Influenza neuraminidase antibodies provide partial protection forchickens against high pathogenic avian influenza infection
Matthew J. Sylte a, Bolyn Hubby b, David L. Suarez a,∗a Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
934 College Station Road, Athens, GA 30605, USAb Alphavax Inc., P.O. Box 110307, Research Triangle Park, NC 27709-0307, USA
Received 29 December 2006; received in revised form 5 February 2007; accepted 7 February 2007Available online 23 February 2007
bstract
Protection of chickens against avian influenza (AI) is mostly attributed to production of antibodies against the viral glycoprotein hemagglu-inin, whereas less is known about the protective role of antibodies to the other surface glycoprotein neuraminidase (NA). Therefore, vaccinesncoding NA antigen (e.g., DNA and alphavirus-based virus like replicon particles (VRP)) or baculovirus-expressed recombinant NA (rN2)ere tested for their ability to protect against highly pathogenic AI (HPAI) in chickens. Vaccination with A/Pheasant/Maryland/4457/93
Ph/MD) rN2 protein produced significantly higher levels of NA-inhibition (NI) activity and 88% protection from HPAI H5N2 challenge thanaccination with Ph/MD N2 DNA (25% protection). Vaccination with Ph/MD N2 VRP a minimum of two times also produced high levelsf NI activity and protection against HPAI challenge (63% protection). Vaccination with VRP encoding an N2 gene that was genetically
istant from the challenge virus N2 failed to protect chickens. Vaccines producing higher levels of NI activity conferred partial protection, butailed to affect viral shedding. Consideration of the homology between vaccine and challenge virus isolate NA genes may provide improvedmmunity if high levels of NI activity are obtained.
2007 Elsevier Ltd. All rights reserved.
sdctiddts(H
eywords: Avian influenza; Neuraminidase; Vaccine
. Introduction
The humoral immune response is vital to protect chick-ns from highly pathogenic avian influenza (HPAI). Therimary protective antibodies are produced against the sur-ace glycoproteins, hemagglutinin (HA). The HA gene isxtremely variable in sequence and 16 defined subtypes haveeen described where by definition antibody to one subtypeill neutralize only virus in the same subtype. Although a
trong antibody response is generated to some of the inter-al proteins, including the nucleoprotein and matrix 1, these
ntibodies are not protective, but are useful for diagnostic pur-oses. The other surface glycoprotein, neuraminidase (NA),lso elicits antibody that can be protective. The amino acid
equence of NA protein is also extremely variable, with nineefined subtypes. The major function of NA protein is toatalyze the release of terminal sialic acid, the receptor forhe virus, from host and viral glycoproteins (e.g., HA). Thiss vital to prevent aggregation of mature influenza virionsuring their release from infected cells [1]. Additional evi-ence suggests that NA may also be involved in viral adhesiono human respiratory epithelial cells [2]. Hemagglutinin-pecific antibodies are known to prevent avian influenzaAI) infection by blocking attachment to host cell receptors.owever, the role of NA antibodies in protection againstI infection is less clear. The balance of humoral immune
esponse to influenza glycoproteins is also skewed towards
A because there is approximately 2.5 times more HA thanA antigen expressed on the surface of influenza virions, andA immunologically out competes NA [3]. Attempts to bal-
nce the humoral immune response by vaccinating with equal
mounts of HA and NA antigen produced approximate anti-A and anti-NA responses [4]. Yet, when purified HA or NAere administered individually, protection against influenza
hallenge in mice was markedly different. For example, miceaccinated with chromatographically purified HA were com-letely protected against influenza morbidity, mortality andulmonary viral shedding, whereas mice vaccinated withurified NA were not protected against infection, but shedignificantly less virus in their lungs [4]. Thus, NA-specificntibodies may produce “permissive immunity” [4], whichunction to limit mortality because they decrease viral releaserom the apical surface of infected cells, but fail to preventiral infection [5].
Numerous studies have used different NA vaccines (e.g.,NA vaccines, chromatographically purified protein, bac-lovirus expressed recombinant protein, reassorted killedirus and others) to examine the role of NA antibodies inrotection against influenza in mice or ferrets, yet much lesss known of their contribution in chickens. Rott et al. wererst to demonstrate a protective role of NA antibodies inhickens infected with AI [6]. McNulty et al. demonstratedhat administration of killed H7N1 virus protected against5N1-associated mortality, but failed to completely protect
gainst clinical disease because most vaccinated birds devel-ped conjunctivitis post-challenge [7]. Webster et al. reportedroduction of serum neuraminidase inhibition (NI) activityn chickens after vaccinating with inactivated virus, vacciniairus encoding NA or purified NA. Vaccination with purifiedA or killed virus completely protected against homologoushallenge (H5N2), but did not protect against heterologoushallenge (H7N7) [8]. Qiao et al. recently demonstratedroduction of NA-specific antibodies in chickens followingdministration of recombinant fowl-pox virus encoding H5nd N1, which were completely protected against H5N1 chal-enge [9]. However, an NA-only control was not included inhis study, which makes it difficult to determine a role ofA-antibodies using this vaccine.
Vaccines engineered to express high levels of NA inivo are needed to better understand the role of NI activ-ty in protection against AI. Recent advances in geneticaccines (e.g., eukaryotic expression plasmid DNA and vec-ored replication-deficient viruses) have made it possibleo specifically characterize the role of antibodies targetedo specific antigens. Although plasmid DNA vaccines areasy to produce in large quantities, major disadvantagesnclude lack of ability to mass administer and the neces-ity to administer with transfection agents or other adjuvantso enhance transgene expression in vivo. Using a needlend syringe, intramuscular injection of large quantities ofNA are required (e.g., 50–200 �g per vaccine) to inducehumoral immune response, yet it is estimated that less
han 0.1% of plasmid DNA is transfected into cells. In
ontrast, replication-deficient viruses (e.g., adenovirus, retro-irus, alphavirus and others) transduce a broad range of hostells for enhanced expression of vaccine genes, and have alsoeen safely used for gene therapy and vaccine delivery in a
ecTo
(2007) 3763–3772
ariety of species, including chickens [10,11]. The alphavirusenus consists of 24 different viruses, including Venezuelanquine encephalitis virus (VEE) [12]. Alphavirus-based virusike replicon particles (VRP) vaccines are engineered withhe foreign target gene replacing structural genes, whereasour non-structural genes are left intact at the 5′ end of theenome to allow for robust transcription and translation of thearget gene [13,14]. Although production of VRP vaccinesequire complex transfections, they have the distinct advan-age of higher rates of target gene expression in vivo [15].ased on the fundamental differences between DNA, VRPnd recombinant protein vaccines, we sought to determinehether different types of vaccines could produce protective
evels of NI activity to protect against HPAI challenge.
. Materials and methods
.1. Animals
Two week old white leghorn chickens from the South-ast Poultry Research Laboratory (SEPRL) specific pathogenree (SPF) flocks were vaccinated and housed for 6 weeks in aiosafety level 2 animal facility, and were moved to biosafetyevel 3 Ag facility for challenge with a highly pathogenic AIHPAI) virus. Care was provided as required by the Institu-ional Animal Care and Use Committee.
.2. Viruses
Stock cultures of HPAI A/chicken/Queretaro/14588-9/95 (Ck/Queretaro; H5N2), passaged in chickens beforerchiving [16], low pathogenic A/chicken/Pennsylvania/3609/93 (Ck/PA; H5N2), low pathogenic A/turkey/Ohio/13053/04 (Tk/OH; H3N2) and A/pheasant/Maryland/457/93 (Ph/MD; H5N2) were maintained at −70 ◦C in theirus repository at SEPRL. The egg infectious dose 50%EID50) of the challenge inocula was calculated using theethod of Reed and Muench [17]. Briefly, day 10 embry-
nating chicken eggs (ECE) were infected with 100 �L of0-fold dilutions in brain heart infusion medium (BHI; Difco,etroit, MI) from stock virus. Eggs candled daily for 3 days
o detect deaths, and were chilled overnight at 4 ◦C beforearvesting amnioallantoic fluid (AAF) and testing for hemag-lutination. Incubation of chicken RBCs with control serumr PBS served as negative controls.
.3. Alphavirus vaccination
Groups of 8 white leghorn chickens at 2 weeks of age wereaccinated subcutaneously in the neck every 2 weeks with07 VEE VRP (Alphavax, Inc., Research Triangle Park, NC)
ncoding the N2 gene from either Ph/MD or Tk/OH. Thosehickens receiving three doses of VRP encoding Ph/MD ork/OH N2 were administered at 2, 4 and 6 weeks of age, andthers vaccinated with two doses of VRP encoding Ph/MD
2 were administered at 2 and 4 weeks of age and PBS atweeks of age. To determine if priming the immune systemith Ph/MD N2 expressed by VRP or DNA vaccine affectedroduction of NI activity, chickens were vaccinated at 2eeks of age in the pectoral muscle with 100 �g of low endo-
oxin (≤100 EU/mg DNA) plasmid (Aldevron; Fargo, ND)n pCI vector (Promega, Madison, WI) with lipofectin trans-ection reagent (Invitrogen, Carlsbad, CA) [18] or 107 VRPncoding Ph/MD N2 administered subcutaneously. After-ards, chickens were boostered twice at 4 and 6 weeks of
ge with 107 VRP encoding Ph/MD N2 or 100 �g of plasmidNA encoding Ph/MD N2, respectively. Control chickens
eceived 100 �L of sterile PBS subcutaneously administeredt 2, 4 and 6 weeks of age. Chickens were bled via the brachialein 2 weeks after each vaccination. Serum was harvested andtored at −20 ◦C prior to analysis. Chickens were challengedith HPAI at 8 weeks of age, as described below.
.4. Baculovirus and DNA vaccination
Recombinant N2 protein (rN2) was prepared by infect-ng Sf9 insect cells with baculovirus encoding the Ph/MD2 gene. Four days after infection the rN2 was harvested
rom Sf9 cells by DEAE column chromatography. Fractionsith high levels of NA activity were pooled and prepared
nto an oil and water emulsion vaccine [19]. Groups of 6–8hite leghorn chickens at 2 weeks of age were vaccinated
ubcutaneously in the neck twice at 3-week intervals with80 �g/0.5 mL of partially purified Ph/MD rN2. Other groupsf 2-week old chickens were vaccinated twice at 3-week inter-als in the pectoral muscle with 150 �g of low endotoxin≤100 EU/mg DNA) plasmid encoding Ph/MD or Tk/OH N2n pCI vector conjugated with lipofectin transfection reagent,s described above. Control chickens received 100 �L of ster-le PBS subcutaneously administered at 2 and 5 weeks of age.hickens were bled via the brachial vein 3 weeks after eachaccination. Serum was harvested and stored at −20 ◦C prioro analysis. Chickens were challenged with HPAI at 8 weeksf age, as described below.
.5. Challenge and determination of mortality and viralhedding
When the chickens were 8 weeks old, they were inocu-ated intranasally via the choanal slit with 106 EID50/0.2 mLf highly pathogenic A/chicken/Queretaro/14588-19/95H5N2) [16]. Chickens were observed daily for morbiditynd mortality for up to 15 days post-infection. Mortality dataere analyzed and presented as Kaplan–Meier survival plots.ropharyngeal, tracheal or cloacal swabs were sampled from
nfected birds at days 2–7 post-infection, suspended in 1 mLf BHI broth, and stored at −70 ◦C. Total RNA was extracted
y using an RNeasy mini kit (Qiagen), and quantitative real-ime RT-PCR was performed with primers and probe specificor type A AI virus matrix RNA [20]. Qiagen one-step RT-CR kit was used under the following conditions: 1 �L of
cwd
(2007) 3763–3772 3765
nzyme mixture including RT and Taq polymerase, 10 pmolf each primer, 0.12 �M probe, 320 �M (each) deoxynucleo-ide triphosphate, 3.75 mM MgCl2, 13 U of RNase inhibitor,× reaction buffer, 8 �L of total RNA per sample and nucle-se free water for a total quantity of 25 �L. The RT reactionas performed for 30 min at 50 ◦C, and 15 min at 94 ◦C to
ctivate the Taq polymerase. The following PCR cycling pro-ocol was used; 45 cycles of denaturation at 94 ◦C for 1 s andnnealing at 60 ◦C for 20 s with fluorescence data acquiredt the end of each annealing step using a Smart Cycler IICepheid Sunnyvale, CA) real-time PCR machine. In somexperiments, relative amounts of viral shedding were deter-ined by cycle threshold (Ct) values. In other experiments,
uantitative viral shedding (e.g., number of copies of viralNA recovered from swab/mL of BHI) was interpolated from
he sample cycle thresholds by using a standard curve gen-rated from known amounts of control Ck/Queretaro RNAe.g., titers were exactly 101–106 EID50/mL).
A fluorescent substrate 2′-(4-methylumbelliferyl)-�-d-N-cetylneuraminic acid sodium salt hydrate (MUN; Sigmahemical Co., St. Louis, MO) was used to measure the NAnzymatic activity [21]. Briefly, 25 �L of Ck/PA or Tk/OHtock virus were diluted 2-fold in 25 �L of calcium salineuffer (CaS; 20 mM sodium borate, 7 mM calcium chloride,54 mM sodium chloride, 12.5 mM sodium acetate; pH 7.2)n 96-well, clear, flat-bottom plates, and 15 �L of 0.5 mM
UN was added per well. Uninfected allantoic fluid or MUNlone served as negative controls. The kinetic production ofuorescent byproduct 4-methylumbelliferone was assayedvery 3 min, for a total of 30 min using a Synergy HT Multi-etection microplate reader (BioTek, Winooski, VT) with
n excitation and emission wavelength of 360 and 440 nm,espectively. The linear range of NA activity was determinedy plotting fluorescence data from 15 min incubation ver-us the inverse of viral dilution (1/x, where x = log2 dilutionactor). Twenty-five microliters of virus at the lowest end ofhe linear range of fluorescence was added to each well, and5 �L of chicken serum was diluted 2-fold to produce a rangef 1:2 to 1:4096. Serum and virus were incubated for 30 mint room temperature. Fifteen microliters of 0.5 mM MUNas added to each well, and fluorescence was immediately
nalyzed, as described above. Samples with little to no flu-rescence were determined to be positive for NI activity, asompared to control chicken serum. The NI titer was deter-ined as one log2 dilution below which fluorescence began
o achieve levels similar to that of SPF control serum.
.7. Sequence analysis
Amino acid percent identity of different NA antigens wasalculated using Megalign v5.08 (DNA Star, Madison, WI)ith Clustal W algorithm. Accession numbers and sequenceata for NA genes are available upon request.
Data were analyzed using Prism v4.03 Software pack-ge (GraphPad Software Inc., San Diego, CA), and arexpressed as mean ± standard deviation from chickens perxperimental group. The Log Rank test was used to analyzeaplan–Meier survival rate data, and one way analysis ofariance (ANOVA) with Tukey–Kramer post-test were usedo analyze NI and viral shedding data. Statistical significanceas set at P < 0.05.
. Results
.1. Neuraminidase inhibition activity produced by rN2,NA or VRP vaccination
To test the hypothesis that different types of vaccines affectroduction of NI activity, chickens were initially vaccinatedwice with either 980 �g of baculovirus expressed Ph/MDN2 protein or 150 �g of Ph/MD N2 DNA. Vaccination withN2 or DNA produced significant levels of NI activity againstk/PA virus after each vaccination compared to control sera
P < 0.01; Table 1). Because of the high degree of amino acid
dentity between Ck/PA and the challenge virus Ck/Queretaro2 protein (97.7%), and Ck/PA is a low pathogenic AI iso-
ate that can be handled in biosafety level 2 facilities, Ck/PAirus was selected as the antigen for NI assays to evaluate the
(tNe
able 1ummary of baculovirus expressed recombinant Ph/MD N2 protein and Ph/MD N2
NI titer (log2) 3 weeksafter 1st vaccine
NI titer (log2) 3 weeks after2nd vaccine (pre-challenge)
ontrol 1.5 ± 0.84a† 1.83 ± 0.75a
h/MD DNA 3.38 ± 1.30b 4 ± 1.52b
h/MD rN2 4.25 ± 0.71b 8.5 ± 1.39c
era from control or vaccinated birds were tested for NI activity against Ck/PA virusI activity. Tracheal swabs were tested for viral shedding, as described in Sectiondays post-infection (p.i.). No statistical significant changes in viral shedding were
hickens vaccinated twice with 150 �g of Ph/MD N2 DNA vaccine; and Ph/MDxpressed recombinant Ph/MD N2 protein.)† NI titer values in each column denoted by different letters (a–c) are statistically
able 2ummary of Ph/MD or Tk/OH DNA vaccination in chickens challenged with HPA
NI titer (log2) 3 weeks after 2ndvaccine (pre-challenge) Ck/PAantigen
NI titer (log2) 3 weeks afvaccine (pre-challenge) Tantigen
ontrol 1.38 ± 0.74a† 1.63 ± 0.74a
h/MD DNA 2.36 ± 0.76b 4.72 ± 1.21b
k/OH DNA 1.36 ± 0.56a 6.92 ± 1.38c
era from control or vaccinated birds were tested for NI activity against Ck/PA oreviation log2 NI activity. Tracheal swabs were tested for viral shedding, as desurviving chickens 3 days post-infection (p.i.). No statistically significant changesBS; Ph/MD DNA: chickens vaccinated twice with 150 �g of Ph/MD N2 DNA va2 DNA vaccine).† NI titer values in each column denoted by different letters (a–c) are statistically* Control chickens all died before sampling at 3 days p.i.
(2007) 3763–3772
mmune response towards Ck/Queretaro N2. Next, the abili-ies of two distant N2 antigens (e.g., Ph/MD and Tk/OH) toroduce NI activity against CK/PA virus were tested. Aminocid sequence similarity between Ph/MD and Tk/OH N2emonstrated a distant relation (85% identical). Chickensere vaccinated intramuscular twice at 3-week intervals with50 �g of either Ph/MD or Tk/OH N2 DNA. Three weeksfter the second DNA vaccine, NI activity was comparedgainst Ck/PA and TK/OH viruses. Vaccination with Ph/MDNA produced a significant increase (P < 0.01) in NI activ-
ty, using CK/PA virus as antigen, as compared to sera fromontrol chickens or chickens vaccinated with Tk/OH DNATable 2). Comparison of amino acids between Ph/MD andk/PA N2 showed 95.9% identity, whereas Tk/OH was dis-
antly related (84.3% identical) to Ck/PA N2. To exclude theossibility that NI activity may differ when using Ck/PA orh/MD virus as antigen (amino acid identity is 95.9%), serumI was tested using Ph/MD virus, and no significant differ-
nce was detected (data not shown). The specificity of NIctivity produced after vaccination was compared by usingk/OH virus as antigen. Vaccination with Tk/OH DNA pro-uced significantly enhanced levels of NI activity againstk/OH virus (P < 0.001), as compared to sera from con-
rols or chickens vaccinated with DNA encoding Ph/MD N2
Table 2). Lastly, chickens were vaccinated with combina-ions of VRP or DNA vaccines encoding Ph/MD or Tk/OH2. Chickens vaccinated two or three times with 1 × 107 VRP
ncoding Ph/MD N2 produced significantly elevated NI titers
DNA vaccination in chickens challenged with HPAI virus
, as described in Section 2. Values represent mean ± standard deviation log2
2. Values represent mean ± standard deviation Ct from surviving chickensdetermined (Control: chickens vaccinated twice with PBS; Ph/MD DNA:rN2: chickens vaccinated twice with 980 �g/0.5 mL Ph/MD baculovirus
different from each other (P < 0.05) as determined by ANOVA.
Tk/OH virus, as described in Section 2. Values represent mean ± standardcribed in Section 2. Values represent mean ± standard deviation Ct fromin viral shedding were detected (Control: chickens vaccinated twice with
ccine; and Tk/OH DNA: chickens vaccinated twice with 150 �g of Tk/OH
different from each other (P < 0.05) as determined by ANOVA.
M.J. Sylte et al. / Vaccine 25 (2007) 3763–3772 3767
Fig. 1. Temporal response of serum neuraminidase inhibition (NI) activity after vaccination with VRP encoding Ph/MD or Tk/OH N2. Serum was collected2 weeks after scheduled vaccinations with Ph/MD (panels A and B: NI after 1st and 2nd vaccination, and C and D after 3rd vaccination) (PBS: PBS mockvaccinated three times; Ph/MD 3×: Ph/MD N2 VRP three times; Ph/MD 2×: Ph/MD N2 VRP two times; Ph/MD 1×DNA 2×: Ph/MD N2 VRP one time andPh/MD N2 DNA two times; DNA 1× Ph/MD 2×: Ph/MD N2 DNA one time and Ph/MD N2 VRP two times; and Tk/OH 3×: Tk/OH N2 VRP three times).S us as and in duplc
(aiTeafiogNiiNTn5td3c3
rNo
3
cwCbpFWca(
erum NI activity was determined using Ck/PA (panels A–C and Tk/OH vireviation log2 NI titer of eight chickens per vaccination group, performedompared to PBS vaccinated controls.
P < 0.01) against Ck/PA virus as antigen as early as 2 weeksfter the 2nd vaccination, as compared to chickens receiv-ng PBS control or VRP encoding Tk/OH N2 (Fig. 1A–C).here was no significant change in NI titer whether the chick-ns were primed with DNA or VRP encoding Ph/MD N2,nd boostered with VRP or DNA, respectively. The speci-city of NI activity produced by administration of Ph/MDr Tk/OH VRP was confirmed using Tk/OH virus as anti-en. Only chickens vaccinated with VRP encoding Tk/OH2 showed a significant elevation in Tk/OH virus NI activ-
ty (P < 0.001) (Fig. 1D). Thus, there is specificity to themmune response to VRP or DNA plasmid encoding different2 antigens as is seen with vaccines with HA protein [18].o determine whether production of NI was dependent on theumber of VRP administered, chickens were vaccinated with× 106 VRP encoding Ph/MD N2 every 2 weeks for a total of
uced significantly lower (P < 0.01) log2 NI activity after therd vaccination (4.00 ± 1.21), as compared to chickens vac-inated three times with 107 Ph/MD VRP (NI activity afterrd vaccination = 7.63 ± 2.5). Overall, baculovirus expressed
tcew
tigen (panel D), as described in Section 2. Data represent mean ± standardicate. Significant changes (*P < 0.01) in log2 NI activity were detected, as
N2 and VRP encoding N2 produced the greatest increase inI activity, whereas the DNA vaccines produced lower levelsf antibody.
.2. Morbidity and mortality after viral challenge
Comparison of amino acids between vaccine Ph/MD andhallenge virus Ck/Queretaro N2 showed 97.7% identity,hereas Tk/OH N2 was 85% identical to Ck/Queretaro.hickens vaccinated twice with 980 �g of partially purifiedaculovirus expressed Ph/MD rN2 protein were significantlyrotected against HPAI challenge (P < 0.01; Table 1 andig. 2A), and survivors showed mild signs of clinical disease.hen compared to baculovirus expressed rN2 protein, vac-
ination with Ph/MD N2 DNA failed to significantly protectgainst HPAI challenge or prevent clinical signs of diseaseFig. 2A), but did increase the mean time to death as compared
o control chickens (Table 1). Similarly, there was no signifi-ant change in survival rate between chickens vaccinated withither Ph/MD or Tk/OH DNA, although their survival ratesere significantly different in comparison to control chick-
3768 M.J. Sylte et al. / Vaccine 25 (2007) 3763–3772
Fig. 2. Comparison of NA vaccines and survival against HPAI challenge. Chickens were vaccinated as described in Section 2. At 8 weeks of age, they wereintranasally challenged via the choanal slit with 100 �L of PBS containing 106 EID50 of high pathogenic A/Ch/Queretaro/14588-19/95 (H5N2). Mortality wasassessed daily for up to 15 days post-infection. Data in panel A (PBS: mock vaccinated twice with PBS; Ph/MD DNA: Ph/MD DNA vaccinated twice; andPh/MD rN2: vaccinated twice with baculovirus expressed recombinant Ph/MD N2 protein), panel B (PBS: mock vaccinated twice with PBS; Ph/MD DNA:vaccinated twice with Ph/MD DNA; and Tk/OH DNA: vaccinated twice with TK/OH DNA), panels C and D (PBS: mock vaccinated three times with PBS;Ph/MD 3×: vaccinated three times with Ph/MD VRP; Ph/MD 2×: vaccinated twice with Ph/MD VRP; Ph/MD 1× DNA 2×: vaccinated once with Ph/MDV with Pht llenge. V(
ewtTw(wnccptacicsvwapoaeovV
iatcs
3
dwAacePNpstdsD
RP and twice with Ph/MD DNA; DNA 1× Ph/MD 2×: vaccinated onceimes with Tk/OH VRP) represent percent of chickens surviving HP AI chaP < 0.01) as determined by Log Rank test.
ns (P < 0.001) (Fig. 2B). Similar to Fig. 2A, vaccinationith either Ph/MD or Tk/OH N2 DNA increased the mean
ime to death in comparison to control chickens (Table 2).he survival rates for chickens vaccinated two or three timesith VRP encoding Ph/MD N2 were significantly increased
P < 0.001) (Fig. 2C), as compared to chickens vaccinatedith PBS or VRP encoding Tk/OH N2. Chickens vacci-ated with VRP encoding Tk/OH N2 showed a significanthange in survival rate (P = 0.0013) as compared to controlhickens. None of the VRP vaccine strategies provided 100%rotection, but the mean time to death for birds vaccinatedwo or three times with VRP encoding Ph/MD N2 was 4.8nd 5 days, respectively, compared to 2.9 days for controlhickens. Likewise, chickens vaccinated with VRP encod-ng Ph/MD N2 which survived infection showed only mildlinical signs of disease such as conjunctivitis, and did nothow the severe clinical signs seen in PBS or Tk/OH VRPaccinated birds which included edematous and cyanoticattles and comb, severe conjunctivitis, depression, lethargy
nd subcutaneous hemorrhage. The survival rate of chickensrimed with Ph/MD DNA or VRP, and boostered with VRPr DNA, respectively, was significantly elevated (P = 0.0043nd 0.0017, respectively) in comparison to control chick-
ns (Fig. 2D), and an increase in the mean death time wasbserved (e.g., 4.4 and 4.9 days, respectively). However, sur-ival rates from chickens vaccinated with combinations ofRP and DNA vaccines encoding Ph/MD N2 were signif-
cndd
/MD DNA and twice with Ph/MD VRP; and Tk/OH 3×: vaccinated threealues denoted by different letters are statistically different from each other
cantly less (P < 0.05) than chickens vaccinated three timeslone with VRP encoding Ph/MD N2. These data indicatehat vaccines which produced the highest levels of NI activitylosely related to the challenge virus significantly enhancedurvival from HPAI.
.3. Viral shedding
The effect of different NA vaccines on viral RNA shed-ing from the oropharynx or trachea of infected chickensas determined by real time RT-PCR for AI matrix RNA.lthough different vaccines affected production of NI activity
nd survival from HPAI infection, there were no significanthanges in tracheal viral RNA shedding Ct values from chick-ns vaccinated with Ph/MD rN2 protein or DNA encodingh/MD N2 (Table 1), or comparing Ph/MD versus TK/OH2 DNA (Table 2). Similarly, vaccination with Ph/MD rN2rotein or DNA encoding Ph/MD N2 had no effect on cloacalhedding of viral RNA; Ct values (mean ± standard devia-ion) 35.79 ± 4.58 and 36.14 ± 3.31, respectively. Significantifferences in viral RNA shedding from the oropharynx wereeen in chickens vaccinated with combinations of VRP orNA encoding Ph/MD N2 (P < 0.05) 2 days post-infection,
ompared to control chickens (Fig. 3A). However, there wereo significant changes in oropharyngeal viral RNA sheddingetected from survivors between different vaccine groups onays 5 and 7 post-infection (Fig. 3B and C).
M.J. Sylte et al. / Vaccine 25 (2007) 3763–3772 3769
Fig. 3. N2 vaccines significantly reduce oropharyngeal viral RNA shedding following HPAI challenge. Chickens were vaccinated as described in Section 2(PBS: mock vaccinated three times with PBS; Ph/MD 3×: vaccinated three times with Ph/MD VRP; Ph/MD 2×: vaccinated twice with Ph/MD VRP; Ph/MD1× DNA 2×: vaccinated once with Ph/MD VRP and twice with Ph/MD DNA; DNA 1× Ph/MD 2×: vaccinated once with Ph/MD DNA and twice with Ph/MDVRP; and Tk/OH 3×: vaccinated three times with Tk/OH VRP). At 8 weeks of age, they were intranasally challenged via the choanal slit with 106 EID50/0.2 mLof high pathogenic A/Ch/Queretaro/14588-19/95 (H5N2). Quantitative viral shedding from the oropharynx was assayed on days 2, 5 and 7 post-infection (p.i.)using real time RT-PCR, as described in Section 2. Bars in panels A–C represent geometric mean and dots represent log10 values of viral RNA shed froms * .05) rec samples
4
iNPNClHyPtd
sAtUltiaVa
urvivors at days 2, 5 and 7 post-infection. All vaccines significantly ( P < 0hickens (ND: none detected; all control birds had died before taking these
. Discussion
The immune response to NA can provide protectionn chickens against HPAI challenge when high levels ofI activity are produced. Vaccination with VRP encodingh/MD N2 or baculovirus expressed recombinant Ph/MD2 protein produced the highest levels of NI activity againstk/PA virus, whereas vaccination with N2 DNA produced
ess NI activity. The former were partially protected againstPAI challenge, and the opposite was true for the latter. Anal-
sis of the N2 vaccine antigens used in the present study,h/MD and Tk/OH, demonstrated 85% amino acid iden-
ity. The inclusion of vaccines encoding TK/OH N2 was toetermine whether a distantly related NA gene of the same
lhfT
duced day 2 p.i. oropharyngeal viral shedding, as compared to PBS treated).
ubtype could protect against heterologous HPAI challenge.recent isolate from turkeys, the origin of this virus is likely
he triple reassortant swine viruses commonly found in thenited States [22]. The swine H3N2 viruses that have circu-
ated in the United States since the late 1990s are thoughto have obtained their HA and NA genes from a humannfluenza virus and the internal genes are of swine, humannd avian influenza origin [23]. Vaccinating chickens withRP encoding Tk/OH N2 produced modest increases in NI
ctivity when using Ck/PA as antigen, but significantly higher
evels of NI activity against the Tk/OH antigen. In spite of theigh levels of NI antibody to Tk/OH, they were not protectedrom HPAI challenge. The poor protection from heterologousk/OH antigen was unexpected since it has been reported that
ice were protected using DNA vaccines encoding NA thathared 89% amino acid sequence similarity compared to theethal influenza challenge [24]. In the same study, protectionas not obtained with less stringently matched vaccine (N1)
nd challenge (N2) virus (e.g., 42% amino acid identity) [3].Mortality results from the present study are consistent
ith those obtained previously in chickens, where vaccina-ion with NA similar to the challenge virus protected against
ortality, but not morbidity [7,8]. Results indicate that vac-ination with Tk/OH N2 does not induce high enough levelsf protective NI activity because of the sequence differenceo the challenge virus. It appears that NI activity produced byaccines should be closely related to the challenge strain NA,ven within the same subtype, to protect against HPAI infec-ion. The level of sequence similarity needed for protectionetween vaccine and challenge strains was not determined inhese studies, but levels of NI activity produced also need toe considered for prediction of protection.
This is the first report describing quantitation of post-accination NI activity using MUN as a substrate. The testrovides rapid, sensitive and reproducible quantitative NIata to evaluate immune response against NA vaccine anti-ens, and may serve to differentiate infected from vaccinatednimals. Vaccination with VRP encoding Ph/MD N2 orh/MD recombinant baculovirus expressed N2 protein wereuperior to DNA vaccines with respect to NI production, andevels of NI activity correlated with protection against HPAIhallenge. This study highlights the need for high levels of NIctivity to provide clinical protection. The most commonlysed vaccine in the poultry industry, adjuvanted killed wholeirus, produce NI activity, but NA is present in much loweruantities than HA. Thus, these vaccines likely result in lit-le protection from the NA component of the vaccine to thehallenge strain. This was previously reported using killed AIaccines generated by reverse genetics that differ only in theA subtype, which demonstrated there was no effect on virus
hedding regardless of the vaccine NA subtype present in theaccine [25]. Supplementation with exogenous NA may besolution to a poor immune response towards vaccine NA,hich has been reported in mice [26].In our studies, baculovirus expressed recombinant N2
rotein produced the greatest level of protection, but alsoequired a large quantity of partially purified protein (e.g.,80 �g). Administration of 72% less (273 �g/0.5 mL) of par-ially purified Ph/MD rN2 produced low levels of NI activity2.13 ± 0.64) levels, albeit significantly elevated above con-rol chickens (0.38 ± 0.74), and failed to significantly protect2/8) against HPAI challenge. These results indicate that aarge quantity of recombinant protein may be required to pro-ect, but the minimum amount necessary to provide protections not known.
This is the first investigation into the protective effect
f VRP encoding NA on HPAI infection in chickens. TheRP vaccine transduces a diverse number of cells in vitro,hich results in a single round of robust transgene tran-
cription [15]. Although we did not determine which cells
teft
(2007) 3763–3772
ere expressing VRP-transduced N2 protein after subcu-aneous administration in vivo, NI activity produced afterhe 1st vaccination suggest that antigen presenting cells, inart, may have been transduced. Vaccination of chickenswo or three times with VRP encoding Ph/MD N2 signifi-antly elevated pre-challenge NI activity as early as after thend vaccination, which further increased after the 3rd vac-ine (Fig. 1C). Seventy-five and 80% of chickens that wereaccinated two or three times, respectively, with VRP encod-ng Ph/MD N2 that survived HPAI challenge had NI titers7 log2. Although a protective NI titer is not clearly identified
n the present study, results indicate that NA vaccines shouldtrive to produce NI levels to the challenge antigen of ≥7 log2o consistently achieve clinical protection. The duration ofrotective NI activity produced in response to VRP vacci-ation was not directly examined. A significant elevation inI activity was detected 4 weeks after the last vaccination in
hickens administered VRP encoding Ph/MD N2 two timesFig. 1C). The production of NI was dependent on the num-er of VRP administered in the vaccine. A 50% reduction inRP encoding Ph/MD N2 produced significantly decreasedI titers (P < 0.01). Similarly, serum HI activity was lower
nd AI mortality increased for chickens vaccinated in ovond boostered with 90% fewer VRP encoding H5 [10].
The results of serum NI response and mortality after vac-ination with DNA encoding Ph/MD N2 were unexpected.accination every 2 or 3 weeks may not have been optimal
or DNA vaccines used in the present studies, since monthlyaccinations are more commonly used for HA DNA vaccines18]. It is not clear why pre-challenge NI titers from chick-ns vaccinated with Ph/MD N2 DNA with Ck/PA virus asntigen were not comparable to those obtained with Tk/OH2 DNA with Tk/OH virus as antigen (e.g., 2.36 ± 0.76
ersus 6.92 ± 1.38, respectively). In previous experiments,riming with DNA encoding various HAs produced modestncreases in HI activity, but boostering twice produced theighest levels of HI activity [18]. Similarly, a single dosef VRP encoding H5 was partially protective against HPAIhallenge, but complete protection and increased HI activityas produced after boostering [10]. Based on these findings,e hypothesized that priming and boostering with combina-
ions of DNA or VRP would affect NI production, but weetected no significantly change in NI activity or mortality.verall, these results indicate that plasmid DNA failed toroduce high enough levels of NI activity to protect againstPAI challenge, and that mixing DNA and VRP were notseful to produce a protective immune response.
The use of the VEE vector in chickens has the advantagever other potential viral vectored vaccines like Newcastleisease virus because chickens are unlikely to be naturallyxposed to this virus. A concern is that multiple vaccina-ions with this vector might generate an immune response
o the vector, and result in immune-interference and poorxpression of the transgene. Although we did not test serumor humoral response to VEE, serum NI levels continuedo increase following each of three administrations. These
esults suggest that immunity was not directly generatedgainst VRP vaccine, and they can be safely administeredo chicken multiple times. Additionally, there is little evi-ence of immune interference from preexisting immunity inumans to VRP, and have been shown to be effective forumans in the face of anti-vector responses (Personal com-unication with B. Hubby, Alphavax Inc.). Mass vaccination
f chickens in ovo is a well established technique to protecthickens against Marek’s disease virus. Likewise, in ovo vac-ination with VRP encoding H5 resulted in partial protectiongainst AI challenge [10]. Therefore, it may be possible toaccinate in ovo with VRP encoding NA alone or in combina-ion with recombinant NA protein to protect chickens againstmerging AI isolates.
The effector mechanism of NI activity responsible for pro-ecting chickens from AI is not clear, but it is clearly differentrom antibodies to the HA protein. One likely mechanisms the NI activity keeps AI trapped on the apical surface ofnfected cells [5], where they may be susceptible to cyto-oxic T lymphocyte (CTL)-mediated killing. Although CTLseadily kill influenza infected cells in the respiratory tractf mice and humans [27,28], their contribution is unknownn chickens. This possibility is strengthened by reports thatRP vaccines encoding tumor antigens to produce specificTL responses [29,30]. Serum NI activity may also activatelassical complement pathway and cause disruption of viralnvelope, and affect infectivity [31]. Finally, the NI activityay help aid viral clearance by an opsonin effect [32].In summary, the immune response to NA can protect
hickens against HPAI challenge when high levels of NIctivity are produced. Killed whole virus vaccines are com-only used for chickens, but do not likely produce NI activity
t high enough levels against NA protein [33–35] to enhanceaccine efficacy. This study, like earlier studies in mice, pro-ides evidence that if vaccines can produce levels of NIctivity over 7 log2, protection against HPAI challenge cane achieved with just NA antigen. New technology like theRP can produce effective levels of NI activity in chickens,
nd additional studies to provide practical recommendationsor inclusion of NA in vaccines are warranted.
cknowledgements
We thank Suzanne DeBlois, Joan Beck, Ronald Grahamnd Roger Brock at SEPRL for their technical assistance and
hitney Ellis at Alphavax Inc. for preparation of VRP. Men-ion of trade names or commercial products is solely for theurpose of providing specific information, and does not implyecommendation or endorsement by the U.S. Department ofgriculture.
eferences
[1] Seto JT, Rott R. Functional significance of sialidose during influenzavirus multiplication. Virology 1966;30(4):731–7.
[
(2007) 3763–3772 3771
[2] Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD.Neuraminidase is important for the initiation of influenza virus infectionin human airway epithelium. J Virol 2004;78(22):12665–7.
[3] Johansson BE, Moran TM, Bona CA, Popple SW, Kilbourne ED.Immunologic response to influenza virus neuraminidase is influ-enced by prior experience with the associated viral hemagglutinin. II.Sequential infection of mice simulates human experience. J Immunol1987;139(6):2010–4.
[4] Johansson BE, Bucher DJ, Kilbourne ED. Purified influenza virushemagglutinin and neuraminidase are equivalent in stimulation of anti-body response but induce contrasting types of immunity to infection. JVirol 1989;63(3):1239–46.
[5] Kilbourne ED, Laver WG, Schulman JL, Webster RG. Antiviral activ-ity of antiserum specific for an influenza virus neuraminidase. J Virol1968;2(4):281–8.
[6] Rott R, Becht H, Orlich M. The significance of influenza virus neu-raminidase in immunity. J Gen Virol 1974;22(1):35–41.
[7] McNulty M, Allan G, McKracken R. Efficacy of avian influenzaneuraminidase-specific vaccines in chickens. Avian Pathol 1986;15(1):107–15.
[9] Qiao CL, Yu KZ, Jiang YP, Jia YQ, Tian GB, Liu M, et al. Protection ofchickens against highly lethal H5N1 and H7N1 avian influenza viruseswith a recombinant fowlpox virus co-expressing H5 haemagglutininand N1 neuraminidase genes. Avian Pathol 2003;32(1):25–32.
10] Schultz-Cherry S, Dybing JK, Davis NL, Williamson C, SuarezDL, Johnston R, et al. Influenza virus (A/HK/156/97) hemagglu-tinin expressed by an alphavirus replicon system protects chickensagainst lethal infection with Hong Kong-origin H5N1 viruses. Virology2000;278(1):55–9.
11] Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, etal. Protection of mice and poultry from lethal H5N1 avian influenzavirus through adenovirus-based immunization. J Virol 2006;80(4):1959–64.
12] Tubulekas I, Berglund P, Fleeton M, Liljestrom P. Alphavirus expres-sion vectors and their use as recombinant vaccines: a minireview. Gene1997;190(1):191–5.
13] Pushko P, Parker M, Ludwig GV, Davis NL, Johnston RE, SmithJF. Replicon-helper systems from attenuated Venezuelan equineencephalitis virus: expression of heterologous genes in vitro andimmunization against heterologous pathogens in vivo. Virology 1997;239(2):389–401.
14] Frolov I, Hoffman TA, Pragai BM, Dryga SA, Huang HV, Schlesinger S,et al. Alphavirus-based expression vectors: strategies and applications.Proc Natl Acad Sci USA 1996;93(21):11371–7.
15] Lundstrom K. Virus based vectors for gene expression in mammaliancells: Semliki forest virus. Elsevier Sciences; 2003.
16] Garcia A, Johnson H, Srivastava DK, Jayawardene DA, Wehr DR, Web-ster RG. Efficacy of inactivated H5N2 influenza vaccines against lethalA/Chicken/Queretaro/19/95 infection. Avian Dis 1998;42(2):248–56.
17] Swayne DE, Senne DA, Beard CW. A laboratory manual for theisolation and identification of avian pathogens. Kennett Square, PA:American Association of Avian Pathologists; 1998. p. 150–5.
18] Lee CW, Senne DA, Suarez DL. Development of hemagglutininsubtype-specific reference antisera by DNA vaccination of chickens.Avian Dis 2003;47(3 Suppl.):1051–6.
19] Stone HD. Newcastle disease oil emulsion vaccines prepared with ani-mal, vegetable, and synthetic oils. Avian Dis 1997;41(3):591–7.
20] Spackman E, Senne DA, Myers TJ, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virusand the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol
2002;40(9):3256–60.
21] Potier M, Mameli L, Belisle M, Dallaire L, Melancon SB. Fluorometricassay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-d-N-acetylneuraminate) substrate. Anal Biochem 1979;94(2):287–96.
22] Tang Y, Lee CW, Zhang Y, Senne DA, Dearth R, Byrum B, et al. Iso-lation and characterization of H3N2 influenza A virus from turkeys.Avian Dis 2005;49(2):207–13.
23] Webby RJ, Swenson SL, Krauss SL, Gerrish PJ, Goyal SM, WebsterRG. Evolution of swine H3N2 influenza viruses in the United States. JVirol 2000;74(18):8243–51.
24] Chen Z, Kadowaki S, Hagiwara Y, Yoshikawa T, Matsuo K, Kurata T,et al. Cross-protection against a lethal influenza virus infection by DNAvaccine to neuraminidase. Vaccine 2000;18(28):3214–22.
25] Lee CW, Senne DA, Suarez DL. Generation of reassortant influenzavaccines by reverse genetics that allows utilization of a DIVA (differ-entiating Infected from Vaccinated Animals) strategy for the control ofavian influenza. Vaccine 2004;22(23–24):3175–81.
26] Johansson BE, Matthews JT, Kilbourne ED. Supplementation ofconventional influenza A vaccine with purified viral neuraminidaseresults in a balanced and broadened immune response. Vaccine1998;16(9–10):1009–15.
27] Wiley JA, Hogan RJ, Woodland DL, Harmsen AG. Antigen-specificCD8(+) T cells persist in the upper respiratory tract following influenzavirus infection. J Immunol 2001;167(6):3293–9.
28] Flynn KJ, Riberdy JM, Christensen JP, Altman JD, Doherty PC. In vivoproliferation of naive and memory influenza-specific CD8(+) T cells.Proc Natl Acad Sci USA 1999;96(15):8597–602.
29] Vidalin O, Fournillier A, Renard N, Chen M, Depla E, Boucreux D, etal. Use of conventional or replicating nucleic acid-based vaccines and
[
(2007) 3763–3772
recombinant Semliki forest virus-derived particles for the inductionof immune responses against hepatitis C virus core and E2 antigens.Virology 2000;276(2):259–70.
30] Colmenero P, Liljestrom P, Jondal M. Induction of P815 tumor immu-nity by recombinant Semliki Forest virus expressing the P1A gene.Gene Ther 1999;6(10):1728–33.
31] Mozdzanowska K, Feng J, Eid M, Zharikova D, Gerhard W. Enhance-ment of neutralizing activity of influenza virus-specific antibodies byserum components. Virology 2006;352(2):418–26.
32] Hartshorn KL, Reid KB, White MR, et al. Neutrophil deactivation byinfluenza A viruses: mechanisms of protection after viral opsoniza-tion with collectins and hemagglutination-inhibiting antibodies. Blood1996;87(8):3450–61.
33] Chen Z, Matsuo K, Asanuma H, Akahashi H, Iwasaki T, Suzuki Y,et al. Enhanced protection against a lethal influenza virus challenge byimmunization with both hemagglutinin- and neuraminidase-expressingDNAs. Vaccine 1999;17(7–8):653–9.
34] Johansson BE. Immunization with influenza A virus hemagglutininand neuraminidase produced in recombinant baculovirus results in abalanced and broadened immune response superior to conventional
vaccine. Vaccine 1999;17(15–16):2073–80.
35] Johansson BE, Pokorny BA, Tiso VA. Supplementation of conven-tional trivalent influenza vaccine with purified viral N1 and N2neuraminidases induces a balanced immune response without antigeniccompetition. Vaccine 2002;20(11–12):1670–4.
