Production and Immune Response Against Pandemic Influenza Candidate Vaccines as Preparedness Against the Circulating H5N1 Influenza Viruses

Abstract

Background/Objectives:H5N1 influenza viruses are spreading worldwide and threaten global public health. Preparedness is necessary to mitigate the worst-case scenario should an H5N1 influenza pandemic occur and justify the development of vaccines against circulating H5N1 viruses of concern.

Methods: The production and characterization of egg-based split and inactivated H5Nx of three distinct monovalent antigens from clades 2.3.4.4b, 2.3.2.1c, and 2.3.4 were performed at an industrial scale. These antigens were formulated and their immune responses, when combined or not with IB160 squalene-based oil-in-water emulsion adjuvant in a rat model, were evaluated in a one- or two-dose immunization schedule. IgG antibodies, hemagglutination inhibitions, and microneutralization titers were measured for vaccine-induced immunity and cross-reactivity.

Results: Three monovalent vaccines from clades 2.3.4.4b, 2.3.2.1c, and 2.3.4 were produced at an industrial scale and characterized. The immune responses against the monovalent vaccines showed a clade-specific antibody response and the need to combine with IB160 adjuvant for a required immune response.

Conclusions: Considering the candidate vaccine viruses (CVVs) with the testing potency reagents available and that the antibody response obtained against the CVVs produced was clade-specific, IDCDC RG-71A is the indicated CVV for the predominant currently circulating H5N1 influenza virus of clade 2.3.4.4b and must be combined with adjuvant to induce a higher and efficacious immune response in a two-dose immunization protocol.

Keywords: H5N1 pandemic influenza vaccine; hemagglutination inhibition; immunogenicity; influenza virus; microneutralization; squalene-based emulsion adjuvant.

Figure 1

Geographical distribution of human HPAI H5N1 infections by clade from July 2021 to 21 April 2025. Total cases (N = 109). Clade 2.3.2.1a (black; N = 3): EPI_ISL_14223821, India, July 2021, 11-year-old male, critical illness, death 12 July 2021; EPI_ISL_19156871, Australia, after travel to India, 6 March 2024, severe illness, 2-year-old female; and EPI_ISL_19836227, India, 7 March 2025, 2-year-old female, death. Clade 2.3.2.1c (blue; N = 19): EPI_ISL_17024123, Cambodia, 21 February 2023, 11-year-old female, death; EPI_ISL_17069010, Cambodia, 23 February 2023, 49-year-old male, mild illness; EPI_ISL_18373263, Cambodia, 6 October 2023, 2-year-old female, death; EPI_ISL_18366401, Cambodia, 7 October 2023, 54-year-old male, death; EPI_ISL_18540514, Cambodia, 23 November 2023, 21-year-old female, death; EPI_ISL_18543642: Cambodia, 24 November 2023, 5-year-old female; EPI_ISL_18823967, Cambodia, 23 January 2024, 3-year-old male; isolate ID not published, Cambodia, 28 January 2024, 69-year-old male; EPI_ISL_18879683, Cambodia, late January, 9-year-old male, death; EPI_ISL_19312044, Cambodia, 8 February 2024, 16-year-old male; EPI_ISL_19031556: Vietnam, 19 March 2024, 21-year-old male, death; and two cases with isolate ID not published in Cambodia (Takeo province), a 3-year-old boy and a 5-year-old girl; EPI_ISL_19312043, Cambodia, 30 July 2024, 4-year male; EPI_ISL_19312044, Cambodia, 2 August 2024, 16-year female; EPI_ISL_19353003, Cambodia, 17 August 2024, 15-year female, death; EPI_ISL_19661054, Cambodia, 8 January 2025, 28-year-old man, death; EPI_ISL_19752030, Cambodia, 24 February 2025, 2-year-old male, death; and EPI_ISL_19791427, Cambodia, 22 March 2025, 3-year-old male, death. Clade 2.3.4.4b (red; N = 85): EPI_ISL_8799552, United Kingdom (UK), 26 Dezember 2021, 79 years old, asymptomatic; ON759331.1, USA (Colorado), 20 April 2022, fatigue only; EPI_ISL_15542438, Spain, 23 September 2022, 19-year-old male, asymptomatic; isolate ID not published (identified by association with poultry outbreaks), China, September 2022, death; EPI_ISL_16813290, Spain, 13 October 2022, 27-year-old male, asymptomatic; EPI_ISL_17021605, Ecuador, 5 January 2023, critical illness; EPI_ISL_17075747, China, 31 January 2023, 53-year-old woman, hospitalized, survived; EPI_ISL_17468386, Chile, 24 March 2023, 53-year-old male, critical illness; EPI_ISL_17736680, UK, 4 May 2023 ; EPI_ISL_17736649, UK, 6 May 2023; EPI_ISL_17980947, UK, 21 June 2023; EPI_ISL_18161874, UK, 7 July 2023; isolate from China, May 2024 (* ID isolate and clinical data not available); EPI_ISL_19548836, Canada, 9 November 2024, 13-year-old girl; EPI_ISL_19695821, UK, January 2025; and isolate from Mexico, 18 March 2025, 3-year-old female, death (isolate ID not published). ** From March 2024 to April 2025 additional A (H5N1) cases have been identified in persons exposed to dairy cows and poultry in the USA. Seventy isolates were reported in ten states, with one death in Louisiana. Not reported clade (orange; N = 2): Vietnam, October 2022, critical illness and Cambodia, 21 February 2024, 17-year-old female (both isolate IDs not published) [9,11,18,19,20,21,22]. The figure was created with Biorender.com.

Figure 2

Phylogenetic relationship of some representative H5N1 circulating isolates combined with antigenic prototype H5Nx CVVs. The analysis was based on the H5 amino acid sequence. It was performed with 49 sequences (39 from the antigenic prototype of H5Nx CVVs and 10 from H5N1 isolates). In red, H5 sequences from six human isolates (GISAID: EPI2437581, EPI2510183, EPI2718820, EPI2419700; GenBank: USE07565, UDV79400); two non-human mammal isolates (GenBank: WDW32886, WDW32934); and two bird isolates (GenBank: UWI70278, WAH70677) (N = 10). In blue, the best CVVs for H5N1 clades 2.3.4.4b or 2.3.2.1c. In black and bold, FDA or EMA-licensed H5N1 vaccines (N = 4). In box, the three CVVs selected for vaccine developments at Butantan Institute. * CVVs with the potency testing reagents available. ^† fatal human case. The phylogenetic tree was created using the Interactive Tree of Life (iTOL) tool (https://itol.embl.de/, accessed on 28 May 2025).

Figure 3

Immunogenicity of the three produced monovalent CVVs. The figure shows (a) the immunization schedule created with Biorender.com, (b) the specific anti-CVV IgG titer measured by ELISA, and (c) the titers of HI antibodies measured by HI assay using pools of animal sera. Groups of rats were immunized with one (white bars, N = 5) or two doses (gray bars, N = 6) of the vaccine formulations intramuscularly according to Table 1. (b) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as coating) and for the heterotypic immune response (using one of the two remaining strains as coating). The bars represent the arithmetic mean of IgG antibody titer in pooled sera and the standard deviation among three replicates. (c) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as hemagglutinating antigen) and for the heterotypic immune response (using one of the two remaining strains as hemagglutinating antigen). The bars represent the arithmetic mean of HI titer in pooled sera and the standard deviation between two replicates. The initial dilution was defined as 1:10; serum samples without detectable HI were assigned a titer of five. The horizontal dashed line represents the seroconversion threshold (when HI titers are equal to or greater than 1:40, these sera are considered seroconverted).

Figure 3

Immunogenicity of the three produced monovalent CVVs. The figure shows (a) the immunization schedule created with Biorender.com, (b) the specific anti-CVV IgG titer measured by ELISA, and (c) the titers of HI antibodies measured by HI assay using pools of animal sera. Groups of rats were immunized with one (white bars, N = 5) or two doses (gray bars, N = 6) of the vaccine formulations intramuscularly according to Table 1. (b) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as coating) and for the heterotypic immune response (using one of the two remaining strains as coating). The bars represent the arithmetic mean of IgG antibody titer in pooled sera and the standard deviation among three replicates. (c) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as hemagglutinating antigen) and for the heterotypic immune response (using one of the two remaining strains as hemagglutinating antigen). The bars represent the arithmetic mean of HI titer in pooled sera and the standard deviation between two replicates. The initial dilution was defined as 1:10; serum samples without detectable HI were assigned a titer of five. The horizontal dashed line represents the seroconversion threshold (when HI titers are equal to or greater than 1:40, these sera are considered seroconverted).

Figure 3

Immunogenicity of the three produced monovalent CVVs. The figure shows (a) the immunization schedule created with Biorender.com, (b) the specific anti-CVV IgG titer measured by ELISA, and (c) the titers of HI antibodies measured by HI assay using pools of animal sera. Groups of rats were immunized with one (white bars, N = 5) or two doses (gray bars, N = 6) of the vaccine formulations intramuscularly according to Table 1. (b) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as coating) and for the heterotypic immune response (using one of the two remaining strains as coating). The bars represent the arithmetic mean of IgG antibody titer in pooled sera and the standard deviation among three replicates. (c) The antibodies elicited by different formulations against each CVV were assayed for the homotypic immune response (using the same strain immunized as hemagglutinating antigen) and for the heterotypic immune response (using one of the two remaining strains as hemagglutinating antigen). The bars represent the arithmetic mean of HI titer in pooled sera and the standard deviation between two replicates. The initial dilution was defined as 1:10; serum samples without detectable HI were assigned a titer of five. The horizontal dashed line represents the seroconversion threshold (when HI titers are equal to or greater than 1:40, these sera are considered seroconverted).

Figure 4

H5Nx-specific hemagglutination inhibition titer of individual serum of animals immunized with one or two doses of vaccine formulations. The presence of specific antibodies in sera of individual animals immunized by vaccine formulations used to evaluate the immunogenicity of each CVV (Astrakhan, Duck/Vietnam, or Anhui)-derived formulation, capable of inhibiting red blood cell hemagglutinations by the corresponding homologous virus strain, was titrated. The sera from animals immunized with formulations of the three monovalent bulks comprising split and inactivated H5Nx antigens were collected 28 days after the last dose of the immunization schedules: one-dose (white bars, N = 5) and two-dose (gray bars, N = 6). The individual serum was from the animals of the same assay as described in Figure 3. The bars represent the arithmetic mean among the animals and the standard deviation. Each black circle symbol represents one animal per group. The horizontal dashed line represents the HI titer threshold; equal or higher than 1:40 is considered seroconverted. Panels (a–c) show hemagglutination induced by Astrakhan CVV and sera evaluated from individual animals immunized with Astrakhan antigen formulations; panels (d–f) show hemagglutination induced by Duck/Vietnam CVV and sera evaluated from individual animals immunized with Duck/Vietnam antigen formulations; and panels (g–i) show hemagglutination induced by Anhui CVV and sera evaluated from individual animals immunized with Anhui antigen formulations (see Table 1). Comparing the vaccination effect between various formulations, an asterisk * indicates a significant difference by one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Figure 5

Microneutralization (MN) titers elicited by the pool of serum from immunized animals with Astrakhan (H5N8) CVV formulations. Rats were immunized (a,c) once (white bars, N = 5) or (b,c) twice (gray bars, N = 6) with different formulations of Astrakhan inactivated vaccines with or without IB160 adjuvant (Figure 3a). The MN assay was performed against a wild-type HPAI H5N1 virus using samples of pooled sera collected at day 28 post the last immunization of each group (see Table 1). The pooled sera used were from the same assay as described in Figure 3. CER cells were infected with 100 TCID50 of the wild-type H5N1 virus pre-incubated with serial dilutions of pooled sera for 1 h at 37 °C. The MN titer was considered the highest serum dilution at which no cytopathic effect (CEP) was observed, indicating no infection. The bars represent the arithmetic mean of the titer of three replicates of the MN assay. ** or **** indicates a significant difference by one-way ANOVA (p ≤ 0.01 and p ≤ 0.0001, respectively). The figure shows the comparison of the pooled sera titer (a) among experimental groups on a one-dose immunization schedule, (b) among experimental groups on a two-dose immunization schedule, and (c) between one and two doses for each experimental group. In panel (c), the MN titer of pooled sera from animals immunized with two doses of 7.5 µg of HA + IB160 is also significantly different from the titers of pooled sera from the remaining animal groups, except for two doses of 15 µg HA + IB160.