Data from: Efficacy of Inactivated and RNA Particle Vaccines in Chickens Against Clade 2.3.4.4b H5 Highly Pathogenic Avian Influenza in North America

Tabulated individual data points for data reported in the associated publication: Spackman E, Suarez DL, Lee CW, Pantin-Jackwood MJ, Lee SA, Youk S, Ibrahim S. Efficacy of inactivated and RNA particle vaccines against a North American Clade 2.3.4.4b H5 highly pathogenic avian influenza virus in chickens. Vaccine. 2023 Nov 30;41(49):7369-7376. doi: 10.1016/j.vaccine.2023.10.070. Epub 2023 Nov 4. PMID: 37932132.

Description of methods

Viruses

The highly pathogenic avian influenza virus (HPAIV) isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) and A/Gyrfalcon/Washington/41088/2014 H5N8 (GF/WA/14) isolate were each propagated and titrated in embryonating specific pathogen free (SPF) chicken eggs using standard procedures and titers were determined using the Reed-Muench method.

Vaccines

An in-house vaccine was produced by de novo synthesizing the HA gene of TK/IN/22 that was modified to be low pathogenic (LP) and placing it in a PR8 backbone using rg methods as described . The vaccine (SEP-22-N9) contained 6 genes from PR8 and a de novo synthesized N9 NA from A/blue winged teal/Wyoming/AH0099021/2016 (H7N9). The rg virus was inactivated by treatment with 0.1% beta-propiolactone. Vaccines were produced with Montanide ISA 71 VG (Seppic Inc., Fairfield, NJ) adjuvant at ambient temperature in a L5M-A high shear mixer (Silverson Machines, Inc., East Longmeadow, MA) for 30sec at 1,000rpm, then for 3min at 4,000rpm using an emulsifying screen in accordance with the adjuvant manufacturer’s instructions.

Sham vaccine was prepared in-house using sterile phosphate buffered saline as described above.

Commercial vaccines were supplied by the manufacturers. The commercial inactivated vaccine (1057.R1 serial 590088) (rgH5N1) (Zoetis Inc., Parsippany, NJ) was produced with the GF/WA/14 (clade 2.3.4.4c HA gene) and the remaining 7 gene segments including the NA from PR8 (1). The Sequivity vaccine (serial V040122NCF) (RP) (Merck and Co. Inc., Rahway, NJ) is an updated version of their replication restricted alphavirus vector vaccine that expresses the TK/IN/22 H5 HA (modified to be low pathogenic LP).

Challenge study design

Three-week-old, mixed sex, SPF white leghorn chickens (Gallus gallus domesticus) were obtained from in-house flocks and were randomly assigned to vaccine groups.

All vaccines were administered by the subcutaneous route at the nape of the neck. Commercial vaccines were given at the volumes instructed by the manufacturer (0.5ml each). In-house vaccine was given at a dose of 512 hemagglutination units per bird in 0.5ml. Three weeks post vaccination chickens were challenged with 6.7 log₁₀ 50% egg infectious doses (EID₅₀) of TK/IN/22 in 0.1ml by the intrachoanal route.

Oropharyngeal (OP) and cloacal (CL) swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized sham vaccinates at 1DPC.

To evaluate antibody-based DIVA-VI tests, blood for serum was collected from the RP and SEP-22-N9 vaccinated groups at 7, 10 and 14DPC because the SEP-22-N9 vaccine does not elicit antibodies to N1 and the RP vaccine does not elicit antibodies to the N1 or NP proteins.

Mortality and morbidity were recorded for 14DPC after which time the remaining birds were euthanized. If birds were severely lethargic or had neurological signs they were euthanized and were counted as mortality at the next observation time for mean death time calculations.

Evaluation of antibody titers based on prime-boost order with the RP and inactivated vaccines

To determine if there was a difference in antibody levels based on the order of vaccination with the RP vaccine and an inactivated vaccine, groups of 20 chickens (hatch-mates of the chickens in the challenge study) were given one dose of each vaccine three weeks apart (Supplementary Table 1). The first dose was administered at three weeks of age using the RP or SEP-22-N9 vaccine as described above. Then a second dose of either the same vaccine or the other vaccine was administered three weeks later (six weeks of age). All birds were bled for serum three weeks after the second vaccination (nine weeks of age). Antibody was quantified by hemagglutination inhibition (HI) assay as described below using the homologous antigen (TK/IN/22).

Quantitative rRT-PCR (qRRT-PCR)

RNA was extracted from OP and CL swabs using the MagMax (Thermo Fisher Scientific, Waltham, MA) magnetic bead extraction kit with the modifications described by Das et al., (2). Quantitative real-time RT-PCR was conducted as described previously (3) on a QuantStudio 5 (Thermo Fisher Scientific). A standard curve was generated from a titrated stock of TK/IN/22 and was used to calculate titer equivalents using the real time PCR instrument’s software.

Hemagglutination inhibition assay

Hemagglutination inhibition assays were run in accordance with standard procedures. All pre-challenge sera were tested against the challenge virus. Sera from birds vaccinated with the rgH5N1 vaccine were also tested against the vaccine antigen, GF/WA/14. Titers of 8 or below were considered non-specific binding, therefore negative.

Commercial ELISA

Pre-vaccination sera from 30 chickens were tested to confirm the absence of antibodies to AIV with a commercial AIV antibody ELISA (IDEXX laboratories, Westbrook, ME) in accordance with the manufacturer’s instructions. Pre- and post-challenge sera from the RP vaccine group (the only vaccine utilized here that does not induce antibodies to the NP) were also tested with this ELISA to characterize the detection of anti-NP antibodies post-challenge.

Enzyme-linked lectin assay (ELLA) and neuraminidase inhibition (NI) to detect N1 antibody in serum from challenged chickens

The ELLA assay was performed in accordance with a previously published protocol with minor modifications (4). Absorbance data were fit to a non-linear regression curve with Prism 9.5 (GraphPad Software LLC, Boston, MA) to determine the effective concentration, and the 98% effective concentration (EC₉₈) of the N1 source virus was subsequently used for NI assays.

To detect N1 antibody with the optimized N1 NA concentrations, serum samples from the sham, SEP-22-N9, and RP vaccinated groups collected pre-challenge, 7, 10 and 14DPC, were heat inactivated at 56°C for one hour and diluted 1:20 and 1:40 using sample dilution buffer. Equal volumes of the N1 NA source virus at a concentration of 2X EC₉₈ was added to each of the diluted serum samples. Then 100µl of the serum-virus mixture was added to the fetuin coated plates after the fetuin plates were washed as described above for the NA assay. Fetuin plates with the serum-virus mixture were then incubated overnight (approximately 17-19hr) at 37°C. The NA assay protocol described above was followed for the remaining NI assay steps.

The percent NI activity of individual serum samples was determined by subtracting percent NA activity from 100. To calculate the percent NA activity, the average background absorbance value was subtracted from the sample absorbance value. The result was then divided by the average value of the NA source virus only (no serum) wells then multiplying by 100. A cut-off value for NI activity for positive detection of N1 antibody from chickens post-challenge was calculated by adding three standard deviations to the mean value obtained from pre-challenge sera of corresponding vaccine group for each dilution tested (1:20 and 1:40).

References

1. Kapczynski DR, Sylte MJ, Killian ML, Torchetti MK, Chrzastek K, Suarez DL. Protection of commercial turkeys following inactivated or recombinant H5 vaccine application against the 2015U.S. H5N2 clade 2.3.4.4 highly pathogenic avian influenza virus. Vet Immunol Immunopathol. 2017;191:74-9. Epub 2017/09/13. doi: 10.1016/j.vetimm.2017.08.001.

2. Das A, Spackman E, Pantin-Jackwood MJ, Suarez DL. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR. Journal of Veterinary Diagnostic Investigation. 2009;21(6):771-8.

3. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Journal of Clinical Microbiology. 2002;40(9):3256-60.

4. Bernard MC, Waldock J, Commandeur S, Strauss L, Trombetta CM, Marchi S, et al. Validation of a Harmonized Enzyme-Linked-Lectin-Assay (ELLA-NI) Based Neuraminidase Inhibition Assay Standard Operating Procedure (SOP) for Quantification of N1 Influenza Antibodies and the Use of a Calibrator to Improve the Reproducibility of the ELLA-NI With Reverse Genetics Viral and Recombinant Neuraminidase Antigens: A FLUCOP Collaborative Study. Front Immunol. 2022;13:909297. Epub 2022/07/06.