Determination of the efficacy of a novel universal influenza vaccine in preventing high pathologic influenza infections in chickens or zoonosis in man

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.