A novel approach for preparation of the antisera reagent for potency determination of inactivated H7N9 influenza vaccines.

BACKGROUND: The potency of inactivated influenza vaccines is determined using a single-radial immunodiffusion (SRID) assay and requires standardized reagents consisting of a Reference Antigen and an influenza strain-specific antiserum. Timely availability of reagents is a critical step in influenza vaccine production, and the need for backup approaches for reagent preparation is an important component of pandemic preparedness. OBJECTIVES: When novel H7N9 viruses emerged in China in 2013, candidate inactivated H7N9 influenza vaccines were developed for evaluation in clinical trials, and reagents were needed to measure vaccine potency. METHODS: We previously described an alternative approach for generating strain-specific potency antisera, utilizing modified vaccinia virus Ankara vectors to produce influenza hemagglutinin (HA)-containing virus-like particles (VLPs) for immunization. Vector-produced HA antigen is not dependent upon the success of the traditional bromelain-digestion and HA purification. RESULTS: Antiserum for H7N9 vaccines, produced after immunization of sheep with preparations of bromelain-HA (br-HA), was not optimal for the SRID assay, and the supply of antiserum was limited. However, antiserum obtained from sheep boosted with VLPs containing H7 HA greatly improved the ring quality in the SRID assay. Importantly, this antiserum worked well with both egg- and cell-derived antigen and was distributed to vaccine manufacturers. CONCLUSIONS: Utilizing a previously developed approach for preparing vaccine potency antiserum, we have addressed a major bottleneck encountered in preparation of H7N9 vaccine reagents. The combination of br-HA and mammalian VLPs for sequential immunization represents the first use of an alternative approach for producing an influenza vaccine potency antiserum.

Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis.

Ethylene is an important regulator of plant growth, development and responses to environmental stresses. Arabidopsis perceives ethylene through five homologous receptors that negatively regulate ethylene responses. RTE1, a novel gene conserved in plants, animals and some protists, was recently identified as a positive regulator of the ETR1 ethylene receptor. Here, we genetically analyze the dependence of ETR1 on RTE1 in order to obtain further insight into RTE1 function. The function of RTE1 was found to be independent and distinct from that of RAN1, which encodes a copper transporter required for ethylene receptor function. We tested the ability of an rte1 loss-of-function mutation to suppress 11 etr1 ethylene-binding domain mis-sense mutations, all of which result in dominant ethylene insensitivity due to constitutive signaling. This suppression test uncovered two classes of etr1 mutations -RTE1-dependent and RTE1-independent. The nature of these mutations suggests that the ethylene-binding domain is a possible target of RTE1 action. Based on these findings, we propose that RTE1 promotes ETR1 signaling through a conformational effect on the ethylene-binding domain.

Subcellular co-localization of Arabidopsis RTE1 and ETR1 supports a regulatory role for RTE1 in ETR1 ethylene signaling.

Ethylene is an important plant growth regulator perceived by membrane-bound ethylene receptors. The ETR1 ethylene receptor is positively regulated by a predicted membrane protein, RTE1, based on genetic studies in Arabidopsis. RTE1 homologs exist in plants, animals and protists, but the molecular function of RTE1 is unknown. Here, we examine RTE1 expression and subcellular protein localization in order to gain a better understanding of RTE1 and its function in relation to ETR1. Arabidopsis plants transformed with the RTE1 promoter fused to the beta-glucuronidase (GUS) reporter gene revealed that RTE1 expression partly correlates with previously described sites of ETR1 expression or sites of ethylene response, such as the seedling root, root hairs and apical hook. RTE1 transcript levels are also enhanced by ethylene treatment, and reduced by the inhibition of ethylene signaling. For subcellular localization of RTE1, a functional RTE1 fusion to red fluorescent protein (RFP) was expressed under the control of the native RTE1 promoter. Using fluorescence microscopy, RTE1 was observed primarily at the Golgi apparatus and partially at the endoplasmic reticulum (ER) in stably transformed Arabidopsis protoplasts, roots and root hairs. Next, a functional ETR1 fusion to a 5xMyc epitope tag was expressed under the control of the native ETR1 promoter. Immunohistochemistry of root hairs not only showed ETR1 residing at the ER as previously reported, but revealed substantial localization of ETR1 at the Golgi apparatus. Lastly, we demonstrated the subcellular co-localization of RTE1 and ETR1. These findings support and enhance the genetic model that RTE1 plays a role in regulating ETR1.

REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis.

Arabidopsis thaliana has five ethylene hormone receptors, which bind ethylene and elicit responses critical for plant growth and development. Here we describe a negative regulator of ethylene responses, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which regulates the function of at least one of the receptors, ETR1, in Arabidopsis. RTE1 was identified based on the ability of rte1 mutations to suppress ethylene insensitivity of the dominant gain-of-function allele etr1-2. rte1 loss-of-function mutants have an enhanced ethylene response that closely resembles the etr1 null phenotype. The etr1 rte1 double null mutant is identical to the etr1 and rte1 single null mutants, suggesting that the two genes act in the same pathway. rte1 is unable to suppress the etr1-1 gain-of-function allele, placing RTE1 at or upstream of ETR1. rte1 also fails to suppress gain-of-function mutations in each of the four other ethylene receptor genes. RTE1 encodes a previously undescribed predicted membrane protein, which is highly conserved in plants, animals [corrected] and protists but absent in fungi and prokaryotes. Ethylene treatment induces RTE1 expression, and overexpression of RTE1 confers reduced ethylene sensitivity that partially depends on ETR1. These findings demonstrate that RTE1 is a negative regulator of ethylene signaling and suggest that RTE1 plays an important role in ETR1 function.