Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian virus

Vaccine 2007

Huoying Shi, Xiu Fan Liu, Xiaorong Zhang, Sujuan Chen, Lei Sun and Jianhong Lu

Influenza virus

http://www.bch.cuhk.edu.hk/influenza/virology.html

(hemagglutinin)

(neuraminidase)

•a globally important respiratory pathogen

•causes a high degree of morbidity and mortality in humans and animals annually

Avian influenza virus

• 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes

• since the late 1990s, some of avian influenza A viruses have transmitted directly from birds to humans

• H5 subtype highly pathogenic avian influenza (HPAI) and H5N1 (HPAIV)

is continuously undergoing antigenic change to escape the host's acquired immunity

Avian influenza pathogenesis and epidemiology

http://www.influenzareport.com/ir/ai.htm

• 2003–2006, a highly pathogenic avian influenza A (H5N1) virus caused poultry disease in Asian countries and infected many people

most of these individuals had close contact with poultry

to development of an H5N1 AIV vaccine

• not all viruses that are closely related are suitable for vaccine production, some grow poorly in eggs

plasmid-based system

http://www.futurasciences.com/fr/print/comprendre/dossiers/doc/t/medecine-1/d/grippe-aviaire-et-prevention-chez-lhomme-33_682/c3/221/p1/

1.live attenuated vaccine2.cold-adaptive strain vaccine3.capable of inducing broad mucosal and systemic responses

Generation of the C4/F reassortant virus and confirmed it in vitro growth properties

↓

Pathogenicity and replication in chickens

↓

Pathogenicity and replication in BALB/c mice

↓

Immunogenicity and protective efficacy of a formalin-inactivated C4/F virus vaccine

Receptor

Basic patches

Cleavage siteHA2

HA1

C4/F

8-plasmid transfection syste

m

HA

NA

PB2 PB1 PA

NP M NS

HA and NA from C4/H5N1

Internal gene from F strain

HA titers: 2048~ 20 passages in embryonated chicken eggs

Pathogenicity and replication in chickens

Eight chickens (4-week-old SPF white leghorn chickens)

Intravenously (i.v.) with 0.2 ml 1:10 dilution of stock virus 1. H5N1 2. C4/F 3. F

Pathogenicity and replication in BALB/c mice

Eight 6–8-week-old female BALB/c mice

Intranasally (i.n.) with 50×106 l 50% eggs infectious dose (EID50) 1. H5N1 2. C4/F 3. F

Immunogenicity and protective efficacy of a formalin-inactivated C4/F virus vaccine

White Leghorn chicken

7-day-old

wt. C4/H5N1 and C4/Fformalin-inactivated vaccineSingle i.m. dose (3 g HA)

Three weeks after immunizationImmunogenicity

challenge with 100 ELD50 of virus Protective efficacySurvival and disease sign

Immunogenicity and protective efficacy of a formalin-inactivated C4/F virus vaccine

3 Weeks PostvaccinationHemagglutination Inhibition

3 Weeks PostvaccinationChallenge with wt. C4/H5N1 Intranasally

3 days 2 weeks

Discussion

• Measures for the control of emerging and reemerging H5N1 influenza include the use of inactivated vaccines and improvement in biosecurity.

1. HPAI viruses are lethal to embryonated eggs, which limits growth in a high titer.

2. The multibasic amino acid motif at the HA cleavage site is believed to contribute to the virulence of these viruses in humans as well as in domestic poultry.

郭懿瑩

• In this study

Developed an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses, a reassortant C4/F converted the basic amino acids of HA gene seen in the C4/H5N1 viruses to the sequence motif seen in avirulent avian influenza viruses, was generated by reverse genetics with two surface genes from C4/H5N1 and the remaining six genes from F, which is not pathogenic and replicate for mice and low pathogenic for chickens.

the C4/F reassortant showed good growth characteristic with the HA titer as high as 1:2048.

• This data indicated that although the C4/F transfectant virus replicated in the trachea and lungs of mice, the attenuation phenotypes in the HA and the accompanying genotype are associated with a loss of virulence for chicken and mice.