Performing Department
Biomedical Sciences
Non Technical Summary
Project SummaryHigh pathogenic influenza virus such as H5, H7, and H9 hemagglutinin bearing strains represent a significant threat to poultry requiring their culling to prevent spread. Furthermore, strains such as H5 (Asia) and H7 (Asia and North America) are zoonotic and associated with human infection with death or high morbidity. Thus, these viruses are of significant concern. Here, we seek to extend our current work on a universal influenza vaccine for humans and swine and extend our work into whether this vaccine is efficacious in poultry. We also further wish to extend our work (currently concentrating on H1 and H3 viruses) protects humans from high pathologic infection of avian influenza viruses. If deemed efficacy, our vaccines could be used to vaccinate chickens/turkeys or stored for further outbreaks. Additionally, our vaccine could then be used in poultry workers and stored for future human pandemic strains. Here, we seek to generate preliminary data on the depth and breadth of protection in chickens and ferrets (human model) in support of obtaining funding for vaccine work in poultry or humans.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Specific AimsInfluenza remains a serious threat to humans, swine, and poultry due to: (1) the vast number of continuously circulating influenza strains within/between populations/species, (2) viral shifts/drifts that occur within the hosts, and the (3) difficulty in designing universal vaccines. Furthermore, while influenza vaccines can limit/prevent morbidity or mortality, they generally fail to prevent infection in swine/poultry or exhibit no to modest efficacy in humans or simply cannot be used in poultry. A significant cause of concern for the poultry industry and a threat to human health are the circulating strains of high pathologic avian influenza (HPAI) with H5, H7, and H9 hemagglutinin. H5N2 caused large-scale culls of poultry throughout the U.S. in 2015 ($1.2 billion loss in Iowa) and H7N9 is beginning to arrive/spread and necessitate new culls this year. Each of these HPAI strains, which exhibit potential zoonotic infection in humans (infections in Asia ongoing), represent a serious potential for severe pandemic infection. Currently available poultry influenza H5 strains may not protect from the circulating strains in poultry and there are no such vaccines available for humans (or H7 or H9 either). Thus, a universal influenza vaccine would certainly help limit the potential for these strains to cause widespread culls or human pandemic given they continue to circulate and are causing zoonosis in man.In mammals, influenza infection can generate strong (and sometimes broadly) neutralizing antibodies (nAbs) after viral clearance as evidenced by: (1) H1N1 A/PR8/34 infection of mice that induces IgM nAbs2, (2) human infections that generate broadly neutralizing hemagglutinin (HA) head binding antibodies (such as antibody clone 5J8 3,4 or C055), or (3) murine infections with H3N2 A/Aichi/2/68 that induce broadly neutralizing head IgG (such as antibody clone s139/16). Broadly neutralizing antibodies (bnAbs) are a key component of a future universal influenza vaccine and while new generation vaccines that target the HA stem can induce bnAbs 7-10, they still might not be comprehensive enough to protect from infection of poultry with H5, H7, and H9 strains (or cheap enough).We have recently discovered that vaccinating two different mammalian species (mice and horses) with equine H3N8 (live attenuated) led to the broadest nAbs profile to date (within strains: H1N1, H3N2 including H3N2v2, and H5N1 with further binding to H7, H9, and H13 HAs) and protected from multiple H1N1 and H3N2 influenza challenges. Moreover, the vast majority of vaccinated animals had protective HAI (HA inhibition) titers across the viral strains, exhibited similar microneutralization titers, and hybridomas derived from vaccinated animals had protective HAI titers to H1N1 and H3N2 viruses. If this vaccine should work in poultry, it could represent a significant advancement toward preventing zoonosis of these strains into humans. Alternatively, vaccination of poultry workers could protect from zoonosis.The underlying principle behind this proposal is to explore the host response to our candidate influenza vaccine and determine the correlates of protection, ways to bolster any afforded protection, and the best delivery platform to achieve robust and lasting immunity in poultry while further examining its potential to protect humans from HPAI. We know that our vaccine (1) elicits protective nAbs that block sialic acid binding in the HA head AND stalk regions, (2) doesn't bind to linear epitopes, and (3) appears to elicit antibodies of the IgM isotype. New preliminary data suggest our vaccine elicits protection through bnAbs directed to the HA receptor binding area (all HAs), nAbs directed to the esterase region 11 (all HA3s), and nAbs binding the stem (HA1, HA3, maybe more). These three sites suggest our vaccine has the broadest protection of ANY known human vaccines and escape mutations may be difficult for the virus. One dogma in the influenza field is that head binding antibodies are not broadly protective although there is ample evidence to dispute this 12 If H3N8 vaccination leads to protection afforded by head binding antibodies, this would be one of the first immunogens capable of eliciting it without first causing disease. However, we do not know what the efficacy would be in poultry or whether the vaccine's elicitation of the bnAbs in mammals could protect from HPAI challenge.The specific hypotheses to be tested are: (1) use of an equine HA3 immunogen will elicit protective bnAbs in vaccinated poultry, (2) and the observed bnAbs we have observed in horses and mice will extend to protection of humans from HPAI. The major innovations of this proposal are the use of a unique immunogen that elicits broadly bnAbs as a potential vaccine candidate while not eliciting antibodies that would limit their use in poultry due to low efficacy or causing an inability to determine if broilers were infected by circulating strains (e.g. H5N2 vaccination would be impossible to determine from natural infection and thus restrict these broilers from being sold overseas).The scope of this proposal comprises the following two specific aims to obtain preliminary data for grants:Aim 1. Optimize influenza antigen for enhancing protective titers in poultry- Here, we propose to test our vaccine in chickens. However, we must first determine the optimal antigen delivery for highest protective Ab titers (route, type such as subunit, and dosage). We will then evaluate the level of protective Ab titers and breadth of neutralization.Aim 2. Examine the durability of anti-influenza immunity in ferrets (human model) against HPAI- Our preliminary data suggest H3N8 induces broad and protective titers of HAI but we don't know whether the protection or its durability against HPAI. We are funded by Merck for examination of protection from H1N1 and H3N2 viruses but demonstrated protection from potential pandemic strains would help us get additional federal funding. We believe we would have a good chance of this given current flock outbreaks of this but first need the supporting preliminary data.
Project Methods
3. Experimental LayoutA.1. AIM1: Optimize antigen delivery for enhancing protective titersA.2 Experimental design, hypotheses, and questions:a. Qualify our IV and control strains for vaccination. Before we can commence our study, we will first need to generate IV strains or rHAs for comparisons along with the necessary control vaccines. Ideally, we would like to compare LAIV (equine) against LAIV (e.g. H3N2 Texas3/12) or IV against IV.b. Optimize for best levels of nAbs or bnAbs for protection. We hypothesize that LAIV H3N8 vaccine will be more efficacious than IV but IV will be efficacy enough to utilize (easier to deliver in poultry). We believe this for a number of reasons: (1) infection and limited replication will induce a stronger inflammatory response than protein alone; (2) LAIV carries additional proteins such as NP that is fairly conserved between strains; (3) infection can lead to prolonged antigen stimulation. However, in humans there is no difference between LAIV and IV and rHA during vaccinations. There are many pros and cons with LAIV versus IV versus rHA to consider. That being said, from regulatory prospective for approval in poultry IV or rHA would be more ideal. Furthermore, the heat tolerance of IV or rHA and cost would certainly make them a better vaccine over a LAIV for poultry. Thus, if we determine that our IV or rHA induces as similar and as broad of protection as LAIV, we will push the best vaccine forward in that format. We could also come back to determine whether LAIV actually does provide better protection if our challenge study results do not match with our observed HAI or microneutralization titers. For the purposes of obtaining additional funding, we need to demonstrate that our vaccine elicits protective nAbs in poultry. If we obtain this data, funding requests will ask to determine for further analysis of how broad the response is, the rigor and how long immunity lasts, expansion to testing in turkeys as well, and further challenge studies against wild-type HPAI. Whether equine HA3 can protect poultry is the fundamental question we are asking in this proposal. Clearly, we have ample preliminary data that suggests this vaccine works in multiple species. Thus, we hypothesize based on early preliminary data and extrapolations from other species that equine HA3 will protect from multiple HPAI. We will first test against lethal challenge since the read-out is immediate. Follow-on studies could then use sublethal infections to determine whether we are obtaining sterilizing immunity or just protection from disease. Lung samples (of equal weight between animals) will be held in RNAlater, extracted, and virus determined by qRT-PCR (HA and NP). While we would certainly love to test multiple challenge strains, we believe given the time, cost, and resources, that protection from a few HPAI candidate viruses would demonstrate proof of principle.A.3 Experimental Detail:a. Optimize the dosage, route, timing, and best vaccine platform for poultryi. Test the immunogenicity of our vaccines in poultry. Chickens (newly hatched and 2 weeks old) will be housed in barrier housing and will be inoculated with 106-8 TCID50 of our equine vaccine, commercial equine vaccine (similar TCID50), or rHAs (25mcg-250mcg) into the nasal flares or IM. Chickens will be bled on day 0, 7, 14, and 28 days post-vaccination. Total specific antibodies will be examined to recombinant H5, H7, and H9s. Hemagglutinin inhibition assay (HAI) will be performed using chemically killed HPAI H5N1. When approved for BSLII use, we will then expand this to testing against other HPAI as well as use microneutralization assays for further rigor.ii. Determine the breadth of bnAb protection. Once we have determined the optimal vaccine type, delivery, and dosage, we will begin to test for protective titers from vaccinated poultry across H5 and H7 HPAI challenges. We will vaccinate 3 chickens in duplicate replications using a H3N8 and control vaccine. Sera will be obtained after the first and second vaccination and tested against a panel of HPAI viruses. Chickens will then be challenged with lethal and then sublethal challenges in the BSLIII at CVM.B.1 Aim 2. Examine the durability of anit-influenza immunity through controlled antigen releaseB.2 Experimental design, hypotheses, and questions:a. Compare our prior H1 and H3 results to HPAI in ferrets. A central dogma in the vaccine field is that ferrets are the accepted testing model for human vaccine efficacy. Here, we not only wish to test our vaccine in ferrets. To that end, we will compare similar dosage optimization studies in ferrets and challenge these with human influenza strains. These data will also help with seeking FDA approval for human clinical trials should our vaccine prove efficacious. nAb/bnAbs responses and protection will be examined followed by challenge studies.b. What are the correlates of protection? Here, we will concentrate our main efforts on HAI or microneutralization titers since they are the generally recognized correlate of protection. However, our vaccine uses a full inactivated equine H3N8 virus and nAbs to other proteins, like NP, could contribute to protection. In addition or if we only use the rHA, the hemagglutinin protein contains multiple T-cell epitopes and thus cellular immunity could certainly play a role in any observed protection. We hypothesize, based on a wealth of data in humans and mice, CD4 T-cells, rather than CD8 T-cells, induced by our vaccine will be critical in the observed protection against heterosubtypic challenge.B.3 Experimental Detaila. Test the durability of protection in using influenza vaccination/infection - In Objective 1: we will optimize the vaccine platform, the dose, and timing for eliciting strong antibody responses to influenza in poultry. We are currently funded to find these data in mice, horses, and ferrets and thus could port these data to application of protection of vaccinee ferrets from HPAI. We will have the data already determined as to what optimal route and dose to use for H1 and H3 and will then extend these to HPAI using this seed funding.i. Examine the depth of protection to HPAI. As with the poultry, sera from vaccinated ferrets will be tested for neutralization titers toward chemically inactivated H5N1. These HAI assays will then be expanded to other HPAI and microneutralization assays after approval to use the BSLIII.ii. Protection from lethal challenge - Survival will be assessed after vaccination (2-3x) and challenge using ferrets. We see no issues with testing these vaccines at Iowa State University and our IACUC protocol is pending approval of these modifications. We will use n=3 for each strain comparing our vaccine candidate against a control vaccine group and a no-vaccine group for preliminary data for grants or expand to two replicates for publications.Timeline- We have the LAIV in house and have generated the recombinant HAs (equine H3 and a control H3 from H3N2 Texas/3/12). We also have the technology to make split vaccines from egg expanded equine and human H3 viruses. We believe that we can begin efficacy trials in poultry and ferrets early within the funding cycle with potential to shift toward active challenge studies when approved for BSLIII use. This would likely happen toward the end of the funding cycle.