key: cord-0334244-z1c0dvkb
authors: Hicks, Philip; Westover, Jonna B.; Manzoni, Tomaz B.; Roper, Brianne; Rock, Gabrielle L.; Boardman, Kirsten M.; Blotbter, Dallan J.; Gowen, Brian B.; Bates, Paul
title: Safety, Immunogenicity and Efficacy of a Recombinant Vesicular Stomatitis Virus Vectored Vaccine Against Severe Fever with Thrombocytopenia Syndrome Virus and Heartland Bandaviruses
date: 2021-11-30
journal: bioRxiv
DOI: 10.1101/2021.11.29.470508
sha: 4e36d29cb967d1bde0f5648f4a59caf8e1698bc2
doc_id: 334244
cord_uid: z1c0dvkb

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a recently emerged tickborne virus in east Asia with over 8,000 confirmed cases. With a high case fatality ratio, SFTSV has been designated a high priority pathogen by the WHO and the NIAID. Despite this, there are currently no approved therapies or vaccines to treat or prevent SFTS. Vesicular stomatitis virus (VSV) represents an FDA-approved vaccine platform that has been considered for numerous viruses due to its low sero-prevalence in humans, ease in genetic manipulation and promiscuity in incorporating foreign glycoproteins into its virions. In this study, we developed a recombinant VSV (rVSV) expressing the SFTSV glycoproteins Gn/Gc (rVSV-SFTSV) and assessed its safety, immunogenicity and efficacy in mice. We demonstrate that rVSV-SFTSV is safe when given to immunocompromised animals and is not neuropathogenic when injected intracranially into young immunocompetent mice. Immunization of Ifnar-/- mice with rVSV-SFTSV resulted in high levels of neutralizing antibodies and protection against lethal SFTSV challenge. Additionally, passive transfer of sera from immunized Ifnar-/- mice into naïve animals was protective when given pre- or post-exposure. Finally, we demonstrate that immunization with rVSV-SFTSV cross protects mice against challenge with the closely related Heartland virus despite low neutralizing titers to the virus. Taken together, these data suggest that rVSV-SFTSV is a promising vaccine candidate. Importance Tick borne diseases are a growing threat to human health. Severe fever with thrombocytopenia syndrome (SFTS) and Heartland viruses are recently recognized, highly-pathogenic, tick-transmitted viruses. The fatality rates for individuals infected with SFTSV or HRTV are high and there are no therapeutics or vaccines available. The recent introduction of the tick vector for SFTSV (Haemaphysalis longicornis) to the eastern half of the United States and Austrailia raises concerns for SFTSV outbreaks outside East Asia. Here we report the development of a potential vaccine for SFTSV and HRTV based on the viral vector platform that has been successfully used for an Ebola vaccine. We demonstrate that the rVSV-SFTSV protects from lethal SFTSV or HRTV challenge when given as a single dose. We evaluated possible pathogenic effects of the vaccine and show that it is safe in immune compromised animlas and when introduced into the central nervous system.

Abstract (213 words) 25 Severe fever with thrombocytopenia syndrome virus (SFTSV) is a recently emerged tickborne 26 virus in east Asia with over 8,000 confirmed cases. With a high case fatality ratio, SFTSV has 27 been designated a high priority pathogen by the WHO and the NIAID. Despite this, there are 28 currently no approved therapies or vaccines to treat or prevent SFTS. Vesicular stomatitis virus 29 (VSV) represents an FDA-approved vaccine platform that has been considered for numerous 30 viruses due to its low sero-prevalence in humans, ease in genetic manipulation and promiscuity 31 in incorporating foreign glycoproteins into its virions. In this study, we developed a recombinant 32 VSV (rVSV) expressing the SFTSV glycoproteins Gn/Gc (rVSV-SFTSV) and assessed its 33 safety, immunogenicity and efficacy in mice. We demonstrate that rVSV-SFTSV is safe when 34 given to immunocompromised animals and is not neuropathogenic when injected intracranially 35 into young immunocompetent mice. Immunization of Ifnar -/mice with rVSV-SFTSV resulted in 36 high levels of neutralizing antibodies and protection against lethal SFTSV challenge. 37 Additionally, passive transfer of sera from immunized Ifnar -/mice into naïve animals was 38 protective when given pre-or post-exposure. Finally, we demonstrate that immunization with 39 rVSV-SFTSV cross protects mice against challenge with the closely related Heartland virus 40 despite low neutralizing titers to the virus. Taken together, these data suggest that rVSV-SFTSV 41 is a promising vaccine candidate. 42 43 Importance (146 words) 44

Tick borne diseases are a growing threat to human health. Severe fever with thrombocytopenia 45 Introduction aged ferrets, Stat2 -/hamsters, and Ifnar -/mice (28, (31) (32) (33) (34) . Despite these difficulties, several 102 groups have designed and tested SFTSV vaccines, primarily using Ifnar -/mice. Tested vaccine 103 platforms include DNA, virus-vectored, and attenuated recombinant SFTSV vaccines (35) (36) (37) (38) (39) . 104 These vaccines vary in their effectiveness and come with drawbacks. Here, we focus on 105 developing and characterizing a recombinant vesicular stomatitis virus (rVSV) vaccine.

The livestock pathogen vesicular stomatitis virus (VSV) is generally non-pathogenic to humans 108 and of low sero-prevalence (40, 41) . Additionally, VSV is a powerful vaccine platform with 109 genetically tractable models and a promiscuity to incorporate foreign glycoproteins in the virion 110 (42) . An often cited detriment of rVSV vaccines is the propensity for VSV to be neurotropic. It 111 is, however, known that neuropathogenicity is conferred by the tropism of the viral glycoprotein 112 (43, 44) . Currently, the rVSV vaccine platform is approved for use against Ebola virus (EBOV) 113 and has been successfully distributed in Africa during recent EBOV outbreaks (45, 46) . Due to 114 the proven nature of the rVSV platform, we made an rVSV-SFTSV virus containing the SFTSV 115 Gn/Gc glycoprotein in place of the cognate VSV glycoprotein VSV-G.

It has been previously reported by another group that rVSV-SFTSV confers protective immunity 118 to Ifnar -/mice (38) . To go beyond what has been previously shown, we demonstrate that our 119 rVSV-SFTSV is non-neurotropic and safe in immunocompromised animals. We also show that a 120 single administration of vaccine virus is sufficient to induce protection against SFTSV challenge.

Additionally, rVSV-SFTSV vaccintion induces high levels of antibodies in wild-type animals 122 suggesting it can effectively be used in immune competent animals. Both therapeutic and 123 prophylactic passive transfer of sera from immunized animals leads to protection upon challenge 124 of unvaccinated animals suggesting antibodies correlate with protection against SFTSV. Finally, 125 we demonstrate that our rVSV-SFTSV vaccine is cross-protective upon lethal HRTV challenge.

Single vaccination with rVSV-SFTSV induces high levels of neutralizing antibodies 166 To functionally characterize humoral responses to rVSV-SFTSV vaccination we assessed Increasing the vaccination dose increased rates of seroconversion and neutralization titers (Fig.   173 2A). These titers are promising given thay previous work on influenza and SARS-CoV-2 suggest 174 that neutralizing titers of 40-80 are sufficient for protection (53, 54) .

The high levels of neutralizing antibodies achieved with vaccination of Ifnar -/mice was 177 somewhat surprising as interferons (IFN)s are important drivers of immune responses. To 178 determine whether mice deficient in both type I and type II IFN receptors also elicit high levels 179 of neutralizing antibodies, we immunized AG129 (IFN-α/β and γ receptor-deficient) mice with 180 10 1 -10 4 PFU of rVSV-SFTSV. Notably, 2 of 4 mice immunized with 10 4 PFU rVSV-SFTSV 181 succumbed to viral infection (Fig. 2B ). Mice receiving 10 PFU rVSV-SFTSV failed to generate a 182 neutralizing antibody response (Fig. 2C ). Animals receiving higher doses had mean neutralizing 183 titers ranging from 60 to 240 with increasing dosage (Fig. 2C) . These results demonstrate that 184 rVSV-SFTSV elicits humoral responses even in highly immunocompromised animals lacking 185 type I and II IFN responses.

It is well documented that VSV infection is highly sensitive to IFN responses (55, 56) . To 188 determine whether rVSV-SFTSV can induce a neutralizing antibody response in 189 immunocompetent mice, we immunized C57BL/6 mice with 10 4 , 10 5 , or 10 6 PFU of rVSV-190 SFTSV. Dosages were increased relative to Ifnar -/mice to account for IFN responses interfering 191 with rVSV-SFTSV replication and thus reducing the humoral immune response in the immune 192 competent mice. In contrast to what was seen with the immune deficient mice, no weight loss 193 was observed in the C57BL/6 mice at any vaccine dose (data not shown). Additionally, despite 194 the increased dosages, neutralizing titers were far lower than those observed in Ifnar -/mice 195 suggesting that rVSV-SFTSV is sensitive to IFN, consistent with previous reports (Fig. 2D) . A 196 dosage dependent increase in FRNT50 titers was observed with mice receiving 10 6 PFU rVSV-197 SFTSV achieving a mean titer of 113 (Fig. 2D) . Notably, all mice immunized with 10 6 PFU 198 rVSV-SFTSV sero-converted (Fig. 2D) . These data indicate that despite VSV's sensitivity to Because of the high neutralizing antibody titers measured in Ifnar -/mice vaccinated with rVSV-205 SFTSV, we hypothesized that the vaccine would protect these mice against lethal SFTSV 206 challenge. To test this hypothesis, vaccinated mice were challenged subcutaneously with 10 PFU 207 of SFTSV 23 days post-vaccination (2 days after blood collection for neutralizing antibody 208 titration). A single group of unvaccinated mice received 8 days of 100 mg/kg/day of favipiravir 209 therapy following SFTSV challenge as a positive control for protection (57) . As expected, all 210 mice vaccinated with PBS succumbed by 8 dpi (Fig. 3A) . In contrast, only 60% of mice 211 vaccinated with 10 2 PFU, and all Ifnar -/mice vaccinated with at least 10 3 PFU, survived the 212 lethal SFTSV challenge. Mice vaccinated with at least 10 3 PFU were protected from weight loss 213 following SFTSV challenge while rapid weight loss was observed in PBS-vaccinated mice 214 beginning by 3 dpi (Fig. 3B ). Mild weight loss occurred post-challenge in mice that received 10 2

PFU of vaccine, but this trend was driven primarily by the three individuals that succumbed to 216 disease. Vaccination-associated weight loss was also observed in this experiment and was 217 consistent in magnitude to that shown previously (Fig. 1E ).

To assess the effect rVSV-SFTSV vaccination has on SFTSV viremia and tissue viral loads, 219 groups of 4 mice were vaccinated and challenged in, parallel following the timeline described 220 above. These subsets of mice were sacrificed 5 days following SFTSV challenge and serum, 221 liver, spleen, and kidney were collected for SFTSV quantification by endpoint titration on Vero 222 E6 cells. All groups of vaccinated mice had significantly reduced SFTSV serum and tissue viral 223 titers (Fig. 3C ). In the liver and kidney, there was a trend towards dose-dependence with mice 224 vaccinated with the highest dose of rVSV-SFTSV having the lowest viral titers. Favipiravir 225 treatment also reduced SFTSV titers compared to mice vaccinated with PBS. These data 226 demonstrate that rVSV-SFTSV does not provide sterilizing immunity to SFTSV challenge but 227 rather reduces replication in the vaccinated animals. 

SFTSV is an emerging public health threat in southeast Asia with case fatality rates ranging from 286 4 to 30%. This high variability in case fatality rates may reflect access to health care, the genetic 287 background of infected populations, and the virulence of the infecting SFTSV strain (58) (59) (60) . 288 Given that no therapeutics or vaccines are available to curtail or prevent an outbreak of SFTS 289 and that the virus is transmitted by multiple tick species with expanding geographic ranges, the 290 threat SFTSV poses to public health is significant (18, 61) . This has caused the WHO to list 291 SFTSV in its prioritized pathogen research blueprint and the NIAID to include it as a category C 292 priority pathogen (26, 27) . In response to the potential threat from SFTSV, many SFTSV 293 vaccines are being developed using a variety of different technologies including protein subunit, 294 DNA, and recombinant viral platforms (35, 36, 38, 39, 62, 63) . responses make to protection from SFTSV challenge. Unfortunately, the age-related changes that render ferrets susceptible to lethal SFTSV challenge are unknown. This makes it difficult to 330 directly compare results obtained from aged ferrets to other SFTSV animal models.

The only currently FDA-approved rVSV vaccine, rVSV-EBOV, is highly pathogenic and lethal 333 in Ifnar -/mice. In contrast, rVSV-SFTSV only caused mild-to-moderate weight loss at doses 334 that elicited protective immunity. Unlike the parental VSV vector, rVSV-SFTSV did not cause 335 neurologic disease when injected intracranially into 4-week-old C57BL/6 mice, suggesting that 336 this vaccine strain is not neurotropic. Despite these promising results, it is possible that rVSV- 

HRTV is an emerging bandavirus closely related to SFTSV that can cause lethal disease (71, 72) .

Previous studies have shown that sera raised by vaccination against SFTSV glycoproteins can 388 cross-neutralize viruses harboring HRTV glycoproteins (38) . In addition, this same study showed 389 that a rVSV-HRTV protects Ifnar -/mice against lethal SFTSV infection. The present study is 390 the first to report that rVSV-SFTSV protects Ifnar -/mice against a lethal HRTV challenge. rVSV-EGFP and were diluted to a total injection volume of 10 μl with PBS. Mice we monitored 479 during anesthesia recovery until they were ambulatory. Mice were weighed daily and were 480 observed for neurologic signs. Neurologic signs were assigned a severity score ranging from 0-4.

Mice scored "0" showed no signs of illness and were bright, alert, and responsive when handled.

Mice scored a "1" showed mild signs of illness without clear signs of neurologic illness 483 including body hunching, depressed activity, or mild grimace. Mice assigned a "1" had normal 484 ambulation and responded normally to being handled. Mice assigned a "2" had clinical signs 485 consistent with mild encephalitis including hyperexcitability or altered gait that did not impair 486 linear locomotion and used all four limbs. Mice assigned a "3" had more severe neurologic signs 487 which included paraparesis of one or two limbs, mild head tilt, and altered gait that did impair 488 linear locomotion (such as spinning). Mice assigned a "4" had severe neurologic signs that were 489 inconsistent with life including complete pelvic limb paraplegia, ataxia, or tremors/seizures.

Mice scored with a "4" were humanely euthanized with CO2. In the other, 45ug of pCAG-SFTSV Gn/Gc expression plasmid was added, tubes were allowed to 506 sit for 5 minutes at room temperature. Lipofectamine and DNA containing tubes of optimum 507 were combined and gently mixed, after 20 minutes incubating at room temperature. Solution was 508 added to flask of 293T cells, after 4 hours cells were fed with fresh media. Thirty hours after 509 transfection, the SFTSV Gn/Gc expressing cells were infected for 2-4 hours with VSV-G 510 pseudotyped VSVΔG-mNeon at an MOI of ~1-3 (Generated by deleting the cognate VSV-G and 511 linking mNeon to the n-terminus of P. Virus was launched as previously described (12)). After serum. The focus reduction neutralization titer 50% (FRNT50) was measured as the greatest 526 serum dilution at which focus count was reduced by at least 50% relative to control cells that 527 were infected with pseudotype virus in the absence of mouse serum. FRNT50 titers for each 528 sample were measured in two to three technical replicates performed on separate days.

Virus titer determination 531 Virus titers were assayed using an infectious cell culture assay as previously described (74) .

Briefly, a specific volume of tissue homogenate or serum was serially diluted and added to 533 triplicate wells of Vero E6 (African green monkey kidney) cell monolayers in 96-well microtiter 534 plates. The viral cytopathic effect (CPE) was determined 11 days after plating and the 50% 535 endpoints calculated as described (75) . The lower limits of detection were 1.67 log10 CCID50/ml 536 serum and 2.43-3.14 log10 CCID50/g tissue. In samples presenting with virus below the limits of 537 detection, a value representative of the limit of detection was assigned for statistical analysis. 

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