KOSEN 한인과학기술자네트워크

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PeptidesThe “peptide“ vaccine concept is based on the identification and chemical synthesisof B cell and T cell epitopes,particularly those that may be immunodominant and induce specific immune functions(neutralization, killing, help).In their simplest form, peptides used as vaccine candidates are linear polymersof ~ 8-24 amino acids, however, in an attempt to mimic conformational structures(particularly those of the viral envelope) cyclic peptides, branched peptides,peptomers (cross-linked peptide polymers) and other complex multimeric structures,as well as peptides conjugated to other molecules have been developed.Several obstacles limit the usefulness of peptide vaccines: their low immunogenicity,the extensive genetic variability of HIV, the dependence of T cell epitopeson the individual's immunogenetic background,the difficulties in translating results from experimental animals (e.g., peptides recognized by mice,rabbits and macaques may be different from those immunogenic in humans);and the lack of knowledge on the structural conformation of the most relevant neutralizationepitopes in HIV. Nonetheless,current efforts are defying these limitations and several promising discoveriesare making their way to improve on this approach.Background The identification of the third variable loop region (V3) of the HIV envelope as the“principal neutralization determinant“ has been heavily exploited to develop peptide vaccines.Because of the large extent of genetic variability in this region,multiple sequences of it corresponding to dissimilar HIV strains have been mixedor linked in various formulations. It is now clear that other regions of the envelopeare more relevant than V3 for the neutralization of primary HIV isolates;they include the V1-V2 region, the CD4 binding site and the C4 region;such regions have also been made into synthetic peptides,however, critical three-dimensional structures of the envelopehave not been replicated by peptides. Consequently,most current peptides, designs and mixtures have resulted in the induction of antibodiescapable of recognizing linear (non-conformational) structures,and none has resulted in neutralization responses to primary HIV isolates. Exceptionally,some peptides have mimicked conformationally-dependent structures,as determined by their reactivity with conformationally-dependent,broadly reactive neutralizing monoclonal antibodies.Such antibodies are also being used to identify relevant “mimotopes“ in combinatorialpeptide libraries that could mimic relevant structuresand potentially induce broad responses. The “mimotope“ approach has been successfullyused in other experimental viral systems (respiratory syncytial virus, measles, hepatitis C, etc.),and is juts now being applied to generating more broadly reactive HIV peptides. The presence of helper epitopes in the V3 loop has been exploited to enhance neutralizationresponses. In addition, other helper T cell determinants added as chimeric peptideshave shown to experimentally enhance the neutralization response to V3 loop peptides. Targets for peptide vaccines are not limited to envelope-based peptides.Nef-, vpr-, reverse transcriptase-, tat-, and particularly gag-based peptideshave been tested in various forms. Optimal results have been observed with complex,conjugated peptides that include multiple CTL and helper epitopes.While most CTL epitopes are recognized on a very MHC class I-dependent manner,class II helper epitopes appear to be, in general less restricted.Several “promiscuous“ helper epitopes have been identified as potential targetsfor induction of broad responses in openly bred populations.It is common that CTL epitopes (and also B cell epitopes) be present in the vicinityof helper epitopes, facilitating the design of multi-determinant peptides. Current Research In addition to the need for inclusion of helper epitopes,the intrinsically low immunogenicity of peptides requiresthat they be administered with strong adjuvants to enhanceand/or modulate the nature of the response generated.Incomplete Freund's adjuvant and other oil-based adjuvants appear to be more effectiveand favor the induction of Th1 responses,while alum results in a preferentially Th2 response.Additional adjuvant approaches to enhance the response to peptidesinclude the covalent association with lipopeptidic immunostimulants,or the encapsidation of the peptides into lyposomes.Strong adjuvant effects have also been observed with the block copolymeradjuvant P1005. Controlled-released microparticlesand iscom matrix technologies have also been applied to peptide vaccines,however, no extended immunogenicity evaluations of the latter approaches have been conducted.In experiments in mice, the immunomodulatory moleculessuch as IL12 and the active fragment of IL-1 beta have greatly enhancedTh1 responses to peptides. Conjugation of peptides to larger molecules results in enhancedimmunogenicity, particularly if the carrier proteins contain strongT helper epitopes. Direct covalent fusion or cross-linking with glutaraldehydeand other chemical processes have been used. Tetanus toxoid,Pseudomonas aeruginosa toxin A, beta-galactosidase,Brucella abortus (killed bacteria), keyhole limpet hemocyanin,influenza virus hemagglutinin and nucleoprotein,hepatitis B core and surface antigens, etc,are examples of carrier proteins that have successfullyimproved the immunogenicity of peptides.Some of them (e.g., brucella) appear to induced T-cell independent responses,however most of them act by providing T cell help or facilitating their presentation,functions that requires their covalent fusion with the peptides.Experimental attempts to further enhance the immunogenicity of synthetic peptidesinclude fusion with plasma alpha-2 macroglobulin(which has specific receptors in macrophages and may enhance antigen presentation),beta-2 microglobulin, light and heavy immunoglobulin chains, etc. Covalent fusion of peptides to lipids has resulted in conjugates of enhancedimmunogenicity, particularly in more efficient induction of CTLs,bypassing the need for other adjuvants. Encouraging results have been reportedfor hepatitis B core antigen lipopeptide-based vaccine linked to a tetanus toxoid helper T epitope:the magnitude and persistence of the CTL response inducedwere comparable to those observed in patients who successfully cleared the infection.In the SIV model a Nef-based lipopeptide was shown to induce strong specific CTLsto a Nef determinant in macaques; however,after challenge with the SIV source of the nef,escape from this epitope occurred, highlighting the need for broad, multi-epitope responses. While current peptide vaccines require multiple boosts and strong adjuvantsto induce measurable immune responses;they may be useful when given in combination (as a boost) to other vaccines.Such has been the experience in animal studies in which envelopesubunit-primed antibody and proliferative responseswere successfully boosted by V2 and V3 env-based peptides,resulting in protection from SIV challenge. Similarly,increases in antibody and cell-mediated responses were observed after peptide boosting of animals primed with a DNA vaccine or with adenovirus-SIV env recombinants. In human studies, peptide boosting of neutralization responses to envelope subunits has also been reported. In no case, however, the boost effect of the peptide resulted in broadening of the neutralization response to make it effective against primary isolates. Mucosally-administered peptides have been immunogenic in mice(i.e., induction of CTL, Th, and antibody),particularly when administered with cholera toxin as adjuvant.In a recent mouse model of rectal immunization / rectal challenge,a multideterminant peptide-based vaccine (containing HIV envelope B and T cell epitopes)was able to induce resistance to a vaccinia recombinant expressing HIV gp160.Protection was dependent on the mucosal presence of CTLs,and was enhanced by co-administration of IL-12 at the time of vaccination.Intestinal induction of HIV neutralizing antibodies has been achievedby oral administration of a multicomponent peptide vaccine when administered with cholera toxin;similarly CTLs have been induced by mucosal co-administration of a CTL epitope-based peptideand cholera toxin. A great deal effort is being placed on the elucidation of additionalimmunodominant T cell determinants associated with CTL and helper responsesacross populations with various genetic backgrounds.Such effort will contribute to the design of additional peptides to coveras many HIV variants as possible with the minimal (or a reasonable)number of peptides to induce broad responses. The identification of peptidescapable of degenerate binding could also alleviate the limitation imposedby HIV restriction of the cellular responses.In order to facilitate the identification of additional T cell epitopes alternative“reverse genetic“ approaches are being pursued by predicting such epitopesfrom the known binding motiffs of class I and class II MHC molecules.Likewise, it might be possible to enhance MHC peptide binding affinityby replacing residues in the peptide sequences. Clinical trials of an octameric V3 MN-based peptide (United Biomedical, Inc.)have been conducted in seronegative volunteers in the U.S., Australia,Thailand, Brazil and China. Homologous neutralizing antibody,but no responses to primary isolates, were induced in 60-90% of the volunteers.The vaccine was well tolerated and was not associated with laboratoryor clinical abnormalities. More recently, UBI has also developed a microparticulate formulationand a multivalent versions of this vaccine (comprising V3 branched peptides from 15 HIV-1 strains)as well as a gag-based lipopeptide vaccine which are being tested in humans.Results from these studies are not yet available. Clinical trials of a multivalent “C4V3“ chimeric peptide(produced by Wyeth Lederle Vaccines and Pediatrics) have been conducted,initially in HIV seropositive individuals and currently in seronegatives.The “C4“ component of the vaccine corresponds to a strong envelope Th determinant,while the V3 represents a 24 amino acid peptide of the V3 loop of each of four HIV-1 strains,which includes B cell epitopes as well as a B7- and an A2-restricted CTL epitopes.Half of the infected volunteers tested developed neutralizing antibodiesand proliferative responses, and 2 of 8 developed a CTL response.The vaccine is being administered with incomplete Freund's adjuvant,which appears to be associated with grade I injection site reactions. A PPD-coupled peptide has also been tested in humans.It consists of V3 MN peptide covalently fused to PPD.At submicrogram amounts, this peptide has been found to induce antibody(including neutralizing antibodies) as well as CTL responsesin PPD-reactive HIV seronegative volunteers.A multivalent version of the same approach is being tested in infected individuals.Preliminary data from the latter study has shown it to be capable of increasingneutralization titers to primary isolates,an effect that was accompanied by a decrease in HIV viral load. A 30 amino acid peptide from the p17 gag protein,known as HGP-30, is being developed by CEL-SCI Corporation.It includes B, CTL and T helper epitopes conjugated to keyhole limpet hemocyaninand has been shown to be immunogenic in rodents.Limited studies in humans (including HIV seropositive volunteers)have confirmed such immunogenicity: antibody responses,proliferative and CTL responses were generated after 3 doses of the vaccine;adoptive transfer of cells from vaccinees have been shown to protect SCID micefrom challenge with HIV-1.In an attempt to improve on the presentation of this peptide,heteroconjugates with beta-2 microglobulin peptides are currently being tested. Future Research PlansDevelop delivery systems to induce and enhance immune responses to peptides,including formulations that may be administered through mucosal routes. Develop better adjuvants, cytokines and other immunomodulators to stimulateand appropriately direct humoral and cellular responses. Determine and mimic the structural conformation of peptide immunogen requiredto induce high levels of neutralizing antibody. Determine if peptides can boost immune responses that have been primed by anotherregimen or by infection with HIV. Identify additional T cell epitopes, particularly those that may be cross-reactiveacross HIV variants, recognized by multiple class I or class II alleles,immunodominant and functionally relevant. Reference List Enhanced immunogenicity of HIV-1 vaccine construct by modification of the nativepeptide sequence. Ahlers JD; Takeshita T; Pendleton CD; Berzofsky JA Proc NatlAcad Sci U S A. 1997 Sep 30;94(20):10856-61. DNA vaccination followed by macromolecular multicomponent peptide vaccinationagainst HIV-1 induces strong antigen-specific immunity. Okuda K; Xin KO; Tsuji T;Bukawa H; Tanaka S; Koff WC; Tani K; Okuda K; Honma K; Kawamoto S; Hamajima K;Fukushima J Vaccine. 1997 Jul;15(10):1049-56. Degenerate and promiscuous recognition by CTL of peptides presented by the MHCclass I A3-like superfamily: implications for vaccine development.Threlkeld SC; Wentworth PA; Kalams SA; Wilkes BM; Ruhl DJ; Keogh E; Sidney J; Southwood S; Walker BD; Sette A. J Immunol. 1997 Aug 15;159(4):1648-57. A dose-ranging study of a prototype synthetic HIV-1MN V3 branched peptide vaccine.The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.Gorse GJ; Keefer MC; Belshe RB; Matthews TJ; Forrest BD; Hsieh RH; Koff WC;Hanson CV; Dolin R; Weinhold KJ; Frey SE; Ketter N; Fast PE. J Infect Dis. 1996 Feb;173(2):330-9. Neutralization of HIV-1 by secretory IgA induced by oral immunization witha new macromolecular multicomponent peptide vaccine candidate. Bukawa H; Sekigawa K;Hamajima K; Fukushima J; Yamada Y; Kiyono H; Okuda K Nat Med. 1995 Jul;1(7):681-5. Safety and immunogenicity of a V3 loop synthetic peptide conjugated topurified protein derivative in HIV-seronegative volunteers. Rubinstein A; Goldstein H; Pettoello-Mantovani M; Mizrachi Y; Bloom BR; Furer E;Althaus B; Que JU; Hasler T; Cryz SJ AIDS. 1995 Mar;9(3):243-51. A universal T cell epitope-containing peptide from hepatitis B surface antigencan enhance antibody specific for HIV gp120.Greenstein JL; Schad VC; Goodwin WH; Brauer AB; Bollinger BK; Chin RD; Kuo MC J Immunol.1992 Jun 15;148(12):3970-7. A phase I study of HGP-30, a 30 amino acid subunit of the human immunodeficiency virus(HIV) p17 synthetic peptide analogue sub-unit vaccine in seronegative subjects.Kahn JO; Stites DP; Scillian J; Murcar N; Stryker R; Volberding PA; Naylor PH; Goldstein AL;Sarin PS; Simmon VF; et al. AIDS Res Hum Retroviruses. 1992 Aug;8(8):1321-5. Safety and immunogenicity of C4V3 polyvalent synthetic peptides in HIV-1 infected HLA B7 positive subjects with CD4 cells greater than 500/mm(3). Bartlett JA; Wasserman S;Hicks C; Dodge R; Tacket C; Weinhold K; Palker T; Virani-Ketter N; Wittek A; Haynes BF.Conf Adv AIDS Vaccine Dev. 1997 May 4-7;:217 (Poster 112). Neutralization of primary HIV isolates post PPD-V3 loop peptide vaccine immunizationin animals and in HIV+ PPD+ humans. Rubinstein A; Mizrachi Y; Petoello-Mantovani M;Goldstein H; Spirer Z; Cryz S Int Conf AIDS. 1996 Jul 7-12;11(2):122 (abstract no. We.B.3375). The importance of local mucosal HIV-specific CD8(+) cytotoxic T lymphocytes for resistanceto mucosal viral transmission in mice and enhancement of resistance by local administrationof IL-12. Belyakov IM; Ahlers JD; Brandwein BY; Earl P Kelsall BL; Moss B; Strober W;Berzofsky JA J Clin Invest 1998 Dec 15;102(12):2072-81. Mucosal immunization with HIV-1 peptide vaccine induces mucosal nd systemiccytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinantHIV-vaccinia challenge. Belyakov IM; Derby MA; Ahlers JD; Kelsall BL; Earl P; Moss B;Strober W; Berzofsky JA Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1709-14. ※첨부자료가 없습니다. URL을 참고하세요.