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 methodsVirusesThe 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.VaccinesAn 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 designThree-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 vaccinesTo 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 assayHemagglutination 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 ELISAPre-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 chickensThe 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).References1. 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.

本数据集收录了相关出版物中报道的个体数据点：Spackman E, Suarez DL, Lee CW, Pantin-Jackwood MJ, Lee SA, Youk S, Ibrahim S. 评估灭活疫苗及RNA颗粒疫苗对北美2.3.4.4b H5高致病性禽流感病毒在鸡群中的免疫效果。Vaccine. 2023年11月30日；41(49):7369-7376. doi: 10.1016/j.vaccine.2023.10.070. Epub 2023年11月4日. PMID: 37932132.方法描述病毒高致病性禽流感病毒（HPAIV）株A/turkey/Indiana/22-003707-003/2022 H5N1（TK/IN/22）和A/Gyrfalcon/Washington/41088/2014 H5N8（GF/WA/14）分别采用标准程序在孵化特定病原体无菌（SPF）鸡胚中繁殖和滴定，滴度通过Reed-Muench方法确定。疫苗一种内部疫苗通过从头合成TK/IN/22的HA基因，并将其修改为低致病性（LP），然后将其置于PR8骨架中，使用rg方法如上所述进行制备。该疫苗（SEP-22-N9）包含PR8的6个基因和一个从头合成的A/blue winged teal/Wyoming/AH0099021/2016（H7N9）的N9 NA。rg病毒通过0.1%β-丙内酯处理灭活。疫苗在室温下使用Montanide ISA 71 VG（Seppic Inc.，Fairfield，NJ）佐剂，在L5M-A高剪切混合器（Silverson Machines, Inc.，East Longmeadow，MA）中以1,000rpm的转速混合30秒，然后以4,000rpm的转速混合3分钟，使用符合佐剂制造商说明书的乳化筛。安慰剂疫苗采用无菌磷酸盐缓冲盐水按上述方法制备。商业疫苗由制造商提供。商业灭活疫苗（1057.R1序列590088）（rgH5N1）（Zoetis Inc.，Parsippany，NJ）使用GF/WA/14（2.3.4.4c HA基因）和包括PR8的NA在内的剩余7个基因片段制备。Sequivity疫苗（序列V040122NCF）（RP）（Merck and Co. Inc.，Rahway，NJ）是他们更新的复制受限的 alphavirus 病毒载体疫苗，表达TK/IN/22 H5 HA（修改为低致病性LP）。挑战研究设计从内部鸡群中获取了3周大的、混合性别的SPF白腿鸡（Gallus gallus domesticus），并将其随机分配到疫苗组。所有疫苗均通过皮下途径在颈部后部给药。商业疫苗按照制造商的说明给予（每个0.5ml）。内部疫苗以每只512个血凝单位在0.5ml的剂量下给予。疫苗接种后3周，鸡群被挑战感染TK/IN/22 6.7 log10 50%卵感染剂量（EID50），通过鼻腔内途径给予0.1ml。在挑战后2天、4天和7天，从所有鸟类收集口腔咽喉（OP）和泄殖腔（CL）拭子。在1DPC时，从死亡的安慰剂接种疫苗者收集拭子。为了评估基于抗体的DIVA-VI测试，在7DPC、10DPC和14DPC时从RP和SEP-22-N9接种疫苗组收集血液以进行血清采集，因为SEP-22-N9疫苗不会诱导N1抗体，而RP疫苗不会诱导N1或NP蛋白的抗体。在14DPC后记录死亡率和发病率，之后剩余的鸟类被安乐死。如果鸟类极度衰弱或出现神经症状，则被安乐死，并在下一次观察时间点被计入死亡率，用于平均死亡时间计算。评估基于prime-boost顺序的RP和灭活疫苗的抗体滴度为了确定基于RP疫苗和灭活疫苗接种顺序的抗体水平是否存在差异，20只鸡（挑战研究中的鸡的后代）在间隔三周的时间内分别给予每种疫苗一次剂量（补充表1）。首次剂量在3周大时使用RP或SEP-22-N9疫苗，如上所述进行给药。然后在三周后（六周大时）再次给予相同疫苗或另一种疫苗的第二次剂量。在第二次疫苗接种后三周（九周大时），对所有鸟类进行血清采集。通过如下的血凝抑制（HI）试验对抗体进行量化，使用同源抗原（TK/IN/22）。定量rRT-PCR（qRRT-PCR）RNA从OP和CL拭子中提取，使用MagMax（Thermo Fisher Scientific，Waltham，MA）磁珠提取试剂盒，并按照Das等人（2）描述的修改进行。在QuantStudio 5（Thermo Fisher Scientific）上进行了如前所述的定量实时RT-PCR。从滴定的TK/IN/22库存中生成标准曲线，并使用实时PCR仪的软件计算滴度等效物。血凝抑制试验按照标准程序进行。所有预挑战血清均测试了挑战病毒。接种rgH5N1疫苗的鸟类的血清也测试了疫苗抗原GF/WA/14。滴度低于8的被认为是非特异性结合，因此为阴性。商业ELISA从30只鸡的预疫苗接种血清中测试以确认不存在禽流感病毒抗体，使用商业AIV抗体ELISA（IDEXX laboratories，Westbrook，ME），按照制造商的说明进行。RP疫苗组（此处使用的唯一一种不会诱导NP抗体的疫苗）的预挑战和挑战后血清也使用此ELISA进行测试，以表征挑战后抗-NP抗体的检测。酶联凝集素测定（ELLA）和神经氨酸酶抑制（NI）用于检测挑战鸡血清中的N1抗体ELLA试验按照先前发表的方案进行，进行了一些小的修改（4）。将吸收数据拟合到Prism 9.5（GraphPad Software LLC，Boston，MA）的非线性回归曲线，以确定有效浓度，并使用98%有效浓度（EC98）的N1源病毒进行NI试验。为了检测N1抗体，使用优化的N1 NA浓度，将预挑战、7DPC、10DPC和14DPC时收集的安慰剂、SEP-22-N9和RP接种疫苗组的血清样本在56°C下热灭活一小时，并使用样品稀释缓冲液进行1:20和1:40的稀释。将浓度为2X EC98的N1源病毒等体积添加到每个稀释的血清样本中。然后将100µl的血清-病毒混合物添加到上述NA试验中描述的卵清蛋白包被板中。然后将卵清蛋白板与血清-病毒混合物在37°C下过夜（约17-19小时）。对于NI试验的剩余步骤，遵循上述NA试验方案。通过从100减去NA活性百分比来计算单个血清样本的NI活性百分比。为了计算NA活性百分比，从样本吸收值中减去平均背景吸收值。然后将结果除以仅含NA源病毒（无血清）的平均值，然后乘以100。通过将每个稀释度测试（1:20和1:40）对应的疫苗组预挑战血清中获得的平均值的三倍标准差加到平均值上，计算挑战后从鸡中检测N1抗体的NI活性截止值。参考文献1. Kapczynski DR, Sylte MJ, Killian ML, Torchetti MK, Chrzastek K, Suarez DL. 商业火鸡在接种灭活或重组H5疫苗后对2015年美国H5N2亚型2.3.4.4高致病性禽流感病毒的免疫保护。兽医免疫学和免疫病理学。2017年；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. 去除与泄殖腔拭子和组织相关的实时逆转录聚合酶链反应（RT-PCR）抑制剂，以改善通过RT-PCR诊断禽流感病毒。兽医诊断研究杂志。2009年；21(6):771-8.3. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. 开发一种实时逆转录聚合酶链反应（RT-PCR）检测A型流感病毒和禽H5和H7血凝素亚型。临床微生物学杂志。2002年；40(9):3256-60.4. Bernard MC, Waldock J, Commandeur S, Strauss L, Trombetta CM, Marchi S, et al. 验证基于酶联凝集素测定（ELLA-NI）的神经氨酸酶抑制（NI）试验标准操作程序（SOP）用于量化N1流感抗体以及使用校准器提高ELLA-NI在反向遗传学病毒和重组神经氨酸酶抗原中的重现性：FLUCOP协作研究。免疫学前沿。2022年；13:909297. Epub 2022/07/06.