Lbachir BenMohamed

Lipopeptide vaccines—yesterday, today, and tomorrow

Peptide-based vaccines offer several potential advantages over the conventional whole proteins (or whole gene, in the case of genetic immunisation) in terms of purity and a high specificity in eliciting immune responses. However, concerns about toxic adjuvants, which are critical for immunogenicity of synthetic peptides, still remain. Lipopeptides, a form of peptide vaccine, discovered more then a decade ago, are currently under intensive investigation because they can generate comprehensive immune responses, without the use of adjuvants. In this review, we address the past of lipopeptide vaccines, highlight the progress made toward their optimisation, and stress future challenges and issues related to their synthesis, formulation, and delivery. In particular, the recent development of mucosal application of lipopeptide vaccines may present an ideal strategy against many pathogens that infect mucosal surfaces.

A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates.

We recently developed an efficient strategy based on a fully synthetic dendrimeric carbohydrate display (multiple antigenic glycopeptide; MAG) to induce anticarbohydrate antibody responses for therapeutic vaccination against cancer. Here, we show the superior efficacy of the MAG strategy over the traditional keyhole limpet hemocyanin glycoconjugate to elicit an anticarbohydrate IgG response against the tumor-associated Tn antigen. We highlight the influence of the aglyconic carrier elements of such a tumor antigen for their recognition by the immune system. Finally, we additionally developed the MAG system by introducing promiscuous HLA-restricted T-helper epitopes and performed its immunological evaluation in nonhuman primates. MAG:Tn vaccines induced in all of the animals strong tumor-specific anti-Tn antibodies that can mediate antibody-dependent cell cytotoxicity against human tumor. Therefore, the preclinical evaluation of the MAG:Tn vaccine demonstrates that it represents a safe and highly promising immunotherapeutic molecularly defined tool for targeting breast, colon, and prostate cancers that express the carbohydrate Tn antigen.

A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8 + T cells and protects against herpes simplex virus type 2 challenge

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2−/−) or myeloid differentiation factor 88 deficient (MyD88−/−) mice with a herpes simplex virus type 2 (HSV-2) CD8+ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8+ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2−/− and MyD88−/− mice developed significantly less HSV-specific CD8+ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.

Lipopeptide epitopes extended by an Nϵ-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity

Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV‐1) gD1–23 peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either Nϵ‐palmitoyl‐lysine (palmitoyl‐lipidated peptide, palmitoyl‐LP) or cholesterol‐lysine (cholesterol‐lipidated peptide, cholesterol‐LP). Immunization of mice with the palmitoyl‐LP, but not with its cholesterol‐LP analog, induced a strong T cell‐dependent protective immunity against lethal HSV‐1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1‐type immune response was stimulated by the palmitoyl‐LP, as opposed to a Th2 response generated by its cholesterol‐LP analog. The palmitoyl‐LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time‐ and dose‐dependent manner, and induced phenotypic maturation and production of pro‐inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl‐LP induced antigen‐specific T cell responses through the Toll‐like receptor‐2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant

We recently reported that parenteral injection of malaria palmitoyl‐tailed peptides without adjuvant efficiently induces B, Th and CTL responses. We now show that intranasal (IN) or sub‐lingual (SL) delivery of such lipopeptides induces strong systemic immune responses, as demonstrated by specific Th cell responses from the spleen as well as inguinal lymph nodes, and by the production ofhigh levels of serum antibodies. Overall, both types of responses were significantly higher than in parallel experiments in which the same lipopeptides were delivered by the subcutaneous (s.c.) route. Moreover, the mucosal route resulted in the preferential induction of IFN‐γ producing T cells and of IgG2a antibody production, as compared to the dominant IL‐4 and IgG1 responses obtained by the s.c. route, thus bringing a distinct advantage in the field of many infectious diseases and allergy. Possibly related to this Th1 response, we found that dendritic cells, the principal immune‐competent cells to encounter antigens within mucosal membranes, take up lipopeptide antigens more efficiently than macrophages. Mucosal immunization by lipidated peptides appears therefore as a novel, noninvasive vaccine approach that does not require the use of extraneous adjuvant and which, besides cost‐effectiveness, has attractive practical and immunological features.

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity.

ABSTRACT The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4+ T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD49-82, gD146-179, gD228-257, and gD332-358), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4+ Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2d mice with the four newly identified CD4+ Th1 peptide epitopes but not with four CD4+ Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.

Towards a Self‐Adjuvanting Multivalent B and T cell Epitope Containing Synthetic Glycolipopeptide Cancer Vaccine

Malignant tumor cells are characterized by the overexpression of altered glycoproteins or glycolipids resulting from the deregulation of glycosylation processes. The identification of these tumor-associated carbohydrate antigens (TACA) has largely contributed to the development of cancer diagnostic and immunotherapy. Particularly, TACA present strong antibody determinants (B cell epitopes) which are primarily targeted by tumor-specific antibodies (Abs). Although TACA are rightly considered as tremendous potential targets for cancer vaccines, their poor immunogenicity still hampers their use as therapeutic vaccines. To address this challenge, both careful rational design and robust chemical procedures should be considered to construct TACA-based vaccine prototypes capable of promoting a strong and selective Ab response against tumor cells. In the last decade, intensive research has focused on the development of molecularly defined TACA-based vaccine prototypes. These studies have clearly defined that not only the display of TACA, but also their nature and molecular formulation are crucial to improve immunity against tumors. First, a multivalent presentation of TACA, either on carrier protein (for example keyhole limpet hemocyanin) or on synthetic delivery systems containing CD4+ T helper (Th) cell epitope (for example multiple antigen glycopeptide) is required to elicit strong B cell responses and raise high affinity tumor-specific Abs. In addition, it was established that priming and sustaining of both Ab and CD8+ cytotoxic T cell (CTL) responses, the latter also crucial in cancer immunity, requires CD4+ Th cell help. This suggests that an ideal cancer vaccine formulation must incorporate B cell, CD4+ , and CD8+ T cell epitopes to ensure both humoral and cellular eradication of tumors. Finally, these synthetic multivalent vaccines should be delivered together with potent and safe external immunoadjuvants to ensure an early and strong immunity. To avoid the potential toxicity related to most of external adjuvants, especially often in immunocompromised cancer patients, recent reports have highlighted that palmitoyl-tailed B and T cell epitope peptides delivered in adjuvant-free saline are clinically safe, eliciting strong, long-lasting, and multivalent protective immunity. However, except for a few studies reporting the synthesis of up to three-component multivalent or “polytope” vaccines, 8] no molecular constructions have been designed so far on the basis of these overall structural features. This is presumably because of inherent difficulties, despite recent progresses in the synthesis, assembly, and formulation of oligosaccharide and glycoconjugate biomolecules. In this communication, we report for the first time on the design, synthesis, safety, immunogenicity, and protective efficacy of a prototype, molecularly defined, fully synthetic, self-adjuvanting multivalent glycolipopeptide (GLP) cancer vaccine. As illustrated on Figure 1, our GLP vaccine prototype associated four essential components displayed on a molecular delivery system: 1) a cluster of TACA B-cell epitope; 2) a CD4+ Th

High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules.

We have investigated the immunogenicity in chimpanzees of twelve synthetic peptides derived from four new Plasmodium falciparum molecules expressed at pre-erythrocytic stages of the human malaria parasite. These parasite molecules were initially selected through their ability to be recognized by stage restricted human antibodies. Twelve 20- to 41-mer peptides representing potential human B- or T-cell epitopes were selected from these proteins, and synthesized. Six of these were modified by a C-terminal lipidic chain in order to re-inforce their immunogenicity. Strong B- and T-helper cell responses were induced in chimpanzees by lipopeptides injected without adjuvant and by peptides in Montanide. All twelve peptides induced CD4(+) T-cell proliferative responses, as well as the secretion of IFN-gamma (some of them at very high levels) and eleven peptides induced antibody responses. The immune responses elicited in this way were reactive with native parasite proteins, as shown by recall studies with sporozoite stage proteins, and proved to be long-lasting (up to 10 months after immunization). Our results support the strategy employed to select these four new malarial antigens and the corresponding peptides, and suggest that the immunizing formulations are both efficient and clinically acceptable.

HLA-A*0201-Restricted CD8+ Cytotoxic T Lymphocyte Epitopes Identified from Herpes Simplex Virus Glycoprotein D

Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53–61, gD70–78, and gD278–286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-γ ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53–61, gD70–78, and gD278–286 epitopes. In addition, CD8+ T cell lines generated by gD53–61, gD70–78, and gD278–286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53–61, gD70–78, and gD278–286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.

Induction of CTL response by a minimal epitope vaccine in HLA A∗0201/DR1 transgenic mice: dependence on HLA class II restricted TH response

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.