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Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert
Vaccine 18 (2000) 1088±1095
www.elsevier.com/locate/vaccine
Protection against diverse highly pathogenic H5 avian in¯uenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian in¯uenza hemagglutinin gene insert David E. Swayne a,*, Maricarmen Garcia a, Joan R. Beck a, Nikki Kinney b, David L. Suarez a a
Southeast Poultry Research Laboratory, United States Department of Agriculture, Agriculture Research Service, 934 College Station Road, Athens, GA 30605, USA b Merial-Select, Inc., P.O. Box 2497, Gainesville, GA 30503, USA Received 22 April 1999; received in revised form 23 July 1999; accepted 23 July 1999
Abstract A recombinant fowlpox vaccine with an H5 hemagglutinin gene insert protected chickens against clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses. The challenge viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant vaccine, and represented diversely geographic and spatial backgrounds; i.e. isolated from four dierent continents over a 38 year period. The recombinant vaccine reduced detectable infection rates and shedding titers by some challenge viruses. There was a signi®cant positive correlation in hemagglutinin sequence similarity between challenge viruses and vaccine, and the ability to reduce titers of challenge virus isolated from the oropharynx (rs=0.783, P = 0.009), but there was no similar correlation for reducing cloacal virus titers (rs=ÿ0.100, P = 0.78). This recombinant fowlpox-H5 avian in¯uenza hemagglutinin vaccine can provide protection against a variety of dierent highly pathogenic H5 avian in¯uenza viruses and frequent optimizing of the hemagglutinin insert to overcome genetic drift in the vaccine may not be necessary to provide adequate ®eld protection. Published by Elsevier Science Ltd. Keywords: In¯uenza vaccines; Recombinant vaccines; Avian in¯uenza
1. Introduction Avian in¯uenza is caused by an infection with type A orthomyxoviruses [1]. Avian in¯uenza viruses can be subtyped serologically into 15 dierent haemagglutinin subtypes (H1±15) [1]. All H1±4, H6 and H8±15 and most H5 and H7 viruses are mildly pathogenic while the remaining H5 and H7 viruses are highly pathogenic. Avian in¯uenza is not an endemic disease in * Corresponding author. Tel.: +706-546-3433; fax: +706-5463161. E-mail address: [email protected] (D.E. Swayne). 0264-410X/99/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 3 6 9 - 2
commercial poultry and vaccines are not approved for routine use. However, infection by avian in¯uenza viruses has been commonly identi®ed in migratory birds and rural poultry of the Live-Bird Market systems throughout the world [2±7]. Sporadically, outbreaks of mildly pathogenic avian in¯uenza have occurred in commercial poultry, and rarely, outbreaks of highly pathogenic or highly lethal avian in¯uenza have occurred [2,3,8]. The highly pathogenic form of avian in¯uenza impacts international trade of poultry and poultry products, and is a List A disease for the Oce International des Epizooties. The latter is an intergovernmental organization of 152 member nations that establishes animal health codes and regulatory
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Table 1 Deduced hemagglutinin amino acid sequence similarity between the recombinant vaccine and highly pathogenic H5 avian in¯uenza challenge viruses Challenge virus strain
Abbreviation
Subtype
Hemagglutinin amino acid similarity with vaccine strain (%)
A/turkey/Ireland/83 A/turkey/England/91 A/tern/South Africa/61 A/chicken/Scotland/59 A/human/Hong Kong/156/97 A/chicken/Queretaro/14588/95 A/turkey/Ontario/77322/66 A/emu/Texas/39924/93 A/chicken/Pennsylvania/1370/83
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83
H5N8 H5N1 H5N3 H5N1 H5N1 H5N2 H5N9 H5N2 H5N2
100 94.2 93.1 92.0 90.2 89.3 89.1 88.8 87.3
requirements for international trade in animals and their products. In¯uenza control programs for commercial poultry have been developed on an outbreak-by-outbreak basis, but generally control has been accomplished through a multifaceted approach utilizing increased surveillance and diagnostics, quarantine of infected areas, enhanced biosecurity, education of poultry workers and some method of eliminating infected poultry [9,10]. In the United States (US), vaccines have been used in some programs to control mildly pathogenic avian in¯uenza during sporadic outbreaks in turkeys [11]. From 1979 to 1997, 22 million doses of inactivated vaccine were used in Minnesota, the major geographic location of mildly pathogenic avian in¯uenza outbreaks within the U.S. [10]. Recently, vaccines have been used in control programs for highly pathogenic avian in¯uenza in chickens in Mexico and Pakistan [12,13]. Experimentally, various vaccines have shown ecacy in chickens against avian in¯uenza and include inactivated whole avian in¯uenza virus [14], recombinant fowlpox vector with H5 avian in¯uenza viral hemagglutinin gene insert [9,15±18], subunit hemagglutinin protein [19] and DNA containing the hemagglutinin gene vaccines [20]. Such vaccines provided immunity that was subtype speci®c against each of the 15 dierent hemagglutinin proteins of the in¯uenza virus [14]. A few inactivated whole avian in¯uenza vaccines and a recombinant fowlpox-avian in¯uenza H5 hemagglutinin vaccine have been licensed by the US Department of Agriculture (USDA) [21]. However, use of the nonH5 and non-H7 avian in¯uenza vaccines in the ®eld requires special approval by individual state governments. Use of the H5 and H7 vaccines requires approval by the federal government and can only be part of an ocial USDA avian in¯uenza disease control program [21]. Individually, avian in¯uenza vaccines are licensed by the USDA and any changes, such
as replacing the virus strain in the vaccine, requires a new license application to demonstrate purity, safety, potency and ecacy of the vaccine [21]. In addition, live recombinant vaccines are required to have a risk analysis and environmental assessment, in accordance with the National Environmental Policy Act, prior to licensure. Therefore, the initial development or the changing of avian in¯uenza vaccines for poultry is a costly, 2±3 year process. Previous studies in chickens have determined the ability of individual inactivated or recombinant vaccines to protect against challenge by one or two highly pathogenic avian in¯uenza viruses. Typically, protection has been against clinical signs and death [14±19]. In a few studies, the vaccines were shown to reduce the number of chickens shedding the highly pathogenic challenge virus from the respiratory and digestive tracts or completely prevent shedding of the challenge virus [15,17,18,22]. In three studies, the avian in¯uenza vaccines reduced the quantity of challenge avian in¯uenza virus detected in the digestive and respiratory tracts of immunized chickens [9,18,23]. The current study was initiated to address the broad issue of whether avian in¯uenza vaccines should be changed frequently in order to provide acceptable protection for highly pathogenic H5 avian in¯uenza eradication programs. Historically, equine in¯uenza vaccines have been changed infrequently while human in¯uenza vaccines have been evaluated and changed on a yearly basis [24,25]. The current study was conducted to determine the impact of genetic relatedness between the hemagglutinin of vaccine and ®eld avian in¯uenza viruses on the ability to protect against clinical signs, death, and infection and shedding of challenge avian in¯uenza virus from immunized chickens. The study demonstrated a recombinant fowlpox vaccine containing an avian in¯uenza H5 gene insert from a 1983 Eurasian lineage avian in¯uenza virus protected chickens against
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clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses. The vaccine also reduced shedding of most of the challenge avian in¯uenza viruses. The challenge H5 avian in¯uenza viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant H5 vaccine.
2. Materials and methods 2.1. Chickens Speci®c-pathogen-free (SPF) White Leghorn (WL) 1-day-old chicks (SPAFAS, Inc., Storrs, CT and Sunrise Farms, Catskill, NY) were housed in brooder cages at Merial-Select, Inc. for the ®rst 2.5 weeks, and transferred to Southeast Poultry Research Laboratory (SEPRL) for challenge studies. At SEPRL, the chickens were housed in negative-pressure ®ltered-air stainless steel isolation cabinets in a Biosafety Level 3 Agriculture facility [26]. Light exposure was continuous. Water and feed were provided ad libitum.
2.2. Viruses Challenge viruses included eight dierent highly pathogenic H5 avian in¯uenza viruses isolated over a period of 36 years and one H5 human in¯uenza virus strain isolated from Hong Kong in 1997 (Table 1). The highly pathogenic ET/93 was derived in the laboratory from a mildly pathogenic ®eld strain after passage in 14-day embryonating chicken eggs and adult laying hens [27]. All viruses used for challenge were propagated and titrated in 10-day embryonating chicken eggs, using standard methods [28].
2.3. Vaccines A recombinant fowlpox virus vaccine (Vector-HA) containing a cDNA copy of the hemagglutinin gene of A/turkey/Ireland/1378/83 (H5N3) (vFP89, MerialSelect Inc., Gainesville, GA) was used [15]. The parent fowlpox virus without the hemagglutinin gene insert (Vector-Control) was used in the sham vaccination group. The vaccines were administered by the subcutaneous route. Each chicken received either 103.0 TCID50 of Vector-HA or 103.5 TCID50 of Vector-Control in 0.2 ml of diluent.
2.4. Experimental design Nine groups of ten 1-day-old WL chickens were vaccinated with the Vector-HA vaccine, and nine groups of ten 1-day-old WL chickens were vaccinated with the Vector-Control vaccine. At 3-weeks post vaccination, one group of Vector-Control chickens and one group of Vector-HA chickens were challenged with each of the nine H5 challenge virus strains. Each chicken received 250 mean chicken lethal doses (CLD50) of challenge virus. Chickens were observed daily for 2 weeks, and morbidity and mortality were observed and recorded. Oropharyngeal and cloacal swabs were taken from chickens on day 2 or 3 post challenge (PC) for virus re-isolation and titration. Chickens were bled and euthanatized 2 weeks PC.
2.5. Genetic analysis of viruses The amino acid sequences of the H5 viruses used in the study were compared by phylogenetic analysis and pairwise alignment. Nucleotide sequences were available from GenBank for all nine isolates, and the putative amino acid sequences for the HA1 and HA2 parts of the hemagglutinin gene were aligned with the program Megalign (DNASTAR, Madison, WI). Amino acid sequence similarity was determined by pairwise alignment of each isolate with TI/83 also with the Megalign program. The aligned sequences were further analyzed by parsimony with PAUP 3.0 software (D. Swoord, Illinois Natural History Survey, Urbana, IL). Phylogenetic trees were generated with 100 bootstraps analysis using a heuristic method and midpoint rooting. 2.6. Virus isolation and titration Oropharyngeal and cloacal swabs were taken at peak of virus shedding, i.e. day 2 or 3 PI as determined in preliminary titration experiments for each challenge virus (unpublished data). Virus was isolated from the swab medium and virus titers were determined and expressed as log10ELD50/ml of swab ¯uid as described [28]. 2.7. Statistical analysis of biological data Morbidity and mortality rates, and frequency of virus isolation results were analyzed for signi®cance (P < 0.05) between Vector-Control and Vector-HA groups by Fisher's exact test on PC-based software (Statgraphics, Manugistics, Inc., Rockville, MD). Virus isolation titers were tested for normal distribution and equal variance. All titers that failed nor-
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Spearman rank correlation (rs) was used to test association between sequence similarity of vaccine and challenge virus hemagglutinin, and reductions in titers of challenge virus shed from cloaca and oropharynx. Spearman rank correlations were performed on PCbased software (SigmaStat, Jandel Scienti®c, San Rafael, CA).
3. Results 3.1. Comparison of sequence similarity between hemagglutinin gene insert of vaccine and hemagglutinin of challenge viruses
Fig. 1. Phylogenetic analysis using parsimony of the amino acid sequence of the dierent highly pathogenic avian in¯uenza H5 viruses used in this study. The tree is the result of 100 bootstrap replicates using a heuristic search, and the tree is midpoint rooted. Branch lengths are included on each tree to de®ne the sequence distance between isolates.
mality or equal variance tests were analyzed for signi®cance (P < 0.05) by Mann±Whitney Rank Sum test on PC-based software (SigmaStat, Jandel Scienti®c, San Rafael, CA). The minimum virus titer detected by virus isolation procedures in this study was 101.0 ELD50/ml. Thus for statistical purposes, all oropharyngeal and cloacal swabs from which virus was not isolated were given a numeric value of 100.9 ELD50/ml which represents the lowest detectable level of virus if the virus isolation procedure were modi®ed to use four instead of three embryonating chicken eggs per sample.
The HA1 and HA2 parts of the hemagglutinin protein for the dierent challenge viruses used in the study were compared by sequence similarity with TI/ 83, the hemagglutinin insert in the recombinant fowlpox vaccine (Fig. 1). Sequence similarity ranged from 100 to 87.3%. The dierent avian in¯uenza virus isolates phylogenetically separated into two distinct groups, the North American and the Eurasian±African lineages. This separation between the North American and Eurasian±African isolates has been observed in other avian in¯uenza viral genes, and is thought to be the result of the general separation of Old World and New World [29]. The hemagglutinin gene of the vaccine strain, highly pathogenic avian in¯uenza virus TI/ 83, is more closely related to the other European, Asian, and African isolates in this study, but the date of isolation of the virus appears to have no eect on sequence similarity. This likely re¯ects that each isolate used in the study was the result of separate introductions of H5 virus into poultry populations [30].
Table 2 Protection against lethal challenge with highly pathogenic H5 avian in¯uenza virus in chickens immunized at 1 day of age with fowlpox vector (Vector-Control) or recombinant fowlpox-H5 avian in¯uenza hemagglutinin (Vector-HA) vaccine. Chicks were challenged 3 weeks following immunization Morbidity
Mortality
Challenge virus
Vector-Control (n = 10)
Vector-HA (n = 10)
Vector-Control (n = 10)
Vector-HA (n = 10)
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83
10a 10a 10a 10a 8a 10a 10a 10a 10a
0b 0b 0b 0b 0b 0b 0b 0b 0b
10a 10a 10a 9a 8a 10a 9a 7a 10a
0b 0b 0b 0b 0b 0b 0b 0b 0b
a,b Dierent lowercase letters indicate signi®cant dierence (P < 0.05) between Vector-Control and Vector-HA vaccination groups for morbidity and mortality as estimated by Fisher Exact Test.
9/9A 5/5A 8/8A 9/10A 8/10A 10/10A 8/9A 5/9A 10/10A 72/80
(No. positive/total) 4.39a 5.22a 4.07a 3.86a 4.00a 5.26a 4.43a 3.17a 6.40a
(log10 EID50/ml)
Vector-Control
0/10B 0/10B 0/10B 1/10B 1/10B 9/10A 2/10B 1/10B 10/10A 24/90
(No. positive/total) NIb NIb NIb 1.14b 1.10b 3.18a 1.07b 1.42b 4.56a
(log10 EID50/ml)
Vector-HA
7/9A 5/5A 8/8A 9/10A 8/10A 8/10A 8/9A 7/9A 10/10A 70/80
(No. positive/total)
2.24a 2.98a 2.93a 2.08a 3.00a 2.42a 2.52a 1.91a 3.88a
(log10 EID50/ml)
Vector Control
0/10B 0/10B 0.10B 2/10B 0/10B 1/10B 0/10B 0/10B 0/10B 3/90
NIb NIb NIb 1.01b NIb 0.91b NIb NIb NIb
(log10 EID50/ml)
Vector-HA (No. positive/total)
Cloacal swabs
a,b
Dierent uppercase letters indicate signi®cant dierence (P < 0.05) between virus isolation of Vector-Control and Vector-HA groups using Fishers Exact Test. Dierent lowercase letters indicate signi®cant dierences (P < 0.05) between titers of virus shed from Vector-Control and Vector-HA groups using Fishers Exact Test. NI=none isolated. For statistical purposes, all oropharyngeal and cloacal swabs from which virus was not isolated were given a numeric value of 100.9 ELD50/ml which represents the lowest detectable level of virus if the virus isolation procedure were modi®ed to use four instead of three embryonating chicken eggs per sample.
A,B
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83 Total
Challenge virus
Oropharyngeal swabs
Table 3 Virus isolation results and infectious titers from chickens vaccinated at 1 day of age with fowlpox (Vector-Control) or recombinant fowlpox-H5 avian in¯uenze hemagglutinin gene (Vector-HA) vaccine and challenged 3 weeks later. Oropharyngeal and cloacal swabs were taken at peak of challenge virus shed
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Fig. 2. Comparison of percent hemagglutination similarity (Hemagglutinin Homology) of vaccine and challenge viruses with reduction in titers of challenge viruses shed between Vector-Control and Vector-HA groups.
3.2. Protection against clinical signs and death in H5 immunized chickens following challenge by highly pathogenic avian in¯uenza viruses In humans, in¯uenza vaccines are changed yearly in order to provide the best protection against current circulating in¯uenza viruses [24] while in horses, the vaccines have been changed infrequently [25]. Previous studies have shown immunization of chickens with whole in¯uenza virus or the major protective antigen of avian in¯uenza virus, the hemagglutinin protein, prevented clinical signs or death following challenge with highly pathogenic avian in¯uenza viruses of the same hemagglutinin subtype [9,14,15,17]. However, these individual studies were limited to one or two challenge viruses, that most frequently were the same as or closely related to the vaccine strain. In the current study, chickens were immunized with a recombinant fowlpox vaccine in order to determine the role the H5 hemagglutinin protein component had on protection against related and unrelated highly pathogenic H5 avian in¯uenza virus strains. Chickens in sham immunized groups (Vector-Control) developed clinical signs (80±100%) and died (70±100%) following challenge by the nine dierent highly pathogenic H5 avian in¯uenza viruses (Table 2). In contrast, chickens immunized with Vector-HA were protected, with no chickens developing clinical signs or death (Table 2). 3.3. Immunization reduced virus detection rates and titers of challenge virus in the oropharynx and cloaca Previous studies varied in demonstrating reductions in virus shedding from immunized chickens challenged with in¯uenza virus which would indicate the potential for reducing environmental contamination and transmission of the avian in¯uenza virus [9,17,22]. These studies did not standardize the quantity of immunizing
antigen or the dose of the challenge virus. In addition, the eciency in reducing avian in¯uenza infections was not consistently measured from either the intestinal or respiratory tracts at the peak of virus replication, nor were the reductions in detectible virus titers quanti®ed. In the current study, we standardized all four of these variables and then determined if vaccination would reduce challenge virus detection rates, and when virus was detected, determined the quantity. Preliminary experiments indicated that day 3 PC was the time of maximal virus shedding from the oropharynx and cloaca of chickens for TI/83, TE/91, TSA/61, CS/59, CQ/95, TO/66, ET93 and CP/83 avian in¯uenza viruses while for HK/97, the peak virus shedding was day 2 PC. In survivors at days 2 or 3 PC, chickens in Vector-Control groups had challenge virus isolated commonly from the oropharynx (90%) and cloaca (88%) (Table 3). Immunization with Vector-HA eliminated or reduced to a minimum challenge virus detection from the cloaca in all groups and from the oropharynx in all groups except CP/83 and CQ/95 (Table 3). Immunization with Vector-HA signi®cantly reduced the titers of challenge virus recovered or was associated with lack of virus recovery from both the oropharynx and cloaca in all groups, except CQ/95 and CP/83 from the oropharynx (Table 3). The deduced amino acid sequence similarity for the hemagglutinin protein of vaccine and challenge viruses, and the ability to reduce virus shedding from oropharynx and cloaca were compared statistically between the nine dierent challenge viruses. There was no correlation between hemagglutinin sequence similarity and reduction in virus titers shed from the cloaca (rs=ÿ0.10, P = 0.78), but there was direct correlation between the sequence similarity of the HA and the ability to reduce virus titers shed from the oropharynx (rs=0.78, P = 0.009) (Fig. 2).
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4. Discussion A recombinant fowlpox vaccine with an H5 hemagglutinin gene insert protected chickens against clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses, including the highly pathogenic H5N1 in¯uenza virus isolated from a child in Hong Kong during 1997. These avian in¯uenza challenge viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant vaccine, and represented diversely geographic and spatial backgrounds; i.e. isolated from four dierent continents over a 38 year period. This broad-based protection against avian in¯uenza isolates in poultry over multiple years is in contrast to human in¯uenza where vaccines are changed yearly to provide optimal protection in the face of antigenic drift in seasonal ®eld viruses [24] and in horses where vaccine strains have been changed infrequently [25]. The current study demonstrated that the hemagglutinin contained within a single recombinant fowlpox vaccine can provide protection against clinical signs and death following exposure to nine dierent highly pathogenic H5 avian in¯uenza viruses, and frequent changing of the hemagglutinin insert within the recombinant vaccine may not be necessary to provide similar protective eects against prospective ®eld viruses. The prevention of clinical disease and death in immunized chickens challenged by various highly pathogenic avian in¯uenza viruses is well documented [1]. Such protection is dependent on a systemic humoral immune response that prevents hematogenous spread of the virus, replication in critical visceral organs and brain, and death of the individual. However, eective vaccine strategies to control or eradicate avian in¯uenza will require prevention of virus replication in the respiratory and gastrointestinal tracts through speci®c immunity at the mucosal level and the resulting prevention of virus shedding and bird-to-bird transmission [9]. Previous experimental studies have reported prevention of in¯uenza virus infection in immunized chickens varying from complete absence of detectable virus [18] to no inhibition of virus replication in the respiratory tract [9]. The recombinant vaccine prevented or decreased virus detection rates and reduced shedding titers of some challenge viruses [9,15,17], but this reduction was not absolute [9]. Various factors aect the mucosal replication of virus including the antigenic relationship between vaccine and challenge strain of the major protective viral protein, the hemagglutinin. In the current study, we demonstrated reduction of in¯uenza virus replication in respiratory and gastrointestinal tracts of immunized chickens. This reduction had a signi®cant positive correlation between hemagglutinin sequence similarity of challenge virus and vaccine, and the ability to reduce
titers of challenge virus detected in the oropharynx (rs=0.783, P = 0.009). However, there was no similar correlation for reducing cloacal virus titers (rs=ÿ0.100, P = 0.78). Further analysis indicated that reduction in virus replication at the mucosal level in chickens immunized with fowlpox-avian in¯uenza TI/83 hemagglutinin gene recombinant vaccine was most consistent for the Eurasian±African lineage of ®eld viruses. Reductions in virus replication were less consistent for the North American H5 avian in¯uenza challenge viruses, indicating that total sequence similarity of the hemagglutinin may be less important than similarity of speci®c antigenic epitopes in preventing virus replication at the mucosal level. For example, ET/93 had 88.8% hemagglutinin similarity with the vaccine and signi®cantly reduced the shedding of challenge virus from the oropharynx, while CQ/95 had 89.1% similarity, but did not reduce challenge virus shed from the oropharynx as compared to the control fowlpox vaccine groups. A previous study using inactivated H5 avian in¯uenza vaccines demonstrated reductions in mucosal viral replication in immunized chickens, but this did not correlate with hemagglutinin sequence similarities between vaccine and challenge virus [23]. This suggests that factors in addition to hemagglutinin sequence similarity between vaccine and challenge strain impact the ability to reduce mucosal replication in immunized chickens, possibly other genes or speci®c segments of the hemagglutinin gene. From a pragmatic view, previous studies have demonstrated the ability of this recombinant vaccine to prevent horizontal transmission of the Mexican CQ/ 95 avian in¯uenza virus (89.3% hemagglutinin similarity with vaccine) to in contact non-immunized chickens [9]. Furthermore, the current along with previous studies [9,15,17,18] have shown that a single recombinant fowlpox-H5 avian in¯uenza hemagglutinin vaccine can provide protection against a variety of highly pathogenic H5 avian in¯uenza viruses and frequent changing of the hemagglutinin insert in the vaccine to overcome genetic drift in ®eld viruses may not be necessary to prevent transmission of the virus, clinical signs and death under ®eld conditions.
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[17] Webster RG, Kawaoka Y, Taylor J, Weinberg R, Paoletti E. Ecacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for in¯uenza in chickens. Vaccine 1991;9:303±8. [18] Webster RG, Taylor J, Pearson J, Rivera E, Paoletti E. Immunity to Mexican H5N2 avian in¯uenza viruses induced by a fowl pox-H5 recombinant. Avian Dis 1996;40:461±5. [19] Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, Perdue ML. Baculovirus-derived hemagglutinin vaccines protect against lethal in¯uenza infections by avian H5 and H7 subtypes. Vaccine 1999;17:2265±74. [20] Robinson HL, Hunt LA, Webster RG. Protection against a lethal in¯uenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 1993;11:957±60. [21] Myers TJ, Morgan AP. Policy and guidance for licensure of avian in¯uenza vaccines in the United States. In: Swayne DE, Slemons RD, editors. Proceedings of the Fourth International Symposium on Avian In¯uenza. Richmond, Virginia: U.S. Animal Health Association, 1998. p. 373±8. [22] Stone HD. Ecacy of avian in¯uenza oil-emulsion vaccines in chickens of various ages. Avian Dis 1987;31:483±90. [23] Swayne DE, Beck JR, Garcia M, Stone HD. In¯uence of virus strain and antigen mass on ecacy of H5 avian in¯uenza inactivated vaccines. Avian Pathology 1999;28:245±56. [24] CDC. Update: In¯uenza activity Ð United States and worldwide, 1997±98 season, and composition of the 1998±99 in¯uenza vaccine. MMWR Morb Mortal Wkly Rep 1998;47:280±4. [25] Cullinane AA, Osborne M, Jackson T, Brennan C, Nelly M. A comparative study of the antigenicity of two equine in¯uenza vaccines in young thoroughbred horses. In: Van Reeth K. editors. Symposium on Animal In¯uenza Viruses. Ghent, Belgium: European Society of Veterinary Virology, 1999;53. [26] Barbeito MS, Abraham G, Best M, Cairns P, Langevin P, Sterritt WG, Barr D, Meulepas W, Sanchez-Vizcaino JM, Saraza M, Requena E, Collado M, Mani P, Breeze R, Brunner H, Mebus CA, Morgan RL, Rusk S, Siegfried LM, Thompson LH. Recommended biocontainment features for research and diagnostic facilities where animal pathogens are used. Rev Sci Tech O Int Epiz 1995;14:873±87. [27] Swayne DE, Beck JR, Perdue ML, Brugh M, Slemons RD. Assessment of the ability of ratite-origin in¯uenza viruses to infect and produce disease in rheas and chickens. Avian Dis 1996;40:438±47. [28] Swayne DE, Senne DA, Beard CW. In¯uenza. In: Swayne DE, Glisson JR, Jackwood MW, Pearson JE, Reed WM, editors. Isolation and identi®cation of avian pathogens. Kennett Square, Pennsylvania: American Association of Avian Pathologists, 1998. p. 150±5. [29] Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of in¯uenza A viruses. Microbiol Rev 1992;56:152±79. [30] Alexander DJ. Orthomyxovirus infections. In: McFerran JB, McNulty MS, editors. Virus infections of birds. London: Elsevier Science, 1993. p. 287±316.
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Protection against diverse highly pathogenic H5 avian in¯uenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian in¯uenza hemagglutinin gene insert David E. Swayne a,*, Maricarmen Garcia a, Joan R. Beck a, Nikki Kinney b, David L. Suarez a a
Southeast Poultry Research Laboratory, United States Department of Agriculture, Agriculture Research Service, 934 College Station Road, Athens, GA 30605, USA b Merial-Select, Inc., P.O. Box 2497, Gainesville, GA 30503, USA Received 22 April 1999; received in revised form 23 July 1999; accepted 23 July 1999
Abstract A recombinant fowlpox vaccine with an H5 hemagglutinin gene insert protected chickens against clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses. The challenge viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant vaccine, and represented diversely geographic and spatial backgrounds; i.e. isolated from four dierent continents over a 38 year period. The recombinant vaccine reduced detectable infection rates and shedding titers by some challenge viruses. There was a signi®cant positive correlation in hemagglutinin sequence similarity between challenge viruses and vaccine, and the ability to reduce titers of challenge virus isolated from the oropharynx (rs=0.783, P = 0.009), but there was no similar correlation for reducing cloacal virus titers (rs=ÿ0.100, P = 0.78). This recombinant fowlpox-H5 avian in¯uenza hemagglutinin vaccine can provide protection against a variety of dierent highly pathogenic H5 avian in¯uenza viruses and frequent optimizing of the hemagglutinin insert to overcome genetic drift in the vaccine may not be necessary to provide adequate ®eld protection. Published by Elsevier Science Ltd. Keywords: In¯uenza vaccines; Recombinant vaccines; Avian in¯uenza
1. Introduction Avian in¯uenza is caused by an infection with type A orthomyxoviruses [1]. Avian in¯uenza viruses can be subtyped serologically into 15 dierent haemagglutinin subtypes (H1±15) [1]. All H1±4, H6 and H8±15 and most H5 and H7 viruses are mildly pathogenic while the remaining H5 and H7 viruses are highly pathogenic. Avian in¯uenza is not an endemic disease in * Corresponding author. Tel.: +706-546-3433; fax: +706-5463161. E-mail address: [email protected] (D.E. Swayne). 0264-410X/99/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 3 6 9 - 2
commercial poultry and vaccines are not approved for routine use. However, infection by avian in¯uenza viruses has been commonly identi®ed in migratory birds and rural poultry of the Live-Bird Market systems throughout the world [2±7]. Sporadically, outbreaks of mildly pathogenic avian in¯uenza have occurred in commercial poultry, and rarely, outbreaks of highly pathogenic or highly lethal avian in¯uenza have occurred [2,3,8]. The highly pathogenic form of avian in¯uenza impacts international trade of poultry and poultry products, and is a List A disease for the Oce International des Epizooties. The latter is an intergovernmental organization of 152 member nations that establishes animal health codes and regulatory
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Table 1 Deduced hemagglutinin amino acid sequence similarity between the recombinant vaccine and highly pathogenic H5 avian in¯uenza challenge viruses Challenge virus strain
Abbreviation
Subtype
Hemagglutinin amino acid similarity with vaccine strain (%)
A/turkey/Ireland/83 A/turkey/England/91 A/tern/South Africa/61 A/chicken/Scotland/59 A/human/Hong Kong/156/97 A/chicken/Queretaro/14588/95 A/turkey/Ontario/77322/66 A/emu/Texas/39924/93 A/chicken/Pennsylvania/1370/83
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83
H5N8 H5N1 H5N3 H5N1 H5N1 H5N2 H5N9 H5N2 H5N2
100 94.2 93.1 92.0 90.2 89.3 89.1 88.8 87.3
requirements for international trade in animals and their products. In¯uenza control programs for commercial poultry have been developed on an outbreak-by-outbreak basis, but generally control has been accomplished through a multifaceted approach utilizing increased surveillance and diagnostics, quarantine of infected areas, enhanced biosecurity, education of poultry workers and some method of eliminating infected poultry [9,10]. In the United States (US), vaccines have been used in some programs to control mildly pathogenic avian in¯uenza during sporadic outbreaks in turkeys [11]. From 1979 to 1997, 22 million doses of inactivated vaccine were used in Minnesota, the major geographic location of mildly pathogenic avian in¯uenza outbreaks within the U.S. [10]. Recently, vaccines have been used in control programs for highly pathogenic avian in¯uenza in chickens in Mexico and Pakistan [12,13]. Experimentally, various vaccines have shown ecacy in chickens against avian in¯uenza and include inactivated whole avian in¯uenza virus [14], recombinant fowlpox vector with H5 avian in¯uenza viral hemagglutinin gene insert [9,15±18], subunit hemagglutinin protein [19] and DNA containing the hemagglutinin gene vaccines [20]. Such vaccines provided immunity that was subtype speci®c against each of the 15 dierent hemagglutinin proteins of the in¯uenza virus [14]. A few inactivated whole avian in¯uenza vaccines and a recombinant fowlpox-avian in¯uenza H5 hemagglutinin vaccine have been licensed by the US Department of Agriculture (USDA) [21]. However, use of the nonH5 and non-H7 avian in¯uenza vaccines in the ®eld requires special approval by individual state governments. Use of the H5 and H7 vaccines requires approval by the federal government and can only be part of an ocial USDA avian in¯uenza disease control program [21]. Individually, avian in¯uenza vaccines are licensed by the USDA and any changes, such
as replacing the virus strain in the vaccine, requires a new license application to demonstrate purity, safety, potency and ecacy of the vaccine [21]. In addition, live recombinant vaccines are required to have a risk analysis and environmental assessment, in accordance with the National Environmental Policy Act, prior to licensure. Therefore, the initial development or the changing of avian in¯uenza vaccines for poultry is a costly, 2±3 year process. Previous studies in chickens have determined the ability of individual inactivated or recombinant vaccines to protect against challenge by one or two highly pathogenic avian in¯uenza viruses. Typically, protection has been against clinical signs and death [14±19]. In a few studies, the vaccines were shown to reduce the number of chickens shedding the highly pathogenic challenge virus from the respiratory and digestive tracts or completely prevent shedding of the challenge virus [15,17,18,22]. In three studies, the avian in¯uenza vaccines reduced the quantity of challenge avian in¯uenza virus detected in the digestive and respiratory tracts of immunized chickens [9,18,23]. The current study was initiated to address the broad issue of whether avian in¯uenza vaccines should be changed frequently in order to provide acceptable protection for highly pathogenic H5 avian in¯uenza eradication programs. Historically, equine in¯uenza vaccines have been changed infrequently while human in¯uenza vaccines have been evaluated and changed on a yearly basis [24,25]. The current study was conducted to determine the impact of genetic relatedness between the hemagglutinin of vaccine and ®eld avian in¯uenza viruses on the ability to protect against clinical signs, death, and infection and shedding of challenge avian in¯uenza virus from immunized chickens. The study demonstrated a recombinant fowlpox vaccine containing an avian in¯uenza H5 gene insert from a 1983 Eurasian lineage avian in¯uenza virus protected chickens against
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clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses. The vaccine also reduced shedding of most of the challenge avian in¯uenza viruses. The challenge H5 avian in¯uenza viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant H5 vaccine.
2. Materials and methods 2.1. Chickens Speci®c-pathogen-free (SPF) White Leghorn (WL) 1-day-old chicks (SPAFAS, Inc., Storrs, CT and Sunrise Farms, Catskill, NY) were housed in brooder cages at Merial-Select, Inc. for the ®rst 2.5 weeks, and transferred to Southeast Poultry Research Laboratory (SEPRL) for challenge studies. At SEPRL, the chickens were housed in negative-pressure ®ltered-air stainless steel isolation cabinets in a Biosafety Level 3 Agriculture facility [26]. Light exposure was continuous. Water and feed were provided ad libitum.
2.2. Viruses Challenge viruses included eight dierent highly pathogenic H5 avian in¯uenza viruses isolated over a period of 36 years and one H5 human in¯uenza virus strain isolated from Hong Kong in 1997 (Table 1). The highly pathogenic ET/93 was derived in the laboratory from a mildly pathogenic ®eld strain after passage in 14-day embryonating chicken eggs and adult laying hens [27]. All viruses used for challenge were propagated and titrated in 10-day embryonating chicken eggs, using standard methods [28].
2.3. Vaccines A recombinant fowlpox virus vaccine (Vector-HA) containing a cDNA copy of the hemagglutinin gene of A/turkey/Ireland/1378/83 (H5N3) (vFP89, MerialSelect Inc., Gainesville, GA) was used [15]. The parent fowlpox virus without the hemagglutinin gene insert (Vector-Control) was used in the sham vaccination group. The vaccines were administered by the subcutaneous route. Each chicken received either 103.0 TCID50 of Vector-HA or 103.5 TCID50 of Vector-Control in 0.2 ml of diluent.
2.4. Experimental design Nine groups of ten 1-day-old WL chickens were vaccinated with the Vector-HA vaccine, and nine groups of ten 1-day-old WL chickens were vaccinated with the Vector-Control vaccine. At 3-weeks post vaccination, one group of Vector-Control chickens and one group of Vector-HA chickens were challenged with each of the nine H5 challenge virus strains. Each chicken received 250 mean chicken lethal doses (CLD50) of challenge virus. Chickens were observed daily for 2 weeks, and morbidity and mortality were observed and recorded. Oropharyngeal and cloacal swabs were taken from chickens on day 2 or 3 post challenge (PC) for virus re-isolation and titration. Chickens were bled and euthanatized 2 weeks PC.
2.5. Genetic analysis of viruses The amino acid sequences of the H5 viruses used in the study were compared by phylogenetic analysis and pairwise alignment. Nucleotide sequences were available from GenBank for all nine isolates, and the putative amino acid sequences for the HA1 and HA2 parts of the hemagglutinin gene were aligned with the program Megalign (DNASTAR, Madison, WI). Amino acid sequence similarity was determined by pairwise alignment of each isolate with TI/83 also with the Megalign program. The aligned sequences were further analyzed by parsimony with PAUP 3.0 software (D. Swoord, Illinois Natural History Survey, Urbana, IL). Phylogenetic trees were generated with 100 bootstraps analysis using a heuristic method and midpoint rooting. 2.6. Virus isolation and titration Oropharyngeal and cloacal swabs were taken at peak of virus shedding, i.e. day 2 or 3 PI as determined in preliminary titration experiments for each challenge virus (unpublished data). Virus was isolated from the swab medium and virus titers were determined and expressed as log10ELD50/ml of swab ¯uid as described [28]. 2.7. Statistical analysis of biological data Morbidity and mortality rates, and frequency of virus isolation results were analyzed for signi®cance (P < 0.05) between Vector-Control and Vector-HA groups by Fisher's exact test on PC-based software (Statgraphics, Manugistics, Inc., Rockville, MD). Virus isolation titers were tested for normal distribution and equal variance. All titers that failed nor-
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Spearman rank correlation (rs) was used to test association between sequence similarity of vaccine and challenge virus hemagglutinin, and reductions in titers of challenge virus shed from cloaca and oropharynx. Spearman rank correlations were performed on PCbased software (SigmaStat, Jandel Scienti®c, San Rafael, CA).
3. Results 3.1. Comparison of sequence similarity between hemagglutinin gene insert of vaccine and hemagglutinin of challenge viruses
Fig. 1. Phylogenetic analysis using parsimony of the amino acid sequence of the dierent highly pathogenic avian in¯uenza H5 viruses used in this study. The tree is the result of 100 bootstrap replicates using a heuristic search, and the tree is midpoint rooted. Branch lengths are included on each tree to de®ne the sequence distance between isolates.
mality or equal variance tests were analyzed for signi®cance (P < 0.05) by Mann±Whitney Rank Sum test on PC-based software (SigmaStat, Jandel Scienti®c, San Rafael, CA). The minimum virus titer detected by virus isolation procedures in this study was 101.0 ELD50/ml. Thus for statistical purposes, all oropharyngeal and cloacal swabs from which virus was not isolated were given a numeric value of 100.9 ELD50/ml which represents the lowest detectable level of virus if the virus isolation procedure were modi®ed to use four instead of three embryonating chicken eggs per sample.
The HA1 and HA2 parts of the hemagglutinin protein for the dierent challenge viruses used in the study were compared by sequence similarity with TI/ 83, the hemagglutinin insert in the recombinant fowlpox vaccine (Fig. 1). Sequence similarity ranged from 100 to 87.3%. The dierent avian in¯uenza virus isolates phylogenetically separated into two distinct groups, the North American and the Eurasian±African lineages. This separation between the North American and Eurasian±African isolates has been observed in other avian in¯uenza viral genes, and is thought to be the result of the general separation of Old World and New World [29]. The hemagglutinin gene of the vaccine strain, highly pathogenic avian in¯uenza virus TI/ 83, is more closely related to the other European, Asian, and African isolates in this study, but the date of isolation of the virus appears to have no eect on sequence similarity. This likely re¯ects that each isolate used in the study was the result of separate introductions of H5 virus into poultry populations [30].
Table 2 Protection against lethal challenge with highly pathogenic H5 avian in¯uenza virus in chickens immunized at 1 day of age with fowlpox vector (Vector-Control) or recombinant fowlpox-H5 avian in¯uenza hemagglutinin (Vector-HA) vaccine. Chicks were challenged 3 weeks following immunization Morbidity
Mortality
Challenge virus
Vector-Control (n = 10)
Vector-HA (n = 10)
Vector-Control (n = 10)
Vector-HA (n = 10)
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83
10a 10a 10a 10a 8a 10a 10a 10a 10a
0b 0b 0b 0b 0b 0b 0b 0b 0b
10a 10a 10a 9a 8a 10a 9a 7a 10a
0b 0b 0b 0b 0b 0b 0b 0b 0b
a,b Dierent lowercase letters indicate signi®cant dierence (P < 0.05) between Vector-Control and Vector-HA vaccination groups for morbidity and mortality as estimated by Fisher Exact Test.
9/9A 5/5A 8/8A 9/10A 8/10A 10/10A 8/9A 5/9A 10/10A 72/80
(No. positive/total) 4.39a 5.22a 4.07a 3.86a 4.00a 5.26a 4.43a 3.17a 6.40a
(log10 EID50/ml)
Vector-Control
0/10B 0/10B 0/10B 1/10B 1/10B 9/10A 2/10B 1/10B 10/10A 24/90
(No. positive/total) NIb NIb NIb 1.14b 1.10b 3.18a 1.07b 1.42b 4.56a
(log10 EID50/ml)
Vector-HA
7/9A 5/5A 8/8A 9/10A 8/10A 8/10A 8/9A 7/9A 10/10A 70/80
(No. positive/total)
2.24a 2.98a 2.93a 2.08a 3.00a 2.42a 2.52a 1.91a 3.88a
(log10 EID50/ml)
Vector Control
0/10B 0/10B 0.10B 2/10B 0/10B 1/10B 0/10B 0/10B 0/10B 3/90
NIb NIb NIb 1.01b NIb 0.91b NIb NIb NIb
(log10 EID50/ml)
Vector-HA (No. positive/total)
Cloacal swabs
a,b
Dierent uppercase letters indicate signi®cant dierence (P < 0.05) between virus isolation of Vector-Control and Vector-HA groups using Fishers Exact Test. Dierent lowercase letters indicate signi®cant dierences (P < 0.05) between titers of virus shed from Vector-Control and Vector-HA groups using Fishers Exact Test. NI=none isolated. For statistical purposes, all oropharyngeal and cloacal swabs from which virus was not isolated were given a numeric value of 100.9 ELD50/ml which represents the lowest detectable level of virus if the virus isolation procedure were modi®ed to use four instead of three embryonating chicken eggs per sample.
A,B
TI/83 TE/91 TSA/61 CS/59 HK/97 CQ/95 TO/66 ET/93 CP/83 Total
Challenge virus
Oropharyngeal swabs
Table 3 Virus isolation results and infectious titers from chickens vaccinated at 1 day of age with fowlpox (Vector-Control) or recombinant fowlpox-H5 avian in¯uenze hemagglutinin gene (Vector-HA) vaccine and challenged 3 weeks later. Oropharyngeal and cloacal swabs were taken at peak of challenge virus shed
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Fig. 2. Comparison of percent hemagglutination similarity (Hemagglutinin Homology) of vaccine and challenge viruses with reduction in titers of challenge viruses shed between Vector-Control and Vector-HA groups.
3.2. Protection against clinical signs and death in H5 immunized chickens following challenge by highly pathogenic avian in¯uenza viruses In humans, in¯uenza vaccines are changed yearly in order to provide the best protection against current circulating in¯uenza viruses [24] while in horses, the vaccines have been changed infrequently [25]. Previous studies have shown immunization of chickens with whole in¯uenza virus or the major protective antigen of avian in¯uenza virus, the hemagglutinin protein, prevented clinical signs or death following challenge with highly pathogenic avian in¯uenza viruses of the same hemagglutinin subtype [9,14,15,17]. However, these individual studies were limited to one or two challenge viruses, that most frequently were the same as or closely related to the vaccine strain. In the current study, chickens were immunized with a recombinant fowlpox vaccine in order to determine the role the H5 hemagglutinin protein component had on protection against related and unrelated highly pathogenic H5 avian in¯uenza virus strains. Chickens in sham immunized groups (Vector-Control) developed clinical signs (80±100%) and died (70±100%) following challenge by the nine dierent highly pathogenic H5 avian in¯uenza viruses (Table 2). In contrast, chickens immunized with Vector-HA were protected, with no chickens developing clinical signs or death (Table 2). 3.3. Immunization reduced virus detection rates and titers of challenge virus in the oropharynx and cloaca Previous studies varied in demonstrating reductions in virus shedding from immunized chickens challenged with in¯uenza virus which would indicate the potential for reducing environmental contamination and transmission of the avian in¯uenza virus [9,17,22]. These studies did not standardize the quantity of immunizing
antigen or the dose of the challenge virus. In addition, the eciency in reducing avian in¯uenza infections was not consistently measured from either the intestinal or respiratory tracts at the peak of virus replication, nor were the reductions in detectible virus titers quanti®ed. In the current study, we standardized all four of these variables and then determined if vaccination would reduce challenge virus detection rates, and when virus was detected, determined the quantity. Preliminary experiments indicated that day 3 PC was the time of maximal virus shedding from the oropharynx and cloaca of chickens for TI/83, TE/91, TSA/61, CS/59, CQ/95, TO/66, ET93 and CP/83 avian in¯uenza viruses while for HK/97, the peak virus shedding was day 2 PC. In survivors at days 2 or 3 PC, chickens in Vector-Control groups had challenge virus isolated commonly from the oropharynx (90%) and cloaca (88%) (Table 3). Immunization with Vector-HA eliminated or reduced to a minimum challenge virus detection from the cloaca in all groups and from the oropharynx in all groups except CP/83 and CQ/95 (Table 3). Immunization with Vector-HA signi®cantly reduced the titers of challenge virus recovered or was associated with lack of virus recovery from both the oropharynx and cloaca in all groups, except CQ/95 and CP/83 from the oropharynx (Table 3). The deduced amino acid sequence similarity for the hemagglutinin protein of vaccine and challenge viruses, and the ability to reduce virus shedding from oropharynx and cloaca were compared statistically between the nine dierent challenge viruses. There was no correlation between hemagglutinin sequence similarity and reduction in virus titers shed from the cloaca (rs=ÿ0.10, P = 0.78), but there was direct correlation between the sequence similarity of the HA and the ability to reduce virus titers shed from the oropharynx (rs=0.78, P = 0.009) (Fig. 2).
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4. Discussion A recombinant fowlpox vaccine with an H5 hemagglutinin gene insert protected chickens against clinical signs and death following challenge by nine dierent highly pathogenic H5 avian in¯uenza viruses, including the highly pathogenic H5N1 in¯uenza virus isolated from a child in Hong Kong during 1997. These avian in¯uenza challenge viruses had 87.3 to 100% deduced hemagglutinin amino acid sequence similarity with the recombinant vaccine, and represented diversely geographic and spatial backgrounds; i.e. isolated from four dierent continents over a 38 year period. This broad-based protection against avian in¯uenza isolates in poultry over multiple years is in contrast to human in¯uenza where vaccines are changed yearly to provide optimal protection in the face of antigenic drift in seasonal ®eld viruses [24] and in horses where vaccine strains have been changed infrequently [25]. The current study demonstrated that the hemagglutinin contained within a single recombinant fowlpox vaccine can provide protection against clinical signs and death following exposure to nine dierent highly pathogenic H5 avian in¯uenza viruses, and frequent changing of the hemagglutinin insert within the recombinant vaccine may not be necessary to provide similar protective eects against prospective ®eld viruses. The prevention of clinical disease and death in immunized chickens challenged by various highly pathogenic avian in¯uenza viruses is well documented [1]. Such protection is dependent on a systemic humoral immune response that prevents hematogenous spread of the virus, replication in critical visceral organs and brain, and death of the individual. However, eective vaccine strategies to control or eradicate avian in¯uenza will require prevention of virus replication in the respiratory and gastrointestinal tracts through speci®c immunity at the mucosal level and the resulting prevention of virus shedding and bird-to-bird transmission [9]. Previous experimental studies have reported prevention of in¯uenza virus infection in immunized chickens varying from complete absence of detectable virus [18] to no inhibition of virus replication in the respiratory tract [9]. The recombinant vaccine prevented or decreased virus detection rates and reduced shedding titers of some challenge viruses [9,15,17], but this reduction was not absolute [9]. Various factors aect the mucosal replication of virus including the antigenic relationship between vaccine and challenge strain of the major protective viral protein, the hemagglutinin. In the current study, we demonstrated reduction of in¯uenza virus replication in respiratory and gastrointestinal tracts of immunized chickens. This reduction had a signi®cant positive correlation between hemagglutinin sequence similarity of challenge virus and vaccine, and the ability to reduce
titers of challenge virus detected in the oropharynx (rs=0.783, P = 0.009). However, there was no similar correlation for reducing cloacal virus titers (rs=ÿ0.100, P = 0.78). Further analysis indicated that reduction in virus replication at the mucosal level in chickens immunized with fowlpox-avian in¯uenza TI/83 hemagglutinin gene recombinant vaccine was most consistent for the Eurasian±African lineage of ®eld viruses. Reductions in virus replication were less consistent for the North American H5 avian in¯uenza challenge viruses, indicating that total sequence similarity of the hemagglutinin may be less important than similarity of speci®c antigenic epitopes in preventing virus replication at the mucosal level. For example, ET/93 had 88.8% hemagglutinin similarity with the vaccine and signi®cantly reduced the shedding of challenge virus from the oropharynx, while CQ/95 had 89.1% similarity, but did not reduce challenge virus shed from the oropharynx as compared to the control fowlpox vaccine groups. A previous study using inactivated H5 avian in¯uenza vaccines demonstrated reductions in mucosal viral replication in immunized chickens, but this did not correlate with hemagglutinin sequence similarities between vaccine and challenge virus [23]. This suggests that factors in addition to hemagglutinin sequence similarity between vaccine and challenge strain impact the ability to reduce mucosal replication in immunized chickens, possibly other genes or speci®c segments of the hemagglutinin gene. From a pragmatic view, previous studies have demonstrated the ability of this recombinant vaccine to prevent horizontal transmission of the Mexican CQ/ 95 avian in¯uenza virus (89.3% hemagglutinin similarity with vaccine) to in contact non-immunized chickens [9]. Furthermore, the current along with previous studies [9,15,17,18] have shown that a single recombinant fowlpox-H5 avian in¯uenza hemagglutinin vaccine can provide protection against a variety of highly pathogenic H5 avian in¯uenza viruses and frequent changing of the hemagglutinin insert in the vaccine to overcome genetic drift in ®eld viruses may not be necessary to prevent transmission of the virus, clinical signs and death under ®eld conditions.
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