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  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • A61P31/12—Antivirals
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  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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  • 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

Definitions

• the present invention relates to influenza vaccines, and particularly to peptide-based vaccines comprising conserved epitopes of both B and T-lymphocytes recognized by the prevalent HLA's in humans.
• ABBREVIATIONS Ab: Antibodies; CTL: Cytotoxic T-lymphocytes; EID: Egg-infective dose; HA: Hemagglutinin; HAU: Hemagglutination unit; i.n.: intranasal; i.p.: intraperitoneal; NP: Nucleoprotein; PMBC: Peripheral blood ononuclear cells; TT: Tetanus toxoid.
• Influenza is a public health concern, it results in economic burden, morbidity and even mortality. Influenza infection may result in a variety of disease states, ranging from sub-clinical infection through a mild upper respiratory infection and tracheobronchitis to a severe occasionally lethal viral pneumonia. The reasons for this wide spectrum of severity are explained by the site of infection and the immune status of the host. The most important characteristic of influenza, from the immunological point of view, is the rapid, unpredictable changes of the surface glycoproteins, haemagglutinin and neuraminidase, referred to as antigenic shifts and drifts. These changes lead eventually to the emergence of new influenza strains, that enable the virus to escape the immune system and are the cause for almost annual epidemics (Laver et al . , 1980 and 1980a; Webster, 1982).
• influenza vaccines currently available and licensed are based either on whole inactive virus, or on viral surface glycoproteins . These influenza vaccines fail to induce complete, long-term and cross-strain immunity.
• Influenza virus comprises two surface antigens: neuraminidase (NA) and hemagglutinin (HA) , which undergo gradual changes (shifts and drifts), leading to the high antigenic variations in influenza.
• NA neuraminidase
• HA hemagglutinin
• the HA molecule (75-80 kD) comprises a plurality of antigenic determinants, several of which are in regions that undergo seguence changes in different strains (strain-specific determinants) and others in regions which are common to many HA molecules (common determinants) .
• US 4,474,757 describes a synthetic vaccine against a plurality of different influenza virus comprising a suitable macromolecular carrier having attached thereto a peptide being an antigenic fragment of HA which is common to a plurality of different influenza virus strains.
• a suitable macromolecular carrier having attached thereto a peptide being an antigenic fragment of HA which is common to a plurality of different influenza virus strains.
• One of the described common determinants is the HA epitope 91-108 which is conserved in all H3 influenza subtype strains.
• the nucleoprotein (NP) is located in the viral core and is one of the group specific antigens which distinguishes between influenza A, B and C viruses. In contrast to the HA, the NP is one of the most conserved viral proteins, being 94% conserved in all influenza A viruses .
• Influenza A virus NP-specific antibody has no virus neutralizing activity, but NP is an important target for cytotoxic T lymphocytes (CTL) which are cross-reactive with all type A viruses (Townsend and Skehel, 1984). CTL recognize short synthetic peptides corresponding to linear regions of the influenza NP molecule (Townsend et al . , 1985 and 1986).
• PCT International Publication WO 93/20846 describes 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 at least one amino acid sequence of an epitope of influenza virus HA or NP, or an aggregate of said chimeric protein.
• a synthetic recombinant anti-influenza vaccine based on three epitopes was found to be highly efficient in mice.
• This vaccine included HA 91-108, a B cell epitope from the HA which is conserved in all H3 strains and elicits anti-influenza neutralizing antibodies, together with a T-helper and CTL epitopes from the NP(NP 55-69 and NP 147-158, respectively), which induce MHC-restricted immune responses.
• Each of these epitopes was expressed in the flagellin of Salmonella vaccine strain. The isolated flagella were administered intranasally to mice, resulting in protection against viral infection (Levi and Arnon, 1996) .
• influenza peptide epitopes reactive with human cells were expressed in Salmonella flagellin and tested for efficacy in a human/mouse radiation chimera in which human PBMC were functionally engrafted. Clearance of the virus after challenge and resistance to lethal infection was found only in the vaccinated mice and production of virus specific human antibodies was also higher m this group. FACS analysis showed that most numan cells in the transplanted mice were CD8+ and CD4+, indicating that the protection was mediated mainly by the cellular immune response.
• the present invention thus relates to a human synthetic peptide-based influenza vaccine for intranasal administration comprising a mixture of flagella containing at least four epitopes of influenza virus each expressed individually in Salmonella flagellin, said influenza virus epitopes being reactive with human cells and being selected from the group consisting of: (i) one B-cell hemagglutinin (HA) epitope;
• HA hemagglutinin
• NP nucleoprotein
• M matrix protein
• the preferred B-cell HA epitope is the influenza virus hemagglutinin epitope 91-108 [HA 91-108] of the sequence: Ser-Lys-Ala-Phe-Ser-Asn-Cys-Tyr-Pro-
• T-helper epitopes are the influenza virus hemagglutinin epitope 307-319 [HA 307-319] of the sequence:
• cytotoxic T-lymphocyte (CTL) epitopes used in the vaccine of the invention will change according to the population type, namely Caucasian or non-Caucasian (of Asian or African origin) .
• the preferred CTL epitopes are the influenza virus nucleoprotein (NP) epitope 335-350 [NP 335-350] of the sequence: Ser-Ala-Ala-Phe-Glu-Asp-Leu-Arg- Val-Leu-Ser-Phe-Ile-Arg-Gly-Tyr and the NP epitope 380-393 [NP 380-393] of the sequence: Glu-Leu-Arg-Ser-Arg-Tyr-Trp- Ala-Ile-Arg-Thr-Arg-Ser-Gly
• the intranasal influenza vaccine consists of a mixture of the four influenza virus epitopes: hemagglutinin epitopes HA91-108 and HA307-319, and nucleoprotein epitopes NP335-350 and NP380-393, expressed individually in Salmonella flagellin.
• other CTL epitopes can be used.
• the present invention also relates to the use of a mixture of flagella containing at least four epitopes of influenza virus each expressed individually in Salmonella flagellin, as described above, for the preparation of a human synthetic influenza vaccine for intranasal administration.
• the present invention further relates to a method for inducing a human immune response and conferring protection against influenza virus in humans, which comprises administering intranasally to human individuals a synthetic peptide-based influenza vaccine comprising a mixture of flagella, as described above.
• tetra construct means a mixture of the flagella expressing the four influenza epitopes HA91-108, HA307-319, NP335-350 and NP380-393, respectively.
• Figs.lA-lB depict typical FACS histograms of human lung lymphocytes in human/mouse radiation chimera, immunized with the tetra construct. The samples were taken 7 days after the immunization. The cells were separated on phicoll gradient and stained with anti-CD45 together with anti-CD3 (Fig. 1A) or together with anti-CD19 (Fig. IB), conjugated to the respective fluorescence dye.
• Fig.2 Lungs homogenates from the immunized and non- immunized mice as well as a group of non-transplanted mice were analyzed for the virus titer 5 days after viral challenge. The mice were immunized with the tetra construct (left block) or native flagellin that does not express the influenza epitopes (middle block) . Another control group did not receive PBMC but were immunized with the tetra construct (right block) . The figure presents mean data from 7 repeated experiments, in which each group consisted of 6-8 animals. In each experiment different donor was employed.
• Fig.3 Human antibodies production (total amount of IgG, Ig and IgA) , in human/mouse radiation chimera (6-8 animals per group in 7 repeated experiments, different donor employed in each experiment) immunized with the tetra construct (left column) or native flagellin that does not express the influenza epitopes (middle column) .
• Another control group did not receive PBMC, but was immunized with the tetra construct (right column).
• Sera samples were diluted 1:10, lungs samples were diluted 1:60.
• Ab production in the group that was transplanted and vaccinated with the tetra construct (left column) was significantly higher than in the other control groups .
• Fig.4 Percent survival of human/mouse radiation chimera from lethal challenge after intranasal vaccination with the tetra construct.
• the mice (5-10 animals per group in 2 repeated experiments, different donor employed in each experiment) were transplanted with PBMC on day 0, vaccinated on day 9 and challenged 7 days later.
• Vaccination with the tetra construct black circles
• native flagellin native flagellin
• non-transplanted mice that were vaccinated with the tetra construct (squares) .
• the survival rate remained the same and all the surviving mice eventually recovered .
• Fig.5 Body weight of surviving mice, which is indicative of disease severity and the potential for a recovery process.
• Human/mouse radiation chimera (5-10 animals per group in 2 repeated experiments, different donor employed in each experiment) were transplanted with PBMC on day 0, vaccinated intranasally on day 9 and challenged intranasally 7 days later with a lethal dose of the virus. Mice vaccinated with the tetra construct (black circles) lost less weight and recovered more quickly than the other groups. Control groups consisted of transplanted mice that were administered with native flagellin (hollow circles) or non-transplanted mice that were vaccinated with the tetra construct (squares) . After day 40, all the surviving mice slowly recovered and gained weight .
• Fig.6 Protective vaccination of human/mouse radiation chimera transplanted with PBMC and immunized intranasally with the tetra construct.
• Each group of human/mouse chimera (5-10 animals per group in 2 repeated experiments, different donor employed in each experiment) transplanted with PBMC obtained by leukapheresis from one donor was infected 7 days after the immunization with one of three different influenza strains: A/PR/8/34 (H1N1) , A/ apanese/57 (H2N2) or A/Texas/1/77 (H3N2). Both transplanted (left column) and non-transplanted (right column) mice were vaccinated with the tetra construct. However, only the transplanted mice were able to resist the infection and the virus titer in their lungs is significantly reduced.
• Fig.7 Serum human antibodies towards influenza virus following immunization of lethally irradiated human/mouse radiation chimera (5-10 per group) radioprotected with 3x10 ⁇
• SCID bone marrow (BM) and transplanted with 70xl0 6 human PBMC All the groups were immunized with the tetra construct and then challenged with sub-lethal dose of H1N1 strain (black lozenges) or H2N2 (black circles) or H3N2 (black squares) .
• the control group consisted of irradiated SCID replenished mice that did not receive PBMC and were immunized with the same vaccine prior to challenge with H1N1 (hollow lozenges) or H2N2 (hollow circles) or H3N2 (hollow squares).
• peptide-based vaccine holds several advantages over traditional vaccines, including safety considerations, the relatively long shelf-life, the ability to target the immune response towards specific epitopes that are not suppressive nor hazardous for the host and the possibility of preparing multi-pathogen vaccine.
• the efficacy of a peptide vaccine is highly dependent on the exact identification of the immunogenic epitopes that confer protection as well as the efficient presentation of these epitopes to the immune system.
• a preferred B-cell influenza epitope is HA 91-108.
• Preferred T-helper influenza epitopes are HA 307-319 and HA 306-324 (Rothbard, 1988), but also NP 206-229 (Brett, 1991) may be used.
• the CTL influenza epitopes are different in the Caucasian, the Asia- or the Africa-originated population.
• the preferred influenza CTL epitopes are NP335-350 and NP380-393 (Dyer and Middleton, 1993; Gulukota and DeLisi, 1996) , that are restricted to the most prevalent HLA molecules in the Caucasian population.
• influenza epitopes that can be used according to the invention for the Caucasian population are the nucleoprotein epitopes: NP305-313 (DiBrino, 1993); NP384-394 (Kvist, 1991); NP89-101 (Cerundolo, 1991); NP91-99 (Silver et al, 1993); NP380-388 (Suhrbier, 1993); NP44-52 and NP265-273 (DiBrino, 1994); and NP365-380 (Townsend, 1986); and the matrix protein
• influenza CTL epitopes that can be used are HA458-467 of the sequence Asn-Val-Lys-Asn-Leu-Tyr-Glu-Lys-Val-Lys (NVKNLYEKVK) , a CTL epitope for allele All with high frequency in Japanese, Chinese, Thais and Indian populations (J. Immunol.
• the three T-cell epitopes used in the vaccine of the present invention were selected due to their specific recognition by the prevalent HLA's in the Caucasian population, and were included in the vaccine together with the HA 91-108 B cell epitope. In order to overcome the problem of antigenic variation of the virus, all these epitopes are derived from conserved regions in the virus proteins and hence, can induce cross-strain protection.
• the two CTL epitopes from the inner nucleoprotein are recognized by the prevalent HLAs of the Caucasian population: the NP 335-350 epitope is restricted to A2, A3, Aw68.1 and B37 HLA haplotypes, and the NP 380-393 epitope is restricted to B8 and B27 HLA haplotypes.
• the T- helper epitope from the hemagglutinin, HA 307-319 is a "universal" epitope restricted to most of MHC class II molecules, including DR1, DR2 , DR , DR5, DR7, DR9, DR52A, and others.
• These T-cell epitopes, together with the B-cell epitope HA 91-108, were expressed individually in flagellin and the mixture of resultant flagella was used without any adjuvant for intranasal vaccination of human/mouse radiation chimera, thus inducing a human immune response and conferring protection.
• the vaccinated mice were also protected from a lethal infection and their recovery was quicker.
• a humanized mouse model was employed.
• human PBMC can be adoptively transferred i.p. into the SCID mouse and that the engrafted cells survive for an extended period of time producing high levels of human Ig, has offered many new possibilities in clinical immunology research (reviewed in Mosier, 1991) .
• many researchers have been utilizing this model for studying the capacity of engrafted lymphocytes to generate primary and secondary human humoral responses, and for viral research studies .
• the human/mouse radiation chimera were immunized with the tetra construct administered by the intranasal route. This is the first report of induction of local immune response in the nasal cavity and lungs following intranasal immunization in the human/mouse radiation chimera.
• the induction of local immune response in the lungs was demonstrated by the presence of specific anti-influenza antibodies in the lungs homogenates (Fig. 3) , by elevation of
• CD8 + lymphocytes proportion and by the viral clearance as a result of immunization with the tetra construct (Fig. 2) .
• the tetra flagellin construct could also protect the mice from a lethal dose challenge of the virus, which is the ultimate demonstration of the protective effect. Under these conditions, in which the challenge dose is orders of magnitude higher than that pertaining in natural infection, all the chimera were infected regardless of their immune state. However, whereas none of the immunized mice that had not been transplanted with the human lymphocytes survived the infection, and only 50% of the transplanted but not immunized mice survived, the transplanted and immunized group was completely protected and showed 100% survival (Fig. 4) .
• the partial protection in the non-vaccinated mice is probably due to polyclonal stimulation and expansion of memory cells originating from the donor. This could be due to either previous exposure of the donor to the antigen or because it is cross-reactive to some extent with other recall antigens, a phenomena that was previously reported for other antigens (Marcus et al, 1995) . However, although such partial protection was indeed observed, a significant difference in the efficacy of the recovery process between the immunized and non-immunized groups was observed as evident both by survival rate and by their weight loss pattern (Figs. 4, 5).
• HLA phenotypes of the PMBC donors were not determined, all of the transplanted mice were protected as a result of the vaccination, indicating that the epitopes used in the present invention are indeed recognized by a wide range of HLA molecules .
• One of the most acute problems related to currently existing influenza vaccines is the narrow range of their specificity and their restricted strain-specific activity.
• the rapid variation in the viral surface glycoproteins leads to appearance of new strains with high variability in their serospecificity, and hence the vaccines containing the outer glycoproteins of some specific strains are limited in their efficacy to these strains .
• mice BALB/c mice (4-8 weeks old) were obtained from Olac Farms (Bicester, U.K.), NOD/SCID mice (4-6 weeks old) from the Weizmann Institute Animal Breeding Center (Rehovot, Israel) . All mice were fed sterile food and acid water containing ciprofloxacin (20 ⁇ g/ml) .
• BALB/c mice were exposed to a split lethal total body irradiation (TBI) of 4 Gy followed 3 days later by 10 Gy .
• TBI lethal total body irradiation
• the source of radiation is a gamma beam
• Bone marrow cells from NOD/SCID mice (4-6 weeks old) were obtained according to Levite et al . , 1991.
• mice Recipient irradiated mice were injected with 2-3x10 ⁇ SCID bone marrow cells (i.v. in 0.2 ml phosphate-buffered saline (PBS)) one day after irradiation.
• PBS phosphate-buffered saline
• Leukapheresis was performed on normal volunteers. Cells were collected by processing 3-4 liters of blood through Haemonetics V50 (USA) during 3-3.5 hours. The Leukapheresis product was centrifuged at 1200 rpm for 10 min. and the plasma removed.
• Oligonucleotides corresponding to the designated influenza epitopes namely NP335-350 (SAAFEDLRVLSFIRGY) , NP380-393 (ELRSRYWAIRTRSG) and two peptides from the H3 subtype haemagglutinin: HA91-108 (SKAFSNCYPYDVPDYASL) and HA307-319 ( PKYVKQNTLKLAT ) were synthesized in a 380B Applied Biosystems DNA Synthesizer, with additional GAT sequence at the 3' of each oligonucleotide in order to preserve the EcoRV restriction site, as described (Levi and Arnon, 1996) .
• the synthetic oligonucleotides were inserted at the EcoRV site of the plasmid pLS408 and eventually transformed into a flagellin negative live vaccine strain (an Aro A mutant) of Salmonella dublin SL5928 by transduction, using the phage P22HT105/1 int. Finally, the flagella were purified after acidic cleavage and a fine suspension was used for immunization
• the desired plasmids were used to transform Salmonella typhimurium LB5000 (a restrictive negative, modification proficient non flagellated) 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 dubl in SL5928 by transduction using the phage P22HT105/1 int (Orbach and Jackson, 1982, and Schmieger, 1972, both herein entirely incorporated by reference) .
• the transformed S . dublin 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 Figure et al . , 1985: Bacterial cells from an overnight culture grown in LB/ampicillin medium were pelleted and suspended in a 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 minutes and the pH was readjusted to 7.4. The flagella were then precipitated by (NH-J 2 S0 4 (35% w/v) and maintained overnight at 4°C.
• the pellet obtained after centrifugation at 10,000 rpm for 10 minutes 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 .
• mice engrafted with human lymphocytes were sacrificed 27-29 days after PBMC transplantation. Lymphocytes from lung homogenates as well as peritoneal washes were separated on ficoll-paque gradient
• Total human Ig was quantified in sera samples by sandwich ELISA using goat F(ab)2 ⁇ purified anti-human Ig (G+M+A) (Sigma) as the capture agent and peroxidase-conjugated purified goat anti-human Ig (G+M+A) (Sigma) as the detection reagent. Human serum of known immunoglobulin concentration was used as the standard. ELISA was performed as described by Marcus et al., 1995.
• influenza strains A/PR/8/34 H1N1
• A/Japanese/57 H2N2
• A/Texas/1/77 H3N2
• Virus amounts were measured in hemagglutination units (HAU) .
• HAU hemagglutination units
• the inactive virus A/Texas/1/77
• sucrose gradient was used.
• Virus growth and purification were according to standard methods (Barret and Inglis, 1985) .
• lung samples were homogenized in PBS containing 0.1% BSA and centrifuged in order to remove debris.
• Virus titers were determined by whole egg titration method (Barret and Inglis, 1985) . The titer was calculated by hemagglutination and presented as Log EID50
• EXAMPLE 1 Response of the chimeric mice to whole inactivated influenza virus .
• Fig. 1 is a FACS histogram depicting the pattern of human lung lymphocytes after immunization with the tetra construct without further challenge infection. The cells were stained with anti-CD45 antibodies together with anti-CD3 or together with anti-CD19.
• CD3+ namely T cells (80%-90%) and only a minor population is CD19+ (3%-10%) .
• Influenza infection is a respiratory disease, hence, a local immune response induced by an intranasal administration of the vaccine could be more efficient than parenteral administration.
• the immunization schedule was modified in order to adapt it for intranasal immunization.
• mice (6-8 per group in 7 repeated experiments) were immunized intranasally (i.n.) 10-12 days after PBMC transplantation, as described in the Methods. Ten days later, they were challenged i.n. with 10 ⁇ 4 HAU in 50 ⁇ l allantoic fluid of live A/Texas/1/77 strain of influenza virus. Five days later they were sacrificed and their lungs were removed for virus titration. As shown in Fig. 2, which depicts the cumulative results, the vaccination with the tetra construct enabled the chimera to clear the virus from their lungs significantly more efficiently than the group vaccinated with the native flagella, or the group which was not transplanted with PMBC but were immunized with the tetra construct.
• Fig. 4 describes the results of two repeated experiments and demonstrates the survival of vaccinated and non-vaccinated mice (both transplanted with human PBMC) , as well as of another control group that was not transplanted but was vaccinated with the tetra construct. As can be seen, while all control mice that were immunized with the tetra construct but had not been transplanted with the human lymphocytes died within 19 days after the infection, 100% survival was observed in the mice that received the PBMC prior to immunization.
• the body weight loss pattern of the challenged mice is depicted: the transplanted group that was immunized with the tetra flagellin construct, shows only a slight reduction in their body weight following the lethal dose infection and a rapid return to normal, while the control group that was transplanted with human PMBC but immunized with the native flagellin, lost more weight (the body weight is significantly different between the experimental group and the control groups on days 22-33 after transplantation) and the surviving mice started to recover weight only on the last days of the experiment. The non-transplanted, vaccinated control group lost weight rapidly and did not recuperate.
• influenza vaccines are effective only against the strains included in the vaccine. Therefore, it was of interest to examine the ability of the flagellin hybrids that express influenza epitopes to protect mice from different influenza strains that carry various hemagglutinin and neuraminidase glycoproteins.
• the B-cell epitope that is expressed in the flagellin is conserved in all influenza H3 subtypes, while the T-cell epitopes are from regions of the hemagglutinin and nucleoprotein highly conserved in other subtypes as well.
• rabbit antibodies towards these epitopes can indeed recognize and react in ELISA with different strains of influenza including
• mice per group were immunized i.n. with the tetra construct. Their resistance to different influenza strains challenge was detected 7 days later and compared to non-transplanted mice that were immunized with the same flagella mixture.
• the influenza strains that were used for infection were: A/Texas/1/77 (H3N2), A/Japanese/57 (H2N2) and A/PR/8/34 (H1N1) .
• Protective immunity was observed against all three strains, as presented in Fig. 6. Human Ig specific for each influenza strain was detected in the sera of all the transplanted and vaccinated mice, but not in the control group, as shown in Fig. 7. Table 1

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  • 1999-11-28 KR KR1020017006639A patent/KR100703571B1/ko not_active Expired - Lifetime
  • 1999-11-28 WO PCT/IL1999/000640 patent/WO2000032228A2/en not_active Ceased
  • 1999-11-28 ES ES10003160T patent/ES2389611T3/es not_active Expired - Lifetime
  • 1999-11-28 EP EP10003160A patent/EP2204187B1/en not_active Expired - Lifetime
  • 1999-11-28 AU AU14066/00A patent/AU766883B2/en not_active Expired
  • 1999-11-28 HK HK02102105.6A patent/HK1040620A1/zh unknown
  • 1999-11-28 IL IL14336799A patent/IL143367A0/xx active IP Right Grant
  • 1999-11-28 CA CA2352454A patent/CA2352454C/en not_active Expired - Lifetime
  • 1999-11-28 NZ NZ511918A patent/NZ511918A/en not_active IP Right Cessation
  • 1999-11-28 AT AT10003160T patent/ATE556719T1/de active
  • 1999-11-28 JP JP2000584919A patent/JP2002531415A/ja active Pending
  • 1999-11-28 US US09/856,920 patent/US6740325B1/en not_active Expired - Lifetime
• 2001
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• 2004
  • 2004-05-17 US US10/846,548 patent/US7192595B2/en not_active Expired - Lifetime
• 2007
  • 2007-02-07 US US11/672,429 patent/US7514086B2/en not_active Expired - Fee Related

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