A replicating RNA vaccine protects against lethal clade 2.3.4.4b influenza A H5N1 virus challenge in cynomolgus macaques

A replicating RNA vaccine protects against lethal clade 2.3.4.4b influenza A H5N1 virus challenge in cynomolgus macaques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article A replicating RNA vaccine protects against lethal clade 2.3.4.4b influenza A H5N1 virus challenge in cynomolgus macaques David Hawman, Amanda Griffin, Atsuhi Okumura, Shanna Leventhal, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5723772/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In early 2024, clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A H5N1 virus was detected in United States dairy cattle. While so far the public health threat of contemporary clade 2.3.4.4b H5N1 virus strains remains low, continued circulation in mammals and frequent spillover into humans poses a threat of pandemic H5N1. The United States and other countries have stockpiled vaccines and have plans in place to rapidly produce vaccines should a pandemic H5N1 virus emerge. However, the continued antigenic drift of clade 2.3.4.4b H5N1 antigens compared to historical antigens used by stockpiled vaccines has raised questions of whether these vaccines will confer protection or if stockpiles need to be updated. We recently evaluated a replicating RNA (repRNA) vaccine against lethal contemporary 2.3.4.4b H5N1 virus challenge in mice and found that a homologous but not historical H5 hemagglutinin (HA)-based vaccine conferred protection. Here we further evaluated the protective capacity of a repRNA expressing the contemporary 2.3.4.4b HA or a repRNA expressing a historical H5 HA (A/Vietnam/1203/2004) in a recently developed lethal non-human primate challenge model. We found that both vaccines conferred robust protection against lethal 2.3.4.4b H5N1 virus challenge, protecting against clinical disease and death, reducing viral loads and signs of respiratory illness. Our data show the repRNA platform can elicit protective immunity against lethal respiratory influenza disease and that historical H5 HAs elicit cross-protective immunity. Biological sciences/Microbiology/Virology/Influenza virus Biological sciences/Microbiology/Vaccines/RNA vaccines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Highly-pathogenic avian influenza (HPAI) A H5N1 was first detected in United States dairy cattle in March 2024 and has continued to spread in the United States. As of late December 2024, HPAI H5N1 has affected 866 herds in 16 states and poultry in all 50 states 1 . In addition to economic impacts of poultry and dairy infections 1 over 61 humans have been infected with HPAI A H5N1 in the United States since March 2024 mostly with mild symptoms 1 . However, a Canadian teen and a person in Louisiana were hospitalized with severe disease in late 2024 2–4 and California declared a state of emergency over continued viral spread within the state 4 . These infections have largely been associated with livestock or poultry exposure but a case in Missouri and the Canadian teen had no known contact with livestock or poultry 3 . The continued circulation of HPAI A H5N1 viruses in mammals raises concerns about viral adaptation that could lead to efficient human-to-human spread. Recently it has been shown that just one amino acid substitution is necessary for clade 2.3.4.4b H5N1 to change from avian to human sialic acid specificity 5 and cows express sialic acids preferred by both avian and human influenzas 6 potentially allowing H5N1 viruses to switch receptor preference. Vaccines are a critical tool to stop an H5N1 influenza pandemic and the United States has stockpiled millions of doses of vaccines with plans to rapidly produce hundreds of millions of doses should the need arise 7–10 . However, the continued evolution and antigenic drift of H5N1 viruses raises concerns about the effectiveness of stockpiled vaccines based on historical H5N1 strains. During the COVID-19 pandemic, nucleic acid vaccines demonstrated their ability to be rapidly updated to match emerging variants of concern 11 . Here we evaluated a replicating RNA (repRNA) vaccine based on an alphavirus replicon that is delivered by a cationic nanocarrier, called LION TM 12 . This vaccine platform has demonstrated pre-clinical efficacy against a variety of pathogens 13–18 along with clinical efficacy against SARS-CoV-2 19 . We recently showed in a lethal mouse challenge model with Influenza virus A/bovine/OH/B24OSU-342/2024 (hereafter A/bovine) that a repRNA expressing the hemagglutinin (HA) of A/bovine but not a repRNA expressing the HA of A/Vietnam/1203/2004 (A/Vietnam) conferred complete protection against challenge. Cumulatively our mouse data suggested that immunity elicited by historical HAs, as utilized by some stockpiled vaccines, may not protect against contemporary H5N1 challenge. To further investigate this question, here we evaluated these vaccines, repHA-Vietnam and repHA-Bovine, in a recently developed lethal challenge model in cynomolgus macaques 20 . In contrast to mice, macaques vaccinated with either repHA-Vietnam or repHA-Bovine were significantly protected against A/bovine challenge, exhibiting little-to-no clinical signs, reduced viral replication and complete survival. Our data demonstrate that our repRNA platform confers robust protection against respiratory infection with A/bovine and that historical H5 HAs can confer cross-protective immunity against contemporary 2.3.4.4b H5N1. Results RepRNAs expressing the HA of influenza A H5N1 A/Vietnam or A/Bovine are immunogenic in cynomolgus macaques. Groups of cynomolgus macaques (n = 6) were sham-vaccinated or vaccinated with either repHA-Vietnam or repHA-Bovine. Four-weeks later animals were boosted with identical vaccinations. Weekly exams were conducted and antibody response to recombinant HAs from A/Vietnam or A/bovine measured by ELISA. Against rHA-Vietnam, we observed a general trend of increasing binding antibody in the repHA-Vietnam vaccinated animals that further increased two-weeks after boosting (Figure 1a) but this was not significant compared to sham-vaccinated animals. However, prior to vaccination, we detected rHA-Vietnam binding antibodies in most animals (Figure 1a) suggesting potential prior exposure to influenza virus(es) in these animals. Against A/bovine HA, pre-existing binding antibodies were largely undetectable (Figure 1a). After vaccination, compared to sham-vaccinated animals, we measured increased binding antibodies to rHA-Bovine in both repHA-Vietnam and repHA-Bovine vaccinated animals after boosting (Figure 1a). Interestingly, we measured a decline in antibody from 3 to 4 weeks after boosting (day -7 to day 0) (Figure 1a) suggesting the effect of boosting may be transient. We also measured homologous and heterologous neutralizing activity by virus neutralization assay at time of challenge. No neutralizing activity against authentic A/Vietnam was measured at time of challenge (Figure 1b) while we detected low neutralizing responses in 3 of 6 repHA-Bovine vaccinated animals against A/bovine (Figure 1b). Cumulatively, these results indicate that repHA-Vietnam elicits antibodies capable of binding both A/Vietnam and A/bovine but with undetectable neutralization activity. In contrast, repHA-Bovine elicited antibodies capable of binding A/bovine and with low homologous neutralizing activity. To more fully characterize the breadth of the HA-binding response elicited by repHA vaccination and to evaluate heterosubtypic binding, we utilized a bead-based assay to evaluate binding to multiple H5 HAs across several clades at time of challenge, four-weeks after boosting. Comparing repHA vaccine groups, repHA-Vietnam vaccinated animals had significantly increased binding antibodies to most non-clade 2.3.4.4b HAs while repHA-Bovine animals had significantly increased binding antibodies to clade 2.3.4.4b HAs (Figure 1c and supplemental figure 1). Against heterosubtypic HAs, in some animals, we measured weak binding against other influenza A H3 and H1 subtypes and other avian influenza viruses H6N1, H10N8, and H9N2 HAs prior to vaccination (Supplemental Figure 1) suggesting previous influenza A virus exposure in these animals. RepHA vaccination increased binding to heterosubtypic HAs in some animals but responses remained largely focused against H5 HAs (Supplemental Figure 1). Cumulatively, these data suggest that repRNA expressed A/Vietnam and A/Bovine HAs elicit distinct antibody responses, with a matched repHA-Bovine eliciting the greatest responses against clade 2.3.4.4b HAs. RepHA vaccination protects against lethal A/bovine challenge. Four-weeks after boosting, animals were challenged with 10 7 median-tissue culture infectious doses (TCID 50 ) of A/bovine via the intratracheal (IT) route. Animals were monitored daily for signs of clinical disease and exams were conducted on days 0, 1, 3, 5, 7, 9, 14 and 21 post-innoculation (PI). Sham-vaccinated animals quickly developed overt clinical signs characterized by lethargy, hunched posture and signs of respiratory distress (Figure 2a). Additional clinical signs such as weight loss and elevated body temperature were also measured (Figure 2b and c). Between days 6 to 8, all sham-vaccinated animals reached endpoint criteria with signs of respiratory distress and were euthanized (Figure 2d). In contrast, both repHA-Vietnam and repHA-Bovine vaccinated animals were protected against clinical disease until study end on d21 (Figure 2a-d). We observed transient elevated body temperature in repHA-vaccinated animals on day 1 PI (Figure 2c), possibly reflecting acute host responses to the large amounts of challenge virus. On exam days, thoracic radiographs were taken and scored for evidence of pneumonia and inflammation. Sham-vaccinated animals developed consistent evidence of pulmonary infiltrates (Figure 2e). RepHA-vaccinated animals had reduced radiographic evidence of pulmonary infiltrates (Figure 2e) consistent with protection from clinical disease. Cumulatively, these data indicate that both repHA-Vietnam and repHA-Bovine confers robust protection against clinical disease upon challenge with A/bovine. RepHA vaccination confers control of viral replication. On exam days or at time of necropsy, we collected blood, as well as oral and nasal swabs for quantification of virus replication by PCR and TCID 50 assay. Overall viral RNA in the blood or swabs was variable (Figure 3a-c) and infectious virus in the blood or oral swabs negligible (Supplemental Figure 1). Sporadic infectious virus was detected in some nasal swabs, with greatest amount detected in the terminal nasal swab of one sham-animal that succumbed on day 6 PI (Supplemental Figure 1). At day 5 PI, during peak detection of virus genome copies in nasal swabs of sham-vaccinated animals, only repHA-Bovine vaccinated animals exhibited a significant reduction in viral load (Figure 3c). We also collected bronchoalveolar brushes (BABs) on days 3 and 9 PI to measure viral loads within the lower respiratory tract. In contrast to upper respiratory swabs, infectious virus was consistently detected in the BABs of sham vaccinated animals at day 3 PI (Figure 3d) suggesting that A/bovine was mostly restricted to the lower respiratory tract in challenged animals. RepHA-Vietnam vaccinated animals exhibited a trend towards lower infectious virus in the day 3 BAB (Figure 3d). However, by day 3, 4 of 6 repHA-Bovine vaccinated animals had undetectable infectious virus in the BABs (Figure 3d) demonstrating prompt control of the virus within the lungs. Consistent with little-to-no clinical disease at day 9, no infectious virus was detected in the BABs of repHA vaccinated animals collected at day 9 (Figure 3d). We next evaluated viral loads in various tissues collected at time of necropsy, days 6 – 8 PI for sham-vaccinated and day 21 for repHA-vaccinated. RepHA vaccination significantly reduced viral RNA in all tissues examined except for the heart, though viral RNA was inconsistently detected in this tissue even in sham-vaccinated animals (Figure 3e). We also detected infectious virus in the lung tissue of sham- but not repHA-vaccinated animals (Figure 3f). Cumulatively, these data demonstrate that repHA vaccination resulted in rapid control of A/bovine virus. RepHA vaccination protects against lung pathology. At time of necropsy, the lungs were weighed and % of total body weight of the lungs calculated as an indicator of pulmonary edema 21 . Both repHA vaccinated groups had reduced lung weights as % of body weight compared to sham-vaccinated animals (Figure 4a). This correlated with consistent presence of lesions across multiple lung lobes in sham-vaccinated animals but no gross lesions in any repHA-vaccinated animal (Figure 4b). These data are consistent with our radiograph data showing evidence of pneumonia in sham-vaccinated animals but not repHA-vaccinated animals at euthanasia. Robust anamnestic antibody responses in repHA vaccinated animals. We evaluated serum collected at day 0, 7, 14 and 21 PI for HA binding antibodies by ELISA and for neutralization against A/Vietnam and A/bovine on day 14 PI. Against both A/Vietnam and A/bovine HA antigen, we measured a rapid increase in binding antibodies by ELISA in both repHA vaccinated groups by day 7 PI (Figure 5a). No increase in HA binding antibodies was measured in sham-vaccinated animals prior to euthanasia (Figure 5a) consistent with poor outcome in these animals. Despite detecting little neutralizing activity prior to challenge in repHA vaccinated animals, by day 14 PI we detected strong neutralizing activity against A/bovine in both repHA-Vietnam and repHA-Bovine vaccinated animals (Figure 5b). In contrast, neutralizing activity against A/Vietnam was only measured in repHA-Vietnam vaccinated animals (Figure 5b). This is consistent with lower levels of A/Vietnam HA binding antibodies in repHA-Bovine vaccinated animals (Figure 5a). Using our bead-based assay, at day 21 PI, serum from repHA-Vietnam-vaccinated animals contained antibodies that bound all H5 HAs tested and with reactivity to H1 and H6 HAs, consistent with H5, H1 and H6 being group 1 HAs 22 (Figure 5c). Serum from repHA-Bovine-vaccinated animals had strong reactivity to clade 2.3.4.4b HAs with reduced binding to other clade H5 HAs (Figure 5c) consistent with our ELISA and VN data (Figure 5a-b). Heterosubtypic binding antibodies against H1N1 and H6N1 viruses also developed post-infection in repHA-Bovine vaccinated animals (Figure 5c). Discussion The fitness and genetic diversity of clade 2.3.4.4b H5NX viruses poses a serious risk of pandemic HPAI 23–25 . While most recent cases of clade 2.3.4.4b H5N1 infections have been mild, a Canadian teenager and Louisiana resident have been hospitalized with severe disease 26,27 and continued circulation in livestock and wild birds will result in further spillover into humans. Vaccines to address pandemic HPAI are stockpiled in many countries but whether these vaccines need to be updated to address current 2.3.4.4b circulation or to address future antigenic drift is unclear. Our data demonstrate that a repRNA vaccine expressing either the A/Vietnam or A/bovine HA can confer robust protection against HPAI A H5N1 infection in a relevant macaque model. Protection was associated with HA binding antibodies but interestingly, at time of challenge, little-to-no VN activity was detected in repHA-vaccinated animals. Although neutralizing antibodies correlate with protection against influenza 28 , it is possible that repHA-elicited non-neutralizing antibodies may have contributed to protection 29,30 . We and others have seen strong protection against 2.3.4.4b H5N1 challenge in mice in the absence of potent neutralizing antibody responses 31 . Alternatively, the rapid anamnestic response we measured in repHA vaccinated animals by day 7 PI may have contributed to protection. However, the significantly reduced viral loads within the lungs by 3 days PI suggests immunity present at time of challenge conferred robust protection on its own. Our data support the hypothesis that the mutations accumulating in the head domain of the A/bovine HA compared to A/Vietnam can lead to functional differences in antibody recognition of HA. In mice vaccinated with repHA-Vietnam or repHA-Bovine we measured significantly reduced binding to the opposite H5 HA and only repHA-Bovine protected against A/bovine challenge. Here, although both repHA vaccines conferred protection, repHA-Vietnam vaccination elicited antibodies with diminished binding to clade 2.3.4.4b HAs. This was associated with delayed control of infectious virus within the lungs and reduced control of viral shedding in the nasal cavity of repHA-Vietnam vaccinated animals. Heterologous repHA-Vietnam vaccination and A/bovine challenge also resulted in cross-clade neutralizing antibodies against both A/Vietnam and A/bovine while homologous vaccination and challenge resulted in neutralizing antibodies against only A/bovine. Our findings that potent neutralizing antibodies against A/bovine can fail to neutralize A/Vietnam demonstrate that the mutations in the HA between A/Vietnam and A/bovine impact epitopes targeted by neutralizing antibodies. Recent structural studies of bovine HA have shown that the receptor binding site of the H5 HA may be shielded by an autoglycan that could shield neutralizing antibody binding to the receptor binding site 32 . While the glycosylated amino acid is highly conserved among H5 HAs, the presence of this glycan has been rarely reported for H5 HA structures and further study is needed to determine whether this glycan impacts pathogenesis. Our data suggest that heterologous A/bovine challenge in repHA-Vietnam animals boosted neutralizing antibodies targeting more conserved epitopes between A/bovine and A/Vietnam and heterologous prime-boosts may be an optimal strategy to elicit broad cross clade immunity. In humans receiving homologous or heterologous H5N1 vaccines, heterologous boosting induced broader cross-clade antibody responses than homologous prime-boost 33 suggesting heterologous vaccination may afford greater breadth of protection against H5N1. Further, humans vaccinated with an H5N1 vaccine had increased numbers of antibodies targeting the more conserved HA stem region suggesting that H5 immunization of individuals with seasonal H1 or H3 HA immunity preferentially recruits B-cells with specificity for more conserved domains of HA 34 . Preexisting H1N1 immunity due to virus infection in ferrets was sufficient to protect against lethal bovine H5N1 infection 35 . Immunity against protective epitopes outside the HA in H1N1-infected ferrets may have contributed to protection against H5N1 36 and suggest inclusion of other antigens than just HA may increase vaccine efficacy. Notably, our data are in contrast to our recent evaluation of these same vaccines in a lethal mouse challenge model in which only repHA-Bovine vaccination protected against A/bovine challenge. This is an important consideration as while updated H5 vaccines are in clinical development, stockpiled vaccines for H5N1 in the United States use historical H5 HAs 8,37 and our data here suggest these vaccines may confer protection against A/bovine and related viruses. It is unclear why we measured distinct protection against A/bovine in mice and non-human primates but could be due to differing H5N1 disease presentation in these models. Whereas non-human primates develop a severe lower respiratory disease following IT challenge, mice rapidly exhibit both respiratory and neurological involvement following IN challenge 38 . It is likely the correlates of protection differ for upper versus lower respiratory tract infection as well as for neurological disease. Indeed, we observed superior control of viral load in the nasal swabs of NHPs vaccinated with homologous repHA-Bovine (Figure 3c), suggesting an antigen-dependent control of virus in the upper respiratory tract. Historically, this tissue has been difficult to protect from respiratory virus infection following peripheral vaccination with conventional RNA vaccine platforms 39–41 . In contrast, we have consistently observed protection of the upper airway in respiratory virus-challenge models vaccinated with repRNA/LION 15,42–44 . We also measured low levels of H5, H3 and H1 HA binding antibodies in some macaques prior to vaccination suggestive of previous influenza exposure. Cross-neutralizing activity against clade 2.3.4.4b viruses was elicited in clade 1 or 2.1 H5N1 vaccinated humans 45 and preexisting influenza A immunity has been shown to improve the breadth of subtypic responses after vaccination 34 . These data suggest that the animal model, vaccine platform and preexisting immunity may influence apparent vaccine efficacy against H5N1 and support the use of both mouse and NHP models in preclinical evaluation of influenza countermeasures. Together our data show that our repRNA/LION platform expressing the H5 HA of either a historical or contemporary H5N1 isolate can protect against lethal H5N1 challenge in non-human primates. 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Clade 2.3.4.4b but not historical clade 1 HA replicating RNA vaccine protects against bovine H5N1 challenge. (2024) doi:10.21203/rs.3.rs-4946897/v1. Methods Animals, Biosafety and Ethics. All infectious work with infectious influenza was performed in the maximum containment laboratory in accordance with standard operating procedures approved by the Rocky Mountain Laboratories Institutional Biosafety Committee, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Hamilton, MT, USA). All animal work was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the Office of Animal Welfare, National Institutes of Health and the Animal Welfare Act of the US Department of Agriculture, in an AAALAC-accredited facility and study protocol approved by the RML Animal Care and Use Committee. Male (n = 15) and female (n = 3) Mauritius origin cynomolgus macaques between the ages of 6.16 to 15.5 years of age were evenly divided by sex and then randomly assigned to study groups. Animals were housed in adjoining individual primate cages that enabled social interaction, under controlled conditions of humidity, temperature and light (12-h light/12-h dark cycles). Water was available ad libitum. Animals were monitored at least twice daily (pre- and post-inoculation) and fed commercial monkey chow, treats and fruit twice a day by trained personnel. Environmental enrichment consisted of manipulanda, visual enrichment and audio enrichment. All procedures on nonhuman primates were performed by board-certified clinical veterinarians. Upon inoculation animals were comprehensively evaluated for disease signs using a score sheet by staff blinded to study groups 20 . Thoracic radiographs were taken on exam days or at time of euthanasia and scored by a trained veterinarian and each lung lobe scored according to 0 = none, 1 = mild, 2 = moderate and 3 = severe interstitial pneumonia. Scores for all the lung lobes were then summed and presented as total lung score per animal. All necropsies were performed by board-certified veterinary pathologists. Vaccination and virus challenge. Vaccines used here were identical to those used previously 46 . Vaccination was performed by a single intramuscular injection consisting of 25μg of RNA. Animals were boosted with identical vaccinations four-weeks later. Vaccination appeared well tolerated with no adverse events observed following vaccinations. A/bovine challenge virus was identical to that previously described 46 . Animals were challenged with 1x10 7 median-tissue culture infectious doses (TCID 50 ) via the intratracheal route as previously described 20 . ELISA and Neutralization assays. The ELISAs against recombinant HAs from A/Vietnam or A/bovine were as previously described 46 . Maxisorp (Nunc) plates were coated with recombinant HA from A/Vietnam/1203/2004 (IBT Bioservices) or A/dairy cow/Texas/24-008749-002 (SinoBiological, homologous to A/bovine) at 100ng/well in phosphate buffered saline (PBS) overnight. Plates were blocked and serial dilutions of mouse sera applied. Bound antibody was detected with an anti-mouse IgG conjugated to horseradish peroxidase (Southern Biotech, Catalog #1030-05, Lot# D1922-YI62C, 1:4000), plates developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (SeraCare) and absorbance was read at 450nM. Endpoints are reported as the reciprocal of the serum dilution to have an absorbance of 3 standard deviations above background absorbance as determined by curve-fit and interpolation of absorbance value. For VN assay, 100 TCID 50 of virus was mixed with serial dilutions of heat inactivated (56°C for 30 minutes) sera for 1 hour at 37C. Residual infectious virus was then measured on MDCK cell monolayers. After three days, supernatant was collected and presence of infectious virus determined by mixing with 0.33% turkey erythrocytes and measurement of hemagglutination. Titers are reported as the reciprocal of the last dilution to exhibit VN activity. qRT-PCR and TCID 50 to measure viral loads. Measurement of viral RNA and infectious virus was as previously described 20 Bead-based multiplex HA binding assay. The bead based binding assay was performed as previously described 46 . Statistics. Indicated statistical comparisons were performed with GraphPad Prism v10.2. Declarations Data availability. The data underlying the figures presented here are posted publicly at FigShare, DOI TBD Acknowledgements. The A/bovine virus was kindly provided by Richard Webby St. Jude’s Children hospital and Andrew Bowman Ohio State University. We also wish to thank Vincent Munster and Emmie De Witt and their laboratory staff, Laboratory of Virology, NIAID for their efforts to obtain, propagate and titer virus stocks. We thank the Rocky Mountain Veterinary Branch biologists and animal care staff for support of the animal study. This study was supported by the Intramural Research Program, NIAID. Funders had no input on study design, data interpretation or decision to publish. Author Contributions. DWH, KR, JHE, HF designed the study. DWH, AG, AO, SSL, NM, MA, MC, EA, ML, TH, ETS, NW, JL, BJS, PH, GS, CS, CC performed the study. DWH, AO and SSL performed data analysis. DWH wrote the manuscript. DWH, JHE, HF edited the manuscript. HF obtained funding. All authors reviewed the manuscript and agree with publication. Conflicts of Interest. JHE, ETS, NW, SP, KH, EH, and KG have equity interest in HDT Bio. JHE is an inventor on patents (US Patent nos. 11,458,209; 11,433,142; 11,752,218; 11,648,321; and 11,654,200) and patent applications (PCT/US22/76787, PCT/US23/60225, and PCT/US2024/010326) pertaining to the LION formulation and repRNA compositions described in the studies. Funders had no role in study design, data interpretation or decision to publish. The remaining authors declare no competing interest. Additional Declarations Yes there is potential Competing Interest. JHE, ETS, NW, SP, KH, EH, and KG have equity interest in HDT Bio. JHE is an inventor on patents (US Patent nos. 11,458,209; 11,433,142; 11,752,218; 11,648,321; and 11,654,200) and patent applications (PCT/US22/76787, PCT/US23/60225, and PCT/US2024/010326) pertaining to the LION formulation and repRNA compositions described in the studies. Funders had no role in study design, data interpretation or decision to publish. The remaining authors declare no competing interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5723772","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":396153859,"identity":"9f64fe43-166e-485a-b7c6-169ff9b3cc17","order_by":0,"name":"David Hawman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDCCA0DEAyJB4GMDMwODBGMD8VoYZzYwSxClhQGmhZkXrIWAu/iOnzE88IbhjrzBtcPPHtvusK5jkG5ufIBPi+SZHIODcxieGW64nWZunHsmXYJB5mCzAT4tBgfSEg7zMBxm3HY7wUw6t+0w0GGJbXjdZnD+GViL/bbb6d+kLYnSciP5AEhL4rbbOWbSjMRokbzx+MDBOQaHk/ffzimT7G1Ll2wj5Be+84nNH95UHLadOTt9m8TPNmt+fun2hw/waYE6D4nNRlj5KBgFo2AUjAJCAADsZlKsFbAtdAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8233-8176","institution":"HDT Bio","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Hawman","suffix":""},{"id":396153860,"identity":"e45c5a57-64a3-47a7-b3fa-cd1fa7a7297b","order_by":1,"name":"Amanda Griffin","email":"","orcid":"","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Griffin","suffix":""},{"id":396153861,"identity":"f58e40a0-93c4-4fce-847c-f9ce0b6358be","order_by":2,"name":"Atsuhi Okumura","email":"","orcid":"","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Atsuhi","middleName":"","lastName":"Okumura","suffix":""},{"id":396153862,"identity":"436826fe-b232-4964-ba7b-b6393de3a538","order_by":3,"name":"Shanna Leventhal","email":"","orcid":"","institution":"Laboratory of Virology, Division of Intramural Research, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Shanna","middleName":"","lastName":"Leventhal","suffix":""},{"id":396153863,"identity":"70e1858a-eca3-4d65-887d-65d893a63c9a","order_by":4,"name":"Natalie McCarthy","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"","lastName":"McCarthy","suffix":""},{"id":396153864,"identity":"21ba45b3-4e08-4dc4-af8c-48fafac41138","order_by":5,"name":"Mahati Agumamidi","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Mahati","middleName":"","lastName":"Agumamidi","suffix":""},{"id":396153865,"identity":"9c5d88d9-3a14-4c3d-b865-11f4ab160e49","order_by":6,"name":"Michael Chorabik Jr","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Chorabik","suffix":"Jr"},{"id":396153866,"identity":"9658e5e1-6f2a-4433-b571-30ad0b43a9bc","order_by":7,"name":"Ekaterina Altynova","email":"","orcid":"","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Altynova","suffix":""},{"id":396153867,"identity":"31d9fafd-6ecc-42fc-b738-12c24b39cd4e","order_by":8,"name":"Matthew Lewis","email":"","orcid":"","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Lewis","suffix":""},{"id":396153868,"identity":"97f12b94-fb68-448f-9488-f58086cf9c27","order_by":9,"name":"Troy Hinkley","email":"","orcid":"","institution":"HDT Bio","correspondingAuthor":false,"prefix":"","firstName":"Troy","middleName":"","lastName":"Hinkley","suffix":""},{"id":396153869,"identity":"9ca1e2db-f658-4dc3-a780-8dc21d887efc","order_by":10,"name":"E. Taylor Stone","email":"","orcid":"","institution":"HDT Bio","correspondingAuthor":false,"prefix":"","firstName":"E.","middleName":"Taylor","lastName":"Stone","suffix":""},{"id":396153870,"identity":"7236cdd9-56a3-4491-8b8b-dcbf2fc77485","order_by":11,"name":"Nikole Warner","email":"","orcid":"https://orcid.org/0000-0002-2307-5529","institution":"HDT Bio","correspondingAuthor":false,"prefix":"","firstName":"Nikole","middleName":"","lastName":"Warner","suffix":""},{"id":396153871,"identity":"5b3017ae-d3db-469f-935b-7596b1a865b6","order_by":12,"name":"Jamie Lovaglio","email":"","orcid":"https://orcid.org/0000-0002-7567-446X","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Jamie","middleName":"","lastName":"Lovaglio","suffix":""},{"id":396153872,"identity":"59cbdebc-940a-4326-bada-d223d704cf80","order_by":13,"name":"Brian Smith","email":"","orcid":"","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Smith","suffix":""},{"id":396153873,"identity":"d51a25da-1ced-4f0c-bf4d-228188c3c2b0","order_by":14,"name":"Patrick Hanley","email":"","orcid":"https://orcid.org/0000-0003-2300-1818","institution":"NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Hanley","suffix":""},{"id":396153874,"identity":"a3c60a5a-e5a5-4c79-98a3-7cfaa6d6a802","order_by":15,"name":"Greg Saturday","email":"","orcid":"https://orcid.org/0000-0002-0803-6177","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Greg","middleName":"","lastName":"Saturday","suffix":""},{"id":396153875,"identity":"03dc22c0-4403-4705-9350-3efa1ce1afae","order_by":16,"name":"Carl Shaia","email":"","orcid":"https://orcid.org/0000-0001-8907-8821","institution":"Rocky Mountain Veterinary Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Carl","middleName":"","lastName":"Shaia","suffix":""},{"id":396153876,"identity":"05e1c324-d7ea-4036-924e-d2330512e721","order_by":17,"name":"Chad Clancy","email":"","orcid":"https://orcid.org/0000-0002-5354-9270","institution":"National Institute of Allergy and Infectious Diseases","correspondingAuthor":false,"prefix":"","firstName":"Chad","middleName":"","lastName":"Clancy","suffix":""},{"id":396153877,"identity":"8f3d0f39-95da-4495-944b-d2ff932f42d8","order_by":18,"name":"Kyle Rosenke","email":"","orcid":"https://orcid.org/0000-0001-8101-4348","institution":"NIH/NIAD/Rocky Mountain Laboratories","correspondingAuthor":false,"prefix":"","firstName":"Kyle","middleName":"","lastName":"Rosenke","suffix":""},{"id":396153878,"identity":"639c6ee2-e8c0-42a0-860b-907e2792598d","order_by":19,"name":"Jesse Erasmus","email":"","orcid":"https://orcid.org/0000-0003-1612-2697","institution":"HDT Bio Corp","correspondingAuthor":false,"prefix":"","firstName":"Jesse","middleName":"","lastName":"Erasmus","suffix":""},{"id":396153879,"identity":"f83c62ef-6036-49aa-9794-3109ca0abe2d","order_by":20,"name":"Heinz Feldmann","email":"","orcid":"https://orcid.org/0000-0001-9448-8227","institution":"Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Heinz","middleName":"","lastName":"Feldmann","suffix":""}],"badges":[],"createdAt":"2024-12-27 23:25:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5723772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5723772/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73113684,"identity":"897bd227-d02e-46d1-a454-38fec5314674","added_by":"auto","created_at":"2025-01-06 23:25:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erepHA is immunogenic in cynomolgus macaques. \u003c/strong\u003eGroups of cynomolgus macaques (n = 6 per group) were vaccinated on day -56 and boosted on day -28 PI with indicated vaccine. (a) Binding antibody to A/Vietnam or A/bovine HA antigen was measured by ELISA. P values between sham and repHA groups calculated with repeated measured two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. (b) Neutralizing antibodies in the sera of vaccinated animals at day 0 PI was measured by virus neutralization assay with authentic infectious A/Vietnam or A/bovine. Dashed line indicates limit of detection and samples without measurable neutralization were set to 1:5 to distinguish from samples with measurable neutralization at limit of detection. P value for neutralization against A/bovine calculated with Kruskal-Wallis and Dunn’s multiple comparisons test. (c) HA-binding antibodies to indicated HAs were measured by a bead-based multiplex assay. P values calculated with a two-way ANOVA and Tukey’s multiple comparisons test. Colored * indicate comparison between repHA group and sham vaccinated animals. (a-c) * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Combined1.png","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/209a20b6b72cead490edc5a4.png"},{"id":73113689,"identity":"9c237935-dc1e-40b2-9c3c-33c2b9e5b76e","added_by":"auto","created_at":"2025-01-06 23:25:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erepHA vaccination protects against clinical disease following H5N1 challenge. \u003c/strong\u003eFour-weeks after boosting, animals were challenged with A/bovine via the intratracheal route. Animals were scored daily (a) and weights and temperature recorded on exam days (b-c). (d) Animals were euthanized once they reached endpoint criteria. P value calculated with Log-Rank test with Bonferroni’s correction for multiple comparisons. Thoracic radiographs were taken on exam days or at time of euthanasia and scored by a trained veterinarian and each lung lobe scored according to 0 = none, 1 = mild, 2 = moderate and 3 = severe interstitial pneumonia and total lung score shown. Maximum score is 18.\u003c/p\u003e","description":"","filename":"Combined2.png","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/581cd85638c64f77f7dee238.png"},{"id":73113685,"identity":"ef68f23e-49c3-41ed-9395-afa910adf93b","added_by":"auto","created_at":"2025-01-06 23:25:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":256254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erepHA vaccination confers control against H5N1 replication. \u003c/strong\u003e(a-c) Viral RNA in the blood, nasal or oral swabs was quantified by qRT-PCR. P values from day 0 to 7 were calculated with a repeated measures two-way ANOVA with Dunnett’s multiple comparisons test. (d) Infectious virus in the lower respiratory tract was measured by bronchoalveolar brush and TCID\u003csub\u003e50\u003c/sub\u003e assay on day 3 and 9 PI. P value for d3 comparisons was calculated by Kruskal-Wallis test with Dunn’s multiple comparisons test. (e) Viral RNA in tissues was quantified by qRT-PCR. P values were calculated with a two-way ANOVA with Tukey’s multiple comparisons test. (f) Infectious virus in each lung lobe was quantified by TCID\u003csub\u003e50\u003c/sub\u003e assay and pooled by vaccination group. P value calculated by Kruskal-Wallis test and Dunn’s multiple comparisons test. (e-f) The left lung lobes for one sham-vaccinated animal were not collected due to technical error.\u0026nbsp;\u0026nbsp; (a-f) * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Combined3.png","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/04340751c87f824c3a295cc6.png"},{"id":73113787,"identity":"8696570a-2486-4f7a-94a8-4a5ff308ff9a","added_by":"auto","created_at":"2025-01-06 23:33:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003erepHA vaccination protects against lung pathology. \u003c/strong\u003e(a) Lungs were weighed at time of necropsy and the weight as percentage of total body weight calculated. P value calculated with Kruskal-Wallace test and Dunn’s multiple comparisons test. * P \u0026lt; 0.05. (b) At necropsy, percentage of each lung lobe showing evidence of lesions was reported in both the dorsal and ventral presentation.\u003c/p\u003e","description":"","filename":"Combined4.png","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/b535218ab2c7237a20407df9.png"},{"id":73113687,"identity":"b00f0628-5b77-48d3-9443-9b9a841f0a29","added_by":"auto","created_at":"2025-01-06 23:25:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":337984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRapid anamnestic antibody responses after H5N1 challenge. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eBinding antibodies to indicated HA antigen was measured by ELISA. (b) Serum neutralization was measured at day 14 PI against infectious virus. P value calculated with a Welch’s t-test. * P \u0026lt;0.05. (c) Binding antibodies to indicate HAs was measured by bead-based multiplex assay.\u003c/p\u003e","description":"","filename":"Combined5.png","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/a2058446290fde885703628c.png"},{"id":73777649,"identity":"2505213a-50ea-4d90-91c4-3b3e3a03ca76","added_by":"auto","created_at":"2025-01-14 14:49:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2169131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/7b8039bf-4996-4c89-8674-d8ed7db6456e.pdf"},{"id":73113688,"identity":"51aaaafd-a0c0-49be-a714-0c127d7e6a9b","added_by":"auto","created_at":"2025-01-06 23:25:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1610533,"visible":true,"origin":"","legend":"Supplemental Figures","description":"","filename":"SupplementalCombined.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5723772/v1/9c9eea9fbf1a929a48ed4174.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJHE, ETS, NW, SP, KH, EH, and KG have equity interest in HDT Bio. JHE is an inventor on patents (US Patent nos. 11,458,209; 11,433,142; 11,752,218; 11,648,321; and 11,654,200) and patent applications (PCT/US22/76787, PCT/US23/60225, and PCT/US2024/010326) pertaining to the LION formulation and repRNA compositions described in the studies. Funders had no role in study design, data interpretation or decision to publish. The remaining authors declare no competing interest.","formattedTitle":"A replicating RNA vaccine protects against lethal clade 2.3.4.4b influenza A H5N1 virus challenge in cynomolgus macaques","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHighly-pathogenic avian influenza (HPAI) A H5N1 was first detected in United States dairy cattle in March 2024 and has continued to spread in the United States. As of late December 2024, HPAI H5N1 has affected 866 herds in 16 states and poultry in all 50 states \u003csup\u003e1\u003c/sup\u003e . In addition to economic impacts of poultry and dairy infections \u003csup\u003e1\u003c/sup\u003eover 61 humans have been infected with HPAI A H5N1 in the United States since March 2024 mostly with mild symptoms \u003csup\u003e1\u003c/sup\u003e . However, a Canadian teen and a person in Louisiana were hospitalized with severe disease in late 2024 \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e and California declared a state of emergency over continued viral spread within the state \u003csup\u003e4\u003c/sup\u003e . These infections have largely been associated with livestock or poultry exposure but a case in Missouri and the Canadian teen had no known contact with livestock or poultry \u003csup\u003e3\u003c/sup\u003e. The continued circulation of HPAI A H5N1 viruses in mammals raises concerns about viral adaptation that could lead to efficient human-to-human spread. Recently it has been shown that just one amino acid substitution is necessary for clade 2.3.4.4b H5N1 to change from avian to human sialic acid specificity \u003csup\u003e5\u003c/sup\u003e and cows express sialic acids preferred by both avian and human influenzas \u003csup\u003e6\u003c/sup\u003e potentially allowing H5N1 viruses to switch receptor preference. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVaccines are a critical tool to stop an H5N1 influenza pandemic and the United States has stockpiled millions of doses of vaccines with plans to rapidly produce hundreds of millions of doses should the need arise \u003csup\u003e7\u0026ndash;10\u003c/sup\u003e. However, the continued evolution and antigenic drift of H5N1 viruses raises concerns about the effectiveness of stockpiled vaccines based on historical H5N1 strains. During the COVID-19 pandemic, nucleic acid vaccines demonstrated their ability to be rapidly updated to match emerging variants of concern \u003csup\u003e11\u003c/sup\u003e. Here we evaluated a replicating RNA (repRNA) vaccine based on an alphavirus replicon that is delivered by a cationic nanocarrier, called LION\u003csup\u003eTM\u003c/sup\u003e \u003csup\u003e12\u003c/sup\u003e. This vaccine platform has demonstrated pre-clinical efficacy against a variety of pathogens \u003csup\u003e13\u0026ndash;18\u003c/sup\u003e\u0026nbsp; along with clinical efficacy against SARS-CoV-2 \u003csup\u003e19\u003c/sup\u003e. We recently showed in a lethal mouse challenge model with Influenza virus A/bovine/OH/B24OSU-342/2024 (hereafter A/bovine) that a repRNA expressing the hemagglutinin (HA) of A/bovine but not a repRNA expressing the HA of A/Vietnam/1203/2004 (A/Vietnam) conferred complete protection against challenge. Cumulatively our mouse data suggested that immunity elicited by historical HAs, as utilized by some stockpiled vaccines, may not protect against contemporary H5N1 challenge. To further investigate this question, here we evaluated these vaccines, repHA-Vietnam and repHA-Bovine, in a recently developed lethal challenge model in cynomolgus macaques \u003csup\u003e20\u003c/sup\u003e. In contrast to mice, macaques vaccinated with either repHA-Vietnam or repHA-Bovine were significantly protected against A/bovine challenge, exhibiting little-to-no clinical signs, reduced viral replication and complete survival. Our data demonstrate that our repRNA platform confers robust protection against respiratory infection with A/bovine and that historical H5 HAs can confer cross-protective immunity against contemporary 2.3.4.4b H5N1. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRepRNAs expressing the HA of influenza A H5N1 A/Vietnam or A/Bovine are immunogenic in cynomolgus macaques.\u0026nbsp;\u003c/strong\u003eGroups of cynomolgus macaques (n = 6) were sham-vaccinated or vaccinated with either repHA-Vietnam or repHA-Bovine. Four-weeks later animals were boosted with identical vaccinations. Weekly exams were conducted and antibody response to recombinant HAs from A/Vietnam or A/bovine measured by ELISA. Against rHA-Vietnam, we observed a general trend of increasing binding antibody in the repHA-Vietnam vaccinated animals that further increased two-weeks after boosting (Figure 1a) but this was not significant compared to sham-vaccinated animals. However, prior to vaccination, we detected rHA-Vietnam binding antibodies in most animals (Figure 1a) suggesting potential prior exposure to influenza virus(es) in these animals. Against A/bovine HA, pre-existing binding antibodies were largely undetectable (Figure 1a). \u0026nbsp;After vaccination, compared to sham-vaccinated animals, we measured increased binding antibodies to rHA-Bovine in both repHA-Vietnam and repHA-Bovine vaccinated animals after boosting (Figure 1a). Interestingly, we measured a decline in antibody from 3 to 4 weeks after boosting (day -7 to day 0) (Figure 1a) suggesting the effect of boosting may be transient.\u003c/p\u003e\n\u003cp\u003eWe also measured homologous and heterologous neutralizing activity by virus neutralization assay at time of challenge. No neutralizing activity against authentic A/Vietnam was measured at time of challenge (Figure 1b) while we detected low neutralizing responses in 3 of 6 repHA-Bovine vaccinated animals against A/bovine (Figure 1b). Cumulatively, these results indicate that repHA-Vietnam elicits antibodies capable of binding both A/Vietnam and A/bovine but with undetectable neutralization activity. In contrast, repHA-Bovine elicited antibodies capable of binding A/bovine and with low homologous neutralizing activity.\u003c/p\u003e\n\u003cp\u003eTo more fully characterize the breadth of the HA-binding response elicited by repHA vaccination and to evaluate heterosubtypic binding, we utilized a bead-based assay to evaluate binding to multiple H5 HAs across several clades at time of challenge, four-weeks after boosting. Comparing repHA vaccine groups, repHA-Vietnam vaccinated animals had significantly increased binding antibodies to most non-clade 2.3.4.4b HAs while repHA-Bovine animals had significantly increased binding antibodies to clade 2.3.4.4b HAs (Figure 1c and supplemental figure 1). Against heterosubtypic HAs, in some animals, we measured weak binding against other influenza A H3 and H1 subtypes and other avian influenza viruses H6N1, H10N8, and H9N2 HAs prior to vaccination (Supplemental Figure 1) suggesting previous influenza A virus exposure in these animals. RepHA vaccination increased binding to heterosubtypic HAs in some animals but responses remained largely focused against H5 HAs (Supplemental Figure 1). Cumulatively, these data suggest that repRNA expressed A/Vietnam and A/Bovine HAs elicit distinct antibody responses, with a matched repHA-Bovine eliciting the greatest responses against clade 2.3.4.4b HAs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepHA vaccination protects against lethal A/bovine challenge.\u0026nbsp;\u003c/strong\u003eFour-weeks after boosting, animals were challenged with 10\u003csup\u003e7\u003c/sup\u003e median-tissue culture infectious doses (TCID\u003csub\u003e50\u003c/sub\u003e) of A/bovine via the intratracheal (IT) route. Animals were monitored daily for signs of clinical disease and exams were conducted on days 0, 1, 3, 5, 7, 9, 14 and 21 post-innoculation (PI). Sham-vaccinated animals quickly developed overt clinical signs characterized by lethargy, hunched posture and signs of respiratory distress (Figure 2a). Additional clinical signs such as weight loss and elevated body temperature were also measured (Figure 2b and c). Between days 6 to 8, all sham-vaccinated animals reached endpoint criteria with signs of respiratory distress and were euthanized (Figure 2d). In contrast, both repHA-Vietnam and repHA-Bovine vaccinated animals were protected against clinical disease until study end on d21 (Figure 2a-d). We observed transient elevated body temperature in repHA-vaccinated animals on day 1 PI (Figure 2c), possibly reflecting acute host responses to the large amounts of challenge virus.\u003c/p\u003e\n\u003cp\u003eOn exam days, thoracic radiographs were taken and scored for evidence of pneumonia and inflammation. Sham-vaccinated animals developed consistent evidence of pulmonary infiltrates (Figure 2e). RepHA-vaccinated animals had reduced radiographic evidence of pulmonary infiltrates (Figure 2e) consistent with protection from clinical disease. Cumulatively, these data indicate that both repHA-Vietnam and repHA-Bovine confers robust protection against clinical disease upon challenge with A/bovine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepHA vaccination confers control of viral replication.\u0026nbsp;\u003c/strong\u003eOn exam days or at time of necropsy, we collected blood, as well as oral and nasal swabs for quantification of virus replication by PCR and TCID\u003csub\u003e50\u003c/sub\u003e assay. Overall viral RNA in the blood or swabs was variable (Figure 3a-c) and infectious virus in the blood or oral swabs negligible (Supplemental Figure 1). Sporadic infectious virus was detected in some nasal swabs, with greatest amount detected in the terminal nasal swab of one sham-animal that succumbed on day 6 PI (Supplemental Figure 1). At day 5 PI, during peak detection of virus genome copies in nasal swabs of sham-vaccinated animals, only repHA-Bovine vaccinated animals exhibited a significant reduction in viral load (Figure 3c). We also collected bronchoalveolar brushes (BABs) on days 3 and 9 PI to measure viral loads within the lower respiratory tract. In contrast to upper respiratory swabs, infectious virus was consistently detected in the BABs of sham vaccinated animals at day 3 PI (Figure 3d) suggesting that A/bovine was mostly restricted to the lower respiratory tract in challenged animals. RepHA-Vietnam vaccinated animals exhibited a trend towards lower infectious virus in the day 3 BAB (Figure 3d). However, by day 3, 4 of 6 repHA-Bovine vaccinated animals had undetectable infectious virus in the BABs (Figure 3d) demonstrating prompt control of the virus within the lungs. Consistent with little-to-no clinical disease at day 9, no infectious virus was detected in the BABs of repHA vaccinated animals collected at day 9 (Figure 3d).\u003c/p\u003e\n\u003cp\u003eWe next evaluated viral loads in various tissues collected at time of necropsy, days 6 \u0026ndash; 8 PI for sham-vaccinated and day 21 for repHA-vaccinated. RepHA vaccination significantly reduced viral RNA in all tissues examined except for the heart, though viral RNA was inconsistently detected in this tissue even in sham-vaccinated animals (Figure 3e). We also detected infectious virus in the lung tissue of sham- but not repHA-vaccinated animals (Figure 3f). Cumulatively, these data demonstrate that repHA vaccination resulted in rapid control of A/bovine virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepHA vaccination protects against lung pathology.\u0026nbsp;\u003c/strong\u003eAt time of necropsy, the lungs were weighed and % of total body weight of the lungs calculated as an indicator of pulmonary edema \u003csup\u003e21\u003c/sup\u003e. Both repHA vaccinated groups had reduced lung weights as % of body weight compared to sham-vaccinated animals (Figure 4a). This correlated with consistent presence of lesions across multiple lung lobes in sham-vaccinated animals but no gross lesions in any repHA-vaccinated animal (Figure 4b). These data are consistent with our radiograph data showing evidence of pneumonia in sham-vaccinated animals but not repHA-vaccinated animals at euthanasia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRobust anamnestic antibody responses in repHA vaccinated animals.\u0026nbsp;\u003c/strong\u003eWe evaluated serum collected at day 0, 7, 14 and 21 PI for HA binding antibodies by ELISA and for neutralization against A/Vietnam and A/bovine on day 14 PI. Against both A/Vietnam and A/bovine HA antigen, we measured a rapid increase in binding antibodies by ELISA in both repHA vaccinated groups by day 7 PI (Figure 5a). No increase in HA binding antibodies was measured in sham-vaccinated animals prior to euthanasia (Figure 5a) consistent with poor outcome in these animals. \u0026nbsp;Despite detecting little neutralizing activity prior to challenge in repHA vaccinated animals, by day 14 PI we detected strong neutralizing activity against A/bovine in both repHA-Vietnam and repHA-Bovine vaccinated animals (Figure 5b). In contrast, neutralizing activity against A/Vietnam was only measured in repHA-Vietnam vaccinated animals (Figure 5b). This is consistent with lower levels of A/Vietnam HA binding antibodies in repHA-Bovine vaccinated animals (Figure 5a). \u0026nbsp;Using our bead-based assay, at day 21 PI, serum from repHA-Vietnam-vaccinated animals contained antibodies that bound all H5 HAs tested and with reactivity to H1 and H6 HAs, consistent with H5, H1 and H6 being group 1 HAs \u003csup\u003e22\u003c/sup\u003e (Figure 5c). Serum from repHA-Bovine-vaccinated animals had strong reactivity to clade 2.3.4.4b HAs with reduced binding to other clade H5 HAs (Figure 5c) consistent with our ELISA and VN data (Figure 5a-b). \u0026nbsp;Heterosubtypic binding antibodies against H1N1 and H6N1 viruses also developed post-infection in repHA-Bovine vaccinated animals (Figure 5c).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe fitness and genetic diversity of clade 2.3.4.4b H5NX viruses poses a serious risk of pandemic HPAI \u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. While most recent cases of clade 2.3.4.4b H5N1 infections have been mild, a Canadian teenager and Louisiana resident have been hospitalized with severe disease \u003csup\u003e26,27\u003c/sup\u003e and continued circulation in livestock and wild birds will result in further spillover into humans. Vaccines to address pandemic HPAI are stockpiled in many countries but whether these vaccines need to be updated to address current 2.3.4.4b circulation or to address future antigenic drift is unclear. \u0026nbsp;Our data demonstrate that a repRNA vaccine expressing either the A/Vietnam or A/bovine HA can confer robust protection against HPAI A H5N1 infection in a relevant macaque model. Protection was associated with HA binding antibodies but interestingly, at time of challenge, little-to-no VN activity was detected in repHA-vaccinated animals. Although neutralizing antibodies correlate with protection against influenza \u003csup\u003e28\u003c/sup\u003e , it is possible that repHA-elicited non-neutralizing antibodies may have contributed to protection \u0026nbsp;\u003csup\u003e29,30\u003c/sup\u003e . We and others have seen strong protection against 2.3.4.4b H5N1 challenge in mice in the absence of potent neutralizing antibody responses \u003csup\u003e31\u003c/sup\u003e. \u0026nbsp;Alternatively, the rapid anamnestic response we measured in repHA vaccinated animals by day 7 PI may have contributed to protection. However, the significantly reduced viral loads within the lungs by 3 days PI suggests immunity present at time of challenge conferred robust protection on its own.\u003c/p\u003e\n\u003cp\u003eOur data support the hypothesis that the mutations accumulating in the head domain of the A/bovine HA compared to A/Vietnam can lead to functional differences in antibody recognition of HA. In mice vaccinated with repHA-Vietnam or repHA-Bovine we measured significantly reduced binding to the opposite H5 HA and only repHA-Bovine protected against A/bovine challenge. Here, although both repHA vaccines conferred protection, repHA-Vietnam vaccination elicited antibodies with diminished binding to clade 2.3.4.4b HAs. This was associated with delayed control of infectious virus within the lungs and reduced control of viral shedding in the nasal cavity of repHA-Vietnam vaccinated animals. \u0026nbsp;Heterologous repHA-Vietnam vaccination and A/bovine challenge also resulted in cross-clade neutralizing antibodies against both A/Vietnam and A/bovine while homologous vaccination and challenge resulted in neutralizing antibodies against only A/bovine. Our findings that potent neutralizing antibodies against A/bovine can fail to neutralize A/Vietnam demonstrate that the mutations in the HA between A/Vietnam and A/bovine impact epitopes targeted by neutralizing antibodies. Recent structural studies of bovine HA have shown that the receptor binding site of the H5 HA may be shielded by an autoglycan that could shield neutralizing antibody binding to the receptor binding site \u003csup\u003e32\u003c/sup\u003e . While the glycosylated amino acid is highly conserved among H5 HAs, the presence of this glycan has been rarely reported for H5 HA structures and further study is needed to determine whether this glycan impacts pathogenesis.\u003c/p\u003e\n\u003cp\u003eOur data suggest that heterologous A/bovine challenge in repHA-Vietnam animals boosted neutralizing antibodies targeting more conserved epitopes between A/bovine and A/Vietnam and heterologous prime-boosts may be an optimal strategy to elicit broad cross clade immunity. In humans receiving homologous or heterologous H5N1 vaccines, heterologous boosting induced broader cross-clade antibody responses than homologous prime-boost \u003csup\u003e33\u003c/sup\u003e suggesting heterologous vaccination may afford greater breadth of protection against H5N1. Further, humans vaccinated with an H5N1 vaccine had increased numbers of antibodies targeting the more conserved HA stem region suggesting that H5 immunization of individuals with seasonal H1 or H3 HA immunity preferentially recruits B-cells with specificity for more conserved domains of HA \u003csup\u003e34\u003c/sup\u003e. Preexisting H1N1 immunity due to virus infection in ferrets was sufficient to protect against lethal bovine H5N1 infection \u003csup\u003e35\u003c/sup\u003e. \u0026nbsp;Immunity against protective epitopes outside the HA in H1N1-infected ferrets may have contributed to protection against H5N1 \u003csup\u003e36\u003c/sup\u003e and suggest inclusion of other antigens than just HA may increase vaccine efficacy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, our data are in contrast to our recent evaluation of these same vaccines in a lethal mouse challenge model in which only repHA-Bovine vaccination protected against A/bovine challenge. This is an important consideration as while updated H5 vaccines are in clinical development, stockpiled vaccines for H5N1 in the United States use historical H5 HAs \u003csup\u003e8,37\u003c/sup\u003e and our data here suggest these vaccines may confer protection against A/bovine and related viruses. It is unclear why we measured distinct protection against A/bovine in mice and non-human primates but could be due to differing H5N1 disease presentation in these models. Whereas non-human primates develop a severe lower respiratory disease following IT challenge, mice rapidly exhibit both respiratory and neurological involvement following IN challenge \u003csup\u003e38\u003c/sup\u003e. It is likely the correlates of protection differ for upper versus lower respiratory tract infection as well as for neurological disease. Indeed, we observed superior control of viral load in the nasal swabs of NHPs vaccinated with homologous repHA-Bovine (Figure 3c), suggesting an antigen-dependent control of virus in the upper respiratory tract. Historically, this tissue has been difficult to protect from respiratory virus infection following peripheral vaccination with conventional RNA vaccine platforms \u003csup\u003e39\u0026ndash;41\u003c/sup\u003e. In contrast, we have consistently observed protection of the upper airway in respiratory virus-challenge models vaccinated with repRNA/LION \u003csup\u003e15,42\u0026ndash;44\u003c/sup\u003e. We also measured low levels of H5, H3 and H1 HA binding antibodies in some macaques prior to vaccination suggestive of previous influenza exposure. Cross-neutralizing activity against clade 2.3.4.4b viruses was elicited in clade 1 or 2.1 H5N1 vaccinated humans \u003csup\u003e45\u003c/sup\u003e and preexisting influenza A immunity has been shown to improve the breadth of subtypic responses after vaccination \u003csup\u003e34\u003c/sup\u003e. These data suggest that the animal model, vaccine platform and preexisting immunity may influence apparent vaccine efficacy against H5N1 and support the use of both mouse and NHP models in preclinical evaluation of influenza countermeasures.\u003c/p\u003e\n\u003cp\u003eTogether our data show that our repRNA/LION platform expressing the H5 HA of either a historical or contemporary H5N1 isolate can protect against lethal H5N1 challenge in non-human primates. These data support the utility of nucleic acid vaccines to be updated to match viral evolution and emergence while also suggesting that stockpiled vaccines based on A/Vietnam HA or other historical H5N1 strains will likely provide protection against contemporary H5N1 infections. Nevertheless, our data also suggest that the HA of clade 2.3.4.4b H5N1s circulating in the United States are accumulating mutations leading to antigenic drift from stockpiled vaccine antigens. Thus, continued genetic surveillance of the circulating strains of H5N1 in the United States is prudent.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1. CDC. H5 Bird Flu: Current Situation. \u003ca href=\"https://www.cdc.gov/bird-flu/situation-summary/index.html\"\u003ehttps://www.cdc.gov/bird-flu/situation-summary/index.html\u003c/a\u003e (2024).\u003c/p\u003e\n\u003cp\u003e2. Canda, P. H. A. of. Statement from the Public Health Agency of Canada: Update on Avian Influenza and Risk to Canadians. \u003ca href=\"https://www.canada.ca/en/public-health/news/2024/11/update-on-avian-influenza-and-risk-to-canadians.html\"\u003ehttps://www.canada.ca/en/public-health/news/2024/11/update-on-avian-influenza-and-risk-to-canadians.html\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e3. Stobbe, M. 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(2024) doi:10.21203/rs.3.rs-4946897/v1.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals, Biosafety and Ethics.\u0026nbsp;\u003c/strong\u003eAll infectious work with infectious influenza was performed in the maximum containment laboratory in accordance with standard operating procedures approved by the Rocky Mountain Laboratories Institutional Biosafety Committee, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Hamilton, MT, USA). All animal work was performed in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the Office of Animal Welfare, National Institutes of Health and the Animal Welfare Act of the US Department of Agriculture, in an AAALAC-accredited facility and study protocol approved by the RML Animal Care and Use Committee. Male (n = 15) and female (n = 3) Mauritius origin cynomolgus macaques between the ages of 6.16 to 15.5 years of age were evenly divided by sex and then randomly assigned to study groups. \u0026nbsp;Animals were housed in adjoining individual primate cages that enabled social interaction, under controlled conditions of humidity, temperature and light (12-h light/12-h dark cycles). Water was available ad libitum. Animals were monitored at least twice daily (pre- and post-inoculation) and fed commercial monkey chow, treats and fruit twice a day by trained personnel. Environmental enrichment consisted of manipulanda, visual enrichment and audio enrichment. All procedures on nonhuman primates were performed by board-certified clinical veterinarians. Upon inoculation animals were comprehensively evaluated for disease signs using a score sheet by staff blinded to study groups \u003csup\u003e20\u003c/sup\u003e . Thoracic radiographs were taken on exam days or at time of euthanasia and scored by a trained veterinarian and each lung lobe scored according to 0 = none, 1 = mild, 2 = moderate and 3 = severe interstitial pneumonia. Scores for all the lung lobes were then summed and presented as total lung score per animal. All necropsies were performed by board-certified veterinary pathologists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVaccination and virus challenge.\u0026nbsp;\u003c/strong\u003eVaccines used here were identical to those used previously \u003csup\u003e46\u003c/sup\u003e. \u0026nbsp;Vaccination was performed by a single intramuscular injection consisting of 25\u0026mu;g of RNA. Animals were boosted with identical vaccinations four-weeks later. Vaccination appeared well tolerated with no adverse events observed following vaccinations. A/bovine challenge virus was identical to that previously described \u003csup\u003e46\u003c/sup\u003e. Animals were challenged with 1x10\u003csup\u003e7\u003c/sup\u003e median-tissue culture infectious doses (TCID\u003csub\u003e50\u003c/sub\u003e) via the intratracheal route as previously described \u003csup\u003e20\u003c/sup\u003e .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA and Neutralization assays.\u0026nbsp;\u003c/strong\u003eThe ELISAs against recombinant HAs from A/Vietnam or A/bovine were as previously described \u003csup\u003e46\u003c/sup\u003e. Maxisorp (Nunc) plates were coated with recombinant HA from A/Vietnam/1203/2004 (IBT Bioservices) or A/dairy cow/Texas/24-008749-002 (SinoBiological, homologous to A/bovine) at 100ng/well in phosphate buffered saline (PBS) overnight. Plates were blocked and serial dilutions of mouse sera applied. Bound antibody was detected with an anti-mouse IgG conjugated to horseradish peroxidase (Southern Biotech, Catalog #1030-05, Lot# D1922-YI62C, 1:4000), plates developed with 3,3\u0026prime;,5,5\u0026prime;-Tetramethylbenzidine (TMB) substrate (SeraCare) and absorbance was read at 450nM. Endpoints are reported as the reciprocal of the serum dilution to have an absorbance of 3 standard deviations above background absorbance as determined by curve-fit and interpolation of absorbance value. For VN assay, 100 TCID\u003csub\u003e50\u003c/sub\u003e of virus was mixed with serial dilutions of heat inactivated (56\u0026deg;C for 30 minutes) sera for 1 hour at 37C. Residual infectious virus was then measured on MDCK cell monolayers. After three days, supernatant was collected and presence of infectious virus determined by mixing with 0.33% turkey erythrocytes and measurement of hemagglutination. Titers are reported as the reciprocal of the last dilution to exhibit VN activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR and TCID\u003csub\u003e50\u003c/sub\u003e to measure viral loads.\u0026nbsp;\u003c/strong\u003eMeasurement of viral RNA and infectious virus was as previously described \u003csup\u003e20\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBead-based multiplex HA binding assay.\u0026nbsp;\u003c/strong\u003eThe bead based binding assay was performed as previously described \u003csup\u003e46\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics.\u0026nbsp;\u003c/strong\u003eIndicated statistical comparisons were performed with GraphPad Prism v10.2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability.\u0026nbsp;\u003c/strong\u003eThe data underlying the figures presented here are posted publicly at FigShare, DOI TBD\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003eThe A/bovine virus was kindly provided by Richard Webby St. Jude\u0026rsquo;s Children hospital and Andrew Bowman Ohio State University. We also wish to thank Vincent Munster and Emmie De Witt and their laboratory staff, Laboratory of Virology, NIAID for their efforts to obtain, propagate and titer virus stocks. We thank the Rocky Mountain Veterinary Branch biologists and animal care staff for support of the animal study. This study was supported by the Intramural Research Program, NIAID. Funders had no input on study design, data interpretation or decision to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions.\u0026nbsp;\u003c/strong\u003eDWH, KR, JHE, HF designed the study. DWH, AG, AO, SSL, NM, MA, MC, EA, ML, TH, ETS, NW, JL, BJS, PH, GS, CS, CC performed the study. DWH, AO and SSL performed data analysis. DWH wrote the manuscript. DWH, JHE, HF edited the manuscript. HF obtained funding. All authors reviewed the manuscript and agree with publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest.\u0026nbsp;\u003c/strong\u003eJHE, ETS, NW, SP, KH, EH, and KG have equity interest in HDT Bio. JHE is an inventor on patents (US Patent nos. 11,458,209; 11,433,142; 11,752,218; 11,648,321; and 11,654,200) and patent applications (PCT/US22/76787, PCT/US23/60225, and PCT/US2024/010326) pertaining to the LION formulation and repRNA compositions described in the studies. Funders had no role in study design, data interpretation or decision to publish. The remaining authors declare no competing interest.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5723772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5723772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In early 2024, clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A H5N1 virus was detected in United States dairy cattle. While so far the public health threat of contemporary clade 2.3.4.4b H5N1 virus strains remains low, continued circulation in mammals and frequent spillover into humans poses a threat of pandemic H5N1. The United States and other countries have stockpiled vaccines and have plans in place to rapidly produce vaccines should a pandemic H5N1 virus emerge. However, the continued antigenic drift of clade 2.3.4.4b H5N1 antigens compared to historical antigens used by stockpiled vaccines has raised questions of whether these vaccines will confer protection or if stockpiles need to be updated. We recently evaluated a replicating RNA (repRNA) vaccine against lethal contemporary 2.3.4.4b H5N1 virus challenge in mice and found that a homologous but not historical H5 hemagglutinin (HA)-based vaccine conferred protection. Here we further evaluated the protective capacity of a repRNA expressing the contemporary 2.3.4.4b HA or a repRNA expressing a historical H5 HA (A/Vietnam/1203/2004) in a recently developed lethal non-human primate challenge model. We found that both vaccines conferred robust protection against lethal 2.3.4.4b H5N1 virus challenge, protecting against clinical disease and death, reducing viral loads and signs of respiratory illness. Our data show the repRNA platform can elicit protective immunity against lethal respiratory influenza disease and that historical H5 HAs elicit cross-protective immunity.","manuscriptTitle":"A replicating RNA vaccine protects against lethal clade 2.3.4.4b influenza A H5N1 virus challenge in cynomolgus macaques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 23:25:32","doi":"10.21203/rs.3.rs-5723772/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64dfd650-8e30-44fe-bea3-e26c519e0528","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42205127,"name":"Biological sciences/Microbiology/Virology/Influenza virus"},{"id":42205128,"name":"Biological sciences/Microbiology/Vaccines/RNA vaccines"}],"tags":[],"updatedAt":"2025-01-14T14:41:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 23:25:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5723772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5723772","identity":"rs-5723772","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}