VIROLOOY

69, 511-522 (1976)

Preparation

and lmmunogenicity of an Influenza Virus Hemagglutinin and Neuraminidase Subunit Vaccine W. G. LAVER

Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601 Australia AND

R. G. WEBSTER St. Jude Children’s Research Hospital, Memphis, Tennessee 38101 Accepted August 4,1975 Vaccines containing hemagglutinin and neuraminidase subunits were prepared from the A/Port Chalmers/l/73 (H3N2) strain of influenza virus. The virus particles were disrupted with ammonium deoxycholate and the matrix protein, which was insoluble in this detergent, was removed by centrifugation. Following removal of deoxycholate, the hemagglutinin and neuraminidase subunits aggregated by their hydrophobic ends, forming mixed clusters. These were then freed from nucleocapsids by electrophoresis. The separated nucleocapsid protein and the matrix protein were thus obtained as byproducts in a highly purified form suitable for the production of antisera and for structural studies. The hemagglutinin and neuraminidase subunits were as effective as intact inactivated virus (at equivalent concentration) in eliciting a late primary antibody response when injected in saline into rabbits. In hamsters, the subunits failed to induce antibody when injected in saline (in contrast to intact virus), but the immune response to the subunits could be potentiated by the simultaneous injection of an intact heterologous influenza A or B virus.

subunits can be isolated on a small scale from certain influenza virus strains by electrophoresis on cellulose acetate strips after disruption of the virus particles with the detergent sodium dodecyl sulphate (SDS) (Laver, 1973). Subunits isolated in this way are highly immunogenic when inoculated with adjuvant into animals and are very useful for the preparation of “monospecific” antisera to the hemagglutinin or neuraminidase, but the use of SDS for their isolation precludes their administration to man. In any case, large-scale preparation of the subunits by this method is not feasible at the present time. Other detergents (e.g., Triton X-100) have been used to isolate hemagglutinin and neuraminidase subunits but, like SDS, these detergents have not been safety-tested in man (Scheid et al., 1972).

INTRODUCTION

Inactivated influenza virus vaccines that contain intact virus particles may produce toxic reactions when inoculated parenterally into man (Salk, 1948; Quilligan et al., 1949). This adverse toxicity can be abolished by disrupting the virus particles with ether or detergents, and ether-split and deoxycholate-disrupted influenza virus vaccines have been used in man for a number of years; for a review, see Neurath and Rubin (1971). Such disrupted virus vaccines contain, however, in addition to the two surface antigens, internal components of the virus that, it is thought, play no role in the stimulation of neutralizing antibody and are therefore undesirable components of the vaccine. Pure hemagglutinin and neuraminidase 511 Copyrigh: All rights

8 1976 by Academic Press, Inc. of reproduction in any form reserved.

512

LAVER

AND WEBSTER

Hemagglutinin and neuraminidase subunits may also be isolated from certain influenza viruses by proteolytic digestion of the virus particles (Brand and Skehel, 1972; No11 et al., 1962; Seto et al., 1966; Drzeniek et al., 1966, 1968). These subunits have also been isolated from deoxycholate-disrupted virus particles by affinity chromatography on lectin columns (Hayman et al., 1973) but have not been tested in man. We are now describing the preparation of hemagglutinin and neuraminidase subunits by a relatively simple method, readily adaptable to large-scale production, using reagents already accepted for administration to man. The paper also describes the preparation, as byproducts, of purified, antigenically active matrix and nucleocapsid proteins. The immunogenicity in animals of the hemagglutinin and neuraminidase is described and a further paper (Webster et al., in preparation) describes their immunogenicity in man. MATERIALS

AND

METHODS

Viruses. The Port Chalmers variant of Hong Kong influenza virus (A/Port Chalmers/l/73; H3N21, the LEE strain of influenza B, and the avian influenza viruses, AlShearwaterlAustl72 and A/Chick Germany/N/49, were grown in the allantoic sac of ll-day-old embryonic chicken eggs. The viruses were purified from the infected allantoic fluids by adsorption and elution on washed chicken erythrocytes and were sedimented from the eluates by centrifugation. The virus particles were purified further by sedimentation through a sucrose gradient (lo-40% sucrose in 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.21, as described (Laver, 19691, and finally suspended in 0.15 M NaCl or a solution containing 2% ammonium chloride and 0.01 M diammonium hydrogen phosphate (pH about 7.5). Ammonium deoxycholate. Deoxycholic acid was recrystallized from acetic acid. Five grams of recrystallized deoxycholic acid was suspended in 80 ml of water, and ammonium hydroxide (sp grav, 0.91) was added slowly, with stirring, until all of the deoxycholic acid dissolved and the pH was

about 7.5. The volume was then made to 100 ml with water to give a 5% solution of ammonium deoxycholate. Disruption of virus and separation of the M (matrix OF membrane) protein. The purified A/Port Chalmers/l/73 virus particles, suspended in 2% NH&l, 0.01 M (NH,),HPO, at a concentration of approximately lo6 HAU/ml, were disrupted with ammonium deoxycholate (HAU, hemagglutinating units). Sodium ions needed to be kept out; otherwise, gelling occurred. Five percent ammonium deoxycholate was added to the suspension of virus in ammonium chloride-diammonium hydrogen phosphate, with stirring at 20”, to give a final ammonium deoxycholate concentration of 0.2%. Sometimes transient clearing of the opalescent virus suspension occurred during the addition of the ammonium deoxycholate, but usually there was no noticeable change in the opacity of the virus suspension after addition of this detergent; virus disrupted with sodium deoxycholate, on the other hand, is almost water-clear. The virus particles disrupted with ammonium deoxycholate were allowed to stand for about 5 hr at room temperature (20”). During this time, the membrane (M) protein flocculated and this was accompanied by a change in opalescence. The flocculated M protein was then removed by low speed centrifugation (10,000 g for 20 min at 20”) and the supernatant fluid was dialysed to remove the ammonium deoxycholate. Removal of ammonium deoxycholate. The supernatant fluid, after removal of the M protein was dialysed against 2% NH&l containing 0.01 M (NH&HPO, for 48 hr at 4”. It was then dialysed against phosphatebuffered saline (0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2) for a further 24 hr. Under these conditions, gel formation did not occur, and the deoxycholate content was reduced to undetectable levels (Szalkowski and Mader, 1952). Sedimentation through sucrose. The dialysed material was centrifuged at low speed (10,000 g, 10 min), and the small amount of precipitate was discarded. The supernatant solution was then layered

INFLUENZA

SUBUNIT

onto an equal volume of 35% (w/v) sucrose in phosphate-buffered saline and centrifuged at 200,000 g for at least 6 hr. The opalescent supernatant fluid was discarded and the clear pellet was resuspended by sonication in phosphatejbuffered saline to give an almost waterlclear solution with a faint blue opalescence. This material contained the hemaglutinin and neuraminidase subunits of iifhe virus, together with the nucleocapsids, #and was stored frozen at -20” or at 4” with sodium aside added to prevent bacterial growth. Separation of nucleocapsids from hepnagglutinin and neuraminidase. Nucleobapsids were separated from hemagglutinin and neuraminidase by electrophoresis on cellulose acetate or paper strips. This was done as described previously (Laver, 1964) except that the buffer did not contain any detergent. Further purification of M protein. The insoluble M protein, removed by centrifugation from ammonium deoxycholate-disrupted virus, contained traces of other virus antigens. The M protein precipitate was therefore dissolved in 1% Sarkosyl NL-97 (Geigy Chemical Co.), and centrifuged on Sarkosyl-sucrose density gradients (5-20% sucrose in 0.15 M NaCl containing 0.01 M sodium phosphate, pH 7.2, and 0.05% Sarkosyl) for 17 hr at 25,000 rpm (Spinco rotor SW 27) and a temperature of 5’. The fractions containing the M protein near the top of the gradient were collected, dialysed, and examined by acrylamide-gel electrophoresis. Acrylamide-gel electrophoresis. This was done as described (Laver et al., 1969). Hemagglutinin and neuraminidase assays. Hemagglutination titrations were done as described by Fazekas de St. Groth and Webster (1966), and neuraminidase assays as described by Aymard-Henry et al. (1973). Single radial-immunodiffusion tests were done according to Schild et al. (1972). Immunization of animals. Young adult New Zealand white rabbits were injected intramuscularly with varying doses of heand magglutinin plus neuraminidase equivalent doses of intact Port Chalmers

513

VACCINE

virus. All antigens were injected in saline without adjuvant. Rabbits were reinjected after 27 days and were bled at 7 day intervals and after the second inoculation. In some experiments, mice were injected intravenously with hemagglutinin plus neuraminidase with or without the addition of a heterologous intact influenza virus. For preparation of matrix protein antisera, the purified M protein was dissolved in Sarkosyl, heated to 100” for 5 min, inoculated with Freund’s complete adjuvant into rabbits, and reinoculated 40 days later. Electron microscopy. Dilute samples of hemagglutinin plus neuraminidase or RNP (ribonucleoprotein) were dried down from saline suspension in 4% aqueous sodium silicotungstate on carbon-coated 400mesh grids and examined in a Philips EM 301 electron microscope at 100 kV at a magnification of 60,000. RESULTS

Disruption of Virus Protein

and Separation

of M

A/Port Chalmers/73 virus particles were disrupted with ammonium deoxycholate and the insoluble M (matrix or membrane) protein was removed by centrifuging. Examination of the supernatant solution by acrylamide-gel electrophoresis in the presence and absence of dithiothreitol (DTT) showed that the M protein was removed completely and that the supernatant fluid contained the hemagglutinin, neuraminidase, and nucleocapsid proteins (Fig. 1). Recovery of hemagglutinin activity varied between 100 and 200%, but hemagglutinin activity may either increase or decrease, depending on the state of aggregation of the subunits as well as the cells used in the hemagglutination tests. Therefore, these values give little idea of the recovery of HA. Single radial-immunodiffusion tests showed 66% recovery of hemagglutinin antigen. Recovery of neuraminidase activity in the supernatant fluid was approximately 70%. Traces of neuraminidase only were found in the M protein precipitate; therefore, it is likely that about 30% of the neuraminidase was inactivated during the disruption process and subsequent dial-

514

LAVER

AND

ysis. Single radial-immunodiffusion tests showed 79% recovery of neuraminidase antigen. The M protein was purified further on Sarkosyl-sucrose density gradients and obtained free from the other virus proteins (Fig. 1). The purified M protein was highly immunogenic when boiled in Sarkosyl and injected in adjuvant into rabbits. Attempts to prepare hemagglutinin and neuraminidase subunits from influenza B virus (LEE strain) using the same method failed: The B/LEE M protein did not precipitate in the ammonium deoxycholate solution. The method also did not seem to work j

WITH ~I’THOUT

WEBSTER

successfully with HONl and HlNl influenza A viruses, but it was successful with all H2N2 and H3N2 strains tried. Further Purification Neuraminidase,

of Hemagglutinin, and Nucleoprotein

The supernatant fluid remaining after removal of the M protein was dialysed to remove ammonium deoxycholate, and the hemagglutinin, neuraminidase, and nuwere sedimented cleocapsid antigens through a layer of 35% sucrose. The supernatant fluid, which was opalescent and probably contained the lipid of the virus, was discarded, and the antigens were resuspended in saline to give an almost water-clear solution with a faint blue opalescence. Examination of this material in the electron microscope showed that it contained small clusters of hemagglutinin and neuraminidase subunits and also free nucleocapsids. Single radial-immunodiffision tests showed that no loss of HA occurred during pelleting. Separation of Hemagglutinin and Neuraminidase from Nucleocapsids

RNP -, w&l-b C HA (HA1 + HA21

AMM. DOC IN$OLUBLE

AMM.

DOC

SOLUBlE

FIG. 1. Stained polyacrylamide gels showing the polypeptides present in the soluble and insoluble fractions obtained after disruption of A/Port Chalmers/73 influenza virus with ammonium deoxycholate. The M (matrix or membrane) protein has about the same mobility as the light polypeptide of the hemagglutinin (HA2). Therefore, in order to show that the soluble fraction contained no M protein, the gels were run in the absence of dithiothreitol (DTT), as well as in its presence. The mobility of the M protein is the same in each case, but in the absence of DTT, HA2 and HA1 are not dissociated. It is thought that the two faint bands just below HA1 on the middle gel represent the neuraminidase polypeptides. The band below these contained carbohydrate and may represent aggregated hemagglutinin or neuraminidase polypeptides.

The nucleocapsids were removed from the clusters of hemagglutinin and neuraminidase subunits by electrophoresis on cellulose acetate strips in buffer that did not contain detergent. The nucleocapsids had a higher electrophoretic mobility at pH 9 than the hemagglutinin and neuraminidase subunits (which migrated together), and acrylamide-gel electrophoresis of the two fractions suggested that complete separation of the nucleocapsids from the other two antigens had been obtained (Fig. 2). Electron

Micrographs

Electron micrographs of the hemagglutinin and neuraminidase fraction eluted from cellulose acetate (or paper) strips showed mixed clusters of hemagglutinin and neuraminidase subunits that had aggregated by their hydrophobic regions in the manner previously described (Laver and Valentine, 1969). The clusters contained, on an average, about 11 subunits, but some larger clusters and some single subunits could be seen (Fig. 3). The nucleocapsid fraction, eluted from

INFLUENZA

SUBUNIT

ACIIYLAMIClE GELS

4-

HAI

CECLULOSE ACETATE

FIG. 2. Cellulose acetate strip stained with Coomassie blue, showing the electrophoretic separation of the nucleocapsids from the hemagglutinin and neuraminidase present in the soluble fraction of ammonium deoxycholate-disrupted A/Port Chalmers/73 influenza virus. Material eluted from the strips was examined by polyacrylamide-gel electrophoresis; stained gels are shown.

cellulose acetate contained coiled structures (Fig. 4), variable in length and similar in appearance to influenza virus nucleocapsids described by others (Pons et al., 1969; Compans et al., 1972). The electron micrographs showed, however, that neither the hemagglutinin and neuraminidase nor the nucleocapsid fractions were completely pure, as traces of contamination of one fraction with the other could be seen (Figs. 3 and 4). 32P-labeling experiments showed that at least some RNA was associated with the isolated nucleocapsids and that none was present in the hemagglutinin and neuraminidase fraction. Immunogenicity of the Hemagglutinin and Neuraminidase Subunits in Rabbits

Different doses of the hemagglutinin and neuraminidase clusters, plus the nu-

VACCINE

515

cleoprotein, were injected in saline intramuscularly into rabbits. Equivalent doses of intact A/Port Chalmers/73 virus were injected into companion rabbits. The doses of subunits were estimated from the original concentration of the virus used in their preparation. Since recovery of hemagglutinin and neuraminidase (measured by single radial-immunodiffusion tests) in the subunit preparation was about 70%, the doses of subunits injected therefore contained about 30% less of the antigens than did the equivalent doses of intact virus. The antibody response to these vaccines is shown in Fig. 5 and Table 1. At low doses of vaccines (316 HAU), those rabbits which received subunits gave the same HI (hemagglutination inhibition) antibody response as those receiving intact virus. At higher doses (31,600 HAU), there was significantly less early antibody produced by the subunits than by the intact virus, but the peak of the primary IgG antibody response was the same for intact virus and subunit vaccines. The intact virus vaccine and the subunit vaccine also induced similar levels of antibodies to neuraminidase (Table 1). Immunogenicity of Hemagglutinin and Neuraminidase in Presence and Absence of RNP

The aim of this work was to prepare an influenza virus hemagglutinin (HA) and neuraminidase (NA) subunit vaccine free from all other viral components. It was necessary to demonstrate therefore whether removal of nucleocapsids and the associated RNA affected the immunogenicity of the subunit vaccine. It has been suggested that RNA and DNA act as adjuvants in induction of immunity (Cowan, 1973); it was therefore important to learn whether the subunit vaccine had altered immunogenicity after removal of nucleocapsids. The immunogenicity of the hemagglutinin and neuraminidase subunits (both with and without RNP antigen) was compared with that of intact A/Port Chalmers/73 virus (Fig. 6). At low doses (100 HAU per rabbit) the subunit vaccines failed to induce detectable antibodies during the primary response period. The sub-

516

LAVER

AND

WEBSTER

FIG. 3. Electron micrograph showing clusters of hemagglutinin and neuraminidase subunits eluted from cellulose acetate strips (Fig. 2). A single contaminating nucleocapsid can be seen. The HA and NA subunits have aggregated by their hydrophobic regions, which previously attached them to the lipid of the virus envelope, in the manner described previously (Laver and Valentine, 1969). Magnification x 225,000. Electron micrograph by Nick Wrigley.

INFLUENZA

SUBUNIT

VACCINE

517

FIG. 4. Electron micrograph of nucleocapsids eluted from cellulose acetate strips (Fig. 2). A single contaminating cluster of neuraminidase subunits can be seen. Magnification x 180,000. Electron micrograph by Nick Wrigley.

unit vaccines at this low dose did however, prime for a secondary response. At higher doses of vaccine (320, 1000, and 10,000 HAU per rabbit), the subunits again induced a lower early primary response than was induced by the intact virus vaccine;

but by the end of the primary response, there was no significant difference between the immunogenicities of the intact virus and subunit vaccines. At two doses of vaccine (320 and 1000 HAU per rabbit) the subunits induced slightly higher second-

LAVER AND WEBSTER

518

DOSE OF VACCINE (LOGw,lO

5ml)

XIINTACT “lR”S VACCINE

sueuw

DAYS

AFTER

“AcClNE

VACCINATION

FIG. 5. Immune response of rabbits to A/Port Chalmers/73 intact influenza virus and subunit vaccines as measured in hemagglutination-inhibition (HI) tests. Groups of three rabbits were injected with intact virus vaccine and hemagglutinin plus neuraminidase vaccines at equivalent doses (this subunit vaccine contained nucleocapsids). The rabbits were reinjected with the same doses after 27 days. The doses of subunits were estimated from the original concentration of the virus used in their preparation. Since recovery of hemagglutinin and neuraminidase (measured by single radial-immunodiffusion tests) in the subunit preparation was about 70%, the doses of subunits injected therefore contained about 30% less of the antigens than did the equivalent doses of intact virus. Values represent the mean HI titers per 0.25 ml of serum and are expressed as the reciprocals of the dilution inhibiting three out of four hemagglutinin doses of virus.

TABLE 1 IMMUNE RESPONSE OF RABBITS TO A/PORT CHALMERS/73 INFLUENZA VIRUS VACCINES AS MEASURED IN NI TESTY

Dose of antigen (logJ0.5)

2.5 3.5 4.5

Neuraminidase inhibition

titers”

Intact virus vaccine

Subunit vactine

22” 48 770

16 28 620

UTiters are expressed as the reciprocal of the dilution of serum giving 50% inhibition of neuraminidase per 0.05 ml of serum. The tests were done by the short procedure (30 min of enzyme action) as described in Materials and Methods. The assays were done on the same groups of rabbits as in Fig. 5, 7 days after receiving the second dose of antigen.

ary antibody titers than the intact virus vaccine. Removal of the RNP antigen from the subunit vaccine did not alter the immunogenicity of the HA and NA subunits. Addition of RNP to the HA plus NA vaccine did not change the immunogenicity of the subunits. Both the intact and subunit vaccines induced antibodies to the neuraminidase subunits, and there were no differences in the

levels of antibodies induced by each kind of vaccine. The sera were tested for the presence of antibodies to RNP using single radialdiffusion plates containing AIChicklGermany/W’/49 virus. The traces of RNP present in the subunit vaccine (Fig. 4) failed to induce detectable levels of antibodies when injected into rabbits. Intact virus vaccine, isolated RNP, and subunit vaccine containing RNP antigen all induced antibodies in rabbits to the RNP antigen. Lack of Zmmunogenicity of Subunit Vaccines in Certain Animals and Potent&ion with Zntact Znfluenza A and B Viruses (a) Response in hamsters to intact and subunit influenza virus uaccines. Comparison of the immunogenicity in hamsters of intact and HA + NA subunit influenza virus vaccines showed that hamsters failed to produce antibody to the subunit vaccines (Table 2). Even at doses of subunits equivalent to 30,000 HAU per hamster, there were no detectable antibodies induced during the primary response; even after reimmunization, the antibody responses were still very low. In comparison, the hamsters produced high levels of anti-

INFLUENZA

SUBUNIT

519

VACCINE

RG. 6. Immune response of rabbits to A/Port Chalmers/73 intact influenza virus and subunit vaccines as -red in HI tests. Conditions were the same as in Fig. 5, except that one of these subunit vaccines did not contain nucleocapsids.

TABLE 2 ANTIBODY RESPONSE OF HAMSTERS TO A/PORT CHALMEFS/~/~~ INFLUENZA VIRUS VACCINES

Material injected

Whole virus

Hemagglutinin and neuraminidase subunits

Days

0 7 14 21 28 B+7 0 7 14 21 28 Bt7

HI response with the following doses” 30,000 HA

3,000 HA

300 HA

< 1200 1800 1800 1650 8380

< 550 410 335 290 4300

230 320 300 300 2000

< < 4 <