Immunogenicity and safety of a rabies-based highly pathogenic influenza A virus H5 vaccine in cattle

Abstract

The circulation of highly pathogenic H5 influenza A viruses in cattle, other mammals, and wildlife threatens animal and human health. To address this, we vaccinated heifer-calves with a deactivated rabies-virus-based H5 vaccine, which was well-tolerated and elicited neutralizing antibodies against both clade-1 and clade-2.3.4.4b H5N1 viruses, comparable to naturally H5-infected and convalescing cows. The immune responses to the vaccine platform were durable for at least 200 days and unaffected by preexisting RABV immunity.

Fig. 1. H5 immunogenicity of RABV-H5 vaccine in cattle.

A Schematic genomic maps of the RABV-H5 vaccine vector and the parental RABV. The R333E-attenuating mutation in the RABV G antigen is indicated. N=nucleoprotein, P=Phosphoprotein, M=matrix protein, G=glycoprotein, L=polymerase. B Immunofluorescent staining of H5 and RABG in RABV-H5 and RABV-infected Vero cells. Cells were infected at MOI 0.01 and fixed after 48 h. Cells were permeabilized and stained with α-HPAI-H5N1 H5 polyclonal antibodies (purple), α-RABV-G 4C12 monoclonal antibody (green), and DAPI to visualize the nuclei (blue). In the merged images, white indicates an overlap between purple and green. Images were taken at 40X magnification. Scale bars represent 100 μm. C, D represent protein composition analysis of 1 μg sucrose purified virions by western blotting (C) and SDS-PAGE followed by SYPRO®-RUBY protein staining (D). Blots were either probed with α- H5 (NR-163), α-RABV-G 4C12 (middle panel) or α-RABV-N polyclonal rabbit sera (bottom panel). E Cattle immunization and blood draw schedule; 3 groups of 4 cattle were immunized once (group A) or twice (B, C) with 100 μg/dose of BPL-inactivated RABV-H5 vaccine, with (A, C) or without (B) SEPPIC-SWE^TM adjuvant. Created with Biorender.com. VSV∆G-H5-GFP virus neutralization in prime only with adjuvant (F), prime-boost without adjuvant (G) and prime-boost with adjuvant (H). Filled circles are NT₅₀ titers. Average (bars) and STDEV (error bars) are shown. Comparison of day 42 NT₅₀ values between the three vaccination groups is shown (I). Day 0 and 42 VNA titers (NT₅₀) against VSV∆G-H5-GFP harboring the H5-cow version (J) or the H5- A/Viet Nam/1203/2004 version (K) and the comparison between them (L). PR8-H5N1 (M) and HPAI-H5N1 cow (N) VNAs (100% neutralization) are shown. Ordinary one-way ANOVA with Tukey’s Multiple Comparison Test was used to determine statistical differences between groups at each time point. Stars indicate significant differences. Error bars indicate the mean with SD for groups of 4 cattle with samples run in duplicate. (****p < 0.0001; ***p = 0.0001; **p ≤ 0.0029; *p < 0.0173; ns not significant).

Fig. 2. Vaccine immunogenicity in the presence and absence of preexisting RABV immunity.

Rabies virus (strain CVS-11) VNAs (IU/ml) in cattle sera were determined by Rapid fluorescent focus inhibition test (RFFIT) in prime only with adjuvant (A), prime- boost without adjuvant (B) and prime-boost with adjuvant (C). Comparison of day 42 VNA titers is shown (D). Cattle immunization and blood draw schedule (E). Six cattle were immunized twice with 100 μg/dose of BPL-inactivated, SEPPIC-SWE^TM adjuvanted RABV-GP38 vaccine. On Day 200, the cattle were vaccinated with the same dose of RABV-H5 with SEPPIC-SWE; and on day 2283 cattle received another dose of the adjuvanted RABV-H5 vaccine. Syringes represent immunizations, and blood tubes indicate the days blood was drawn. Created with Biorender.com. VSV∆G-H5-GFP virus-neutralization titers (NT₅₀) (F), PR8-H5N1 virus-neutralization titers (100% neutralization) (G), A/cattle/Texas/56283/2024 (H5N1) virus-neutralization titers (100% neutralization) (H) and Rabies virus-neutralization titers (IU/ml) (I) are shown. Open circles in (I) refer to cows receiving 1 dose of the H5N1 vaccine 3 total doses of the RABV vector while closed circles refer to 2 H5 doses and 4 total RABV doses. Bars represent the mean values, and error bars indicate standard deviation. Ordinary one-way ANOVA with Tukey’s Multiple Comparison Test was used to determine statistical differences between groups (****p < 0.0001; ***p ≤ 0.0008; **p ≤ 0.0024; *p ≤ 0.047; ns not significant). The dotted line indicates 0.5IU/mL, the WHO-accepted protective antibody level against RABV.