Brief Communication
Efficacy of oseltamivir and baloxavir against A(H5N1)-contaminated bovine milk in mice
https://doi.org/10.21203/rs.3.rs-5119512/v1
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Understanding the reliability of FDA-approved antivirals against A(H5N1) influenza viruses circulating in peridomestic species including dairy cows is critical to public health and pre-pandemic planning, but it cannot be modeled clinically due to A(H5N1) disease severity. In mice, we demonstrate baloxavir treatment mediates greater disease outcomes (survival, viral dissemination) than oseltamivir after lethal challenge (orally, intranasally, ocularly) with A(H5N1)-contaminated cow milk.
Highly pathogenic avian influenza(HPAI) A(H5N1) HA clade 2.3.4.4b viruses emerged in North America in 2021 and have been detected in wild birds, terrestrial mesocarnivores, and aquatic mammals1. Zoonotic exposure has caused 13 human cases. Recent identification of A(H5N1) in 208 dairy cow herds in 14 states represents additional risk for virus evolution. Multiple routes of potential human exposure exist including respiratory and ocular(aerosols/splashes during milking or cleaning procedures) and oral(consumption of contaminated raw milk)2. Occupational exposure to infected cows has caused 4 confirmed human infections resulting in conjunctivitis and/or mild respiratory symptoms. All were treated with neuraminidase inhibitor(NAI) oseltamivir phosphate(Tamiflu)3,4. Baseline oseltamivir carboxylate(OSE) 50% inhibitory concentrations(IC50) among clade 2.3.4.4b avian viruses were reported as 15-fold higher than those of seasonal A(H1N1)pdm09 reference5. It is unclear if this indicates actual decreased OSE efficacy or if a new baseline comparison value is needed for this subtype. Understanding in vivo activity of existing influenza antivirals after multi-route exposure to A(H5N1) contaminated milk therefore becomes of substantial public health interest.
Here, we tested US FDA-approved antivirals OSE and cap-dependent endonuclease inhibitor baloxavir(Xofluza) following oral, intranasal, or ocular inoculation(to mimic potential routes of human exposure) of mice with milk collected from an A(H5N1) infected dairy cow. 50% mouse lethal doses (MLD50) were determined for each route and used to deliver a lethal challenge dose(Fig. S1). Drug treatment was initiated 24 hpi. OSE(oral, 20mg/kg/day, 5 days, BID) was administered to mimic human dosing regimens and/or pre-clinical mouse studies6,7. BXA(5mg/kg, 1 dose, subcutaneous) was administered to provide similar PK/PD and elimination half-life values to pre-clinical animal studies8. Due to the severity of HPAI infection, a high dose of each drug(OSE 200mg/kg, BXA 25mg/kg) was also included. Non-drug treated animals served as negative controls. Viral titers in multiple tissues were determined near peak clinical disease(Fig. S2).
All orally inoculated, untreated animals succumbed to infection with a mean time-to-death(MTTD) of 4.0 days. Virus(≥ 3.1 log10TCID50/mL) was detected in all collected organs except the stomach. OSE provided 13–25% survival(Fig. 1A). Statistical significance between drug-treated animals and non-treated were compared for organ titers. Only high dose OSE lowered any organ titer(nasal turbinate [NT] only, P ≤ 0.01)(Fig. 1D). BXA treatment provided 0–25% survival(Fig. 1A). However, both BXA doses decreased viral titers in the blood, eye, NT, and intestine(P ≤ 0.01), while high dose BXA also decreased lung titers(P < 0.01)(Fig. 1C).
All intranasally inoculated, untreated animals succumbed to infection(MTTD 5.0 days), with virus(2.2–7.6 log10TCID50/mL) detected in all organs. No low dose OSE animals survived, while high dose provided 40% survival(Fig. 1D). Low dose OSE decreased blood titers(P ≤ 0.01), while high dose OSE additionally decreased intestine titers(P ≤ 0.01)(Fig. 1E). BXA treatment provided 50–75% survival and decreased viral titers in all tissues(P ≤ 0.05)(Fig. 1F).
All ocularly inoculated, untreated animals succumbed to infection(MTTD 8.5 days), and virus(≥ 1.1–6.1 log10TCID50/mL) was detected in all organs except the stomach. OSE treatment provided 25–63% survival(Fig. 1G). Both OSE doses decreased viral titers in the brain and lung(P ≤ 0.0001), and a trend towards fewer animals with ocular titers was observed. High dose OSE additionally decreased NT, and intestine titers (P ≤ 0.0001)(Fig. 1H). Both BXA doses provided 100% protection and decreased all tissue titers including the eye(P ≤ 0.05)(Fig. 1I).
We next tested the phenotypic susceptibility of our inocula to BXA and OSE and compared the results to median avian A(H5N1) 2.3.4.4b IC50 values reported by the US Centers for Disease Control and Prevention5. Virus present in our milk inocula had a BXA IC50 of 0.41 nM, a ≤ 1.4-fold change compared to A(H5N1) 2.3.4.4b median or A(H1N1)pdm09 references, indicating normal inhibition9. The OSE IC50 was 2.24 nM, a ≤ 1.5-fold change in susceptibility compared to the A(H5N1) 2.3.4.4b median, but a 13-fold decrease compared to seasonal A(H1N1)pdm09 reference(Table 1), within the range indicating reduced drug inhibition10 and perhaps contributing towards our results. Correlation of these NAI IC50 values and treatment outcomes remains somewhat challenging due to low numbers of relevant published studies11,12. Importantly, avian A(H5N1) 2.3.4.4b IC50 values for other NAIs (zanamivir and peramivir) were not significantly different from seasonal references, suggesting an OSE-specific effect5.
| Virus sample | Oseltamivir | Baloxavir |
|---|---|---|
| IC50 ± SD (nM) | EC50 ± SD (nM) | |
| Virus-contaminated milk | ||
| A/bovine/Ohio/B24OSU-439/2024 | 2.24 ± 0.07 | 0.41 ± 0.16 |
| References | ||
| A(H5N1) HA clade 2.3.4.4b | ||
| Median (n = 15 viruses, 2022)1 | 2.66 | 0.34 |
| Median (n = 12 viruses, 2022–2023)2 | 1.46 ± 0.18 | 0.29 ± 0.06 |
| A(H1N1)pdm09 | ||
| A/Illinois/45/20191 | 0.17 ± 0.03 | - |
| A/Illinois/08/20181 | - | 1.12 ± 0.32 |
| A/Denmark/524/20092 | 0.37 ± 0.25 | - |
| rg-A/California/04/20092 | - | 0.61 ± 0.06 |
| 1 Nguyen et al., Antiviral Research, 2023, PMID: 37494978; Centers for Disease Control and Prevention | ||
| 2 Andreev et al. JID, 2024, PMID: 37770028, St. Jude Children's Research Hospital | ||
| rg, reverse genetics; (-) not performed for this isolate | ||
Clinical relevance of these increased OSE IC50 values is not fully understood and extrapolation of our data to clinical care requires caution. Neurological pathology associated with 2.3.4.4b virus in animal models13 has not been observed in humans. We cannot rule out that extra-respiratory spread contributes toward the high mortality we observed. Nevertheless, the inability to conduct human A(H5N1) clinical trials means animal modeling and associated therapeutic interventions is currently the only option to begin to understand the relationships between in vitro susceptibilities and in vivo efficacy. While our data indicate oral inoculation with HPAI virus-contaminated milk is difficult to treat with either drug, disease outcomes after intranasal and ocular inoculation were broadly better with BXA vs. OSE. It may be important to consider BXA, in addition to NAIs like OSE, for treatment of severe infections and/or stockpiling for pandemic preparedness. Given BXA’s low barrier to resistance14 monitoring of patient shedding, ideally with sequence analysis, may be needed. Combination therapy with baloxavir, OSE, or approved nucleoside analogues like molnupiravir may be useful strategies to decrease treatment-emergent resistance and improve clinical HPAI outcomes. Extending OSE dosing may also be relevant for A(H5N1) infection, as significant survival outcomes and decreased tissue titers were observed in A(H5N1)-challenged mice given 8 days of OSE treatment vs. only 515. Testing such strategies are again largely dependent on pre-clinical animal modeling. Additional monotherapy and combination therapy studies for avian and bovine HPAI A(H5N1) are critically needed. Performing these studies in mice and other influenza animal models such as ferrets may provide critical data to inform clinical implementation of these antiviral drugs during localized outbreaks or a pandemic.
This study was conducted with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, contract #75N93021C00016(R.W.)
There is NO Competing Interest.
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