WO1993020846A1 - Recombinant influenza vaccines - Google Patents

Recombinant influenza vaccines Download PDF

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Publication number
WO1993020846A1
WO1993020846A1 PCT/US1993/003606 US9303606W WO9320846A1 WO 1993020846 A1 WO1993020846 A1 WO 1993020846A1 US 9303606 W US9303606 W US 9303606W WO 9320846 A1 WO9320846 A1 WO 9320846A1
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Prior art keywords
epitope
influenza virus
fla
chimeric protein
influenza
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PCT/US1993/003606
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French (fr)
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Ruth Arnon
Raphael Levi
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Yeda Research And Development Co. Ltd
Arnon, Yoram
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Publication of WO1993020846A1 publication Critical patent/WO1993020846A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • 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
    • 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
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to synthetic recombinant vaccines, particularly to synthetic recombinant influenza vaccines in which one or more of the influenza virus hemagglutinin or nucleoprotein epitopes are expressed in Salmonella flagellin, and their use for mucosal and systemic immunization.
  • Influenza virus appears as three subtypes designated A, B and C, of which subtype A comprises the major antigenic compounds that are associated with pandemics. Since subtype A is capable of changing its antigenic identity in a remarkable way, the specific immunity established in response to infection or vaccination by a particular strain may give little or no protection against subsequent infections by virus, this being an explanation for influenza still being a major epidemic disease in man.
  • Influenza virus comprises two surface antigens: the neuraminidase (NA) , which is common to many influenza strains and antibodies against which are almost non- neutralizing and non-protective, and the hemagglutinin (HA) , which undergoes gradual changes (drifts) , is a strong immunogen and is the most significant antigen in defining the serological specificity of the different viral strains. Antibodies to the HA neutralize virus infectivity, and resistance to influenza infection was shown to correlate with serum anti-HA antibodies levels.
  • NA neuraminidase
  • HA hemagglutinin
  • the HA is a molecule of molecular weight of 75-80 kD. It comprises a plurality of antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are common for the various known hemagglutinins (common determinants) .
  • strain-specific determinants are in regions that undergo sequence changes in different strains
  • hemagglutinins common determinants
  • the " nucleoprotein (NP) is located in the viral core which consists of RNA segments, the three different polymerase proteins and the NP. It is one of the group specific antigens which distinguishes between influenza A, B and C viruses. Influenza A viruses can be classified into at least five groups based on RNA- NA hybridization studies of NP genes. NP genes of several influenza A viruses isolated from different species have been sequenced and the derived amino acid sequences were compared. The results suggest that only two different NP subtypes exist: subtype 1, found with all human strains, and subtype 2, found with all avian strains. However, there are no significant differences between these two groups concerning secondary structure predictions (Gammelin et al., 1989). Indeed, in contrast to the HA, the NP is one of the most conserved viral proteins, being 94% conserved in all influenza A viruses (Townsend & Skehel, 1984).
  • Influenza A virus NP-specific antibody has no virus neutralizing activity, but NP is an important target for cytotoxic lymphocytes (CTL) which are cross-reactive for all type A viruses (Townsend & Skehel, 1984; Yewdell et al., 1985) .
  • CTL cytotoxic lymphocytes
  • NP-specific CTL had a role in protection against influenza infection in vivo until Taylor and Askonas (1986) showed that a series of NP-specific CTL transferred into syngeneic mice were capable of limiting virus replication in the lungs and trachea of influenza infected hosts and could protect it against lethal infection. This and other studies have emphasized the potential of NP as an alternative vaccine that could overcome the limitations of inactivated influenza vaccines which do not induce A-virus cross-reactive T cells (Webster & Askonas, 1980) .
  • These can be prepared by: a) synthesis of antigenically relevant proteins of the organism by recombinant DNA technology in which such proteins are expressed in bacteria, yeasts or mammalian cells, followed by isolation of the proteins (Woodrow, 1990) , or production of bacteria (Salmonella) or viruses
  • Attenuated Salmonella which induce a broad immune response after oral administration (Dougan et al., 1987), offer a convenient way of presenting heterologous antigens to the immune system.
  • cholera toxin epitope Newton et al., 1989
  • r malaria antigens Sepatitis B surface antigen
  • Wu et al., 1989 Hepatitis B surface antigen
  • tetanus toxin Fairweather et al., 1990
  • Streptococcal M protein Piereptococcal M protein
  • influenza vaccines are administered parenterally and consist of whole virus vaccine, subunit or "split" vaccines, or live attenuated virus vaccines. Each of these types has problems concerning efficacy and safety.
  • the live vaccine is more potent, but it is not considered safe enough.
  • novel approaches for influenza vaccines are being sought.
  • influenza vaccines it would be most advantageous to obtain influenza vaccines to be administered by oral or intranasal route that provide effective mucosal immunization and more efficient protection against infection by several different types of influenza virus.
  • Figure 1 A-D depicts structure of pLS408 vector (1A) and the base sequence of the insert (IB, SEQ ID NO:l; 1C, SEQ ID NO:2; ID, SEQ ID NO:3).
  • Hl-d is the flagellin gene from Salmonella munchen.
  • the oligonucleotide sequences shown at the bottom were designed to code for epitope 91-108 of the influenza A H3 subtype HA, and epitopes 55-69 and 147- 158 from the nucleoprotein. Codon usage was according to the sequence of the flagellin gene, with minor modifications in order to create the Mlul restriction site, and a stop codon for the inserts which are in the wrong orientation.
  • Figure 2 shows shift in molecular weight of the recombinant flagellins Fla-91, Fla-55 and Fla-147 when compared to the native flagellin (Fla-control) , as demonstrated in SDS-PAGE (10%) of the purified chimeric flagellins-
  • Figure 3A-B shows detection of influenza epitope in the recombinant flagellin. Binding of anti-flagellin antibodies (filled lozenges) , antipeptide 91-108 antibodies (filled circles) and preimmune serum (empty lozenges) , to: 3A- Pure native flagellin. ; 3B- Purified chimeric flagellin Fla-91.
  • Figure 4A-B shows binding in ELISA of antisera from rabbits immunized with: Salmo-91 (live bacteria i.m.) (filled squares) , or Fla-91 (purified hybrid flagellin, s.c. with CFA) (filled circles) , as compared to preimmune sera (empty circles) .
  • Salmo-91 live bacteria i.m.
  • Fla-91 purified hybrid flagellin, s.c. with CFA
  • FIG. 5 shows humoral response of CD1 mice immunized with Salmo-91 (filled triangles) and Salmo-control
  • Figure 6 shows the protective effect of recombinant vaccines in BALB/c mice, expressed by percent of lung homogenates that exhibit viral growth in embryonated eggs, as a function of the homogenate dilution. Immunizations were with: Untreated control (empty squares) , Fla-control (empty lozenges) and Fla-91 (empty circles) , given intranasally. Error bars represent the SEM values.
  • Figure 7 shows immunization (three times, at three week intervals) of BALB/c mice with Fla-91, Fla-147 and a combination of the two preparations, intranasally, and the protective capacity of these vaccinations, as compared to controls, against viral challenge.
  • Figure 8 shows the effect of combination of the various chimeric flagellins, on the protection of the immunized mice from viral challenge.
  • the immunizations were given i.n. three times at three week intervals.
  • the challenge was done one month after the last boost.
  • Figure 9A-C shows cross protection of BALB/c mice vaccinated with combinations of recombinant flagellins against various influenza virus strains.
  • Figure 10A-B shows binding in ELISA of IgA antibodies in the lung homogenates of the BALB/c mice from the protection experiment, to peptide HA 91-108 (10A) and the intact virus (10B) . Immunization was intranasally with: Untreated control (empty squares) , Fla-control (empty circles) , or Fla-91 (filled circles) .
  • Figure 11A-F shows staining of lymphocyte infiltrations (indicated by arrows) in lung sections taken from mice immunized with untreated and Fla-control (11A-B) , Fla-55 and Fla-147 (11C-D) , combination of Fla-55 + Fla-147 and infected mice (11E-F) . Each section represents the general condition of 4-5 animals per treatment. Magnification xl50.
  • the chimeric protein of the invention comprises the amino acid sequence of flagellin and at least one amino acid sequence of an influenza virus hemagglutinin and/or nucleoprotein epitope.
  • the aggregates, comprising several subunits of the chimeric protein, are represented by the bacterial flagella.
  • hemagglutinin epitope 91-108 may be used, which is a highly conserved region in all type H3 strains and confers partial protection against challenge with several strains.
  • Hemagglutinin epitope 91-108 has the following sequence:
  • a chimeric protein comprising flagellin and the influenza virus hemagglutinin epitope 91-108, expressed in Salmonella flagellin, is useful as a component of a vaccine of the present invention.
  • a synthetic oligonucleotide comprising 54 bases coding for the corresponding sequence was inserted into the plasmid pLS408 and transformed into E. coll JM101.
  • Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative "live vaccine" Aro A" mutant of Salmonella dublin.
  • Mice immunized with the same preparations developed influenza-specific IgG antibodies in the blood and secreted IgA antibodies in their lungs.
  • NP 55-69 has the following amino acid sequence: Arg-Leu-Ile-Gln-Asn-Ser-Leu-Thr-Ile-Glu-Arg-Met- Val-Leu-Ser (SEQ ID NO:5) .
  • NP 147-158 has the following amino acid sequence: Thr-Tyr-Gln-Arg-Thr-Arg-Ala-Leu-Val-Arg-Thr-Gln
  • an oligonucleotide coding for the respective epitope is synthesized and inserted into a suitable expression vector, i.e., plasmid pLS408, depicted in Fig. 1.
  • a suitable expression vector i.e., plasmid pLS408, depicted in Fig. 1.
  • Suitable bacterial cells e.g., E. coll JM 101 cells, are transformed with the expression vector containing the desired oligonucleotide, and positive colonies are used to transform a virulent pathogenic strain of Salmonella, such as Salmonella typhimurium LB 5000 (Smith et al.
  • the flagella comprising the chimeric protein may be isolated and purified for use in the vaccine, or the intact bacteria may be used as a vaccine, as a non-limiting example.
  • a synthetic recombinant vaccine against a plurality of different influenza virus strains comprising at least one chimeric protein comprising the amino acid sequence of flagellin and the amino acid sequence of influenza virus hemagglutinin or nucleoprotein epitope, and aggregates thereof.
  • Preferred epitopes according to the invention are the influenza epitopes HA-91-108 (SEQ ID N0:4) , NP 55-69 (SEQ ID NO:5) and NP-147-158 (SEQ ID NO:6) .
  • combinations of HA 91-108 and NP 147-158 or of the three epitopes are used. While the double combination increased the protective capacity of the HA 91-108 epitope, the triple combination seems to be superior to all other preparations.
  • the invention provides a synthetic recombinant vaccine against a plurality of different influenza virus strains comprising a non-virulent Salmonella strain into which an expression vector comprising a nucleotide sequence encoding an epitope of influenza virus hemagglutinin or nucleoprotein was introduced, said strain being capable of expressing said epitope in the flagella within the flagellin sequence as a chimeric protein.
  • the Salmonella strain is genetically engineered Salmonella dublln expressing one of the epitopes HA 91-108 (SEQ ID N0:4), NP 55-69 (SEQ ID N0:5) or NP 147-158 (SEQ ID NO:6) .
  • a humoral immune response is shown following oral immunization with the recombinant bacteria, or intranasal immunization with the purified chimeric flagellin. Intranasal vaccination with the hybrid flagellin also conferred partial protection against viral challenge.
  • a preferred embodiment of the invention is the effective immunization afforded by using the intranasal immunization route, which delivers the antigens to the nasal cavity, and the upper part of the lungs, thereby leading to a local immune response toward a respiratory disease agent.
  • Oral immunization which is directed to other parts of the secretory system, is less preferred. Higher efficacy of the intranasal route has been observed when using the intact influenza hemagglutinin for vaccination (Hirabayachi et al., 1990; Tamura et al. , 1990).
  • the intranasal route of administration may be used employing a recombinant chimeric protein of the present invention to achieve effective protection with no added adjuvant.
  • oligonucleotides coding for several epitopes from one or several proteins of the same virus, which are expressed in either the same or separate constructs.
  • mice inbred strains BALB/c and C57BL/6 were from Olac, Blackthorn, Bicester, Oxon U.K.
  • Outbred strain mice CD1 were from the Animal Breeding Center of the Weizmann Institute of Science. All mice were used at the age of 8-12 weeks. Nine to eleven day embryonated hen eggs were obtained from the Kfar Bilu Hatchery, Israel.
  • H3N2 A/England/42/72 (H3N2) , A/Japanese/57 (H2N2) and A/PR/8/34 (H1N1) were grown in the allantoic cavity of 9-11 day old embryonates eggs and used as infectious allantoic fluid.
  • Virus was grown and purified according to standard methods (Barrett and Inglis, 1985) . Titration of virus in the allantoic fluid was performed by the hemagglutination assay using 50 ⁇ l allantoic fluid serial dilutions and 50 ⁇ l of 0.5% chicken red blood cells (RBC) in saline. The titres were expressed as hemagglutination units (HAU) .
  • RBC chicken red blood cells
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • mice were immunized either with Salmonella orally, or with the purified flagellin intranasally. Live bacteria from an overnight culture were washed in PBS and diluted to 2.5 O.D. 600 nm/ml. Each animal received 200 ⁇ l orally, using animal feeding needles (Popper & Sons, Inc. N.Y., USA). Flagellin, 50 ⁇ g per animal in 25-50 ⁇ l PBS, was administered i.n. to mice anesthetized with ether. in immunogenicity experiments, mice immunized orally with whole bacteria received 5 boosts, 7 days apart. Those immunized i.n. with purified flagellin were boosted 3 times, 14 days apart. Boosts were given with the same amounts of antigen used for the initial immunization. The animals were sacrificed 7 days after the last boost and antibody titres were evaluated.
  • mice were inoculated i.n. with 3-6 HAU
  • Virus titres were assayed by either of two methods (1) or (2) : (1) The allantoic on shell method, described by Fazekas De St. Groth and White, 1958. The assay was performed in U bottom microplates, each well containing 150 ⁇ l standard medium, a 4 x 4 mm piece of 11 days embryonated egg shell with the chorioallantoic membrane attached, and 15 ⁇ l of sample. The plates were incubated at 37°C with agitation for 48 hrs. The egg shells were then removed and 80 ⁇ l 1% RBC solution in saline was added to assay for virus presence by hemagglutination, as indicated above.
  • Peptides HA 91-108 (SEQ ID NO: , NP 55-69 (SEQ ID NO:5) and NP 147-158 (SEQ ID NO:6) were ithesized by the solid phase technique in a 430A peptide nthesizer (Applied Biosystems) , as described by Shapira et al. , 1985. After cleavage from the resin, the peptides were purified on a Sephad ⁇ x G-25 column. Synthetic oligonucleotides were prepared in a 38OB Applied Biosystems DNA synthesizer or equivalent.
  • the desired plasmids were used to transform Salmonella typhimurium LB5000 (a restrictive negative, modification proficient nonflagellated) competent cells (Bullas and Ryu, 1983, herein entirely incorporated by reference) and were then transferred to a flagellin negative live vaccine strain (an Aro A mutant) of Salmonella dublin SL5928 by transduction using the phage P22HT105/1 int (Orbach and Jackxon, 1982, and Schmieger, 1972, both herein entirely incorporated by reference) .
  • the transformed S. dublln were selected for ampicillin resistance, motility under the light microscope and growth in semisolid LB agar plates, supplemented with Oxoid nutrient broth #2. Selected clones were grown overnight in 2 liters of LB amp. medium and the flagellin was purified by acidic cleavage, according to the technique described by Bennett et al., 1985, herein entirely incorporated by reference.
  • Flagella were isolated according to Bennett et al. (1985) : Bacterial cells from an overnight culture grown in LB/ampicillin medium were pelleted and suspended in small volume of PBS. The pH was reduced with 1M HC1 to 2.0 and the suspension was incubated at room temperature for 30 minutes with gentle agitation. The stripped cells were removed by centrifugation at 5000 rpm for 15 min and the pH was readjusted to 7.4. The flagella were then precipitated by (NH 4 ) 2 S0 4 (35% w/v) and maintained overnight at 4°C. The pellet obtained after centrifugation at 10,000 rpm for 10 min at 4°C was dissolved in PBS, dialyzed against a large volume of PBS at 4°C and any formed precipitate was discarded.
  • the resultant protein was stored at -20°C.
  • This resulting flagella is an aggregate of the flagellin protein and may be used as such for a vaccine. Presence of the chimeric flagellin HA and NP epitope protein of the invention are shown in Fig. 2 after SDS-PAGE of the flagella.
  • ELISA was used for determining the presence of the inserted peptide in the chimeric flagellin, using rabbit antibodies against the synthetic peptide HA 91-108, prepared as described by Shapira et al., 1985, herein entirely incorporated by reference. The plates were pretreated with 0.2% glutaraldehyde to allow better adsorption of the peptide. ELISA was also used in the assay for specific IgA or IgG antibodies, respectively, in lung washes and sera. In this case, the antigens adsorbed to the plate were the HA peptide 91-108 (1 ⁇ l/well) or intact purified A/Texas/l/77 virus (20 HAU/well) , in 100 ⁇ l PBS.
  • Synthetic oligonucleotides (SEQ ID N0S:l-3) (Fig. 1, bottom) , coding for amino acids of epitopes HA 91-108 (SEQ ID N0:4), NP 55-69 (SEQ ID N0:5) and NP 147-158 (SEQ ID N0:6) were prepared, with the codon usage corresponding to that in the sequence of Salmonella flagellin gene (Wei and Joys, 1985), as illustrated in Fig. 1. As shown, a Mlul restriction site was created, that together with the Hindlll and Alul sites already present in the sequence, facilitate analysis of the product.
  • An inverted stop codon was also chosen so as to present expression in case of insertion of the synthetic oligonucleotide in the wrong orientation (Fig. 1) .
  • the synthetic oligonucleotide was inserted into the plasmid pLS408 and transformed into E. coll JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative "live vaccine" Aro A" mutant of Salmonella dublln .
  • the recombinant bacteria carrying the epitopes HA The synthetic oligonucleotide was inserted into the plasmid pLS408 and transformed into E. coll JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative "live vaccine" Aro A" mutant of Salmonella dublln .
  • 91-108 (SEQ ID N0:4) , NP 55-69 (SEQ ID N0:5) and NP 147-158 (SEQ ID NO:6) were designated Salmo-91, Salmo-55 and Salmo- 147, respectively, and the flagellin purified from them were denoted Fla-91, Fla-55 and Fla-147, respectively.
  • Salmo- control and Fla-control are the terms given to the bacteria expressing flagellin coded by plasmid pLS408 and the flagellin purified from them, respectively.
  • influenza sequences in the flagellin chimeric proteins were demonstrated by the following criteria: (a) a shift in molecular weight as observed in SDS-PAGE (Fig. 2) , as the recombinant flagellin has a slightly higher molecular weight than the native one; (b) recognition of the recombinant flagellin by anti-HA 91- 108 antibodies, as demonstrated by ELISA (Fig. 3) .
  • the i munogenicity of a recombinant bacteria of the present invention was first tested in rabbits, which were immunized either i.m. with the live recombinant bacteria, or s.c. with the isolated Fla-91 in the presence of CFA. Both immunizations resulted in a specific IgG response against the synthetic peptide, as well as against the intact A/Texas/l/77 virus (Fig. 4A-B) . In the latter case, the immunization with whole bacteria elicited higher antibody levels.
  • mice were immunized and given 3-5 boosters, in order to ensure a positive response.
  • a specific humoral response was observed in all these strains, manifested in both IgG antibodies in the blood and IgA antibodies in the lungs.
  • the antibodies reacted with the synthetic peptide, as well as with the intact virus (Fig. 5A- D) .
  • the protection was evaluated by comparing the lung virus titre, as manifested by the hemagglutination capacity, in the immunized mice, with that of untreated mice or those immunized with the salmo- or Fla- controls, which do not express the HA epitope.
  • the isolated hybrid flagellin Fla- 91 preparation (empty circles) provided better protection, as manifested not only by the lower incidence of infection, but also by the egg-infective dose of the lung homogenates.
  • Fla-91 and Fla-147 administered i.n. individually or in various combinations were compared. As shown in Fig. 7. Fla- 91 administered intranasally is effective, but it appears that a combination of Fla-91 (designed to produce antibodies) and Fla-147 (for CTL activity) is superior to Fla-91 or Fla- 147 alone.
  • the BALB/c mice were challenged with 6 HAU A/Texas/l/77.
  • Figure 9 shows the protective effect of the two combinations Fla-91 + Fla-147 and Fla-55 + Fla-91 + Fla-147 administered i.n. to BALB/c mice challenged with 1 HAU of A/England/42/72 (H3N2) (A) , A/Japanese/57 (H2N2) (B) and A/PR/8/34 (H1N1) (C) .
  • H3N2 A/England/42/72
  • H2N2N2N2 A/Japanese/57
  • B A/PR/8/34
  • H1N1N1N1 A/PR/8/34
  • mice were immunized i.n. with Fla-control, Fla-55, Fla-147 and a combination of Fla-55 + Fla-147.
  • the animals (four per group) were boosted once after 3 weeks, and 3 weeks later the lungs were removed, fixed and stained using the light green staining procedure.
  • the stained lung sections from the immunized mice were compared to those from untreated animals and from an infected mouse. The sections were examined under a light microscope for the presence of infiltrating lymphocytes.
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Abstract

Synthetic recombinant vaccines comprising one or more chimeric proteins comprising an amino acid sequence of flagellin and an amino acid sequence of an epitope of influenza virus hemagglutinin (HA) or nucleoprotein (NP), and aggregates thereof, are provided for immunization orally or intranasally against a plurality of different influenza virus strains. Preferred epitopes are HA 91-108, NP 55-69 and NP 147-158 or combination thereof. Such chimeric proteins can be administered individually or in combination. Further provided are vaccines comprising a genetically engineered non-virulent Salmonella strain expressing at least one of said epitopes in the flagella.

Description

RECOMBINANT INFLUENZA VACCINES
FIELD OF THE INVENTION
The present invention relates to synthetic recombinant vaccines, particularly to synthetic recombinant influenza vaccines in which one or more of the influenza virus hemagglutinin or nucleoprotein epitopes are expressed in Salmonella flagellin, and their use for mucosal and systemic immunization.
BACKGROUND OF THE INVENTION
Influenza virus appears as three subtypes designated A, B and C, of which subtype A comprises the major antigenic compounds that are associated with pandemics. Since subtype A is capable of changing its antigenic identity in a remarkable way, the specific immunity established in response to infection or vaccination by a particular strain may give little or no protection against subsequent infections by virus, this being an explanation for influenza still being a major epidemic disease in man.
Influenza virus comprises two surface antigens: the neuraminidase (NA) , which is common to many influenza strains and antibodies against which are almost non- neutralizing and non-protective, and the hemagglutinin (HA) , which undergoes gradual changes (drifts) , is a strong immunogen and is the most significant antigen in defining the serological specificity of the different viral strains. Antibodies to the HA neutralize virus infectivity, and resistance to influenza infection was shown to correlate with serum anti-HA antibodies levels.
The HA is a molecule of molecular weight of 75-80 kD. It comprises a plurality of antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are common for the various known hemagglutinins (common determinants) . U.S. Patent No. 4,474,757, herein entirely- incorporated by reference, describes several synthetic peptides corresponding to antigenic fragments of the hemagglutinin component of the influenza viruses, which antigenic fragments are common to a plurality of different influenza virus strains and are capable of eliciting antibodies capable of neutralizing each of said intact differing influenza virus strains, when bound to a suitable macromolecular carrier, such as purified tetanus toxoid. These conjugates are useful as syntheticvaccines for vaccination of mammals against each of a plurality of differing influenza virus strains, when administered by subcutaneous or intraperitoneal injection.
One of these peptides, corresponding to the HA epitope 91-108 of H3 influenza subtype, was shown to be immunogenic when coupled to tetanus toxoid (Muller et al. , 1982) . When this antigen was administered parenterally to mice, either in complete Freundrs adjuvant or as a conjugate with the synthetic adjuvant muramyl dipeptide, it led to partial protection against challenge infection (Shapira et al., 1985) .
The"nucleoprotein (NP) is located in the viral core which consists of RNA segments, the three different polymerase proteins and the NP. It is one of the group specific antigens which distinguishes between influenza A, B and C viruses. Influenza A viruses can be classified into at least five groups based on RNA- NA hybridization studies of NP genes. NP genes of several influenza A viruses isolated from different species have been sequenced and the derived amino acid sequences were compared. The results suggest that only two different NP subtypes exist: subtype 1, found with all human strains, and subtype 2, found with all avian strains. However, there are no significant differences between these two groups concerning secondary structure predictions (Gammelin et al., 1989). Indeed, in contrast to the HA, the NP is one of the most conserved viral proteins, being 94% conserved in all influenza A viruses (Townsend & Skehel, 1984).
Influenza A virus NP-specific antibody has no virus neutralizing activity, but NP is an important target for cytotoxic lymphocytes (CTL) which are cross-reactive for all type A viruses (Townsend & Skehel, 1984; Yewdell et al., 1985) . This emerged from experiments using target cells transfected with a cloned NP gene (Townsend et al., 1984) and also cells infected with a NP-vaccinia virus recombinant (Yewdell et al., 1985). However, it was not clear whether NP-specific CTL had a role in protection against influenza infection in vivo until Taylor and Askonas (1986) showed that a series of NP-specific CTL transferred into syngeneic mice were capable of limiting virus replication in the lungs and trachea of influenza infected hosts and could protect it against lethal infection. This and other studies have emphasized the potential of NP as an alternative vaccine that could overcome the limitations of inactivated influenza vaccines which do not induce A-virus cross-reactive T cells (Webster & Askonas, 1980) .
Townsend et al. (1985 and 1986) demonstrated that CTL recognize fragments of influenza NP rather than the whole protein, showing that CTL recognize short synthetic pep ides corresponding to linear regions of the NP molecule. The last decade has seen the development of new approaches to vaccine preparation. In contrast to the conventional vaccines, which are based on the entire disease causing organism, in either killed or live attenuated form, or on the intact detoxified bacterial toxins, some new approaches are based on synthetic products. These can be prepared by: a) synthesis of antigenically relevant proteins of the organism by recombinant DNA technology in which such proteins are expressed in bacteria, yeasts or mammalian cells, followed by isolation of the proteins (Woodrow, 1990) , or production of bacteria (Salmonella) or viruses
(vaccinia) expressing such proteins which can be used as live vaccines (WHO Meeting, 1989); (b) chemical synthesis of peptide epitopes of such proteins, and their conjugation to appropriate carriers; or (c) a combination of both approaches, namely insertion of oligonucleotides coding for these epitopes in suitable vectors, for their expression by genetic engineering procedures.
The latter approach was recently demonstrated for peptides of two bacterial toxins, cholera toxin (Newton et al., 1989) and Shiga toxin. In the case of Shiga toxin, a fusion protein in E. coll expressing the N-terminal region of the toxin led to a humoral response (McEwen et al., 1989) .
In the case of cholera .toxin, both an isolated fusion protein and live Salmonella (Newton et al., 1989) expressing a critical epitoper CTP3, were shown to induce neutralizing anti-toxin effect. There was no attempt in either case to test the efficacy against bacterial challenge infection.
Attenuated Salmonella, which induce a broad immune response after oral administration (Dougan et al., 1987), offer a convenient way of presenting heterologous antigens to the immune system. In recent studies, using living or formalin-inactivated Salmonella as a carrier, it was possible to induce an immune response directed against several foreign antigens, including cholera toxin epitope (Newton et al., 1989) r malaria antigens (Sadoff et al., 1988), Hepatitis B surface antigen (Wu et al., 1989), tetanus toxin (Fairweather et al., 1990) and Streptococcal M protein (Poirier et al., 1988) .
Currently available influenza vaccines are administered parenterally and consist of whole virus vaccine, subunit or "split" vaccines, or live attenuated virus vaccines. Each of these types has problems concerning efficacy and safety. The live vaccine is more potent, but it is not considered safe enough. In view of these limitations, novel approaches for influenza vaccines are being sought.
Various studies have suggested that resistance to respiratory viral infections is mainly mediated by antiviral IgA antibodies which are generated in respiratory mucosa, rather than in the serum (Liew et al., 1984; Perkins et al., 1989; Scott and Sydiskis, 1976). Indeed, both oral and intranasal administration of influenza antigens were shown to provoke in mice humoral IgA response in the lungs, that protected them from viral challenge (Chen and Ominnau, 1989; Wright et al., 1983).
It would be most advantageous to obtain influenza vaccines to be administered by oral or intranasal route that provide effective mucosal immunization and more efficient protection against infection by several different types of influenza virus.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome one or more deficiencies of the background art. It is also an object of the present invention to provide a chimeric protein comprising the amino acid sequence of flagellin and the amino acid sequence of an epitope of an influenza virus hemagglutinin or nucleoprotein, and aggregates thereof. It is another object of the present invention to provide a synthetic recombinant vaccine against a plurality of different influenza virus comprising a chimeric protein of the invention or an aggregate thereof, with or without any adjuvant. A further object of the invention is to provide synthetic recombinant influenza vaccines in which one or a few predominant epitope(s) of the influenza virus are expressed by appropriate non-virulent Salmonella strains, for the induction of mucosal and systemic immunity.
It is yet another object of the invention to prepare recombinant vaccines expressing various epitopes of influenza virus hemagglutinin and/or nucleoprotein, or combinations thereof, in a non-virulent Salmonella vaccine strain. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A-D depicts structure of pLS408 vector (1A) and the base sequence of the insert (IB, SEQ ID NO:l; 1C, SEQ ID NO:2; ID, SEQ ID NO:3). Hl-d is the flagellin gene from Salmonella munchen. The oligonucleotide sequences shown at the bottom were designed to code for epitope 91-108 of the influenza A H3 subtype HA, and epitopes 55-69 and 147- 158 from the nucleoprotein. Codon usage was according to the sequence of the flagellin gene, with minor modifications in order to create the Mlul restriction site, and a stop codon for the inserts which are in the wrong orientation.
Figure 2 shows shift in molecular weight of the recombinant flagellins Fla-91, Fla-55 and Fla-147 when compared to the native flagellin (Fla-control) , as demonstrated in SDS-PAGE (10%) of the purified chimeric flagellins-
Figure 3A-B shows detection of influenza epitope in the recombinant flagellin. Binding of anti-flagellin antibodies (filled lozenges) , antipeptide 91-108 antibodies (filled circles) and preimmune serum (empty lozenges) , to: 3A- Pure native flagellin. ; 3B- Purified chimeric flagellin Fla-91.
Figure 4A-B shows binding in ELISA of antisera from rabbits immunized with: Salmo-91 (live bacteria i.m.) (filled squares) , or Fla-91 (purified hybrid flagellin, s.c. with CFA) (filled circles) , as compared to preimmune sera (empty circles) . A- Binding to synthetic peptide HA 91-108; B- Binding to intact influenza A/Texas/77 virus.
Figure 5 shows humoral response of CD1 mice immunized with Salmo-91 (filled triangles) and Salmo-control
(empty triangles) , given orally, or Fla-91 (filled circles) and Fla-control (empty circles), given intranasally (i.n.), measured by binding in ELISA of IgA from lung washes (A and
B) and IgG from serum (C and D) to the synthetic peptide HA 91-108 (A, C) or the intact virus (B, D) .
Figure 6 shows the protective effect of recombinant vaccines in BALB/c mice, expressed by percent of lung homogenates that exhibit viral growth in embryonated eggs, as a function of the homogenate dilution. Immunizations were with: Untreated control (empty squares) , Fla-control (empty lozenges) and Fla-91 (empty circles) , given intranasally. Error bars represent the SEM values.
Figure 7 shows immunization (three times, at three week intervals) of BALB/c mice with Fla-91, Fla-147 and a combination of the two preparations, intranasally, and the protective capacity of these vaccinations, as compared to controls, against viral challenge.
Figure 8 shows the effect of combination of the various chimeric flagellins, on the protection of the immunized mice from viral challenge. The immunizations were given i.n. three times at three week intervals. The challenge was done one month after the last boost.
Figure 9A-C shows cross protection of BALB/c mice vaccinated with combinations of recombinant flagellins against various influenza virus strains.
Figure 10A-B shows binding in ELISA of IgA antibodies in the lung homogenates of the BALB/c mice from the protection experiment, to peptide HA 91-108 (10A) and the intact virus (10B) . Immunization was intranasally with: Untreated control (empty squares) , Fla-control (empty circles) , or Fla-91 (filled circles) . Figure 11A-F shows staining of lymphocyte infiltrations (indicated by arrows) in lung sections taken from mice immunized with untreated and Fla-control (11A-B) , Fla-55 and Fla-147 (11C-D) , combination of Fla-55 + Fla-147 and infected mice (11E-F) . Each section represents the general condition of 4-5 animals per treatment. Magnification xl50.
DETAILED DESCRIPTION OF THE INVENTION
The chimeric protein of the invention comprises the amino acid sequence of flagellin and at least one amino acid sequence of an influenza virus hemagglutinin and/or nucleoprotein epitope. The aggregates, comprising several subunits of the chimeric protein, are represented by the bacterial flagella.
Any of the known influenza virus hemagglutinin epitopes, such as those described in US 4,474,757, herein incorporated by reference, may be used in the present invention. In a preferred embodiment, epitope 91-108 may be used, which is a highly conserved region in all type H3 strains and confers partial protection against challenge with several strains. Hemagglutinin epitope 91-108 has the following sequence:
Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-Tyr-Asp-Val- Pro-Asp-Tyr-Ala-Ser-Leu. (SEQ ID NO:4) According to a non-limiting example of the present invention, a chimeric protein comprising flagellin and the influenza virus hemagglutinin epitope 91-108, expressed in Salmonella flagellin, is useful as a component of a vaccine of the present invention. For that purpose, a synthetic oligonucleotide comprising 54 bases coding for the corresponding sequence was inserted into the plasmid pLS408 and transformed into E. coll JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative "live vaccine" Aro A" mutant of Salmonella dublin. Rabbits immunized either with che live recombinant S. dublln or with the flagellin isolated from it, showed significant levels of IgG response against the synthetic peptide 91-108 as well as against the intact A/Texas/77 influenza virus. Mice immunized with the same preparations developed influenza-specific IgG antibodies in the blood and secreted IgA antibodies in their lungs.
Furthermore, these mice showed about 50% protection against challenge infection with the virus. Intranasal immunization with the isolated recombinant chimeric flagellin, when employed without the aid of adjuvant, is a preferred method. Any nucleoprotein epitope may be used in the present invention, such as the non-limiting examples of NP 55-69, a T-helper epitope and NP 147-158. NP 55-69 has the following amino acid sequence: Arg-Leu-Ile-Gln-Asn-Ser-Leu-Thr-Ile-Glu-Arg-Met- Val-Leu-Ser (SEQ ID NO:5) .
NP 147-158 has the following amino acid sequence: Thr-Tyr-Gln-Arg-Thr-Arg-Ala-Leu-Val-Arg-Thr-Gln
(SEQ ID NO:6) .
In order to obtain the chimeric protein or the genetically engineered Salmonella for use as a vaccine, an oligonucleotide coding for the respective epitope is synthesized and inserted into a suitable expression vector, i.e., plasmid pLS408, depicted in Fig. 1. Suitable bacterial cells, e.g., E. coll JM 101 cells, are transformed with the expression vector containing the desired oligonucleotide, and positive colonies are used to transform a virulent pathogenic strain of Salmonella, such as Salmonella typhimurium LB 5000 (Smith et al. , 1983) and are then transduced to a nonvirulent Salmonella vaccine strain, such as a flagellin negative live vaccine Aro A" mutant of Salmonella dublin (Smith et al. , 1984). The epitope is then expressed as a chimeric protein with flagellin in the flagella, which is constituted of subunits of flagellin. The flagella comprising the chimeric protein may be isolated and purified for use in the vaccine, or the intact bacteria may be used as a vaccine, as a non-limiting example. According to the present invention, a synthetic recombinant vaccine against a plurality of different influenza virus strains is provided comprising at least one chimeric protein comprising the amino acid sequence of flagellin and the amino acid sequence of influenza virus hemagglutinin or nucleoprotein epitope, and aggregates thereof. Preferred epitopes according to the invention are the influenza epitopes HA-91-108 (SEQ ID N0:4) , NP 55-69 (SEQ ID NO:5) and NP-147-158 (SEQ ID NO:6) . In preferred embodiments, combinations of HA 91-108 and NP 147-158 or of the three epitopes, are used. While the double combination increased the protective capacity of the HA 91-108 epitope, the triple combination seems to be superior to all other preparations.
In another embodiment, the invention provides a synthetic recombinant vaccine against a plurality of different influenza virus strains comprising a non-virulent Salmonella strain into which an expression vector comprising a nucleotide sequence encoding an epitope of influenza virus hemagglutinin or nucleoprotein was introduced, said strain being capable of expressing said epitope in the flagella within the flagellin sequence as a chimeric protein. In one embodiment, the Salmonella strain is genetically engineered Salmonella dublln expressing one of the epitopes HA 91-108 (SEQ ID N0:4), NP 55-69 (SEQ ID N0:5) or NP 147-158 (SEQ ID NO:6) . Several routes of administration are envisaged for the vaccines of the present invention. A humoral immune response is shown following oral immunization with the recombinant bacteria, or intranasal immunization with the purified chimeric flagellin. Intranasal vaccination with the hybrid flagellin also conferred partial protection against viral challenge.
A preferred embodiment of the invention is the effective immunization afforded by using the intranasal immunization route, which delivers the antigens to the nasal cavity, and the upper part of the lungs, thereby leading to a local immune response toward a respiratory disease agent. Oral immunization, which is directed to other parts of the secretory system, is less preferred. Higher efficacy of the intranasal route has been observed when using the intact influenza hemagglutinin for vaccination (Hirabayachi et al., 1990; Tamura et al. , 1990).
As a non-limiting example, the intranasal route of administration may be used employing a recombinant chimeric protein of the present invention to achieve effective protection with no added adjuvant. These results are of commercial significance since, in the design of human vaccines, one of the biggest obstacles is the development of an adequate adjuvant. With the exclusion of alum, no adjuvant for human use has been approved as yet for safety. It is therefore an advantage of the present invention that the vaccine preparations do not require adjuvants, and in addition may be administered by the intranasal route.
In order to augment the protective effect of a vaccine of the present invention, it is possible to provide simultaneously oligonucleotides coding for several epitopes, from one or several proteins of the same virus, which are expressed in either the same or separate constructs.
In the following description, the invention will be illustrated with reference to specific methods and materials for preparing the synthetic recombinant vaccine and for testing its antiviral effects in animal models, as also used in the following examples.
MATERIALS AND METHODS Animals
Rabbits, New Zealand strain, 3-4 months old were obtained from the Animal Breeding Center of the Weizmann
Institute of Science, Rehovot, Israel. Mice inbred strains BALB/c and C57BL/6, were from Olac, Blackthorn, Bicester, Oxon U.K. Outbred strain mice CD1, were from the Animal Breeding Center of the Weizmann Institute of Science. All mice were used at the age of 8-12 weeks. Nine to eleven day embryonated hen eggs were obtained from the Kfar Bilu Hatchery, Israel.
Virus Influenza virus strains A/Texas/1/77 (H3N2) ,
A/England/42/72 (H3N2) , A/Japanese/57 (H2N2) and A/PR/8/34 (H1N1) were grown in the allantoic cavity of 9-11 day old embryonates eggs and used as infectious allantoic fluid. Virus was grown and purified according to standard methods (Barrett and Inglis, 1985) . Titration of virus in the allantoic fluid was performed by the hemagglutination assay using 50 μl allantoic fluid serial dilutions and 50 μl of 0.5% chicken red blood cells (RBC) in saline. The titres were expressed as hemagglutination units (HAU) .
Immunization and infection procedures Rabbits were injected either with intact
Salmonella intramuscularly (i.m.) or with the purified flagellin subcutaneously (s.c). The flagellin was injected in complete Freund's adjuvant (CFA) 1 mg/animal, in 1 ml emulsion. Three booster injections were given at 3-4 week intervals, with half the amounts of the antigen. In the case of the flagellin, incomplete Freund's adjuvant (IFA) was used. Blood was collected 4-7 days after boosts.
Mice were immunized either with Salmonella orally, or with the purified flagellin intranasally. Live bacteria from an overnight culture were washed in PBS and diluted to 2.5 O.D.600 nm/ml. Each animal received 200 μl orally, using animal feeding needles (Popper & Sons, Inc. N.Y., USA). Flagellin, 50μg per animal in 25-50 μl PBS, was administered i.n. to mice anesthetized with ether. in immunogenicity experiments, mice immunized orally with whole bacteria received 5 boosts, 7 days apart. Those immunized i.n. with purified flagellin were boosted 3 times, 14 days apart. Boosts were given with the same amounts of antigen used for the initial immunization. The animals were sacrificed 7 days after the last boost and antibody titres were evaluated.
Lung washes and blood collection
In order to assay the production of IgA (in lungs) or IgG (in serum) in the immunized mice, a procedure essentially as described by Liew et al., 1984 was employed. Briefly, a canula was inserted into the trachea, and the lungs were washed with 5 ml PBS per animal, using a three-way stopcock. The fluid obtained was centrifuged in order to remove cells and debris, concentrated and assayed for IgA. Blood accumulated in the thoracic cavity was assayed for IgG. Viral challenge and virus titration
In challenge studies to assess the antiviral activity elicited by the immunization, seven days after the last boost, mice were inoculated i.n. with 3-6 HAU
A/Texas/1/77 or with 1 HAU of the other viruses used, and three days later the animals were sacrificed, their lungs removed, and blood collected. All samples were stored at 70°C. Immediately prior to the assay, lungs were thawed, homogenized in PBS (10% w/v) and centrifuged in order to remove debris.
Virus titres were assayed by either of two methods (1) or (2) : (1) The allantoic on shell method, described by Fazekas De St. Groth and White, 1958. The assay was performed in U bottom microplates, each well containing 150 μl standard medium, a 4 x 4 mm piece of 11 days embryonated egg shell with the chorioallantoic membrane attached, and 15 μl of sample. The plates were incubated at 37°C with agitation for 48 hrs. The egg shells were then removed and 80 μl 1% RBC solution in saline was added to assay for virus presence by hemagglutination, as indicated above. (2) Whole egg titration, by injection of 100 μl of the lung homogenate dilutions into the allantoic cavity of 9-11 day old embryonated eggs. Following incubation for 48 hours at 37°C and overnight at 4°C, allantoic fluid was removed. Virus presence was assayed by hemagglutination in microtitre plates containing 50 μl of the allantoic fluid and 50 μl of 0.5% RBC in saline.
Synthetic peptides and oligonucleotides
Peptides HA 91-108 (SEQ ID NO: , NP 55-69 (SEQ ID NO:5) and NP 147-158 (SEQ ID NO:6) were ithesized by the solid phase technique in a 430A peptide nthesizer (Applied Biosystems) , as described by Shapira et al. , 1985. After cleavage from the resin, the peptides were purified on a Sephadεx G-25 column. Synthetic oligonucleotides were prepared in a 38OB Applied Biosystems DNA synthesizer or equivalent.
Preparation of recombinant bacteria The construction of the expression vector pLS408 is described by Newton et al., 1989, herein incorporated entirely by reference. The synthesized oligonucleotides were inserted at the EcoRV site of the plasmid pLS408, and transformed into E. coli JM101 competent cells. Colonies containing the recombinant plasmid were selected by probing them with one of the oligonucleotides labeled with 32P-ATP. Plasmids from positive colonies were purified and the insert orientation was determined using restriction analysis. The desired plasmids were used to transform Salmonella typhimurium LB5000 (a restrictive negative, modification proficient nonflagellated) competent cells (Bullas and Ryu, 1983, herein entirely incorporated by reference) and were then transferred to a flagellin negative live vaccine strain (an Aro A mutant) of Salmonella dublin SL5928 by transduction using the phage P22HT105/1 int (Orbach and Jackxon, 1982, and Schmieger, 1972, both herein entirely incorporated by reference) . The transformed S. dublln were selected for ampicillin resistance, motility under the light microscope and growth in semisolid LB agar plates, supplemented with Oxoid nutrient broth #2. Selected clones were grown overnight in 2 liters of LB amp. medium and the flagellin was purified by acidic cleavage, according to the technique described by Ibrahim et al., 1985, herein entirely incorporated by reference.
Isolation of flagella
Flagella were isolated according to Ibrahim et al. (1985) : Bacterial cells from an overnight culture grown in LB/ampicillin medium were pelleted and suspended in small volume of PBS. The pH was reduced with 1M HC1 to 2.0 and the suspension was incubated at room temperature for 30 minutes with gentle agitation. The stripped cells were removed by centrifugation at 5000 rpm for 15 min and the pH was readjusted to 7.4. The flagella were then precipitated by (NH4)2S04 (35% w/v) and maintained overnight at 4°C. The pellet obtained after centrifugation at 10,000 rpm for 10 min at 4°C was dissolved in PBS, dialyzed against a large volume of PBS at 4°C and any formed precipitate was discarded. The resultant protein was stored at -20°C. This resulting flagella is an aggregate of the flagellin protein and may be used as such for a vaccine. Presence of the chimeric flagellin HA and NP epitope protein of the invention are shown in Fig. 2 after SDS-PAGE of the flagella.
ELISA
A slight modification of the method described by Engvall et al. , 1972, was used, by employing ELISA immunoplates (Nunc) instead of tubes.
ELISA was used for determining the presence of the inserted peptide in the chimeric flagellin, using rabbit antibodies against the synthetic peptide HA 91-108, prepared as described by Shapira et al., 1985, herein entirely incorporated by reference. The plates were pretreated with 0.2% glutaraldehyde to allow better adsorption of the peptide. ELISA was also used in the assay for specific IgA or IgG antibodies, respectively, in lung washes and sera. In this case, the antigens adsorbed to the plate were the HA peptide 91-108 (1 μl/well) or intact purified A/Texas/l/77 virus (20 HAU/well) , in 100 μl PBS. Goat anti-mouse IgA antibodies conjugated to alkaline phosphatase (Sigma) and sheep antimouse IgG antibodies conjugated to β- galactosidase (Amersham) were used as second antibodies. Para- itrophenol (PNP) or ortho-nitrophenyl-/J-D- galactopyranoside (ONPG) , 1 mg/ml solutions (100 μl/well) , were used as substrates for the alkaline phosphatase and β- galactosidase, respectively, and the plates were read at 405 nm. Example 1- Expression of Influenza epitopes in
Salmonella flagellin
Synthetic oligonucleotides (SEQ ID N0S:l-3) (Fig. 1, bottom) , coding for amino acids of epitopes HA 91-108 (SEQ ID N0:4), NP 55-69 (SEQ ID N0:5) and NP 147-158 (SEQ ID N0:6) were prepared, with the codon usage corresponding to that in the sequence of Salmonella flagellin gene (Wei and Joys, 1985), as illustrated in Fig. 1. As shown, a Mlul restriction site was created, that together with the Hindlll and Alul sites already present in the sequence, facilitate analysis of the product. An inverted stop codon was also chosen so as to present expression in case of insertion of the synthetic oligonucleotide in the wrong orientation (Fig. 1) . The synthetic oligonucleotide was inserted into the plasmid pLS408 and transformed into E. coll JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative "live vaccine" Aro A" mutant of Salmonella dublln . The recombinant bacteria carrying the epitopes HA
91-108 (SEQ ID N0:4) , NP 55-69 (SEQ ID N0:5) and NP 147-158 (SEQ ID NO:6) were designated Salmo-91, Salmo-55 and Salmo- 147, respectively, and the flagellin purified from them were denoted Fla-91, Fla-55 and Fla-147, respectively. Salmo- control and Fla-control are the terms given to the bacteria expressing flagellin coded by plasmid pLS408 and the flagellin purified from them, respectively.
The presence of the influenza sequences in the flagellin chimeric proteins were demonstrated by the following criteria: (a) a shift in molecular weight as observed in SDS-PAGE (Fig. 2) , as the recombinant flagellin has a slightly higher molecular weight than the native one; (b) recognition of the recombinant flagellin by anti-HA 91- 108 antibodies, as demonstrated by ELISA (Fig. 3) . Example 2. Humoral Immune Response
The i munogenicity of a recombinant bacteria of the present invention was first tested in rabbits, which were immunized either i.m. with the live recombinant bacteria, or s.c. with the isolated Fla-91 in the presence of CFA. Both immunizations resulted in a specific IgG response against the synthetic peptide, as well as against the intact A/Texas/l/77 virus (Fig. 4A-B) . In the latter case, the immunization with whole bacteria elicited higher antibody levels. Based on the results obtained in rabbits which showed that the recombinant bacteria were immunogenic, these preparations were used for immunizations of three mice strains, namely C57BL/6, BALB/c and CD1, by either the oral or the intranasal route. The mice were immunized and given 3-5 boosters, in order to ensure a positive response. A specific humoral response was observed in all these strains, manifested in both IgG antibodies in the blood and IgA antibodies in the lungs. The antibodies reacted with the synthetic peptide, as well as with the intact virus (Fig. 5A- D) . As shown in this experiment, intranasal immunization with Fla-91 induced a better response to the viral epitope than oral immunization with the live Salmonella recombinant, which was proven to be ineffective. These results were obtained with CD1 mice. Similar patterns of humoral response were obtained in the two other strains.
Example 3. Protection against viral challenge
In view of the positive humoral response, the ability of the recombinant products to induce protective immunity against viral challenge was investigated.
Protection experiments were conducted in C57BL/6 and BALB/c mice. Following oral or intranasal immunization with each of Fla-55, Fla-91 and Fla-147 and combinations thereof according to the schedule described in "Immunization and infection procedures" hereinabove, the animals were challenged with 3-6 HAU of influenza A/Texas/l/77 virus or with 1 HAU of A/England/24/72, A/Japanese/57 and A/PR/8/34 virus. Three days later (assumed peak of infection) the animals were sacrificed and the presence of infectious virus in the lungs was determined by injecting lung homogenates into the allantoic cavity of embryonated eggs. The protection was evaluated by comparing the lung virus titre, as manifested by the hemagglutination capacity, in the immunized mice, with that of untreated mice or those immunized with the salmo- or Fla- controls, which do not express the HA epitope. As shown in Fig. 6, in BALB/c mice, the isolated hybrid flagellin Fla- 91 preparation (empty circles) provided better protection, as manifested not only by the lower incidence of infection, but also by the egg-infective dose of the lung homogenates. In this experiment, the control flagellin also reduced the viral titre as compared to untreated control, but to a lower extent, probably because of a non-specific immunoresponse due to the fact that the mice were challenged with the virus seven days after the last boost. This effect disappeared when the animals were challenged one month af er the last boost, as shown in the experiment of Fig. 8. Oral immunization with Salmo-91 was ineffective. In a similar experiment performed in C57BL/6 mice, essentially the same results were obtained, namely, partial protection elicited by Fla-91 intranasal immunization, while oral immunization with intact bacteria failed to protect the mice. n the following experiments, the effects of Fla-
55, Fla-91 and Fla-147 administered i.n. individually or in various combinations were compared. As shown in Fig. 7. Fla- 91 administered intranasally is effective, but it appears that a combination of Fla-91 (designed to produce antibodies) and Fla-147 (for CTL activity) is superior to Fla-91 or Fla- 147 alone. The BALB/c mice were challenged with 6 HAU A/Texas/l/77.
In another comprehensive experiment, all the possible combinations of the three epitopes were studied. The results shown in Fig. 8 exhibit a similar pattern as the previous experiments regarding Fla-91 and the Fla-91 + Fla- 147 combinations. The new combinations tested in this experiment, Fla-147 + Fla-55 (carrying a T-helper epitope) and the triple combination, namely, Fla-55 + Fla-91 + Fla-147 gave the best protection. In this experiment, BALB/c mice were challenged with 4 HAU A/Texas/1/77 four weeks after the last boost.
Figure 9 shows the protective effect of the two combinations Fla-91 + Fla-147 and Fla-55 + Fla-91 + Fla-147 administered i.n. to BALB/c mice challenged with 1 HAU of A/England/42/72 (H3N2) (A) , A/Japanese/57 (H2N2) (B) and A/PR/8/34 (H1N1) (C) . The triple combination was again shown to be superior, even against the more virulent A/PR/8/34 influenza strain.
Since the assumption was that the immunization by the intranasal route is effective due to the induction of local immunity, it was of interest to correlate the protection level with the titre of IgA antibodies in the lung homogenates of the mice which were used in the experiment described in Fig. 6. The antibody titre was determined by ELISA, using plates coated with either the synthetic peptide 91-108 or the intact virus, using alkaline phosphatase labeled specific anti-mouse IgA antibodies. As shown in Fig. 7, specific anti-peptide and anti-influenza IgA antibodies were present in the Fla-91 immunized mice, and not in the non-immunized mice. In the mice immunized with the Fla control, a low titre of anti-influenza IgA antibodies can be detected, much lower than the response in the Fla-91 immunized mice.
Example 4. Cellular Response in order to evaluate the in vivo cellular response to the recombinant vaccines, mice were immunized i.n. with Fla-control, Fla-55, Fla-147 and a combination of Fla-55 + Fla-147. The animals (four per group) were boosted once after 3 weeks, and 3 weeks later the lungs were removed, fixed and stained using the light green staining procedure. The stained lung sections from the immunized mice were compared to those from untreated animals and from an infected mouse. The sections were examined under a light microscope for the presence of infiltrating lymphocytes.
From the results presented in Fig. 11A-F, it can be seen that immunizations with Fla-55 resulted in mile infiltration of lymphocytes, while Fla-147 and the combination gave extensive infiltration. The response was less than in the case of viral infection, yet significant when compared to the untreated and Fla-control samples.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and" guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
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Townsend, A.R.M. et al., Cell, 1984, 39, 13-25 Townsend, A.R.M. et al., Cell, 1985, 42, 457-467 Townsend, A.R.M. et al.. Cell, 1986, 44, 959-968 Webster, R.G. and Askonas, B.A. , Eur. J. Immunol. 1980, 10, 396-401
Wei, L.N. and Joys, T.M. J. Mol. Biol. 1985, 186, 791-803 WHO Meeting Geneva 19-22 June 1989. Vaccine 1990, 8, 425-437 Woodrow, G.C In : New generation vaccines (Ed. Woodrow
G.C. and Levine M.M. ) Marcel Dekker, Inc. New York 1990, pp. ' 31-41
Wright, P.F. et-al., Infect. Immun. 1983, 40, 1092-1095 Wu, J.Y. et al., PNAS USA 1989, 86, 4726-4730 Yewdell, J.W. , et al. PNAS USA 1985, 82, 1785-1789
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ARNON, Ruth
LEVI, Raphael
(ii) TITLE OF INVENTION: RECOMBINANT INFLUENZA VACCINES
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BROWDY & NEIMARK (B) STREET: 419 Seventh Street, N.W.
(C) CITY: Washington
(D) STATE: D.C
(E) COUNTRY: U.S.A.
(F) ZIP: 20004
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Browdy, Roger L.
(B) REGISTRATION NUMBER: 25,618 (C) REFERENCE/DOCKET NUMBER: ARNON=5PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-628-5197
(B) TELEFAX: 202-737-3528
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TCT AAA GCT TTC TCT AAC TGT TAT CCT TAT GAT GTT CCG GAT TAC GCG 48 Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 15
TCT TTA
54 Ser Leu
{2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..48
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ATC CGT CTG ATT CAG AAT TCT CTG ACT ATT GAA CGT ATG GTC CTA TCT
48 lie Arg Leu lie Gin Asn Ser Leu Thr lie Glu Arg Met Val Leu Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:3: (±) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ACT TAT CAG CGG ACC CGT GCT TTA GTT CGT ACT GGT GAT 39 Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Asp 1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Al 1 5 10 15
Ser Leu
(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: lie Arg Leu lie Gin Asn Ser Leu Thr lie Glu Arg Met Val Leu Se i 5 10 15
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Asp 5 10

Claims

1. A chimeric protein comprising the amino acid sequence of flagellin and at least one amino acid sequence of an epitope of influenza virus hemagglutinin or nucleoprotein, and aggregates thereof.
2. A chimeric protein according to claim 1 comprising an influenza virus hemagglutinin epitope, and aggregates thereof.
3. A chimeric protein according to claim 2 comprising the influenza virus hemagglutinin epitope 91-108 of the sequence
Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-Tyr-Asp-Val- Pro-Asp-Tyr-Ala-Ser-Leu (SEQ ID N0:4) .
4. A chimeric protein according to claim l comprising an influenza virus nucleoprotein epitope.
5. A chimeric protein according to claim 4, comprising the influenza virus nucleoprotein epitope 55-69 of the sequence:
Arg-Leu-Ile-Gln-Asn-Ser-Leu-Thr-Ile-Glu-Arg-Met- Val-Leu-Ser (SEQ ID N0:5) .
6. A chimeric protein according to claim 4, comprising the influenza virus nucleoprotein epitope 147-158 of the sequence:
Thr-Tyr-Gln-Arg-Thr-Arg-Ala-Leu-Val-Arg-Thr-Gln (SEQ ID NO:6) .
7. A synthetic recombinant vaccine against a plurality of different influenza virus strains comprising at least one chimeric protein according to any of claims 1 to 6, or an aggregate thereof.
8. A synthetic recombinant vaccine according to claim 7, comprising the chimeric protein of claim 3.
9. A synthetic recombinant vaccine according to claim 7, comprising a combination of the chimeric proteins of any of claims 3, 5 and 6.
10. A recombinant vaccine against a plurality of different influenza virus strains, comprising a non- virulent Salmonella strain into which an expression vector comprising a nucleotide sequence encoding an epitope of influenza virus hemagglutinin or nucleoprotein has been introduced, said strain being capable of expressing said epitope in the flagella within the flagellin sequence as a chimeric protein.
11. A recombinant vaccine according to claim 10, comprising genetically engineered Salmonella dublin expressing an influenza virus hemagglutinin nucleoprotein or epitope.
12. A synthetic'recombinant influenza vaccine, according to any of claims 7 to 11, for oral immunization.
13. A synthetic recombinant influenza vaccine, according to any of claims 7 to 11, for intranasal immunization.
14. A synthetic recombinant vaccine, according to claim 13, for administration without adjuvant.
PCT/US1993/003606 1992-04-17 1993-04-19 Recombinant influenza vaccines WO1993020846A1 (en)

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