CROSS-REACTIVE INFLUENZA A IMMUNIZATION

Abstract: This disclosure relates to methods and compositions for stimulating in an individual an influenza A virus protective response which is subtype cross-protective. Influenza A virus NS1 protein, or a T cell epitope thereof, is administered to the individual in an amount sufficient to stimulate the virus protective response.

Description: CROSS-REACTIVE INFLUENZA A IMMUNIZATION _££._iaΞ°HΞ-?_2£_l_2_c_lBZ_-E-zi£Σ! Influenza A virus is a large RNA- containing animal virus. The protein capsid of the virus is further enclosed in a lipid bila er-based envelope containing protruding spikes of viral glycoprotei . Three influenza A serotypes have been identified (HI, H2 and H3) ; the classification based upon differences in the viral glycoprotei . Upon infection by the Influenza A virus, the body produces antibodies to variable regions of the surface glycoproteins hemagglutinin (HA) and neura inidase (NA) . This response results in the production of virus-specific antibodies which constitute the primary defense of the immune system. These antibodies provide immunological pressure which leads to antigenic d ift within viral subtypes, as well as shifts between viral subtypes. This relatively high rate of mutagenesis can render vaccine preparations ineffective because the antigenic determinants of the mutated viral proteins can differ significantly from those of the protein used as immunogen resulting in the failure of the body to effectively deal with the infection. Two central components of the immune system are the B cells and T cells, both of which are lymphocytes. The lymphocyte lineage diverges at the prelymphoblast stage into distinct sublineages. B cells produce and secrete antibody molecules; a process generally referred to as the humoral response. T cells are responsible for a variety of cellular responses referred to generally as cell mediated immune responses. B cells develop antigen specificity even in the absence of antigen stimulation. It has been estimated, for example, that the preimmune repertoire of a mouse comprises a class having many millions of different antibody molecules. This preimmune repertoire is apparently large enough to insure B cell specificity for almost any potential antigenic determinant. Current inactivated whole or subunit influenza vaccines provide B cell mediated (humoral) immunity in that they induce antibodies which are directed toward antigenic determinants of the surface glycoproteins of the virus. The first presentation of an influenza antigen to a B cell specific for the antigen (e.g. , at the time of vaccination) results in the maturation of the B cell into a plasma cell which is highly specialized for antibody production. Upon a second encounter with the same antigen, a rapid and increased secondary response results. The foreign antigen is bound by the specific antibody followed by clearance of the bound antigen from the bloodstream. However, in the case of influenza A, the production of virus-neutralizing antibodies provides immunological pressure which leads to antigenic drift within viral subtypes, as well as shifts between viral subtypes. Vaccines which are directed against antigenic sites do not elicit a broadly cross -reactive (i.e. protective against all influenza A virus subtypes) B cell response. Furthermore, the mutations which result from this immunological pressure can render current vaccines ineffective . T cells comprise a class of cells which, although they do not produce circulating antibodies, do play a central role in the immune system. The T cell class includes helper T cells, cytotoxic T cells and suppressor T cells. Helper T cells function, in part, by augmenting the response of other lymphocytes. For example, helper T cells stimulate activated T lymphocytes, in addition to stimulating B cell activation, by secreting interleukins as well as other soluble factors. Cytotoxic T cells (also referred to as killer T cells) , on the other hand, function by destroying cells marked with a particular antigen (e.g. cells infected by virus). T cells are stimulated by the recognition of a T cell epitope, in combination with a class I or a class II major histocompatiblity (MHC) antigen, on the surface of an antigen presenting cell. Macrophages belong to the class of antigen presenting cells. Macrophages are phagocytes which ingest foreign particles in the body. These cells are capable of ingesting even large micro¬ organisms such as protozoa. Following ingestion, the antigen presenting cell digests the foreign particle and fragments of the foreign particle are displayed on the surface of the cell. T cell epitopes differ fundamentally from B cell epitopes. B cell epitopes are antigenic determinants found in the native antigen molecule and not represented in the denatured antigen or fragments thereof. T cell epitopes, on the other hand, are found on unfolded molecules or fragments thereof. Furthermore, the T cell epitopes comprise helper T cell epitopes and cytotoxic T cell epitopes. These epitopes are thought to be con- tained by distinct, albeit possibly overlapping, portions of the antigen molecule. Influenza A virus infection continues to cause epidemics of death and tremendous morbidity throughout the world today even though the etiological agent is known. A great deal of effort has been devoted to the development of a vaccine, to little avail. A need exists for an effective influenza A vaccination strategy which could provide cross - subtype immunity from Influenza A viral infection. ^H IBi?;_IΣ_£-I_-_2 _i_lΣ££_ci£I- This invention relates to Applicants' finding that T cell epitopes of the influenza A NSl protein are capable of stimulating an influenza A virus protective response, in an individual, which is subtype cross -protective . In a first aspect, the method comprises administering an effective amount of influenza A virus NSl protein, in combination with a pharmaceutically acceptable carrier, thereby stimulating a T cell response against an NSl epitope in the individual resulting in an influenza A virus protective response which is subtype cross- protective. A homologue of the NSl protein in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof, is also effective for this purpose. In another aspect, the invention relates to a method and composition for immunizing an individual against infection by influenza A virus subtypes by the adminis¬ tration of an effective amount of an influenza A virus T cell epitope in combination with a pharmaceutically acceptable carrier, thereby stimulating a T cell response against the NSl epitope in the individual resulting in an influenza A protective response which is subtype cross- protective. The T cell epitope can stimulate a cytotoxic T cell response, a helper T cell response, or both. A homologue of the NSl T cell epitope in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof, is also effective for this purpose. The invention also relates to an essentially pure oligopeptide having an amino acid sequence corresponding to a T cell epitope of the influenza A NSl protein. This T cell epitope can stimulate a cytotoxic T cell response, and/or a helper T cell response. Again, a homologue of the NSl T cell epitope in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof, is also effective for this purpose. Also disclosed is a method for immunizing an indivi¬ dual against infection by influenza A virus subtypes comprising administering an effective amount of a recombinant virus which expresses the influenza A virus NSl protein. The individual can also be immunized by administering an effective amount of a recombinant virus which expresses an influenza A virus T cell epitope. These methods are limited to the administration of a recombinant virus which expresses the NSl protein or a T cell epitope thereof, thereby stimulating a T cell response against a T cell epitope resulting in an influenza A virus protective response which subtype cross -protective . The methods and compositions described herein provide for a broadly cross -reactive vaccination scheme which is protective against HI, H2 and H3 subtypes of influenza A virus. ^£____Ll l5_5£ £_liE_ϊ .£I__£___i_l _l.IlZ£.Σ£i£I. As discussed previously, influenza A virus comprises three subtypes, HI, H2 and H3. Applicants' invention relates to methods for immunizing an individual, particularly a human, against infection by any of these subtypes. Although the methods described herein are particularly useful for human immunization, the methods are equally applicable to other mammals. The term "cross-protective" is used in this application to describe immunity against HI, H2 and H3 subtypes. The gene encoding the influenza A NSl protein has been isolated, cloned, and expressed in a recombinant vaccinia system (see e.g. , Bennink e__t a 1 . , J__. _[___[_ , ____ 61:1098-1102 (1987)) . Using standard biochemical techniques (e.g. column chromatographv) the NSl protein, having a known molecular weight, can be isolated from cells in which it is expressed. If necessary to attain the desired purity, a hybridoma producing monoclonal antibody specific for NSl can be generated. Monoclonal antibody produced by this hybridoma can then be used in an affinity capture purification scheme. Homologues of the NSl protein in which amino acids have been deleted, inserted or substituted without essentialy detracting from the immunological properties thereof can be generated in a variety of ways . For example, _in vitro mutagenic techniques can be used to modify the cloned gene encoding the NSl protein. Such methods, which are well known to one skilled in the art, can be used to delete, insert or substitute nucleotides in the gene resulting in the deletion, insertion or substitution of amino acids in the encoded product. The immunological properties of the mutagenized encoded product can be assayed using methods such as those described in the Exemplification which follows. Effective dosages fo the stimulation of an influ¬ enza A virus protective response are determined empiri¬ cally with initial dosage ranges based upon historical data for peptide/protein vaccine compositions. As used herein, the term virus protective refers to an immuno¬ logical response in the individual resulting in the successful control or limitation of infection by influenza A virus subtypes which is clinically observed. For example, individuals can be administered dosages of NSl protein ranging from 0.5-500 icrograms. Whether a particular dosage is effective can be determined using well known T cell prolifera ion and c totoxicity assays. For example, following adminis ation of the protein to an individual blood is drawn. Cytotoxic T cells are identifiable by Cr release assay (see e.g. Kuwano e_t _λ - > l_.-lil2.l_. I____ : 1264-1268 (1988)) . Helper T cells are identifiable by a standard T cell proliferation assa (see e.g. Kurane et a ^ 1. , _L j __CiA2__ , _Iϋ_-_____t:__ E53_: 506-513 (1989)). The results from these studies are compared with results from the same experiments conducted with T cells from the same individual prior to administration of the antigen. By comparing this data, effective dosage ranges can be determined. A wide variety of pharmaceutically acceptable carriers are useful. Pharmaceutically acceptable carriers include, for example, water, physiological saline, ethanol polyols (e.g. glycerol or administration is typically parenteral (i.e. , intravenous, intra¬ muscular, intraperitoneal or subcutaneous) . An adjuvant (e.g. , alum) can also be included in the vaccine mixture. The invention also pertains to a method for im¬ munizing an individual against infection by influenza A virus subtypes by administering a vaccine composition comprising at least one essentially pure T cell epitope of the NSl protein in combination with a pharmaceutically acceptable carrier. Due to genetic variability between individuals, a single T cell epitope may not stimulate a virus protective response in all individuals to whom it is administered. Therefore, by combining two or more distinct T cell epitopes, the vaccine is more broadly effective. As indicated above, helper T cell epitopes and cytotoxic T cell epitopes are thought to comprise distinct (albeit possibly overlapping) regions of pro¬ teins. Cytotoxic T cell epitopes can be dis inguished from helper T cell epitopes experimentally using the cytoxicity and proliferation assays described above (helper T cells stimulate proliferation but do not possess cytotoxic activity) . The T cell epitope will be administered as an oligopeptide . Such oligopeptides can be synthesized chemically following identification of the portion of the protein containing the T cell epitope. Alternatively, a truncated portion of the gene encoding the NSl protein which contains a T cell epitope can be expressed in a cell, and the encoded product can be isolated using known methods (e.g. column chromatography , gel eleetrophoresis , etc.). In addition, the intact NSl protein can be treated chemically or enzymatically to generate fragments which contain a T cell epitope. Such fragments can be isolated as described above. As used herein, the term oligopeptide means any amino acid sequence which is identical or substantially homologous to a portion of the NSl protein. The expression substantially homologous refers to oligopeptides having a am no acid seque ce of an Sl T cell epitope in which amino acids have been deleted, inserted or substituted without essentailly detracting form the immunological properties thereof. This definition includes amino acid sequences of sufficient length to be classified as polypeptides (these terms are not used consistently or with great precision in the 1iterature ) . In a preferred embodiment, both a helper T cell epitope and a cytotoxic T cell epitope are administered to the individual. The stimulation of cytotoxic T cells is desirable in that these cells ill kill cells infected by influenza A virus. The stimulation of helper T cells is beneficial in that they secrete soluble factors which have a stimulatory effect on other T cells, as well as B cells. As discussed above, due to the genetic vari¬ ability between individuals, it is preferable to include two or more cytotoxic T cell epitopes and two or more helper T cell epitopes. Several methods are described in the literature which are useful for the identification of T cell epi¬ topes. For example, DeLisi e t _ 1_ . have suggested that potential epitopic sites may be located by identification of potential amphipathic alpha helical regions in the molecule. DeLisi e_t a . , j? £ _Na.t_L__A ad_* ϋ£_l_* U^A !B2:7048 (1987) . Bixler et a1_. describe a strategy of synthesizing overlapping synthetic peptides encompassing an entire protein molecule for delineation of T cell epitopes. Bixler et al. , l_nmH_2£l._._._-£_!_!__, 1 . * 593 (1983) ; Bixler et a1. , ^_Immunogenet^ 3.1:339 (1984). A synthetic method described by Gysen (C_i.ba._Founda:i_£n _5 _5££s._L__!E 11 *130 (1986)) permits synthesis of a large variety of peptides thereby mimicking of a variety of potential binding sites, in turn allowing rapid scanning of a molecule . More traditional methods, such as enzymatic or chemical digestion of proteins provide peptide fragments which may be readily tested for T cell activity. For example, enzymes such as chy otrypsin , elastase, ficin, papain, pepsin, or trypsin provide limited and predict¬ able fragments by cleavage of specified amino acid linkages; similarly chemical compounds such as N-chloro¬ succinimide BPNS -skatole , cyanogen bromide, formic acid, or hydroxylamine , also produce defineable fragments by their action on proteins. The presence of the desired T cell stimulating activity in any given fragment can be readily determined by subjecting purified fragments to a standard T cell proliferation assay, or by anlayzing unpurified fragments with a T cell Western Assay. Young £.£ l* . lϋ__Q:_.I_£i• 11 : 67 (1986). In another embodiment, the gene encoding the NSl protein, or a portion thereof which contains a T cell epitope, can be cloned into a recombinant virus which expresses the NSl protein, or T cell epitope contai-.ing portion thereof, in the individual to be immunized. An example of such a recombinant virus system is the vaccinia system described by Paoletti et a.1_ . (U.S. Patent No. 4,603,112), the disclosure of which is incorporated herein by reference. Other viruses have been described in the literature which have a genome which can accom¬ modate the insertion of foreign DNA such that a protein encoded by the DNA is expressed i_n yiy_o . Any such recombinant virus is useful for the practice of this invention. One skilled in the art will recognize that the compositions described herein can be combined with the components of influenza A vaccines currently in use, thereby resulting in an i poved vaccine. The invention is illustrated further by the following Exemplification. _._S______rlZl_; A ϊl____ ATERI.A S_AND_METHODS _U£_- BALB/c mice (H-2 ) were purchased from Charles River Breeding Laboratories (Stone Ridge, NY) . They were used at 5 to 9 weeks of age. lDUli_£B_l-___i∑H_.£_i Influenza A viruses, A/PR/8 (H1N1) , A/BZ (H1N1), A/JAP (H2N2), and A/PC (H3N2) , or B/HK, were propagated in 10-day-old embryonated chicken eggs. Infected allantoic fluids were harvested 2 days after infection, aliquoted, and stored at -80° (Kuwano, K. et l . , J^ Immunol. 140:1264-1268 (1988)) . Vaccinia recombinant viruses containing genes (HA, NP, NSl, and PB2) for A/PR/8 virus were kindly provided by Dr. B. Moss (Bethesda, MD) . They were constructed and propagated as previously described (Smith, G.L. e t _ _ . , Yl££l££Z 160:336-345 (1987)) . Briefly, HeLa cells were infected with virus for 3 days at 37° . Infected cells were pelleted by centrifugation , and resuspended in MEM containing 2% FCS . Three cycles of freezing and thawing were performed and the suspensions were gently sonicated in water for 1 min followed by tr psinization for 30 min at 37° . After centrifugation at 500 rpm for 5 min, supernatants were aliquoted and stored at -80° . Celiϋ The cell lines used in this study, P815 cells (H-2 ; mastocytoma) derived from DBA/2 mice, Class 1 MHC molecules, H-2L - or H-2D -tranεfected L929 cell line, and LM1 (K , L d , D k ) or DM1 (K k , L k , D d ) , were as described by Weis, J.H. and J.G. Seidman ( _. Immu ^].^ 134:1999-2003 (1985)) . Fu i ^ £rι_Pro£ei.n D proteins were produced in E. ££U as described previously (Yamada, A. e t a ] .. , Eϋ£h - £_l ___ £li^ _._.___._._, Med^ 162:663-674 (1985) ; Kuwano , K. et al. , l__._l_D!_ . £ϊ_£l__. 1.40:1264-1268 (1988)) . Briefly, plasmids containing DNA fragments complementary to the viral RNA of A/PR/8 virus were manipulated to achieve expression of D proteins, which are hybrids of the first 81 amino acids of NSl fused to the 157 amino acids from the C-terminal end of HA2 through a linker of glutamine - iεoleucine-proline . After lysis of the bacteria, two 0.1% deoxycholate extractions and one 1% Triton X-100 extraction were performed to remove contaminating E. ££U proteins, and the D protein was solubilized with 4 M urea at 4 β for 30 min. The urea was removed by dialysis at 4 β . The proteins were stored in 50 mM Tris-HCl, pH 8.0, and 1 mM EDTA. D protein was provided by J.F. Young (Smith, Kline and French Laboratories, Philadelphia, PA) . CTL_Cl£ne CTL clones were established as described pre iousl (Kuwano, K. et a1. , J___l_nmun£]_ 140:1264-1268 (1988)) . Briefly, CTL responder cells were stimulated weekly with A/PR/8 or D protein-pulsed normal syngeneic 7 -irradiated spleen cells in the presence of 10% Con A stimulated rat IL2 for several weeks. A limiting dilution was carried out to isolate CTL clones. The B-7 clone was established by stimulation of D protein (Kuwano, K. et a I . , ^ I un£l^ 140:1264-1268 (1988)) . The A-ll clone was stimulated by A/PR/8 virus and grew, at a frequency of growth 2 out of 96 wells, from a well where two responder cells had been seeded. For routine passage of clones, 2 X 10 of clone cell were stimulated weekly by 30 X 10 of A/PR/8 virus or D protein treated 7 -irradiated spleen cells in the presence of 10% rat IL2 and 5 X 10 M 2-ME. CTL_A a P815, LM1 , or DM1 cells (2 X 10 ) were incubated 7 8 with 0.5 ml of virus (10 -10 PFU) in the presence of Cr at 37° for 60 min. After three washings, target 4 cells were incubated for another 1 hr . Then 1 X 10 51Cr-labeled target cells were incubated with 1 X 104 effector cells in a total volume of 200 μl in 96-well round bottom microplates for 4 hr at 37°. The supernatant fluids were harvested and specific lysis was determined as percentage specific lysis - 100 X [(release by CTL - spontaneous release)/(maximum release - spontaneous release)] . Ad££t_iv _Tran_sfer_£f_CTL_C] 1 one Cells of the CTL clone (3 X 10 ) were suspended in 0.5 ml of RPMI 1640 and injected into mice via the tail vein. A preliminary experiment indicated that transfer of 1.0 X 10 cells resulted in significant reductions in mean pulmonary virus titers (0.6 - 0.8 log. -PFU) in recipients of clone A- 11. Six hours after adoptive transfer of the CTL clone, mice were infected intra- 3 nasally with 10 PFU of virus under ether anesthesia. The lungs of four mice per group were harvested 3 days later for measurement of virus titers. Pu]. ona _V_iru _Ti.trat!£ Virus titrations were performed by plaque formation using MDCK cells as previously described (Kuwano, K. e t i- > _L__l_!!I!!HΣ>£l_- lf+0 • 1264- 1268 (1988)) . Briefly, infected lungs taken from recipient mice were manually homogenized in 1.5 ml of PBS containing 0.1% BSA. After centrifugation, the lung supernatants were serially 10-fold diluted in PBS. Diluted virus samples (100 μl) were added to confluent MDCK cells in 24-well tissue culture plates and incubated at 37° for 1 hr. Each well then received 1 ml of 1% agar prepared as described earlier (Kuwano, K. et a1_ . , ±___∑_l_ }_r_o l_ . 140:1264-1268 (1988)). After 2 days of incubation, 1 ml of 10% neutral red (GIBCO, Chagrin Falls, OH) in PBS was overlaid on the agar in the wells. Plaques were counted 8 hr later. The results were expressed as the mean log- n PFU/ml of duplicate samples. RESULTS C££££_ιR a£__iχi t y_£f___l£2£_ A _ιil____ilBHl -ϊ£_-____ϊ_ ZZ Z-__Y Four CTL clones were established that were derived from A/PR/8 virus-immune spleen cells of BALB/c mice (H-2 ) stimulated by A/PR/8 virus (H1N1) . Two of the CTL clones demonstrated HI subtype - specific lysis of virus- infected target cells. These CTL clones were PB2 protein specific as determined using target cells infected with a vaccinia recombinant virus containing the gene for PB2 of A/PR/8 virus. Clone 1E8 , representative of two subtype Hl-specific clones, is shown as a negative control in Table 2. Clone A-ll, which is representative of the other two CTL clones, demonstrated cross-reactive lysis of target cells which were infected with A/PR/8 (HIND , A/BZ (H1N1) , A/JAP (H2N2) , or A/PC (H3N2) viruses, but failed to lyse B/HK- infected target cells (Table 1) . The B-7 CTL clone (Kuwano, K. et a1. , l__ , _ mun£].^ 1.40:1264- 1268 (1988)) was used as a control. B-7 had been stimulated by a fusion protein containing part of the HA2 subunit of A/PR/8 virus and showed subtype H1H2 cross- reactive lysis of target cells that had been infected with A/PR/8 (HI) , A/BZ (HI) , or A/JAP (H2) viruses. The phenotypes of the cell surface antigens of both the A-ll a nd B-7 clones were Thyl+, Lyt-2+, and L3T . TABLE 1 Virus Specificity of Clone A-ll Stimulated by A/PR/8 Virus *___E££i£i£_lϊ£i _£f_Z!li_ϊ__I£ £_Ce^l. A/PR/8 A/BZ A/JAP A/PC £l£ne E/T_Ratio J.H1N12 J . H1N12 XH2N22 J . H3N2 B/HK Uninfected Clone A-ll expresses 94% of Thyl.2, 86% of Lyt-2, and 5% of L3T4 surface Ag. Clone B-7 expresses 97% of Thyl.2, 95% of Lyt-2, and 0% of L3T4 surface Ag. CI1_Cl£ne_A :ι ll_i _N l_ Pr£tein_St)e£i.fi.£ To examine the influenza protein recognized by clone A-ll, target cells were infected with recombinant vaccinia viruses containing various influenza genes of A/PR/8 virus and were used in CTL assays. As shown in Table 2, clone A-ll significantly lysed NSl -VAC-infected and A/PR/8 virus - infected P815 target cells as positive control. However, clone A-ll failed to recognize HA-VAC, NP-VAC, PB2-VAC, or parental VAC-infected P815 target cells. Clone B-7 as a negative control lysed HA-VAC- infected target cells as well as A/PR/8 virus - infected target cells, but did not lyse NP-VAC-or VAC-infected target cells. Clone 1E8 , also derived from A/PR/8 virus-immune spleen cells by repeated stimulation with A/PR/8 virus as described above, recognized PB2-VAC- infected target cells or A/PR/8 virus - infected target cells, but failed to recognize NSl-VAC or HA-VAC- infected target cells; it is also included as a control. These results indicate that CTL clone A-ll recognizes the NSl protein on influenza A virus - infected cells. Table 2 Recognition of NSl Protein of A/PR/8 Virus by Clone A-ll i__i£ £ifi£_lY£l£_£l_]_ilI_Ii_I__ £___£ll__ E/T Unin- l££e Ratio A/PR £ HA^VAC NP_-_VAC NSl-VAC PB^VAC VAC fected A-ll 1.0 55 -4 0.5 47 -2 1E8 1.0 61 62 0.5 53 47 A-ll 3.0 78 ND 0 1.0 75 ND 0 B-7 3.0 91 ND -2 1.0 72 ND -1 _^H££i£S_£__ Hi!S£Ξ _ΪZ_ i∑H _Σi_: __ _ _i_l£ f££_ f N£l_:S2ecific_CTL_Clone To examine whether adoptive transfer of NSl protein- specific CTL clone A-ll would reduce virus titers in the lungs of mice infected with influenza viruses, 3 X 10 cells of clone A-ll were adoptively transferred to BALB/c mice 6 hr prior to influenza infection. Three days later, lungs were removed for titration of influenza viruses. Virus titrations were performed by plaque formation assays in MDCK cells. Similar results were obtained in two experiments with mean decreases in pulmonary virus titers of about 1.0 log.^. As shown in Table 3, adoptive transfer of CTL clone A-ll signifi¬ cantly reduced the virus titers in the lungs of mice infected with A/PR/8 , A/JAP, or A/PC viruses, but did not reduce the virus titer in the lungs of mice infected with B/HK virus. These results reflect the i.n vitro cross- reactivity of CTL clone A-ll shown in Table 1. Table 3 Reduction of Pulmonary Virus Titers by Adoptive Transfer of Clone A-ll RECIPIENTS a 6 Cells (3 X 10 ) were transferred 6 hr before virus challenge; + , transferred; -, no cells transferred Lungs were taken and virus titers were examined by plaque assays in MDCK cells 3 days after virus challenge . C P<0.01, Student's t test. P<0.02, Student's t test. e P<0.005, Student's t test. P<0.005, Student's t test. S P<0.0005, Student's t test. P<0.005, Student's t test. MHC_Re tr i£t i_£n_£f_Tar£e t_Ce l l_Ly_ Ϊ _b ~ "" k ~ L929 cells (H-2 ) transfected with genes encoding H-2D d (DM1 cells) and H-2L d (LMl cells) were used to examine the MHC restriction of target cells lysis by CTL clone A-ll. As shown in Table 4, CTL clone A-ll signifi¬ cantly lysed A/PR/8 virus- infected LMl (H-2L ) target cells, but failed to lyse A/PR/8 virus - infected DM1 (H-2D d ) or A/PR/8 virus - infected DAP(H-2 k ) target cells. As a control, CTL derived from bulk cultures of A/PR/8 virus-immune BALB/c (H-2d) spleen cells that had been stimulated by A/PR/8 virus in the presence of IL2 for several weeks were also used in this experiment. These virus-stimulated CTL lysed LMl or DM1 target cells infected with A/PR/8 virus, but did not kill A/PR/8 virus - infected DAP target cells. It was also observed that the CTL clone A-ll was unable to recognize A/PR/8 virus - infected peritoneal exudate cells of C3H.0L mice d k (H-2K , D ). These results indicate that recognition by the CTL clone A-ll of NSl on A/PR/8 virus - infected target cells is restricted by the H-2L allele. Table 4 MHC Restriction of CTL Recognition by CTL Clone A-ll £l ll^lϋ _°H_. h.l ?-li *_£££ A-ll 9 2 0 0 2 2 2 0 A/PR/8 s imu- lated CTL 5.0 36 2 24 2 1 1 2.5 30 1 11 1 1 1

Claims: CLAIMS A method for immunizing an individual against infection by influenza A virus subtypes, the method comprising administering to the individual an effective amount of influenza A virus NSl protein, or a homologue thereof in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof, in combination with a pharma¬ ceutically acceptable carrier, thereby stimulating a T cell response against NS-1 epitope in the indi¬ vidual, resulting in an influenza A virus protective response which is subtype cross-reactive . A method for immunizing an individual against infection by influenza A virus subtypes, the method comprising administering an effective amount of a recombinant virus which expresses influenza A virus NSl protein, or a homologue thereof in which amino acids have been deleted, inserted of substituted without essentially detracting from the immuno¬ logical properties thereof, thereby stimulating a T cell response against an NSl epitope in the indi¬ vidual resulting in an influenza A virus protective response which is subtype cross-reactive . A vaccine composition which stimulates an influenza A virus protective response when administered to an individual, the vaccine composition comprising an effective amount of influenza A virus NSl protein, in combination with a pharmaceutically acceptable carrier . 4. A method for immunizing an individual against infection by influenza A virus subtypes, the method comprising administering an effective amount of an influenza A virus NSl protein T cell epitope, or a homologue thereof in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties there¬ of, in combination with a pharmaceutically accept¬ able carrier, thereby stimulating a T cell response against the NSl protein T cell epitope in the individual resulting in an influenza A virus protective response which is subtype cross- protective . 5. A method of Claim 4 wherein the T cell epitope stim_-_.ates a cytotoxic T cell response. 6. A method of Claim 4 wherein the T cell epitope stimulates a helper T cell response. 7. A method for immunizing an individual against infection by influenza A virus subtypes, the method comprising administering an effective amount of a recombinant virus which expresses an influenza A virus NSl protein T cell epitope, or a homologue thereof in which amino acids have been deleted, inserted or substituted without essentially de- tracting from the immunological properties thereof, thereby stimulating a T cell response against the T cell epitope in the individual resulting in an influenza A virus protective response which is "subtype cross -protective . 8. A method of Claim 7 wherein the T cell epitope stimulates a cytotoxic T cell response. 9. A method of Claim 7 wherein the T cell epitope stimulates a helper T cell response. 10. A vaccine composition which stimulates an influenza A virus protective response when administered to an individual, the vaccine composition comprising an effective amount of an isolated influenza NSl protein T cell epitope, or a homologue thereof in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof, in combination with a pharmaceutically acceptable carrier. 11. A vaccine composition of Claim 10 which stimulates a cytotoxic T cell response. 12. A vaccine composition of Claim 10 which stimulates a helper T cell response. 13. A vaccine composition of Claim 10 which stimulates a cytotoxic T cell response and a helper T cell response . 14. An essentially pure oligopeptide having an amino acid sequence corresponding to a T cell epitope of the influenza A NSl protein, or a homologue thereof in which amino acids have been deleted, inserted or substituted without essentially detracting from the immunological properties thereof. 15. An essentially pure oligopeptide of Claim 14 which corresponds to a cytotoxic T cell epitope. 16. An essentially pure oligopeptide of Claim 14 which corresponds to a helper T cell epitope.

Inventors: ENNIS FRANCIS A, ENNIS, FRANCIS, A.
Applicants: UNIV MASSACHUSETTS MEDICAL, UNIVERSITY OF MASSACHUSETTS MEDICAL CENTER
Application year: 1991
Main classification: A61K39
IPC codes: A61K39, C07K14