Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/145—Orthomyxoviridae, e.g. influenza virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16161—Methods of inactivation or attenuation

Definitions

• 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.
• HA/NA vaccines 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.
• NA neuraminidase
• 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.
• Tc cytotoxic T
• Th helper T
• 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.
• 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.
• 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.
• HA haemagglutinin
• 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.
• 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.
• 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.
• 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.
• 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.
• a method of inducing a T-cell response in a mammal and a method of protecting a mammal against infection by influenza virus 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.
• a parenterally acceptable aqueous carrier such as physiological saline
• the carrier being pyrogen-free when the vaccine is intended for clinical use.
• the incorporation of vaccine adjuvants could be advantageous.
• 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.
• 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.
• 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.
• 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.
• 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- ⁇ ells _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.
• 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 ⁇ 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 NH 4 C1 and 0.01 M (NH 4 ) 2 HP0 4 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.
• the clear supernatant was dialysed against 2% w/v aqueous NH 4 C1 containing 0.01M (NH 4 ) 2 HP0 4 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.
• NP did not contain infectious virus particles
• aliquots (10 ⁇ g) were injected into triplicate 11-day embryonated chicken eggs. These were incubated at 33°C for 3 days and 100 ⁇ l 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 ⁇ g/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 ⁇ g/ml NP; (b) 10 ⁇ g/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 51 Cr 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%.
• 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 ⁇ g of the M protein fraction from the same ammonium deoxycholate preparation.
• PBS/A phosphate buffered saline
• Tc cells were generated from Balb/c mice either injected with (a) 10 ⁇ g NP intramuscularly, (b) two intramuscular doses of 10 ⁇ g NP with one month interval between doses, (c) 10 ⁇ g 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 ( ⁇ ) 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 51 Cr was less than 10%.
• 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) .
• NP was administered subcutaneously to prime mice for A virus crossreactive Th cells.
• the procedure and results are set out in Table 1 below.
• Purified NP induces influenza A virus crossreactive T-helper cells
• mice 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.
• 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.
• 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.
• mice were injected with 2 x 10 ⁇ g doses of NP subcutaneously (s.c.) at four week intervals or with 1 x 50 ⁇ g 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 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.

Applications Claiming Priority (2)

Publications (1)

Family

ID=10573520

Family Applications (1)

Country Status (5)

Cited By (3)

Citations (2)

• 1985
  • 1985-01-28 GB GB858502096A patent/GB8502096D0/en active Pending
• 1986
  • 1986-01-23 WO PCT/GB1986/000044 patent/WO1986004242A1/en not_active Application Discontinuation
  • 1986-01-23 EP EP86900813A patent/EP0211029A1/en not_active Withdrawn
  • 1986-01-23 JP JP61500850A patent/JPS62501973A/ja active Pending
  • 1986-01-28 CA CA000500552A patent/CA1270438A/en not_active Expired - Fee Related

Patent Citations (2)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events