CA2117181C - Induction of cytolytic t-lymphocytes with cytomegalovirus polypeptides - Google Patents

Abstract

Immunogens for use in human cytomegalovirus vaccines or skin tests which comprise human cytomegalovirus matrix proteins or fractions thereof are disclosed. It is also disclosed that such matrix proteins are target antigens for human cytomegalovirus specific cytotoxic T-lymphocytes.

Description

Induction of Cytolytic T-lymphocytes with Cytomegalovirus Polypeptides This invention was made with government support under Grants No. P01 CA30206 and P30 CA33572 awarded by the National Cancer Institute and by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION
This invention relates to human cytomegalovirus (HCMV) vaccines in which the immunogen is an HCMV
matrix protein or a fragment of such a protein. This invention also relates to immunologic cytomegalovirus proteins or peptides that induce cytolytic T-lymphocytes effective.to lyse cytomegalovirus infected cells, to the production of such proteins or peptides and to vaccines and skin tests in which such proteins or peptides are utilized.

ABBREVIATIONS
CMV Cytomegalovirus HCNV Human Cytomegalovirus CTL Cytotoxic T-lymphocytes MHC Major Histocompatibility Complex IE Immediate Early gB Glycoprotein B
CNBr Cyanogen Bromide HF Human Foreskin Fibroblasts FCS Fetal Calf Serum PBL Peripheral Blood Lymphocytes APC Antigen Presenting Cell ActD Actinomycin D
pp65 An HCMV Matrix Protein TFA Trifluoroacetic acid REFERENCES

1. Sissons, J.G.P., et al., Immunol Today 7:57 (1986).
2. Alford, C.A., et al., Virology, 2d ed., B.N.
Fields, D.M. knipe, et a1.,,Eds., Raven press, New York (1990).

3. Ho, M. Cytomegalovirus: Biology and Infection, 2nd edition, pp. 249-300, Plenum medical Book Co., New York (1991).

5. Neiman, P., et al., Transplantation 15:478 (1973).
6. Quinnan, G.V., et al., N.Engl. J.Med. 307:7 (1982).

8. Borysiewicz, L.K., et al. Eur. J. Immunol.
18:269 (1988).
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Wr '4/00150 211 71, g1 PCT/US92/05432 11. Riddell, S.R., et al., J.Immunol. 146:2795 (1991).
12. Gooding, L.R., et al., J.Immunol. 131:2580 (1983).
13. Townsend, A.R.M., et al., Cell 42:457 (1985).
14. Nuchtern, J.G., et al., Nature 339:223 (1989).
15. Moore, M.W., et al'., Cell 54:777 (1988).
16. Townsend, A.R.M., et al., Cell 44:959 (1986).
17. Maryanski, J.L., et al., Nature 324:578 (1986). 1 18. Reddehase, M.J., et al., Nature 337:651 (1989).
19. Carbone, F.R., et al., J. Exp. Med. 167:1767 (1988).
20. Wabuke-Bunoti, M., J. Immunol. 127:1122 (1981).
21. Clark, B.R., et al., J. Virol. 49:279 (1984).
22. Riddell, S.R., et al., J. Immunol. Methods 128:189 (1990).
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Y
24. Borysiewicz, L.K., et al., Eur.J.Immunol.
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30. Reddehause, M.J., et al., Nature 312:369 (1984).
31. Reddehause, M.J., et al., J.Virol. 61:3102 (1987).

33. Townsend, A., et al., Nature 340: 433 (1989).
34. Braciale, T.J., et al., Immunol.Rev. 98:95 (1987).

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39. Plotkins, et al., Infection and Immunity 12:521-527 (1975).

BACKGROUND OF THE INVENTION
Cytomegalorius (CMV) infection is a frequent pathogen in humans. It is usually associated with asymptomatic primary invention, followed by a state of viral persistence or latency (1,2). In patients with congenital or acquired immune deficiencies and those undergoing solid organ or bone marrow transplantation, primary CMV infection and reactivation of persistent CMV
have frequently been associated with life-threatening invasive visceral disease (3,5).

T-lymphocyte reactivity to the matrix protein of HCMV following natural infection of an individual has been reported. Peripheral blood mononuclearleukocytes were separated using Ficol-hypaque, washed, suspended in RPMI-1640 medium with ten percent (10%) AB+, Rh+ human serum and were then culturedat 37 C. in microtiter plates (105 cells/well). Different samples of the peripheral blood mononuclear leukocytes were then stimulated wr -94/00150 2117181 PCT/US92/05432 with optimal dilutions of individual ones of (a) the matrix protein of human cytomegalovirus (HCMVgp64) such as HCMVgp64, (b) HCMV-infected cell lysate and (c) control cell lysate, and cultures were analyzed for proliferation and for lymphokine production.
The matrix protein of human cytomegalovirus (4 g/ml) stimulated H3-thymidine in HCMV seropositive donor peripheral blood mononuclear leukocytes but not in seronegative donor peripheral blood mononuclear leukocytes. The peripheral blood monoclear leukocytes produced the same stimulation with HCMV-infected cell lysate as when the matrix protein of human cytomegalovirus (HCMVgp64) was used. The matrix protein of human cytomegalovirus (HCMVgp64) induced interleukin-2 (IL-2) production, IL-2 receptor expression and interferon production. The addition of antibody.to the IL-2 receptor blocked the reactivity of the peripheral blood mononuclear leukocytes to HCMVgp64.
The development of cell-mediated immunity, particularly that involving CD8+ class I
MIIiC-restricted CMV-specific CTL, represents'an essential host factor in the control of persistent infection and the recovery from CMV disease (6, 9).
The importance of CD8+ CTL responses in providing protective immunity in CMV seropositive individuals has fostered studies to elucidate the specificity of this response for selected CMV antigens with the objective of developing methods to augment CTL
responses in immunocompromised hosts. Studies using vaccinia recombinant viruses to express the non-structural 72 KD CMV immediate early protein (IE) and the major envelope glycoprotein, gB, have demonstrated that in two CMV seropositive individuals, a subset of CTL recognized IE and a minor subset recognized gB (10). However, the majority of CMV-specific CTL in these individuals recognized other undefined viral antigens (10).
It is now established that CD8+ CTL recognize peptide fragments of antigens, derived as a result of intracellular processing of cytoplasmic protein and subsequently association with class I MHC molecules =
in the endoplasmic reticulum prior to cell surface presentation (12, 13, 14, 15). Consequently, small protein fragments or synthetic viral peptides can sensitize uninfected target cells by direct association with MHC molecules at the cell surface (16, 17, 18). Thus, an alternative approach to using recombinant expression vectors for examining the specificity of CTL recognition is to directly sensitize target cells by incubation with synthetic peptides or peptides derived from native protein by proteolytic digestion (19, 20).

DESCRIPTION OF THE FIGURES
Figure 1 demonstrates that polyclonal CMV-specific CTL lyse autologous cells from five different donors pulsed with CNBr digest of'the CMV
matrix protein pp65. CMV-specific CTL lines were generated from 5 CMV seropositive donors and assayed in a 4 hour chromium release assay for recognition of autologous target cells either infected for 24 hours with CMV (a), pulsed for 1 hour with CNBr cleaved pp65 (A), or mock infected (e). Control MHC
mismatched target cells were either infected=for 24 hours with CMV ( ), or pulsed for 1 hour with CNBr cleaved pp65 (n) .
Figure 2 demonstrates in vitro stimulation of peripheral blood lymphocytes with CNBr cleaved pp65 elicits class I MHC restricted CTL that lyse CMV
infected target cells. PBL from 4 donors were wr 14/00150. 2117181 PCI'/US92/05432 stimulated with CNBr cleaved pp65 either directly (donor MW) or presented on autologous fibroblasts (donors CM, MR, and DH). Effector-cells from these cultures were assayed in a 4 hour chromium release assay for lysis of autologous fibroblast target cells either infected with CMV for 24 hours (M), pulsed for 1 hour with CNBr cleaved pp65 (A), or mocked infected (o). Control MHC mismatched target cells infected with CMV for 24 hours (p) and pulsed with CNBR cleaved pp65 (e) were assayed to demonstrate that lysis in class I MHC restricted.
Figure 3 demonstrates pp65-specific CTL clones lyse CMV infected cells at all stages of the viral replicative cycle. CD3+, CD8+, CTL clones were derived from the pp65-specific CTL line generated from donor CM. Clones 1C8 (a) and 2C8 (M) were assayed for recognition of autologous target cells either mock infected or infected with CMV at an MOI
of 5 for 2- 66 hours prior to use in a 4 hour chromium release assay. The effector to target ratio in this assay was 5:1. Lysis of MHC mismatched target cells infected with CMV was <5% at all time points (data not shown).
Figure 4 is a map of a pp65-CNBr digest.
SUMMARY OF THE INVENTION
This invention includes the discovery that HCMV
matrix protein pp65 is a target antigen for HCMV
specific CTL. CNBr cleavage of pp65 yields other CTL
specific target antigens. Immunogens for use in HCMV
vaccines or skin tests are provided by pp65 and by identification and synthesis of cytolytic pp65 sequences, e.g., the sequences 1 to 6 (Sequence ID
Nos. 1-6), =infra. An important advantage of this aspect of the invention is the simplicity and rapidly with which it can be determined whether an individual viral protein functions as a CTL target antigen without the construction of recombinant vectors.
The need to synthesize substantial amounts of a large protein is avoided.
In accordance with an aspect of the present invention, there are provided cytotoxic T-lymphocytes generated by stimulation with isolated or purified HCMVpp65, or fragments thereof, such that said lymphocytes are enriched for cytotoxic T-lymphocytes capable of lysing HCMV (human cytomegalovirus) infected cells.
In accordance with another aspect of the present invention, there is provided use of T cells capable of being exposed to an isolated or purified pp65 or an isolated or purified immunologically effective fragment thereof for the induction of cytotoxic T-lymphocytes (CTLs).
In accordance with a further aspect of the present invention, there is provided an isolated purified peptide having a sequence selected from the group consisting of Sequence ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No.
5, and ID No. 6.
In accordance with a further aspect of the present invention there is provided a man made peptide having a sequence selected from the group consisting of Sequence ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5 and ID
No. 6.
In accordance with another aspect of the present invention there is provided use of ppGS specific cytotoxic T cell effector cells for the lysis of HMCV
infected target cells wherein stimulator cells are - 8a -capable of being pulsed with an isolated or purified pp65 or an isolated or purified immunologically effective fragment thereof, said stimulator cells are capable of being co-cultured with peripheral blood lymphocytes to generate said effector cells and said effector cells are capable of interaction with the HCMV infected target cells.
In accordance with a further aspect of the present invention there is provided a human cytomegalovirus vaccine in which the immunogen is a peptide having an amino acid sequence according to SEQ ID No. 1, SEQ ID No.
2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID
No. 6.
In accordance with a further aspect of the present invention, there is provided a human cytomegalovirus vaccine in which the immunogen is a mixture of peptides having amino acid sequences according to Sequence ID Nos.
1 to 6.

In accordance with another aspect of the present invention, there is provided use of an isolated or purified pp65 or an isolated or purified immunologically effective fragment thereof which generates a pp65 specific cytotoxic T cell response for delivery to a host to increase the host's immune response to HCMV.
In accordance with a further aspect of the present invention, there is provided cytotoxic T-lymphocytes generated by stimulation with isolated or purified HCMVpp65 fragments having a sequence selected from the group consisting of SEQ ID NO: 1, S:EQ ID NO.3; SEQ ID NO:
5, and SEQ ID NO: 6, such that said lymphocytes are enriched for cytotoxic T-lymphocytes capable of lysing HCMV (human cytomegalovirus) infected cells.

- 8b -In accordance with a another aspect of the present invention, there is provided use of T cells capable of being exposed to an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO.3; SEQ ID NO: 5, and SEQ ID NO:

6, for the induction of cytotoxic T-lymphocytes (CTLs).
In accordance with a further aspect of the present invention, there is provided use of pp65 specific cytotoxic T cell effector cells for the lysis of HCMV
infected target cells wherein stimulator cells are capable of being pulsed with an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO.3; SEQ ID NO: 5, and SEQ ID NO: 6, said stimulator cells are capable of being co-cultured with peripheral blood lymphocytes to generate said effector cells and said effector cells are capable of interaction with the HCMV infected target cells.

In accordance with another aspect of the present invention, there is provided use of an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO.3; SEQ ID
NO: 5, and SEQ ID NO: 6 generates a pp65 specific cytotoxic T cell response, for delivery to a host to increase the host's immune response to HCMV.
EXPERIMENTAL
Virus and Cell Lines CMV Towns strain was grown in human foreskin fibroblast (HF) monolayer cultures and harvested for virus antigen production 120 yours post infection as described by Clark, et al. (21). AD 169 strain CMV was obtained from - 8c-ATCC and propagated by three serial passages onto confluent 150 cm2 flasks of HF in DMEM supplemnted with 2%
FCS. Viral stocks were prepared from infectious supernatants when HF cells showed 100% cytopathic effect.

Primary dermal fibroblast lines were established by explant culture of a skin biopsy obtained using a standard procedure from 5 CMV seropositive individuals with the following HLA types: MW - Al, A24, B7, B8; CM 0 Al, A2, B34, B57; TM - A3, A24, B8, 351; MR - A24, A25, B18, B35: DH - Al, A31, B38, B44. These cells were maintained in DMEM o4 Waymouths media supplemented with 10-15% FCS (Hyclone Logan, UT), 2 mM L-glutamine, and penicillin/streptomycin. Fibroblasts were used as target cells after 4-12 passages. Bone marrow derived fibroblasts were obtained from a CMV seropositive transplant donor (SA) of HLA type A28, A30, B18, B35, and maintained in culture for 6 passages prior to use. EBV
transformed B lymphoblastoid cell lies (LCL) were generated and maintained as previously described (22).

CMV pp65 purification and CNBr Cleavage CMV pp65 was purified from extracellular virion plus dense bodies of CMV by reverse phase high performance liquid chromatography as previously described (23). Purity was assessed by amino acid analysis. For chemical cleavage purified pp65 was dissolved in 60% formic acid and treated with a 100 fold molar excess of CNBr (over methionine residues) overnight. The solvent was evaporated at room temperature overnight under a stream of argon, the residue resuspended in water and lyophilized.
Generation of Cytotoxic Effector Cells CMV-specific polyclonal CTL were generated from the PBL of CMV seropositive, asymptomatic donors (CM, MR, TM, MW, and DH) using described methods (11, 22, 8). Briefly, PBL were isolated by HistopaqueTM
(Sigma, St. Louis, MO) gradient Gentrifugation and suspended in RPMI medium supplemented with 12% human CMV seronegative AB serum, 4 mM L-glutamine, 25 M 2 mercaptoethanol (Sigma), penicillin and streptomycin (referred to as culture media). 1 x 107 PBL were cultured with 5 x 105 autologous fibroblasts infected two hours previously with AD169 or Towne strain of CMV at an MOI of 5 in individual wells of a 6 well plate or in 25 cm2 flasks in a final volume of 6 ml culture media. After 7 days, the cultures were restimulated with both autologous CMV-infected fibroblasts, y-irradiated (3000 rads) autologous PBL, and then supplemented with recombinant IL2 (2-5 U/ml) 2 and 4 days after restimulation. After a total of 14 days in culture the effector cells were harvested and used in a standard Cr51 release assay.
To generate pp65-specific CTL, 5 x 105 autologous fibroblasts were cultured for 1 hour with 100 pg/ml peptides from CNBr digested pp65 (CNBr pp65) in 0.2 ml DMEM and used to stimulate 1 x 107 PBL. These cultures were restimulated at 7 days with CNBr pp65 pulsed fibroblasts, and supplemented.with IL2 (2-5 U/ml), 2 and 4 days following restimulation. After a total of 14 days in culture, the effector cells were harvested and used in a Cr51 release assay. For a single donor, PBL were cultured in the presence of 75 g/ml CNBr pp65 for 5 days and then assayed for CTL
activity.
CD8+ pp65-specific CTL clones were generated from 14 day CTL lines by first depleting CD4+ T cells with OKT4 mAb (Ortho, Raritan, New Jersey) and rabbit complement (Pel freeze, Brown Deer, WI) and then plating the enriched CD8+ T cells in 96 well round bottom plates at 0.5 cells/well with autologous irradiated (5000 rads) LCL (2 x 104 cells/well) as feeder cells, and autologous CNBr pp65 pulsed fibroblasts (2 x 103 cells/well) as stimulators in 0.2 ml culture media with 50 U/ml of IL2. Wells positive for growth at 14 days were transferred to larger wells and the clones restimulated every 7 days with autologous CNBr pp65 pulsed fibroblasts or CMV-infected fibroblasts, supplemented with irradiated feeder cells and fed with IL2 (60 U/ml) 2 and 4 days following each restimulation.
Chromium Release Assay A 4 hour chromium release assay was used to assay T cell lines and clones for cytolytic activity.
Effector cells were harvested 5 to 7 days after restimulation, washed once in cold PBS and resuspended in RPMI with 10% FCS. Autologous and MHC-mismatched fibroblast target cells were infected with CMV at an MOI = 5 for 24 hours or incubated in the presence of CNBr-pp65 (1 x 107 cells/100 pg of pp65) for 1 hour. In some experiments, BSA was used as a soluble protein control, and cells were treated as for CNBr pp65 cells. All target cells were then labelled with 200 pCi of Cr51 for 2 hours at 37 C.
Metabolic blockade with Actinomycin D (Act D) was used to prepare target cells such that they were infected with CMV but not endogenously synthesizing any CMV gene products, as previously described (il).
Briefly, Cr51 labelled fibroblast monolayers were preincubated in medium containing 20 g/ml Act D for 30 minutes, infected with CMV (MOI of 5) in the continued presence ot Act D for 4 hours, and maintained in Act D containing media during two washes and the 4 hour chromium release assay. In these experiments, control fibroblasts were infected with CMV for 4 hours in the absence of Act D.
For experiments examining the kinetics of pp65 presentation to CTL, fibroblasts were infected with CMV at an MOI = 5 for 2, 4, 12, 24, 36, 48 or 66 hours and labelled with Cr51 for 2 hours prior to use as target cells.
To prepare targets for plating, the Cr51 labelled -fibroblasts were washed three times in coldliBSS and resuspended in RPMI 10% FCS for counting. For all assays; target cells were plated in triplicates in-96 =
well round bottom plates at 1 x 104 cells/well, and effector cells were added to achieve various E/T
ratios in a final volume of 200 l. Following a 4 hour incubation, plates were centrifuged at 70 x g for 10 minutes and 100 pl of supernatant harvested for gamma counting. Spontaneous release was determined from target cells incubated with media alone, maximal release was determined from target cells incubated with 1% NP-40 (Sigma), and percent specific lysis was calculated using the standard formula. Spontaneous release was less than 15% for , -=~,.
21.17181 -12-CMV-infected cells and less than 20% for CNBr-pp65 labelled cells.
Monoclonal Antibodies and Cell Surface Phenotype Depletion of CD4 and CD8+ T cells from effector cell populations was done as previously described (11). T cell clones were phenotyped for CD3, CD4, and CD8 expression by indirect immunofluorescence using OKT3 (aCD3 ), OKT4 (aCD4 ) and OKT8 (aCD8) mAb (Ortho, Raritan, New Jersey) and FITC conjugated goat antimouse Ig (Tago, Burlingame, CA).
Results Recognition of pp65 by Polyclonal CD8+ CMV-specific CTL
To determine whether pp65 would serve as a significant target antigen for a component of the CMV-specific CTL derived from CMV seropositive individuals with protective immunity, polyclonal CMV-speca;fic CTL were activated by stimulating peripheralblood lymphocytes with autologous CMV-infected fibroblasts in short term cultures.
These effector cell populations were assayed for the ability to lyse autologous and MHC mismatched' fibroblasts either infected with CMV, mock-infected, or pulsed with CNBr digested pp65. Polyclonal =
CMV-specific CTL lines generated from four of five consecutive donors studied lysed both autologous CNBr pp65 pulsed target cells and CMV-infected cells but not mock infected targets (Figure 1). Lysis of both CMV-infected and CNBr pp65 pulsed autologous targets was class I MHC-restricted since target cells that were mismatched for class I NgiC antigens were not lysed (Figure 1). Lysis was predominantly mediated by CD8+ T cells since selective depletion of the CD8+
T cell subset but not.the CD4+ subset abrogated lytic activity (Table 1).

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Fibroblasts pulsed with native pp65 were not lysed (data not shown). These results demonstrated that in the majority of CMV seropositive individuals a component of the memory CD8+ CMV-specific CTL
response activated by stimulation with autologous CMV-infected fibroblasts, recognize epitopes of the CMV matrix protein pp65. In these individuals, the lytic profile of polyclonal CTL for CNBr pp65 pulsed autologous fibroblasts suggests that pp65-specific CTL comprise a significant subset of the CMV-specific CTL response. The inability to identify CTL that recognize CNBr pp65 pulsed fibroblasts in 1 of the 5 individuals may be due to an absence of a memory CTL
response specific for pp65 in this individualor to a failure of CNBr cleavage of pp65 to produce peptides similar enough to the epitopes generated by intracellular antigen processing that are recognized in the context of class I MHC by CTL of this individual.
CD8+ pp65-Specific CTL Clones Recognize CMV-Infected Cells in the Absence of Viral Gene Expression pp65 is one of the most abundant structufal virion proteins (25), and thus represents an attractive candidate as a target antigen for this response. To determine if class I MHC restricted presentation of pp65 in CMV-infected cells was independent of viral gene expression, it was necessary to selectively isolate pp65-specific CTL
and then determine whether those could lyse CMV
infected target cells with and without viral gene blockage. The pp65 specific CTLs were isolated by culturing PBL directly with CNBr digested pp65 or by pulsing autologous I irradiated fibroblasts with CNBr pp65 and using these cells as APC to stimulate PBL in short term cultures. In 3 of 4 donors, these . ...: .............. : .. , , , .

stimulations consistently elicited CTL that lysed autologous CMV-infected or CNBr pp65 pulsed fibroblasts in a class I MHC-restricted fashion (Figure 2).
CD8+ pp65-specific T cell clones were then generated from one of these lines by plating CD8+ T
cells in limiting dilution cultures with 7 irradiated autologous CNBr pp65 pulsed fibroblasts as both stimulators and irradiated feeder cells. From these cultures, a panel of CD3+, CD8+, CD4- T cell clones were identified that lysed autologous CMV-infected and CNBr pp65 pulsed fibroblasts but not mock infected or MHC mismatched target cells. The cytolytic reactivity of 7 representative clones is shown in Table 2.

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synthesis inhibitor ActD. The concentration of ActD
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.. . .,,. ., . ... ... , .. .t~.hw .. . . .... . ... .. . .. . . .. 2117181 WQ,94/00150 PCT/US92/05432 pp65-Specific CTL Recoanize CMV-Infected Cells at all Stages of The Viral Replicative Cycle To determine if pp65 is efficiently presented by CMV-infected cells throughout the replicative cycle, the recognition of autologous cells infected with CMV
for various periods of time (2-66 hours) prior to use as targets in the chromium release assay was examined. These experiments demonstrated that pp65-specific T cell clones recognized target cells infected with CMV at all stages of the viral replicative cycle. Representative data is shown for two clones in Figure 3. Lysis of CMV-infected cells by these clones is evident at the earliest time point (2 hours) after infection, and peaks at 36 and 48 hours after infection. Thus, presentation of the pp65 matrix protein in CMV infected cells for class I
MHC restricted T cell. recognition occurs promptly after viral entry and during all stages of the viral replicative cycle.
The structural matrix protein pp65 has been identified as a target antigen for CMV-specific class I MHC restricted.CTL derived from the periph6ral blood of 4 of 5 asymptomatic CMV seropositive individuals. The lytic profile observed for polyclonal CMV-specific CTL on CNBr pp65 sensitized target cells suggests that pp65 represents a significant target antigen, among others, recognized by CMV-specific CTL. The described method to identify pp65 specific CTL is based on the many observations that synthetic peptides and chemically or enzymatically cleaved protein fragments can substitute for endogenously expressed protein antigens when presented to CTL in the context of MHC
on an appropriate target cell (12, 16, 17, 18, 19, 20). This approach circumvents the need to generate .....

recombinant viral vectors expressing single CMV genes when the putative antigen can be obtained as a soluble protein. Synthetic viral peptide fragments may sensitize uninfected cells for lysis either by directly associating with class I molecules, thus bypassing the cellular processing requirement altogether, or by being internalized by the cell in an already degraded form that enables the peptide to enter the intracellular trafficking and processing pathways leading to class I NgiC association and presentation (33). In either case, the result is the presence at the cell surface of class I MHC peptide complexes of sufficient density for recognition by CTL. The CNBr-pp65 digest used in the described experiments apparently works in a similar manner, except that several protein fragments are present, one or more of which would be similar to a naturally processed fragment produced in CMV-infected cells.
This is consistent with observations that antigen processing events for native proteins include internalization and degradation within lysosomal compartments resulting in association with class II
and not class I MiC molecules for transport to the cell surface (34). The advantage of this aspect of =
the invention is the simplicity and rapidity with which it can be determined whether an individual virion protein is able to serve as a CTL target antigen without the construction of recombinant vectors. The disadvantages are that not all viral proteins can be readily purified from infected cells, and chemical or enzymatic cleavage may not produce protein fragments capable of mimicking all the epitopes produced by endogenous processing in the virally infected cell.

This invention also demonstrates that CD8+ class I MHC restricted CTL specific for the matrix protein pp65, are readily isolated from the majority of healthy CMV seropositive individuals. These CTL will recognize CMV-infected cells without the requirement for viral gene expression and thus may provide an important effector cell for limiting CMV reactivation as well as for the resolution of active CMV disease.
A strong CTL response to virion proteins such as the pp65 matrix protein which are available for class I antigen processing immediately following virion entry and prior to the onset of viral replication in the infected cell is biologically attractive since such a CTL response may be particularly effective at limiting the potential for viral reactivation to progress to clinical disease. If the pp65-specific CTL response is also a major contributor to the resolution of active CMV infection, it would be expected that CMV-infected cells would be targets for CTL throughout the duration of the viral replicative cycle. Following CMV entry and uncoating, the viral genoma is expressed in a cascade fashion wwithR
sequential expression of immediate early (0-2 hours), early (2-24 hours) and late (>24 hours) viral proteins (26). Abundant expression of pp65 does not occur until late stages of viral replication (27).
Nevertheless, analysis of CTL induced target cell lysis after CMV infection indicated that pp65 specific lysis was present at all times post infection with a peak activity at 36-48 hours consistent with peak synthesis of this protein (25).
Studies in immunocompromised bone marrow transplant patients have identified that recovery of CD8+ CMV-specific CTL responses correlates with protection,from developing CMV disease, but that ~9t~"@''"RSi ?'a ' : . . .. . .. . . . .. . . . . . . . , --' ,... .4 ...zdac:. , n:: . . . . . . . . WO 94/00150 PCT/US92/05432 greater than 50% of BMT recipients have delayed recovery of, or absent CMV-specific CTL responses for prolonged periods of time following transplant. This group of patients is at particularly high risk for developing CMV pneumonia (9). Thus, the identification of specific CTL target antigens provided by this invention is useful in the design of methods to augment CMV-specific immunity in BMT
recipients. Synthetic viral peptides have recently been shown to be effective in specifically priming naive animals to generate high affinity virus-specific CTL when the peptide is conjugated to a lipoprotein or contained within a liposome (36, 37). Such an approach has the advantage of potentially eliciting CTL responses to CMV in immunosuppressed patients without the requirement for exposure to infectious virus. The denonstrati,on that pp65 is a target antigen for CTL in the majority of seropositive individuals throughout the viral replicative cycle suggests that pp65 is an appropriate antigen to target in efforts to provide immunocompromised hosts with immunity to CMV."
Isolation and Purification pp65-CNBr Digest Peptides This aspect of the invention includes the preparation of CNBr digests of pp65 and the isolation and purification of immunologically significant peptides from such digests.
For chemical cleavage with CNBr, pp65 was dissolved in 60% formic acid and treated overnight under argon with a 100 fold molar excess of CNBr (over methionine residues). The solvent was evaporated at room temperature under a stream of ' argon, the residue resuspended in water and lyophilized. The peptides were fractionated by HPLC

WO 94/00150 2117181 Pt"T/US92/05432 on a Vydac C4 reverse phase column, using a 90-minute linear gradient from 100% solvent A (0.1% TFA) to 100% solvent B (TFA/H20/CH3CN, 0.1/9.9/90, v/v/v/).
The HPLC purified peaks from map A were subjected to amino acid sequencing (performed by City of Hope) and from map B were used for CTL analysis (performed at the Fred Hutchinson Cancer Center).
Figure 4 is a map produced by the HPLC analysis.
The HPLC purified peptide peaks designated by asterisks in Figure 4 were sequenced. These peptides correspond to sequences 1 through 6 (Sequence ID
Nos. 1-6), of this application. The invention includes the use of these six peptides or portions thereof to lyse HCMV infected cells, and as immunogens in HCMV vaccines and skin test formulations.

SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: Pande, Hema Riggs, Arthur Zaia, John A.

(ii) TITLE OF INVENTION:

Induction of Cytolytic T-Lymphocytes With Cytomegalovirus Proteins and Peptides (iii) NUMBER OF SEQUENCES: Six (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: City of Hope (B) STREET: 1500 East Duarte Road (C) CITY: Duarte (D) STATE: California (E) COUNTRY: United States of America (F) ZIP: 91010-0269 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3M Double Density 1/411 diskette (B) COMPUTER: Wang PC

(C) OPERATING SYSTEM: MS DOS Version 3.20 (D) SOFTWARE: Microsoft (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown (B) FILING DATE:

(C) CLASSIFICATION: Unknown (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER,: US 07/307,526 WO 94/00150 Pi'T/US92/05432 (B) FILING DATE: 8 February 1989 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Irons, Edward S.

(B) REGISTRATION NUMBER: 16,541 (C) REFERENCE/DOCKET NUMBER: None (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 785-6938 (B) TELEFAX: (202) 785-5351 (C) TELEX: 440087 LM WSH

(2) INFORMATION FOR SEQ ID NO:1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 98 amino acid residues (B) TYPE: Amino Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of pp65 =
protein expressed by HCMV (Towne) (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not applicable (v) FRAGMENT TYPE: Internal Fracgment (vi) ORIGINAL SOURCE:

(A) ORGANISM: HCMV
(B) STRAIN: Towne 21171.81 -26-(C) INDIVIDUAL ISOLATE

HCMV (Towne strain) was obtained from S. Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: None (viii) POSITION IN GENOME: Not applicable (ix) FEATURE:

(A) NAME/KEY: Induces CTLs that lyse HCMV infected cells (B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV (AD169) sequence by Chee, et al.
in (HCMV sequence-in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 12-109 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces rCTLs that lyse HCMV infected cells (x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 ISVLGPISGH VLKAVFSRGD TPVLPHETRL LQTFIHVRVS QPSLILVSQY
TPDSTPCHRG DNQLQVQHTY FTGSEVENVS VNVHNPTGRS ICPSQEPM

INFORMATION FOR SEQ ID NO:2 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acid residues (B) TYPE: Amino Acid (C) STRANDEDNESS: Single 2117tS1 (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of HCMV pp65 protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not available (v) FRAGMENT TYPE: Internal Fragment (vi) ORIGINAL SOURCE:

(A) ORGANISM: HCMV
(B) STRAIN: Towne (C) INDIVIDUAL ISOLATE:

HCMV (Towne strain) was obtained from S.
Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: Not available (viii) POSITION IN GENOME: Not available (ix) FEATURE: =

(A) NAME/KEY: Induces CTLs that lyse HCMV
infected cells =
(B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV
(AD169) sequence by Chee, et al. in (HCMV
sequence in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 110-120 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces CTLs that lyse HCMV infected cells (x) PUBLICATION INFORMATION: None {{..:\=. , ,.- . y (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 S I Y V Y A L P L K M
INFORMATION FOR SEQ ID NO:3 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acid residues (B) TYPE: Amino Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of HCMV pp65 protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not available (v) FRAGMENT TYPE: Internal Fragment (vi) ORIGINAL SOURCE:

(A) ORGANISM: HCMV -(B) STRAIN: Towne =
(C) INDIVIDUAL ISOLATE

HCMV (Towne strain) was obtained from S.
Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: Not available (viii) POSITION IN GENOME: Not applicable (ix) FEATURE:

(A) NAME/KEY: Induces CTLs that lyse HCMV
infected cells yi~r'.. .. . .. , , . ... ;... _ . .. . . . . .. ..

21 1'71g1 (B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV
(AD169) sequence by Chee, et al. in (HCMV
sequence in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 176-208 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces CTLs that lyse HCMV infected cells (x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 KEPDVYYTSA FVFPTKDVAL RHVVCAHELV CSM

INFORMATION FOR SEQ ID NO:4 (i) SEQUENCE CHARACTERISTICS:

.(A) LENGTH: 8 amino acid residues (B) TYPE: Amino Acid v (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of HCMV pp65 protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not applicable (v) FRAGMENT TYPE: Internal Fragment (vi) ORIGINAL SOURCE:

(A) ORGANISM: HCMV
(B) STRAIN: Towne (C) INDIVIDUAL ISOLATE

HCMV (Towne strain) was obtained from S. Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: Not available (viii) POSITION IN GENOME: Not Applicable (ix) FEATURE:

(A) NAME/KEY: Induces CTLs that lyse HCMV infected cells (B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV (AD169) sequence by Chee, et al.
in (HCMV sequence in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 209-216 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces CTLs that lyse HCMV infected cells (x) PUBLICATION INFORMATION: None ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 E N T R A T K M
INFORMATION FOR SEQ ID NO:5 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acid residues (B) TYPE: Amino Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of HCMV pp65 protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not available (v) FRAGMENT TYPE: Internal Fragment (vi) ORIGINAL SOURCE:

(A) ORGANISM: HCMV
(B) STRAIN: Towne (C) INDIVIDUAL ISOLATE

HCMV (Towne strain) was obtained from S. Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: Not available (viii) POSITION IN GENOME: Not Applicable (ix) FEATURE: (A) NAME/KEY: Induces CTLs that lyse HCMV infected cells (B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV (AD169) sequence by Chee, et al.
in (HCMV sequence in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 485-499 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces CTLs that lyse HCMV infected cells ~.

(x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 P P W Q A G I L A R N L V P M

INFORMATION FOR SEQ ID NO:6 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 62 amino acid residues (B) TYPE: Amino Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE:

(A) DESCRIPTION: Fragment of HCMV pp65 protein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: Not available (v) FRAGMENT TYPE: C-terminal fragment (vi) ORIGINAL SOURCE: (A) ORGANISM: HCMV

(B) STRAIN: Towne (C) INDIVIDUAL ISOLATE

HCMV (Towne strain) was obtained from S. Star (Childrens Hospital, Philadelphia) (39) and passaged for <10 times in the lab. The resulting virus was used as inoculum.

(vii) IMMEDIATE SOURCE: Not available (viii) POSITION IN GENOME: Not Applicable (ix) FEATURE:

(A) NAME/KEY: Induces CTLs that lyse HCMV infected cells Wa.94/00150 2117181 PC'1'/US92/05432 (B) LOCATION:

The sequence of pp65 corresponds to nucleotides (121037-119355) of the HCMV (AD169) sequence by Chee, et al.
in (HCMV sequence in EMBL Release 23, May 1990 accession no. X17403). The protein pp65 is 561 amino acid in size. The peptide sequence ID No. 1 corresponds to amino acid 500-561 in this protein.

(C) IDENTIFICATION METHOD: By experiment (D) OTHER INFORMATION: Induces CTLs that lyse HCMV infected cells (x) PUBLICATION INFORMATION: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 VATVQGQNLK YQEFFWDAND IYRIFAELEG VWQPAAQPKR RRHRQDALPG
PCIASTPKKH RG

, =

Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Cytotoxic T-lymphocytes generated by stimulation with isolated or purified HCMVpp65 fragments having a sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO.3; SEQ ID NO: 5, and SEQ ID NO: 6, such that said lymphocytes are enriched for cytotoxic T-lymphocytes capable of lysing HCMV (human cytomegalovirus) infected cells.

2. Use of an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO.3; SEQ ID NO: 5, and SEQ ID NO: 6, for the induction of cytotoxic T-lymphocytes (CTLs).

3. An isolated purified peptide having a sequence selected from the group consisting of SEQ ID No. 1, SEQ
ID No. 3, SEQ ID No. 5, and SEQ ID No. 6.

4. A man made peptide having a sequence selected from the group consisting of SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5 and SEQ ID No: 6.

5. Use of pp65 specific cytotoxic T cell effector cells for the lysis of HCMV infected target cells wherein stimulator cells are capable of being pulsed with an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ

ID NO.3; SEQ ID NO: 5, and SEQ ID NO: 6, said stimulator cells are capable of being co-cultured with peripheral blood lymphocytes to generate said effector cells and said effector cells are capable of interaction with the HCMV infected target cells.

6. The use of claim 5 wherein said fragment of pp65 is present in CNBr digest of pp65.

7. The use of claim 5, wherein said stimulator cells are capable of being pulsed with a peptide having a sequence selected from the group consisting of SEQ ID
No: 1, SEQ ID No: 3, SEQ ID No: 5 and SEQ ID No: 6.

8. A human cytomegalovirus vaccine in which the immunogen is a peptide having an amino acid sequence according to SEQ ID No: 1.

9. A human cytomegalovirus vaccine in which the immunogen is a peptide having an amino acid sequence according to SEQ ID No: 3.

10. A human cytomegalovirus vaccine in which the immunogen is a peptide having an ami:no acid sequence according to SEQ ID No: 5.

11. A human cytomegalovirus vaccine in which the immunogen is a peptide having an amino acid sequence according to SEQ ID No. 6.

12. A human cytomegalovirus vaccine in which the immunogen is a mixture of peptides having amino acid sequences selected from the group consisting of SEQ ID
No: 1, SEQ ID No: 3, SEQ ID No: 5 and SEQ ID No: 6.

13. Use of an isolated or purified pp65 fragment having a sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO.3; SEQ ID NO: 5, and SEQ ID NO: 6 to generate a pp65 specific cytotoxic T cell response, for delivery to a host to increase the host's immune response to HCMV.

14. The use of claim 13 wherein the host is a mammal.

15. The use of claim 14 wherein the mammal is human.