DOI: 10.5061/dryad.9cnp5hqtq
title: IgM and IgY CDR3 sequences from naive, vaccinated and/or infected chickens

description: Avian influenza viruses (AIVs) are a major economic burden to the poultry
 industry, and pose serious zoonotic risk, with human infections being
 reported every year. To date, the vaccination of birds remains the most
 important method for the prevention and control of AIV outbreaks. Most
 national vaccination strategies against AIV infection use whole-virus
 inactivated vaccines, which predominantly trigger a systemic
 antibody-mediated immune response. There are currently no studies that
 have examined the antibody repertoire of birds that were infected with
 and/or vaccinated against AIV. To this end, we evaluate the changes in the
 H9N2-specific IgM and IgY repertoires in chickens subjected to
 vaccination(s) and/or infectious challenge. We show that a large
 proportion of the IgM and IgY clones were shared across multiple
 individuals, and these public clonal responses are dependent on both the
 immunisation status of the birds and the specific tissue that was
 examined. Furthermore, analysis reveals specific clonal expansions which
 are restricted to particular H9N2 immunisation regimes. These results
 indicate that both the nature and number of immunisations are important
 drivers of the antibody responses and repertoire profiles in chickens
 following H9N2 antigenic stimulation. We discuss how the repertoire
 biology of avian B cell responses may affect the success of AIV
 vaccination in chickens, in particular the implications of public versus
 private clonal selection.
description: <strong>1.1. </strong><strong>Experimental
 design and tissue samples</strong> White leghorn
 chickens (Valo breed, n=70) were hatched and reared in the poultry
 facility at the Pirbright Institute according to national and
 institute-specific regulations (licence number P68D44CF4). Birds were
 divided into 6 treatment groups (G1-6) and maintained together until
 infection, then transferred to isolators at day 21 post-hatching.
 Uninfected birds remained together for the duration of the experiment.
 Chickens from G1 (n=10), G2 (n=10), G4 (n=10), and G5 (n=10) received a
 single subcutaneous injection of 0.2ml of 1024 HAU/dose inactivated H9N2
 vaccine immediately after hatching. Individuals from G2 and G5 received an
 additional dose of the vaccine at 14 days after hatching. Birds from G3
 (n=15) and G6 (n=15) did not receive the vaccine. Weekly blood samples
 were collected from all birds immediately after the first intervention
 (i.e. infection or vaccination). At day 21 post-hatching, birds from G1-3
 received a single intranasal inoculation of 0.1ml of 10<sup>6
 </sup>pfu/ml H9N2 low pathogenic avian influenza virus (50 µl in
 each nostril). Following infection, birds (G1-3) were weighed daily and
 buccal and cloacal swabs were collected each day for 10 consecutive days
 to confirm infection status via qRT-PCR (see below). At day 3 and at day
 14 post-infection, 5 birds from each group (G1-G6) were culled and tissue
 samples (trachea, spleen and bursa) were harvested and stored in RNAlater
 (Thermo Fisher Scientific). Additionally, at day 7 post-infection, 5 birds
 from G3 and G6 were culled and their tissues were harvested as for the
 rest of the time points. A diagram of the experimental design is provided
 in Supplementary Figure 1. The tissues used for repertoire analysis were
 the ones harvested at the last time point of the experiment (14 days
 post-infection). <strong>1.2.
 </strong><strong>Haemagglutination and haemagglutination
 inhibition assays</strong> The HA titre of virus
 and the haemagglutination inhibition (HI) titre of chicken antisera were
 determined using established protocols (17). Briefly, to determine HA
 titre, a two-fold dilution series of virus in PBS was prepared in V-bottom
 96-well plates (Thermo Fisher Scientific) and incubated 1:1 with 1%
 chicken red blood cells (RBCs) in PBS for one hour at 4°C. The HA titre
 was recorded as the reciprocal of the highest dilution that virus caused
 complete haemagglutination of RBCs. To determine the HI
 titre, a series of 25 μl two-fold dilutions of post-infection chicken
 polyclonal antisera was incubated with 4 haemagglutinating units (HAU) per
 25 µl of virus for one hour at room temperature then incubated 1:1 with
 chicken 1% RBCs in PBS for one hour at 4°C. The HI titre was recorded as
 the reciprocal of the highest dilution of chicken antiserum that
 completely inhibited haemagglutination of RBCs.
 <strong>1.3.
 </strong><strong>ELISA</strong> To
 assess the levels of H9N2-reactive IgM and IgY in vaccinated and/or
 challenged birds, enzyme-linked immunosorbent assays (ELISAs) were
 developed using inactivated virus. Nunc MaxiSorp (Thermo Fisher
 Scientific) flat-bottom 96-well plates were coated with 50 µl per well of
 0.25 µg/ml inactivated virus and incubated overnight at 4°C. The following
 day, plates were washed 4 times using 0.1% Tween 20 (Sigma-Aldrich) in
 PBS. Subsequently, the plates were incubated for 30 minutes at room
 temperature using 100 µl of blocking buffer (0.25% BSA in 0.1% Tween-20 in
 PBS). Following another wash step, 50 µl of 1:800 serum dilution was added
 to duplicate wells and incubated for 1h at room temperature. The plates
 were then washed again and incubated for 30 minutes with 50 µl of either a
 1:7000 dilution of goat IgG conjugated with horseradish peroxidase (HRP)
 having anti-chicken IgY specificity (Biorad), or 1:3000 dilution of goat
 IgG conjugated with horseradish peroxidase (HRP) having anti-chicken IgM
 specificity (Invitrogen, cat. PA1-84676). After another wash step, 50 µl
 of OptEIA TMB Substrate (BD Biosciences) was added to each well of the
 plate and incubated at room temperature. The reaction was stopped using 50
 µl of 2M H<sub>2</sub>SO<sub>4</sub> after 20
 minutes of incubation. The optical density of the wells was then read
 using a 450/630 nm setting of a ELx808 plate reader (BioTek). The raw
 optical density (OD) values were then standardised using the sample to
 positive ratio. For this, a sample of pooled sera from three 24-day-old
 naïve birds from a separate experiment was used as a negative control on
 the ELISA plates. Similarly, a sample of pooled sera from three 24-day-old
 birds that were vaccinated at day 1 and 14 post-hatching during a separate
 experiment was used as a positive control. Because of the high number of
 serum samples, multiple 96-well plates were used for the ELISA procedure.
 Cross-plate standardisation was performed by calculating the
 sample-to-positive ratio, using the following formula:
 <strong>1.4.
 </strong><strong>qRT-PCR</strong>
 Total RNA from buccal and cloacal swab samples from H9N2 infected
 birds was extracted using the QIAamp Virus BioRobot MDx Kit (Qiagen) on a
 Biorobot Universal (Qiagen). Total RNA concentration was then measured
 using the NanoPhotometer NP80 (IMPLEN GmbH) to standardise across samples.
 A quantitative real-time PCR (qRT-PCR) using H9N2 matrix (M) gene primers
 was performed on samples using an M gene of known concentration (8.5 x
 10<sup>7</sup> copies/µl) as a standard, which was previously
 generated within the AIV group at the Pirbright Institute. For the qRT-PCR
 reactions, the Superscript III Platinum One-Step qRT-PCR Kit (Invitrogen)
 was used following the manufacturer’s instructions on a Quantstudio 5
 Real-Time PCR System (Thermo Fisher Scientific). The results were then
 analysed using the Quantstudio 5 software (Thermo Fisher
 Scientific). <strong>1.5.
 </strong><strong>RNA extraction from
 tissues</strong> RNA isolation from chicken
 tissue samples were carried as described in our previous work (18). 15 mg
 of tissue samples were transferred to tubes containing 600 µl of ice cold
 RLT lysis buffer (Qiagen) and 100 µl of 0.2 mm silica beads. Tissues were
 homogenised in a Mini-Beadbeater-24 (BioSpec) using 5 cycles of 1 minute
 each. Homogenate was then cooled on ice for 30 seconds, and RNA was
 extracted using the RNeasy Mini kit (Qiagen). On-column genomic DNA
 digestion was carried out using the RNase-Free DNase Set (Qiagen).
 Extracted RNA was eluted in 40 µl of nuclease-free water and RNA quality
 and integrity were assessed using an RNA ScreenTape (Agilent Technologies)
 and a 4200 TapeStation (Agilent Technologies), respectively. Samples were
 immediately stored at -80°C. <strong>1.6.
 </strong><strong>cDNA generation and 5’RACE
 PCR</strong> 5’RACE-ready cDNA was generated
 using the SMARTer kit (Takara) according to the manufacturer’s
 instructions, and then 5’RACE PCR was carried out to amplify B cell
 receptor genes. 5’RACE PCR involved the use of 7-bp barcoded forward
 primers for immunoglobulin C<sub>μ</sub>
 (NNNNNNNCACAGAACCAACGGGAAG), and immunoglobulin C<sub>γ</sub>
 (NNNNNNNCGGAACAACAGGCGGATAG). The 5’RACE PCR was carried out in accordance
 with our previous protocol (18). This involved, universal SMARTer kit
 reverse primers that were specific to the common 5’ adapter added during
 5’RACE cDNA synthesis. 5’RACE PCR reaction mixes contained: 5 µl of
 Phusion 5X Buffer (New England Biolabs), 0.5 µl of 10 mM dNTP, 0.5 µl of
 10 µM UPA-short primer, 0.5 µl of 2 µM UPA-long and 0.25 µl Phusion Hot
 Start Flex DNA Polymerase (New England Biolabs) were added to 15.25 µl
 nuclease-free water, for a total of 22 µl volume. To this, 0.5 µl of the
 10 µM gene-specific 7bp-barcoded primer and 2.5 µl of cDNA were added. The
 individual 25 µl volume 5’RACE PCR reactions were then carried out in
 96-well plates using the thermocycler program recommended by the 5’RACE
 kit (Takara), with 35 cycles of gene-specific amplification with an
 annealing temperature of 60°C. Barcoded PCR products were pooled and
 subjected to electrophoresis on a 1.4% agarose in 45 mM Tris-Borate/ 1mM
 EDTA (TBE) buffer gel containing 1:10,000 SYBR green (Sigma-Aldrich) at
 120 V for 35 minutes. The bands of the expected lengths were gel extracted
 and purified using the QIAquick Gel Extraction Kit (Qiagen).
 <strong>1.7. </strong><strong>DNA library
 preparation and sequencing</strong> Pooled
 barcoded PCR products were used to generate DNA libraries using the
 NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs).
 A NEBNext Library Quant Kit for Illumina (New England Biolabs) and a D1000
 DNA tape (Agilent Technologies) for the 4200 TapeStation (Agilent
 Technologies) were used to assess the quantity and quality of the DNA
 libraries. Library preparation and sequencing was carried out using an
 Illumina MiSeq platform by the Bioinformatics, Sequencing &
 Proteomics group at the Pirbright Institute.
 <strong>2.1. </strong><strong>Repertoire
 sequence data processing and analysis</strong> An
 in-house python package (available on GitHub:
 https://github.com/sgp79/reptools) was used to process the raw sequencing
 data using the same workflow established previously (18,19). Briefly, the
 software identifies the V and J gene ID(5)s to the sequences by BLAST
 (20). The algorithm then extracts the CDR3 sequences after a
 Smith-Waterman alignment (21), allowing for higher precision at the
 junctions (22). The output was then analysed using R (23) and unique
 clonal IDs were assigned to individual IgM and IgY sequences based on CDR3
 nucleotide identity alone. As chickens only exhibit one copy of the J
 genes, and BCR diversification occurs through the process of gene
 conversion, V and J identities were not used for assigning clonal identity
 (5).
description: # IgM and IgY CDR3 sequences from naive, vaccinated and/or infected white
 VALO chickens
 [https://doi.org/10.5061/dryad.9cnp5hqtq](https://doi.org/10.5061/dryad.9cnp5hqtq) ## Description of the data and file structure ### Files and variables #### File: IgY\_data\_submission.csv **Description:**  ##### Variables * bird: unique ID of individual bird from which the sequence was derived from * status: immunisation group identifier * tissue: tissue origin for the specific sequence * CDR3nt: IgY CDR3 nucleotide sequence identified in the specific tissue of the specific bird * CDR3aa: corresponding (translated) CDR3 amino acid sequence * count: number of sequence reads identified in the tissue sample #### File: IgM\_data\_submission.csv **Description:**  ##### Variables * bird: unique ID of individual bird from which the sequence was derived from * status: immunisation group identifier * tissue: tissue origin for the specific sequence * CDR3nt: IgM CDR3 nucleotide sequence identified in the specific tissue of the specific bird * CDR3aa: corresponding (translated) CDR3 amino acid sequence * count: number of sequence reads identified in the tissue sample