Vaccine-induced cytotoxic T lymphocytes protect against retroviral ...

ARTICLES

Vaccine-induced cytotoxic T lymphocytes protect against retroviral challenge ANDREW D. HISLOP1, MICHAEL F. GOOD1, LUIS MATEO1, JOY GARDNER1, MAGTOUF H. GATEI1, RICHARD C.W. DANIEL2, BARRY V. MEYERS1, MARTIN F. LAVIN1 & ANDREAS SUHRBIER1 1

The Co-operative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia 2 School of Veterinary Science and Animal Production, University of Queensland, Queensland 4067, Australia Correspondence should be addressed to A.S.

NATURE MEDICINE • VOLUME 4 • NUMBER 10 • OCTOBER 1998

a

BLV1 rVV.env

BLV1 rVV.gag. pro. pol

BLV2 rVV.env

BLV2 rVV.gag. pro. pol

E:T ratio

b % specific lysis

BLV is a member of the human T-cell leukemia virus (HTLV)/BLV genus of retroviruses and is the etiological agent of a chronic Bcell leukemia in cattle and sheep. After infection with BLV, a brief viremia rapidly establishes a largely asymptomatic latent infection in B cells, where proviral gene expression is transcriptionally repressed9. To identify a target of ovine CTLs specific for BLV, we cultured peripheral blood mononuclear cells (PBMC) from BLV-infected sheep in vitro, which resulted in BLV antigen expression and expansion of BLV-specific CTLs (data not shown). Bulk CTL effectors from two BLV-infected sheep, BLV1 and BLV2, were found to recognize autologous ovine skin fibroblasts (OSF) infected with a recombinant vaccinia virus expressing the env gene of BLV (rVV.env) (Fig. 1a), which codes for the surface and transmembrane glycoproteins gp51 and gp30 (ref. 9). A major histocompatibility complex (MHC)-matched, BLV-negative sheep, whose fibroblasts were capable of presenting products of the env gene to BLV1 CTL effectors, was then identified (Fig. 1b, sheep 661). Such an animal was required as a source of Conconavalin A (ConA) blast target cells for peptide mapping studies, as blasts could be readily sensitized with peptide (in contrast to OSF, which were difficult to sensitize efficiently with peptide; data not shown). Blasts derived from BLV-infected sheep were unsuitable because Con A activated BLV antigen expression9 and the PBMC became targets for BLV-specific CTLs without the addition of potential target peptide epitopes (data not shown). A set of peptides 20 amino acids long (20-mers), overlapping each other by 10 amino acids and spanning the gp51 sequence, was used to sensitize Con A blasts from sheep 661. Bulk CTL effectors from BLV1 were found to efficiently lyse only cells sensi-

tized with the peptide KFSISIDQILEAHNQSPFCA, which represented amino acids 21–40 of gp51 (data not shown). The CTLs specific for KFSISIDQILEAHNQSPFCA were determined to be CD8+ by depletion experiments using an anti-ovine CD8 antibody11,12 (data not shown). To identify the minimal CTL epitope, all possible 8-mers, 9-mers and 10-mers spanning KFSISIDQILEAHNQSPFCA were tested; ISIDQILEA (amino acids 24–31) was the shortest, most active peptide (Fig. 2). This epitope is well conserved between different BLV isolates, with eight of the ten BLV isolates listed in GenBank sharing the same sequence in this region. The minimal epitope, ISIDQILEA, was formulated as a peptide vaccine and used to immunize a cohort of sheep, which were matched by CTL-based MHC typing for the allele restricting ISIDQILEA. BLV-negative sheep (n = 46) were typed by sensitizing their Con A blasts with peptide and using these cells as targets for BLV1 CTL effectors. Con A blasts from 12 sheep were shown to be capable of presenting ISIDQILEA (data not shown). Eight sheep were immunized with ISIDQILEA peptide (plus tetanus toxoid as a source of CD4 T-cell help13) in incomplete Freund’s adjuvant (IFA), and four control sheep were vaccinated with the same formulation without peptide. Before vaccination, all sheep were shown to have no ISIDQILEA-specific CTLs as judged by the inability of PBMC re-stimulated in vitro with peptide to generate ISIDQILEA-specific effectors (Fig. 3, PRE-IMMUNE). After vaccination, the presence of ISIDQILEA-specific CTLs was determined by re-stimulating PBMC in vitro with pep-

% specific lysis

The development of prophylactic vaccines against retroviral diseases has been impeded by the lack of obvious immune correlates for protection1,2. Cytotoxic T-lymphocyte (CTL)3,4, CD4-lymphocyte5, chemokine6 and/or antibody responses7 have all been associated with protection against HIV and AIDS; however, effective and safe vaccination strategies remain elusive1,8. Here we show that vaccination with a minimal ovine CTL peptide epitope identified within gp51 of the retrovirus bovine leukemia virus (BLV), consistently induced peptide-specific CTLs. Only sheep whose CTLs were also capable of recognizing retrovirus-infected cells were fully protected when challenged with BLV. This retrovirus displays limited sequence variation9; thus, in the relative absence of confounding CTL escape variants10, virus-specific CTLs targeting a single epitope were able to prevent the establishment of a latent retroviral infection.

E:T ratio

Fig. 1 a, Env is a target of BLV-specific CTLs. Specific lysis of BLV1- and BLV2-derived effector CTLs used against autologous ovine skin fibroblast infected with either control VV (m), or rVV.env (L), or rVV.gag.pro.pol (L), as indicated. b, Identification of an MHC-matched, BLV-negative sheep. BLV1 CTL effectors were used against ovine skin fibroblasts infected with either control VV (m) or rVV.env (L). Examples of one matched (661) and one unmatched animal (972) are shown. E:T Ratio, effector-to-target ratio. 1193

ARTICLES Fig. 2 The minimal CTL epitope recognized by env-specific CTLs is ISIDQILEA. BLV1 CTL effectors were used against Con A blasts from sheep 661, sensitized with the 20-mer peptide identified by mapping studies, KFSISIDQILEAHNQSPFCA (gp51 amino acids 21–40), and all possible 8-, 9and 10-mers spanning this sequence. The titration curves of selected peptides are shown. Peptide-specific lysis (vertical axis) refers to the ‘percent specific lysis of targets sensitized with peptide’ minus ‘percent specific lysis of targets not sensitized with peptide’. The effector-to-target ratio was 50:1.

% peptide specific lysis

ISIDQILEA Peptides

8-mers

9-mers

20-mer

Peptide concentration (ng/ml)

tide (Fig. 3, POST-IMMUNIZATION, PEPTIDE) as for pre-immune, and by re-stimulating with BLV-infected cells (Fig. 3, POST-IMMUNIZATION, BLV). Re-stimulation in the absence of added antigen (Fig. 3, POST-IMMUNIZATION, MOCK) was included to demonstrate the antigen specificity of these re-stimulation protocols. By using the peptide re-stimulation protocol, ISIDQILEA-specific CTLs could be detected in all eight peptidevaccinated sheep, but not in sheep that had received the control vaccine (Fig. 3, POST-IMMUNIZATION, PEPTIDE). After re-stimulation with BLV, however, CTLs capable of killing ISIDQILEAsensitized target cells could only be generated from four sheep (81, 90, 104 and 123; called BLV-responders). Why the CTLs Animal number

Pre-immune

Animal number

Post-immunization Peptide

Mock

raised in sheep 80, 86, 116 and 661 were unable to recognize and be re-stimulated by BLV-infected cells is unclear, but this may reflect the induction of low-affinity CTLs in these sheep (called BLV-nonresponders). Such CTLs may be capable of recognizing the high epitope density of peptide sensitized stimulator cells but be unable to recognize the much lower epitope density presented after physiological processing of antigen by BLV-infected cells13. Alternatively, the BLV-nonresponders may have a subtly different processing machinery or MHC subtype(s), although the ability of rVV.env-infected fibroblasts from sheep 661 (a BLVnonresponder) to present this epitope to BLV1 CTLs (Fig. 1b) would argue against this contention. PBMC from all animals except sheep 967, 106 and 81 had significant vaccine-induced proliferative responses to tetanus toxoid (stimulation index >2; data not shown). No BLV-specific antibodies or CD4 lymphocyte responses could be detected after vaccination, using ELISA and proliferation assays, respectively (data not shown). Fourteen weeks after vaccination, all 12 sheep were challenged Pre-immune

Post-immunization Peptide

BLV

Mock

BLV

% specific lysis

% specific lysis

ND

CONT.

CONT.

CONT.

CONT.

E:T ratio

Fig. 3 CTL responses after peptide immunization. Percent specific lysis of re-stimulated PBMC, from peptide- and control- (CONT.) immunized sheep, used against autologous Con A blasts sensitized with (L) and without (l) ISIDQILEA peptide. Pre-immunization PBMC, taken just before vaccination, re-stimulated with peptide (PRE-IMMUNE). Mock re-stimulated pre-immunization PBMC showed no CTL activity (data not shown). Post-immunization PBMC, taken 9–11 days after immunization, were restimulated with peptide (POST-IMMUNIZATION, PEPTIDE), without pep1194

E:T ratio

tide (POST-IMMUNIZATION, MOCK), and with BLV1 PBMC (POST-IMMUNIZATION, BLV). Sheep 81, 90, 104 and 123 are BLV-responders, and 80, 86, 116 and 661 are BLV-nonresponders (based on their post-immunization CTL responses after BLV restimulation). All BLV-responders have significantly higher lysis values than all BLV-nonresponders and all the controls, using comparison of triplicate wells at a 30:1 effector-to-target ratio (P < 0.001). The BLV-nonresponders are not significantly different from the controls (P > 0.27). NATURE MEDICINE • VOLUME 4 • NUMBER 10 • OCTOBER 1998

ARTICLES Peptide immunized

Control

Weeks after challenge

Fig. 4 Sheep with BLV-specific CTLs were protected against retroviral challenge. At the indicated times after challenge (vertical axis), PCR analysis using env-specific primers12 was done on DNA extracted from PBMC and the PCR products were resolved by agarose gel electrophoresis. Arrowheads indicate the specific BLV-derived 443-bp products. The 550-bp marker is in the first and last lanes. DNA from BLV1 was used as a positive control, and DNA from a BLV-negative sheep was used as a negative control (BLV-ve).

with BLV-infected peripheral blood lymphocytes. PBMC from the challenged sheep were monitored for the presence of proviral DNA by polymerase chain reaction12 (PCR) 2, 4, 6 and 9 weeks after challenge. Control-immunized sheep and the BLV-nonresponder sheep became BLV-PCR-positive by 4 weeks after challenge. In contrast, BLV-responder sheep (81, 90, 104 and 123) remained PCR-negative for the entire study (Fig. 4). An established latent infection can usually be detected in sheep within 3 weeks, even after inoculation with the minimum number of PBMC from a BLV-infected sheep (about 1,000 cells) required to establish such an infection14. As the sheep here were challenged with an inoculum more than 50 times larger than this, the lack of proviral DNA in sheep 81, 90, 104 and 123 (Fig. 4) provided compelling evidence that these sheep were protected against a substantial BLV challenge. To confirm the BLV-negative status, 107 PBMC from the protected sheep were inoculated into naive recipient sheep 13 weeks after challenge. After 4 weeks, these recipient sheep remained PCR-negative, whereas a positive control recipient sheep (inoculated with PBMC from animal 80) became PCR-positive (data not shown). This highly sensitive assay for detecting BLV-infected cells9 further demonstrated that vaccination of sheep 81, 90, 104 and 123 had prevented the establishment of a latent infection. The data presented here support previous correlations between the presence of retrovirus-specific CTLs and protection3,4, and indicate that CTLs alone, in the absence of antibody or CD4 responses, can prevent the establishment of a latent retroviral infection. Because CTLs are usually only activated by infected cells, vaccine-induced CTLs are unlikely to prevent the initial infections after challenge. However, retrovirus-specific CTLs seem to have a ‘window of opportunity’ to abort the infection before the establishment of a latent infection. Prevention of infection of any cells (sterile immunity), therefore, does not seem to be a necessary pre-requisite for a protective prophylactic retroviral vaccine15. The demonstration of protection in this BLV system may NATURE MEDICINE • VOLUME 4 • NUMBER 10 • OCTOBER 1998

have depended on at least two factors. First, the limited sequence variation, characteristic of the HTLV/BLV genus, meant that the protective activity of vaccine-induced (epitope-specific) CTLs would not be obscured by epitope-escape variants10. Second, the vaccine-induced CTLs in the protected sheep were capable of recognizing cells infected with retrovirus13,16. The ability of CTLs to prevent the establishment of a latent BLV infection may have implications for both HTLV (ref. 17) and HIV (ref. 1) vaccination strategies in humans. A prophylactic HIV vaccine might induce CTLs against multiple epitopes or epitope variants and thereby generate responses specific for the spectrum of HIV isolates present in a challenge inocula and/or preempt early escape mutants10. Polyepitope- or ‘polytope’-based vaccination would represent a suitable strategy for inducing such polyspecific CTL responses18. The challenge may be the design of human vaccination modalities that can generate CTLs of sufficient affinity to recognize retrovirus infected cells: this is an important consideration given the nef-mediated down regulation of class I MHC by HIV16. Methods Animals. Merino ewes (BLV1/BLV2) were infected with an Australian bovine isolate of BLV several years before these experiments. Infection was confirmed by an agar gel immunodiffusion assay and PCR (ref. 12). BLV-negative Merino wethers (2–5 years old) were used for vaccination studies. BLV-infected and BLV-negative sheep were housed separately. Effector CTL generation from BLV1/2. Blood was collected by jugular venipuncture into heparin-containing tubes, and PBMC were prepared using standard Ficoll-Paque (Pharmacia). PBMC (3 × 106 per well of a 24well plate) were cultured in medium, supplemented after two days with MLA-144 supernatant (as a source of IL-2) to 25% (volume/volume). The medium was RPMI-1640 supplemented with 10% FCS, 50 µM 2-mercaptoethanol (Sigma), 55 µg/ml penicillin, 90 µg/ml streptomycin (CSL, Melbourne, Australia) and 2 mM glutamine (Life Technologies). On day 9, 9 × 106 of these cells were restimulated by co-culture with 80 × 106 γ-irradiated (25 Gy) autologous PBMC and maintained for 5 days in 40 ml medium supplemented with 25% MLA-144 supernatant. These bulk effectors usually contained more than 80% CD8+ cells (data not shown). Restimulation of PBMC from vaccinated sheep. For peptide restimulation, PBMC (3 × 106) were co-cultured with 0.3 × 106 peptide-sensitized (0.5 µg/ml ISIDQILEA peptide for 90 min at 37 °C in 100 µl medium, followed by one wash) autologous PBMC stimulators in 24-well plates. On day 2, MLA-144 supernatant was added to 25%. On day 7, the cells were used as effectors. BLV restimulation was done in a similar way, except the stimulators were γ-irradiated (25 Gy) PBMC from the BLV-infected animal BLV1. Recombinant vaccinia viruses. The rVV were provided by D. Boyle of CSIRO, AAHL is Commowealth Scientific and Industrial Research Organization, Australian Animal Health Laboratories (Geelong, Victoria, Australia). The rVV.env coded for the envelope proteins of an Australian isolate of BLV under the control of the early late promotor of fowlpox virus 12, and rVV.gag.pro.pol coded for the gag, protease and polymerase proteins from the same virus. The control vaccinia was the parent TK – vaccinia virus VV-WR-L929. Target cells. Two types of ovine target cells were used. Ovine skin fibroblasts (OSF), prepared as described19 without addition of epidermal growth factor, were infected with rVV (multiplicity of infection = 20) at the same time as 51 Chromium (51Cr) labelling, and were used in 8-hour 51Cr release assays. Con A blasts were generated by culturing PBMC for 4–5 days in medium containing 5 µg/ml Con A (ICN, Sydney, Australia), after which the cells were maintained in medium containing 30% MLA-144 supernatant. The Con A blasts (5–21 days old) were sensitized by incubation overnight with peptide (0.1 µg/ml unless stated otherwise) before 51Cr labelling and use in standard 6hour 51Cr release assays. 1195

ARTICLES Peptide mapping studies. Twenty-six 20-mer peptides overlapping each other by 10 amino acids and spanning gp51 of the Australian isolate of BLV (ref. 20) (data not shown), and overlapping 10-mers, 9-mers and 8-mers covering the active 20-mer (peptide 21-40) (Chiron Technologies, Emeryville, California) were used to sensitize Con A blasts from sheep 661 before 51Cr release assays using BLV1 effectors. Peptide vaccination and challenge. Each sheep received 200 µg of ISIDQILEA peptide (>95% pure, Chiron Technologies, Emeryville, California) and 6 Lyme facotrs (Lf) tetanus toxoid (CSL, Melbourne, Australia) emulsified with 1 ml of incomplete Freund’s adjuvant (1:1 volume/volume; Sigma). Sheep were given two 1-ml subcutaneous injections on each inner thigh. Control sheep received the same formulation without peptide. Two months after vaccination, sheep were challenged intravenously with 5 × 104 PBMC from BLV1. BLV1 had developed a frank BLV lymphocytic leukemia at this time and was killed after blood was taken.

Acknowledgments We thank R. Verrall and B. Shirley (School of Veterinary Science and Animal Production, University of Queensland) for care of the animals in this study. This work was supported by the Cooperative Research Centre for Vaccine Technology, the Australian Commonwealth AIDS Research Grants Program and Related Diseases, and the Australian Centre for International & Tropical Health & Nutrition.

RECEIVED 6 AUGUST; ACCEPTED 8 SEPTEMBER 1998 1. Burton, D.R. & Moore, J.P. Why do we not have an HIV vaccine and how can we make one? Nature Med. S4, 495–498 (1998) 2. de The, G. & Kazanji, M.J. An HTLV-I/II vaccine: from animal models to clinical trials? Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13, S191–S198 (1996). 3. Rowland-Jones, S., Tan, R. & McMichael, A. Role of cellular immunity in protection against HIV infection. Adv. Immunol. 65, 277–346 (1997). 4. Gallimore, A. et al. Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques. Nature Med. 1, 1167–1173 (1995).

1196

5. Rosenberg, E.S. et al. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science 278, 1447–1450 (1997). 6. Garzino-Demo, A., Devico, A.L. & Gallo, R.C. Chemokine receptors and chemokines in HIV infection. J. Clin. Immunol. 18, 243–255 (1998) 7. Burton, D.R. A vaccine for HIV type 1: the antibody perspective. Proc. Natl. Acad. Sci. USA 94, 10018–10023 (1997). 8. Yasutomi, Y. et al. A vaccine-elicited, single viral epitope-specific cytotoxic T lymphocyte response does not protect against intravenous, cell-free simian immunodeficiency virus challenge. J. Virol. 69, 2279–2284 (1995). 9. Kettmann, R.A. et al. in The Retroviridae Vol. 3 (ed. Levy, J.A.) 39–81 (Plenum, New York, 1994). 10. Mortara, L. et al. Selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine. J. Virol. 72, 1403–1410 (1998). 11. McClure, S.J., Davey, R.J., Lloyd, J.B. & Emery, D.L. Depletion of IFN-gamma, CD8+ or TCR gamma delta+ cells in vivo during primary infection with an enteric parasite (Trichostrongylus colubriformis) enhances protective immunity. Immunol. Cell Biol. 73, 552–555 (1995). 12. Gatei, M.H. et al. Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70. J. Virol. 67, 1803–1810 (1993). 13. Scalzo, A.A. et al. Induction of protective cytotoxic T cells to murine cytomegalovirus by using a nonapeptide and a human-compatible adjuvant (Montanide ISA 720). J. Virol. 69, 1306–1309 (1995). 14. Mammerickx, M., Palm, R., Portetelle, D. & Burny, A. Experimental transmission of enzootic bovine leukosis to sheep: latency period of the tumoral disease. Leukemia 2, 103–107 (1988). 15. Kent, S.J., Hu, S.L., Corey, L., Morton, W.R. & Greenberg, P.D. Detection of simian immunodeficiency virus (SIV)-specific CD8+ T cells in macaques protected from SIV challenge by prior SIV subunit vaccination. J. Virol. 70, 4941–4947 (1996). 16. Collins, K.L., Chen, B.K., Kalams, S.A., Walker, B.D. & Baltimore, D. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397–401 (1998). 17. Dalgleish, A.G. Human T-cell lymphotropic virus type 1: infections and pathogenesis. Curr. Opin. Infect. Dis. 11, 195–199 (1998). 18. Thomson, S.A. et al. Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination. J. Immunol. 160, 1717–1723 (1998). 19. Andrew, M. et al. Antigen specificity of the ovine cytotoxic T lymphocyte response to bluetongue virus. Vet. Immunol. Immunopathol. 47, 311–322 (1995). 20. Coulston, J. et al. Molecular cloning and sequencing of an Australian isolate of proviral bovine leukaemia virus DNA: Comparison with other isolates. J. Gen. Virol. 71, 1737–1746 (1990).

NATURE MEDICINE • VOLUME 4 • NUMBER 10 • OCTOBER 1998