We have previously found that the acquired protection against malaria implicates a mechanism of defense that relies on the cooperation between cytophilic antibodies and monocytes. Accordingly, an assay of antibody-dependent cellular inhibition (ADCI) of parasite growth was used as a means of selecting for molecules capable of inducing protective immunity to malaria. This allowed us to identify in the sera of clinically protected subjects an antibody specificity that promotes parasite killing mediated by monocytes. This antibody is directed to a novel merozoite surface protein (MSP-3) of a molecular mass of 48 kD. Purified IgG from protected subjects are effective in ADCI and those directed against MSP-3 are predominantly cytophilic. In contrast, in nonprotected individuals, whose antibodies are not effective in ADCI, anti-MSP-3 antibodies are mostly noncytophilic. A region in MSP-3 targetted by antibodies effective in the ADCI assay was identified and its sequence was determined; it contains an epitope not defined by a repetitive structure and does not appear to be polymorphic. Antibodies raised in mice against a peptide containing this epitope, as well as human antibodies immunopurified on this peptide, elicit a strong inhibition of Plasmodium falciparum growth in ADCI assay, whereas control antibodies, directed to peptides from other molecules, do not. The correlation between isotypes of antibodies produced against the 48- kD epitopes, clinical protection, and the ability of specific anti-MSP- 3 antibodies to block the parasite schizogony in the ADCI assay suggests that this molecule is involved in eliciting protective mechanisms.

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) gD 1-23 peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either N epalmitoyl-lysine (palmitoyl-lipidated peptide, palmitoyl-LP) or cholesterol-lysine (cholesterollipidated 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 Tolllike receptor-2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.

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.

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 (gD 49-82 , gD 146-179 , gD 228-257 , and gD 332-358 ), 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-2 d 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.Genital, dermal, and ocular herpes simplex virus (HSV) infections cause prevalent, lifelong recurrent infections, with a spectrum of clinical manifestations, including cold sores, genital lesions, corneal blindness, and encephalitis (41,58,60,64,81). Despite the availability of many interventional strategies, there has been a constant increase of HSV prevalence during the last 3 decades (27,41,47). Several challenges face the development of an effective herpes vaccine that could help control this epidemic, including the uncertainty about the exact immune correlates of protection, the identification of immunogenic epitopes, and the development of an effective and safe immunization strategy (9,13,33,35,58,60).Despite previous emphasis on antibody (Ab) and CD8 ϩ T-cell responses (34, 42), there is growing evidence to support a pivotal role for the T-helper type 1 (Th1) subset of CD4 ϩ T cells in antiherpesvirus immunity (29,37,43,46,54,63,71). CD4 ϩ T cells are required for the protection of mice from HSV challenge (32,55,66). In humans, CD4 ϩ T cells are stimulated in vivo following an HSV infection and the integrated CD4 memory response to HSV type 1 (HSV-1) appears to occur in up to 0.2% of circulating CD4 ϩ T cells (2,45,67,70). Severe herpetic infections are often seen in immunocompromised individuals with impaired T-cell immunity, such as AIDS and transplant patients, where the immune defect is predominantly displayed in CD4 ϩ T cells (16). While it is believed that CD4 ϩ T-cell responses are important for protection in general, the importance of Th1-versus Th2-type immune responses for prot...

؉ T cells were also obtained. These CD8 ؉ T cells were able to recognize naturally processed viral proteins. Finally, specific gamma interferon-secreting CD8 ؉ T cells were also detected ex vivo.

We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.

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