International Immunology International Immunology Advance Access published December 4, 2014

Sendai virus recombinant vaccine expressing a secreted, unconstrained respiratory syncytial virus fusion protein protects against RSV in cotton rats

Manuscript ID: Manuscript Type:

Complete List of Authors:

INTIMM-14-0098.R1

Original Research

12-Oct-2014

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Date Submitted by the Author:

International Immunology

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Zhan, Xiaoyan; St. Jude Children's Research Hospital, Department of Infectious Diseases Slobod, Karen; St. Jude Children's Research Hospital, Infectious Diseases Jones, Bart; St. Jude Children's Research Hospital, Department of Infectious Diseases Sealy, Robert; St. Jude Children's Research Hospital, Department of Infectious Diseases Takimoto, Toru; St. Jude Children's Research Hospital, Department of Infectious Diseases Boyd, Kelli; St. Jude Children's Research Hospital, Animal Resource Center Surman, Sherri; St. Jude Children's Research Hospital, Department of Infectious Diseases Russell, Charles; St. Jude Children's Research Hospital, Department of Infectious Diseases Portner, Allen; St. Jude Children's Research Hospital, Department of Infectious Diseases Hurwitz, Julia; St. Jude Children's Research Hospital, Infectious Diseases

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Keywords:

Sendai virus , Respiratory syncytial virus, Unconstrained F, secreted fusion protein, protection, vaccine

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International Immunology

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REVISED INTIMM-14-0098

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Sendai virus recombinant vaccine expressing a secreted, unconstrained respiratory

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syncytial virus fusion protein protects against RSV in cotton rats

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Xiaoyan Zhan1, Karen S. Slobod1, Bart G. Jones1, Robert E. Sealy1,Toru Takimoto1, Kelli Boyd2,

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Sherri Surman1, Charles J. Russell1, Allen Portner1, and Julia L. Hurwitz1,3

6 Department of Infectious Diseases and 2Animal Resource Center, St. Jude Children’s

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Research Hospital; 3Department of Microbiology, Immunology and Biochemistry, University of

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Tennessee Health Science Center, Memphis, Tennessee

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Running title: SeV-expressed RSV F secreted protein confers protection against RSV

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Keywords: protection, respiratory syncytial virus, secreted fusion protein, Sendai virus,

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unconstrained F, vaccine

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Current Addresses:

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Xiaoyan Zhan: Department of Pharmacology, Vanderbilt University, Nashville, TN; Karen

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Slobod: Infectious Diseases and Pediatrics, Department of Technical Development, Novartis

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Vaccines, 350 Mass Ave, Cambridge, MA; Toru Takimoto: Department of Microbiology and

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Immunology, University of Rochester, Rochester, New York; Kelli Boyd: Vanderbilt University

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School of Medicine, Division of Animal Care, AA-6206 MCN, Nashville, TN;

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*

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Julia L. Hurwitz

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Department of Infectious Diseases

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St. Jude Children’s Research Hospital

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262 Danny Thomas Place

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Memphis, TN 38105

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Phone: 901-595-2464

To whom correspondence should be sent:

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Fax: 901-595-3099

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e-mail: [email protected]

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The respiratory syncytial virus (RSV) is responsible for as many as 199,000 annual deaths

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worldwide. Currently there is no standard treatment for RSV disease, and no vaccine. Sendai

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virus is an attractive pediatric vaccine candidate because it elicits robust and long-lasting virus-

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specific B cell and T cell activities in systemic and mucosal tissues. The virus serves as a gene

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delivery system as well as a Jennerian vaccine against its close cousin, human parainfluenza

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virus type 1. Here is described the testing of a recombinant SeV (SeVRSV-Fs) that expresses

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an unconstrained, secreted RSV-F protein as a vaccine against RSV in cotton rats. After a

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single intranasal immunization of cotton rats with SeVRSV-Fs, RSV-specific binding and

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neutralizing antibodies were generated. These antibodies exhibited cross-reactivity with both

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RSV A and B isolates. RSV-F-specific interferon-γ-producing T cells were also activated. The

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SeVRSV-Fs vaccine conferred complete protection against RSV challenge without enhanced

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immunopathology. In total, results showed that an SeV recombinant that expresses RSV F in

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an unconstrained, soluble form can induce humoral and cellular immunity that protects against

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infection with RSV.

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ABSTRACT

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Respiratory syncytial virus (RSV) can cause close to 200,000 deaths in a single year worldwide

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(1-6). The most vulnerable individuals are premature infants, children with congenital heart/lung

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disease, and immunocompromised patients. Currently, there is only one form of successful RSV

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prophylaxis, which involves the injection of preformed anti-RSV antibodies into vulnerable

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infants (7). However, because of the high cost and logistical difficulty associated with

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treatments, pre-formed antibody prophylaxis is rarely available to the children who need it most

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(8;9). RSV vaccines have been studied for decades, but there remains no licensed product.

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Clearly, the development of a successful RSV vaccine is a critical, unmet need in the pediatric

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healthcare arena (10;11).

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Here we describe the testing of a Sendai virus-based vaccine that expresses an unconstrained,

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soluble, RSV F protein. Sendai virus was chosen as a vector, because it induces rapid and

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durable B cell and T cell responses in blood and in mucosal tissues of the respiratory tract. The

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virus has already been tested in adults and 3-6 year old children, and shown to be immunogenic

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and well tolerated.

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This study addresses a question pertinent to recent literature: can an unconstrained, soluble

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RSV F protein, truncated to remove transmembrane and intracellular protein regions, suffice as

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a recombinant vaccine target? The question is based on an understanding that in the absence

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of its transmembrane region, the pre-fusion form of RSV F protein is metastable and often

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converts to a post-fusion form, a low-energy six helix bundle that lacks certain antibody

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neutralizing determinants (12;13). Our data show that even though the RSV F protein expressed

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by SeVRSV-Fs is unconstrained, the vaccine elicits RSV-specific binding and neutralizing

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antibodies and T cell responses in cotton rats. The vaccine fully protects against RSV challenge

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without enhanced immunopathology.

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INTRODUCTION

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MATERIALS AND METHODS

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To make SeVRSV-Fs cDNA, we inserted a His tag sequence and a stop codon after the codon

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for residue 524 in the RSV F gene, using QuickChange Site-directed Mutagenesis Kit

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(Stratagene). The gene encodes a a protein truncated by 50 amino acids at its C-terminus and

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therefore lacking transmembrane and intracellular regions The mutated gene was inserted into

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the NotI site of pSV(E), Figure 1A (14;15).

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Reverse genetics rescue was performed as described previously(16). The 293T cell line was

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infected with a UV-inactivated, T7 RNA polymerase-expressing recombinant vaccinia virus

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(vTF7.3) for 1 hr at 37°C. Cells were then co-transfected with plasmids containing recombinant

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SeV cDNA plasmid and with supporting T7-driven plasmids respectively expressing the NP, P,

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and L genes of SeV (pTF1SVNP, pTF1SVP, and pTF1SVL) in the presence of Lipofectamine

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(Life Technologies, Grand Island, NY). After 40 hrs, cell lysates were prepared and amplified by

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inoculation into embryonated hens eggs. Allantoic fluids were harvested after three days and

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virus was cloned by plaque purification on LLC-MK2 cells. Cloned recombinant virus (termed

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SeVRSV-Fs) was amplified in hen’s eggs to prepare stocks for further testing.

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Purification and testing of RSV F protein

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Allantoic fluids were harvested three days after the inoculation of eggs with SeVRSV-Fs . Fluids

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were centrifuged (approximately 300 x g, 10 min) to remove debris and filtered (0.45 µM filter).

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Samples were passed over a PBS-equilibrated Sepharose column bound by the anti-F

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monoclonal antibody Palivizumab (Synagis; MedImmune Inc., Gaithersburg, MD). The column

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was washed with PBS and eluted with 0.2 M glycine (pH 2.8), after which pooled protein

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fractions were dialyzed overnight against PBS. As controls for RSV F protein analysis, RSV-

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Construct design

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infected cell lysates were used. These were from HEp-2 cells that were infected with RSV-A2 in

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PBS at room temperature for 1 hr. After washing, cells were cultured in Dulbecco’s modified

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eagle’s medium (Cambrex Bio Science Walkersville, Inc, Walkersville, MD) supplemented with

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lysed with TNE buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA).

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Supernatants were clarified by centrifugation (15,000 x g, 10 min).Western blots were

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performed with test and control samples by separating proteins on SDS polyacrylamide gels

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under non-reducing conditions and transferring proteins to an Immobilon membrane (Millipore,

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Danvers, Mass.). Membranes were treated with 5% milk/TBST (Tris buffered saline plus 0.5%

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Tween), incubated with an RSV F-specific monoclonal antibody (antibody 1269, kindly provided

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by Dr. Coelingh, NIAID(17)). Development was with a goat anti-mouse IgG (H+L) horse radish

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peroxidase (HRP) conjugate (BioRad, Hercules, CA) followed by a SuperSignal West Pico

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Chemiluminescent (HRP) Substrate (Pierce, Rockford, Il).

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Immunizations and RSV challenges

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Immunizations and RSV challenges were conducted as described previously (16;18-20). Cotton

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rats (Sigmodon hispidus; Harlan Sprague Dawley, Indianapolis, IN) were grouped (up to 5

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animals per group) to receive SeVRSV-Fs or unmanipulated SeV intranasally (i.n., 2 x106

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PFU/animal). Additional controls were with PBS alone. After five weeks, animals were

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challenged i.n. with RSV-A2 at a dose of 1.5 x 106 PFU/cotton rat. Unless described as

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preliminary, experiments were repeated to ensure reproducibility.

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Enzyme-linked immunosorbent assay (ELISA)

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ELISAs were described previously (16). Briefly, ELISA microtiter plates were coated with 1

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ug/ml purified F protein for overnight incubation. Plates were blocked with PBS/3% bovine

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serum albumin (BSA, Sigma, St Louis, MO), after which serially diluted serum samples from the

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glutamine, gentamicin and 10% fetal calf serum. Three days later, cells were harvested and

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test and control cotton rats were added and incubated for at least 1 hr at 37 °C. Plates were

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washed and incubated with rabbit anti–cotton rat antibody (Virion Systems, Rockville, MD) for

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30 min at room temperature. Plates were developed by incubation with an anti-rabbit IgG-

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horseradish peroxidase conjugate (diluted in PBS/1% BSA, Bio-Rad, Hercules, CA, Cat# 170-

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6515) for 30 min, followed by 2,2′-azino-bis-(3-ethylbenzthiazolinesulfonic acid) (ABTS,

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Southern Biotechnology Associates, Inc, Birmingham, AL). Plates were read at O.D. 405 nm.

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Neutralizing activity was measured using an RSV plaque assay as described previously (16).

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Serially diluted test and control sera were mixed with RSV (100-150 PFU/well) in EMEM

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(Cambrex Bio Science Walkersville, Inc, Walkersville, MD) for 1 hr at 37°C. The virus-serum

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mixtures were inoculated onto HEp-2 cell monolayers in 12-well plates, except for virus RSV B1

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that was inoculated onto Vero cells. Plates were incubated for 1 hr (37°C, 5% CO2) and then

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overlaid with medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75%

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methylcellulose (Fisher Scientific, Fair Lawn, NJ). After culture for 5-6 days (37°C, 5% CO2), the

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methylcellulose was removed. Cells were treated with formalin phosphate and stained with

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hematoxylin and eosin. Plaques were counted visually. Inhibition was defined as the percentage

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reduction of plaques comparing post-vaccine sera with pre-vaccine sera. Viruses included RSV

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A2 (Laboratory isolate, Type A RSV, American type culture collection, ATCC, Rockville, MD),

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RSV B1 (Laboratory isolate, Type B RSV, ATCC), VR 1580 (Type B RSV, ATCC), K1014, and

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K1016 (primary isolates, Type A RSV, kindly provided by Dr. J. DeVincenzo, Le Bonheur

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Children’s Hospital, Memphis, TN).

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Peptide synthesis and IFN-gamma ELISPOT assays

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The T cell assays were conducted as described previously (16). Briefly, overlapping F peptides

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(derived from the RSV A2 F sequence) were prepared by the Hartwell Center for Bioinformatics

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Neutralization assays

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and Biotechnology at St. Jude Children’s Research Hospital. Peptides were generally 15 amino

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acids in length. Peptide sequences were initiated at 5 amino acid intervals to cover the entire

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length of the RSV F protein. Twenty two peptide pools were made for T cell testing.

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goat anti-cotton rat IFN-gamma antibody (R & D Systems, Minneapolis, MN) and incubated

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overnight at 4°C. Plates were washed, and incubated for at least 1 hr at 37°C with complete

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tumor medium (CTM (21;22), a modified Eagle’s medium [Invitrogen, Grand Island, NY]

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supplemented with 10% fetal calf serum, dextrose [500 µg/m], glutamine [2 mM], 2-

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mercaptoethanol [3 x 10-5 M], essential and non-essential amino acids, sodium pyruvate,

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sodium bicarbonate, and antibiotics). Mediastinal lymph node cells were harvested from cotton

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rats 10 days after vaccination. Cells were suspended in CTM and added to wells (106 cells/well)

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containing the peptide pools. The final concentration of each peptide was approximately 10 µM.

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As positive controls, cells were stimulated with 4 µg/ml Con A (Sigma-Aldrich, St. Louis, MO).

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The plates were incubated for 48 hr at 37°C. Plates were then washed with PBS followed by

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PBS wash buffer (PBS with 0.05% Tween 20). Plates were next incubated with biotinylated goat

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anti-cotton rat IFN-gamma antibody (100 µl of 0.5 µg/ml; R & D Systems, Minneapolis, MN) in

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PBS containing 0.05% Tween 20 and 1% FCS (incubated at 37°C for at least 2 hr) followed by

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streptavidin-conjugated alkaline phosphatase (Cat# D0396, DAKO, Copenhagen, Denmark)

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diluted 1:500 in PBS wash buffer. After 1 hr, plates were rinsed with wash buffer and water and

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spots were developed with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium alkaline

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phosphatase substrate (Sigma-Aldrich). Spots were counted with an Axioplan 2 microscope

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and software (Carl Zeiss, Munich-Hallbergmoos, Germany).

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Histopathology studies

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Multiscreen-hemagglutinin filtration plates (Millipore, Bedford, MA) were coated with 3.3 µg/ml

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described (16;18;20;23). Briefly, lungs were inflated via the trachea with 10% neutral buffered

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formalin. They were then submerged in formalin for overnight fixation. Tissue was embedded in

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paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin. Lung pathology was

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scored on the basis of three parameters: peribronchiolitis (inflammatory cells around small

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airways), alveolitis (inflammatory cells within alveolar spaces), and interstitial pneumonitis

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(inflammatory cell infiltrates and thickening of alveolar walls). Scoring was performed by a

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pathologist using a scale of 0 (no inflammation) to 4 (maximum inflammation within the

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experiment), defined independently for each parameter. The pathologist was blinded to the

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origin of tissues from test or control groups while scoring.

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RSV titration in cotton rat lungs after challenge

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RSV measurement in cotton rat lungs has been described previously (16). Three days after i.n.

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RSV challenge, cotton rats were sacrificed. Lungs were harvested and homogenized on ice with

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a mechanical Dounce homogenizer (PowerGen125 PCR Tissue Homogenizing kit; Fisher

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Scientific). Homogenates were centrifuged (approximately 1500 x g, 10 min) and supernatants

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were collected. They were stored frozen prior to testing. For virus titration, serially diluted

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supernatants were inoculated onto HEp-2 cell monolayers (except for virus B1 that was

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measured on Vero cells) in 12-well plates in EMEM. After 1 hr at 37 °C, 5% CO2, the wells were

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overlaid with medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75%

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methylcellulose. Cells were incubated for 5-6 days (37°C, 5% CO2), after which the

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methylcellulose was removed. Cells were fixed with formalin phosphate and the plates were

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stained with hematoxylin and eosin for plaque counting. The total pulmonary virus burden was

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expressed as the plaque count (PFU) per cotton rat.

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SeVRSV measurement in cotton rat lungs after vaccination

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Five days after RSV challenge, lungs were prepared for histological analyses as previously

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Replication-competent SeVRSV was monitored in cotton rat lungs by plaque count as described

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previously (24) after i.n. vaccination with 2 x 106 PFU virus. Briefly, lungs were homogenized in

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5 ml PBS, centrifuged briefly to remove debris, and plated using serial 10-fold dilutions on

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adherent LLC-MK2 cell monolayers in six-well plates. After incubation at 33°C for 1 hour, the

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inoculum was removed and replaced with an overlay containing acetylated trypsin (5 µg/ml).

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Plates were inverted and cultured for 4-6 days at 33°C. A second overlay was added containing

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neutral red. Plaques were visualized and counted after an additional incubation of cultures for 1-

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3 days.

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RESULTS

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Recombinant SeV particles were designed to express a truncated RSV F protein as described

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in the Materials and Methods section and as illustrated in Figure 1A. To confirm F protein

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secretion by infected cells, gel electrophoresis and Western blotting was performed with

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allantoic fluids from infected eggs. The secreted F protein from SeVRSV-Fs was predictably

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smaller than the full-length RSV F from RSV particles (data not shown). In a preliminary

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experiment to ensure vaccine growth, three cotton rats received SeVRSV-Fs by the i.n. route

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and were examined on days 1, 3, 5, and 7 for vaccine amplification. As shown in Figure 1B,

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virus peaked on day 3 after inoculation and was cleared by day 7 in all animals.

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Vaccination with SeVRSV-Fs elicits RSV-specific binding and neutralizing antibodies in

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cotton rats.

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To determine whether the SeVRSV-Fs vaccine induced an antibody response, we inoculated

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groups of five cotton rats by the i.n. route (2 x 106 PFU/cotton rat). Animals injected with PBS or

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unmodified SeV were used as controls to define baseline activities. Blood was collected before

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and four weeks after vaccination for testing of RSV- specific antibodies. Post-vaccine antibodies

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bound antigen in an ELISA (Figure 2A). Pooled serially diluted serum samples were also tested

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for neutralization by incubating with RSV-A2 prior to infection of cultured cells. Sera were found

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to be neutralizing against this homologous virus (Figure 2B). Neutralizing antibodies were also

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detected against non-homologous viruses, including clinical isolates of the RSV A subtype

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(Figures 2C and 2D), a laboratory isolate of the RSV B subtype (B1, Figure 2E), and a clinical

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isolate of the RSV B subtype (Figure 2F, VR1580).

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RSV F protein secreted by SeVRSV-Fs-infected cells.

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To examine T cell responses, we performed IFNγ-ELISPOT assays. Mediastinal lymph nodes

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(MLN) were harvested from cotton rats ten days after vaccination with SeVRSV-Fs or

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unmanipulated, non-recombinant virus (Sendai). Cell suspensions prepared from MLN were

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combined for each experimental group (3 or more animals per group). To examine IFNγ

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expression, we incubated cell suspensions with pools of overlapping peptides representing the

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entire RSV F protein (listed in Table 1). As shown in Figure 3, SeVRSV-Fs induced IFNγ-

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producing T cells against RSV peptide pools.

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SeVRSV-Fs confers complete protection against RSV challenge in the cotton rat model.

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SeVRSV-Fs was next tested for protection of animals from RSV challenge. Five weeks after

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vaccination, animals were challenged i.n. with RSV-A2. Three days after challenge, animals

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were sacrificed and lungs were collected for measurement of RSV burden. As shown in Figure

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4, animals vaccinated with a single dose of SeVRSV-Fs were completely protected from RSV

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challenge. In contrast, all animals inoculated with control SeV and subsequently challenged with

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RSV were infected.

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No enhanced immunopathology upon RSV challenge following vaccination with SeVRSV-

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Fs.

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Cotton rats inoculated with SeVRSV-Fs or PBS and were sacrificed five days after challenge

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with RSV to test for immunopathology. The lungs were harvested, perfused with formalin,

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sectioned, and stained with hematoxylin and eosin for histologic examination. Peribronchiolitis,

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interstitial pneumonitis, and alveolitis were detected in control cotton rats five days after i.n. RSV

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challenge, and were not enhanced in vaccinated animals. Scores for test and control animals

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are shown in Table 2.

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SeVRSV-Fs vaccination elicits RSV-specific T cell responses

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This report describes the immunization of cotton rats with SeVRSV-Fs, an SeV-based vaccine

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produced by reverse genetics to deliver a truncated RSV F gene to mammalian cells. Cells

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infected with SeVRSV-Fs expressed a secreted, unconstrained RSV F protein. Vaccination

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generated binding and neutralizing antibody responses as well as T cell responses toward RSV.

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Following a single i.n. inoculation with SeVRSV-F, cotton rats were fully protected from RSV

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infection after challenge. Immunopathology was not enhanced after RSV challenge in animals

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that were vaccinated with SeVRSV-Fs compared to controls.

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Our previous studies with SeV vaccines have demonstrated the induction of rapid and durable B

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and T cell responses systemically and at upper respiratory tract (URT) and lower respiratory

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tract (LRT) mucosal surfaces (25;26). An unmanipulated SeV was previously demonstrated to

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confer complete protection against challenge with its cousin, human parainfluenza virus-type 1

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(hPIV-1), in African green monkeys (24). Unmanipulated SeV is currently in clinical trials. It has

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been well tolerated in adults (27) and 3-6 year old children (data not shown), paving the way for

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similar studies with recombinant SeV vaccine products.

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A recombinant SeV that expresses the full length RSV F has already been tested in cotton rats

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and has been shown to protect against challenges with both RSV A and B isolates (16).

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Protection is demonstrated even in the presence of passively transferred RSV-specific and SeV-

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specific neutralizing antibodies in a cotton rat maternal antibody model (28). The vaccine safely

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protects against RSV challenge in African green monkeys (29). Sendai viruses are attractive

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vaccine candidates, in part because of their appealing immunogenicity and safety profiles, and

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in part because they maintain genetic stability when successively passaged and amplified to

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high titers in hen’s eggs or mammalian (e.g. Vero) cell cultures (data not shown). The study

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DISCUSSION

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described here demonstrates that an SeV recombinant expressing a truncated, secreted RSV F

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protein can serve as a successful vaccine against RSV.

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protein in a vaccine. Some researchers suggest that RSV F should be constrained in its pre-

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fusion form, based on arguments that: (i) there are important antibody determinants (e.g. D25,

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AM22, 5C4) that exist only within the pre-fusion form of F, and (ii) the majority of neutralizing

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antibodies in RSV immune human sera bind to the pre-fusion F molecule (12;13). Opposing

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arguments are that: (i) several important antigenic determinants including 101F exist on post-

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fusion F proteins (30;31), (ii) the Palivizumab neutralizing monoclonal antibody binds post-fusion

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F and provides excellent prophylaxis against RSV A and B isolates in clinical practice (7;9), (iii)

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antibodies clear viruses by a variety of mechanisms that function both before and after the

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fusion of virus to its target cell (32-34), and (iv) the artifactual manipulations required to

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constrain soluble F protein in its pre-fusion form may inadvertently alter important native

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antigenic epitopes, a situation that was associated with significant morbidity and mortality when

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a formalin-treated RSV vaccine was tested in children (11;35). Our results support the argument

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that RSV F constraint is not required, consistent with data from Swanson et. al. who tested a

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subunit F protein as an RSV vaccine in mice (36).

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It is important to consider that antibodies protect against virus infections using sophisticated

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mechanisms that function before and after virus fusion. In the context of HIV-1 infection, for

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example, antibodies can inhibit infection well after virus has penetrated the cell. This is in part

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due to the shuttling of virus particles to proteasomes when antibodies bind viral antigens and

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TRIM-21 (32). Antibodies are also well known for their support of antibody-dependent cell-

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mediated cytotoxicity (ADCC), a mechanism by which cytotoxic cells (NK or CD8+ T cells) are

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Our results address a recent question in the RSV field that concerns the configuration of RSV F

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linked to their virus-infected targets (37;38). Moreover, antibody-antigen complexes and free

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antibodies modulate inflammation by up- and down-regulation of innate and adaptive immune

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cells (39-41). Together, there are a plethora of antibody-mediated mechanisms that counter

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virus infection and disease, and that are clearly not dependent on the pre-fusion structure of

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RSV F. To date, the most clinically-relevant propohylactic regimen against RSV infections is

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with an antibody that binds the post-fusion form of RSV F (8).

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In conclusion, we have demonstrated that SeVRSV-Fs confers protection against RSV

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challenge in a cotton rat model and that constraint of the secreted F protein in its pre-fusion

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form is not required. Clearly, a number of vaccine products that present soluble F in an

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unconstrained form remain viable vaccine candidates for the protection of humans from RSV

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infection.

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ACKNOWLEDGEMENTS

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We thank Virion Systems for providing cotton rat antibody reagents. We thank Dr. J.

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DeVincenzo, Le Bonheur Children’s Hospital, Memphis, TN for kindly providing primary RSV

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isolates for neutralization assays. This work was supported by NIH NIAID P01 AI054955, NIH

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NIAID R01 AI088729, NIH NCI P30-CA21765, and the American-Lebanese Syrian Associated

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Charities (ALSAC).

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Conflict of interest statement: The authors declared no conflict of interests.

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Table 1. RSV F peptide sequences for T cell testing

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Overlapping peptides were synthesized to cover the full sequence of secreted F. The

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synthesized peptides were generally 15 amino acids in length and were initiated at 5-amino acid

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intervals to cover the entire length of the RSV F protein. Amino acids represented in each

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peptide pool are shown. Pools 21 and 22 included amino acids in the transmembrane and

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cytoplasmic regions of F that were not included in the vaccine (as indicated by dashed lines).

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Each pool comprised approximately 10 peptides. Seven peptides were not synthesized due to

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technical difficulties: ITTILTAVTFCFASG (pool 1); KIKSALLSTNKAVVS (pool 7);

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NFYDPLVFPSDEFDA (pool 20); and STTNIMITTIIIVII, MITTIIIVIIVILLS, IIVIIVILLSLIAVG, and

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VILLSLIAVGLLLYC (pool 21).

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RSV F Sequences MELLILKANAITTILTAVTFCFASGQNITEEFYQS QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE GWYTSVITIELSNIKENKCNGTDAKVKLIKQELDK VKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRE PPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKR NVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHL GVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGV KAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQ KQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITR KNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSL MLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQS NNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVID VQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLT TKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV FFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVD PSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSL DVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTF NKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVN SVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPS NFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSD QSLAFIRKSDELLHNVNAGKSTTN---------------------------------------------

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Table 2. Histopathology following vaccination and subsequent RSV challenge

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SeVRSV-Fs SeVRSV-Fs SeVRSV-Fs SeVRSV-Fs SeVRSV-Fs

2 2 2 0 1

2 1 1 0 1

6 7 8 9 10

PBS control PBS control PBS control PBS control PBS control

4 4 2 4 3

2 3 3 4 3

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Bronchiolits

Alveolitis

Interstitial Pnemonitis 1 1 1 0 1 2 2 3 4 3

Cotton rats were vaccinated with SeVRSV-Fs or PBS and then challenged with RSV.

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Five days later, cotton rats were scored for immunopathology. The most severe disease

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in each category within the experiment was scored as ‘4’ and the absence of disease

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was scored as ‘0’.

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Vaccine Group

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FIGURE LEGENDS

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Figure 1. Design and characterization of recombinant SeV expressing RSV F as a

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secreted protein.

362 A. The diagram illustrates the design of SeVRSV-Fs. A unique NotI restriction enzyme site was

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created in the non-coding region of the HN gene of the full genome SeV cDNA for insertion of

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the RSV Fs gene. The RSV gene was isolated from RSV-A2 with RT-PCR, digested with NotI

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and cloned into the NotI site of pSV(E). T7=T7 promoter; ribo=hepatitis delta virus ribozyme

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sequence.

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B. In a preliminary experiment, cotton rats were vaccinated i.n. with 2 x 106 PFU SeVRSV-Fs

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and examined throughout a one week period for vaccine virus amplification in the lower

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respiratory tract, by testing homogenized lungs in plaque assays on LLC-MK2 cells. Each

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symbol represents virus measurements from a different animal. ND=Not detected.

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Figure 2. Cotton rats primed with SeVRSV-Fs generate RSV-specific neutralizing

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antibodies.

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Groups of five cotton rats were inoculated with 2 x 106 SeVRSV-Fs or unmodified SeV. Sera

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were collected after four weeks and pooled for each group of animals. (A) Sera were diluted

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1:250 and 1:1000 for testing in an ELISA for RSV-F-specific binding antibody. (B-F) Pooled

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serum samples from cotton rats inoculated with SeVRSV-Fs or unmodified SeV were tested for

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inhibition of RSV growth in a plaque assay. Results are the mean percentage plaque reduction

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for post-vaccine test sera compared to pre-vaccine control sera from the same animal set.

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Standard error bars are shown. Neutralization experiments were conducted with different

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viruses including RSV A2 (laboratory type A isolate, panel B), K1014 (primary type A isolate,

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panel C), K1016 (primary type A isolate panel D), B1 (laboratory type B isolate, panel E), and

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VR1580 (primary type B isolate, panel F). Test and control samples were plated in triplicate in

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ELISAs and in neutralization assays. All ELISAs and neutralization assays revealed statistically

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significant differences between vaccinated and control samples using unpaired T tests (p3 animals per group) were tested against each of 22

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peptide pools or against no peptide. Spot-forming cells (SFC) were counted per 106 plated

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cells. Values are means of duplicate wells with standard errors. Asterisks indicate peptide pools

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for which mean test results were ≥4x control values. Unpaired T tests were conducted using

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GraphPad Prism software and showed that results for pools 2, 3, and 10 were all statistically

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significant (p

Sendai virus recombinant vaccine expressing a secreted, unconstrained respiratory syncytial virus fusion protein protects against RSV in cotton rats.

The respiratory syncytial virus (RSV) is responsible for as many as 199000 annual deaths worldwide. Currently, there is no standard treatment for RSV ...
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