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Avian influenza vaccines against H5N1 ‘bird flu’ Chengjun Li, Zhigao Bu, and Hualan Chen State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin, China 150001

H5N1 avian influenza viruses (AIVs) have spread widely to more than 60 countries spanning three continents. To control the disease, vaccination of poultry is implemented in many of the affected countries, especially in those where H5N1 viruses have become enzootic in poultry and wild birds. Recently, considerable progress has been made toward the development of novel avian influenza (AI) vaccines, especially recombinant virus vector vaccines and DNA vaccines. Here, we will discuss the recent advances in vaccine development and use against H5N1 AIV in poultry. Understanding the properties of the available, novel vaccines will allow for the establishment of rational vaccination protocols, which in turn will help the effective control and prevention of H5N1 AI. H5N1 AIV Influenza virus has a segmented, single-stranded, negative-sense RNA genome and is divided into three genera, A, B, and C, within the family Orthomyxoviridae. To date, AI is caused only by influenza A virus, which is further categorized into subtypes based on the antigenicity of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Currently, 18 HA (H1–H18) subtypes and 11 NA (N1–N11) subtypes of influenza A viruses have been identified [1]. Among them, the H1–H16 and N1–N9 subtypes were all isolated from aquatic birds, whereas the H17N10 and H18N11 viruses were identified in bats [1,2]. The H5N1 AIV has been detected in domestic poultry and wild birds from more than 60 countries across three continents [3,4]. Of note, the H5N1 AIVs have become enzootic (see Glossary) in poultry and wild birds in Bangladesh, China, Egypt, India, Indonesia, and Vietnam [5]. The poultry farming systems in these developing countries are outmoded, with very little or no biosecurity measures in place in the backyard farms, small scale poultry farms, water poultry farms, and live bird markets. This situation has increased the virus diversity through reassortment between viruses of different sources and facilitated the spreading of H5N1 AIVs around the world. Corresponding author: Chen, H. ([email protected]). Keywords: H5N1; avian influenza; vaccine. 0167-7799/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.01.001

H5N1 AIVs have caused huge economic losses for the poultry industry and even the overall national economy of some countries. Outbreaks of H5N1 AIVs in poultry and wild birds also pose a great threat to public health. Their wide dissemination has resulted in 641 confirmed cases of human infection, of which 380 cases were lethal [see the World Health Organization (WHO) website, http://www. who.int]. Recently, several studies have revealed that H5N1 AIVs could attain aerosol transmissibility in mammalians by acquiring a2,6-linked sialic acid (human-type receptor) binding ability or by reassorting with the 2009 H1N1 pandemic virus [6–8]. It is therefore important to control and prevent the infection and spread of H5N1 AIVs to protect the health of both animals and humans. To deal with the global threat caused by the H5N1 viruses, international organizations and national governments of affected countries have developed a comprehensive strategy for the effective control and prevention of H5N1 AI, including biosecurity, depopulation of infected poultry, diagnostics and surveillance, notification and education, and vaccination. Of the more than 60 countries affected by H5N1 AIVs, 13 countries have sporadically or routinely utilized vaccination to control H5N1 AIVs [3,4]. This review focuses on the advances in vaccine development and use for the control and prevention of H5N1 AIVs in poultry.

Glossary Allantoic fluid: refers to the fluid within the allantoic membrane of the chick embryo. The influenza virus is released into the allantoic fluid when it replicates in the chick embryo. Antigenic drift: is caused by the accumulation of mutations in the antigenic sites of the protective surface antigen of an infectious agent during its circulation, resulting in the mismatch of antigenicity between the mutant strain and the parent strain. Enzootic: the infectious disease can be termed as ‘enzootic’ when it is maintained in a human (or animal) population of a certain geographical region without the need of external inputs. Live attenuated vaccine: is a genetically modified vaccine such that its virulence is lost or reduced but it still maintains its viability. When administered, the live attenuated vaccine can still replicate in the host with a reduced magnitude and can stimulate stronger immune responses compared with the inactivated vaccine. Neutralizing antibody: is an antibody that can protect the host cell by inhibiting or neutralizing infection by a certain infectious agent. Seroconversion: is a term denoting the detection of specific antibody against a certain microorganism in the blood serum upon infection or immunization. Vaccine seed virus: refers to the first batch of virus culture stock that is used in the subsequent inoculation of embryonated chicken eggs or cell culture to amplify the virus in large quantities for vaccine production.

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Inactivated whole virus vaccines The inactivated whole virus vaccine was first developed in the 1940s for the control and prevention of human influenza [9]. Over the past 30 years, it has also been the major type of vaccine used to control AIVs in poultry. To combat H5N1 AI outbreaks in Indonesia, an inactivated vaccine developed from a highly pathogenic vaccine seed virus, A/chicken/Indonesia (Legok)/03 (H5N1), was used to vaccinate chickens [10]. However, there are issues with selecting a highly pathogenic virus as a vaccine seed virus. For example, in terms of biosafety, it would be potentially hazardous to the vaccine production personnel. In addition, the highly pathogenic H5N1 vaccine seed viruses often kill eggs within 48 h, resulting in a low titer of viral antigen in the allantoic fluid. A low pathogenic H5N1 influenza vaccine seed virus cannot be developed by using conventional techniques. The invention and application of reverse genetics technology of influenza virus has revolutionized the practice of H5N1 influenza vaccine development [11–13]. In this technology, the viral RNA (vRNA) expressing plasmids of HA and NA of the H5N1 virus and the six internal genes of the highyield donor virus A/Puerto Rico/8/1934 (H1N1) (PR8), together with four PR8 protein expression plasmids encoding the polymerases and nucleoprotein are cotransfected into Vero cells, where the vRNAs and proteins are produced, resulting in the formation of a H5N1/PR8 vaccine seed virus (Figure 1). During construction of the vRNA expressing plasmids, the H5N1 HA segment is mutagenized to remove the multiple basic amino acids at the HA cleavage site, which resembled that of the low pathogenic

influenza viruses [14,15]. By using reverse genetics technology, Tian et al. developed an inactivated H5N1 AI vaccine based on A/goose/Guangdong/1/96, the first highly pathogenic H5N1 virus isolated in China [16,17], which was designated H5N1/PR8 (2+6) [18]. The vaccine seed virus displayed an excellent biosafety profile. It was attenuated in chickens and was nonpathogenic to embryonated chicken eggs. The vaccine seed virus replicated efficiently in embryonated chicken eggs with a four- to sixfold increase in virus titer in allantoic fluid compared with the parental highly pathogenic A/goose/Guangdong/1/ 96 (H5N1) virus. With this high yield, the inactivated vaccine could be directly prepared from allantoic fluid without the need for a concentration process. After vaccination, the chickens, ducks, and geese were completely protected from challenges with homologous H5N1 virus and early isolates of heterologous H5N1 viruses. Numerous other studies have also determined the immune efficacy of inactivated whole virus vaccine against H5N1 virus infection in chickens and ducks. The vaccine strains used in these studies were developed from the naturally low pathogenic H5N2 and H5N9 viruses [19– 24], or were developed from H5N1 or H5N3 vaccine seed viruses rescued by reverse genetics in the backbone of PR8 virus [25–29]. The protective effectiveness of the vaccines differed across the studies, ranging from complete protection to partial protection from virus shedding, disease signs, and death. Vaccine immune efficacy was influenced by the antigenic relatedness of the vaccine and the challenge strains, the amount of HA antigen, and the vaccination schedule. Of these factors, high antigenic relatedness

H5N1

PR8

Four protein expression plasmids

PB2

PB1

PA

NP

NA

PB2

PB1

PA

vRNA expression plasmids for HA and NA

NP

M

NS

HA

vRNA expression plasmids for the six internal genes

H5N1/PR8 vaccine seed virus TRENDS in Biotechnology

Figure 1. Schematic diagram of the reverse genetics system to generate H5N1 vaccine seed virus. The viral RNA (vRNA) expression plasmid for HA from the wild type H5N1 virus was modified to remove the multiple basic amino acids at the cleavage site, thus converting it from virulent to avirulent. Eight vRNA expression plasmids, including the modified HA and NA of H5N1 virus and the six internal genes from the high-yield donor A/Puerto Rico/8/1934 (H1N1) (PR8) virus, together with four protein expression plasmids encoding the polymerase basic protein (PB)2, PB1, polymerase acidic protein (PA), and nucleoprotein (NP) of PR8 virus, were cotransfected into Vero cells where the vRNAs and proteins were produced, resulting in the formation of H5N1/PR8 vaccine seed virus. The rescued vaccine seed virus possesses the desirable properties of high yield in eggs, avirulence for poultry, and antigenicity match with H5N1 field strains.

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Box 1. Vaccination strategies Emergency vaccination is usually applied as ring vaccination around a focus of infectious disease to prevent its outward spread or as barrier vaccination along the border of a country or region when it is at risk due to the prevalence of infectious disease in an adjacent country or region [76]. A routine or mass vaccination strategy is implemented to protect the maximum number of humans or animals when a certain infection disease is prevalent in a large geographical region and cannot be eliminated within the short term [77]. The application of mass vaccination can rapidly increase the population immunity, thereby limiting the morbidity and mortality caused by the infectious disease. DIVA is the abbreviated term denoting ‘differentiating infected from vaccinated animals’ [78]. This strategy is achieved by the application of a vaccine lacking antigens that are only present in the field strains of an infectious agent. By employing a diagnostic test to detect the specific antigen that is lacking in the vaccine, the infected animals and vaccinated animals can be differentiated [79].

between the vaccine and the field strains was critical for optimal immune protection against H5N1 virus challenge. The vaccine cannot be fully effective when antigenic variants emerge in the field [30]. Therefore, it is essential to conduct intensive surveillance and antigenic analysis of field strains during the vaccine use to allow the timely

update of the vaccine strain to best match the antigenicity of the field strains. Recombinant virus vector vaccines Compared with inactivated vaccines, recombinant virus vector vaccines are able to induce more balanced T helper 1 (Th1) and T helper 2 (Th2) immune responses. In general, recombinant H5N1 influenza vaccines developed using other virus vectors can provide protection to two kinds of viral diseases, which is superior in terms of administration convenience and cost reduction. Moreover recombinant vaccines only express the HA gene of H5N1 viruses; therefore, a serological test could be easily established for differentiating infected from vaccinated animals (DIVA) (Box 1). To date, a variety of recombinant H5N1 influenza vaccines have been developed by using fowl pox virus (FPV), Newcastle disease virus (NDV), turkey herpes virus (HVT), duck enteritis virus (DEV), and infectious laryngotracheitis virus (ILTV) (Table 1). Recombinant FPV vector vaccine Recombinant FPV vaccine was one of the first live virus vector vaccines to be successfully developed by using genetic engineering [31]. A recombinant FPV influenza

Table 1. Recombinant virus vector vaccines developed against highly pathogenic H5N1 AIV Vaccine

Vaccine strain

Recombinant FPV vector vaccine Expressing HA and NA A/goose/Guangdong/ 1/96 (H5N1)

Expressing HA and IL-6 A/mallard/Huadong/ SY/2005 (H5N1) A/turkey/Ireland/ Expressing HA 1378/83 (H5N3) Recombinant NDV vector vaccine A/bar headed goose/ Expressing HA Qinghai/3/05 (H5N1)

Expressing HA

Animal model Mode of immunization Chickens

Ducks Chickens

Oculonasal

A/Vietnam/1203/2004 (H5N1) Chickens

Oculonasal

Chickens

Intramuscular

Chickens

Subcutaneous

Recombinant DEV vector vaccine A/duck/Anhui/1/06 (H5N1) or Ducks Expressing HA A/duck/Guangdong/ Broilers S1322/2010 (H5N1)

Recombinant ILTV vector vaccine A/chicken/Vietnam/ Expressing HA P41/2005 (H5N1)

Chickens

Refs

[34,35] Wing-web, Provided complete protection against lethal intramuscular, challenges with homologous and or subcutaneous heterologous H5N1 viruses and 100% survival rate against a lethal challenge with H7N1 virus Subcutaneous 80–90% survival rate against a lethal challenge [37] with homologous H5N1 virus Subcutaneous 100% survival rate and reduced virus shedding [38,39] in lethal challenges with diverse highly pathogenic H5 viruses including H5N1 viruses

Chickens

Recombinant HVT vector vaccine A/goose/Guangdong/ Expressing HA 3/96 (H5N1) A/swan/Hungary/ Expressing HA 4999/2006 (H5N1)

Vaccine efficacy

Conferred complete protection against lethal [44] challenge with homologous virus and a 100% survival rate following challenge with A/goose/ Guangdong/1/96 (H5N1) virus Chickens were completely protected against [43] lethal challenges with homologous virus and A/egret/Egypt/01/2006 (H5N1) virus 60% survival rate in a lethal challenge with A/ [53] goose/HLJ/QFY/2003 (H5N1) virus 70–100% survival rate in chickens with or [55] without maternal antibodies following lethal challenges with A/chicken/Egypt/1709-1VIR08/ 2007 (H5N1) or A/chicken/Egypt/1709-6/2008 (H5N1) viruses

Intramuscular

Conferred complete protection for ducks [57] against lethal challenge with A/duck/Hubei/49/ [58] 05 virus as early as 1 week post-vaccination and provided complete protection for broilers against lethal challenge with homologous A/ duck/Guangdong/S1322/2010 virus

Ocular

100% survival rate in lethal challenges with homologous and heterologous H5N1 viruses as well as A/chicken/Italy/8/98 (H5N2) virus

[60,61]

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Review vaccine was first developed in 1988 and was registered and used in Mexico for the control of H5N2 AIVs [32,33]. Chickens vaccinated with this vaccine can be protected even when the hemagglutination inhibition (HI) antibody response is weak or undetectable, suggesting that the elicited cellular immunity may play an important role for immune protection. A recombinant FPV vaccine expressing the HA and NA genes of A/goose/Guangdong/1/96 (H5N1) has been licensed for use in China [34] (Table 1). It can protect chickens from lethal challenges with both H5N1 and H7N1 highly pathogenic viruses. In one study, the protective neutralizing antibody it induced against the homologous H5N1 virus lasted for up to 40 weeks [35] (Table 1). As early as 1 week post-vaccination, the recombinant FPV vaccine provided protection from a lethal challenge with homologous influenza virus [35,36]. By contrast, there are several disadvantages to recombinant FPV vaccines. First, vaccination with this vaccine will often not confer sterile immunity, as demonstrated by virus shedding in tracheal and cloacal swabs [37–39] (Table 1). Second, the vaccination efficacy can be compromised by maternal antibodies against FPV. Prior use of the conventional FPV vaccine ahead of the recombinant FPV vaccine could also interfere with vaccine efficacy [40]. Third, recombinant FPV vaccine application is not convenient due to the high labor cost of immunizing individual chickens via web-wing puncture or subcutaneous injection. Last, there is no advantage to using this vaccine in broilers because it is not necessary to vaccinate broilers against FPV. Thus, the aim of protecting against two infectious diseases with only one vaccine would not be achieved. Recombinant NDV vector vaccine Newcastle disease (ND) is a highly contagious disease caused by NDV. In many countries, vaccination of live attenuated NDV vaccine is indispensable in farms of layers and broilers. In China alone, over 30 billion doses of live attenuated NDV vaccine are given each year [41]. Therefore, the use of a live attenuated NDV strain to develop a recombinant NDV vector H5 influenza vaccine could achieve the goal of simultaneously preventing two deadly infectious diseases, AI and ND, with a single live virus vaccine. Swayne et al. constructed a recombinant NDV virus to express the HA gene of H7 AIV by using the highly attenuated B1 strain, and showed that it only provided partial protection against challenge with highly pathogenic H7 AIV and NDV [42]. By contrast, recombinant NDV vaccines constructed using the less attenuated Lasota strain or its derivative, Clone 30, were better able to elicit antibody responses and confer immune protection [43–45] (Table 1). In studies by Ge et al. [44] and Nayak et al. [43], recombinant NDV viruses expressing the HA gene of H5N1 AIV that were constructed using the Lasota strain induced significant HI antibody responses against NDV and H5N1 AIV and provided complete immune protection from challenges with NDV as well as from lethal challenges with both homologous and heterologous H5N1 AIV. As with inactivated vaccines, the effectiveness of the immune protection afforded by recombinant NDV vaccines 4

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was reduced when chickens were challenged with antigenically distinct H5N1 viruses [46]. Therefore, it is essential to update the H5 HA gene expressed in the recombinant NDV vaccine when significant antigenic variation is found for the H5N1 field strains. In addition, maternal NDV antibodies could interfere with the immune effectiveness of the recombinant NDV live vaccine [47]. Therefore, it is very important to optimize the immunization schedule according to the levels of maternal antibodies. The recombinant NDV vaccine and the recombinant FPV vaccine, when used in combination, can provide long-lasting protection against challenges with H5N1 viruses [48]. The bivalent recombinant NDV vector H5 HA vaccine has several advantages, including ease of production, high production yield, ease of administration to poultry in the field, and the ability to induce mucosal immunity and allow the application of the DIVA strategy. Recombinant HVT vector vaccine HVT has been widely used for the prevention of Marek’s disease and has also been used to develop bivalent recombinant vaccines against infectious bursal disease and ND [49–51]. The HVT vector vaccine is very convenient because it can be applied via in ovo vaccination of 18day-old chicken embryos or hatchery vaccination of 1day-old chicks. Using infectious bacterial artificial chromosome (BAC) technology, the recombinant HVT vector H7 AI vaccine was constructed by inserting the H7 HA gene into the unique long 45 (UL45)–UL46 region of the HVT genome. Immunization of 1-day-old chicks provided complete protection against challenge with homologous H7N1 virus without virus shedding, clinical signs, and death [52]. Several other studies have examined the immune efficacy of recombinant HVT vector H5 HA vaccines [53–55] (Table 1). The recombinant HVT-H5 HA vaccine provides better protection when the H5 HA gene is inserted into the uniqe short 2 (US2) site than the US10 site [53]. It can confer clinical protection against challenges with antigenically related H5N1 viruses, but the protective efficacy decreases when chickens are challenged with antigenic variants [54]. Of interest, the recombinant HVT-H5 HA vaccine still provides 70%–90% clinical protection against H5N1 AIV challenge of commercial broilers carrying maternal antibodies against both HVT and H5N1 viruses [55]. To maximize the immune efficacy of HVT vector vaccine, its future development should test different vaccination programs, such as the vaccine doses and the inclusion of booster dose. Recombinant DEV vector vaccine Ducks infected with most of the H5N1 AIVs usually show no signs of disease or death, but can release large amounts of virus into the environment. Therefore, ducks serve as the ‘Trojan horse’ to spread H5N1 viruses to susceptible animals and humans [16]. A large number of ducks are bred in many Asian countries. In China alone, up to 4 billion ducks are reared annually accounting for about 75% of the duck population worldwide [56]. Currently, the vaccination coverage rate is considerably low in ducks mostly because duck farmers are unwilling to use the inactivated H5N1 vaccine because H5 viruses usually cause no observable disease in ducks.

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(A)

(A1) 0

20

40

60

80

120 irs

100

ul

ul42

D

us8

us7 H

103945 Q 143040

77208

44511

68314

160 trs

us

ul41 ul40 38245

1

140

J

109887

119308

Poly aatB2

ul41

T

158091

D-ul41HA

(A2) ul41 aatB1 sv40

HA

T-us78HA

(A3) us7

aat

Poly

aat

Survival (%)

(B)

(C)

HA

sv40

us8

Virus shedding

Day 0

rDEV-ul41HA

rDEV-us78HA rDEV-ul41HA

Day 3

62.5

75

Low ter

Low ter

Day 7

100

100

None

None

Day 21

100

100

None

None

Survival (%)

Virus shedding

Day 0

rDEV-re6

rDEV-re6

Day 3

40

Low ter

Day 7

82.5

Low ter

Day 14

100

None

Day 21

100

None

Day 35

100

None

rDEV-us78HA

Key: Vaccinaon Challenge TRENDS in Biotechnology

Figure 2. Development of a novel recombinant duck enteritis virus (DEV) vector vaccine against H5N1 avian influenza (AI). (A) Schematic diagram of the strategy for developing recombinant DEV vector vaccine. Adapted from Liu et al. [57]. (A1) The genomic structure of DEV and its recovery from five fosmid DNAs. (A2) The construction of recombinant DEV vector vaccine rDEV-ul41HA in which the hemagglutinin (HA) gene of A/duck/Anhui/1/2006 (AH/1) (H5N1) was inserted within the u41 gene. (A3) The construction of recombinant DEV vector vaccine rDEV-us78HA in which the AH/1 HA gene was inserted between the us7 and us8 genes. (B) The vaccine efficacy of rDEVul41HA and rDEV-us78HA in ducks against challenge with highly pathogenic A/duck/Hubei/49/05 (HB/49) (H5N1) virus [57]. The ducks were intramuscularly immunized with 105 plaque-forming units (PFU) of recombinant rDEV-ul41HA or rDEV-us78HA vaccine. They were then intranasally infected with a 100-fold 50% duck lethal dose of HB/49 virus at different time points. The ducks were monitored for survival and tested for virus shedding in oropharygeal and cloacal swabs over a 2-week experimental period. (C) The vaccine efficacy of recombinant DEV vector vaccine rDEV-re6 in broilers [58]. The rDEV-re6 vaccine was constructed by inserting the HA gene of A/duck/Guangdong/ S1322/2010 (H5N1) between the us7 and us8 genes of DEV. The broilers were intramuscularly immunized with 106 PFU of rDEV-re6 vaccine. They were then intranasally infected with a 100-fold 50% chicken lethal dose of A/duck/Guangdong/S1322/2010 (H5N1) virus at different time points. The broilers were monitored for survival and tested for virus shedding in oropharygeal and cloacal swabs over a 2-week experimental period.

Duck viral enteritis is a deadly infectious disease caused by DEV. Since the 1960s, a live attenuated DEV vaccine has been developed and routinely used for the control of duck viral enteritis in China with an annual usage of several billion doses [57]. Therefore, the development of a recombinant DEV vector H5 AI vaccine is highly desirable because it could be used to control both duck viral enteritis and H5N1 AI in ducks. To this end, Liu et al. generated two recombinant DEVs with the H5 HA gene inserted at different sites in the DEV vaccine strain genome [57] (Figure 2A and Table 1). Experiments in ducks demonstrated that these DEV vectored live vaccines

were immunogenic and provided solid protection against challenges with H5N1 AIV and DEV (Figure 2B). Of particular value is the speed with which the recombinant DEV virus induces a protective immune response; it can completely protect ducks against a challenge with DEV on the same day of vaccination and provides complete protection from lethal challenge with H5N1 AIV as early as 1 week post-vaccination [57]. The application of this novel vaccine could successfully reduce the susceptibility of ducks to H5N1 AIVs and reduce or eliminate the source of infection while still effectively protect ducks against DEV. 5

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Review The recombinant DEV vector H5 vaccine is also safe and effective in chickens. It can provide solid protection as early as 1 week post-vaccination when administered to 7-day-old broilers, and this protection is sustainable for the entire growing period of broilers [58] (Figure 2C and Table 1). Unlike the use of other recombinant vaccines, the use of the recombinant DEV vector H5 vaccine in chickens can completely avoid the interference of maternal antibodies on the replication of the vector virus in vivo. The recombinant DEV vector H5 vaccine virus can grow to high titers in chicken embryo fibroblast, thus reducing production costs. In addition, the recombinant DEV vector H5 vaccine seed virus can be generated within 2–3 weeks, which ensures the rapid update of new vaccine seed viruses in response to the antigenic drift of H5N1 field strains. Recombinant ILTV vector vaccine Using the homologous recombination approach, a recombinant ILTV vector H7 AIV vaccine strain was constructed by inserting the H7 HA gene into the UL0 gene of ILTV. This vaccine provided protection against challenge with a virulent ILTV strain and against lethal challenge with H7 AIV [59]. In addition, vaccination with H5 HA expressing recombinant ILTV vaccines that are constructed by inserting the H5 HA gene into the UL50 gene locus can confer effective protection against challenges with both homologous and heterologous H5 viruses [60–62] (Table 1). The efficacy of the H5 HA expressing ILTV vaccine is enhanced when the chickens are co-immunized with a recombinant ILTV expressing the NA gene of the N1 subtype [61] (Table 1). The recombinant ILTV vaccine can be produced in large quantities in embryonated chicken eggs or primary chicken cell cultures, and can induce mucosal immunity when administered through drinking water or spray [63]. However, the application of the recombinant ILTV vaccine is limited because it is only needed in older layers in the few areas where ILTV is circulating. Therefore, the bivalent ILTV vector H5 vaccine is of limited significance in the control of H5N1 AIVs compared with other live virus vector vaccines. DNA vaccines DNA vaccines offer a number of advantages over conventional vaccines [64]. First, a DNA vaccine can elicit both humoral and cellular immunity after administration. Second, the construction of DNA vaccines is easy and their manufacture and storage is cost-effective. Third, DNA vaccines can be administered multiple times to enhance the immune efficacy because no immune response is raised against the DNA vaccine vector. Fourth, the use of DNA vaccines still permits the application of the DIVA strategy. Although different influenza viral genes have been tested, the HA gene has shown the best protection in studies to develop a DNA vaccine. Several early studies demonstrated the efficacy of an HA-expressing DNA vaccine against challenges with highly pathogenic H5 or H7 viruses in chickens [65–67]. More recently, efforts have focused on developing more effective DNA vaccines against H5N1 AIV in chickens. Jiang et al. constructed an H5 HA gene, optiHA, containing chicken-biased codons [68]. A DNA vaccine, pCAGGoptiHA, was constructed by inserting 6

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optiHA into the pCAGGS vector under the control of the chicken b-actin promoter. When intramuscularly administered to chickens, this DNA vaccine elicited strong HI and neutralizing antibodies. Chickens immunized with 100 or 10 mg of pCAGGoptiHA vaccine were completely protected from disease signs and death after challenge with a lethal H5N1 AIV. Further investigation demonstrated that the protective immune response lasted for more than a year when two doses of 10 mg pCAGGoptiHA were administered to chickens. This DNA vaccine has gone through the clinical field trials and its registration and licensure are currently under evaluation in China. Studies have also explored the generation of broadspectrum immune responses to the HA–DNA vaccine. When the DNA vaccine is developed as a mixture expressing different clades of the HA gene of the H5N1 viruses, it can provide protection to challenges with heterologous H5N1 viruses in mice and chickens [69,70]. In addition, the inclusion of adjuvants, such as MDA5 and CD154, can enhance the immune efficacy of HA-based DNA vaccines against H5N1 viruses [71,72]. DNA vaccines have also been shown to be effective against the challenge of a lethal H5N1 virus in quail [73]. More evaluation of its efficacy in other poultry, such as ducks and geese, is needed in the future. AI vaccines used in the field Given the global spread of H5N1 viruses, effective control solely by culling is not feasible, even in the short term. Vaccination strategies are therefore implemented as part of comprehensive control programs in many affected countries (Box 1) [4]. In most of these countries, vaccine is used in emergency vaccination programs or as a preventive measure when the risk of an H5N1 virus endemic is high. However, in China, Egypt, Indonesia, and Vietnam, where H5N1 influenza viruses have become enzootic in poultry and wild birds, mass vaccination campaigns are being conducted as a routine measure for the control of AI, accounting for over 99% of the vaccine usage worldwide [3,4]. A vaccine program was first implemented in Hong Kong in 2002. An inactivated vaccine prepared from A/chicken/ Mexico/232-CPA/94 virus, was used within a small area [74]. Since 2003, a large-scale vaccination campaign has been continuously implemented in Hong Kong. At the end of 2003, the H5N1 viruses caused extensive outbreaks in many countries of Southeast Asia. In mainland China, an inactivated H5N2 vaccine prepared from the A/turkey/ England/N28/1973 vaccine strain was used from 2004 to 2006 [41] (Table 2). Also, from 2004, the reverse geneticsbased inactivated vaccines were routinely used in China to control H5N1 AI. The initial reassortant 1 (Re-1) vaccine strain was developed with the HA and NA genes of A/goose/ Guangdong/1/96 (H5N1) virus. In China, a nationwide surveillance program is in place for H5N1 AIV. Once the antigenicity of the vaccine strain does not match that of the majority of the field strains, a new vaccine strain will be developed and quickly updated. To date, the vaccine strain seed virus for the reverse genetics-based inactivated H5N1 vaccine has been updated three times in mainland China, including Re-1, Re-4, Re-5, and Re-6. Of note, three

Vaccine

Seed name Inactivated vaccine

H5N2 subtype H5N1 subtype

a

Recombinant FPV vaccine Recombinant NDV vaccine

Total

Country where used

/

10.18

China

/

/

22.64

0.95 6.80

0.24 6.77

0.025 3.71

3.605 28.88

/ 9.08 /

/ 7.51 /

/ 7.67 /

/ 4.03 3.66

3.7 29.79 3.66

/ / /

/ / /

/ / /

/ 0.25 /

4.23 0.2 /

4.23 0.45 0.615

China, Vietnam, Mongolia, Indonesia, Egypt China China, Vietnam, Indonesia, Burma, Egypt China China China, Indonesia, Egypt China Egypt China

0.5 0.7 /

/ 1.47 /

/ 1.74 /

/ 1.71 /

/ 1.03 0.72

4.4 6.65 0.72

China China China

HA and/or NA gene donor virus (HA clade) Not applicable

2004

2005

2006

2007

2008

2009

2010

2011

2012

2.5

4.08

3.6

/

/

/

/

/

A/goose/Guangdong/1/1996 (0)

0.57

3.3

4.57

9.6

4.6

/

/

H5N1/PR8 (Re-4) H5N1/PR8 (Re-5)

A/chicken/Shanxi/2/2006 (7.2) A/duck/Anhui/1/2006 (2.3.4)

/ /

/ /

0.84 /

0.42 /

0.59 4.4

0.54 7.20

Re-1/Re-4 Re-4/Re-5 H5N1/PR8 (Re-6)

– – A/duck/Guangdong/ S1322/2006 (2.3.2)

/ / /

/ / /

/ / /

2.2 / /

1.5 1.5 /

Re-4/Re-6 H5N1/PR8 (Egypt-1) rFPV-HA-NA

– A/chicken/Egypt/18-H/09 (2.2.1.1) A/goose/Guangdong/1/1996 (0)

/ / /

/ / 0.615

/ / /

/ / /

rLH5-1 rLH5-5 rLH5-6

A/goose/Guangdong/1/1996 (0) A/duck/Anhui/1/2006 (2.3.4) A/duck/Guangdong/S1322/2006 (2.3.2)

/ / /

/ / /

2.6 / /

1.3 / /

A/Turkey/England/N-28/73 (H5N2) (N-28) H5N1/PR8 (Re-1)

/, denotes that the vaccine was not used in that particular year.

7

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Live virus vector vaccine

Doses used in each year (billions) a

Seed virus generated

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Review

Table 2. Vaccines developed in China for H5N1 AI control

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Review combinations of two different vaccine seed viruses, including Re-1/Re-4, Re-4/Re-5, and Re-4/Re-6, were produced and used in the areas where both clades of H5N1 viruses had been detected. The inactivated vaccines used in China were also exported to several other countries, including Egypt, Indonesia, Vietnam, Bangladesh, Burma, and Mongolia, to support the effective control of H5N1 AI. In 2011, a vaccine with the seed virus H5N1/PR8 (Egypt-1), bearing the HA and NA genes from a native Egyptian H5N1 isolate, A/chicken/Egypt/18-H/09, was developed and used in Egypt (Table 2). To date, over 100 billion doses of inactivated vaccine have been used (Table 2). In addition to the inactivated vaccine, recombinant virus vector vaccines were also used in mainland China (Table 2). A small amount of recombinant FPV vector vaccine was used in 2005. In early 2006, a bivalent recombinant NDV vector H5N1 AI vaccine, rLH5-1, was approved for use in chickens in China [75]. It is the first industrialized live vector vaccine worldwide to be developed from an RNA virus. So far, the recombinant NDV vector vaccine seed virus has been updated twice with the HA gene from A/duck/Anhui/1/06 in 2008 and that from A/ duck/Guangdong/S1322/2006 in 2012 (Table 2). In total, over 11 billion doses of the bivalent recombinant NDV vector H5N1 AI vaccine were manufactured and used in chickens in China between 2006 and 2012. Concluding remarks H5N1 AIV has caused the largest enzootic disease in poultry and wild birds. It is clear that the vaccination strategy has played an important role in limiting the devastating effects of H5N1 AI on the poultry industry, decreasing the virus load in the environment, securing food security, and reducing virus transmission from poultry to humans. Our experience with vaccines against H5N1 viruses has attested that modern biotechnology, exemplified by reverse genetics technology, has made it easier to develop influenza vaccines with desirable properties such as high growth, low pathogenicity, and high immunogenicity. To ensure that vaccination programs continue to be effective in the future, optimal protocols that make use of different types of vaccines must be carefully developed. Protocol development should consider the species, age, and immune status of the poultry, as well as the potential for combination usage of different vaccines. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of H5N1 viruses in poultry. Of note, without a comprehensive control strategy at the national and international level, the effective control of H5N1 viruses is an impossible task. In China, control strategies are established and in place at both national and provincial levels. The government provides financial compensation for slaughtered poultry. With respect to H5N1 vaccination, the central government covers the cost of the vaccine, which is then distributed to the farmers [41]. Coupled with vaccination, the comprehensive strategy 8

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includes rapid diagnostics and surveillance, depopulation of infected poultry, notification and information sharing, and improved biosecurity. Acknowledgments We thank S. Watson for editing the manuscript. This work was supported by the Ministry of Agriculture (CARS-42-G08) and by the Ministry of Science and Technology (2012ZX10004214).

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