Immunology Letters66(1999)177–181
Pre-clinical development of a multi-CTL epitope-bad DNA prime
MVA boost vaccine for AIDS
Toma´s Hanke*,Andrew McMichael
Institute of Molecular Medicine,Uni6ersity of Oxford,The John Radcliffe Hospital,Headley Way,Oxford OX39DS,UK
Received19October1998;accepted18November1998
2020语文高考
Abstract
Reliable and effective methods for induction of cytotoxic T-lymphocytes(CTL)are constantly persued.Central to this arch is work in animal models,which allow to test novel vaccine strategies and ultimately lead to a more efficient planning of clinical trials.Here,human immunodeficiency virus(HIV)vaccine candidates were constructed as a string of partially overlapping CTL epitopes(20human,3macaque and1mou)delivered and expresd using plasmid DNA and modified virus Ankara(MVA;an attenuated vaccinia virus),which are both vaccine vehicles acceptable for u in humans.In mice,the vaccines were shown to induce virus-specific interferon-g-producing
and cytolytic CD8+T-cells after a single intramuscular needle injection.When immunization protocols were sought which would improve the level of induced HIV-specific T-cells,DNA priming-MVA boosting was found to be the most potent protocol.The multi-epitope DNA also elicited CTL when delivered intradermally using the Accell®gene delivery device(gene gun).Finally,a combined intradermal gene gun DNA–MVA vaccination regimen induced in macaques high frequencies of circulating CTL,which were comparable to tho obrved in simian immunodeficiency virus (SIV)-infected monkeys.Further optimization of this method in non-human primates is under way.Thus,a vaccination regimen for an effective elicitation of CTL has been developed which might facilitate evaluation of the role(s)that the lymphocytes play in the control of SIV and HIV infections.©1999Elvier Science B.V.All rights rerved.
Keywords:HIV vaccines;CTL polyepitope;DNA prime–MVA boost
1.Introduction
An effective vaccine is the best hope for controlling the acquired-immunodeficiency syndrome(AIDS)pan-demic.The development of such a vaccine would be greatly assisted by understanding of the requirements for protective immunity against human immunodefi-ciency virus(H
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IV)infection.Although an HIV vaccine may have to induce CD8+and CD4+T-cell respons, and neutralizing antibody,the induction of one in the abnce of the others would help to elucidate the roles of the components in protection.The major obstacles to this approach have been the difficulties in eliciting sufficiently strong CD8+T cell respons,and eliciting antibody neutralizing primary HIV isolates.Identifica-tion of vaccines that elicit the respons may repre-nt significant progress towards vaccination procedures that either prevent HIV infection or attenu-ate it and thereby postpone,perhaps indefinitely,the ont of AIDS.
什么是pcCD8+T-cells participate in the organism’s defence in more than one way:they prevent virus reproduction by killing infected cells and also crete a variety of cytokines such as interferon(IFN)-g and tumor necro-sis factors along with chemokines,which directly or indirectly contribute to the control of viral infections. There is an emerging body of data suggesting that CD8+T-cells play a central protective role in all stages of HIV infection:their prence rather than that of neutralizing antibodies correlates with the control of viraemia following primary infection[1,2];they are
*Corresponding author:Tel.:+1865-222334;fax:+1865-222502;
e-mail:ac.uk.
0165-2478/99/$-e front matter©1999Elvier Science B.V.All rights rerved. PII:S0165-2478(98)00164-3
T.Hanke,A.McMichael/Immunology Letters66(1999)177–181 178
剑桥少儿英语下载readily detected in many asymptomatic individuals and circulating cytotoxic T-lymphocyte(CTL)fre-quency correlates inverly with plasma virus load[3]; HIV CTL-escape mutants have been isolated and in some situations associated with deterioration of the clinical status[4,5];cytotoxic activity decreas with dia progression[6–8];CTL were detected in unin-fected,but HIV-expod women[9]and in uninfected infants born to HIV-infected mothers[10].
It is recognized that for practical and safety rea-sons,viral subunits rather than whole inactivated or attenuated HIV preparations are likely to be chon for AIDS vaccines.The u of antigen epitopes derived from veral different proteins reduces the amount of protein or genetic material that needs to be delivered during vaccination,enables the immune respons to be focud towards important or con-rved protein regions,reduces the chance of incorpo-rating undesired(immunopathogenic or immunosuppressive)regions,and facilitates construc-tion of combined multi-isolate/clade vaccines.An ob-stacle to epitope vaccines is the polymorphism of major histocompatibility complex(
MHC)molecules, which caus different individuals to prent different ts of peptides to T-cells.However,it has been esti-mated that optimally lected epitopes prented by thefive commonest HLA types could cover80–90% of the Caucasian and Oriental populations and epi-topes prented by nine HLA molecules would be re-quired for coverage of general population irrespective of ethnic descent[11].If more than one relevant epi-tope for each person is required,the complexity of a broadly efficacious epitope-bad vaccine would in-crea,but would still be feasible.
Central to the evaluation of new vaccine strategies is the u of animal models.Small animal models are uful for initial immunogenicity studies as the cost of using larger numbers of animals is not prohibitive. For HIV and AIDS,infection of non-human primates with immunodeficiency virus offers a spectrum of models in terms of dia verity and difficulty in preventing virus infection.The models range from the infection of chimpanzees with HIV-1SF2,which can be easily protected by neutralizing antibodies [12–14],to infection of rhesus macaques with SIV-mac,where partial or complete protection appears to require cell-mediated immune respons[15–19]. The models permit important aspects of vaccine de-sign and vaccination protocol to be addresd in a faster and cheaper way compared to studies in hu-mans and ultimately provide a basis for a more effi-cient planning of clinical trials.Here,a pre-clinical development of a candidate AIDS vaccine is de-scribed.2.Experimental tting
2.1.DNA multi–CTL epitope6accine
A HIV vaccine prototype was constructed.This was bad on a synthetic gene coding for a string of partially overlapping epitopes,designated H.This multi-CTL epitope protein consists of20human epi-topes(restricted by12different HLA alleles),3 macaque epitopes and1murine epitope.Thus the very same vaccines can be tested for their CTL im-munogenicity in mice,macaque and humans.To be able to follow the induction of T-cells specific for two different epitopes in mice,another polyepitope gene containing one Plasmodium-derived murine epitope was coupled to the3%-end of the H gene to generate gene HM[20].
Direct DNA inoculation is a means of active spe-cific immunization that has been successfully ud for in vivo induction of both humoral and cell-mediated immune respons,including MHC class I-restricted CD8+T-cells[21].A novel vector pTH designed for DNA immunization was constructed,into which the H and HM genes were inrted.The vectors ex-presd high levels of recombinant polypeptides in a transient transfection of cultured cells.Mice needle injected once )with the DNA vaccine generated cytotoxic(23%killing at effector-to-target ratio of100:1)and IFN-g-producing(300 per106splenocytes)CD8+T-cells specific for both murine epitopes[20].
2.2.MVA multi-CTL epitope6accine
MVA is an attenuated vaccinia virus strain which grows well in chicken embryofibroblasts,but has vir-tually lost its ability to replicate in mammalian cells. In addition,MVA has lost veral genes coding for immunomodulatory proteins such are the soluble re-ceptors for IFN-g,IFN a/b,tumor necrosis factor and CC-chemokines[22].Importantly,MVA was proven to be safe when ud in over120000humans [23].
Recombinant MVA carrying the H and HM genes were constructed.Mice were immunized intravenously (i.v.)using a single do of106plaque-form-ing units(pfu)of MVA and the induction of CTL was assd.It was demonstrated that both adminis-tration routes induced specific CTL respons that lasted at least55days and that the ute was moderately more immunogenic than ute(42 and33%killing,respectively,against the more effi-cient HIV-derived epitope at effector-to-target ratio of 100:1).The frequencies of ex vivo splenocytes produc-ing IFN-g upon MHC class I-restricted peptide stimu-lation were determined using an ELISPOT assay to
T.Hanke,A.McMichael/Immunology Letters66(1999)177–181179
be an average of283and227cells per106splenocytes for the HIV-derived epitope following the i.v.and
< administrations,respectively[24].
3.Results
3.1.Enhancement of T-cell induction by a DNA prime-MVA boost6accination regimen
齐东野语翻译Immunization protocols were sought which would improve the levels of induced HIV-specific T-cells.It was found that previous immunological exposure to MVA reduced the efficiency of subquent priming and boosting using the same vaccine vehicle.However,a combined regimen whereby the animals werefirst primed with the DNA vaccine and then boosted with MVA was the most potent protocol for the induction of both IFN-g-producing(1000cells per106splenocytes) and cytolytic(55%killing at effector-to-target ratio of 100:1)CD8+T-cells against the two CTL epitopes[25].
3.2.Accell®gene gun deli6ery can substitute needle injection of DNA in a bimodal6accination regimen Gene gun-mediated immunization delivers DNA-coated gold particles into the epidermis,a major im-munological inductive site,and requires in primates 100–1000-fold less DNA[26].The are important advantages over the intramuscular route.An effective induction of CTL with the multi-epitope DNA vector was achieved by immunization using a gene gun(Pow-derJect Vaccines)alone or in a combined vaccination regimen(70%killing at effector-to-target ratio of100:1) [27].Thus,a gene gu
n-mediated immunization with the multi-CTL epitope gene did induce CTL and could substitute for the needle injection of DNA in the DNA prime-MVA boost immunization protocol.
3.3.Induction of high le6els of CTL in non-human primates
In the past,some vaccines immunogenic in mice failed to induce immune respons in humans.There-fore,it was important to test prior to clinical trials the immunogenicities of the multi-epitope vaccines in pri-mates.The vaccines contained an MHC allele Mamu-A*01-restricted SIV gag-derived epitope[28],thus three Mamu-A*01-positive rhesus macaques were lected us-ing PCR-SSP[29]and immunized twice with the DNA vaccine using the Accell®gene delivery device(weeks 0–8)followed by two intradermal immunizations with 5×108pfu of MVA(weeks17–22).PBL were isolated 1week after the last immunization,peptide-restimu-lated in vitro and assayed for specific CTL lysis in a 51Cr-relea assay.The vaccination regimen induced CTL respons,which resulted in48,50and66% peptide-specific lys at effector-to-target ratios of37:1, 45:1and14:1,respectively.Lymphocytes from week-35 bleed were capable of lysing PHA-stimulated SIV-in-fected PBL.The frequencies of circulated SIV-specific T-cells induced by vaccination were determined using soluble tetrameric Mamu-A*01-peptide complexes and ranged at peak1–5%of CD8+lymphocytes(unpub-lished results).Thus the DNA prime-MVA boost vacci-nation protocol induc
ed effectively and consistently high frequencies of CTL precursors not only in mice, but also in non-human primates which are clor in size and immune responsiveness to humans,and in which the vaccine efficacy against SIV infection can be tested. Human CTL clones and lines for four HLA-re-stricted HIV epitopes were available and ud to test processing and prentation of the epitopes in human cells infected with MVA.H or MVA.HM.All four epitopes were correctly generated in human cells and nsitized infected target-cells to CTL lysis[24].
4.Discussion
4.1.Why does the DNA-MVA regimen induce CTL effecti6ely?
There are veral possible reasons for the effective induction of CTL by the candidate HIV vaccines de-scribed in this work.Firstly,both vaccines were deliv-ered into the epidermis,where the multi-epitope protein is most likely produced by keratinocytes and shed into immediate vicinity of Langerhans cells.The antigen prenting cells are the most efficient initiators and modulators of immune reactions specialized at this site in antigen capture[30].In mice,this route of immuniza-tion is more efficient than the intramuscular delivery [31].Secondly,priming of immune respons using DNA as a vaccine vehicle allows for focusing of the initial CTL on the recombinant immunogen,simpl
y becau that is the only foreign protein expresd. Although recombinant poxvirus may be intrinsically more immunogenic than DNA vectors as vaccine vehi-cles,virus-infected cells produce a large number of virus-derived peptide epitopes prented by MHC class I that compete with the recombinant immunogen for CTL immunodominance.Previously,only a minority of vaccinees immunized with recombinant vaccinia re-sponded to the inrted protein,although all responded to the vaccinia antigens[16,32].Thus,by the time of MVA boost,DNA-primed animals already have in-cread frequencies of immunogen-specific CTL precur-sors.Thirdly,using MVA rather than other vaccinia virus strains or poxvirus may be relevant[31]possibly becau of the unique t of the immunomodulatory molecules that MVA express[22].MVA also gener-
T.Hanke,A.McMichael/Immunology Letters66(1999)177–181 180
ates a strong type I interferon respon,which is likely to contribute to the CTL induction[33].Fourthly,this particular expresd multi-CTL epitope is non-toxic to the transfected cells,which may be due to the ques-tration of the polypeptide into large cytoplasmic vesi-cles[20].This may allow the cells to sustain the production of the immunogen over longer until their destruction by the immune respons.Fi-nally,there is now an emerging body of data suggesting that combined vaccination protocols involving different vaccine vehicles or means of anti
gen prentation to the immune system induce immune respons more effi-ciently[25,27,33–40].
4.2.Can CTL protect against infection with immunodeficiency6irus?亲人的英文
CTL per cannot prevent incoming cell-free virus from infecting host T-cells.However,if there are high levels of memory CTL prent in the relevant tissue or circulation and the virus‘challenge’is low,CTL might clear the small number of infected cells before the virus spreads further and establishes generalized infection.In humans,identification of HIV-specific CTL respons in expod but uninfected individuals raid the possi-bility that CTL may be able to protect or contribute to the protection against HIV infection[9,10,41].In the SIV/macaque model,control of the SIV infection has been associated with the MHC class I Mamu-A*26 allele[15].Using tetrameric complexes,the same Mamu-A*01-restricted gag epitope ud in this study was shown to be immunodominant in SIV-infected monkeys[42],however,there have been two studies so far in which animals with vaccine-induced CTL against this gag epitope were challenged and not completely protected[43],although a decrea in virus load was obrved(S.Norley,Paul-Ehlrich Institute,personal communication).The levels of CTL in the studies were likely lower than found in our animals.Protection against SIV infection may be dependent on the strength of CTL induced by vaccination[16]and the12-mer peptide p11C ud for boosts in the former study may have induced sub-optimal CTL respons[44].
It is also possible that high levels of CTL against proteins ex-presd early in the replication f)may have a better chance to prevent infection than tho against late structural ag)[16].
5.Conclusion
Prototype multi-CTL epitope AIDS vaccines were constructed using vaccine vehicles lected for their suitability for u in humans.Their immunogenicities were confirmed in mice and a combined vaccination regimen was identified that effectively induced CTL respons in mice and non-human primates.The method for induction of CTL,u of individual epi-topes,and a simple and nsitive assay for detection of CTL using tetrameric complexes available in rhesus macaques provide powerful tools for the evaluation of the role(s)of CTL and the importance of individual CTL epitopes in the control of SIV infection.Finally, results from the rhesus monkey model may lead to more rationally designed vaccines and vaccination regi-mens for humans,which can be ud either prophylacti-cally or therapeutically in combination with highly active anti-retroviral therapy(HAART).In turn,results from human trials may indicate which is the most relevant primate model for evalution of candidate AIDS vaccines.
Acknowledgements
Grant support from MRC UK is fully acknowledged.
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