Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.

Malaria vaccine development using synthetic peptides as a technical platform.

The review covers the development of synthetic peptides as vaccine candidates for Plasmodium falciparum- and Plasmodium vivax-induced malaria from its beginning up to date and the concomitant progress of solid phase peptide synthesis (SPPS) that enables the production of long peptides in a routine fashion. The review also stresses the development of other complementary tools and actions in order to achieve the long sought goal of an efficacious malaria vaccine.

Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. Evidence that two peptide residues are buried in the MHC molecule.

In the present study, we have explored ways of inducing a CTL response to a previously defined H-2Kd MHC class I restricted epitope in the circumsporozoite (CS) protein of Plasmodium berghei, and studied in detail the fine specificity of the response. We found that the s.c. injection of a variety of synthetic peptides emulsified in Freund's adjuvant efficiently induced a specific CTL response in (BALB/c x C57BL/6)F1 (H-2d x H-2b) mice. In contrast, BALB/c mice responded only marginally, consistent with the possible requirement for a concomitant Th response that would be provided by the C57BL/6 strain. Similar to our previous observations in analyzing CTL clones from sporozoite-immunized mice, the CTL response induced by peptide immunization was in part cross-reactive with an epitope from the Plasmodium yoelii species. The minimal P. berghei CS epitope, the octapeptide PbCS 253-260, was studied in detail by the analysis of a series of variant CS peptides containing single Ala substitutions. The relative antigenic activity for each variant peptide was calculated for 28 different CTL clones. Overall, the response to this P. berghei CTL epitope appeared to be extremely diverse in terms of fine specificity. This was evident among the CTL derived from sporozoite-immunized mice, as well as among those from peptide-immunized animals. The heterogeneity found at the functional level correlates with the highly diverse TCR repertoire that we have found for the same series of CTL clones in a study that is reported separately. The relative competitor activity for each Ala-substituted peptide was also determined in a quantitative functional competition assay. For the residues (Tyr253 and Ile260) within the 8-mer CS peptide, substitution with Ala reduced competitor activity by at least 40-fold, and for two others the reduction was 5- to 10-fold. When the relative antigenic activity for each CTL/peptide combination was normalized to the relative competitor activity of the peptide, a striking pattern emerged. The two residues that most affected competitor activity showed no additional effect on recognition beyond that observed for competition. In marked contrast, Ala substitutions at the other five positions tested varied widely, depending on the CTL/peptide combination. This pattern not only supports a model whereby the Tyr253 and Ile260 residues anchor the peptide to the Kd molecule, but also implies that they are virtually inaccessible to the TCR.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

Long synthetic peptides for the production of vaccines and drugs: a technological platform coming of age.

Long synthetic peptides (LSPs) have a variety of important clinical uses as synthetic vaccines and drugs. Techniques for peptide synthesis were revolutionized in the 1960s and 1980s, after which efficient techniques for purification and characterization of the product were developed. These improved techniques allowed the stepwise synthesis of increasingly longer products at a faster rate, greater purity, and lower cost for clinical use. A synthetic peptide approach, coupled with bioinformatics analysis of genomes, can tremendously expand the search for clinically relevant products. In this Review, we discuss efforts to develop a malaria vaccine from LSPs, among other clinically directed work.

Long synthetic peptides as biologically active proteins: the example of the chemokines.

Chemokines constitute an expanding protein family of over 40 members which exhibit a wide variety of biological activities and are involved in many normal physiological processes, such as cellular migration, differentiation and activation, but also in pathological situations, such as inflammation and metastasis. Over the last few years, we have developed methods to manufacture long synthetic peptides of up to 130 residues, and to achieve the formation of native-like cysteine pairings. This ability prompted us to undertake the total chemical synthesis of chemokines. So far, we have successfully produced over 30 chemokine species, which exhibit biological activities similar to, or greater than, those reported by others. Chemical synthesis offers a clear advantage over recombinant technologies for the introduction of fluorochromes and haptens at molecularly defined positions. In addition, approval of chemically synthesized products for use in humans is straightforward compared with material produced by biological methods.

Evaluation of two long synthetic merozoite surface protein 2 peptides as malaria vaccine candidates.

Merozoite surface protein 2 (MSP2) is a promising vaccine candidate against Plasmodium falciparum blood stages. A recombinant 3D7 form of MSP2 was a subunit of Combination B, a blood stage vaccine tested in the field in Papua New Guinea. A selective effect in favour of the allelic family not represented by the vaccine argued for a MSP2 vaccine consisting of both dimorphic variants. An alternative approach to recombinant manufacture of vaccines is the production of long synthetic peptides (LSP). LSP exceeding a length of well over 100 amino acids can now be routinely synthesized. Synthetic production of vaccine antigens cuts the often time-consuming steps of protein expression and purification short. This considerably reduces the time for a candidate to reach the phase of clinical trials. Here we present the evaluation of two long synthetic peptides representing both allelic families of MSP2 as potential vaccine candidates. The constructs were well recognized by human immune sera from different locations and different age groups. Furthermore, peptide-specific antibodies in human immune sera were associated with protection from clinical malaria. The synthetic fragments share major antigenic properties with native MSP2. Immunization of mice with these antigens yielded high titre antibody responses and monoclonal antibodies recognized parasite-derived MSP2. Our results justify taking these candidate poly-peptides into further vaccine development.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

H-2Kd-restricted antigenic peptides share a simple binding motif.

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

Malaria vaccines - The long synthetic peptide approach: Technical and conceptual advancements.

This review describes the advances in malaria antigen discovery and vaccine development using the long synthetic peptide platforms that have been made available during the past 5 years. The most recent technical developments regarding peptide synthesis with the optimized production of large synthetic fragments are discussed. Clinical trials of long synthetic peptides are also reviewed. These trials demonstrated that long synthetic peptides are safe and immunogenic when formulated with various adjuvants. In addition, long synthetic peptides can elicit an antibody response in humans and have demonstrated inhibitory activity against parasite growth in vitro. Finally, new approaches to exploit the abundance of genomic data and the flexibility and speed of peptide synthesis are proposed.

Antiangiogenic peptides and proteins: from experimental tools to clinical drugs.

The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is a stromal reaction essential for tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models, thereby indicating that tumor-associated vasculature may be a relevant target to inhibit tumor progression. Among the antiangiogenic molecules reported to date many are peptides and proteins. They include cytokines, chemokines, antibodies to vascular growth factors and growth factor receptors, soluble receptors, fragments derived from extracellular matrix proteins and small synthetic peptides. The polypeptide tumor necrosis factor (TNF, Beromun) was the first drug registered for the regional treatment of human cancer, whose mechanisms of action involved selective disruption of the tumor vasculature. More recently, bevacizumab (Avastin), an antibody against vascular endothelial growth factor (VEGF)-A, was approved as the first systemic antiangiogenic drug that had a significant impact on the survival of patients with advanced colorectal cancer, in combination with chemotherapy. Several additional peptides and antibodies with antiangiogenic activity are currently tested in clinical trials for their therapeutic efficacy. Thus, peptides, polypeptides and antibodies are emerging as leading molecules among the plethora of compounds with antiangiogenic activity. In this article, we will review some of these molecules and discuss their mechanism of action and their potential therapeutic use as anticancer agents in humans.

Elicitation of specific cytotoxic T cells by immunization with malaria soluble synthetic polypeptides.

We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of the P. falciparum 7G8 isolate CS protein expressed on the sporozoite surface. They comprise what is believed to be the mature protein, except for the central repetitive B cell domain. BALB/c (H-2d) mice were immunized s.c. with 50 micrograms soluble CS polypeptides emulsified in IFA. After a single immunization, CS-specific helper and cytotoxic T lymphocytes (CTLs) could be obtained. The resultant CTLs obtained by in vitro restimulation of primed lymph node (LN) cells recognized H-2Kd target cells in the presence of short synthetic peptides defined in the present study. These epitopes are contained within the N- and C-terminal regions of the CS protein, and correspond to sequences 39-47 and 333-342. In addition, these CTLs can specifically lyse H-2d target cells transfected with the CS gene. These results suggest that, by immunization of mice with large soluble CS synthetic polypeptides in IFA, it is possible to obtain MHC class I-restricted T cell responses specific for the CS protein. This approach might be advantageous in the formulation of efficient malaria subunit vaccines.

Longitudinal analyses of immune responses to Plasmodium falciparum derived peptides corresponding to novel blood stage antigens in coastal Kenya

We have recently described 95 predicted alpha-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1-6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p<0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11_0424 as another likely target of protective antibodies against P. falciparum malaria