A single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus H5N1

Abstract

Background: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission.

Methodology/principal findings: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1.

Conclusions/significance: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses.

Figure 1. Expression, purification and biological activity of recombinant, soluble trimeric H5 protein.

(A) Schematic representation of the H5 expression cassettes used. The H5 ectodomain encoding sequence (H5) was cloned in frame with DNA sequences coding for a signal peptide (SP), the GCN4 isoleucine zipper trimerization motif (GCN4) and the Strep-tag II (ST) under the control of a CMV promoter. (B) H5 expression and secretion into the culture media was analyzed by SDS-PAGE followed by western blotting. The recombinant protein was detected using a mouse anti-Strep-tag antibody. (C) Analysis of purified recombinant H5 proteins by gel filtration. Shown is the elution profile of a H5 protein preparation using a Superdex200GL 10–300 column. The elution of a 232 kDa catalase control is indicated by the line. (D) Blue native-PAGE analysis of the recombinant H5 protein. The position in the gel of the momomeric, dimeric and trimeric ectodomain species observed after heating of the HA sample prior to electrophoresis is indicated. (E) Recombinant soluble H5 trimers were complexed with a HRP-conjugated mouse antibody directed against the Strep-tag prior to their application in a fetuin binding assay. HA binding was also assessed after treatment of fetuin with VCNA (fetuin+VCNA).

Figure 2. sH5³ vaccination in chickens.

Ten chickens were immunized two times with 10 ug sH5³ (on day 0 and 21). As a challenge control, chickens were mock-treated (PBS). Three weeks after the vaccination, all birds were challenged with 10⁵ TCID₅₀ of HPAI H5N1 A/Viet Nam/1194/04 (A) Kaplan-Meier survival curves, indicating percentage mortality on each day for each group (B–C–D) Blood samples were collected 3 weeks after the first immunization (sH5³ 1×) and 3 weeks after the second vaccination (sH5³ 2×). The sH5³ antibody levels as determined by ELISA (B), the HI titers against H5N1 (NIBRG-14) (C) and the HI titers against sH5³ (D) in serum for each bird. Bars represent geometric means per group. The dotted lines indicate the lowest antibody levels correlating with protection in this experiment.

Figure 3. sH5³ dose titration after a single or boost vaccination in chickens.

Seven groups of 10 chickens were immunized i.m. with 10, 2 or 0.4 µg sH5³ either once or twice with 3 weeks interval. As a challenge control, one group was mock-treated (PBS). Four weeks after the vaccination, all chickens were challenged with 10⁵ TCID₅₀ of HPAI H5N1 A/Viet Nam/1194/04. Kaplan-Meier survival curves, indicating percentage mortality on each day for each group that was (mock-)vaccinated twice (A) or once (B). (C–D) The sH5³ antibody levels at the day of challenge as determined by ELISA for each chicken that was (mock-)vaccinated twice (C) or once (D). (E–F) Serum HI titers in the same sera, measured against sH5³ for the chickens that were (mock-)vaccinated twice (E) or once (F). Bars represent the geometric means for the test groups.

Figure 4. sH5³ vaccination of mice.

Groups of 10 BALB/c mice were immunized i.m. with 2 µg sH5³ either once (day 21) or twice with a 3-week interval (day 0 and day 21). As a challenge control, one group of mice was mock-treated (PBS) twice (day 0 and day 21). Three weeks after the vaccination, mice were infected with ∼10 LD₅₀ of A/Viet Nam/1194/04 and monitored daily for clinical signs and body weight during the next 14 days. (A) Kaplan-Meier survival curve indicating percentage mortality on each day for each group. (B) Median clinical scores per group. (C) Mean body weights per group expressed as percentage of starting body weight, plotted as a function of time. Error bars represent the standard deviation. (D–E) Blood samples were collected at the day of challenge. sH5³ antibody levels as determined by ELISA (D). HI titers against sH5³ (E). Bars represent geometric means.