Performing Department
Biomedical Sciences
Non Technical Summary
Influenza virus is a significant pathogen and causes substantial suffering and high death rates each year. While vaccines do exist for influenza, they have a lot of limitations as they must be re-derived each year and cannot prevent infections of influenza with late variations that allow for viral escape from the vaccines. While most vaccines have used the difficult target of the HA stalk region as immunogens, progress has been slow although there are some early hints of success. We recently discovered an alternative immunogen using equine HA3 antigen that generates strong antibody mediated protection in horses and experiment mice by targeting the more variable HA head. Antibodies from these animals can neutralizing multiple influenza strains. This vaccine antigen appears to have a unique conformation that may allow us to use this as a future vaccine.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
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. While we understand 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, we don't quite yet know the protective site(s). 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(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 or swine vaccine 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. 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. Very early studies also suggest swine generate protective heterosubtypic HAI after vaccination. However, more qualifying work in protection efficacy, VAERD, vaccine delivery, and correlates of protection need to be done. Thus, we have a series of objectives for this qualification of this vaccine:Objective 1: Develop and optimize an influenza vaccine based on the equine HA3 antigen We know from preliminary data that vaccination with a H3N8 based equine vaccine generates strong HAI/neutralizing antibodies in horses and protects mice from challenge after vaccination. Swine are also susceptible to VAERD when using inactivated vaccines. Thus, we believe that a LAIV that displays the equine HA3 antigen could be efficacious in swine but IV, if determined to be safe, would be more cost effective for the industry. To develop this, we will test our original vaccine formulation as a LAIV or IV vaccine against a LAIV using a seed stock with internal swine influenza proteins but containing HA3 or HA1 from a circulating strain.Objective 2: Test the efficacy of the vaccine against multiple strains of swine influenza We will test the bnAb profile from the sera in the prior objective. Here, our vaccine candidates will be tested for protection from disease versus protection from infection after vaccination and challenge. We will also assess for any VAERD and further develop the vaccine candidate that protects from the most strains with no VARED.Objective 3: Determine the correlates of protection for our vaccine We know from preliminary data that cross-protective bnAbs occur toward the head and stem regions of the hemagglutinin but the exact locations are not known (no complete inhibition of known bnAbs with our Abs). Furthermore, our LAIV could induce important cellular immunity as Abs toward NP are known to coordinate with antiviral CD4 T-cells for heterosubtypic immunity in humans. Thus, if our recombinant HA vaccine induces similar levels of Abs as our LAIV, missing or reduced cellular immunity could impair the protection afforded. Thus, we will dissect the correlates of protection in swine with concentration on defining location of bnAbs (with comparisons to mice) and on cellular immunity using adoptive transfers of memory populations to better isolate their contributions to any protection. Understanding these variables, could allow for even better second-generation vaccinations while also allowing us to examine how immunological maturation alters the immune response/level of protection afforded.Objective 4: Examine whether antigen delivery can enhance or prolong the level of observed broadly protective immunity As we previously stated, we know that our vaccine induces mainly bnAbs that are IgM and that influenza vaccination generally wanes after a year. Here, we will seek to determine whether we can induce isotype switching of bnAbs by manipulation of antigen loads to alter humoral somatic hypermutation or cellular responses while also examining the effect of the duration of immunity (while potentially bolstering it). Our delivery device senses waning immunity to delivery a boosting device. While our first generation system is large, we are making great strides in reducing the size of the device for future use in humans. Our device is biodegradable, can be placed directly under the skin similar to a Norplant device, and is not immunogenic by itself.
Project Methods
Experimental MethodsA.3 Experimental Detail:a. Swine vaccine stocks-i. Development of control vaccine stocks. For comparisons to the H3N8 vaccine, we will obtain 1-2 currently licensed swine influenza vaccine(s) for comparisons to IV or rHA (ISU Veterinary Pharmacy). We will also clone the swine HA3 protein from A/Missouri/14 and insert the gene into the backbone of a LAIV vaccine strain (A/turkey/Ohio/04 or A/swine/Texas/9861) with point mutations in the PA genes thus rendering it cold adaptive. If this is delayed, we will commence our studies first with equine HA3 versus commerical IV/rHA.ii. Development of an inactivated or recombinant equine HA3 vaccine. We have already generated the stock recombinant proteins for this. We will seek to qualify our protein in that it is trimeric before use. To do so, we will run native protein gels and look for the correct size of a trimeric HA (fold-on domain added). Correct HA folding will be determined similar to 62. We will initially test whether cleaved or uncleaved rHA yields the highest Ab and nAb responses in mice and use that formulation for our swine studies. We will grow our H3N8 at 30 degrees in eggs, inactivate with triton x-100, and purify for IV vaccines on sucrose cushions using high-speed centrifugation.b. Optimize the dosage, route, timing, and best vaccine platform for swinei. Test the immunogenicity of our vaccines in swine. Swine (5wks old) will be housed in barrier housing and will be inoculated with 106-8 TCID50 of our reassorted control vaccine, commercial equine vaccine (similar TCID50), or rHAs (25mcg-250mcg) into the nasal flares of swine, subQ, IM, polyanhydride delivery device, or IP (if no Ab response is noted in other routes). Swine will be bled on day 0, 7, 14, and 28 days post-vaccination. Total specific antibodies will be examined to recombinant H3 and H1 antigens. Hemagglutinin inhibition assay (HAI) will be performed using 1 prototypical H1N1 and 1 prototypical H3N2 swine virus. Small numbers of swine 2-3 will be used for these pilot studies before expansion into replicates using larger animal numbers.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 swine (identified in bi above) across multiple influenza strains. We will vaccine 6 swine 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 swine H1N1, H1N2, and H3N2 viruses. We will also test for reactivity to other HAs such as H7 and H9 by ELISA. The same sera will also be examined for viral neutralization in microneutralization assays since this also measures stem Abs.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. Although we will eventually test our vaccine against potential pandemic strains under BSLIII; here, our focus will be on using H1N1 and H3N2 strains that cause swine disease (sw-H1N1 pnd09, H1N1 A/Texas/14/2008, and H3N2 MO/A01476459/12). Protection from lethal challenge (survival) will be assessed after vaccination (2-3x) and challenge. 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 2 replications of n=3 for each strain comparing our vaccine candidate against a control vaccine group and a no-vaccine group. We will also compare the duration of immunity induced by our polyanhydride delivery platform in swine, ferrets, and horses. This will occur after we have fully explored whether this platform is more immunogenic as traditional methods (bnAbs levels will be compared with concentration on titer/activity and breadth neutralization).b. Examine the T-cell profile before and after vaccination/challenge- To determine the contributions of T-cells toward any protection afforded by our vaccine, we will isolate PBMCs and lung mucosal T-cells (collagenase digestions) from vaccinated and from vaccinated/challenged swine. We will stimulate T-cells using autologous APCs (mitomycin C arrested) preincubated with influenza H3N8 or swine H1N1 or swine H3N2. Proliferation will be determined in CD4 of CD8 T-cell populations by inclusion of BrdU in the cell culture media. Additionally, we will use intracellular staining to determine the antiviral response (IL-2, IFNg, IL-4, and TNFa) by flow cytometry. These antibodies are all commercially available although we may have to conjugate them to fluorophores using commercial kits. Alternatively, we could use ELISAs to determine concentrations of cytokines in cell culture supernatants or use ELISOPT assays for IFNg for quantification of antiviral T-cells.b1. Confirm cellular immunity contributes to protection in swine- While a finding of cellular responses are activated after vaccination and/or challenge would suggest they are protective, heterosubtypic protection mediated by adaptive cells (outside of immunity) has not been well described in humans (outside limited studies of NP antibodies plus CD4 T-cells). Thus, if we find either CD4 T-cells or CD8 T-cells are playing a role in protection (suggested in our preliminary data in mice (not shown)), we will use adoptive transfers of either cell population or both (from lungs or draining nodes or both) to confirm their contributions to protection. We assess this, transfers by IV will be done to obtain physiological levels of influenza-specific memory will be done followed by challenge and examination of cellular responses after BrdU administration by ICS assay of GFP+ (memory) or negative populations (host). Viral burdens will be assessed as will lung pathology. These data will serve multiple additional purposes outside just dissection of correlates of protection: (1) if VAERD is found with our vaccine, this would help to determine cellular cause; (2) these data will allow us to confirm the role of vaccine induced resident versus non-resident T-cell populations to protection; and (3) inclusion of vaccinations of older swine (1yr old) will allow for comparisons between young pigs with more limited immunological maturation to be compared with older mature swine fostering another model of human infant studies available. As shown in Table 2, we have preliminary data that suggests we can transfer and track CD4 T-cell memory in adoptive swine hosts. Alternatively, we could use in vivo antibody depletion of cell populations similar to murine studies although this will take optimization.c. Determine the extent, if any, of VAERD occurs after vaccination or challenge- Protection against sublethal (lower viral loads, HAI titers, microneutralization titers, lung pathology, clinical signs) will be assessed using strains similar to our lethal challenge but also including the use of H1N2 virus. We will examine for any associated VAERD by determining the levels of viral loads by qRT-PCR after challenge, tissue histopathology, and clinical signs.