Jay A, BerzofskyJeffrey D. AhlersMichael A, DerbyC. David PendletonTatsumi ArichiIgor M, Belyakov

Approaches to improve engineeredvaccines for humanimmunodeficiency virusand other viruses that causechronic infections

Atjthors' addresses

Jay A. Bcnofeky, Jeffrey D, Ahlers, Michael A, Derby,

C, David Pendleton, Tiit,«umi Arichi, iyor M, Belyukov,

Molecular fminunogenetics and Vaccine

Research Section, Metabolism Branch, National

Cancer Institute, National Institutes of Heahh,

Bethesda. Maryland, USA,

Coirespondence to:

Joy A, Berzofsky

Molecular Inimunogenetics andVaccine Research SectionMetabolism BranchNational Cancer InstituteBuilding 10, Room 6B-I2 (MSC#1S78)NIHBethesda MD 20892-1578USAFax: I 301 496 9956

Acknowledgements

Seep, 167

Summary: We used several approacfics to develop enlianced vaccines forchronic viral infections such as human itntnunodeficiency virus (HIV) andhepatitis C virus (HCV), I) Selected epitopes were used to avoid poten-tially harmful immune responses. 2) Linkage between helper and cyto-toxic T-lymphocyte (CTL) epitopes was found to be important, 3) Wedeveloped an "epitope enhancement" approach modifying the sequencesof epitopes to tnake more potent vaccines, including example,s for HIV andHCV epitopes presented by murine class II and human class I major histo-compatibility complex (MHC) molecules, 4) CTL avidity was found to beimportant for clearing viral infections in vivo, and the mechanism wasexamined. High-avidity CTLs, however, were found to undergo apoptosiswhen confronted wich high-density antigen, through a mechanisminvolving tumor necrosis factor (TNF), TNF-RII, and a permissive stateinduced tiirough the T-cell receptor. 5) We employed cytokines in theadjuvant to steer immune responses toward desired phenotypes, andshowed synergy between cytokines, 6) Intrarectal immunization with pep-tide vaccine induced mucosal and systemic CTL, Local mucosal CTL werefonnd to be critical for resistance to mucosal viral transmission and thisresistance was enhanced with mucosally delivered interleukin-12, 7) Weused an asymmetry in inductioti of mucosal and systemic immuneresponses to circumvent pre-existing vaccinia immunity for use of recom-binant vaccinia vaccines.

Immonotagicof Revieivs 1999Vol. 170: 151-172Printed in Denmark. All rights reservec

Copyright © Maaks^mid 1999

Immunological ReviewsISSN 0105-2896

Introduction

Viruses chat cause chronic infections, such as human immuno-deficiency virus (HIV) and hepatitis C virus (HCV), usuallyehcit immune responses in the infected individual, but theseare often inadequate to dear the virus. The result is acquiredimmunodeficiency syndrome (AIDS) or chronic hepatitis. Sim-ilarly, HCV infection may not produce adequate immunity toprevent reinfection (1), No successful vaccine is yet availablefor either of these viruses. In contrast, successful vaccines areavailable for many viruses, but almost exclusively ones that pro-duce acute, self-limited infections (2). Unless the patient diesof acute overwhelming infection, the immune system clears thevirus and then the recovered individual is immune for manyyears, often a lifetime. It is the latter fact thac led to the para-digm thac the best vaccine is the one that most closely mimicsnatural infection, which in turn led to the widespread itse of

lSl

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

1, Strength or magnitude

2, Breadth: specilicity for dominant eprtopes

3, Type: classes of antibodies and T lymphocytes

4, Affinit/: tightness of binding; detection of low levels of virus

5, Balance: harmful vs helpful responses

6, Location: effector cells or antibodies at the site of virus entry (e,g. in the mucosa)

7, Speed: rate of development of immune response a, vis-^-vis rate of establishment of latent infection

b, vis-a-vis rate of diversification of virus

Table 1. Areas in whichthe immune response tonatural viral infectionmay be inadequate, andwhich could beimproved with anengineered vaccine

livc attenuated viral vaccines (2). These vaccines have heeneminently successful because they ehcit protective immunitysimilar to that elicited by the natural viral infection. However,for viruses that cause chronic infections, for which immunityto che natural infection is inadequate, this paradigm may notapply, A vaccine may have to do better than the natural infec-tion.

In what ways might the immune responses elicited by nat-ural infection be inadequate, and can these be improved with avaccine? One can make a list of several possibihties from firstprinciples and from wbat we know ahout the natural immuneresponse to these Infections (Table 1). 1) The strength or mag-nitude of the responses may be insufficient, 2) The breadth oftbe response may be inadequate, e.g. it may be focused on toolimited an array of dominant antigenic determinants, allowingescape mutations to occur (3-9). 3) The type of immuneresponse may not be appropriate, for example tbe wrongclasses of antibodies or subtypes ofT lymphocytes, such as T-helper (Tli) 2 vs Th 1, might be elicited (10-14). 4) The affm-ity of the antibodies or cytotoxic T lymphocytes (CTL) may beinsufficient. The need for high-affmity antibodies is self-evi-dent, but we will sbow here the importance of high-avidityCTL in clearing viral infection, 5) The halance between helpfuland harmful immune responses may be inappropriate. Forexample, some immune responses to HIV have been suggestedto contribute to immunodeficiency (15-20), or to enhance oraccelerate virus infection or disease, e.g. enhancing antibodiesthai facihtate uptake of virus into cells via Fc or complementreceptors, as has been documented as important in denguevirus infection (21, 22) and suggested by in vitro evidence inHIV infection (23-25). Similarly, some immune responses aretbought to contribute to immunopathological damage to theliver in chronic hepatitis C infection (26-28). 6) The locationof the immune response may not be optimal, for example one,may need to generate antibodies or effector cells at mucosalsites of transmission. 7) Tbe speed of the response may be inad-equate, both with respect to establishment of latent infectionand witb respect to the rate of diversification of the virus.

We would like to address all of tbese issues in an engi-neered vaccine in order to improve on the natural immuneresponse. Our approach has beeu a reductionist one, exatniningindividual epitopes or combinations of epitopes in the form ofsynthetic peptides, ro induce all three arms of the immune sys-tem: antibodies, helper T cells, and CTL. However, the resultscan be extrapolated to other forms of engineered vaccines,including recombinant proteins, recombinant viral vectors,DNA or RNA vaccines, or even modified live attenuated viruses.

Selection of epitopes and design of peptide vaccine

constructs

One goal of an engineered vaccine is the selective use ofepitopes. We attempted to avoid epitopes that might producepotentially harmful immune responses, such as enhancingantibodies (23-25) or autoimmune responses that might con-tribute to immunodeficiency (15-20). Instead, we havefocused on epitopes that could elicit helper T cells, cytotoxicT cells, and neutralizing antibodies (Fig. i).

We first identified several helper epitopes in the HIV-1envelope protein recognized by murine, macaque, and humanhelper T cells (29-32), The TI epitope in the CD4-bindingdomain also elicited help for a neutralizing antibody B-cellresponse to tbe principal neutrahzing antibody epitope (33),To achieve a broader response in individuals of diverse majorhistocompatibihty complex (MHC) types, we first used themouse as a model system and scanned the HIV-1 envelope pro-tein with a series of synthetic peptides to identify ones thatwould be presented by multiple different class II MHC mole-cules (34). We identified six multideterminant regions eachcontaining several overlapping antigenic determinants recog-nized hy mice of different MHC types (34), We reasoned that aslighdy longer peptide spanning a multideterminant regionmight be recognized by helper T cells of mice that could recog-nize each of the overlapping determinants contained therein.Accordingly, we synthesized these "cluster peptides" each con-taining a cluster of overlapping determinants, and found that

152 Immunological Reviews 170/1999

Berzofsk/ et al • Enhancing vaccines for HIV and hepatitis C

Enhancitig

antibody Autoimmune

epitope epitope

Neutralizingantibody epitopeand CTL epitope

PI 8

OverlappingT-helper

epitopes =multideterminant

region 3

Citjsterpeptide

PCLUS 3

Multivalent constructs for increased potency

OverlappingT-helper

epitopes =multideterminant

region 6

Clusterpeptide

PCLUS 6

Second generationvaccines

Modifiedsequences to enhance efficacy

(epitope enhancement)

Fig. I. Engineering of peptide vaccines withselected epitopes. A vir,il ])r<)ieiii, such as the HIV-1envelope protein gp 160. is depicted as a linear barwith segments representing various epitopes. Forantibodies, only segmental or continuous epitopescan be represented in this way, VirUtally all T-ceilepitopes cati be represented a;; continuous segmentsof sequence. As detailed with references in the text,the intent was to avoid epilopes inducing pot:entiallyharmful immune responses, such as enhancingantibodies or autoimmune responses, as shown inmagenta and red. and lo use selecdvely epitopes thatwould induce neutralizing antibodies and CTL.shown in yellow, and T-cell help for these, shown ingreen and blue. One segment. P18 of the V3 loopregion, serves to induce both neutralizing antibodiesand CTL. For T-cell help, multideterminant regionswere identified that contain overlapping antigenicdeterminants presented by different class II MHCmolecules, Peptides spanning tbese clusters ofoverlapping epitopes. called duster peptides, weresynthesized and shown to induce CD4 T-cellresponses in mice and humans of multiple class IIMHC types. The cluster peptides PCLUS3 and PCLUS6were synthesized as continuous peptides on the N-terminal side oi P18. to make first generation vaccineconstructs, Covalent linkage of the belper and CTLepitopes was shown to be important. The sequencesof the belper or CTL determinants could then bemodified to increase their binding to class II or class IMHC tnoiecules and thus increase theirimmunogenicity, or to broaden tbe responses theyelicited. We call this process of sequence modificationto improve vaccine function epitope enhancement.Alternatively, tbe constructs could be mademultivalent to increase immunogenicity.

indeed three of these clusters were recognized by helper T cells

from mice of all four MHC types examined (35). Three of the

cluster peptides did not fulfil! this prediction, indicating that

the whole is not always as good as the sum of the parts, perhaps

because of interference with each other's processing or binding

to MHC molecules. However, we also found that several of these

cluster peptides were widely recognized by human T cells

(35), For this reason, we have used two of these as the helper

portion ofa synthetic vaccine. One of these, PCLUS3, is within

the CD4-binding domain and contains the original TI epitope.

The other, PCLUS6. is near the C-terminns of gp41 and con-

tains the original helper epitope termed TH4.1 or HP53

(Fig. I),

For effector neutralizing antibody and CTL responses, we

were fortunate that the same segment could be used for both

(Fig, 1). The V3 loop of HIV-I gp] 20 had been identified as the

principal neutralizing determinant by several other labs

(36-38), In screening a large nnmber of synthetic peptides

from the HIV-1 envelope protein, we had identified a portion

of the V3 loop, called peptide 18 or P18 (RIQRGPGRAFVTIGK

in the IIIB strain, RIHIGPGRAFYTTKN in the MN strain) as an

immunodominant CTL antigenic determinant in H-2'' mice (6,

39, 40). Indeed, this same epitope was immunodominant in

four strains of mice with at least three unrelated class I MHC

molecules (41), Murine CTL responses to this epitope pre-

sented by H-ZD** have been confirmed in several other labora-

tories (42-48), This epitope w as also recognized by human

CTL with several common human leukocyte antigen (HLA)

class I molecnles, such as HLA-A2,1. -A3, -All and -B27

(49-52). Interestingly, in some mice this was presented not

only by the class I molecule H-2D''. but also by the class II MHC

moiecuie I-A'', and could provide help in vitro for induction of

CTL against itself (53), although an additional source of help

was much more effective in vivo (54). Moreover, surprisingly,

we found that the amino acid residues involved in binding to

the three MHC rnolecules. murine class II I-A**, murine class I

rmmunoiogical Reviews J70/ I999 153

Berzofsk/ et al • Enhancing vaccines for HIV and hepatitis C

H-2D'', and human class I HLA-A2.], were unexpectedly con-cordant (52, 55), Thus, this same region could be used as a tar-get for both neutralizing antibodies and CTL, It also had tbeadvantage of being able to elicit neutralizing antibodies whengiven as a short peptide immunogen, not requiring the intactnative protein structure for eliciting neutralizing activity incontrast to many other neutralizing antibody epitopes (56),This was an essential quality for function in a short syntheticpeptide vaccine. For these reasons, we used it as a prototypeepitope for eliciting both neutralizing antibodies and CTL.despite tbe disadvantage of its hypervariability.

Our original experience with malaria (57) and HIV (33)constructs showed that a helper determinant coupled on tbe N-terminal side of an antibody epitope was effective at eliciting astrong antibody response, consistent witb tbe results of otbers(58-60), We compared several orientations of different belperepitopes coupled to the P18 neutralizing antibody epitope andfound that the vaccine constructs PCLUS3-PI8IIIB andPCLUS6-18IIIB were most effective, eliciting bigh titers of neu-tralizing antibodies, up to 1:16.000 after just two immuniza-tions with complete Freund's adjuvant (CFA) in mice of multi-ple MHC baplotypes (61). Tbese consisted of one oftbe clusterpeptides, either PCLUS3 or PCLUS6, synthesized on the N-ter-minal side of P1 8 of tbe IIIB strain of HIV-1. as one continuoussynthetic peptide of 39-41 residues. These constructs also elic-ited CTL in mice of several MHC haplotypes (54),

We asked whether covalent linkage of CTL and helperepitopes in these constructs was also important for elicitationof CTL against PI8, It had been known for decades tbat hap-ten-carrier linkage was important for antibody production(62-64), but it was not clear if the same applied to CTL induc-tion, because the same mechanism was not possible (seebelow), and because it had not been possible to test untilrecently, since CTL had in tbe past been induced by liveviruses, cells, or tissue grafts. In tbe non-etnulsion adjuvantQS21, we found that only the covalent construct PCLUS3-i8inB induced CTL at all, whereas the PI8 peptide alone, orPI8 mixed with tbe cluster peptide but not coupled, did not(54), In contrast, when the unlinked but mixed peptides weretrapped together in a water-in-oil emulsion of incomplete Fre-und's adjuvant (IFA). then the mixture also worked to someextent, consistent with tbe results of otbers (59, 65), We con-cluded that the helper and CTL epitope segments had to be atleast physically associated and preferably covalently linked toeffectively elicit CTL (54, 66), The explanation could not bethe same as for hap ten-carrier linkage for antibody produc-tion, in which the hapten-specific B cell binds tbe hapten viaits specific surface immunoglobulin, internalizes it and carries

along the carrier, which can then be processed and presentedwith ciass II MHC molecules to helper T cells (67-69). In thecase of CTL, the ligand is on another cell and not internalizedby the T cell, and murine CTLs do not even express class IIMHC molecnles and thus could not present the peptide to aclass II MHC-restricted helper cell. We reasoned that thisrequirement for physical or chemical association betweenhelper and CTL epitope was to allow them to stay togetherwben injected in vivo and get into the same antigen-presentingcell (APC). If tbe helper cell is activated by the same APC as theCTL, then it can potentially deliver cytokines more effectively,and aiso, and perhaps more importantly, can activate the APCto make it more effective at stimulating the CTL (54, 70-72),A likely mechanism is that the helper T cell upregulates co-stimulatory molecules such as B7.1 or B7,2 on the APC, thatinteract with CD28 on tbe CTL, This mechanism is consistentwith the findings that constitutive expression of B7,l allowsinduction of CD8+ CTL without CD4+ help (73), and thatCD40L on helper T cells can activate APCs to make them moreeffective at stimulating CTL (74-76),

Based on these results, we prepared to test these first gen-eration vaccine constructs in humans. To do so, we needed tomake several changes. First, we substituted the MN strainsequence of P18 for that of the IIIB strain, since the former wasmore representative of clade B strains of HIV-1 prevalent inNorth America, Europe, and certain other parts of the world(77), and was also a CTL and neutralizing antibody epitope (3,39, 40), Secondly, we truncated the PCLUS6 helper epitope bya few residues at the N terminus to avoid overlap with thedeterminant eliciting antibodies crossreactive with HLA-DR(15, 16), forming the new helper peptide PCLUS6,1 (78).Third, we had to find an adjuvant acceptable for human use thatwould indnce both neutralizing antibodies and CTL, We badused CFA for inducing antibodies in mice (61), but that wasnot acceptable for use in humans. Further, we had used a dif-ferent adjuvant, QS21, for inducing CTL (54), It was not clearif a single adjuvant wonld be optimal for both types ofresponses, or if conditions optimal for one would be detrimen-tal for the other. Comparing several adjuvants, we found thatmontanide ISA51, a human grade of incomplete Freund's adju-vant, with a purified mannide monooleate substituting for theArlacel A emulsifier, which can be an irritant, was most effec-tive at ehciting neutrahzing antibodies, and also elicited CTLabout as well as the others (78), Thus, we chose this adjuvantfor our human trials as well as future studies in mice.

A phase I trial of the two constructs, PCLUS3-I8MN andPCLUS6.1-18MN, in montanide ISA51, given subcutaneouslyto HIV-1-infected individuals with high CD4 counts and no

154 Immunological Revims 170/1999

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

symptoms, is in progress (carried out by R, Yarchoan's groupat NIH). Initial results, using PCLUS3-18MN in the first 8 indi-viduals, showed that a new CTL response not present beforeitnmunization could be induced by the vaccine in one individ-ual, establishing at least proof of principle of induction of CTLby a peptide vaccine in an HlV-infected human (79). HelperT-cell responses and neutralizing antibody titers were alsoincreased in a number of subjects. Related constructs, using theTl-helper peptide attached to an overlapping portion of the V3loop called SP 10(A), are being tested in preclinical and humanphase I trials in parallel by the group of Haynes and co-workers(33, 46, 80-82), Further collaborative studies are in progressto test the combination of both PCLUS3-1 8MN and PCLUS6.1-18MN given together in individuals on triple drug highlyactive antiretrovirai therapy (HAART).

The immunogenicity of these vaccine constructs could bepotentially increased by several approaches to make secondgeneration vaccines, depicted in Fig. I. One approach, reviewedelsewhere (83-85), is to tnake the constructs multivalent byrepeating copies of individual epitopes. Another is to modifythe amino acid sequences, as will be described below.

Epitope enhancement: improving efficacy of vaccine

epitopes by sequence modification

Tbe remainder of this review will deal with our severalapproaches to improve on first generation vaccine constructs asstudied in murine model systems. Although carried out withsynthetic peptides, the experiments also provide principles thatcan be applied to many types of vaccines.

The first approach, which we call epitope enhancement(84—88), is based on the concept that peptide side ebains inter-acting with the MHC molectile in a peptide-MHC complex areoften distinct from those interacting with the T-celi receptor(TCR). Thus, it may be possible to modify tbe peptide sequencein such a way as to improve the binding affmity of the peptidefor the MHC molecule without altering the surface of the pep-tide-MHC complex which the TCR binds. Such a modificationwould require that no alterations be made that affect either theside chains directly interacting with the TCR, or the conforma-tion of the peptide as it is bound in the MHC groove. If onecould accomplish such an improvement in afilnity. the resultmight be a more potent vaccine (84-88), We will show proofof principle in two model systems, one involving enhancedCD4^ T-ceii heip for induction of CTL tiirough improving thebinding of an HIV-1 peptide to a murine class II MHC mole-cule, and the other involving enhanced induction of CTLthrough improving the binding of an HCV core peptide to

Table 2. Increased potency by removing an adverse interaction

SubstitLition Side chain Potency

T1(436Glu) -CHiC

436Ala -CH3

-CH,CH:CONH;

436Asp -CH.COO-

human HLA-A2, i, and studied with human T cells in vitro andHLA-A2,1-transgenic mice in vivo.

The first observations were based on a study of the originalTl-helper peptide (29) from the CD4-binding region of theHIV-1 envelope protein gpl20. With Boehncke in the lab ofGermain, we attempted to examine the role of each residue inthe peptide in binding to the MHC moiecuie or the TCR (89).We made three different substitutions at each of 1 2 positions inthe sequence, and tested their ability to stimulate T cells spe-cific for Tl. We found that substitutions at most positionseither had no effect or decreased activity, but at one position,residue 436 in the numbering of Ratner et al, (90), sitbstitutionof the natural Glu residue with Ala or Gin resulted in increasedpotency of the peptide by several orders of magnitude. Thus,the substituted peptides stimulated T celis at concentrations atleast 100 to 1.000-fold lower than the natural sequence peptide(89), Since the Ala substitution decreases the physical bulk ofthe side chain as well as removing the negative charge, whereasthe Gin removes only the charge, it seemed as though removalof the charge was the critical factor (Table 2). This interpretationwas supported by the finding that substituting Asp, which issmaller but retains the negative charge, had no effect on pep-tide potency. By using competition for binchng to the murineclass II MHC molecule I-E'' with the cytochrome c peptide thatbinds to this molecule (91), we sbowed that tbe increasedpotency was at least in part attributable to increased affinity forthe class II MHC molecule (89).

We therefore reasoned that this peptide, which binds withhigher affinity to the class II MHC molecule tban the naturalpeptide but is still seen by helper T cells specific for the naturalsequence, might be a more immunogenic vaccine for elicitingT cells against the natural viral sequence. Indeed, we found firstthat the Ala-substituted peptide was more potent in vira forinducing T ceils proliferating in vitro to the natural viral peptidethan was the viral peptide itself (92), To see if this modifiedsequence would be more effective at providing help for a CTLresponse, we took advantage of the fact that T1 is a portion of

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Berrofsky et al • Enhancing vaccines for HIV and hepatitis C

100 n

80-

•t 60 -

20

Snmol

PCLUS3-18IIIB

PCLUS3(A)-18IIIB

0,78:1 1,5:1 3:1 6:1 12.5:1 25:1 50:1 100:1

E:T

Fig. 2. Epitope enhancement for CTL indnction can be accomplishedby modification oftbe T-helper epitope without modifying the CTLepitope. CTL respDiisf iirA,AL jiucu iinmtinized with S nmol of thenative construct PCUJS3-1 8 IIIB (O), or PCLUS3 (A)-18 IIIB, comprisedof lhe mcudified T-lielper epitope and full-lengih CTL deiermiiianiPI 8IIIB (A). Non-specific lysis of Rbroblasi largeis is indicated bydashed lines. Error bars representing SEM are visiblii cmly when greaterthan lilt' size of the symbol, Aniinal,s immimized wiili constructscontaining lhe modified mulddeterminant helper peptide PCLUS3(A)had significantly greaier specific lysis of P18IIIB ptiised Pibroblast targetcells ihan animals iniiiuinized with vaccine constructs containing thenative mullidetermiiiani helper pepiide (33-fold more lytic units){p<0.05). Comparable results were obtained in two independentexperiments,[Modified from (92) with permission]

the PCLUS3-heiper peptide. Thus, we made die Glu to Ala sub-stitution in tbe PCLUS3 portion of the vaccine constructPCLUS3- i 8niB. and compared this with the original construct(Fig, 2). The modified peptide was more effective at inducingCTL, in that it produced 33-fold more lytic units tban tbeunmodified peptide (92), That tbis enbancement was due tothe improved interaction with the class I! molecule was con-firmed by a genetic experiment, in wbit:li two strains of micewere compared that shared the class I moiecuie H-ID^ whichpresents the CTL epitope, but differ in their class LI molecules.One (A.AL) has I-E": that shows improved binding of the mod-ified peptide, and it showed the enhancement in CTL induc-tion. The other (A.TH) uses l-A' to present both peptidesequally, and it showed no enhancement in CTL induction (92).We conclude that the enhancement in induction of class 1MHC-restricted CD8+ CTL was linked to the class 11 MHC mol-ecule. These results demonstrate two points. First, they showagain tbe importance of optimizing class II-MHC-restricted

" T-cell help for induction of class I MHC-restricted CD8+CTL, Second, they provide proof of principle of the approachof epitope enhancement. It is feasible to modify the sequenceofa helper epitope to increase the level of help achieved whenthat epitope is used as part ofa vaccine construct. However, thisproof of principle was obtained in an animal model system,using a murine class II MHC molecule. We wanted to extendthis to human MHC molecules and class I MHC molecules.

To apply epitope enhancement to a peptide binding to abuman class I MHC molecule, we turned to the case of anepitope (C7A2, DLMGYTPLV) from the HCV core protein thatwe bad shown to be presented by HLA-A2,1, the most com-mon human class I MHC molecule, in both HCV-infectedbutiiaiis and HLA-A2.1 transgenic mice (93-95), This peptidehas an HLA-A2.1 binding motif (96, 97), with Leu at position2 and Val at position 9, but it binds with only moderate affmity.Thus, tbere was room for improvement by modifying second-ary anchor residues (98). We screened a series of peptides withsubstitutions at different positions and identified several withup to 10-fold higher affmity for HLA-A2,] than the wild-typepeptide (99), However, only two peptides, I Ala and 8Ala, wererecognized by human CTL from an HCV-infected individual at5 to 10-fold lower concentrations than tbe wild-type peptide(Fig, 3) (99), Otber substituted peptides with higher affinity inthe binding assay, such as IAsn and IThr, did not sensitize tar-gets for lysis hy the human CTL, or, like 4Ala, had reducedactivity for CTL recognition, perhaps because they altered thesurface of the peptide-MHC complex interfacing with the TCR,To test immunogenicity in vivo, we used HLA-A2.1-transgenicmice as a surrogate for humans, having previously found thatthey were a good predictor of what epitopes would be seen byhutnan CTL with HLA-A2,1 (95), We foimd that only the 8Aia-subsiituted peptide was more immunogenic than the wild-typeC7A2 peptide for inducing CTL (99) (when given in incom-plete Freund's adjuvant along wiih a helper epitope peptidefrom tbe hepatitis B core protein that is presented by I-A'' andthe cytokines granulocyte-macrophage colony-stimulating fac-tor (GM-CSF) and interleukin (IL)-12, based on our resultswith cytokines in adjuvant below (100)) (Fig, 3). Moreover,when tested for lysis of targets pulsed with the wild type C7A2peptide. the CTL raised by immunization with tbe 8Ala-substi-tuted peptide actually recognized the wild-type peptide atlower concentration than did the CTL raised by immunizationwith the wild-type peptide (99). We conclude that epitopeenhancement works for human class I MHC molecules as well.

Another approach to epitope enhancement that we carriedout was hased on the observation that CTL specific for thehomologous epitopes (PI 8) of two strains of HIV-1, IIIB and

156 Immunologicol Reviews 170/1999

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

tn

10-6 10-5 10-4 10-3 10-2 10-1Peptide concentration

10 100E/T ratio

1000

Fig. 3. Epitope enhancement ofa CTL epitope firom HCVpresented by a human class I HLA molecule, HLA~A2.1:enhancement of in vitro potency and in vivoimmunogenicity by sequence modification.

A. Recognition of alaiiinc-subsdnned peptides based on (lieHCV core epitope peptide C7A2 by human CTL line ViT2,CIR,A2,I target cells were incubated for 2 h with 'Cr,washed three times and plated at 3.000 cells per well in 96-well round bottom plates with different peptideconcentrations hidicated. After 2 h effector cells were added{E/T ratio: 5/1} and the plates were incubated for 4h, Thewild-type C7A2 is shown in green squares, and the twopeptides with increased potency, active at almost 10-foldlower concentrations, are highlighted in color (blue solidtriangles for lAla and red solid circles for 8Ala),

B. Immunogenicity in vivo of C7A2-substituced peptides inAAD iransgenic mice. Mice were immunized with 50 nmoiCTL epitope plus 50 nmol HBVc 128-140 helper epitopeand GM-CSI'' and IL-12 in incomplete Freund's adjuvant.Two weeks lateT they were boosted, and 10-14 days afterthe boost spleen cells were removed and stimulated in viirowith the CTL epitope. After one week of in vitro stimulationCTL assay was performed with target cells (AAD Con Ahlasts) incubated with or without 10 )JM CTL peptide, CTLfrom each group were tested only with the peptide used toimmunize those mice, but in similar experiments, the CTLraised by immunization with 8Ala killed targets with wild-type C7A2 at similar or higher levels, Lytic units were about5-fold higher m mice immunized with the 8A peptide (redcircles) than in mice immunized with the wild-type C7A2(green squares), whereas the IA peptide (blue triangles)was not immunogenic. Only tlie results for peptide pulsedtarget cells are shown, hi alt cases, the percentages ofspecific lysis against pepiide unpulsed target cells werebelow 5%,

[Both panels are modified from (99) with permission]

MN, did not cross-react, but both focused on a residue at thesame position as the key residue interacting with the TCR (39).By examining multiple substitutions at this position, we foundthat CTL specific for the IIIB strain responded to the PI8 pep-tide with any aliphatic amino acid at that position (Val, Leii.He), whereas the MN-specific CTL responded to the same pep-tides but with any ring structure at that position, including thearomatic amino acids Tyr, Phe, Trp, and His, as well as Pro(101). By making a chimeric peptide with the sequence of theMN version of PI 8 but with an aliphatic amino acid such as Valinstead of the Tyr found in MN, we were able to restimuiate CTLfrom mice immunized with the IIIB version of the envelopeprotein and induce CTL that were hroadly cross-reactive withmultiple strains of HIV Including both IIIB and MN, andresponding to peptides with both aliphatic and aromatic aminoacids at this key position, as well as other residues such as Lysor Gin at this position (101), Thus, chimeric peptides thatbroaden cross-reactivity for multiple viral strains or variants

represent another type of epitope enhancement that could beuseful in developing a broadly effective vaccine.

One caution is raised by studies of an epitope we had iden-tified in the HIV-] reverse transcriptase protein (102) pre-sented by the mnrine class I molecule. H-2K , In studies inwhich we mapped the minimal epitope to the nonapeptideTEMEKEGKI and identified residues involved in interactingwith the MHC moiecuie or the TCR (103), we found that thecentral Lys at position 5 seemed to be critical for neither func-tion, and could be replaced by Ala, If we immunized with this5Ala-substituted peptide, however, we obtained CTL that didnot crossreact with the original 5Lys peptide. This loss of activ-ity could have been due to simple mechanisms such as theintroduction of a large bulky and charged side chain whereonly a methyl group had been before, or alternatively the inter-action of the Lys with the adjacent Glu at position 6. However,neither of these mechanisms could explain the data becausemaking an additional replacement of the Glu at position 6 with

Inimunologicai Reviews 170/1999 157

Berrofsky et al • Enhancing vaccines for HIV and hepatitis C

Ala, resulting in peptide 5Lys-6Ala. restored activity, that is rec-

ognition by the CTL specific for the 5Ala-6Glu peptide. The

doubly substituted peptide 5Lys-6Ala has neither of the origi-

nal residues (5Ala-6Glu), Thus, a second substitution adjacent

to the first restored the loss of recognition caused by the first

substitution (103), This result emphasizes the importance of

pairwise interactions hetween nearhy residues in the peptide in

determining activity. The assumption, frequently made, that

each position can be altered independently, used in analyzing

tbe function of different residues and in predicting binding to

MHC molecules, is clearly only a first approximation. At this

point in time, however, we cannot predict these pairwise inter-

actions in cases such as the example given, and these must be

determined empirically.

In conclusion, not all viral epitopes are optimal in the nat-

ural viral sequence, perhaps not surprisingly given that viruses

evolve by escaping from immune selective pressure. In some

cases, we have shown that it is possible lo improve on these to

make more potent vaccines. Several other labs have made

related observations of sequence modifications that enhance in

vitro or in vivo potency of epitopes from viruses such as HIV

(104) or tumor antigens (105, 106), It is important to note

that the method of epitope enhancement, while studied in each

case with synthetic peptides, can he applied to virtually any

form of engineered vaccine, by modifying the epitope in

recombinant proteins, DNA or RNA vaccines, recomhinant

viral vectors, or even live attenuated viruses. We are currently

applying the 8Ala modification to a DNA vaccine (107) con-

taining the whole HCV core gene, which we have found to he

very ininiun<D genie for inducing CTL responses to several HLA-

A2, l-presented epitopes in HLA-A2.1-transgenic mice, and

which protects these mice against infection with a recombinant

vaccinia virus expressing the HCV core gene via a CD 8" cell-

dependent mechanism (T, Arichi, T. Saito, M, E, Major,

I, M. Belyakov, M, Shirai, V H. Engelhard, S, M, Feinstone,

J, A, Berzofsky, manuscript in preparation).

The role of CTL avidity in virus clearance and clonal

exhaustion

A second area in which immune responses may be inadequate

is the avidity of the antihodies or cells in the effector arm. Avid-

ity of antibodies has long been accepted as important in virus

neutralization, but little was known about the role of CTL avid-

ity. To address this issue, we immunized mice with a recombi-

nant vaccinia virus, vPE16, expressing the HIV-1 IIIB gpl 60

envelope protein (108), and then restimulated the immmie

spleen cells in vitro with syngeneic spleen cells pulsed with

either exquisitely low concentrations of the minimal epitope

peptide. P18-I10 at 0,0001 jaM, or very high concentrations,

100 jiM, or concentrations in hetween (109), We thereby grew

oui a series of CTL hnes with graded avidities, ranging from the

highest avidity stimulated with the lowest concentration of

antigen lo the lowest avidity stimulated with the highest con-

centration of antigen (Fig, 4), The differences in avidity w-ere

shown to be unrelated to differences in the cell surface levels of

TCR, CD8. or adhesion molecules that we could measure. By

default, we presume that the difference is primarily one of

intrinsic affinity of the TCR, but that cannot be proven directly

without cloning the TCR genes, expressing recombinant TCR

molecules, and measuring affmity directly (M, Derby, K, Nata-

rajan. D, H, Margulies, J. A. Berzofsky, work in progress). We

asked whether the different T-cell lines could kill target cells

infected with recombinant vaccinia expressing HIV-] gpl 60,

and found that the low-avidity lines killed the target cells

infected overnight almost as well as the high-avidity CTL hnes

(109), Erom that result, one might predict that they would have

similar efficacy for clearing virus in vivo. To test this question, we

adoptively transferred high or low-avidity CTL hnes into

BALB/c severe combined immunodeficiency (SCID) mice and

then infected the mice with the gpl60-recombinant vaccinia,

vPE16, We measured viral plaque-forming units (pfu) per

gram of ovary, the site at which the virus replicates most effi-

ciently. Surprisingly, only the high avidity CTL produced a

reduction in viral pfu, more than 1.000-fold, whereas the low-

avidity CTL had almost no effect, compared to mice that

received no CTL (Fig, 4). Since the SCID mice have no T or

B cells of their OWTI, the clearance differences must be due to

the CTL lines transferred. This result was reproducible with sev-

eral sets of independently derived high- and low-avidity CTL

lines. We conclude that high-avidity CTL are much more effec-

tive at clearing virus than low-avidity CTL (109). Thus, the

quality of the CTL is as important as the quantity of CTL, and in

vitro efficacy is not always a good predictor of in vivo efficacy.

This finding was subsequently confirmed in another viral sys-

tem, that of lymphocytic choriomeningitis virus, hy Gallimore

et al, ( n o ) . This may have importance for adoptive immuno-

therapy of viral infections and cancer, as well as for designing

vaccines to attempt to maximize CTL avidity. The prediction

that high avidity CTL might be more effective in the adoptive

immunotherapy of cancer was recently confirmed (111), Eur-

ther, an approach to isolate such high-avidity human tumor-

antigen-specific CTL by staining with peptide-MHC tetramers

has been recently described by Greenberg and co-workers

(112). The high-avidity CTL selected by brighter tetramer

staining were more effective at killing human tumor cells in viiro

than the low avidity CTL selected by less bright staining.

158 Immunoiogical Rei'ievvs 170/1999

zofsk/ et al • Enhancing vaccines for HIV and hepatitis C

1.0 |IM Free 110100.0 MM Pulsed0.1 MM Pulsed0.0001 p.M Pulsed

0.1 1 10Concentration of 110 (nM)

100

No CTL1.0 [IM Free 110100 jJM Pulsed0-0001 \iM Pulsed

CTL Line for Reconscitution

Fig. 4. High-avidity CTL can be grown selectively oncells presenting low densities of antigen, and are moreeffective at clearing virus infection in vivo.

A. The concentration of peptide antigen used forstimulation determines the determinant densityrequirement of the resulting CTL line. CTL lines weregenerated by restimulation of splenocytes from BALB/cmice immunized with vPE-16. Lines were generated withstimulators pulsed wirh 100 |aM, O.I ^M. 0.0001 ,LIM ofPI 8-110 peptide or by addition of 1 jiMllO directly intothe cultures, as indicated in the inset legend. Followingmultiple stimulation cycles, CTL lines were assayed forlysis of P815 target cells pulsed with each often differentconcentrations of 110 peptide at an E;T of 10:1. Thehighest avidiiy CTL killed targets at about 200-folc] lowerconcentrations of peptide than did the lowest avidity CTL,but were specific for the same peptide-MHC complex.

B. In vivo, high avidity lines efficiently clear virus whilelow avidity lines are ineffective. 1x10' CTL were injectedi.v. and 5x10' pfu of vPE 16 recombinant vacciniaexpressing gpl60 were injected i.p. into H-2'' SCID mice.On day 3, mice were sacrificed and ovaries harvested, asthe virns has a tropism for the ovaries. Vaccinia present inthe tissues was determined by titering on BSC-1 cells. CTLhnes tested were generated by stimulation with 0.0001^M or 100 |j.M-pulsed splenocytes or in the presence of 1|JM soluble peptide, with varying avidities shown in panelA, and are denoted by the concentration of peptide usedto generate them. Results shown are the titers obtained ina representative experiment with 3-5 mice per group.Similar results were obtained in two additionalindependent experiments. P-values calculated usingStudent's l-test were as follows: mice receiving the linegenerated on O.OODi \iU IlO-pulsed stimulators versusmice receiving no CTL, p = 0.00002 or mice receiving100 UM stimulated CTL, p - 0.00005, or mice receivingCTL generated on 1 fXM soluble II0, p - 0.004. Resultsobtained from mice receiving low avidity lines vs no CTLhad p-values of >0.05.

[Both panels are modified from (109) with permission]

Our working hypothesis to explain the difference in effi-cacy at clearing virus was that the high-avidity CTL could detectcells early in viral infection before much viral protein was syn-thesized, because they were sensitive to very low levels of pep-tide-MHC complex on the cell surface, whereas low-avidityCTL should kill cells only after much viral protein was made, toachieve a higher peptide-MHC density on the surface. In thelatter case, viral progeny may also have already been made.Therefore, the high-avidity cells, by killing cells early in infec-tion before viral progeny was made, should be more effectiveat clearing the viral infection. To test this hypothesis, we stud-ied the time course of viral infection of target cells in vitro, anddetermined the ahility of target cells to be Iysed by the high orlow-avidity CTL at increasing times after viral infection. Thehigh-avidity CTL could kill targets as early as 2 h after infection.

whereas no kiUing by the low-avidity CTL was observed until8 h after infection (M. Derby, M. A. Alexander-Miller, R. Tse,J. A. Berzofsky, manuscript in preparation). This result supportsthe working hypothesis.

Finally, we asked why high concentrations of peptide selec-tively stimulated only low-avidity CTL. In the case of low con-centrations of peptide, it is clear that only the high-avidity CTLcould detect the stimulator cells and respond. However, it wasnot clear why high concentrations of peptide did not stimulateboth types of cells. We hypothesized that the high concentra-tions of antigen might inhibit or kill high-avidity CTL. To testthis hypothesis, we titrated peptide for stimulation of prolifer-ation of high and low-avidity CTL. As predicted, the high con-centrations of peptide inhibited the proliferation of high-avid-ity CTL, whereas they were necessary for the proliferation of

Immunoiojjicui RCTieivs 170/1999 159

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

30-

25 -

20 -

15

10-

5 -

0

None -4 - 3 - 2 - 1 0 1 2

Log Concentration of 110 (MM)

r16I

- 1 4 •§:

-10 l ^ '

-8 ^ J

-4 I

-0 T

Fig. 5. Inhibition of proliferation of high-avidity CTI by APCpulsed with high concentrations of peptide antigen. High-and low-avidity CTL lines were assayed for proliferation on dayfour foUowing routine siitnnlation. (Similar results wereobtained with the lines after 11 days of rest (113)). CTL werestimulated with APC pulsed with various concentrations ofpeptide. Cultures were pulsed with 'H-thymidine at 48 h andharvested at 72 h. Splenic APC pulsed with 100 pM or 10 JJMpeptide inhibited proliferation of the high-avidity line (circles)while maximally stimulating the low-avidicy line (squares). As acontrol for residual free peptide (171), supernatant from thefinal wash of the 100 jiM and 10 |iM-pulsed cells was added at1:1 diluiioninio wells with 0.001 j^M-pulsed APC. Counts fromaddition of the 100 |.iM-pulsed APC wash were 91741270 andfrom the 10 tiM-pulsed wash APC were 9982+409, i.e. noinhibition relative to wells in the absence of the washsupernatant.[Modified from (113) with permission]

low-avidity CTL (Fig. 5) (113). Indeed, the total strength of the

signal from the TCR and CD8 was what seemed to determine

this inhibition, because, at high antigen concentrations, the

proliferation of the high-avidity CTL was paradoxically

increased by blocking with anti-CD8, whereas anti-CD8 inhib-

ited proliferation of the high- and low-avidity CTL at the opti-

mal antigen concentration for each. In other words, the anti-

CD8 reduced the strength of the supra-optimal signal and

pushed the high-avidity CTL back on the titration curve, as if

they had been given less antigen (113). We found that this

inhihition was due to induction of apoptosis, mediated by

tumor necrosis factor (TNF)-a, and the TNF receptor II, not by

fas (Fig. 6) (113, 114), consistent with findings for bulk lymph

node CD8+ cells stimulated with anti-CD3 antibodies (115),

and dependent on caspases (114). However, the mechanism

was different from that of antigen-indnced cell death (AICD) of

CD4+ T cells in that it was TNF rather than Fas-mediated (Fig. 6)

and in that a single exposure of resting CTL to high concentra-

tions of antigen for as little as 2 h, withotit cell cycling or IL-2,

was sufficient to initiate the apoptotic mechanism (Fig. 6), and

removing the cells after 2 li and placing them on optimal con-

centrations of antigen or no antigen did not rescue them (114).

In contrast, AICD of CD4+ T cells was shown to require a re-

exposure to antigen after the cells were induced to undergo cell

cycling from the first exposure to antigen (116, 117).

Surprisingly, although both TNF and the TNF-RII were

necessary for high-dose antigen induction of apoptosis of CTL,

both of them were upregulated at the optimal concentration of

antigen at which death did not occur, and remained high at the

supra-optimal concentration of antigen. (114). This was true

for both secreted and membrane-bound TNF. Thns, hoth TNF

and its receptor together were not sufficient for apoptosis.

High-dose antigen must do something else also, either upreg-

ulate another molecnle necessary for cell death, or decrease a

molecule that prevents cell death. Indeed, we found that Bcl-2

levels were decreased by high but not optimal doses of antigen

(1 14). The supra-optimal signal through the TCR creates a

permissive state that allows the TNF-TNF-RII signal to kill the

cells (1 14). We are currently workitig on the nature of this

permissive state and the difference in signal transduction

between optimal and supra-optimal peptide-MHC density that

accounts for the difference between proliferation and apopto-

sis. The induction of apoptc^sis by high concentrations of pep-

tide-MHC complex may accoutit for the clonal exhaustion that

has heen descrihed as occuring in viral infections with high

levels of viremia (118, 119). Since high-avidity CTL are selec-

tively deleted hy high levels of antigen, and these are the most

effective at clearing virus, the effect on immune control of

virus infection would be expected to be proportionately

greater than the reduction in the total number of virus-specific

CTL.

Steering of the immune response to vaccines with

cytokines incorporated in the adjuvant

The third approach to improving engineered vaccines involves

the use of cytokines incorporated in the adjuvant with the anti-

gen, not only to increase the magnitude of the immune

response, but also to steer it toward a desired phenotype (1 00).

This approach actually dates back to our original attempts to

overcome low responsiveness of the Ir gene by incorporating

recombinant IL-2 in the adjuvant with the antigen (120).

Genetic low responders to myoglobin inade antibodies at

almost the same level as congenic high responders if IL-2 was

160 Immunological Reviews 170/1999

U

irzofsky et ai • Eniiancing vaccines for HIV and i^epatitis C

0.001 |JM 100Concentration of 110

0 ^ 40

30

i 20

10

0.001 MM 100Concentration of 110

aTNFRI

aTNFR2

NoAb

Hours Exposed to Antigen

48

Fig. 6. Apoptosis of high-avidity CDS* CTL induced by supra-optimal

antigeu differs from that of CD4'* T cells in that the CTL apoptosis is

mediated by TNF-a and TNF-RII, not by fas, and requires only a single

brief (2 h) exposure to antigen. A. Inhibition of pToliferaUcin of high

avidity CTL hy Mipia-optinial puptide/MHC density is reversed by

neiUralir.ing aiHi-TNF-a, not by anti-fas. Fonr days following routine

siitnulation, high-avidity CTL were assayed Ibr proliferation resulting

from stimulation with irradiated splenocytes pulsed with various

concentrations of peptide antigen as in Fig, 5. No antibody (yellow bars),

neutralizing anti-TNF-a antibody (I:SO) (green bars), or Jo2 anti-fas

antibody (10 jig/ml) (red bars) was added to determine the role of tbese

two molecules in tlieinhibitiun observed. In the case of anti-fas antibody.

FcBlock (S Jig/ml) was added to prevent possible crosslinking of tlie

antibody by APC. (In other experiments the presence of FcBlock alone did

not affect the response.) No significant effect was observed in the

presence of ami-fas antibody. As a positive control for the function of the

anti-fas antibody, we tested its ability to inhibit lysis of fas-expressing

LI 2 10 transfectants by the fas-ligand-expres.sing d l lS CTL hybridoma

(172). At a 15:1 E:T ratio, the anti-fas antibody (iO ug/mL) inhibited

specific 'Cr release from 78 ± 2% in the absence of antibody to I 5 ± 2%

in the presence of the antibody (81%Lnhibition). [Modiiied from (113)

with permission]. B. The apoptotic death resulting from stimulation with

supra-optimal peptide/MHC determinant densiry is mediated by binding

of TNF-a to TNF-Rn. To determine whether TNF-a transduces the death

signal via TNF-RI or TNF-RII. high-avidity CTL were stimulated with

high (100 [.iM) or low (0.001 JIM) concentrations of peptide in the

presence of blocking antibodies to either TNF-RI or TNF-RII (each at 5

Hg/well). Proliferation following stimulation with APC pulsed with liigh

concentrations of peptide antigen was restored only in the presence of

antibodies to TNF-RII. The inabihty of the anti-TNF-Rl antibody to

prevent death was not due to an inability to block signaling through this

receptor as this aniibody could block TNF-OL mediated death in a TNF-a-

sensitive L929 cell line (data not shown). [Modified from (114) with

permission]. C. The trigger for apoptotic death is a result of the initial

encounter with supra-optimal peptide/MHC determinant density. Wells

of an Immulon IV plate were coated with 0.5 fig of recombinant H-2D'^

blocked, and pulsed with 50 )iM 110 peptide. Following extensive

washing, 1x10^ resting CTL were added to each well. CTL were

transferred to wells without D''/IIO at the hmes indicated in the figure

aEid the culture period continued. At 4S h following initiation of the

culture. CTL were harvested and assessed for apoptotic nuclear

morphology by Hoechst 33342 staining. The CTL were irreversibly

triggered to undergo apoptotic death following as little as 2 h of antigen

exposure. [Modified from (114) with permi.ssion].

ImmunolDjjicuI Reviews 170/1999 161

berzotsky et al • fcnhancing vaccines for HIV and hepatitis C

Cyto-kine

GM-CSFIL-IpIL 2IL-4

IL-7IL-12

TNFaIFN-Y

CTL

t

*

*

t

Prolif-eration

t

-I-t/-f

IL-2

•

IL-4

i -

• •

t

-rs i . *•

t

IFN-Y

•4

••

Si. t

t

• *

-t-

AbIsotype

IgGl,2b

IgGllgG2aIgGl,

2bIgGligGi,2a, 2b

NDlgG2a,

2b

Neutral-izingAb

t

t-t

••

t

ND

Two peptide vaccine candidates: PCLUS 3-18MN, PCLUS6.1-18-MNTwo mouse genetic backgrounds: BALB/c, BIO congenics

Fig. 7. Cytokines incorporated into the emulsionadjuvant with the antigen can enhance and steerimmune responses toward desired phenotypes.Shown is a summary of results from a matrixcomparison of 8 different cytokines incorporateditidividually into incomplete Fretind's adjuvant(Montanide ISA51, Seppic) emulsion with thePCLUS3-18MN or PCLUS6.1 -1 8MN synthetic HIVvacchie constructs, and tested for 7 differentimmune responses. The matrix is mademultidimensional hy the nse of 2 different peptidevaccines and mice of 2 different geneticbackgrounds, BALB/c and B10 congenics. Thus, thearrows indicate only the predominant effect of eachcytokine but cannot encompass the more suhtledifferences between peptides and mouse strains,reported in detail in (100). Upward, downward andhorizontal arrows indicate increased, decreased andunchanged responses, respectively, si. — slight. ND= not determined, Resnhs for cytokine mRNA (notshown) parallel the results for secreted cytokine.Synergy was also seen for CTL induction hetweenGM-CSF and IL-12 and between TNF-a and IL-12(see (JOO)). Results are summarized from (100).

incorporated in the Freund's adjuvant emulsion with the myo-globin, IL-2 administered i.v, three times per day did not haveany effect. Similarly, we found that antibody responses to therepeat sequence neutrahzing antibody epitope of the malariaPlasmodium falciparuni circumsporozoite protein wtire enhancedhy immunizing with IL-2 along with the antigen in the emul-sion adjuvant (121). In the latter study, use ofa mutated IL-2that lacked cytokine activity confirmed that the enhancementrequired the cytokine function of the IL-2, and it was not sim-ply functioning as a carrier protein. We concluded that incor-porating IL-2 in the adjuvant was more effective than systemicadministration because it provided a local depot of the cytokineat the site of the antigen depot, and both were released togetherslowly and went to the same draining lymph nodes where theimmune response was being initiated. A further advantage wasthat only a single dose was sufficient, so one avoided the diffi-culties and potential side effects of repeated systemic adminis-tration of the cytokine.

To extend this approach to other cytokines, not just toincrease the magnitude of the immune response but also tosteer it toward desired phenotypes, such as CTL, Thl or Th2cellular responses or different antibody isotypes, we carried outa comprehensive study of 8 cytokines in the adjuvant with theHIV peptide vaccine constructs, measuring 8 different immuneresponses (Fij|. 7) (100). This matrix of comparisons was madefurther multidimensional by using two different peptide con-structs. PCLUS3-18MN, and PCLUS6,1-18MN, and by usingtwo different backgrounds of mice, BALB/c and BI 0 congenics

(100). The reason for using the different mice is that BALB/chas a predisposition toward Th2 type responses, whereas theB10 congenics have more of a tendency toward Th 1 responses(10, 122-12S),

We found that GM-CSF had the broadest activity in increas-ing the magnitude of most of the immune responses measured,CTL, Thl and Th2 cytokines, and neutralizing antibodies,without altering the balance of the response between Thl andTh2 or between different immunoglobulin (Ig) isotypes (100),In contrast, most of the other cytokines were more selective.For example, IL-12 enhanced the CTL and Thl responses butdecreased Th2 responses, and steered antihody isotopes toIgG2a, as might be expected, as well as increasing neutralizingantibodies, and IL-4 more selectively enhanced the Th2responses and neutralizing antibodies, but decreased CTLresponses (100). IL-2 and interferon (IFN)-y increased T-cellproliferation and IFN-y production, and shifted the antibodyresponses toward IgG2a, but did not substantially increase theCTL responses, somewhat surprisingly Further, GM-CSFincreased neutralizing antibodies in all strains, whereas IL-12increased neutralizing antibody production primarily in BIOcongenic strains of mice and IL-4 increased iieutraiizing anti-bodies primarily in BALB/c mice.

Since GM-CSF and IL-12 each increased CTL responses, hutprobably by different mechanisms, the former acting on anti-gen-presenting cells and the latter perhaps on cytokine balance,we asked whether the two would synergize. Indeed, after a sin-gle immunization, when neither GM-CSF nor IL-12 alone had

162 Immunological Reviews 170/1999

Berzofsk/ et al • Enbancing vaccines for HIV and Hepatitis L.

a marked effect on CTL response, the combination of the twocytokines synergistically enhanced the CTL response to the pep-tide vaccine (100). In the majority of experiments, the effectwas statistically significandy more than additive, that is syner-gistic. Interestingly, although TNF-a did not enhance the CTLresponse by itself, it also synergized with IL-12 to produce ahigher CTL response than with either cytokine alone, and alsomarkedly enhanced antigen-specific production of IFN-y iuBALB/c mice (by more than 10-fold) (100). We are currentlyexploring the mechanism of synergy in these combinations.Furthermore, we have recently found that all three cytokinestogether. GM-CSF, IL-12, and TNF-a. enhance the CTLresponse more than the combination of any two ofthem. Thistriple combiiiati<m of cytokines incorporated with thePCLUS6.I-18MN peptide vaccine construct also markedlyincreases protection of BALB/c rnice against challenge with arecombinant vaccinia virus expressing the HIV-1 MN envelopeprotein (Ahlers et al. manuscript in preparation).

Thus, cytokines can be extremely useful components ofadjuvant formulations because they serve not only to increasethe magnitude of the immune response, hut also to steer theresponse selectively toward desired types of immunity. Localdelivery with the antigen in the adjuvant allows a slow releaseto the draining lymph nodes where the immune response isoccurring, and reduces the risk of side effects frorn repeatedsystemic administration of cytokines. Other groups have usedan analogous approach of incorporating cytokine genes in DNAvaccines (126-128), We are currently developing clinical pro-tocols to incorporate some of these cytokines in montanideISA51, a human grade of incomplete Freund's adjuvant (IFA),for administration of peptide vaccines to patients.

Importance of local mucosal CDS* T lymphocytes

in resistance to mucosal viral transmission

Because natural transmission of HIV is often via a mucosalroute, either genital or gastrointestinal, achievement of protec-tion by a vaccine may require induction of mucosal immunity(129-132). Much mucosal immunology has focused on anti-body responses, such as secretory IgA (133), but only limitedinformation is available about the role of mucosal CTL (134).We therefore asked whether our HIV-1 peptide vaccine con-struct, PCLUS3-18IIIB, could he given via a mucosal route toelicit niucosal CTL.

The Peyer's patches are minilymphoid organs in the intes-tinal mucosa that are believed to be one of the major intestinalinductive sites of mticosal immunity, whereas the lamina pro-pria is thought to be an effector site (135). We asked whether

peptide immunization through different mucosal routes elic-ited CTL in the Peyer's patches or lamina propria. As an adju-vant, we used cholera toxin (CT) mixed with the peptide, hutthere was no linkage of the peptide to the CT, nor was there anyemulsion or other depot form of adjuvant, Peptide with CT wasgiven intrarectally, intragastrically, and intranasally, and com-pared with subcutaneous immunization in IFA (136). Subcuta-neous immunization gave CTL in the spleen, but not in theniucosal sites (136). Of the three mucosal routes, the intrarec-tal immunization gave by far the highest levels of CTL in boththe Peyer's patches and the lamina propria, and also producedCTL in the spleen at levels similar to those induced by the sub-cutaneous immunization. Thus, there was an asymmetry, inthat systemic immunization gave only systemic CTL, whereasmucosal immunization gave both mucosal and systemic CTL(Fig. 8) (136), This asymmetry turned out to be very useful, asnoted below.

The CTL immunity in the mucosai sites and spleen lasted atleast 6 months after immunization, and the CTL were able tokill targets endogenously expressing HIV-1 IIIB gpl60 (136),Furthermore, although the response was enhanced by CT, wefound that we could induce CTL in the mucosal sites and spleenwith peptide alone given intrarectally, w-ithout CT or any otheradjuvant (136), Recent data indicate that a mutant Escherichio colilabile toxin (LT) that is safer for human use (137) may beequally effective as a mucosal adjuvant for HlV-specific CTLinduction in mice (I. M. Belyakov. J. D. Clements. ]. D. Ahlers,W Stroher, J. A. Berzofsky, manuscript in preparation).

To determine the role of these CTL in protection againstmucosal viral transmission, we could not challenge the micewith HIV, so we used a surrogate virus, recombinant vacciniavirus vPE16 expressing HIV-1 gp 160 (a gift of P Earl and B.Moss) (108). This virus has the advantage that, unlike HIV, itdoes not incorporate gpI60 into the virus particle, and there-fore is not susceptible to anti-gpl60 neutralizing antibodies.Therefore, the only place the gpl60 is expressed and theimmunity could be acting is in the infected cells. We challengedthe mice immunized intrarectally with the recombinant vac-cinia virus expressing gpl60 and tneasured viral pfu in theovary (where this virus replicates most) after 6 days. Theintrarectally immtmized mice consistently had a virus titerahout 10,000-fold lower than unimmunized mice (136), Theprotection lasted at ieast 6 months, even though the antigenwas not given in depot form, and was specific for gpl60,because there was no protection against a control vaccinia virusexpressing only p-galactosidase (138).

Although a number of studies have detected mucosal CTL(134, 139—142) or have shown vaccine protection against viral

Immunological Reviews 170/J 999 163

Berzotsky et al • Enhancing vaccines for HIV and hepatitis C

Systemic Immunization 1E+09

Lamina propria

0 10 20 30 40 SO 10 20 30 40 50

% Specific lysis at S0:^

Fig. 8. Asymmetry in CTL response to mucosal vs systemicimmimization: intrarectal immimization with HIV peptide vaccineconstrnct PCLUS3-18IIIB indnces both mucosal and systemic P18-specific CTL responses, whereas, systemic (s.c.) immunization inducesonly systemic CTL. BALB/c mice were immunized intrarectally with 50|.ig peptide and 10 fig cholera toxin, without other adjuvant, or s.c. inincomplete Freund's adjuvant, on daysO, 7, 14, and 21, and the indicatedtissues were harvested on day 35. The lymphocytes from these Ussueswere stimulated for 7 days with PI 8-110 peptide and IL-2 as described(I 36), and then assayed for lytic activity on P8 15 targets. Kilhng of PI 8-II0 peptide-pulsed targets (red bars) is compared with killing ofunpulsed targets {green bars) at an effector-to-target ratio of 50:1. Similarresults were obtained at ratios of 25 and 12.5:1.[Modified from (I 36) with permission]

transmission (!42-149), it has been hard to prove cause andeffect. Even when protection against mucosal transmission hasbeen shown to depend on CTL, it has not been clear if the CTLneed to be in the mucosa to be effective (150-152). Our ani-mal model with the recombinant vaccinia that was not suscep-tible to neutralizing antibodies specific for gpl60 gave us theopportunity to address this question. First, we treated theimmunized mice with anti-CD8 antihodies to deplete CD8^CTL in vivo, and found that all protection against intrarectal chal-lenge was abrogated (Fig. 9) (138). Thus, the protection isdependent on CD8* cells, and not on other mechanisms thatmight attack infected cells, such as antihody-dependent cellularcytotoxicity (ADCC) or complement-mediated lysis. However.since the intrarectal immunization elicits CTL in the spleen aswell as in the Peyer's patches and lamina propria, and the pro-tection was measured as viral pfu in the ovary, this resuit didnot distinguish between CTL protection at the mucosal site andCTL protection due to systemic CTL acting in the ovary or else-where. To distinguish these possibilities, we took advantage ofthe asymmetry noted earlier (Fig. 8). Mice immunized subcuta-neously with peptide had levels of CTL in the spleen just as highas those immunized intrarectally, hut they did not have detect-able mucosal CTL. Therefore, we reasoned that if splenic CTLwere sufficient to protect, the subcutaneously immunized miceshould be as resistant against mucosal viral challenge as theintrarectally immunized mice. However, this was not the case

1E+04

Immunization

Treatment

NONE IR IR

NONE NONE ANTI-CD8

1E+09n

1E+04

Immunizaiion

Challenge

NONE SC IR

vPE16 vPE16 vPE16

Fig. 9. Intrarectal peptide immunization indnces resistance againstmucosal challenge with virus that is CDS dependent and dependent onthe presence of CTL in the mucosal sites. A. Protection induced bymucosa! immunization with HIV-peptide is dependent on CDS positiveT celis. BALB/c mice were immunized as in Fig. 8, and challenged on day35 intrarectally with 2 x 10'pfu of vPE16 vaccinia virus expressing gp160IIIB. One group was treated i.p. with 0.5 mg monoclonal anti-CD8antibody (clone 2.43; NIH, Frederick, MD, USA) 2 days before and 3 daysafter the virus challenge. Bars show means of 5 mice per group. Thedifference between the middle and right bars is significant at p < 0.01 byStudent's t-test. B. Only intrarectally (IR) immunized mice, which haveCTL in the Peyer's patches, lamina propria, and spleen (Fig. 8) areprotected, but mice immunized subcutaneously (SC). which have splenicbut not mucosal CTL. are not. Mice were immunized as for Fig. 8 and forpanel A. On day 35. IR (bar 3) or SC (bar 2) immunized BALB/c micewere challenged intrarectally with 2.5 x 10'plaque-forming units (pfu)of vaccinia virus expressing gp 160IIIB, and compared withunimmunized mice (bar I). Bars show means of 5 mice per group. Thedifference between bar 3 and either of the others is signiOcaiU at p< 0.01by Student's t-test.[Both panels are modified from (138) with permission]

(Fig. 9). We found that only the intrarectally immunized micewere protected against intrarectal challenge, and the subcutane-ously immunized mice were not protected at all againstintrarectal challenge (138). Thus, we conclude that systemicCTL are not sufficient, and CTL must be present in the localmucosal site to mediate protection against mucosal transmis-sion.

164 Immunological Reviews 170/1999

Berzofsky et al - Enhancing vaccines for HIV and hepatitis C

12,5:1 25:1 50:1 100:1 12.5:1 15:1 50:1 100:!

—a— vSCS MVA8g.6

—D— vSCS MVA89.6

—A— vSC8 VPE16

— i — vSCB VPE16

-%— none MVA89.8

—O— none MVA8S.6

—y— nono vPE16

—V— none vPElB

[ Second,atlon

\fSC8 MVA8S.6

vSCS MVA8e.8

vSC8 WPE16

vSCe VPE18

none MVABe.6

none MVA8e.6

none vPE18

nofl« vPEie

P18-89.BR10

none

PI 8-110

none

pie-8g.eRio

nono

PIWIO

nona

P1B-SS.eR10

none

PI 8-110

none

P18-89.6R10

none

PI 8-110

none

12.5:1 U:l 50;l 100:1

E:T

12.5:1 25:1 SO:1 100:1

E:T

Fig. 10. Mucosal immnnization circumvents the barrier torecombinant vaccinia immunization caused by pre-existing poxvirnsimmunity. Absence of PI 8-89.6R10- and P18-I10-speciPic CTLresponses in the spleen (SP) and Peyer's patch (PP) in mice withpreexisting immunity to vaccinia after subcutaneous (SC) immunizationwith recombinant viruses (A, B) and induction ofP18-89,6R10- andP i 8-110-specific CTL responses in the SP and PP in mice with pre-existing immunity to vaccinia after intrarectal (IR) immunization withrecombinant viruses (C. D). PI 8-89.6R10 is the homolog of PI 8-110.but from the strain 89.6 of HIV-1, corresponding to the strain of HIV-1envelope expressed in the recombinant replication incompetent vacciniaMVA89.6. vPE16 is the replication competent recombinant vacciniaexpressing HIV-1 envelope protein gp 160 from strain IIIB. BALB/c mice

weTe immuni7ed subcutaneously with vSC8 vaccinia virus at 5 x 10' pfu(A. B. C, D). One month later these mice and control naive mice werereimmunized either subcutaneously (A, B) or intrarectally (C, D) witheither recombinanc vaccinia MVA89.6 or vPEl6 at I x lO^pfu. Threeweeks later the induction of PI8-89.6R10- and Pl8-I10-specific CTLresponses were studied in the SP (A. C) and PP (B, D) after 7 daysrestimulation with the corresponding peptide. Resuhs are means of 10mice per group, with SP tested individually and PP pooled to obtainsufficient cell numbers. Groups preimmunized with vSC8 are coded red.and those not preimmunized are coded green, for clarity. Closed symbolsare results on peptide-pulsed targets and open symbols are results onunpulsed targets, E:T, effector-co-target ratio.[Modified from (170) with permission]

Although this conclusion best fit the data, it remained pos-sible that the CTL induced in the spleen by subcutaneousimmunization were qualitatively different from the CTLinduced in the spleen by intrarectai immunization, and that thisdifference explained the inability of the former to protect. Toaddress this possibility, we compared results from challengingthe mice intraperitoneally We reasoned that if the protectionwas mediated by systemic CTL acting, for example, in theovary, they should protect equally well against intraperitonealchallenge. However, mice immunized intrarectally were pro-tected only against intrarectal challenge, not against intraperi-toneal challenge, indicating that the CTL likely acted at themucosal site, and not in the ovary. Of course, these are immu-nocompetent mice, and they eventually clear the virus from theovary, but the titers early in infection are greatly reduced by theintrarectal immnnization only when the transmission ismucosal. It still remained possible that the difference was due

to the delay in transmission of virus from the mucosal site tothe ovary, allowing time for the CTL memory cells to be reacti-vated by the infection. However, two other results arguedagainst this hypothesis. First, the protection was seen as early asday 2 in the ovary, before the virus had time to rephcate in theovary, and therefore was likely mediated by reduction of thevirus inoculum at the initial mucosal site of infection, reducingthe level of virus that could reach the ovary (138). Second, pro-tection was also seen in the gastrointestinal mucosa itself, fromaround 10* pfu in unimmunized mice to undetectable (< 10pfu) in the intrarectally immunized mice (138). Thus, theamount of virus in the mucosa is severely reduced, althoughthe numhers are much smaller than found in the ovary. We con-clude that protection occurs at the mucosal site, where localCTL clear infected cells in the mucosa before virus can bereleased and spread to systemic sites such as the ovary. Whenthe transmission is mucosal and the CTL are present locally, the

Immunologica! Reviews 170/1999 165

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

CTL have a place to make their stand to prevent the virus fromgetting beyond this initial barrier, whereas when the virus isgiven intraperitoneally. the CTL have no place to contain thevirus before it reaches the ovary. This conclusion is also consis-tent with the correlation recently reported between the pres-ence of mucosal CTL and resistance to intrarectal challenge ofmacaques with simian immunodeficiency virus (142).

Given the critical role of mucosal CTL, it was important tomaximize the mucosal CTL response. A due to one approachcame from our observation that the intrarectal peptide vaccineinduction of CTL was greatly reduced or eliminated by treat-ment of the mice in vivo with antibodies to IL-12, given beforeaud after eacb immunization (136). We reasoned that if endog-enous IL-12 was necessary, and probably limiting in quantitysince it could be blocked with the limited amount of antibodywe could administer in vivo, additional IL-12 might enhance theresponse. To deliver IL-12 iutrarectally with che peptide, wemixed it with DOTAP (Boehringer-Mannheim, Mannheim,Germany), a cationic lipofection agent, and compared mice soimtiitmized with mice that received only peptide with DOTAP,but without the cytokine. Ln hoth cases, CT was also included.The mice given IL-12 had a significant enhancement of CTLinduction in botb tbe spleen and the mucosal sites compared tothose immunized without cytokine (138). In contrast, if theIL-12 was administered intraperitoneaily at the time ofintrarectal immunization with peptide, it had no effect. Thus,the enhancement required local dehvery of the cytokine. Theanimals immunized with peptide plus IL-12 intrarectally alsohad an increase in the amount of IFN-y made in vitro when theircells were stimulated with antigen. Most importantly, the IL-1 2also increased the degree of protection, in that mice immu-nized intrarectally with peptide alone had a 4-log reduction invirus titer in the ovaries after intrarectal challenge, whereasthose immunized with peptide plus IL-12 intrarectally had a 6-log reduction in virus titer (138). Thtis, we have learned howto protect mice against colorectal mucosal transmission ofvirus, the importance of CTL in that protection, and how toincrease the level of protection against mucosal transmission.

Overcoming the problem of pre-existing poxvirus immunityin use of recombinant vaccinia vectors as vaccines bymucosal immunization

Recombinant vaccinia virus vectors have been among the mosteffective vaccine constructs in animal studies and have beenimmunogenic in humans (1S3, 1 54). The problem of safetyhas been overcome by the deveiopment of highiy attenuatedstrains of vaccinia, such as NYVAC (155) and modified vaccinia

Ankara (MVA) (156-159), as vectors. MVA is replicationincompetent in most mammalian cells, hut we and others havefound that as a retombinant vector, it is at least as immuno-genic as replication competent strains of vaccinia (148,156-158, 160-167). One problem that remains, however, isthat a Iaige proportion of the adult population has been immu-nized against smallpox with a vaccinia vaccine, and there is evi-dence in mice and humans tiiat pre-existing immunity to vac-cinia can limit the effectiveness of a recombinant vaccinia vac-cine (168, 169).

The asymmetry in mucosal and systemic immuneresponses that we observed after mucosal versus systemic infec-tion (136, 138, 160) suggested to us that we might take advan-tage of this asymmetry to circumvent the problem of pre-exist-ing systemic immunity to vaccinia (170). We reasoned that ifthe mucosal immune system remained naive after systemicimmunization, it might stili be amenable to immunizationwith recombinant vaccinia vectors that would not be effectivesystemically in the face of pre-existing systemic immunity tovaccinia (170). Indeed, we bad ohserved the same asymmetrywhen we immunized mice incrarectaily or systemicaliy with arecombinant MVA or recombinant replication competent vac-cinia expressing HIV-1 gpl60 as antigen (160). Thus, micethat were immunized intrarectally with cither recombinantvirus developed CTL in both the spleen and the mucosal sites.Peyer's patches and lamina propria, whereas mice immunizedintraperitoneally with either virus developed CTL only in thespleen, and had marginal or no response in the Peyer's patchesand no response in the lamina propria (160).

Since previous studies of the effect of pre-existing vacciniaimmtmity focused on antihody responses, we first confirmedwhether systemic (s.c.) immunization with a controi vacciniavSC8 expressing only P-galactosidase would interfere with CTLinduction by subsequent s.c. immunization witb a recombinantvaccinia expressing HIV-1 gp 160, either the replication compe-tent vPE16 or the replication incompetent MVA89.6 strain. Incontrast to nnimniunized mice that made a good CTL responseto each virus, tnice previously immunized s.c. with vSC8 failedto make a CTL response to gpl60 when immunized s.c. witheither vPEl 6 or MVA89.6 viruses (170). Thtts, the effect of pre-existing immunity to vaccinia extends to CTL as well as anti-bodies (Fig. 10).

To determine whether mucosal immunization would cir-cumvent this probiem, we gave the second immunizationintrarectally rather than s.c. In contrast to the subcutaneousimmunization, the intrarecta] immunization with eitherrecomhinant vaccinia virus induced CTL specific for gpl60 inboth the spleen and the mucosal sites in mice that had been

166 Immimologiciil Rcviewi 170/1999

Berzofsky et al • Enhancing vaccines for HIV and hepatitis C

pre-immunized with vSC8 s.c. as well as mice that had not beenpre-immunized (17 0). In the Peyer's patches, this CTL responsewas comparable to that in mice that had not heen pre-immu-nized, and in the spleen, it was still substantial (Fig. 10) Intra-nasal immunization also overcame the adverse effects of pre-existing immunity. Similar results were found for the antibodyresponse (170). In addition, we found that boosting with a sec-ond dose of the same recombinant vaccinia, MVA89.6. by theintrarectal route was more effective than hoosting with thesame vaccinia by the subcutaneous route. These results supportour hypothesis that the mucosal immune system remains naiveto vaccinia (as well as any recombinant protein genes itexpresses) when it is given subcutaneously, and tbus that pre-existing vaccinia immunity can be circumvented by mucosalimmunization with recomhinant vaccinia vector vaccines. Theyalso emphasize the importance of the compartmentalizationbetween systemic and mucosal immune systems and demon-strate the functionai significance of the asymmetry in traffick-ing of iympiiocytes between tbese systems.

Conclusions

To come full circie to the question with which we opened, canwe engineer a vaccine to improve on the immune responseselicited by naturai infection witii viruses that cause ciironicinfections, such as HIV and HCV? We have examined severalapproaches to address each of the points raised in the introduc-tion.

First, the strength or magnitude of the response can beincreased by modifying the epitope to improve binding to theMHC molecule, or by incorporation of appropriate cytokines.Second, the breadth of the response may be expanded hyincreasing the strength of the response to suhdominantepitopes, either by modifying these epitopes to increase theiraffmity for the MHC moiecuie, or hy increasing the ievei ofresponse with cytokines or potentially co-stimulatory mole-cules. Third, the type of immune response may be altered orsteered by selective use of cytokines in the adjuvant witii theantigen, selectively increasing CTL, Thl vs Th2 responses, ordifferent antibody isotypes. Fourth, we have seen that che avid-ity of CTL may be critical in clearing viral infection. This resulthas immediate applicabiiity to adoptive immunotherapy ofvirai infections and possibiy cancer. For vaccine deveiopment,we are currentiy expioring ways to seiectiveiy induce bigheravidity CTL. Fifth, the baiance between helpfui and barmfuiimmune responses can be aitered by use of engineered vaccinescontaining oniy a subset of epitopes from the virus, avoidingones that induce harmful responses. Sixth, we have shown that

iocai mucosai CTL are criticai in providing resistance tomucosal viral transmission, and thus a vaccine for viruses suchas HIV that are frequently transmitted through a mucosai routemust induce mucosal CTL immunity. We have seen that an engi-neered peptide vaccine construct or recomhinant viral vector,given intrarectaiiy, can induce a strong protective mucosal CTLresponse, and that response can be increased hy iocai deliveryof IL-12 with the vaccine antigen. Seventh, the speed of theresponse can be increased hy priming the immune system inadvance with a prophylactic vaccine. An excellent vaccine forsuch priming is a recombinant vaccinia vector, such as theattenuated replication incompetent MVA recombinants. Wehave discovered that the problem of interference by pre-exist-ing immunity to vaccinia witb induction ofa new response toa recombinant vaccinia vector vaccine can be circumvented byimmunizing through a mucosal route. This approach takesadvantage of our finding that the mucosal immune systemremains naive after systemic immunization, whereas mucosalimmunization can induce both systemic as weii as mucosalimmunity. Thus, tiie asymmetric trafficking of immune cellswe bave found between mucosai and systemic compartmentscan be effectiveiy expioited.

Aithough there is not yet a successfui vaccine for suchchronic virai infections as HIV and HCV, we hope tiiat tiieseresuits wili contribute btiiiding biocks for constructing such avaccine. Since viruses have evoived to escape the immune sys-tem, an attenuated but otherwise natural virus may not be thebest vaccine in all cases. We ought to be able to make a moreeffective vaccine, based in part on the principles outlinedherein. In the case of viruses that cause chronic infections, suchimprovements may prove essential.

Acknov/ledgements

This review describes work done principally in the MolecularImmunogenetics and Vaccine Research Section. MetabohsmBranch, NCI. over more than a dozen years. Needless to say. itrepresents the efforts ofa multitude of individuals, both mem-bers of the section and a host of extremely helpful collabora-tors, both at the NIH and elsewhere. We would iike to thankeach ofthem immensely for their contributions, referenced inthe text. The principal former members of our lab who con-trihuted to the work described include, alphaheticaily, MarthaA. Aiexander-Miller, Kemp B. Cease, Anne S. DeGroot. PaulaHale, Anne Hosmalin, Kazutaka Kurokohchi, Graham R. Leg-gatt, Pahlo Sarobe. Mutsunori Shirai, Hidemi Takahashi. andToshiyuki Takeshita. The principal outside coiiaborators with-out whom this work wouid not have been possible include, al-

Immunoiogical Reviews 170/J 999 167

Berzofsk/ et al • bnhancing vaccines for HIV and hepatitis C

phabetically. Toshitaka Akatsuka, Manuel Battegay, Mario Cleri-

ci, James L. Cornette, Charles DeLisi, Patricia Earl, Victor H. En-

gelhard, Stephen M. Feinstone, Robert C. Gaiio, Ronaid N. Ger-

main. Michaei A. Good, Pierre Henkart, Stephen Hoffman,

Brian Keisall, Steven Kozlowski, Anita Malik Kumar, Sanjai Ku-

mar, Richard Litde, Marian Major, Hanah Margalit, David H.

Margulies, Louis H. Milier. Bernard Moss, Peter L. Nara, Ligia

Pinto, Apurva Sarin, Gene M. Shearer, Alan Sher, Warren Stro-

ber, Robert Yarchoan, Linda Wyatt, Daniei Zagury, aiong with

many of tiieir co-workers who are too numerous to list, btit are

included in the references. We apologize for any inadvertent

omissions. It bas been a great privilege to work with each of

these wonderful colleagues over the years. It is only through

such a network of great collaborators tbat our laboratory has

been able to make whatever progress we have achieved.

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