- Email: [email protected]
Design of peptide and polypeptide vaccines
442
Design of peptide and polypeptide vaccines Tamar Ben-Yedidia Advances
and Ruth Arnon*
have been made in the development
based on synthetic peptides tumor-associated
antigens and protective
and parasites. Advances design of vaccines
and polypeptides
of vaccines representing
epitopes
of viruses
within the past year include the
based on artificial proteins, for example
multiantigen
peptides,
branched
recombinant
peptides,
as well as single T cell epitopes
tumor antigen peptides.
polypeptides,
fusion and
Although peptide vaccines
and
are not in
use as yet, their potential is being explored.
Addresses Department of Immunology, Weizmann Institute of Science, Rehovot, Israel *a-mail: [email protected] Current Opinion in Biotechnology
1997, 61442-440
http://biomednet.com/elecre1/0958166900000442 0 Current Biology Ltd ISSN 0958-l
669
Abbreviations BSA
bovine serum albumin
CSP CTL
circumsporozoite
GST HLA MAP
protein
cytotoxic T lymphocyte glutathione_S-transferase human leukocyte antigen multiple antigen peptide
Advantages of peptide- and polypeptidebased vaccines Both humoral and cellular arms of the immune system recognize and react with only specific regions of pathogens. This led to the design of vaccines based on subunits of the pathogen, either naturally occurring immunogenic polypeptide(s) or synthetic peptides that correspond to highly conserved regions required for the pathogen’s function. The goal of developing this strategy is to vaccinate with a minimal structure, consisting of a well-defined antigen, in order to stimulate an effective specific immune response, while avoiding potential hazardous effects. This strategy must take into consideration, however, the essential presence of epitopes recognized by both B cells and T cells, as well as the MHC restriction of the T cell response. Since the cellular immune response in humans is restricted to specific HLAs, any single epitope-based vaccine will probably not be effective in a broad population. This can be overcome by the use of vaccines comprising several peptides which would be effective in inducing both arms of the immune response. Furthermore, this approach allows the selection of those epitopes restricted to the HLAs which are most frequent in the population of interest.
Delivery systems for small antigens Introduction
Carriers
The development of vaccines has been one of the most important achievements of immunology and medicine to date. The traditional vaccines are based on the intact disease-inducing agents - inactivated or live attenuated (aimed at maintaining the immunogenicity of the pathogen while eliminating its infectivity and/or toxic effects)-and have led to diminished incidence in morbidity and mortality of a large number of infectious diseases, including major killers such as smallpox and polio. Yet there are crucial drawbacks incurred by the current procedures for vaccine preparation, such as the difficulty of irr vitro culturing of many viruses and parasites, biohazard and safety considerations, as well as the loss of efficacy due to the genetic variation of many viruses. New approaches for vaccine development are aimed at overcoming these shortcomings. They comprise on one hand the use of recombinant DNA technology, including naked DNA vaccines, and on the other hand the utilization of synthetic peptides which constitute epitopes that induce protection against infection. Although no peptide or polypeptide vaccine is being used as yet, several such experimental vaccines are undergoing clinical trials. Recent progress in this field is described below. This review will demonstrate the recent advances in the development and design of peptide-based vaccines and appropriate delivery systems.
For peptides that are small and nonimmunogenic, coupling to a carrier is essential to endow them with the capacity to induce a response of from T helper cells. Large proteins that contain sufficient reactive groups are suitable as carriers and bacterial proteins (tetanus toxoid, diphtheria toxoid) are widely used for chemical conjugation of peptides. Incidentally, proteins of mammalian origin, such as BSA, have been reported to be less efficient [l]. An alternative method of peptide presentation involves recombinant DNA methods. A plasmid containing the carrier protein gene is used and the epitope DNA sequence can be inserted into it. The expressed fusion protein can be of bacterial origin, for example the flagellin of Salmonella [Z], or of viral origin [3].
Viruses
viruses also serve as carriers-one of the vectors most frequently used for expressing foreign antigens is vaccinia [4,.5]. Among the other viruses used for this purpose are the retroviruses and fowlpox virus, adenoviruses and polio [6--81. Recently, the use of virus particles as carriers for peptide has been proposed: the use of empty capsids of retroviruses [9] and hepatitis B virus has been reported [9,10]. Intact
Design of peptide and polypeptide vaccines Ben-Yedidia and Arnon
Artificial proteins
The multiple antigen peptide (MAP) approach for immunization [11’,12] offers several advantages over the conventional carrier-antigen construct. MAP comprises multiple clusters of antigenic peptide epitopes synthesized on a branched oligolysine core. The greater part of the molecule consists of the immunogen in a well-defined orientation, avoiding unnecessary or even suppressor epitopes that might be present in a conventional carrier. The MAP is highly immunogenic and can be constructed as a multivalent vaccine, carrying any selected peptide. The application of MAP for vaccination was studied in model systems, and was found to protect animals from malaria [13] and to stimulate both humoral and cellular responses against HIV [14]. Another artificial protein that is even purer than MAP and can be produced in higher yields is the polyoxime vaccine, which was recently shown to induce high levels of antibody production against the peptides that are presented on it [15].
Liposomes and immunostimulating
complexes
These lipid-based particles enable the introduction of lipid-soluble molecules or peptides to the immune system. Liposomes have been shown to induce both humoral and cell-mediated immune responses to a wide spectrum of antigens [13,16]. Immunostimulating complex technology efficiently elicits a protective T cell response specific for the peptide and bypasses the need for an adjuvant [16,17]. It delivers soluble antigens both to the cytosolic and to the endosomal pathways of antigen processing, the natural pathway of the induction of a class I immune response. Cytosolic delivery can prime a cytotoxic T lymphocyte (CTL) response specific for the peptide, as was shown with a peptide from the measles nucleoprotein [18].
Peptide and polypeptide against viruses
vaccines
Influenza
Influenza is a major public health concern: it occurs in recurrent epidemics that start abruptly, spread rapidly and are frequently distributed worldwide. Usually the influenza infection is a mild disease, responsible for many millions of infected individuals, causing an economic burden. Mortality is high in infants and the elderly leading to tens of thousands of deaths annually. The influenza virus undergoes frequent and unpredictable changes of the surface glycoproteins hemagglutinin and neuraminidase. These antigenic variations enable the virus to escape the immune system and reduce the effectiveness of vaccines. The currently available vaccines are of several types, namely whole virus vaccines, subunit vaccines and live-attenuated influenza virus vaccines. Only killed (inactivated) and subunit vaccines are currently licensed for human use. These vaccines fail to induce local and cellular immunity and their efficacy is limited; hence,
novel approaches are being considered, of peptide-based vaccines.
including
443
the use
To develop such vaccines, protective epitopes were selected for vaccination, while avoiding those that are responsible for damaging the host cells or inducing suppressor T cells. Both a chimeric flagellin from Salmonella [19] and proteosome constructs [ZO] carrying three epitopes from the heamagglutinin and nucleoprotein were successfully used in a mouse model. Using recombinant methods, a B-cell epitope from hemagglutinin together with T helper and CTL epitopes from the nucleoprotein of the influenza virus were individually inserted into the protein. A combination of the three constructs was used for intranasal immunization. It conferred MHC-restricted protection against lethal challenge and produced efficient long-term immunity as well as cross strain protection in mice [Zl]. Carrying this approach one step further, an effort to construct a human vaccine is currently underway using the same methodology: expression in flagellin of B-cell epitopes from the heamagglutinin surface antigen and three T-cells epitopes that are restricted to the most prevalent HLA molecules. Presenting influenza epitopes to the immune system was also carried out using different delivery systems in order to enhance the immunogenicity of the peptides. One of the systems is the artificial protein previously mentioned, the polyoxime, which upon administration to mice induced a specific humoral response [15]. Another approach is genetic immunization: many studies used whole nucleoprotein or hemagglutinin expressed in a plasmid for vaccination which conferred protection in model animals ([Z&23] and reviewed in [24]). Expanding this approach to an epitope-based vaccine, Nomura eta/. [25] constructed a plasmid with an epitope recognised by CTLs, which indeed led to induction of specific CTLs in mice. HIV
Successful vaccination against HIV presents several difficulties: firstly, the virus itself attacks and destroys the T helper lymphocytes: secondly, the proviral DNA of the HIV integrates into the host genome and may remain there, unrecognizable; thirdly, the virus spreads by transmission of infected cells in which proviral DNA is integrated into the genome; and finally, the virus undergoes antigenic variation. Hence, a successful vaccine must prevent the first virus-host interaction. Since early regulatory proteins of HIV are expressed by the infected cells before the initiation of the synthesis of structural proteins, immunization of seropositive individuals with epitopes from these proteins was attempted. An HIV-l p17 synthetic peptide vaccine, HGP30, was evaluated in phase 1 clinical trials and both cellular (Thl) and humoral responses were observed. In severe combined immune deficiency @CID) mice, reconstitution of mice with cells from HGP-30-vaccinated individuals resulted in protection from a viral challenge [26]. Similarly, four
444
Protein engineering
epitopes from the HIV-l regulatory protein Rev were used for in vitro immunization of human lymphocytes and were found to elicit specific CTLs that could destroy the infected cells before the release of infectious virions [27]. In another study, the immunogenicity of a CTL-recognized epitope from the V3 region of HIV-1 gp120 was demonstrated: immunization of mice resulted in highly specific, long-term CTL response [28]. From the same protein, an 18 amino acid peptide that contains epitopes recognized by both B cells and CTLs was defined [29]. It was further conjugated to inactivated Bnccda abortus and used for immunization of mice. It was found that such immunization induced a humoral response and virus-specific CTLs in both normal and CD4+ T cell-depleted mice [29]. A branched peptide vaccine from the V3 loop was also tested in humans. It induced neutralizing antibodies as well as a proliferation response in 75% of the vaccinated people. This prototype vaccine was safe and is being further evaluated [14]. Yet another approach, using mixotopes, aims to overcome one of the major problems that synthetic peptide vaccines face, namely that peptide antigens usually associate with only one or a few variants of the MHC molecules. Mixotopes are mixtures of synthetic peptide containing different amino acid residues at each position of the epitope. Using combinatorial synthesis surrounding a conserved peptide of the V3 loop of HIV Env protein, a library of the relevant sequences was generated [30,31]. Such a mixture of different but closely related peptides should be recognized by T cells, bypassing their MHC restriction. Indeed, immunization of various rodents with this mixotope elicited both a humoral response and cellular response. The profile of cytokine production indicates the induction of both type 1 helper T cell and type 2 helper T cell subsets [30,31]. This cumulative evidence indicates the potential of vaccines based on synthetic peptides in the case of HIV
Other viruses
Using similar approaches, T and B cell epitopes were defined for several other viruses, including measles virus [32-361, hepatitis B virus [37], respiratory syncytial virus [38-391, morbillivirus [40], polio [41] and others. Such epitopes are currently being investigated as candidates for peptide based vaccines.
Peptide and polypeptide parasites
vaccines against
The culturing of parasitic pathogens in the large amounts needed for vaccine preparation is completely impractical, and so the development of synthetic peptide vaccines is particularly suitable. This issue will be discussed below by two examples, malaria and schistosomiasis.
Malaria
The causative agent of malaria in humans is four species of Plasmodium protozoa; I! falcipanrm is responsible for the brain form of the disease which accounts for the great majority of deaths. The disease is further transmitted from human to human by the female Anopheles mosquito and also by blood transfusion. Malaria has been successfully curbed, or even eradicated, in many parts of the world but the existing endemic areas are inhabited by 2.5 billion people. The parasite’s life cycle is complex and this leads to complications in designing a vaccine against it. This fact led the investigators studying subunit-based vaccines to try to define proteins from early stages of the infection that would control it as early as possible. One such protein is the circumsporozoite protein (CSP) of the sporozoite stage, which was shown to be protective [42]. The sporozoites are infective needle-shaped uninucleated cells present in infected mosquito saliva. A related peptide vaccine for malaria is a synthetic peptide containing up to 40 repetitions of the CSP repeat sequence, NANP (amino acid single letter code). Immunization of human volunteers with this recombinant product resulted in humoral and cellular responses to the CSP with good protective effect [43]. The only antimalarial vaccine that has been subjected to large-scale clinical trials is Spf66, a synthetic peptide based vaccine. This is a synthetic hybrid molecule containing the protective epitopes of three blood stage antigens combined with the NANP from the sporozoite stage. It is safe and was shown to confer significant protection when tested in South America [44]. A subsequent trial in Africa was not so effective [45].
Another peptide vaccine that was evaluated in humans is a selection of six epitopes of pflSS/RESA, the blood stage antigen, which is an important vaccine candidate. The blood stage of the parasite is used as it is maintained in the host for a longer duration than the sporozoite stage. When investigated in women from an area in which malaria is endemic, most individuals reacted to at least one epitope and only 23% of the vaccinated women failed to respond, with a cellular response, to any peptide [46].
Another polypegtide that is under evaluation is the 15 kDa carboxy-terminal region of the merozoite surface antigen. Expressed with glutathione-S-transferase (GST) as a fusion protein, it induced protective immune responses in rodents, involving a humoral response and probably cellular mechanisms as well [47,48]. There are several additional recent studies aimed at identifying epitopes of malaria and testing them in animal models, either for direct immunization against the sporozoite and merozoite stages or for the sexual stages that mediate transmission of the parasite from man to mosquito. The latter approach is designed to inhibit parasite development in the mosquito vector by using antipeptide monoclonal antibodies, constituting ‘transmission blocking immunity’ [49].
Design of peptide and polypeptide vaccines Ben-Yedidia and Arnon
Schistosomiasis
Infection by at least one of several strains of Sckistosoma, mainly S. mansoni, S. kematobuh and S. japonicum, is a major parasitic disease which afflicts around 200 million people, mostly in developing countries. Sckistosoma is also an important pathogen for several domestic animal species and causes economic losses in endemic areas. The disease is associated with daily production of eggs by the adult worm; the eggs that fail to escape the body are deposited into the liver, intestine and genito-urinary tract where they stimulate a strong inflammatory reaction and granuloma formation, which eventually leads to death.
It is obvious today that drug therapy is not effective in endemic areas and that development of a vaccine is the only practical measure for disease control. Several immunodominant molecules have been described as candidates for a vaccine against schistosomiasis. These molecules induce a substantial degree of protection (see [50] for review). A possible protective antigen is one that is common to the larval and adult stages: for example, the recombinant polypeptide 28 kDa GST from S. mansoni. Immunization of rodents and baboons with this polypeptide resulted in protection against experimental infection [51]. Recently, immunization with a Bacillus Calmette-Guerin recombinant strain expressing the GST also induced a humoral response, as well as neutralization of the enzyme activity which correlates with protection against schistosomiasis in humans [52]. Another recombinant polypeptide (62 kDa), which is part of a protein expressed on newly transformed schistosomula, was also used for vaccination of mice and baboons [53]. It elicited high antibody titers and significant resistance to challenge infection [53]. In murine schistosomiasis, the highest levels of resistance to cercarial challenge were obtained by vaccination with radiated cercaria. Interestingly, both the humoral response and cellular response of the vaccinated mice was directed towards the same integral membrane protein, Sm23. A synthetic peptide corresponding to a segment of this antigen contains epitopes recognised by both B and T cells and is therefore also a candidate vaccine [54,55]. Our laboratory described a protective surface antigen [56,57], denoted 9B, which contains two major polypeptide chains of 45 kDa and 30 kDa. It is an abundant protein in the cercaria and schistosomula and very rare in the adult worm. It is important during parasite invasion into the mammalian host: antibodies specific towards the 9B antigen prevent infection. Immunization of mice with 9B complexed to proteosomes resulted in 62% protection. Furthermore, a protective immunopeptide of this antigen was defined that led to 42% reduction of adult worm burden in mice that were immunized with its BSA conjugate (unpublished data). This defined epitope is under further study as a vaccine candidate. The considerable progress that has been made in several laboratories renders the synthetic peptide approach a promising one.
Peptide and polypeptide cancer
445
vaccines against
The central hypothesis behind active vaccination for cancer treatment is that tumor cells express unique antigens that are capable of inducing a specific response. A proposed vaccine would have to deliver this antigen to the immune system, which would recognize it as foreign and destroy any cell bearing this antigen. Many of the tumor associated antigens in humans, however, are nonmutated self proteins. Overcoming the tolerance of the immune system towards them is crucial for their utilization as anti-tumor vaccines, and this could be achieved by employing tumor-specific peptides. A rat model was used for immunization with peptides derived from an overexpressed oncogenic protein (HER-Z/neu), which is highly homologous to the human HER-Z/neu protein. The immunization resulted in a CD4+ T cell response and antibody production [58]. With the recent identification of several tumor associated antigens and antigenic peptide epitopes in melanoma and other cancers, immunotherapy researchers are now investigating new strategies for anticancer vaccines, namely, to immunize with such peptides that are recognized by tumor-specific CTLs. Regrettably, however, even though promising results were obtained in animal models [59,60] there has been very little success to date in parallel clinical trials [61,62]. Another cancer-associated antigen is the protein mucin; therefore, it is of interest that a 16 residue peptide derived from synthetic mucin induced an immune response highly specific in recognizing the native mucin [63]. The same conclusions were arrived at when a very short hexapeptide of mucin was studied. This peptide also induced antibodies that reacted with human ovarian and breast cancer cells but not with normal cells. Since no significant cytotoxicity was observed in the presence of these specific antibodies, however, it seems that they can be used for drug targeting and/or diagnosis but that the peptide is probably not promising for the purpose of vaccination [64]. A novel strategy employing peptides for cell vaccination is based on a synthetic Ras peptide as a cancer vaccine in patients with advanced pancreatic carcinoma. The treatment principle relies on loading professional antigen presenting cells from peripheral blood with a synthetic Ras peptide ex vtio and re-injecting the cells into the patient. The procedure was found to be safe, with no side effects, and a cellular response specific for the cancer cells was observed. Although in all patients the tumor eventually progressed, it seemed that the vaccinated ones had some pain relief [65*]. A recent approach for vaccination is the injection of naked DNA as an epitope-based anticancer vaccine. A minigene coding for a specific epitope from a mutant p53
446
Protein
engineering
was inserted into the plasmid and used for vaccination: the immunized mice survived a tumor challenge [66], most probably by CTLs attacking the tumor. These promising preliminary results are paving the way for further development of anti cancer peptide vaccines.
Conclusions The design of peptide- or polypeptide-based vaccines is an attractive concept for vaccination since it will lead to elimination of the toxic effects of the pathogens, while emphasizing their immunogenic elements that are sometimes hidden in the native organism. Some genera1 conclusions are that a successful synthetic vaccine will probably consist of a cocktail of peptides in order to overcome the problem of hypervariable sequences and MHC restriction. These vaccines will contain both B and T cell epitopes, and possibly CTL-inducing epitope(s). Immunization with such peptides using appropriate delivery systems indeed leads to induction of specific immune responsesboth humoral and cellular and conferred protection. With the encouraging results of preliminary clinical trials and the development of more efficient delivery systems, either for the peptides or the corresponding DNA, it is likely that the first generation of peptide vaccines might become a reality in the not too distant future.
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Mendlovic F, Arnon R, Tarrab HR, Puri J: Genetic control of immune response to a purified Schistosoma mansoni antigen. II. Establishment and characterization of specific I-A and I-E restricted T-cell clones. Parasite lmmunol 1989, II :683-694.
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Disis ML, Gralow JR, Bernhard H, Hand SL, Rubin WD, Cheever MA: Peptide-based. but not whole protein, vaccines elicit immunity to HER-S/neu, oncogenic self-protein. J lmmunol 1996, 156:3151-3158.
59.
Toes RE. Blom RJ. Offrinoa R. Kast WM. Melief CJ: Enhanced tumor outgrowth’after peptide vaccination. Functional deletion of tumor-specific CTL induced by peptide vaccination can lead to the inability to reject tumors. J lmmunol 1996, 156:39113918.
60.
Ossevoort MA, Dendritic cells epitope-based papillomavirus Tumor lmmunol
61.
Linehan DC, Goedegebuure PS, Eberlein TJ: Vaccine therapy for cancer. Ann Surg Oncol1996, 3:219-226.
62.
Maeurer MJ, Storkus WJ, Kirkwood JM, Lotze MT: New treatment options for patients with melanoma: review of melanomaderived T-cell epitope-based peptide vaccines. Melanoma Res 1996, 6:l l-24.
63.
Liu X. Sejbal J, Kotovych G, Kogerrty RR, Reddish MA, Jackson L, Gandhi SS, Mendonca Al, Longenecker BM: Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides. Glycoconj J 1995, 12:607-617.
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Obeid OE, Partidos CD, Howard CR, Steward MW: Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus. J Viral 1995, 69:1420-l 428. Sedlik C. Sarraseca J. Rueda P. Leclerc C. Casel I: Immunogenicity of poliovirus B and T’cell epitopes presented by hybrid porcine parvovirus particles. J Gen Viral 1995, 76:23612368,
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Paterroyo G, Franc0 L, Amador R, Morillo La, Rocha CA, Rogha M, Patarroyo ME: Study of the safety and immunogenicity of the synthetic malaria Spf66 vaccine in children age l-14 years. Vaccine 1992, 10:175-l 78.
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Ballou WR, Blood J, Chongsuphajaissidhi T, Gordon DM, Heppner DG, Kyle DE, Luxemburger C, Nosten F, Sadoff JC, Sioghasivanon P et a/.: Field triah of an asexual blood stage malaria vaccine: studies of the synthetic peptide polymer SPf66 in Thailand and the analytic plan for a phase Ilb efficacy study. Parasitology 1995, 107:25-36.
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Gjertsen MK, Bakka A, BreivikJ, Saeterdal I, Gedde DT Ill, Stokke KT, Solheim BG, Egge TS, Soreide 0, Thorsby E, Gaudernack G: Gr viva ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase VII study. lnt J Cancer 1996, 65:450-453.
.
A oilot ohase VII studvwith a svntheticras oeotide used as a cancer vaccine. The principle is based on loadjng profess&l antigen presentingcells from peripheral blood with the peptide correspondingto the specific mutationin the tumor tissue of the patient.
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Design of peptide and polypeptide vaccines Tamar Ben-Yedidia Advances
and Ruth Arnon*
have been made in the development
based on synthetic peptides tumor-associated
antigens and protective
and parasites. Advances design of vaccines
and polypeptides
of vaccines representing
epitopes
of viruses
within the past year include the
based on artificial proteins, for example
multiantigen
peptides,
branched
recombinant
peptides,
as well as single T cell epitopes
tumor antigen peptides.
polypeptides,
fusion and
Although peptide vaccines
and
are not in
use as yet, their potential is being explored.
Addresses Department of Immunology, Weizmann Institute of Science, Rehovot, Israel *a-mail: [email protected] Current Opinion in Biotechnology
1997, 61442-440
http://biomednet.com/elecre1/0958166900000442 0 Current Biology Ltd ISSN 0958-l
669
Abbreviations BSA
bovine serum albumin
CSP CTL
circumsporozoite
GST HLA MAP
protein
cytotoxic T lymphocyte glutathione_S-transferase human leukocyte antigen multiple antigen peptide
Advantages of peptide- and polypeptidebased vaccines Both humoral and cellular arms of the immune system recognize and react with only specific regions of pathogens. This led to the design of vaccines based on subunits of the pathogen, either naturally occurring immunogenic polypeptide(s) or synthetic peptides that correspond to highly conserved regions required for the pathogen’s function. The goal of developing this strategy is to vaccinate with a minimal structure, consisting of a well-defined antigen, in order to stimulate an effective specific immune response, while avoiding potential hazardous effects. This strategy must take into consideration, however, the essential presence of epitopes recognized by both B cells and T cells, as well as the MHC restriction of the T cell response. Since the cellular immune response in humans is restricted to specific HLAs, any single epitope-based vaccine will probably not be effective in a broad population. This can be overcome by the use of vaccines comprising several peptides which would be effective in inducing both arms of the immune response. Furthermore, this approach allows the selection of those epitopes restricted to the HLAs which are most frequent in the population of interest.
Delivery systems for small antigens Introduction
Carriers
The development of vaccines has been one of the most important achievements of immunology and medicine to date. The traditional vaccines are based on the intact disease-inducing agents - inactivated or live attenuated (aimed at maintaining the immunogenicity of the pathogen while eliminating its infectivity and/or toxic effects)-and have led to diminished incidence in morbidity and mortality of a large number of infectious diseases, including major killers such as smallpox and polio. Yet there are crucial drawbacks incurred by the current procedures for vaccine preparation, such as the difficulty of irr vitro culturing of many viruses and parasites, biohazard and safety considerations, as well as the loss of efficacy due to the genetic variation of many viruses. New approaches for vaccine development are aimed at overcoming these shortcomings. They comprise on one hand the use of recombinant DNA technology, including naked DNA vaccines, and on the other hand the utilization of synthetic peptides which constitute epitopes that induce protection against infection. Although no peptide or polypeptide vaccine is being used as yet, several such experimental vaccines are undergoing clinical trials. Recent progress in this field is described below. This review will demonstrate the recent advances in the development and design of peptide-based vaccines and appropriate delivery systems.
For peptides that are small and nonimmunogenic, coupling to a carrier is essential to endow them with the capacity to induce a response of from T helper cells. Large proteins that contain sufficient reactive groups are suitable as carriers and bacterial proteins (tetanus toxoid, diphtheria toxoid) are widely used for chemical conjugation of peptides. Incidentally, proteins of mammalian origin, such as BSA, have been reported to be less efficient [l]. An alternative method of peptide presentation involves recombinant DNA methods. A plasmid containing the carrier protein gene is used and the epitope DNA sequence can be inserted into it. The expressed fusion protein can be of bacterial origin, for example the flagellin of Salmonella [Z], or of viral origin [3].
Viruses
viruses also serve as carriers-one of the vectors most frequently used for expressing foreign antigens is vaccinia [4,.5]. Among the other viruses used for this purpose are the retroviruses and fowlpox virus, adenoviruses and polio [6--81. Recently, the use of virus particles as carriers for peptide has been proposed: the use of empty capsids of retroviruses [9] and hepatitis B virus has been reported [9,10]. Intact
Design of peptide and polypeptide vaccines Ben-Yedidia and Arnon
Artificial proteins
The multiple antigen peptide (MAP) approach for immunization [11’,12] offers several advantages over the conventional carrier-antigen construct. MAP comprises multiple clusters of antigenic peptide epitopes synthesized on a branched oligolysine core. The greater part of the molecule consists of the immunogen in a well-defined orientation, avoiding unnecessary or even suppressor epitopes that might be present in a conventional carrier. The MAP is highly immunogenic and can be constructed as a multivalent vaccine, carrying any selected peptide. The application of MAP for vaccination was studied in model systems, and was found to protect animals from malaria [13] and to stimulate both humoral and cellular responses against HIV [14]. Another artificial protein that is even purer than MAP and can be produced in higher yields is the polyoxime vaccine, which was recently shown to induce high levels of antibody production against the peptides that are presented on it [15].
Liposomes and immunostimulating
complexes
These lipid-based particles enable the introduction of lipid-soluble molecules or peptides to the immune system. Liposomes have been shown to induce both humoral and cell-mediated immune responses to a wide spectrum of antigens [13,16]. Immunostimulating complex technology efficiently elicits a protective T cell response specific for the peptide and bypasses the need for an adjuvant [16,17]. It delivers soluble antigens both to the cytosolic and to the endosomal pathways of antigen processing, the natural pathway of the induction of a class I immune response. Cytosolic delivery can prime a cytotoxic T lymphocyte (CTL) response specific for the peptide, as was shown with a peptide from the measles nucleoprotein [18].
Peptide and polypeptide against viruses
vaccines
Influenza
Influenza is a major public health concern: it occurs in recurrent epidemics that start abruptly, spread rapidly and are frequently distributed worldwide. Usually the influenza infection is a mild disease, responsible for many millions of infected individuals, causing an economic burden. Mortality is high in infants and the elderly leading to tens of thousands of deaths annually. The influenza virus undergoes frequent and unpredictable changes of the surface glycoproteins hemagglutinin and neuraminidase. These antigenic variations enable the virus to escape the immune system and reduce the effectiveness of vaccines. The currently available vaccines are of several types, namely whole virus vaccines, subunit vaccines and live-attenuated influenza virus vaccines. Only killed (inactivated) and subunit vaccines are currently licensed for human use. These vaccines fail to induce local and cellular immunity and their efficacy is limited; hence,
novel approaches are being considered, of peptide-based vaccines.
including
443
the use
To develop such vaccines, protective epitopes were selected for vaccination, while avoiding those that are responsible for damaging the host cells or inducing suppressor T cells. Both a chimeric flagellin from Salmonella [19] and proteosome constructs [ZO] carrying three epitopes from the heamagglutinin and nucleoprotein were successfully used in a mouse model. Using recombinant methods, a B-cell epitope from hemagglutinin together with T helper and CTL epitopes from the nucleoprotein of the influenza virus were individually inserted into the protein. A combination of the three constructs was used for intranasal immunization. It conferred MHC-restricted protection against lethal challenge and produced efficient long-term immunity as well as cross strain protection in mice [Zl]. Carrying this approach one step further, an effort to construct a human vaccine is currently underway using the same methodology: expression in flagellin of B-cell epitopes from the heamagglutinin surface antigen and three T-cells epitopes that are restricted to the most prevalent HLA molecules. Presenting influenza epitopes to the immune system was also carried out using different delivery systems in order to enhance the immunogenicity of the peptides. One of the systems is the artificial protein previously mentioned, the polyoxime, which upon administration to mice induced a specific humoral response [15]. Another approach is genetic immunization: many studies used whole nucleoprotein or hemagglutinin expressed in a plasmid for vaccination which conferred protection in model animals ([Z&23] and reviewed in [24]). Expanding this approach to an epitope-based vaccine, Nomura eta/. [25] constructed a plasmid with an epitope recognised by CTLs, which indeed led to induction of specific CTLs in mice. HIV
Successful vaccination against HIV presents several difficulties: firstly, the virus itself attacks and destroys the T helper lymphocytes: secondly, the proviral DNA of the HIV integrates into the host genome and may remain there, unrecognizable; thirdly, the virus spreads by transmission of infected cells in which proviral DNA is integrated into the genome; and finally, the virus undergoes antigenic variation. Hence, a successful vaccine must prevent the first virus-host interaction. Since early regulatory proteins of HIV are expressed by the infected cells before the initiation of the synthesis of structural proteins, immunization of seropositive individuals with epitopes from these proteins was attempted. An HIV-l p17 synthetic peptide vaccine, HGP30, was evaluated in phase 1 clinical trials and both cellular (Thl) and humoral responses were observed. In severe combined immune deficiency @CID) mice, reconstitution of mice with cells from HGP-30-vaccinated individuals resulted in protection from a viral challenge [26]. Similarly, four
444
Protein engineering
epitopes from the HIV-l regulatory protein Rev were used for in vitro immunization of human lymphocytes and were found to elicit specific CTLs that could destroy the infected cells before the release of infectious virions [27]. In another study, the immunogenicity of a CTL-recognized epitope from the V3 region of HIV-1 gp120 was demonstrated: immunization of mice resulted in highly specific, long-term CTL response [28]. From the same protein, an 18 amino acid peptide that contains epitopes recognized by both B cells and CTLs was defined [29]. It was further conjugated to inactivated Bnccda abortus and used for immunization of mice. It was found that such immunization induced a humoral response and virus-specific CTLs in both normal and CD4+ T cell-depleted mice [29]. A branched peptide vaccine from the V3 loop was also tested in humans. It induced neutralizing antibodies as well as a proliferation response in 75% of the vaccinated people. This prototype vaccine was safe and is being further evaluated [14]. Yet another approach, using mixotopes, aims to overcome one of the major problems that synthetic peptide vaccines face, namely that peptide antigens usually associate with only one or a few variants of the MHC molecules. Mixotopes are mixtures of synthetic peptide containing different amino acid residues at each position of the epitope. Using combinatorial synthesis surrounding a conserved peptide of the V3 loop of HIV Env protein, a library of the relevant sequences was generated [30,31]. Such a mixture of different but closely related peptides should be recognized by T cells, bypassing their MHC restriction. Indeed, immunization of various rodents with this mixotope elicited both a humoral response and cellular response. The profile of cytokine production indicates the induction of both type 1 helper T cell and type 2 helper T cell subsets [30,31]. This cumulative evidence indicates the potential of vaccines based on synthetic peptides in the case of HIV
Other viruses
Using similar approaches, T and B cell epitopes were defined for several other viruses, including measles virus [32-361, hepatitis B virus [37], respiratory syncytial virus [38-391, morbillivirus [40], polio [41] and others. Such epitopes are currently being investigated as candidates for peptide based vaccines.
Peptide and polypeptide parasites
vaccines against
The culturing of parasitic pathogens in the large amounts needed for vaccine preparation is completely impractical, and so the development of synthetic peptide vaccines is particularly suitable. This issue will be discussed below by two examples, malaria and schistosomiasis.
Malaria
The causative agent of malaria in humans is four species of Plasmodium protozoa; I! falcipanrm is responsible for the brain form of the disease which accounts for the great majority of deaths. The disease is further transmitted from human to human by the female Anopheles mosquito and also by blood transfusion. Malaria has been successfully curbed, or even eradicated, in many parts of the world but the existing endemic areas are inhabited by 2.5 billion people. The parasite’s life cycle is complex and this leads to complications in designing a vaccine against it. This fact led the investigators studying subunit-based vaccines to try to define proteins from early stages of the infection that would control it as early as possible. One such protein is the circumsporozoite protein (CSP) of the sporozoite stage, which was shown to be protective [42]. The sporozoites are infective needle-shaped uninucleated cells present in infected mosquito saliva. A related peptide vaccine for malaria is a synthetic peptide containing up to 40 repetitions of the CSP repeat sequence, NANP (amino acid single letter code). Immunization of human volunteers with this recombinant product resulted in humoral and cellular responses to the CSP with good protective effect [43]. The only antimalarial vaccine that has been subjected to large-scale clinical trials is Spf66, a synthetic peptide based vaccine. This is a synthetic hybrid molecule containing the protective epitopes of three blood stage antigens combined with the NANP from the sporozoite stage. It is safe and was shown to confer significant protection when tested in South America [44]. A subsequent trial in Africa was not so effective [45].
Another peptide vaccine that was evaluated in humans is a selection of six epitopes of pflSS/RESA, the blood stage antigen, which is an important vaccine candidate. The blood stage of the parasite is used as it is maintained in the host for a longer duration than the sporozoite stage. When investigated in women from an area in which malaria is endemic, most individuals reacted to at least one epitope and only 23% of the vaccinated women failed to respond, with a cellular response, to any peptide [46].
Another polypegtide that is under evaluation is the 15 kDa carboxy-terminal region of the merozoite surface antigen. Expressed with glutathione-S-transferase (GST) as a fusion protein, it induced protective immune responses in rodents, involving a humoral response and probably cellular mechanisms as well [47,48]. There are several additional recent studies aimed at identifying epitopes of malaria and testing them in animal models, either for direct immunization against the sporozoite and merozoite stages or for the sexual stages that mediate transmission of the parasite from man to mosquito. The latter approach is designed to inhibit parasite development in the mosquito vector by using antipeptide monoclonal antibodies, constituting ‘transmission blocking immunity’ [49].
Design of peptide and polypeptide vaccines Ben-Yedidia and Arnon
Schistosomiasis
Infection by at least one of several strains of Sckistosoma, mainly S. mansoni, S. kematobuh and S. japonicum, is a major parasitic disease which afflicts around 200 million people, mostly in developing countries. Sckistosoma is also an important pathogen for several domestic animal species and causes economic losses in endemic areas. The disease is associated with daily production of eggs by the adult worm; the eggs that fail to escape the body are deposited into the liver, intestine and genito-urinary tract where they stimulate a strong inflammatory reaction and granuloma formation, which eventually leads to death.
It is obvious today that drug therapy is not effective in endemic areas and that development of a vaccine is the only practical measure for disease control. Several immunodominant molecules have been described as candidates for a vaccine against schistosomiasis. These molecules induce a substantial degree of protection (see [50] for review). A possible protective antigen is one that is common to the larval and adult stages: for example, the recombinant polypeptide 28 kDa GST from S. mansoni. Immunization of rodents and baboons with this polypeptide resulted in protection against experimental infection [51]. Recently, immunization with a Bacillus Calmette-Guerin recombinant strain expressing the GST also induced a humoral response, as well as neutralization of the enzyme activity which correlates with protection against schistosomiasis in humans [52]. Another recombinant polypeptide (62 kDa), which is part of a protein expressed on newly transformed schistosomula, was also used for vaccination of mice and baboons [53]. It elicited high antibody titers and significant resistance to challenge infection [53]. In murine schistosomiasis, the highest levels of resistance to cercarial challenge were obtained by vaccination with radiated cercaria. Interestingly, both the humoral response and cellular response of the vaccinated mice was directed towards the same integral membrane protein, Sm23. A synthetic peptide corresponding to a segment of this antigen contains epitopes recognised by both B and T cells and is therefore also a candidate vaccine [54,55]. Our laboratory described a protective surface antigen [56,57], denoted 9B, which contains two major polypeptide chains of 45 kDa and 30 kDa. It is an abundant protein in the cercaria and schistosomula and very rare in the adult worm. It is important during parasite invasion into the mammalian host: antibodies specific towards the 9B antigen prevent infection. Immunization of mice with 9B complexed to proteosomes resulted in 62% protection. Furthermore, a protective immunopeptide of this antigen was defined that led to 42% reduction of adult worm burden in mice that were immunized with its BSA conjugate (unpublished data). This defined epitope is under further study as a vaccine candidate. The considerable progress that has been made in several laboratories renders the synthetic peptide approach a promising one.
Peptide and polypeptide cancer
445
vaccines against
The central hypothesis behind active vaccination for cancer treatment is that tumor cells express unique antigens that are capable of inducing a specific response. A proposed vaccine would have to deliver this antigen to the immune system, which would recognize it as foreign and destroy any cell bearing this antigen. Many of the tumor associated antigens in humans, however, are nonmutated self proteins. Overcoming the tolerance of the immune system towards them is crucial for their utilization as anti-tumor vaccines, and this could be achieved by employing tumor-specific peptides. A rat model was used for immunization with peptides derived from an overexpressed oncogenic protein (HER-Z/neu), which is highly homologous to the human HER-Z/neu protein. The immunization resulted in a CD4+ T cell response and antibody production [58]. With the recent identification of several tumor associated antigens and antigenic peptide epitopes in melanoma and other cancers, immunotherapy researchers are now investigating new strategies for anticancer vaccines, namely, to immunize with such peptides that are recognized by tumor-specific CTLs. Regrettably, however, even though promising results were obtained in animal models [59,60] there has been very little success to date in parallel clinical trials [61,62]. Another cancer-associated antigen is the protein mucin; therefore, it is of interest that a 16 residue peptide derived from synthetic mucin induced an immune response highly specific in recognizing the native mucin [63]. The same conclusions were arrived at when a very short hexapeptide of mucin was studied. This peptide also induced antibodies that reacted with human ovarian and breast cancer cells but not with normal cells. Since no significant cytotoxicity was observed in the presence of these specific antibodies, however, it seems that they can be used for drug targeting and/or diagnosis but that the peptide is probably not promising for the purpose of vaccination [64]. A novel strategy employing peptides for cell vaccination is based on a synthetic Ras peptide as a cancer vaccine in patients with advanced pancreatic carcinoma. The treatment principle relies on loading professional antigen presenting cells from peripheral blood with a synthetic Ras peptide ex vtio and re-injecting the cells into the patient. The procedure was found to be safe, with no side effects, and a cellular response specific for the cancer cells was observed. Although in all patients the tumor eventually progressed, it seemed that the vaccinated ones had some pain relief [65*]. A recent approach for vaccination is the injection of naked DNA as an epitope-based anticancer vaccine. A minigene coding for a specific epitope from a mutant p53
446
Protein
engineering
was inserted into the plasmid and used for vaccination: the immunized mice survived a tumor challenge [66], most probably by CTLs attacking the tumor. These promising preliminary results are paving the way for further development of anti cancer peptide vaccines.
Conclusions The design of peptide- or polypeptide-based vaccines is an attractive concept for vaccination since it will lead to elimination of the toxic effects of the pathogens, while emphasizing their immunogenic elements that are sometimes hidden in the native organism. Some genera1 conclusions are that a successful synthetic vaccine will probably consist of a cocktail of peptides in order to overcome the problem of hypervariable sequences and MHC restriction. These vaccines will contain both B and T cell epitopes, and possibly CTL-inducing epitope(s). Immunization with such peptides using appropriate delivery systems indeed leads to induction of specific immune responsesboth humoral and cellular and conferred protection. With the encouraging results of preliminary clinical trials and the development of more efficient delivery systems, either for the peptides or the corresponding DNA, it is likely that the first generation of peptide vaccines might become a reality in the not too distant future.
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