EP0211029A1

EP0211029A1 - Improvements relating to influenza vaccine

Info

Publication number: EP0211029A1
Application number: EP86900813A
Authority: EP
Inventors: Brigitte Alice Askonas; David Cameron Wraith
Original assignee: Medical Research Council
Current assignee: Medical Research Council
Priority date: 1985-01-28
Filing date: 1986-01-23
Publication date: 1987-02-25
Also published as: CA1270438A; GB8502096D0; WO1986004242A1; JPS62501973A

Abstract

Une cellule T productrice d'une substance comprenant une nucléoprotéine du virus de la grippe, utile dans la protection des mammifères contre ledit virus, est obtenue par fragmentation du virus de la grippe. Le virus entier est brisé avec un détergent, de préférence après un traitement préliminaire avec une protéase, et la nucléoprotéine résultante est séparée du virus infectieux entier et d'autres substances, puis formulée pour administration parentérale. Cette nucléoprotéine est capable de protéger contre une attaque par des souches de virus différentes de la souche à partir de laquelle elle a été obtenue.A T cell producing a substance comprising an influenza virus nucleoprotein, useful in the protection of mammals against said virus, is obtained by fragmentation of the influenza virus. The whole virus is broken up with a detergent, preferably after preliminary treatment with a protease, and the resulting nucleoprotein is separated from the whole infectious virus and other substances, then formulated for parenteral administration. This nucleoprotein is capable of protecting against attack by strains of viruses different from the strain from which it was obtained.

Description

DESCRIPTION

TITLE: "IMPROVEMENTS RELATING TO INFLUENZA VACCINE"

THIS INVENTION relates to influenza vaccine and is specifically concerned with antigenic materials that can provide protection against infection by influenza virus and to methods of producing and using such antigenic materials.

The existing influenza vaccines usually fall into one of two categories, inactivated whole virus vaccines or sub-unit vaccines known as HA/NA vaccines. HA/NA sub-unit vaccine is obtained by disruption of whole virus with a detergent followed by the recovery of the neuraminidase (NA) fraction together with part of the haemagglutinin fraction as it was believed that this was the fraction which, on introduction into a host, would provoke formation of antibodies that would protect the host against subsequent infection by a virulent virus.

Both whole virus vaccine and HA/NA sub-unit vaccine suffer from the same disadvantage that the protective antibodies they induce are directed against variable regions of the HA and NA molecules. This means that such vaccines tend to be effective against subsequent challenge by a virulent virus of the same strain from which the inactivated virus or sub-unit virus was obtained but they do not protect against virulent virus of a different strain. There is often considerable variation from year to year in the strain type of influenza virus which means that existing vaccines, being strain-specific, are of limited value. Interest has centered recently upon the role of cytotoxic T (Tc) and helper T (Th) cells in the immune response to and protection against viral infections. Anti-influenza Tc cells have been found capable of lysing cells infected with any influenza virus within a type and Th cells can similarly proliferate and produce lymphokines in response to such infected cells. The behaviour of T-cells thus is to be contrasted with that of antibodies which are almost always influenza strain specific. Available whole virus vaccines and HA/NA sub-unit virus vaccines stimulate production of influenza strain specific antibodies in a host but they induce only insignificant quantities of A virus crossreactive Th and Tc cells. We have now found that a different type of influenza virus sub-unit preparation can induce significant formation of Tc and Th cells in a host, which opens up the possibility of producing a vaccine from one strain of influenza virus that will protect against subsequent infection by a different strain of influenza virus within a particular type. The present invention provides a T-cell inducing material comprising influenza virus nucleoprotein which is substantially free from whole infectious virus, sucrose and free detergent. The nucleoprotein (NP) should be substantially free from each of the three above-mentioned products and preferably also free from matrix protein, as shown by Coomassie Blue staining of a polyacrylamide gel, in order that it may safely be used as an effective vaccine material which will induce T-cell formation in a host.

However the NP material may still contain a haemagglutinin (HA) fraction originating from the virus but usually the HA level will be below 3% by weight.

Preliminary indications show that it is desirable that the NP material of the present invention should not be excessively contaminated by viral glycoproteins and, as will be explained in more detail below, if the NP material is to be derived from whole virus, it is possible to treat the whole virus with a suitable enzyme prior to disruption of the virus to ensure relatively low levels of HA and insignificant levels of NA in the NP material that is isolated. This does not exclude the possibility of preparing NP from whole virus by other means. In the preparations of the present invention we find that the presence of NP and HA in weight ratios of about 5:1 to 100:1 are effective but that the ratio, which appears to influence T-cell induction, can vary outside these limits.

The NP material of the present invention may conveniently be obtained from whole virus by methods to be described below but it is also possible to produce the NP material synthetically or biosynthetically. By synthetically, we mean by chemical synthesis of appropriate polypeptides and by biosynthetically we infer expression of a gene that encodes a desired polypeptide cloned into a suitable expression system, e.g. microbial, yeast or mammalian cells. The desired polypeptide, whether it is to be produced by chemical synthesis or by gene expression, means a polypeptide of structure analogous to the structure of the NP fraction obtained from the influenza virus or a fraction which contains variation in the amino acid sequence but which is an immunogenic equivalent of the natural product.

The influenza virus with which the present invention is concerned and from which the NP sub-unit can be derived, will normally be human influenza A virus but the principle of the present invention is applicable to any human influenza virus type A, B or C or virus strains including those specific to animals such as horses or poultry, for example chickens. Preliminary experiments indicate that the NP material we have obtained from whole A strain virus by the methods described in the Examples below contain 498 amino acid units.

According to a further feature of the present invention, we provide a method of inducing a T-cell response in a mammal and a method of protecting a mammal against infection by influenza virus, the methods comprising parenteral administration to a mammal of an NP material in accordance with the present invention or a composition containing it. Parenteral administration in accordance with the present invention can be carried out subcutaneously, intraperitoneally or intramuscularly but in specific applications, other parenteral routes may be used.

The NP material of the present invention can be used as the basis for vaccine production, e.g. -by presentation in sterile form e.g. a lyophilised material for formulation in a parenterally acceptable aqueous carrier such as physiological saline, the carrier being pyrogen-free when the vaccine is intended for clinical use. In certain situations, the incorporation of vaccine adjuvants could be advantageous.

As indicated above, the NP material of the present invention has been found to induce a Tc and Th cell response in a host so that vaccines incorporating such NP materials find application in protecting a host against subsequent infection by influenza virus within a type different from the strain that was used as the starting material for the NP or the model for the NP when the NP has been prepared synthetically or biosynthetically.

At the practical level, it is convenient to prepare the NP material of the present invention by disruption of whole virulent virus by methods known per se. The disruption of influenza virus with detergent has been known for many years and any of the methods customarily used for the disruption of whole virus for the production of the existing HA/NA sub-unit vaccines could be suitable for producing the NP material of the present invention. One detergent that has become widely accepted for this use is ammonium deoxycholate. When ammonium deoxycholate is used, virus disruption can be brought about by suspending the virus particles in a buffer solution, e.g. ammonium chloride and ammonium hydrogen phosphate and introducing an aqueous solution of the detergent. The mixture is then allowed to stand for several hours, e.g. 3 to 24 hours at 15 to 25°C. As a result of virus disruption, the matrix protein precipitates and can be easily separated by low-speed centrifugation or other methods for separating insoluble protein material.

The supernatant solution recovered after separation of the matrix from non-enzyme treated virus would contain the nucleoprotein, the neuraminidase and the haemagglutinin and these materials could be separated from one another by any of the conventional methods used in biochemistry for separation of material of differing molecular sizes. When it is desired to produce the NP fraction with reduced levels of HA and NA, the intact virus, prior to disruption, can be treated with an enzyme and we have found that the enzyme bromelain is particularly effective. NP material, obtained from enzyme-treated virus by the method described, has been found to be capable of restimulating murine memory T-σells _in vitro, although generally not as effectively as infectious virus, giving T-cells which were cross-reactive for type A influenza viruses. The M protein fraction gave no such stimulation of Tc cells. Mice which were injected either intraperitoneally, intramuscularly or subcutaneously with the NP material were found to be effectively primed for anti-influenza A-type cross-reactive T-cells. Induction of Tc cells was not observed with the M protein fraction or with suitable controls.

In the accompanying Drawings:

Figure 1 shows a gel electrophoretic profile of enzyme-treated A/X31 influenza virus NP fractions. Figure 2 shows the extent of _in vitro Tc memory stimulation by an NP fraction according to the invention. Figure 3 shows an ^ιι vivo Tc response induced by the NP fraction of the invention administered intraperitoneally to mice.

Figure 4 shows an ^Lri vivo Tc induction following i.m. and s.c. administration of the NP fraction of the invention.

Table 1 represents the induction of Th cells both _in vivo and _in vitro with the NP fraction of the invention.

Table 2 represents a series of experiments to investigate the.ability of mice primed by injection with the NP fraction of the invention to combat a lethal influenza virus infection.

EXAMPLE 1 A/X31 influenza virus was treated with bromelain to release the majority of the surface glycoproteins.

Virus at 2 mg/ml in lOmM Tris buffer (pH 8) containing 1 mM EDTA and 50 mM 2-mercaptoethanol was treated with 1 mg/ml bromelain at 35°C for 16 hours. Virus core was recovered by centrifugation; the enzymic process could be repeated at this stage or the viral cores stored at +4°C. Usually >95% of virus haemagglutinating activity would be reduced by this procedure. Cores were resuspended in 2% w/v NH₄C1 and 0.01 M (NH₄)₂HP0₄ to about 5 mg/ml and an amount of 5% w/v aqueous ammonium deoxycholate added to the suspended cores with stirring at 20°C to give a final ammonium deoxycholate concentration of 0.2% w/v. After 5 hours at 20°C, the sample was centrifuged at 10,000 g for 30 minutes during which time the flocculated matrix protein was spun down as the pellet.

Supernatants from three separate preparations were analysed by SDS-polyacrylamide gel electrophoresis on 12.5% gels. Gels were run- using the buffer system of Laemlli, (Nature, 227, 680-685, 1970) , under non-reducing conditions and stained with Coomassie Blue. The results are shown in Figure 1 of the accompanying drawings where the four columns are:

(a) whole A/X31 virus;

(b) - (d) three preparations of ammonium deoxycholate (Am.DOC) NP of .the invention derived from A/X31 virus. Influenza proteins were identified by comparison of their relative mobility to molecular weight standards and by immunoblotting.

The clear supernatant was dialysed against 2% w/v aqueous NH₄C1 containing 0.01M (NH₄)₂HP0₄ for 2 days at 4°c to remove detergent, then against phosphate buffered saline overnight, frozen at -70°C in small aliquots and will be referred to below and in the following Examples as NP. The matrix (M) protein which sediments at 10,000 xg was solubilised in 1% w/v aqueous Sarkosyl NL-97 and centrifuged at 100,000 xg for 1 hour, the supernatant collected and dialysed as above to remove detergent.

To ensure that NP did not contain infectious virus particles, aliquots (10 μg) were injected into triplicate 11-day embryonated chicken eggs. These were incubated at 33°C for 3 days and 100 μl of allantoic fluid passaged in fresh 11-day eggs for a further 3 days. In controlled experiments, no HA activity was recovered from either passage indicating that the NP fraction did not contain infectious virus particles.

The A/X31 strain, A/USSR/90/77 and B/Hong Kong/8/73 strain of influenza virus used in this and subsequent Examples are widely available from virus Collections around the world including The World Health Organisation, World Influenza Centre, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England.

EXAMPLE 2 The NP (Figure lb-d) was used at 1 to 10 μg/ml and was able to generate influenza A virus crossreactive Tc cells _in vitro. Tc cells were generated (see Figure 2) by culturing spleen cells from mice primed intranasally with A/X31 virus with either (a) 1 μg/ml NP; (b) 10 μg/ml ^"NP or (c) A/X31 virus infected cells, for 5 days. P815 target cells were infected with virus (either the homologous A/X31 (H3N2) , the heterologous A/USSR/90/77 (H1N1) or B/Hong Kong/8/73 virus) and labelled with ⁵¹Cr for 1 hour, washed, incubated in RPMI/10 for 3 hours, washed again and these served as targets in 3 hour cytotoxicity assays (Wraith e_t l, Eur. J. Immunol., 13, 762-766, 1983). % Lysis was calculated as described previously (Zweerink e_t a_l, Eur. J. Immunol., ]_, 630-635, 1977. Background release of Cr labelled targets was less than 10%.

The results are shown in Figure 2 in which K/T = killer to target cell ratio at 2 x 10 targets per well. 0 - P815 infected with A/X31, Δ -infected with A/USSR, LJ - infected with B/Hong Kong.

In this experiment the NP stimulated Tc cells lysed both homologous and heterologous A type virus infected targets similarly to (Figures 2a & b) Tc cells stimulated with infective virus (Figure 2c) . There was no significant lysis of B virus infected targets.

EXAMPLE 3 In order to examine whether the NP could prime hosts _in vivo for A virus crossreactive anti-influenza Tc cells, 3 month old Balb/c mice, bred under specific pathogen-free conditions, were immunised by intraperitoneal injection with 50 ug of the NP suspended in phosphate buffered saline (pH 7.2) (PBS/A). Similarly mice were injected with either PBS/A alone or 50 μg of the M protein fraction from the same ammonium deoxycholate preparation. After 3 weeks, spleen cells from the NP, M or control immunised mice were resti ulated j^ vitro at 1 x 10⁶ cells/ml in RPMI/10 by the addition of 2 x 10 syngeneic spleen cells/ml infected with A/X31 virus (Zweerink e_t a_l, loc. cit.). After 5 days T-cell mediated cytotoxicity was tested as above. The results are set out in Figure 3 (Targets were 0 - P815 infected with A/X31,

/\ - infected with A/USSR and I )- infected with B/Hong

Kong. Assay was as for Figure 2) . - Mice injected with NP (Figure 3c) clearly were primed for A virus crossreactive Tc, whereas mice primed with M protein (Figure 3b) or PBS alone (Figure 3a) showed no such priming.

EXAMPLE 4 Crossreactive Tc response _irι vitro after vaccination with the NP of influenza A virus. Tc cells were generated from Balb/c mice either injected with (a) 10 μg NP intramuscularly, (b) two intramuscular doses of 10 μg NP with one month interval between doses, (c) 10 μg NP subcutaneously or (d) primed intranasally with A/X31 virus. After at least one month, spleen cells were co-cultured with A/X31 infected syngeneic spleen cells for 5 days prior to testing for cytotoxicity. Targets were^*P815 (H-2d) cells infected with A/X31 (0), P815 infected with A/USSR (Δ) and P815 infected with B/Hong Kong (D) . K/T = killer to target cell ratio at 2 x 10 targets per well. Percentage lysis was calculated as previously described. Background release of ⁵¹Cr was less than 10%.

As Figure 4 shows, mice were clearly primed for A virus crossreactive Tc by either i.m. (Figure 4a) or s.c. (Figure 4b) administration of NP and this was enhanced by further boosting (Figure 4c) . Levels of Tc priming were not as high as following intranasal infection (Figure 4d) . EXAMPLE 5

NP was administered subcutaneously to prime mice for A virus crossreactive Th cells. The procedure and results are set out in Table 1 below.

TABLE 1

Purified NP induces influenza A virus crossreactive T-helper cells

IL-2 assay

Priming of donors Antigen -Η-Thymidme Stimulation stimulation CPM (CTLL) index

— 229

A/JAP infection 307 1.3

1 μg/ml NP 194 <1

A/X31 I.N. — 239

!l A/JAP infection 52,413 219

If 1 μg/ml NP 19,184 86

20 μg NP

(from X31)S.C.2x 312 n A/JAP infection 53,242 170

II 1 μg/ml NP 26,899 86

Donors primed rn vivo develop T memory cells which on stimulation by antigen _ir vitro release interleukin-2 (IL-2) within 48 hours. IL-2 production was assayed using the CTLL cell line. This IL-2 release is inhibited by antibody to the T-helper cell marker L3T4. A/X31 virus has the 1934 NP gene while A/JAP carries a changed NP gene isolated in 1957. We conclude from these experiments that our NP material effectively primes mice for both A virus crossreactive Tc and Th cells and also restimulates crossreactive memory Tc and Th jLn vitro. Furthermore we have shown that mice primed with NP are significantly protected against lethal influenza virus infection. The procedure and results obtained are set out in Table 2 below. We favour the view that recovery from infection, in this case, is mediated by Tc cells.

TABLE 2

Expt. Mice per urvivors Dose (ug) Route group (day 20)

1 4 3 50 2 4 4 50 3 4 2 50 4 4 3 20 5 4 2 20 6 4 3 20 7 5 4 20 8 5 4 20 9 5 4 20 10 6 5 20

Total 45 34

Recovery from a lethal dose of influenza A virus after NP vaccination.

Balb/c mice were injected with 2 x 10 μg doses of NP subcutaneously (s.c.) at four week intervals or with 1 x 50 μg dose intraperitoneally (i.p.). After one month mice were challenged with 0.1 HAU of A/PR/8/34 virus intranasally. In each experiment this dose was lathal for an identical group of age and batch matched mice previously injected with isotonic PBS alone; of 45 infected controls, there were 2 survivors at dl4 and none at d20. For vaccination purposes one would ultimately select a preparation effective in stimulating both humoral and cellular immune responses. While it is accepted that NP can stimulate antibody production the role of such antibody in protection is not clear. Antibody to HA, on the other hand, can neutralise free virus particles, albeit only of the same subtype. Priming for anti-HA antibody is thus advantageous in naive hosts, such as young children, and we would favour the inclusion of HA in a vaccine preparation whether or not it contributes to a Tc response.

Claims

1. A T-cell inducing material comprising influenza virus nucleoprotein, the nucleoprotein being substantially free from whole infectious virus, sucrose and free detergent.

2. A material according to claim 1 which is substantially free from neuraminidase and matrix protein.

3. A material according to claim 1 or 2 containing not more than 3% by weight haemagglutinin.

4. A material according to any one of the preceding claims isolated from detergent disrupted influenza virus.

5. A material according to any one of the preceding claims obtained by expression of a gene that encodes the desired polypeptide sequence and that is cloned into an expression system.

6. A material according to any one of the preceding claims wherein the nucleoprotein is strain A virus nucleoprotein.

7. A material according to claim 6 in which the nucleoprotein comprises 498 amino acid units.

8. A T-cell inducing composition in form suitable for parenteral administration comprising sterile influenza-virus nucleoprotein.

9. A composition according to claim 8 which is an aqueous pyrogen-free preparation.

10. A method of producing a T-cell inducing nucleoprotein from influenza virus which comprises disrupting whole influenza virus by treatment with a surfactant to give a disruption product comprising nucleoprotein with other virus proteins and isolating the nucleoprotein in a form stimulatory for T-cells.

11. A method according to claim 10 wherein the surfactant is cationic.

12. A method according to claim 11 wherein the surfactant is ammonium^*deoxycholate.

13. A method according to any one of claims 10 to 12 wherein the disrupted virus suspension is centrifuged and the supernatant containing nucleoprotein ^{* ' ■} separated from the pellet containing matrix protein.

14. A method according to any one of claims 10 to 13 wherein, prior- to disruption of the whole virus, the whole virus is treated with a protease and the viral core, after separation from the protease, subjected to disruption with the surfactant whereby nucleoprotein substantially free from neuraminidase is isolated.

15. A method according to claim 14 wherein the protease is bromelain.

16. A composition suitable for parenteral administration comprising a sterile material according to any one of claims 1 to 7.

17. A composition according to claim 16 which is in the form of an aqueous pyrogen-free suspension or solution.

18. A material according to any one of claims 1 to 7 or a composition according to claim 8, 9, 16 or 17 for use in a method of treatment of the human or animal body by surgery or therapy or in a method of diagnosis practised on the human or animal body.

19. A material according to any one of claims 1 to 7 or a composition according to claim 8, 9, 16 or 17 for use in a method of preparing a medicament for the treatment of the human or animal body in a method of surgery or therapy or for use in a method of diagnosis practised on the human or animal body.

20. A method of inducing a T-cell response in a mammal by parenteral administration to the mammal of a material according to any one of claims 1 to 7 or a composition according to claim 8, 9, 16 or 17.

21. A method of protecting a mammal against infection by influenza virus which comprises parenteral administration to the mammal of a material according to any one of claims 1 to 7 or a composition according to claim 8, 9, 16 or 17.

22. A method according to claim 20 or 21 wherein the material is administered by the subcutaneous, intraperitoneal or intramuscular route.