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

<p>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.</p

Virus detection in tracheal and cloacal swabs collected from vaccinated chickens after challenge with H5N1.

a<p>Amount of sH5³ per immunization dose is indicated in µg. Day post infection (D) on which the tracheal and cloacal swabs were collected are indicated.</p>b<p>+ = positive; ± = inconclusive (low fluorescence [<0.07] after more than 31 cycles); − = negative; x = not tested.</p>†<p> = chicken did not survive the challenge with HPAI H5N1.</p

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

<p>(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).</p

sH5³ vaccination of mice.

<p>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.</p

Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles

A highly conserved neutralizing epitope on group 2 influenza A viruses

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the V(H)1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapie

Vaccination of mice.

<p>Groups of 10 BALB/c mice were vaccinated with 10^6.5 TCID₅₀ of NDV-H5 or NDV-sH5³, either via the ON or IM route. One group of mice was mock-vaccinated (PBS) as challenge control. Three weeks after the vaccination, mice were infected with ∼10 LD₅₀ of HPAIV H5N1 and weighed daily and observed for clinical signs during 14 days. Graphed for each group are A and B) Kaplan-Meier survival curves indicating percentage of survival p.c., C and D) mean percentage of body weight changes relative to starting weights measured on the day of challenge (day 0) with error bars representing the standard deviation, and E and F) median clinical scores observed after challenge. For reference, each panel includes the same PBS group.</p

Serum antibody responses in vaccinated chickens.

<p>HI antibody titers against A) NDV Herts/33 or B) AIV measured in individual serum samples collected on day 21 p.v. via ON/IT or IM route. Titres are expressed as the reciprocal of the highest serum dilution showing HI. The dashed line indicates the lower limit of detection. Titers below the detection limit were assigned a value of 2 or 4, respectively for calculation of the mean. Horizontal bars represent the geometric mean titers of the groups and vertical bars represent 95% confidence intervals. Serum titers from chickens that died after the challenge are indicated by filled circles, the open circles correspond to animals that survived the challenge. For reference, each panel includes the same PBS group.</p