key: cord-0297552-kv7q67ef
authors: Stubbs, Sarah Hulsey; Pontelli, Marjorie Cornejo; Mishra, Nischay; Zhou, Changhong; de Paula Souza, Juliano; Mendes Viana, Rosa Maria; Lipkin, W. Ian; Knipe, David M.; Arruda, Eurico; Whelan, Sean P. J.
title: Vesicular stomatitis virus chimeras expressing the Oropouche virus glycoproteins elicit protective immune responses in mice
date: 2021-02-19
journal: bioRxiv
DOI: 10.1101/2021.02.19.432025
sha: d6a572f996d543d0d180a42eb04ab0bb9219359c
doc_id: 297552
cord_uid: kv7q67ef

Oropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins Gn and Gc responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized with VSV-OROV specifically neutralized wild type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild type OROV shows that the VSV-OROV chimeras reduce wild type viral infection and suggest that antibodies that recognize the variable N-terminus of Gc afford less protection than those that target more conserved regions of Gc. Importance Oropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wildtype virus. This data provides the first evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

Oropouche virus (OROV), first isolated in 1959 from a forest worker in Trinidad and 58 Tobago, causes a debilitating febrile illness in humans that can progress to meningitis or 59 encephalitis (1, 2). OROV is the most prevalent arbovirus after dengue in Brazil (3, 4) , and is 60 currently circulating in Argentina, Bolivia, Colombia, Ecuador, . In urban 61 areas across the Amazon region seroprevalence rates of up to 15-33%, suggest that OROV 62 infection is underappreciated (8, 9) . The virus infects a broad range of species and has both a 63 sylvatic and urban cycle. During the sylvatic cycle, the virus infects sloths, monkeys, rodents, and 64 birds, with Coquillettidia venezuelensis and Ochlerotatus serratus serving as vectors (1). In the 65 urban cycle, Culicoides paraensis and Culex quinquefasciatus serve as vectors of OROV (1), with 66 human infections paralleling increases in the vector population during the rainy season (10).

Infection is predominantly seen in individuals returning from forested areas (11) 

Oropouche virus, a member of the family Peribunyaviridae, contains a single-stranded, 73 negative-sense RNA genome divided on three segments providing a total genome size of 11, 985 74 nucleotides. The large segment (l) encodes the large protein (L) which acts as the RNA dependent 75 RNA polymerase, the medium segment (m) encodes the viral glycoprotein complex (GPC), and 76 the small segment (s) encodes the nucleocapsid protein that sheaths the genomic and 77 antigenomic RNA. Two non-structural proteins, NSs and NSm are coded by the small and medium 78 segments, respectively. The GPC is synthesized as a single polyprotein that undergoes co-79 translational cleavage by host-cell proteases into the two glycoproteins, Gn and Gc and liberates 80 the intervening NSm protein (14) . The Gc protein acts as the viral fusogen (15), and Gn mediates 81 attachment and shields and protects Gc from premature triggering (16) . Extrapolating from studies with La Crosse (17) (18) (19) (20) and Schamllenberg (16, 21) 

Building on this proven approach, we generate a VSV-chimera in which the native 96 glycoprotein gene is replaced by the GPC of OROV. The resulting VSV-OROV chimera replicates 97 to high-titers in BSRT7 cells in culture, and efficiently incorporates the Gn and Gc of OROV into 98 particles. Following single dose or prime-boost intramuscular injection of mice, VSV-OROV elicits 99 the production of immune specific sera that neutralize wild-type OROV. Using peptide arrays 100 corresponding to the OROV glycoproteins we find significant reactivity to the more variable N-101 terminal domain of Gc that precedes the domains that are structurally homologous to other class 102 II fusion proteins. We generate two additional VSV-OROV chimeras lacking portions of the 103 variable N-terminus of Gc, termed the "head" and "stalk" domain. Although both variants yielded 104 replication competent virus deletion of the stalk domain led to the accumulation of mutations in 105 Gc. Immunization of mice with the VSV-OROV lacking the head domain of Gc generated immune 106 sera reactive with new regions of Gc. Challenge studies demonstrate that immunization with VSV-

OROV or the chimera lacking the head domain of Gc offer protection against wild type OROV infection as evidenced by weight loss, temperature, and viral burden. This study demonstrates 109 that VSV-OROV immunization induces neutralizing serum responses that can protect mice 110 against challenge with wild type OROV.

Orthobunyavirus virions incorporate two viral glycoproteins on their surface, Gn and Gc 114 that mediate binding and entry into the cell. Gn and Gc are synthesized as part of a polyprotein 115 precursor GPC. We engineered an infectious molecular clone of VSV that expresses eGFP as a 116 marker of infection (39), to replace the native attachment and fusion glycoprotein with the GPC 117 polyprotein of OROV ( Figure 1A ). Using established procedures, we recovered a chimeric virus,

VSV-OROV, capable of autonomous replication as evidenced by plaque formation ( Figure 1A ).

Kinetic analysis of the yields of infectious VSV-OROV identify a 1 log reduction in viral growth 120 compared to VSV-eGFP ( Figure 1A ). Analysis of the protein composition of purified virions by 121 SDS-PAGE demonstrates that Gn and Gc are incorporated into VSV-OROV in place of VSV G 122 ( Figure 1B ). VSV-OROV particles retain the classic bullet shape of VSV as evidenced by 123 negative-stain electron microscopy ( Figure 1C ) and are visually decorated with spikes consistent 124 with incorporation of the OROV glycoproteins ( Figure 1C in a further reduction in viral yield, although the mechanism underlying this is unclear (40). Using 136 the infectious cDNA of VSV-OROV we engineered the analogous variants lacking the "head" domain of Gc (VSV-OROVΔ4) or the "head" and "stalk" domains (VSV-OROVΔ8). Autonomously 138 replicating viruses were recovered from both variants (Figure 2A) , although they exhibit a growth 139 defect in cell culture compared to VSV-OROV. Although growth of both truncated variants was 140 attenuated compared to VSV-OROV, VSV-OROVΔ4 reached comparable titers ( Figure 2A ).

Sequence analysis demonstrates that no other changes were present in the GPC gene of VSV-

OROV, or VSV-OROVΔ4, but each of the VSV-OROVΔ8 isolates contained additional mutations 143 in Gc ( Figure 2B ). This result demonstrates that as for BUNV, at least a portion of the N-terminus 

To examine the ability of the mouse sera to neutralize wild-type OROV we used a plaque 160 reduction neutralization titer assay ( Figure 3D ). Consistent with the ability of the sera to neutralize Figure 5D ). Analysis of brain 212 tissue demonstrates that mice immunized with VSV-OROV and VSV-OROVΔ4 have reduced viral 213 RNA levels ( Figure 5D ) consistent with reduced OROV infection.

We report three VSV-chimeric viruses expressing the surface proteins Gn and Gc of the 

Length and width of viral particles was measured using ImageJ. 

Inoculation schedule of male, 6-week old C57Bl/6 mice. Mice were inoculated on day zero with 418 10 6 FFU, boosted on day 28 with the same dose, and then challenged one week later with 10 6 419 TCID 50 OROV (five mice per group). One week after challenge mice were sacrificed. (B) Body 420 weight (top) and temperature (bottom) were assessed each day following challenge with OROV.

(C) One day before challenge, VSV-OROV, VSV-OROVΔ4, and VSV sera was collected from 422 mice and tested for neutralization of OROV. (D) Viral loads were assessed by measuring S 423 segment copies in blood and brain samples from mice following sacrifice.

Oropouche Fever: A 427 Review

Oropouche fever, an emergent disease 429 from the

Generation of

Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

Emergent arboviruses in Brazil

Arboviral etiologies of acute febrile illnesses in Western 439 South America

Isolation of Madre de Dios Virus 443 (Orthobunyavirus; Bunyaviridae), an Oropouche Virus Species Reassortant, from 444 a Monkey in Venezuela

Etiology of acute undifferentiated 448 febrile illness in the Amazon basin of Ecuador

Iquitos 451 virus: a novel reassortant Orthobunyavirus associated with human illness in Peru

Epidemiology of 454 endemic Oropouche virus transmission in upper Amazonian Peru

Oropouche virus. I. A review of clinical, 458 epidemiological, and ecological findings

Oropouche Virus: Clinical, Epidemiological, and 461 Molecular Aspects of a Neglected Orthobunyavirus

464 Transmission of Oropouche virus from man to hamster by the midge Culicoides 465 paraensis

Vegetation loss and the 2016 Oropouche 467 fever outbreak in Peru

Molecular biology of orthobunyaviruses

Proteomics computational analyses suggest that the 471 carboxyl terminal glycoproteins of Bunyaviruses are class II viral fusion protein 472 (beta-penetrenes)

Orthobunyavirus spike architecture and recognition by 475 neutralizing antibodies

Two monoclonal antibodies against La Crosse virus 477 show host-dependent neutralizing activity

Monoclonal 479 antibodies directed against the envelope glycoproteins of La Crosse virus

DNA-based vaccine against La 482 Crosse virus: protective immune response mediated by neutralizing antibodies and 483 CD4+ T cells

Biological activities of monoclonal 485 antibodies reactive with antigenic sites mapped on the G1 glycoprotein of La 486 Crosse virus

488 Analysis of the humoral immune response against the envelope glycoprotein Gc 489 of Schmallenberg virus reveals a domain located at the amino terminus targeted 490 by mAbs with neutralizing activity

Fields virology

Efficient recovery of infectious 494 vesicular stomatitis virus entirely from cDNA clones

Vesicular Stomatitis Virus: Actions in the Brain

Development of a new vaccine for the 502 prevention of Lassa fever

Properties of replication-competent 505 vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and 506 arenaviruses

Vaccination with a recombinant vesicular stomatitis virus 512 expressing an influenza virus hemagglutinin provides complete protection from 513 influenza virus challenge

Efficacy and effectiveness of an rVSV-vectored vaccine 519 expressing Ebola surface glycoprotein: interim results from the Guinea ring 520 vaccination cluster-randomised trial

Efficacy and 526 effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final 527 results from the Guinea ring vaccination, open-label, cluster-randomised trial 528 (Ebola Ça Suffit!)

The effect of dose on the safety and immunogenicity of the VSV 533 Ebola candidate vaccine: a randomised double-blind, placebo-controlled phase 534 1/2 trial

A single dose of a vesicular stomatitis virus-based 537 influenza vaccine confers rapid protection against H5 viruses from different clades

Competent Vesicular Stomatitis Virus Vaccine Vector Protects against SARS-543

CoV-2-Mediated Pathogenesis in Mice

Ebola virus entry requires the cholesterol 547 transporter Niemann-Pick C1

Rabies Internalizes into Primary Peripheral 549 Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body

Reconstruction of the cell entry 553 pathway of an extinct virus

556 Virus entry. Lassa virus entry requires a trigger-induced receptor switch

Live attenuated recombinant vaccine protects nonhuman 561 primates against Ebola and Marburg viruses

A forward 563 genetic strategy reveals destabilizing mutations in the Ebolavirus glycoprotein that 564 alter its protease dependence during cell entry

Bunyamwera orthobunyavirus Gc glycoprotein

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-571 Linked Immunosorbent Assay. MBio 9

In vitro and in vivo 573 studies of ribavirin action on Brazilian Orthobunyavirus

Oropouche virus infection and pathogenesis are 577 restricted by MAVS, IRF-3, IRF-7, and type I interferon signaling pathways in 578 nonmyeloid cells

In vitro and in vivo 580 studies of the Interferon-alpha action on distinct Orthobunyavirus

583 Deletion mutants of Schmallenberg virus are avirulent and protect from virus 584 challenge

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne 587 encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus

The N-terminal domain 590 of Schmallenberg virus envelope protein Gc is highly immunogenic and can 591 provide protection from infection

Generation of bovine respiratory 593 syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication 594 in tissue culture, and the human RSV leader region acts as a functional BRSV 595 genome promoter

Vesicular 597 stomatitis virus enters cells through vesicles incompletely coated with clathrin that 598 depend upon actin for internalization

A multiplex serologic platform for diagnosis of tick-borne 602 diseases

Dependent Signaling Restricts Orthobunyavirus Dissemination to the Central 606 Nervous System