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.

1.1. Experimental design and tissue samples

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 10⁶ 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).

1.2. Haemagglutination and haemagglutination inhibition assays

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.

1.3. ELISA

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 H₂SO₄ 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:

1.4. qRT-PCR

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 10⁷ 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).

1.5. RNA extraction from tissues

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.

1.6. cDNA generation and 5’RACE PCR

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_μ (NNNNNNNCACAGAACCAACGGGAAG), and immunoglobulin C_γ (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).

1.7. DNA library preparation and sequencing

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.

2.1. Repertoire sequence data processing and analysis

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).