Airway epithelial cells enhance the immunogenicity of human myeloid dendritic cells under steady state.

Dendritic cells (DCs) and airway epithelial cells (AECs) are in close proximity, and AECs secrete factors such as retinoic acid which induce tolerance in DCs at homeostasis. However, the question remains as to how DCs in the lung are able to respond to pathogens in the immunosuppressive environment. Using an in vitro human myeloid DC (mDC)-AEC co-culture system, we demonstrate that AECs induced several gene changes in the mDCs cultured with AECs compared to the mDCs not cultured with AECs. Analysis revealed that several chemokine genes were altered. These chemokine genes could serve to attract neutrophils, natural killer (NK) T as well as T helper type 1 (Th1)/Th2 cells to the airways. Genes priming lipid and major histocompatibility complex (MHC) class II antigen presentation were also up-regulated, along with certain anti-microbial protein genes. In addition, the expression and function of pathogen-sensing Toll-like receptors (TLRs) as well as Nod-like receptors (NLRs) and their downstream signalling molecules were up-regulated in mDCs cultured with AECs. Moreover, murine mucosal DCs from the lung expressed significantly higher levels of TLRs and NLRs compared to peripheral DCs from the spleen. These results indicate that AECs prime mDCs to enhance their immunogenicity, which could be one of the mechanisms that compensates for the immunosuppressive mucosal environment.

Dendritic cells from aged subjects contribute to chronic airway inflammation by activating bronchial epithelial cells under steady state.

The mechanisms underlying the increased susceptibility of the elderly to respiratory infections are not well understood. The crosstalk between the dendritic cells (DCs) and epithelial cells is essential in maintaining tolerance as well as in generating immunity in the respiratory mucosa. DCs from aged subjects display an enhanced basal level of activation, which can affect the function of epithelial cells. Our results suggest that this is indeed the scenario as exposure of primary bronchial epithelial cells (PBECs) to supernatants from unstimulated DCs of aged subjects resulted in activation of PBECs. The expression of CCL20, CCL26, CXCL10, mucin, and CD54 was significantly increased in the PBECs exposed to aged DC supernatants, but not to young DC supernatants. Furthermore, aged DC supernatants also enhanced the permeability of the PBEC barrier. We also found that DCs from aged subjects spontaneously secreted increased levels of pro-inflammatory mediators, interleukin-6, tumor necrosis factor (TNF)-α, and metalloproteinase A disintegrin family of metalloproteinase 10, which can affect the functions of PBECs. Finally, we demonstrated that TNF-α, present in the supernatant of DCs from aged subjects, was the primary pro-inflammatory mediator that affected PBEC functions. Thus, age-associated alterations in DC-epithelial interactions contribute to chronic airway inflammation in the elderly, increasing their susceptibility to respiratory diseases.

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.

Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use.

Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor-associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte-associated antigens (HLA). In order to stimulate a T-cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B-cell epitope (Tn antigen: alpha-GalNAc-Ser) covalently linked to peptides corresponding to the Pan DR 'universal' T-helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA-DR1, and HLA-DR4). A strong T-cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo.

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.

Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3.

In humans, sterile immunity against malaria can be consistently induced through exposure to the bites of thousands of irradiated infected mosquitoes. The same level of protection has yet to be achieved using subunit vaccines. Recent studies have indicated an essential function for intrahepatic parasites, the stage after the mosquito bite, and thus for antigens expressed during this stage. We report here the identification of liver-stage antigen 3, which is expressed both in the mosquito and liver-stage parasites. This Plasmodium falciparum 200-kilodalton protein is highly conserved, and showed promising antigenic and immunogenic properties. In chimpanzees (Pan troglodytes), the primates most closely related to humans and that share a similar susceptibility to P. falciparum liver-stage infection, immunization with LSA-3 induced protection against successive heterologous challenges with large numbers of P. falciparum sporozoites.

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) 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.

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.

Lipopeptide immunization without adjuvant induces potent and long-lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees.

We have employed a 26-amino-acid synthetic peptide based on Plasmodium falciparum liver stage antigen-3 to evaluate improvements in immunogenicity mediated by the inclusion of a simple lipid-conjugated amino acid during peptide synthesis. Comparative immunization by the peptide in Freund's adjuvant or by the lipopeptide in saline shows that the addition of a palmitoyl chain can dramatically increase T helper (Th) cell responses in a wide range of major histocompatibility complex (MHC) class II haplotypes, to the extent that responses were induced in mice otherwise unable to respond to the non-modified peptide injected with Freund's adjuvant, and that the increased immunogenicity of the lipopeptide led to high and longer lasting antibody production (studied up to 8 months). B and T cell responses induced by the lipopeptide were reactive with native parasite protein epitopes, and a lipopeptide longer than ten amino acids was endogenously processed to associate with MHC class I and elicit cytotoxic T lymphocyte (CTL) responses. Finally, the lipopeptide was safe and highly immunogenic in chimpanzees, whose immune system is very similar to that of humans. Our results suggest that relatively large synthetic peptides, carefully chosen from pertinent areas of proteins and incorporating a simple palmitoyl-lysine, can induce not only CTL, but also strong Th and antibody responses in genetically diverse populations. Lipopeptides engineered in this way are simple to produce and purify under GMP conditions, they are well tolerated by apes, and with the enhanced immunogenicity without the need for adjuvant that we report here, they offer a quick and relatively low-cost route to provide material for human malaria vaccination trials.

Improved detection of human antibodies to a Plasmodium antigen using a peptide modified with Aib residues.

A 17-mer sequence was selected as a model to study the influence of modifications of terminal ends both on the conformational of a peptide and on its antigenicity towards naturally developing antibodies. This sequence corresponded to a tandemly repeated motif, found in a long repetitive region, with high helical propensity, of a Plasmodium falciparum liver-stage antigen (LSA-1), immunogenic in man. Our model peptide was synthesized with ionizable or non-ionizable ends, or modified in both extremities by introduction of the helix-promoting residue alpha-aminoisobutyric acid (Aib). Helical contribution, absent in the 17 amino-acid sequence possessing ionizable ends, was detectable when non-ionizable ends were introduced, and dramatically increased in the Aib-modified analogue. The presence of ionizable ends totally abolished reactivity towards human sera, otherwise detectable with the peptide possessing non-ionizable ends. While modification by Aib residues was neither detrimental nor beneficial to antigenicity in solution, it clearly resulted in an improved sensitivity of the specific antibody detection when used as solid-phase antigen in ELISA.

A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes.

In the search for subunit vaccines that are able to induce the type of sterile, protective immunity achieved by irradiated sporozoites, there is increasing evidence that defense mechanisms directed at the intrahepatic stage and Ags expressed at this stage are critical. We have initiated a systematic search for such molecules and report here the identification and partial characterization of a novel Plasmodium falciparum gene encoding a 70-kDa protein, expressed in both sporozoite and liver stages (SALSA), with a vaccine potential that stems from its antigenic features. Antigenicity and immunogenicity studies were conducted in individuals exposed to malaria, in immunized mice, and in chimpanzees, using a recombinant protein and two synthetic peptides. Results show that the SALSA nonrepetitive sequence defines 1) major B cell epitopes, as shown by a high prevalence of Abs to each peptide in three African areas differing in their level of endemicity; 2) Th epitopes, as demonstrated by lymphoproliferation and IFN-gamma secretion in cells from the individuals from one of the low transmission areas, as well as helper effect upon Ab secretion in mice; and 3) epitopes for cytolytic lymphocytes, demonstrated in immunized and sporozoite-challenged chimpanzees, and associated with MHC class I leukocyte Ags. The latter are of particular importance, because this is the only part of the malaria life cycle in which the parasite is located in a cell expressing class I Ags and because CD8+ lymphocytes were found to be responsible for protection in experimental models.

Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants.

We have previously identified a Plasmodium falciparum liver stage-specific Ag (LSA-1) found to encode tandem 17 amino acid repeats harboring B cell determinants. Here we extend this study in terms of sequence analysis, protein localization, and immunologic properties. Analysis of the N- and C-terminal regions of LSA-1 from the T9/96 clone reveals high sequence conservation with LSA-1 from NF54. This 200-kDa protein is detected throughout liver schizogony and accumulates in the parasitophorous vacuole space. In our investigation of T and B cell responses to LSA-1, we have focused on both the area of the C-terminal, nonrepetitive "hinge" region and the conserved repetitive region and derived synthetic peptides. These were found to contain major B and T cell determinants. High prevalences and elevated Ab levels to LSA-1, directed primarily, although not exclusively, to the repetitive region, were detected in sera of individuals from one moderately high and two low transmission malaria-endemic areas (prevalences of 97%, 75, and 77%, respectively). In one of these low transmission areas, secretion of the cytokine IFN-gamma, known to inhibit malaria liver stages, and T cell proliferation were detected in PBMC of 22 to 48% and 6 to 20%, respectively, of individuals in response to separate LSA-1 peptides. These results complement the recent finding of conserved CTL epitopes in LSA-1 and support the assertion that immune responses to LS Ag are involved in protection against malaria pre-erythrocytic stages.