysis revealed that binding was significantly 612 reduced for sACE2-Fc and CR3022, and had a trend to reduction for EY6A (Fig. 8B) . 613 Interestingly, the reduction for sACE2 and CR3022 was almost precisely 2-fold, 614 suggesting either that the formaldehyde treatment had reduced the binding activity of 615 a subset of RBD domains, or that half of the formaldehyde-treated S trimers were in a 616 non-RBD available conformation. To differentiate between these two possibilities, we 617 tested binding directly to the isolated untreated or formaldehyde-treated RBD (Fig. 618 8C) . Strikingly, formaldehyde treatment had no effect on RBD-ligand binding, with 619 AUC analysis showing near identical values for formaldehyde-treated and untreated 620 RBD (Fig. 8D) . It therefore seems most likely that formaldehyde treatment is stabilising The SARS-CoV-2 infection in the Ad-GFP-or FIV-vaccinated ferrets followed a 677 similar course to that observed in our previous study 24 with peak viral RNA shedding 678 between 2 and 4 days pc. It was interesting to note that higher viral loads were The presence of inflammatory infiltrates, and particularly perivascular cuffing, has 715 been described as a feature potentially related to VED in SARS-CoV-1 preclinical 716 vaccine trials 19 21 22 . In our study these infiltrates were always of mild to moderate 717 severity. IgG was quantified by ELISA to recombinant nucleocapsid protein (NP), receptor binding domain (RBD) and full-length trimeric and stabilised spike protein (Spike). Bars are geometric mean titre. The significance of any difference from pre-to postvaccination is shown, determined by a paired t-test. The micronutralisation 50% titre (ND50) is also shown with samples obtained pre-and post-vaccination and following SARS-CoV-2 challenge. Bars are geometric mean ND50 titre. Developing covid-19 vaccines at 804 pandemic speed SARS-CoV-2 vaccines in development An mRNA Vaccine against SARS-CoV-2 -Preliminary 809 Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in 811 adults Safety and immunogenicity of ChAdOx1 nCoV-19 813 vaccine administered in a prime-boost regimen in young and old adults 814 (COV002): a single-blind, randomised, controlled, phase 2/3 trial Development of an Inactivated Vaccine Candidate with Potent Protection against SARS-CoV-2 Development of an inactivated vaccine candidate for SARS-820 Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety 822 and Immunogenicity Outcomes: Interim Analysis of 2 Randomized Clinical 823 ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 825 pneumonia in rhesus macaques Evaluation of the mRNA-1273 Vaccine against SARS-CoV-828 2 in Nonhuman Primates Viral-induced enhanced disease 831 illness A perspective on potential antibody-dependent enhancement 833 of SARS-CoV-2 The potential danger of suboptimal antibody responses 835 in COVID-19 An epidemiologic study of altered clinical reactivity to Respiratory Syncytial 838 (RS) virus infection in children previously vaccinated with an inactivated RS 839 virus vaccine A role for 841 nonprotective complement-fixing antibodies with low avidity for measles virus 842 in atypical measles Antibody-dependent enhancement of severe dengue 844 disease in humans. Science (80-. ) A Double-Inactivated Severe Acute Respiratory Syndrome 846 Coronavirus Vaccine Provides Incomplete Protection in Mice and Induces 847 Increased Eosinophilic Proinflammatory Pulmonary Response upon 848 Immunization with SARS coronavirus vaccines leads to 850 pulmonary immunopathology on challenge with the SARS virus Evaluation of modified 853 vaccinia virus Ankara based recombinant SARS vaccine in ferrets Immunogenicity, safety, and protective efficacy of an inactivated 856 SARS-associated coronavirus vaccine in rhesus monkeys Immunodominant SARS coronavirus epitopes in humans 859 elicited both enhancing and neutralizing effects on infection in non-human 860 primates Recombinant Modified Vaccinia Virus Ankara Expressing the 862 Induces Protective Neutralizing Antibodies Primarily Targeting the Receptor 864 Binding Region Dose-dependent response to infection with SARS-CoV-2 in 866 the ferret model: evidence of protection to re-challenge Comparison of Rhesus and Cynomolgus macaques as an 869 authentic model for Isolation and rapid sharing of the 2019 novel coronavirus CoV-2) from the first patient diagnosed with COVID-19 in Australia A single dose of ChAdOx1 MERS provides protective 875 immunity in rhesus macaques Safety and immunogenicity of the analysis, processing of in vivo samples Preparation of in vivo samples and sera PCR data analysis PRNT assays and analysis Leonie Alden, in vivo study management Marilyn Aram, performed PCR assays Natalie Baker, virus inactivation and confirmation; Emily Brunt, performed PRNT 988 assays Rebecca Cobb, processing of in vivo samples Susan Fotheringham, in vivo 989 study management Debbie Harris, in vivo study management; Chelsea Kennard, 990 histology analysis including RNAscope Stephanie Leung, performed PRNT assay processed in vivo samples; Howard Tolley, performed electron 992 microscopy; Nadina Wand, RNA extraction and PCR assays Sarfas designed and performed the macaques ELISpot assays Xiaochao Xue, prepared and characterised proteins Yper Hall, 995 preparation and characterisation of inactivated virus in vivo study 996 design and provision of control vaccine; Sue Charlton, in vivo study design and 997 preparation of inactivated vaccine Simon Funnell, study design and manuscript 998 preparation in vivo study design and provision of control vaccine designed and performed experiments to characterise the effect 1000 of formaldehyde on SARS-CoV-2 spike protein Salguero, lead the histopathology analysis and reporting, drafted manuscript CT scanning and analysis, manuscript preparation; Fergus Gleeson, 1004 analysis of CT scans scientific direction and lead preparation of 1005 the manuscript; Miles Carroll, established the project, in vivo study design, scientific 1006 direction and manuscript preparation Data availability statement: The data that support the findings of this study are 1009 available from the corresponding authors upon reasonable request The authors acknowledge the contributions of all staff within the PHE Biological Investigations Group for assistance with the delivery of the in vivo study and B