key: cord-0692842-hpe536gl
authors: Li, Li-Hsin; Liesenborghs, Laurens; Wang, Lanjiao; Lox, Marleen; Yakass, Michael Bright; Jansen, Sander; Rosales Rosas, Ana Lucia; Zhang, Xin; Thibaut, Hendrik Jan; Teuwen, Dirk; Neyts, Johan; Delang, Leen; Dallmeier, Kai
title: Biodistribution and Environmental Safety of a Live-attenuated YF17D-vectored SARS-CoV-2 Vaccine Candidate
date: 2022-03-16
journal: Mol Ther Methods Clin Dev
DOI: 10.1016/j.omtm.2022.03.010
sha: d152d76541814c71c4abbe45825dd60ea57d5ec8
doc_id: 692842
cord_uid: hpe536gl

New platforms are needed for the design of novel prophylactic vaccines and advanced immune therapies. Live-attenuated yellow fever vaccine YF17D serves as vector for several licensed vaccines and platform for novel candidates. Based on YF17D, we developed an exceptionally potent COVID-19 vaccine candidate called YF-S0. However, use of such live RNA viruses raises safety concerns, i.e., adverse events linked to original YF17D (yellow fever vaccine-associated neurotropic; YEL-AND, and viscerotropic disease; YEL-AVD). In this study, we investigated the biodistribution and shedding of YF-S0 in hamsters. Likewise, we introduced hamsters deficient in Signal Transducer and Activator of Transcription 2 (STAT2) signaling as new preclinical model of YEL-AND/AVD. Compared to YF17D, YF-S0 showed an improved safety with limited dissemination to brain and visceral tissues, absent or low viremia, and no shedding of infectious virus. Considering yellow fever virus is transmitted by Aedes mosquitoes, any inadvertent exposure to the live recombinant vector via mosquito bites is to be excluded. The transmission risk of YF-S0 was hence compared to readily transmitting YF-Asibi strain and non-transmitting YF17D vaccine, with no evidence for productive infection of mosquitoes. The overall favorable safety profile of YF-S0 is expected to translate to other vaccines based on the same YF17D platform.

Unfortunately, long-term effectiveness of current SARS-CoV-2 vaccines is waning due to the 56 combined effect of (i) a rapid decay of virus-neutralizing antibodies (nAb) over time and (ii) 57 emergence of new variants escaping vaccine-induced immunity. 7-9 Furthermore, several first-58 generation COVID-19 vaccines have a rather high reactogenicity. With the growing number of 59 vaccinated people, more cases and a wider spectrum of adverse effects following immunization 60 (AEFI), including severe adverse effects (SAE) such as myocarditis or life-threatening deep-61 venous thrombosis are described. [10] [11] [12] [13] [14] [15] In summary, there is an urgent to develop new and 62 improved second-generation COVID-19 vaccines to quench the pandemic. 63

Recently, we used an alternative vaccine platform that uses the fully replication competent live-64 attenuated yellow fever vaccine YF17D as vector 16 

CoV-2 vaccine candidate (YF-S0) that expresses a stabilized prefusion form of SARS-CoV-2 66 spike protein (S0). 17 YF-S0 was shown to induce vigorous humoral and cellular immune 67 responses in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques 68 (Macaca fascicularis) and was able to prevent COVID-19-like disease after single-dose 69 vaccination in a stringent hamster model. Due to its YF17D backbone, YF-S0 could serve as 70 dual vaccine to also prevent yellow fever virus (YFV) infections, which should provide an 71 added benefit for populations living in regions at risk of YFV outbreaks. 18 72

In addition to preclinical efficacy, development of such a new vaccine requires in-depth 73 evaluations of its safety to support progression from preclinical study to clinical trials. In 74 particular for live-attenuated viral vaccines such as YF-S0, the biodistribution of the vaccine 75 virus after administration needs to be assessed 19 to understand the viral organ tropism and hence 76 J o u r n a l P r e -p r o o f to exclude potential direct harm to specific tissues. Our vaccine candidate YF-S0 showed an 77 excellent safety profile in multiple preclinical models, including in NHP as well as in interferon-78 deficient mice and hamsters. 17 However, use of such a recombinant YF17D vaccine entails 79 some potential concerns. 19 Particularly, replication and persistence of YF-S0 in tissues and 80 body fluids poses a theoretical risk of YF vaccine-associated viscerotropic disease (YEL-AVD) 81 and YF vaccine-associated neurotropic disease (YEL-AND), which are originally linked to 82 parental YF17D. 20 Furthermore, YFV is a mosquito-borne virus, and YF-S0 employs YFV-derived YF17D as a 91

vector. Recombinant YF-S0 might hence, though highly attenuated, pose an environmental risk 92 due to unforeseen phenotypical changes. Taking this theoretical consideration into account, we 93 tested the infectivity of YF-S0 on Aedes aegypti (Ae. aegypti) mosquitoes to assess its 94 transmission potential. Ae. aegypti was selected as target mosquito species because of its well-95 known high vector competence for YFV. 21 It is well documented that wild-type YF-Asibi can 96 infect and disseminate in Ae. aegypti while YF17D only occasionally infects the midgut and is 97 unable to disseminate to secondary organs. 22,23 Therefore, these two YFV strains were used as 98 controls to assess transmission of YF-S0 by a competent vector. 99

Finally, we corroborate the favorable safety profile of YF-S0 by reporting limited dissemination 100 and shedding in vaccinated hamsters, nor any risk of mosquito-borne transmission. 101 102 J o u r n a l P r e -p r o o f

Tissue distribution of YF-S0 and parental YF17D in hamsters 104

For our assessment, we chose wild-type (WT) Syrian golden hamsters as preferred small animal 105 model of YFV infection 24 and injected them with a high dose (10 4 PFU) of either YF17D (n=6) 106 or YF-S0 (n=6) via intraperitoneal (i.p.) route to achieve maximal exposure; with primary 107 pharmacodynamics documented before 17 and confirmed here by consistently high 108 seroconversion rates (at least 80%) to YFV-specific nAb (Fig. S1 ). Likewise, a 10 4 PFU i.p. 109 dose had been shown to elicit saturating levels of SARS-CoV-2 nAb in the hamster model, 110 similar to a two-dose regiment using a 10-fold lower inoculum. 17 As methods control, we 111 inoculated STAT2 knockout (STAT2 -/-) hamsters with 10 4 PFU of YF17D (n=2). STAT2 -/-112 hamsters are deficient in antiviral type I and type III interferon responses 25 and therefore prone 113 to uncontrolled flavivirus replication. 26 Tissues sampled for analysis were chosen based on 114 biodistribution data available from non-human primates and humans. In macaques, detection 115 of YF17D RNA has been reported in lymph nodes, spleen and liver at 7 days post subcutaneous 116 inoculation. 27 Likewise, viral RNA is widespread and abundantly found in spleen, liver, brain, 117 kidney, and other organs in patients who developed YEL-AVD. 20,28 Based on this knowledge, 118 we collected spleen, liver, brain, and kidney as most common target organs to assess the risks 119 for YEL-AVD and YEL-AND. Ileum and parotid gland were collected as additional excretory 120 tissues, and lung as main target of COVID-19 (Fig. 1A) . From our previous experience, 17 we 121 observed that the replication of YF17D or YF-S0 is transient and well tolerated in WT hamsters. 122

Tissue analysis in hamsters was thus performed 7 days post inoculation (dpi), i.e., few days 123 after peak of viremia, in line with similar studies performed for chimeric YF17D vaccine in 124 macaques before 27 and at a timepoint at which STAT2 -/hamsters needed to be euthanized for 125 humane reasons. 17 126 Viral RNA above detection limits in YF17D vaccinated WT hamsters was mostly limited to 127 spleen (RNA detected in 4 out of 6 animals, 4/6), with exception of a single hamster in which 128 viral RNA was widespread to brain, parotid gland, and lung ( Fig. 1B and Suppl Table 1 ). 129

Detection of YF-S0 was markedly less frequent and restricted to only kidney (2/6) and lung 130

(1/6) (Fig. 1B) . Overall, in either group RNA level was low and barely detectable by sensitive 131 RT-qPCR, indicative for limited replication in WT hamsters. In contrast, unrestricted 132 replication of virus to high viral loads was observed in STAT2 -/hamsters ( Fig. 1B and Fig.  133 1C). Importantly, no viral RNA nor infectious virus could be detected in brains of YF-S0 134 vaccinated hamsters, suggesting a low associated YEL-AND risk ( Fig. 1D and Fig. 1E ). 135 J o u r n a l P r e -p r o o f Viremia is considered a key indicator for the risk of developing YEL-AVD. Thus, longitudinal 136 blood sampling was conducted as Fig. 2A . Kinetics of viral RNA in serum as proxy for viremia 137 have been reported earlier for WT hamsters vaccinated with YF17D or YF-S0 17 and are 138 discussed here in comparison to respective data from STAT2 -/controls (Fig. 2B ). Viremia can 139 be detected consistently in all YF17D vaccinated WT hamsters (6/6) starting at 1 dpi and lasting 140 for 2.5 (1-4) days in median (95% confidence interval); by contrast, viral RNA was detected 141 only once at 3 dpi in a single YF-S0 vaccinated hamster (1/6) ( Fig. 2B and Suppl. Table 2 ). In 142 STAT2 -/hamsters, YF17D grew unrestrictedly to markedly increased viral RNA levels ( Experimental feeding was equally efficient for all three virus groups regarding both viral RNA 178 and infectious virus recovered (Fig. 3B&C ). However, 14 days after feeding, viral RNA was 179 detected exclusively in specimens from the YF17D group (8/15) and YF-Asibi group (8/23); 180 yet none from the YF-S0 group. Importantly, infectious viral particles were only detectable in 181 the YF-Asibi group, with virus loads as high as about 10 6 TCID50/body on average (Fig. 3C) . 182

For dissemination beyond the MEB, the remaining head, legs and wings of each six virus-183 positive mosquitoes with highest body virus loads from the YF17D and YF-Asibi groups, 184 respectively, and six randomly chosen specimens from the YF-S0 group were evaluated. All 185 these specimens from the YF-Asibi group (6/6) scored positive for dissemination, while none 186 from the YF-S0 or YF17D groups (Fig. 3B&C ). These results suggest that YF-S0 is neither 187 able to pass the MIB for midgut infection, nor to escape from the midgut (MEB) for 188 dissemination ( The live-attenuated YF17D vaccine is considered as one of the most powerful and successful 192 vaccines and has been used on humans for decades. 33 Its well-known characteristics of 193 stimulating both vigorous humoral and cellular immune responses, as well as favorable innate 194 responses is of interest for other vaccine targets using the YF17D genome as a backbone. 16 To temper the safety concerns, the viscerotropism and neurovirulence of YF-S0 was compared 217 head-to-head with parental YF17D virus by investigating the biodistribution and viremia 218 following administration of either vaccine virus in hamsters. We demonstrate that parental 219 YF17D can spread systemically and viral RNA can be detected in spleen, brain, parotid gland, 220 and lung in YF17D vaccinated WT hamsters. However, replication of YF17D remains 221 restricted, resulting in infectious virus loads below detection limits. Compared to YF17D, 222 J o u r n a l P r e -p r o o f detection of YF-S0 was further limited, with minute amounts of viral RNA in kidney and lung. 223

Unrestricted virus replication to high viral loads as cause of viscerotropic or neurotropic disease 224 was observed only in STAT2 -/hamsters, in line with the essential role innate interferon 225 signaling plays in live vaccines 30,46 and control of viral infections in general. 47 In addition, in 226 YF-S0 vaccinated WT hamsters, detection of viremia was rare (Fig. 2B) and importantly, less 227 frequent (1/6) and markedly lower in magnitude (AUC) and duration (1 day) compared to 228 parental YF17D (6/6 for >2 days). A limitation of our study may be the relatively low number 229 of animals enrolled per group and the finite number of time points selected for analysis. 230

However, taken together, the overall limited tissue distribution of YF-S0 as well as the low 231 abundance of its RNA in blood, below detection limits for infectious virus, suggest a further 232 lowered risk of YEL-AVD/AND for YF-S0 than that reported parental YF17D. It is thus to be WT hamsters (6-8 weeks old, female) were inoculated intraperitoneally with 10 4 PFU/mL dose 294 of YF17D (n = 6) or YF-S0 (n = 6). STAT2 -/hamster (6-8 weeks old, female) were inoculated 295 intraperitoneally with 10 4 PFU/mL of YF17D (n = 2). At 7 dpi, blood, spleen, liver, brain, 296 kidney, ileum, parotid gland, and lung were collected. 297

Shedding 298 WT hamsters (6-8 weeks old, female) were inoculated intraperitoneally with 10 4 PFU/mL of 299 YF17D (n = 6) or YF-S0 (n = 6). STAT2 -/hamsters (6-8 weeks old, male) were inoculated 300 intraperitoneally with 10 4 PFU/mL of YF17D (n = 3). Blood, urine, faces, and buccal swab 301 were collected daily for the first 5 dpi, then every other day until 11 dpi and 15, 22 (except for 302 the blood) and 29 dpi, and afterwards once a week until 29 dpi. 303

Ae.

aegypti Paea 51 were obtained via the Infravec2 consortium 306 (https://infravec2.eu/product/live-eggs-or-adult-females-of-aedes-aegypti-strain-paea-2/) 307 from Institute Pasteur of Paris. Mosquitoes were maintained at the insectary of Rega Institute, 308 and the fourth generation was used for this study. In brief, larvae were fed with yeast tablets 309 (Gayelord Hauser, France) until the pupae stage prior to transfer to cages for emergence. Adults 310 were maintained with cotton soaked in 10% sucrose solution under standard conditions (28˚C, 311 80% relative humidity, and 14h:10h light/dark cycle). 312

7-day-old female mosquitoes were starved 24 h prior to infection. Infectious blood meals 314 contained rabbit erythrocytes plus 5 mM adenosine triphosphate as phagostimulant, 315 supplemented with virus stocks to final titers of 2x10 5 PFU/mL for both YF17D and YF-S0, 316 and 5x10 6 PFU/mL for YF-Asibi, respectively. After 45 minutes, 5 full engorged females from 317 each group were frozen for viral input assessment (ingestion check, Fig. 3A) , and the rest kept 318 J o u r n a l P r e -p r o o f with 10% of sugar solution under both controlled conditions (28 ± 1°C, relative humidity of 319 80%, light/dark cycle of 14h/10h, supplied with 10% sucrose solution) and BSL-3 containment 320 conditions. At 14 dpi, mosquitoes were dissected into two parts; main body (thorax and 321 abdomen) and remainder, collected individually in tubes containing PBS and 2.8 mm ceramic 322 beads (Precellys). The samples were homogenized and pass through 0.8µm column filters 323 (Sartorius, Germany). Thus, cleared supernatants were used for TCID50 assay or keep at -80°C 324 for RNA extraction and subsequent RT-qPCR analysis. 325

Solid tissues (organs), faeces and buccal swabs were homogenized in a bead mill (Precellys) in 327 lysis buffer (Macherey-Nagel; cat no. 740984.10). After homogenization, samples were 328 centrifuged at 10,000 rpm for 5 min to remove cell debris, and total RNA was extracted by 329 

RT-qPCR for YFV detection was performed as previously described 17 using primers and probe 334 targeting the YFV NS3 gene 23 on an ABI 7500 Fast Real-Time PCR System (Applied 335 Biosystems). Absolute quantification was based on standard curves generated from 5-fold serial 336 dilutions of YF17D cDNA with a known concentration. 337

For virus isolation and quantification BHK21 cells were infected with 10-fold serial dilutions 339 in 96-well plates, and incubated at 37˚C for 6 days using DMEM with 2% fetal bovine serum 340 (Hyclone), 2 mM L-glutamine (Gibco), 1% sodium bicarbonate (Gibco), and 1% antibiotics 341 (PenStrep) as assay medium. Solid tissues were homogenized in a bead mill (Precellys) in assay 342 medium, and centrifuged at 10,000 rpm for 5 min (4˚C) to remove debris. Resulting viral titers 343 were calculated by the Reed and Muench method. 344

Titers of YFV-specific neutralizing antibodies were determined using BHK21 cells and a 346 mCherry-tagged variant of YF17D virus (YFV-mCherry) as described. 17 Rafferty, E., Duclos, P., Yactayo, S., and Schuster, M. (2013) . Risk of yellow fever 538 vaccine-associated viscerotropic disease among the elderly: a systematic review. 539

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