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Lee et al HVT HPAI vaccine in chickens data.xlsx (47.55 kB)

Data from: Efficacy of commercial recombinant HVT vaccines against a North American clade H5N1 Highly Pathogenic Avian Influenza Virus in chickens

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posted on 2024-04-24, 21:36 authored by Erica Spackman, Jiho Lee, David L. Suarez, Scott A. Lee, Chang-Won Lee, Taejoong Kim

Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both breeds in the COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken breeds. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both breeds of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.


Viruses. All procedures using infectious material were reviewed and approved by the Institutional Biosafety Committee of US National Poultry Research Center (USNPRC), US Department of Agriculture-Agricultural Research Service, Athens, GA. The HPAI virus isolate A/turkey/Indiana/22-003707-003/2022 H5N1 (TK/IN/22) was provided by Dr. Mia Torchetti, National Veterinary Services Laboratories, US Department of Agriculture-Animal and Plant Health Inspection Service, Ames, IA. The A/Vietnam/1203/2004 H5N1 HPAI virus (Viet/04), A/Whooper Swan/Mongolia/244/2005 H5N1 (WS/Mongolia/05) HPAI virus, and A/Flycatcher/CA/14875-1/1994 H7N1 low pathogenic avian influenza virus isolates were provided by the repository at the USNPRC. Virus isolates were propagated and titrated in SPF embryonating chicken eggs using standard procedures [1]. Titers were determined using the Reed-Muench method [2].

Vaccines. Two commercial rHVT-H5 vaccines were selected because they are licensed in the US (and may be licensed elsewhere) and were supplied by the manufacturers: 2.2-HVT (Vectormune HVT AIV, Ceva Animal Health LLC, Lenexa, KS) (serial 395-134); and COBRA-HVT (Vaxxitek HVT+IBD+H5, Boehringer-Ingelheim Animal Health USA, Ridgefield, CT) (serial EW003). The amino acid similarity between the vaccine antigens and the challenge virus HA1 was 91.7% (COBRA-HVT) and 91.2% (2.2-HVT).

Challenge study design. All animal work was reviewed and approved by the USNPRC Institutional Animal Care and Use Committee. Mixed sex, SPF WL chickens (Gallus gallus domesticus) were obtained at hatch from in-house flocks. Broiler chicken eggs were obtained from a commercial hatchery at 18 days of incubation prior to administration of any in ovo vaccines and were hatched on-site. All birds were randomly assigned to vaccine groups based on breed. Vaccine groups are shown in Table 1. All vaccines were prepared and administered on the day of hatch by the subcutaneous route at the nape of the neck in accordance with the manufacturer’s instructions (0.2ml per chicken). Serum was collected from all chickens 25 days post vaccination to evaluate the antibody response to the vaccines.

Four weeks post vaccination (four weeks of age) chickens were challenged with a target dose 6.0log10 50% egg infectious doses (EID50) per bird of TK/IN/22 in 0.1ml by the intrachoanal route (titration of the challenge virus after dilution confirmed the challenge dose to be 6.7log10 EID50 per bird). Oropharyngeal and CL swabs were collected from all birds at 2-, 4-, and 7-days post challenge (DPC). Swabs were also collected from dead and euthanized birds.

To evaluate antibody-based DIVA-VI tests, serum was collected at 7-, 10- and 14DPC. Mortality and morbidity were recorded for 14DPC. Surviving birds were euthanized at 14DPC. If birds were severely lethargic or presented with neurological signs, they were euthanized and were counted as mortality at the next observation time for mean death time calculations. Euthanasia was performed by cervical dislocation in accordance with American Veterinary Medical Association guidelines.

Quantitative rRT-PCR (qRRT-PCR). RNA was extracted from OP and CL swabs using the MagMax magnetic bead extraction kit (Thermo Fisher Scientific, Waltham, MA) with the wash modifications as described by Das et al., [3]. Quantitative real-time RT-PCR was conducted as described previously [4] on a QuantStudio 5 (Thermo Fisher Scientific) instrument. 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 (HI) assays were run in accordance with standard procedures [5]. All pre-challenge sera collected at 25 days post vaccination were tested against the challenge virus and the closest isolates available to the vaccine antigens. The serum from the 2.2-HVT group was tested against WS/Mongolia/05 (99.3% similarity) and the serum from the COBRA-HVT group was tested against Viet/04 (98.2% similarity). Titers of eight or below were considered negative.

Commercial ELISA. A commercial AIV antibody ELISA (AI Ab Test, IDEXX laboratories, Westbrook, ME) was used in accordance with the manufacturer’s instructions. Sera were tested to detect anti-NP antibodies pre-challenge (25days pos-vaccination) and at 7-, 10- and 14DPC.

Enzyme-linked lectin assay (ELLA) for detection of neuraminidase inhibition (NI) antibody. The ELLA was performed as previously described with minor modifications [6, 7]. Briefly, the NA activity of a beta-propiolactone inactivated H7N1 virus (A/Flycatcher/CA/14875-1/1994) was quantified to determine the effective concentration (EC) of antigen. The 98% EC (EC98) of antigen was subsequently used for the ELLA-NI assays. For ELLA-NI assay, the antigen and serum mixture was incubated overnight (approximately18hr) at 37°C and the NA activity was determined following the procedure as described in Spackman et al. [7]. The average background absorbance value was subtracted from the sample absorbance value then that value was divided by the average values of wells with only NA antigen. This value was multiplied by a factor of 100 to calculate the percent NA activity. The percent NI activity of individual serum samples was determined by subtracting the percent NA activity from 100%. A cut-off value for positive NI activity was determined by adding three standard deviations to the mean NI activity of pre-challenge sera (i.e., NA antibody negative sera) of each corresponding group of chickens at 7-, 10- and 14DPC. Each serum was tested at dilutions of 1:20 and 1:40.


1. Spackman E, Killian ML. Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs. Methods Mol Biol. 2020;2123:149-64. Epub 2020/03/15.

2. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. American Journal of Hygiene. 1938;27:493-7.

3. 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.

4. 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.

5. Spackman E, Sitaras I. Hemagglutination Inhibition Assay. Methods Mol Biol. 2020;2123:11-28. Epub 2020/03/15.

6. 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 20220617.

7. 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 H5 highly pathogenic avian influenza virus in chickens. Vaccine. 2023. Epub 20231104.


USDA-ARS: 6040-32000-081-00D

USDA-APHIS: #60-6040-2-008


Data contact name

Spackman, Erica

Data contact email


Ag Data Commons

Intended use

Data may be used in meta-studies on vaccines for avian influenza virus. Serological data can be used to validate antibody-based tests for identifying animals that have been infected after vaccination (DIVA). Data can be used for disease spread modeling after vaccination and/or to model detection of infected animals post vaccination.

Use limitations

Comparison with other data should ensure that methods are compatible. Parameters in modeling studies should account for variability among avian influenza virus isolate infectivity in the field as well as lower vaccine efficacy in field conditions.

Temporal Extent Start Date



  • Non-geospatial

Geographic location - description

North America

ISO Topic Category

  • farming
  • health

National Agricultural Library Thesaurus terms

vaccines; Influenza A virus; viruses; turkeys; specific pathogen-free animals; White Leghorn; broiler chickens; hemagglutinins; genes; antigens; viral load; cloaca; vaccination; lectins; enzyme inhibition; sialidase; antibody detection; enzyme-linked immunosorbent assay; antibodies; biosafety; veterinary services; plant health; chicken eggs; animal health; amino acids; experimental design; Institutional Animal Care and Use Committee; Gallus gallus; flocks; hatcheries; neck; blood serum; antibody formation; eggs; titration; mortality; morbidity; death; cervical dislocation; guidelines; RNA; magnetism; quantitative polymerase chain reaction; reverse transcriptase polymerase chain reaction; computer software; hemagglutination inhibition test; hemagglutination; lactones; absorbance; wells; standard deviation; models; pathogenicity

OMB Bureau Code

  • 005:18 - Agricultural Research Service

OMB Program Code

  • 005:040 - National Research

ARS National Program Number

  • 103

ARIS Log Number


Pending citation

  • Yes

Public Access Level

  • Public

Preferred dataset citation

Pending. Manuscript has been submitted and is in review.