key: cord-0999787-j65v41jy authors: Rauch, Susanne; Gooch, Karen; Hall, Yper; Salguero, Francisco J.; Dennis, Mike J.; Gleeson, Fergus V.; Harris, Debbie; Ho, Catherine; Humphries, Holly E.; Longet, Stephanie; Ngabo, Didier; Paterson, Jemma; Rayner, Emma L.; Ryan, Kathryn A.; Sharpe, Sally; Watson, Robert J.; Mueller, Stefan O.; Petsch, Benjamin; Carroll, Miles W. title: mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection date: 2020-12-23 journal: bioRxiv DOI: 10.1101/2020.12.23.424138 sha: 8e692a997351cf6c99f52e18952036c9145cd156 doc_id: 999787 cord_uid: j65v41jy The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines to meet a worldwide need. mRNA-based vaccines are the most promising technology for rapid and safe SARS-CoV-2 vaccine development and production. We have designed CVnCoV, a lipid-nanoparticle (LNP) encapsulated, sequence optimised mRNA-based SARS-CoV-2 vaccine that encodes for full length, pre-fusion stabilised Spike protein. Unlike other mRNA-based approaches, CVnCoV exclusively consists of non-chemically modified nucleotides and can be applied at comparatively low doses. Here we demonstrate that CVnCoV induces robust humoral and cellular responses in non-human primates (NHPs). Animals vaccinated with 8 μg of CVnCoV were protected from challenge infection with SARS-CoV-2. Comprehensive analyses of pathological changes in challenged animals via lung histopathology and Computed Tomography (CT) scans gave no indication of enhanced disease upon CVnCoV vaccination. These results demonstrate safety, immunogenicity, and protective efficacy of CVnCoV in NHPs that extend our previously published preclinical data and provide strong support for further clinical testing in ongoing phase 2b/3 efficacy studies. One year after the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified, a pandemic of >70 million confirmed infections has spread worldwide 1 . Coronavirus infectious disease 2019 (COVID-19) has so far resulted in 1.7 million deaths 1 , highlighting the urgent need for rapid vaccine development. Fifty-six vaccine candidates are currently in clinical development 2 , with the first highly promising clinical efficacy data available 3, 4 . mRNA vaccine technologies allow both rapid development and large-scale production 5 and have recently emerged as the most promising technology to tackle this worldwide crisis 6 7 8 9, 10 . CureVac's mRNA technology termed RNActive ® is based on non-chemically modified, sequence engineered mRNA, and is designed to allow rapid development of human vaccines to emerging diseases. RNActive ® vaccines formulated with protamine have been employed in the development of cancer immunotherapy 11 12 13 14 as well as in the context of prophylactic vaccines 15 16 17 18 . A first-in-human phase 1 study using this formulation in a rabies mRNA vaccine demonstrated proof of concept of chemically unmodified mRNA vaccines in humans and found significant levels of rabies neutralizing antibodies 19 . Further studies established significant improvements of vaccine efficacy by encapsulating the mRNA in lipid nanoparticles (LNPs) . These improvements were demonstrated in preclinical models 16 and a phase 1 clinical trial, where two 1 or 2 μg doses elicited immune responses comparable to a three-dose regimen of a licensed rabies vaccine 20 . RNActive ® technology has now been applied to the development of CVnCoV; an LNP formulated mRNA-based vaccine against SARS-CoV-2. The mRNA component of the vaccine consists of non-chemically modified nucleotides and has been optimised for high expression of the encoded protein and moderate activation of innate immune responses. CVnCoV encodes for full-length Spike (S) protein with two proline mutations (S-2P) aimed to stabilise the protein in the pre-fusion conformation, as previously described for MERS-and SARS-CoV-1 21 22 . This protein, a trimeric glycoprotein on the viral surface, is responsible for receptor binding and viral entry 23 24, 25 and represents a critical target for viral neutralizing antibodies 26 27 . Preclinical results have shown the induction of robust cellular and humoral immune responses in CVnCoV vaccinated rodents, as well as the ability to protect against challenge infection in a hamster model 5 . CVnCoV testing in phase 1 clinical trials demonstrated safety and tolerability as well as full seroconversion in the 12 µg vaccine group two weeks post injection of the second dose. Importantly, this group exhibited comparable virus neutralizing antibody titres to the median titres observed in convalescent sera from COVID-19 patients exhibiting multiple symptoms 28 . These results supported further clinical testing of the vaccine currently in phase 2b/3 studies that will investigate the efficacy, safety, and immunogenicity of the candidate vaccine CVnCoV. Here, we evaluate CVnCoV efficacy in a rhesus macaque SARS-CoV-2 challenge model. Nonhuman primates develop mild clinical disease with high levels of viral replication in both the upper and lower respiratory tract and pathological changes indicative of viral pneumonia upon infection with SARS-CoV-2 29 30 . In this study we show that CVnCoV had protective impact against challenge with 5 x 10 6 PFU via the IN and IT routes in an NHP in vivo model of COVID- 19 . Protective endpoints include significantly reduced virus load and protection against lung pathology. Eighteen rhesus macaques of Indian origin were divided into three groups of six, each comprising three males and three females. Animals were vaccinated twice with either 0.5 µg or 8 µg LNP-formulated mRNA encoding SARS-CoV-2 S-2P (CVnCoV) or remained unvaccinated prior to challenge 30 with wild type SARS-CoV-2 (Victoria/1/2020) four weeks after the second vaccination ( Figure 1A ). Analysis of binding titres to either a trimeric form of the S protein or the isolated receptor binding domain (RBD) showed a measurable increase in spike ( Figure 1B ) and RBD-specific IgG titres ( Figure 1C ) in animals vaccinated with 8 µg after a single vaccination. A significant increase was observed in IgG titres after the second vaccination on study day 42, with animals exhibiting median endpoint titres of 1.6 x 10 3 and 3.2 x 10 3 for S and RBD reactive antibodies, respectively (Figures 1 B and C) . A further increase of spike-and RBD specific IgG titres was seen upon challenge in this group, particularly in serum collected at the time of termination (study days 62, 63, and 64). As expected, no significant increase in spike or RBD-specific IgG antibodies was seen in the 0.5 µg CVnCoV (intentionally sub-optimal) dose or in the unvaccinated control group during the vaccination phase. However, a gradual increase in Spike and RBD specific IgG titres was observed at each of the sampling points in animals vaccinated with 0.5 µg CVnCoV after challenge (Figures 1 B and C) . No increase in Spike-or RBD specific IgG titres was observed in the unvaccinated controls ( Figure 1B and C). In agreement with the induction of binding antibodies, robust levels of virus neutralising titres (VNTs) were detectable after the second vaccination in the 8 µg group ( Figure 1D ). VNTs peaked on d42 at median titres of 2.7 x 10 4 . Neutralising antibody titres remained relatively unchanged upon challenge until day 62, 63, and 64 of the experiment. Animals in the 0.5 µg and unvaccinated control groups remained negative before challenge, while SARS-CoV-2 infection induced small increases in antibody titres in 4/6 and 5/6 animals in the 0.5 µg and unvaccinated group, respectively. In order to assess CVnCoV induced cellular responses, peripheral blood mononuclear cells values of 2.9 x 10 6 cp/ml in nasal swabs, respectively, were detectable on d59. However, the difference between the study groups was not statistically significant. Comparable results were generated in throat swabs (Supplementary Figure 1A) . RNA levels in BAL were below the lower limit of quantification for all but one animal in the 8 µg CVnCoV group on d59, which featured low RNA counts. Total viral RNA levels in 0.5 µg CVnCoV vaccinated animals were comparable to the control group. Of note, BAL analyses on d59 only depict female animals and one male animal of the unvaccinated group. The remaining animals were excluded from this analysis since suboptimal BAL sampling conditions prevented further evaluation. The analysis of lung tissue collected at necropsy confirmed results gained in BAL samples. Median titres of 2.9 x 10 8 cp/g were detectable in the unvaccinated group while all animals in the CVnCoV 8 µg vaccinated groups remained below the lower limit of quantification ( Figure 3C ). There was no statistically significant difference between animals in the 0.5 µg CVnCoV and the unvaccinated group. Subgenomic viral RNA analysis in BAL and lung tissue samples yielded comparable results: RNA indicative of replicating virus was detectable in BAL and lung samples of unvaccinated and 0.5 µg CVnCoV vaccinated animals on d59 and d62-d64, respectively. All animals in the 8 µg CVnCoV group were negative in these analyses ( Figure 3E and F). Evaluation of further tissue samples collected at necropsy revealed low but detectable signals of SARS-CoV-2 total RNA in trachea and tonsils of 0.5 µg CVnCoV and unvaccinated animals, Histopathological analyses of lung samples taken at necropsy showed lesions consistent with infection with SARS-CoV-2 in the lungs of challenged animals ( Figure 4 ). Briefly, the lung parenchyma showed multifocal to coalescing areas of pneumonia surrounded by unaffected parenchyma. Alveolar damage, with necrosis of pneumocytes was a prominent feature in the affected areas. The alveolar spaces within these areas were often thickened. Damaged alveolar walls contained mixed inflammatory cells including macrophages, lymphocytes, viable and degenerated neutrophils, and occasional eosinophils. Alveolar oedema and alveolar type II pneumocyte hyperplasia were also observed. In distal bronchioles and bronchiolo-alveolar junctions, degeneration and sloughing of epithelial cells were present. In the respiratory epithelium of larger airways, occasional focal, epithelial degeneration, and sloughing were observed. Low numbers of mixed inflammatory cells, comprising neutrophils, lymphoid cells, and occasional eosinophils, infiltrated bronchial and bronchiolar walls. In the lumen of some airways, mucus admixed with degenerated cells, mainly neutrophils and epithelial cells, was seen. Within the parenchyma, perivascular and peribronchiolar cuffing were also observed, with mostly lymphoid cells comprising the infiltrates. No remarkable changes were observed in non-pulmonary tissues. In agreement with reduced levels of viral RNA, the evaluation of lung samples using a histopathology scoring system showed a significant reduction in severity of lung lesions in CVnCoV vaccinated animals compared to 0.5 µg CVnCoV vaccinated and unvaccinated groups ( Figure 5A Figure 5D ). Of note, highest scores were seen in the 0.5 µg CVnCoV group in this analysis. However, values were not statistically different to the control group. Our data demonstrate that CVnCoV is safe and highly immunogenic in rhesus macaques. Vaccination with 8 µg CVnCoV elicited robust humoral responses, known to correlate with protection in this model as RBD-specific monoclonal antibodies 31 and purified IgGs from convalescent animals 32 are able to protect naïve rhesus macaques from challenge infection. Here we show the induction of high levels of S and RBD specific binding and virus neutralising antibodies upon two 8 µg injections of CVnCoV. While a sub-optimal dose of 0.5 µg was unable to elicit detectable levels of antibodies before challenge, infected animals raised specific antibody responses faster than the unvaccinated control group, indicative of a previously induced priming response in these animals. These data extend our preclinical finding in mice and hamsters, that elicited robust, dose-dependent humoral immune responses upon vaccination with CVnCoV using comparable or lower doses of CVnCoV 5 Another cause of disease enhancement may be vaccine-associated enhanced respiratory disease (VAERD) that is hallmarked by increased inflammation due to Th2-biased immune responses and high ratios of non-neutralising to neutralising antibodies (reviewed in 43 41 44 ). CVnCoV and gave no indication for increased inflammation and pathological changes in suboptimally dosed animals. Results presented here extend our knowledge of CVnCoV safety, immunogenicity and protective efficacy in a highly relevant model system for SARS-CoV-2. The overall outcome of the study in non-human primates in terms of immunogenicity, protective efficacy, and pathology are comparable to results in the hamster model, providing support for hamsters as a model system for SARS-CoV. We conclude that CVnCoV is highly efficacious at a low dose of 8 µg in a COVID-19 NHP challenge model while being safe at both doses tested with lack of any indication of disease enhancement. In alignment with the phase I interim safety and immunogenicity data of CVnCoV 28 , these results provide strong support for further evaluation in the ongoing phase 2b/3 HERALD clinical trial. The mRNA vaccine is based on the RNActive® platform (claimed and described in e.g. Rhesus macaques (Macaca mulatta), Indian origin, were obtained from a UK Home Office approved colony. In total, this study included 18 animals (9 male, 9 female) with a weight of > 4.5 kg and an age of 3-6 years. The challenge agent used in this study was SARS-CoV-2 virus, VERO/hSLAM cell passage 3 (Victoria/1/2020) 45 , titre 2.4 x 10 7 PFU/ml, passaged from material (P1) generously provided to PHE by The Doherty Institute, Melbourne, Australia. All animals were challenged with a dose of 5.0 x 10 6 PFU of SARS-CoV-2 by applying 2 ml of virus preparation to the pre-carinal section of the trachea using a bronchoscope followed by 1 ml applied intranasally (0.5 ml/nostril). Two animals of each group were followed for 6, 7 or 8 days post challenge (p.c.) and euthanised on day 62, 63 or 63 of the experiment. Virus neutralising titres were measured in heat-inactivated serum samples (56°C for 30 min). In-life BAL washes were performed using 10 ml PBS using a bronchioscope inserted to the right side of the lung above the second bifurcation. BAL washes performed post-mortem were conducted on the right lung lobes, after ligation of the left primary bronchus using 20 ml PBS. RNA was isolated from nasal swab, throat swabs, EDTA treated whole blood, BAL and tissue samples (spleen, kidney, liver, colon, duodenum, tonsil, trachea and lung). For tissue samples this equates to an LLOQ of 5.71x10 4 copies/g and LLOD of 5.14 x 10 3 copies/g. Tissue samples from left cranial and caudal lung lobes, trachea, larynx, mediastinal lymph node, tonsil, heart, thymus, pancreas, spleen, liver, kidney, duodenum, colon, brain, vaccinating site (skin including subcutis and underlying muscle) and draining lymph node (left and right) were fixed in 10% neutral-buffered formalin and embedded into paraffin wax. 4 µm thick sections were cut and stained with haematoxylin and eosin (HE). Tissue slides were scanned and examined independently by two veterinary pathologists blinded to the treatment and group details. For the lung, three sections from each left lung lobe were sampled from different locations: proximal, medial and distal to the primary lobar bronchus. A scoring system 30 CT scans were collected from sedated animals using a 16 slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI, USA) in the prone and supine position. All axial scans were performed at 120KVp, with Auto mA (ranging between 10 and 120) and were acquired using a small scan field of view. Rotation speed was 0.8s. Images were displayed as an 11cm field of view. To WHO. 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