key: cord-0331967-igkwgpnq
authors: Lemon, J.; Douglas, A.; Power, U.; McMenamy, MJ.
title: Identification of antigenic epitopes on the F and G glycoproteins of bovine respiratory syncytial virus and in vitro assessment of their synthetic peptide vaccine potential
date: 2021-06-25
journal: bioRxiv
DOI: 10.1101/2021.06.25.449873
sha: d2a7dff6d3b33b7ba25355a3a3e4353d6d08eed5
doc_id: 331967
cord_uid: igkwgpnq

Globally, bovine respiratory disease (BRD) remains the principal reason for mortality of calves over one month of age despite the availability of various vaccines on the UK market. Bovine respiratory syncytial virus (BRSV) was first discovered in the 1970s and is now considered a principal pathogen implicated in the disease complex. Outbreaks occur annually and re-infections are common even in the presence of maternal antibodies. Difficulties have arisen from using both live-attenuated and inactivated vaccines and recent efforts have focused on the development of sub-unit vaccines that are suitable for use in neonatal calves with maternally-derived circulating antibodies. This study was undertaken to identify antigenic epitopes on two of the surface glycoproteins of BRSV, the fusion (F) and attachment (G) proteins, the major surface viral antigens, for inclusion into a novel subunit peptide vaccine. Sequencing and antigenicity prediction of the F and G genes of BRSV revealed 21 areas of potential antigenicity; of which genuine peptide/antisera binding occurred with 4 peptides. Identification of the antigenic components of a vaccine is an important first step in the development of novel BRSV vaccines and this data, therefore, provides the basis for the generation of such vaccines.

The two major glycoproteins of BRSV are the major attachment protein (G) and the 33 fusion protein (F), both of which are targets for virus neutralising antibodies (VNA). The 34 G protein is heavily involved with the modulation of the host immune response, 35 through mechanisms such as glycosylation, receptor mimicry and antibody 36 interference (2-4) while the F protein is known to confer cross-protection between 37 strains due to its high degree of sequence conservation. The F2 subunit has also been 38 suggested as being responsible for the species-specificity witnessed with RSV infection 39 (5). Epitope mapping has also been used to define immunopathological residues to avoid; 54 information which could complement epitopes presented for use in a subunit vaccine. 55 Enhanced lung eosinophilia has been noted after vaccination using FI-RSV antigen and 56 subsequent infection. Using two pGEM4-derived plasmids encoding the G proteins of 57 frameshift mutants, Sparer et al identified residues G193-203, located in the carboxyl 58 terminal of RSV G, as responsible for this. In the same study, mice which had been 59 primed intranasally with recombinant Vaccinia virus incorporating the same residues 60 displayed similar weight loss to those vaccinated with wild-type G, both of which groups 61 demonstrated statistically greater weight loss than negative control vaccines (7). 

Obtaining BRSV F and G gene sequence: Endpoint RT-PCR was performed on extracted viral RNA using 97 primers designed in-house amplifying areas outside the 3' and 5' ends of the F and G genes. These are 98 detailed in Appendix 1. For RT-PCR, primers were used at a concentration of 10 μM and primer sets 99 G1/G2, F1/F3, F2/F4 were used. All reactions were set up in triplicate using a One-Step RT-PCR kit (Qiagen, 100 UK) as per the following run conditions: 30 mins @ 50°C; 15 mins @ 95°C followed by 30 cycles of 30 sec 101 @ 94°C; 30 sec @ 51°C; 2 mins @ 72°C followed by 10 mins @ 72°C. Amplicons were electrophoresed on 102 a 1 % agarose gel at 120 V for 60 min. Amplicons of appropriate size were excised and purified using an 103 Isolate II PCR and Gel kit (Bioline, UK). The purified amplicons were included in cycle sequence reactions, 104 prepared using primers as detailed before, only at the concentration of 3.2pmol and a Big Dye 

Peptide synthesis: Using current literature and prediction software, 21 peptides were chosen for 117 further analysis in this study. A single amino acid residue substitution between the sequence for peptides 118 1, 2 and 3 allowed us include a sequences from 2 Danish strains of BRSV (strain 9402022, strain 9416116) 119 providing some heterogeneity for the study. Peptides were synthesized commercially by Mimotopes, 120 Australis and were synthesised at >70 % purity. All peptides, except peptide 4, were labelled with biotin 121 at the N terminus and capped with an amide group (NH2) at the C terminus. All peptides contained a 4 122 mer serine-glycine-serine-glycine (SGSG) spacer following the biotin tag at the N terminus to reduce the 123 potential for steric hindrance, thus allowing the necessary epitope for antibody binding to be accessible.

As peptide 4 was the starting sequence of the G protein, synthesized without a biotin tag, it was 125 hypothesised that a free amine group at the N terminus would more closely represent an N-terminal 126 native epitope and thus might be more beneficial for antibody binding than a biotin tag. The selected 

Optimisation of ELISA protocol prior to screening: Optimisation work was performed to determine an 138 appropriate plate type and peptide coating concentration. All dilutions were prepared in PBS 1X pH 7.4. with the other test methods. All peptides were coated at 5 μg/mL or 1 μg/mL in and left to incubate at 147 +4°C overnight. The following morning, the ELISA protocol, as described in Appendix 3, was performed.

 Coating optimisation: The lowest molecular weight peptide was coated at 10, 5, 1 and 0.1 μg/mL 149 across the chosen plate, which was then sealed and left overnight at +4°C. The following morning a 150 washing step was performed and biotinylated HRP, containing 1 % bovine serum albumin (v/v) was 151 dispensed at 50 μL/well in decreasing concentrations of 1:80,000, 1:160,000, 1:320,000 to 1:640,000 down 152 the plate. Blocking buffer was removed to reduce any associated non-specific binding and the ELISA 153 protocol, detailed in Appendix 3, was followed from step 6.

In vitro antigenicity screening using bovine sera: All 21 peptides were coated at 5 μg/mL and screened 156 using a pool of BRSV antibody-positive and bovine negative sera, using the ELISA protocol detailed in Repeat testing of peptides using anti-BRSV antibody negative sera: All peptides were tested using 160 bovine sera which did not contain antibodies against BRSV to ensure any reactivity observed during initial 161 screening was specific for BRSV and not as a result of non-specific binding.

Acceptability criteria: At initial analysis using antibody positive sera, any peptide which produced 164 optical densities (OD) less than or equal to twice the negative control value was eliminated, as reactivity 165 was considered too low to inform any further investigations. Corrected averages were then obtained by 166 removing the negative control value, using both antibody negative and antibody positive sera from the 167 respective optical density results, to negate background optical density. A ratio was calculated between 168 the optical density obtained for an individual peptide using antibody positive sera and antibody negative 169 sera and peptides were considered as displaying genuine reactivity if this value was >2. information on the specific disease-causing and protective epitopes of the virus.

Although the immunogenicity of the BRSV epitopes defined herein, and their associated 544 protective efficacy in a vaccine is yet to be defined, they provide the rationale and a 545 solid basis for future investigation into a safer, more efficacious, sub-unit BRSV vaccine. 

A 554 bovine model of vaccine enhanced respiratory syncytial virus pathophysiology

Structural homology of the central 557 conserved region of the attachment protein G of respiratory syncytial virus with the fourth subdomain 558 of 55-kDa tumor necrosis factor receptor

The secreted form of 560 respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of 561 replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes

Immunology of bovine respiratory syncytial virus in calves

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Recombinant vaccinia viruses 569 expressing the F, G or N, but not M2, protein of bovine respiratory syncytial virus (BRSV) induce 570 resistance to BRSV challenge in the calf and protect against the development of pneumonic lesions

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Vaccination 580 against foot-and-mouth disease virus using peptides conjugated to nano-beads

Candidate peptide vaccine induced protection against 583 classical swine fever virus

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Epitope-based peptide vaccines predicted 592 against novel coronavirus disease caused by SARS-CoV-2

Strategies for 598 differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever 599 and avian influenza

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Antigenic and molecular 608 analyses of the variability of bovine respiratory syncytial virus G glycoprotein

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Identification of 620 multiple protective epitopes (protectopes) in the central conserved domain of a prototype human 621 respiratory syncytial virus G protein

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Alteration of the 633 N-linked glycosylation condition in E1 glycoprotein of Classical Swine Fever Virus strain Brescia alters 634 virulence in swine

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4 and left to incubate at +4℃ overnight. The 749 following morning liquid was removed and the plates washed 5 times with 300 μL/well washing 750 buffer. The final wash was left in the plate for 1 minute before discarding

Two hundred μL/well of 4 % (w/v) milk solid

Plates were placed in an incubator set at 37 ˚C for 1 h after which time the excess blocking buffer 754 was shaken off and plates were washed as before

Dilutions of bovine sera and conjugated secondary antibody were prepared in PBS

Fifty μL/well of a 1:200 bovine sera dilution was added and plates were incubated at 37 ˚C for 1

which time the excess serum was shaken off and plates were washed. Fifty μL/well of 758 rabbit anti-bovine IgG (Sigma, UK) was added, used at a dilution of 1:20,000 and plates were 759 incubated at 37 ˚C for 1 h

For a final time, the excess liquid was shaken off and plates were washed as previously described, 761 before the addition of 50 μL/well of TMB substrate

Plates were sealed and left to incubate at room temperature (RT) in the dark

After 15 min, 50 μL/well 1 M H3PO4 was added to stop the reaction. Optical density (OD) was 764 read at 450 nm using a Sunrise plate reader with Magellan software

C denotes the cytoplasmic domain and TM the transmembrane domain. Variable regions, corresponding to 791 mucin-like regions are highlighted in red and amino acid numbers to delimit each domain are noted

 Sequence   G1  For  4600  TCT TTT TAG TCT ACA AAT GCT GCA  G2  Rev  5833  CTG AAG GAG GCC GGT TCA TT  F1  For  5468  ACA GAT CTA ACA ACA CAC CTC CA  F2  For  6040  ACT ACA CCT GGA GGG AGA GG  F3  Rev  6320  TGT ATG TAC TGA GGG GTG TGG  F4  Rev  7393 AAA AAT GCA GGT CTT GGC GGA T 739 740