Neutralizing activity against bovine H5N1 HPAIV (clade 2.3.4.4b) in human plasma after seasonal influenza vaccination

Abstract

In 2024, a clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) emerged in dairy cattle in the United States and spread rapidly to over 1,000 herds across multiple states. At least 41 human infections have occurred through contact with infected cattle, though no fatalities have been reported so far. This raises questions about whether the human innate immune system provides a barrier to bovine H5N1 HPAIV and whether seasonal influenza vaccines offer cross-protection. To address these questions, we used pseudoviruses bearing hemagglutinin (HA) and neuraminidase (NA) from seasonal influenza A or various H5Ny HPAIV strains (from cattle, duck, and seal). Pseudoviruses bearing H5N1 HPAIV HA and NA entered a wide range of mammalian and avian cell lines, including multiple cell lines from the human respiratory tract, while entry into A549 human lung cells was reduced when IFITM proteins were expressed. Additionally, preincubation of pseudovirus particles bearing H5N1 HPAIV HA and NA with plasma from individuals vaccinated with seasonal influenza vaccines inhibited viral entry. Collectively, these results suggest that the human innate immune system imposes a barrier against bovine H5N1 HPAIV infection and that seasonal influenza vaccines can induce cross-neutralizing activity against bovine H5N1 HPAIV.

Keywords: H5N1; bovine; clade 2.3.4.4b; neutralization; seasonal influenza vaccination.

Figure 1.

Cell line tropism, IFITM inhibition and neutralization sensitivity of particles bearing clade 2.3.4.4b bovine H5N1 HPAIV HA and NA. (A) Pseudoviruses were trypsin-treated or mock-treated, and inoculated onto the indicated cell lines. Cell entry was analyzed by quantification of luciferase activity in cell lysates. Data represent the mean of six experiments (four technical replicates), and entry was normalized against the background (signals obtained from particles without viral glycoprotein, set as 1). Error bars represent the standard error of the mean (SEM). (B) A549 cells stably expressing IFITM1, IFITM2, IFITM3, or CAT (chloramphenicol acetyltransferase) were pre-treated with amphotericin B (AmphoB, 2.5 µM) or mock-treated, before being inoculated with pseudoviruses. Data represent the mean of three experiments (four technical replicates), and cell entry was either normalized against the background (upper panels) or against signals obtained for A549-CAT cells (set as 100%, lower panel). Error bars represent the SEM. (C) Pseudoviruses were preincubated with human plasma (n = 50) and inoculated onto Caco-2 cells. Entry was normalized against entry in the absence of plasma (0% inhibition) and the neutralizing titre 50 values were determined. Data represent individual NT50 values and geometric mean titres (GMT) from a single experiment (four technical replicates). Information below the graphs indicate reactivity rates and GMT values. For graphical reasons samples yielding an NT50 value below the limit of detection (LOD, 12.5) were assigned a value of 1. The dashed line indicates the lowest plasma dilution tested. For panels A and B, statistical significance was analyzed by two-tailed Student's t-test with Welch correction (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).