Performing Department
(N/A)
Non Technical Summary
The Influenza virus has been responsible for several pandemics, and highly pathogenic avian influenza (HPAI) viruses remain a significant threat to public health. A recent spillover of the HPAI H5N1 virus to dairy cows has led to human infections, highlighting the need for safe and effective vaccines to prevent influenza airway infections in cows and reduce spread between animals and humans. Current intramuscular vaccines, such as mRNA vaccines, are highly effective in reducing severe diseases and deaths but do not prevent respiratory infections and transmission. This limitation arises because intramuscular vaccines primarily stimulate systemic immunity, which is insufficient for protecting the respiratory mucosa. To address this challenge, developing mucosal vaccines that elicit long-lasting mucosal immunity is essential. These vaccines could prevent both initial and recurrent influenza virus infections and their transmission. Recent studies have demonstrated that using an FcRn-based platform to deliver influenza hemagglutinin (HA) and SARS-CoV-2 spike antigens through the airway can induce protective mucosal immunity in both the nasal and lung compartments. This nasal vaccine effectively prevents virus replication in animals and limits transmission among them. These findings led us to reason that FcRn-mediated nasal spray vaccines for influenza can trigger recall responses that protect against initial and recurrent H5N1 infections in the airway and reduce animal-to-animal and animal-to-human transmission. Given the rapidly evolving nature of influenza viruses, we aim to develop universal mucosal vaccines. The vaccines delivered through the FcRn platform can be easily engineered, manufactured, and adapted for use against other mucosal infections in livestock.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
Influenza A virus has caused several pandemics. The highly pathogenic avian influenza (HPAI) viruses continue to pose significant threats to public health. A recent spillover of the HPAI H5N1 to dairy cows has resulted in human infections. Thus, safe and effective vaccines are urgently needed to prevent H5N1 respiratory infections in cows and to abrogate transmission between animals and from animals to humans. While intramuscular vaccines (e.g. mRNA vaccines) are highly efficacious in reducing severe diseases and deaths, they are not effective in preventing respiratory infections and transmission. That is because intramuscular vaccines primarily trigger a systemic, but not mucosal, immunity which is insufficient to protect the respiratory mucosa. To address this issue, mucosal vaccines eliciting long-lasting mucosal immunity must be developed, which will be able to prevent initial and recurrent influenza virus infection and transmission. Our recent studies have shown that vaccination using the FcRn-based platform to deliver influenza hemagglutinin (HA) and SARS-CoV-2 spike antigens across the airway induced protective mucosal immunity in the nasal and lung compartments. This nasal vaccine effectively prevents virus replication in animals and transmission among animals. These results support our hypothesis that FcRn-mediated nasal spray influenza vaccines elicit recall responses preventing initial/recurrent infections by influenza H5N1 in the airway and animal-to-animal and animal-to-human transmissions. Given the rapidly evolving nature of influenza viruses, we also aim to develop universal influenza mucosal vaccines. Vaccines delivered by the FcRn platform can be easily engineered, manufactured, implemented, and adapted to other mucosal infections in livestock.
Project Methods
Objective 1: Production of bovine IgG Fc-fused influenza HA and HA Stalk antigens and examination of its immunogenicity in mice.1.1 Expressions of the HA-Fc and HA Stalk-Fc fusion proteins. In this study, we will use bovine IgG Fc fragment. Because IgG Fc normally forms a disulfide-bonded dimer, we created a monomeric Fc by substituting cysteines 224, 227, and 229 with serine to eliminate the disulfide bonds in the hinge region. We will also generate an Fc mutant, where the complement C1q-binding motif is eliminated to abrogate C1q binding.The bovine H5N1 clade 2.3.4.4b virus and the HA sequence will be acquired from USDA APHIS National Veterinary Services Laboratories. For the influenza virus to infect cells, the HA precursor HA is cleaved into HA1 and HA2. To produce the non-cleavable HA protein, we will perform mutagenesis at the cleavage sites of HA to preserve the HA pre-cleavage state. The native HA protein exists as a trimer. Maintaining a native conformational structure of the HA antigen is critical for maximizing the immunogenicity during i.n. vaccination. To facilitate the trimerization of HA protein, we will engineer a foldon domain from T4 bacteriophage fibritin protein to the C-terminus of HA. As described above, we will fuse the monomeric bovine IgG Fc in the open reading frame with the HA-Foldon.The influenza virus HA stalk is conserved across influenza strains; it is a promising target for eliciting broadly neutralizing Abs. During infection or vaccination, the HA stalk is thought to be masked by a highly immunogenic HA head domain. To induce high levels of stalk-reactive Abs, we will construct a headless HA Stalk-Fc, which consists of the HA2 domain and the partial regions of HA1 contributing to the stalk folding but lacking the head. In this construct, HA1 Cys52 and Cys277 residues are retained, and the linker peptide is inserted to bridge Cys52 and Cys277. To stabilize the low-pH conformation of HA, we will introduce aspartate mutations (V66D and L73D) in the HA stalk. After confirming plasmids by sequencing, we will transfect the plasmids encoding the HA-Fc or Stalk-Fc into CHO cells.1.2 Characterizations of the HA-Fc and HA Stalk-Fc fusion proteins. A trimeric HA fused to an Fc will most closely mimic the native HA structure by maintaining conformation-dependent B cell epitopes. We next test whether the HA portion of HA-Fc or the Stalk portion of Stalk-Fc maintains its trimeric conformation. We will perfoorm HA specific mAbs, FcRn, and FcgRI binding assays.1.3. Immunization and challenge in the mouse.Immunizations. Groups of eight-week-old wild-type (WT) female mice are listed in Table 1. Ten mice per group are required to show statistical significance between treatment groups and test efficacy in the challenge. The HA-Fc or Stalk-Fc (10 mg) will be i.n. administered into lightly anesthetized (i.p.) mice along with 10 mg CpG in a 30 ml volume. Control mice will be treated with PBS plus 10 mg CpG. All mice will be boosted in a 2-3-week interval, sera will be collected 2 weeks after boost.Challenges. The immunized mice will be challenged in our ABSL-3+ facility. To measure protection, 3 weeks or 6 months (for immune memory) following a boost, mice will be i.n. inoculated with 50 ml 5 LD50 of cow H5N1 clade 2.3.4.4b, B3.13 strain. The infection doses and schedules are chosen from our pilot studies. Lethal dose 50 values will be calculated according to the method of Reed and Muench. Mouse challenge, scoring of the morbidity, mortality, loss of body weight, and virus titration are described in our publication. Infected mice will be euthanized if they lose more than 25% of their initial body weight. At days 3 and 6 post-infection, groups of 5 mice will be euthanized and the whole blood, tissues will be collected, and frozen at -80°C. Each tissue will be sampled individually to prevent cross-contamination by virus. Blood will be snap-frozen on dry ice immediately after collection without anticoagulant. Later, frozen tissue samples will be thawed, mixed with 1 ml of MEM medium containing 0.3% BSA, and homogenized using a TissueLyser II. Homogenates will be centrifugated at 14,000 rpm for 10 minutes and used for plaque assays in MDCK cells.1.4. Probing the immune mechanisms of FcRn-targeted nasal vaccination in mice.We will further analyze T and B cell immune responses that ae induced by bovine Fc-fused HA or Stalk in mice. Memory B cells in the spleen or IgG-secreting plasma cells in bone marrow will be enumerated by flow cytometry or ELISpot. Antibody titers in the sera are measured 6 months after the boost by ELISA. We will measure memory B cell and antibody affinity and longevity in the study. To show that FcRn mucosal delivery-induced memory immune responses confer protection, we will i.n. challenge these mice 6 months after the boost with bovine H5N1.Objective 2: Define the ability of FcRn to transport HA-Fc or Stalk-Fc subunit vaccine across the mucosal barrier and induce protective immune responses in cows.1). Immunogenicity testing in cows. Adult dairy cows (e.g. Holstein cows) will be used in this experiment to ensure that the results are directly applicable to the field problem. Milking cows 2.5-3 years of age and free from influenza virus D and HPIV H5N1 and specific antibodies) will be acquired from local dairy farms or BARC Dairy Unit. The immunization of the cows will be performed at the Dairy Unit of BARC, USDA/ARS, Beltsville, MD. Three groups of cows with 5 cows per group will be used. Based on our recent vaccine studies, a minimum of 5 cows per group are required to show statistical significance between groups. The HA-Fc or Stalk-Fc (100 mg) will be i.n.-sprayed into the nostrils along with 30 mg CpG in a 2-ml volume. Bovine CpG oligonucleotide will be purchased from InvivoGen. The vaccine and CpG dosage will be further optimized in the pilot experiment. Control cows will be i.n.-immunized with PBS plus 30 mg CpG. All cows will be i.n.-boosted at a 3- or 4-week interval.2). Challenge infection of immunized cows. The immunized dairy cows will be challenged at the BSL-3+ facility of the USDA/ARS National Animal Disease Center (NADC), Ames, IA. Drs. Kaitlyn Sarlo Davila and Mark R. Ackermann at NADC have provided the letter of support (see attached letter). Dairy cows will receive nasal vaccinations with the HA-Fc or Stalk-Fc in the presence of CpG adjuvant followed by challenge infection with 5 x 105 PFU of H5N1 given as intranasal aerosol infection (Table 3, n=5). Following infection, animals would be monitored for signs of disease (increased rectal temperature, nasal discharge, conjunctivitis). Nasal and pharyngeal swabs will be collected daily for 14 days from each animal and examined for virus excretion. The whole blood will be snap-frozen on dry ice immediately after collection without anticoagulant. The virus load in nasal secretions or blood will be measured by plaque assay on MDCK cells in the BSL-3 facility. Given the high titers of the H5N1 virus have been detected in the milk (46), milk samples from each animal will be collected daily for 14 days after infection, the virus load in the milk will be measured by RT-PCR for H5N1 clade 2.3.4.4 viruses. Protection is defined as statistical significant (P<0.05) reduction in nasal virus titers in 80% of immunized dairy cows over control animals. The levels of influenza virus-specific IgA and IgG antibodies in sera and nasal will be monitored by ELISA before, during, and after the challenge.All experimental cows will be euthanized 15 days after the challenge infections. Postmortem examinations of the immunized or control cows will be thoroughly performed.