Recombinant neuraminidase vaccine protects against lethal influenza

Elsevier 0264-410X(95)00157-3

ELSEVIER

Vaccine,Vol. 14, No. 6, pp. 561-569, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/96 $15+0.00

Recombinant neuraminidase vaccine protects against lethal influenza Tom Deroo*,

Willy Min Jou* and Walter

Fiers*j-

The N2 neuraminidase gene of AlVictoriai3i75 influenza virus was engineered to encode a secretable protein (NAs) by replacing the natural N-terminal membrane anchor sequence with the cleavable signal sequence of the corresponding influenza hemagglutinin gene. Soluble NAs was expressed by a baculoviruslinsect cell system and accumulated in the medium at levels between 6 and 8 pg ml-‘. A combination of biochemical and standard chromatographic techniques allowed the purtjication of NAs to homogeneity. Crosslinking analysis indicated that NAs was partly recovered as an authentic tetrameric protein, while the remaining fraction was composed of dim’eric molecules and small amounts of monomeric NAs. Purtjied NAs was supplemented with low-reactogenic adjuvants and used to immunize mice. After a challenge infection with a lethal dose of homologous mouse-adapted X47 influenza virus, vaccinated animals showed resistance against severe disease symptoms and were protected from lethality. Based on the results of a passive immunization experiment, it may be concluded that preformed antibody plays a central role in the mechanism by which vaccination with NAs confers viral protection. Copyright 0 1996 Elsevier Science Ltd. Keywords:

Influenza;

recombinant

neuraminidase;

immunization;

protection;

The neuraminidase (NA) of influenza virus is a glycosylated type II integral membrane protein composed of four identical, single-chain polypeptide units. It catalyses the removal of terminal sialic acid residues, thereby destroying potential receptors for the accompanying viral hemagglutinin (HA)‘.2. Each NA tetramer (M,~240000) has a mushroom-like morphology in which the individual monomers are linked as a pair of dimers by disulfide bonds3-6. Unlike HA, NA is anchored in the membrane by an unprocessed, N-terminal lipophilic sequence7s. By far the largest part of the total structure extends above the membrane, forming a distal box-like “head” domain located on top of an elongated “stalk” region5*9. Because of their external location, HA and NA represent the major viral target structures for the host immune system. Administration of equal doses of purified HA or NA antigen to naive subjects does not reveal significant differences in immunogenicity as both proteins can induce similar levels of specific antibody during primary or secondary responses”. However, unlike the neutralizing effect of HA antibodies, antibodies against NA are generally unable to prevent a primary infection across a wide range of concentrationsiO-‘-. Instead, immunity to NA appears to interfere with the release of progeny virus from infected cell surfaces, affecting the yield and spread of virus. When NA *Laboratory of Molecular Biology, University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium. j-To whom correspondence should be addressed. (Received 20 December 1994; revised 10 July 1995; accepted 14 July 1995)

baculovirus

expression

antiserum was incorporated in an agar overlay, the size but not the number of viral plaques was reduced13,14. In tissue culture supernatant or in ovo, the continuous presence of NA antibodies affected virus recovery, although direct neutralization did not occur13. When challenged, subjects immunized with NA show reduced viral 1unF titers and diminished development of lung lesions”. “13. NA immunity also exerts a marked effect on the severity of the clinical disease symptoms, even to an extent that infection seems to be completely silenced12315~17. We here describe a procedure leading to the production and purification of a recombinant, secreted form of NA (NAs), followed by its application as a potent vaccine to induce protective immunity against a lethal dose of influenza virus in a mouse-model system.

MATERIALS

AND METHODS

Products and chemicals TClOO growth medium was supplied Recombinant N-glycanase enzyme

by Gibco BRL. (cloned from Flavobacterium meningosepticum) was purchased from Genzyme. The synthetic NA substrate 2’-(4methylumbelliferyl)-a-D-N-acetylneuraminic acid and the affinity matrix N-(p-aminophenyl)oxamic acidagarose were obtained from Sigma Chemical Co. Sepharose Q and Superdex 200 chromatographic media were from Pharmacia LKB. The chemical cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3) was a product from Pierce. Ribi adjuvant [containing monophosphoryl lipid A (MPLA), trehalose-6,6-dimycolate

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Influenza protection with recombinant neuraminidase: T: Deroo et al. (TDM), squalene and Tween 801 and Salmonella typhimurium MPLA vials were reconstituted according to the instructions of the manufacturer (Ribi Immunothem Research). Muramyl dipeptide (MDP) was purchased from Sigma Chemical Co. All other chemicals were of the highest purity grade available.

To produce NAs protein, cells were infected with recombinant baculovirus at an m.o.i. of 10, resuspended under serum-free conditions (2 x lo6 cells ml - ‘) and incubated for 48 h before harvesting the medium. Purification

Animals

Inbred Balb/c female mice (SCK Mol, Belgium) were 8 weeks old at the beginning of the immunization regimen. For passive immunization experiments, recipient mice were 12 weeks of age. Mice were housed in groups of three animals per cage (350 cm2) and were allowed access to food and water ad libitum. Influenza virus

Influenza virus X47 is a reassortant strain with a H3N2 subtype derived from the wild strain A/Victoria/ 3/7518. For studies involving a lethal challenge in the mouse, the X47 virus was adapted to cause lethality by a series of lun passages in mice (1 LD,, corresponded to 103-7EIDSO)A . Construction of a gene coding for secretable neuraminidase and its integration in a baculovirus expression vector

The plasmid pV6/21 is a pBR322 derivative containing a cDNA copy of the NA gene from A/Victoria/3175 (H3N2) influenza virus2’. pSRAS-8 is a pPLa2311 based plasmid carrying the cDNA start sequence of the HA gene from AJVictoria/3/75 (H3N2)2’. pSV51, and both pSV23m and pSV24m are late and early SV40 replacement vectors, respectively2’. The baculovirus transfer vector pVL941 was constructed by Luckow and Summers**. A flow diagram of the cloning procedure is presented in Figure I. pV6l21 was linearized with PvuI, and, after digestion with Ba131 nuclease and addition of HinDIII linkers, the NA sequence was cloned into the HinDIII site of pSV23m, resulting in pSV23mIVNA. The sequence coding for the HA signal peptide was recovered as a PvuI/PvuII fragment from pSRAS-8 by ligating PvuII linkers to the BstNI site (Klenow enzyme was used to fill in 3’ recessed ends) followed by digestion with PvuI and PvuII. Ligation of this fragment with the 1669 bp PvuI/PvuII fragment of pBR322 yielded pIVpreHA. Next, a plasmid, designated pATIVNAs, containing a chimeric gene (NAs) composed of the HA signal peptide fused to the 5’ region of the NA gene, was constructed: the 861 bp PstIlPvuII fragment of pIVpreHA, the 1291 bp FnuDII/SaI fragment of pSV23mIVNA, and the 2253 bp SaWPstI fragment of pAT153 were ligated together. In order to clone the NAs gene as a BamHI fragment behind the baculovirus polyhedrin promoter of pVL941, an intermediate vector, pSVIVNAs1, was created by ligation of the XbaUSalI NAs sequence from pATIVNAs with the 5562 bp SalI/ EcoRI fragment of pSV51 and the 624 bp EcoRUXbaI fragment derived from pSV24m. The final construction, referred to as pAQIVNAs, was used to produce recombinant baculovirus according to the technique described by Summers and Smith23. CeII culture-production of NAs Spodoptera frugiperda insect cells (S’) were cultured in TClOO medium according to standard procedures23.

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of NAs

Commonly used buffers were as follows: buffer A, 20 mM diethanolamine-HCl (PH 8.5); buffer B, 50 mM NaAc (pH 5.5); buffer C, 20 mM Hepes-HCl (pH 7.4), 150 mM NaCl. All contained 4% butanol (except where contra-indicated) and 2 mM CaCl,. Sf9 suspension cultures (usually about 1 1) were harvested 2 days after inoculation and cellular remnants were pelleted by centrifugation at 4000g for 15 min. The clarified crude medium was subjected to ammonium sulfate fractionation at pH 7.5. Material precipitating between 20% and 60% (NHJ2S04 was collected by centrifugation (lOOOOg,60 min) and dissolved in 100th of the initial culture volume, using buffer A (without butanol), 20 mM NaCl. Following dialysis against the same buffer, insoluble components were removed by centrifugation at 20000g for 15 min. The solution was then supplemented with 4% butanol and loaded on a Sepharose Q column, equilibrated with buffer A, 20 mM NaCl. After washing the column with the same buffer, elution was carried out by applying a linear NaCl concentration gradient in wash buffer up to 250 mM (250 ml). Fractions containing NAs were pooled based on enzyme activity and ELISA levels, and an equal volume of 200 mM NaAc (pH 5.5) was added, before loading the solution on a N-(paminophenyl)oxamic acid-agarose column equilibrated in buffer B, 100 mM NaCl. The column was then washed using equilibration buffer, and subsequently desalted with buffer B. After a second wash step with buffer A, bound NAs was eluted by applying buffer A supplemented with 1 M NaCl. The eluate of the affinity column was concentrated to 2.0 ml by ultrafiltration (30 kDa cut-off). The concentrate was then chromatographed in subfractions of 1.Oml sample volume on a Superdex 200 gel filtration column using buffer C. For long-term storage at - 2O”C, related fractions were pooled, concentrated as above, and then supplemented with glycerol to 50% final concentration. NA enzymatic

assay

The assay of NA catalytic activity was based on the method described by Potier et a1.24Briefly, enzyme tests were done in a 100 ~1 reaction volume containing 200 mM NaAc (pH 6.5), 2 mM CaCl, and 1% butanol in the of 1 mM of the substrate 2’-(4presence methylumbelliferyl)-a-D-N-acetylneuraminic acid. After incubation at 37°C for 30-60 min, the reaction was stopped by addition of 0.5 ml 133 mM glycine, 83 mM 60 mM NaCl (pH 10.7). Free NaHCO,, 4-methylumbelliferone was measured by reading the absorbance at 365 nm. One unit was defined as that amount of enzyme which releases 1 nmol of 4-methylumbelliferone per min. ELISA

Pronase cleaved NA was prepared from egggrown X47 virus25, and was used to raise a polyclonal

Influenza protection with recombinant neuraminidase: Sal1

T: Deroo et al.

BstNI

pSV24m BcoRI/XbaI

Figure1 Strategy for the construction of a secretable NA gene and its integration in a baculovirus transfer vector. Only restriction sites relevant to the cloning procedure have been indicated (see text for details). Single lines refer to bacterial plasmid sequences; heavy segments represent HA (filled) or NA (dotted) specific sequences. The HA signal sequence and NA signal sequence/membrane anchor are indicated with single hatches and cross-hatches, respectively

antiserum in a rabbit, New Zealand strain, 3 months of age (Freund’s adjuvant was supplemented). Purified IgG was used to coat wells of ELISA microtiter plates. Binding of NAs was detected with biotin labeled antiserum followed by the administration of streptavidinalkaline phosphatase conjugate. Plates were developed with p-nitrophenylphosphate. Absorbance values were read at 405 nm.

Analytical methods Discontinuous SDS-PAGE was carried out according to Laemmli26. Gels were subsequently silver stained by a modification of the method described by Morrisey27. Protein concentration was assayed by the method of Bradford”. Cross-linking of NAs samples was carried out in 30 ,~l reaction volumes using BS3 (0.5 mM). The reaction was

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Influenza protection with recombinant neuraminidase:

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allowed to continue for 1 h at room temperature and was then quenched with 5 yl 1.O M Tris-HCl (pH 8.0). Polypeptide patterns were analyzed by SDS-PAGE. To remove N-linked glycosyl groups from NAs, samples (between 0.1 and 1 pg) were pretreated by boiling in 500 mM Tris-HCl (pH 8.0) 0.5% SDS, 50 mM P-mercaptoethanol. After adding N-octylglucoside to a sevenfold excess over the final SDS concentration, N-glycanase was added (enzyme dose according to the instructions of the manufacturer) and the reaction mixture was incubated for 16 h at 37°C. Digested NAs was then analyzed by SDS-PAGE. Immunization

of mice

Groups of 12 mice were injected subcutaneously with three 200 ~1 doses of 1 ,ug NAs each, given at 3-weekly intervals. Adjuvants were chosen according to an immunization study with recombinant influenza HA19. For the first immunization, NAs was emulsified in half the amount of a normal Ribi mouse dose (corresponding to 25 pg MPLA, 25 ,ug TDM, 2 ~1 squalene and 0.1% Tween 80). Booster injections were given by supplementing NAs with 25 pug MPLA and 25 pg MDP. Control mice received adjuvant solubilized in phosphate-buffered saline. For passive immunization, immunized mice were bled by heart puncture 3 weeks after receiving their last immunization. Serum preparations of similarly treated mice were pooled. Naive recipient mice received a single intraperitoneal injection of 400 ~1 immune or control serum. Influenza challenge

Mice were inoculated intranasally under light ether anaesthesia with 20 LD,, of mouse-adapted X47 virus either 3 weeks after the last booster injection with NAs or one day after passive immunization. The course of infection was then monitored by measuring the rectal temperature and body weight for a post-inoculation period of 10 days.

RESULTS Construction

and expression of NAs

The NA gene of the influenza N2 strain AIVictorial 3/75 was severed from its N-terminal membrane-anchor and fused instead to the 5’ sequence of the corresponding HA gene, which contains a signal peptidase cleavage site that permits the synthesis of a secreted protein. The resulting chimeric construct, referred to as NAs, consists of the HA signal sequence plus a short extension of four intact and one mutated codon, followed by the NA sequence lacking the transmembrane section and a part of the stalk region (amino acids l-45) (Figure 2). A gene copy of NAs was then integrated behind the polyhedrin promoter of a baculovirus host. Upon inoculation of 5” insect cells with recombinant baculovirus, NA enzyme activity was readily detected in the medium, indicating that a soluble protein was indeed produced. In order to purify NAs, the medium was harvested 48 h after infection, when the enzymatic activity level reached a plateau (data not shown). Based on the results of several purification experiments, we found that NAs was expressed at levels ranging from 6 to 8 ,ug ml - I, a yield

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A 16 ; 17 HA

N-A&lGh

NA

N

20

21

Asp Leu Pro GCC CAA GAC ClllGA

16

19

Gly

22

23

ASI Aq AAT GAC

C

B 44

45

46

Ser

Pro

Ala

TCC CCC,GCG

4146

49

Asn Asn G!n

50 Val-

C

AAC CAA GTA

C 16 ; 17 NAs

N-AAblGln

16 19 Asp Lw

20 Pro

21 Ah

22 Ah

23 24 Asn Am

25 Gin

26 M-C

GCC CAA GAG CTT CCA GCA GCG AAC AAC CAA GTA ....

Figure 2 Nucleotide (positive cDNA strand) and amino acid sequence flanking the site of ligation between the HA signal peptide and the NA released from its membrane anchor (the drawings do not reflect the relative chain lengths). (A) Diagram of unprocessed HA indicating the signal peptidase cleavage site located between Ala,, and Gin,, (dashed vertical line)“. The N-terminal fragment used to direct secretion of NAs is marked by an arrow. (B) Detail of the stalk region of NA. The truncated sequence involved in the construction of NAs is indicated by an arrow. (C) Sequence of NAs, as deduced by combining A and B. Here given in detail is the fusion region between HA and NA specific sequences. NAs starts with the four N-terminal amino acids of mature HA, followed by a mutated codon (underlined by dots)

comparable to those reported for other secreted, complex glycoproteins produced in this system3’. Purification

of NAs

The different steps of NAs purification are summarized in Table 1. The use of N-(p-aminophenyl)oxamic acid-agarose as a selective adsorbent for influenza or bacterial neuraminidases has already been demonstrated in other studies31,32. In the following Superdex 200 gel filtration step, A2s0 monitoring revealed three peaks, eluting at ~220, z 130, and z 54 kDa, respectively (Figure 3). The immunoreactivity pattern of the eluate as measured by ELISA proved to be a faithful reflection of the A2s0 profile for each of the three peaks registered. SDS-PAGE analysis of the peak fractions revealed an intense band in the expected range of ~55 kDa, although some minor decrease in molecular weight with increasing fraction number was noted (Figure 4A). Further confirmation of the NAs specific nature of the band was obtained by Western analysis (data not shown). Cross-linking with BS3 followed by SDS-PAGE demonstrated that the 220-kDa gel filtration peak was composed of tetrameric NAs, whereas the two peaks corresponding with smaller molecular size were shown to represent dimeric and monomeric NAs, respectively, the latter form being of minor quantitative importance (Figure 49. Due to its rod-like morphology, dimeric NAs is believed to elute somewhat above its actual molecular weight as compared to tetrameric and monomeric NAs, which have a more globular shape. Tetramerit and dimeric NAs were purified to homogeneity as judged from silver stained SDS-PAGE gels, and, after pooling the peak fractions, this material was used for vaccination purposes. The quality of monomeric NAs was somewhat less as some trace contaminants were also detectable. Properties of NAs

Denaturation by boiling with SDS in the presence of /3-mercaptoethanol caused complete dissociation of NAs

Influenza protection with recombinant neuraminidase:

T: Deroo et al.

Table 1 Purification of NAs produced by SfQ insect cells. The results refer to a single, typical purification experiment (see text for details). The specified volumes after Superdex 200 gel filtration represent pools of NAs fractions collected from two chromatographic runs

Steps

Volume (ml)

Protein (mg)

Total activity (U)

Yield (%)

Specific activity (U mg-‘)

Purification (-fold)

Crude medium 20-60% (NH&SO, precipitate Sepharose Q N-(paminophenyl)oxamic acid-agarose Superdex 200 tetramer dimer monomer

995 99.5 60.0 54.0 8.0 8.0 6.0

281 97.5 7.27 2.63 0.66 0.98 0.15

144000 117000 70300 49100 36100 -

100 81.3 48.9 34.1 25.1 -

510 1200 9670 18700 54700 -

1.0 2.4 19.0 36.7 107 -

A 555657 58 5960616263646566676669

71 73 75 6

A -

1.2

-

1.0 ”

-

3

09

002

08

-g

0.7

<

06

‘:

0.5

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z

03 02

.= 2 c

01

:

00

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0.6

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0.4

.$

-

0.2

e

s

c 0.0

0.00 30

40

50

60 70 elut~on volume (ml)

60

90

100

Figure 3 Gel filtration of NAs on Superdex 200. The eluate after K(paminophenyl)oxamic acid agarose separation (2.63 mg protein, 49100 U) was concentrated and a 1.0 ml sample volume was chromatographed on a Superdex 200 column. A,,, was monitored continuously (-). Individual fractions (1.0 ml) were scored both for enzymatic (0) and antigenic activity (A). Arrows (on top) indicate the elution volume of calibration proteins: 443 kDa (l), 200 kDa (2), 150 kDa (3) 67 kDa (4) and 29 kDa (5)

into monomeric chains with a molecular weight close to 55 kDa (Figure 4A). When denatured in the absence of a reducing agent, tetrameric and dimeric NAs migrated as dimeric chains of z 110 kDa (data not shown). These results indicate that NAs dimers are internally linked by disulfide bridges, and can further associate by noncovalent interactions to form a tetrameric protein, consistent with the structural organization of natural NA. It was observed that enzymatic activity was only associated with the tetrameric form of NAs. We had purified previously natural NA from A/Victoria/3/75 by pronase cleavage”. Comparing specific activities, it was found that the enzyme properties of recombinant, tetrameric NAs closely resembled those of its natural counterpart. It has been repeatedly reported that insect cells generate N-glycosyl groups that differ to some extent from those produced by mammalian and other higher cells33~3s. This urged us to examine NAs samples by N-glycanase treatment in combination with SDS-PAGE analysis (Figure 5). As expected, the monomeric chains of deglycosylated NAs migrated with the same electrophoretic mobility irrespective of their oligomeric origin, indicating that NAs was properly synthesized as a polypeptide of uniform chain length (Figure 5, compare lanes 3, 5, and 7). The molecular weight of the N-glycanase treated polypeptide chain was estimated at 47.5 kDa, which is in good agreement with the theoretical mass of 47717 Da, calculated from the predicted amino acid sequence. Although it was clear from the

029 *

29 *

57 665660616263646566676~7671 73 747577

B

6-a ^SL

,/;

6743-

Figure 4

SDS-PAGE analysis of purified NAs. Each lane corresponds to the indicated fraction number after Superdex 200 gel filtration. (A) Reducing SDS-PAGE of Superdex 200 fractions (10 pl samples). Lanes A and B represent marker proteins. (B) Protein samples were cross-linked with BS3 and then separated by electrophoresis on a 5.0-7.5% gradient gel under nonreducing conditions. Fractions 57-68, 10 pl samples; fractions 70-77, 30 ~1 samples. Tetrameric NAs gives rise to bands at ~220 kDa (tetramer) and ~110 kDa (dimer). Dimeric and monomeric NAs remain as bands of =llO and =55 kDa, respectively

band displacement pattern that all NAs forms were glycosylated, a slightly higher amount of carbohydrate could apparently be assigned to tetrameric NAs (Figure 5, lane 2 vs lanes 4 and 6; see also Figure 4A). This finding suggests the possibility of a relationship between the glycosylation properties of NAs and the tetrameric structure of the protein.

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123456

78

94

94

67

67

43

43

10

0

401'

29

29

Figure 5 Comparative analysis of carbohydrate content associated with NAs corresponding to different oligomeric forms as estimated from Nglycanase digestion and reducing SDS-PAGE. The enzyme N-glycanase is visible as an intense band of =35 kDa. Lanes 1 and 8, marker proteins; lane 2, undigested tetrameric NAs; lane 3, N-glycanase treated tetrameric NAs; lane 4, undigested dimeric NAs; lane 5, N-glycanase treated dimeric NAs; lane 6, undigested monomeric NAs; lane 7, N-glycanase treated monomeric NAs

Immunization with NAs protects against a lethal influenza infection It was our prime interest to test whether vaccination with purified recombinant NAs could provide protection against a lethal dose of the homologous influenza virus. To this end, mice were immunized with a mixed population of tetrameric and dimeric NAs following the regimen described under Materials and Methods, and were then challenged with a lethal dose of mouseadapted X47 virus. From Figure 6 it can be seen that NAs vaccinees only experienced a transient and very moderate depression of clinical parameters and all animals survived. By contrast, control mice suffered severe illness as measured by a progressive loss of temperature and body weight. Lethality started at day 4 post-infection and reached 100% within the next 5 days. Protective immunity can be obtained by passive transfer of NAs immune serum By means of a passive immunization experiment, it was examined whether NAs vaccination was able to induce humoral defense mechanisms. To this end, donor mice were immunized following the standard procedure. Bleeding of the animals yielded on average about 400 ~1 of serum per individual. As tested by ELISA, specific antibody to NA could be readily detected in the serum of immunized mice (data not shown). After separate pooling of control and immune sera, recipient mice were injected intraperitoneally with a single dose of 400 ,~l serum. A period of 24 h was interposed to allow systemic spread of antibody molecules before challenging the mice with 20 LD,, mouse-adapted X47 virus. Figure 7 illustrates the course of infection. Whereas animals that had received a dose of control serum subsequently developed acute hypothermia and suffered severe loss of weight ending in death, administration of NAs immune serum protected mice to a similar degree as was demon-

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Vaccine 1996 Volume 14 Number 6

B

G

30

‘2 3 iij z

36

Q E $z

'

'

'

'

'

'r

34 32 30 26 26 0

2

10

days fGst in6fectiot

Figure 6 Protection against lethal influenza infection by immunization with NAs. Vaccinees (n=12) [- - in (A); v in (B) and (C)] and controls (-12) [-- in (A); 0 in (B) and (C)] were challenged with 20 LD,, of homologous mouse-adapted X47 virus. The course of infection was followed by registering the survival number (A) and by measurement of the rectal temperature (B) and body weight (C) of the mice (see text for experimental details). Data points represent mean values+S.D.

strated for actively immunized animals. Hence, that antibody free in circulation is an important tor in the mechanism by which NAs vaccination viral resistance.

it seems mediaconfers

DISCUSSION The baculovirus-insect cell expression system has proved its usefulness for the production of a wide variety of foreign proteins, including viral glycoproteins, which are expressed in a biologically and immunologically active form. In the present study, a baculovirus vector based on the potent polyhedrin promoter served to express the NA of the N2 influenza strain ANictorial31 75. In order to obtain synthesis of a secreted NA molecule (NAs), the combined signal sequence/ membrane anchor region of the NA gene was substituted with the cleavable signal sequence of the corresponding influenza HA. Soluble NAs accumulated

Influenza protection with recombinant neuraminidase:

A 0

2

4

6

8

10

days post infection

B

z

30 1

5

28

1

f

26 0

2

4

6

8

10

8

10

days post infection

fi c

12 10 0

2

,

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’

,

IL

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6

days post infection Figure 7 Passive immunization protects against lethal influenza infection. Groups of mice (n=12) were passively immunized by intraperitoneal injection of NAs immune serum [- - in (A); Y in (B) and (C)l or control serum [- in (A); 0 in (B) and (C)l. Twenty-four hours later, they received a challenge of 20 LD,, of mouse-adapted X47 virus (see text for experimental details). Survival rates, rectal temperatures and body weight are represented in (A), (B), and (C), respectively. Data points refer to mean values&.D.

in the medium at levels between 6 and 8 pug ml ~ ‘, and then was preparatively purified to homogeneity so that it could be used for immunization studies. Purified NAs was demonstrated to consist of tetramers, which were the only enzymatically active molecules, disulfide-linked dimers, and small amounts of monomers. Other work, in which the NA was modified such that its signal/anchor domain was replaced with the hydrophobic fusion related external domain of the paramyxovirus F protein, also led to the secretion of a mixed population of NA oligomers with only the tetrameric structure being enzymatically active36. Similar studies aimed at the construction of chimeric genes or based on the introduction of mutations in the signal/anchor domain of the NA revealed that the N-terminal part has an important contribution in determining the tetrameric structure of the NA molecule37-39. The evidence obtained here, however, suggests that stabilization of the tetrameric structure is also dependent on appropriate

T Deroo et al.

N-glycosylation. Upon digestion with N-glycanase, it was found that the amount of N-glycosylation associated with tetrameric NAs was somewhat larger as compared to dimeric and monomeric NAs. These observations are related to published results. Partial inhibition of glycosylation of viral glycoproteins resulting from either cytochalasin B-induced inhibition of hexose transport or from glucose starvation resulted in the formation of influenza virions that were inactive in their NA40,41 Similarly, purified virions derived from tunicamycin treated cells were found to have a ca 30-fold decrease in NA specific activity42. According to our results, this inactivity of NA might be explained by the fact that under those culture conditions the NA was unable to form a stable tetrameric structure necessary for enzymatic activity due to absence of appropriate glycosylation. Indeed, X-ray diffraction studies have revealed the presence of a glycosyl group that is possibly involved in stabilizing the tetrameric structure by its interaction with a neighbouring subunit5. Most studies dealing with NA immunity are based on the isolation of the natural NA protein’ 1,12,43,or, alternatively, rely on the combined administration of a series of influenza strains with serologically related NA antigens but different HA antigens15. By contrast, few investigators have used recombinant NA for their immunization studies. Thus far, only vaccinia based expression systems have been applied for this purpose, and NA was expressed as an authentic, integral membrane molecule, without subsequent purification’6*44,45. In the present study protective immunity was obtained by means of a purified, secreted NA protein, produced by a baculovirus expression system. Other reports have been dealing with the expression of influenza NA in baculovirus-infected insect cells or larvae, but the resulting, membrane-anchored proteins were not used in vaccination studies4ti8. Mice vaccinated with purified NAs not only were able to fully survive a lethal challenge with homologous mouse-adapted X47 influenza virus, but also showed few disease symptoms. It is important to note that the adjuvants administered in conjunction with NAs are considered to have low-reactogenic properties, such that the immunization strategy might also be relevant to human vaccination. Passive transfer of serum from mice immunized with NAs to naive recipient mice led to comparable levels of protection, indicating that preformed antibody is presumably an important mediator of NAs-induced resistance against viral challenge. Recombinant NA has also been demonstrated by others to be effective at protecting against influenza challenge, Birds immunized by scarification of the comb with a vaccinia-NA recombinant virus were able to survive a lethal influenza infection16. Similarly, inoculation of mice with a vaccinia-NA recombinant virus led to diminished influenza replication in the lungs, and this effect was demonstrated to be mainly antibody mediated45. To our knowledge, the present study is the first report that documents the use of a purified, secreted recombinant NA preparation, capable of inducing protective immunity against a lethal dose of influenza virus. The vaccine production system, as described here, exploits the high level of heterologous gene expression in insect cells and is amenable to considerable scale-up. In addition, a procedure based on recombinant DNA

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technology offers the possibility to modify the antigenic structure of NA at the genetic level, a feature which may become valuable for future vaccine development.

Deroo

et al. 18

19

ACKNOWLEDGEMENTS The authors are grateful to Dr J. Skehel and Dr A. Douglas for kindly providing X47 influenza virus. The plasmid pVL941 was a generous gift from Dr M.D. Summers. We thank G. Maertens and P. Vanlandschoot for mouse-adapted X47 virus. M. Vandecasteele and W. Drijvers are acknowledged for editorial assistance and preparation of photographs. T.D. is a research assistant with the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek. Research was supported by the FGWO, the GOA, and the National Lottery.

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