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

Abstract

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.

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

“…The specificity of the response was demonstrated by negative results obtained using lymphocytes from either preimmunization samples or control monkeys. The high and specific IFN-g secretion against the LSP-NRP and LSA3-729 shorter peptides is in agreement with previous results obtained in Aotus, mice, and chimpanzee immunized with LSA3-729-derived lipopeptides without adjuvant [10,11] and recombinant proteins [4]. It indicates that the immunization with LSP-NRP is an efficient means of induction of strong T-cell responses.…”

“…Antibody titers were lower in the RP region, in contrast to results obtained in exposed populations, and non-significant in the group immunized with NRP alone, as expected. These results are similar to those observed in immunized mice [5] and chimpanzees [10], confirming that the sequences from the N-terminal region of the protein contain potent B-cell epitopes. …”

“…Antibody titers were lower in the RP region, in contrast to results obtained in exposed populations, and non-significant in the group immunized with NRP alone, as expected. These results are similar to those observed in immunized mice [5] and chimpanzees [10], confirming that the sequences from the N-terminal region of the protein contain potent B-cell epitopes. The challenge was performed 60 days after the last immunization in order to avoid a possible nonspecific effect upon liver stages development of pro-inflammatory mediators, such as IFN-a, TNF-a, free oxygen and nitrogen radicals, induced by the adjuvant [2].…”

“…Its role in protection has been observed in vitro in P. falciparum-infected human hepatocytes, at concentrations ranging from 1 to 10 I U [8] as well as in vivo in mice immunized by irradiated sporozoites [9] and is supported by studies in chimpanzees immunized either with irradiated sporozoites or with LSA3-derived antigens [2,10], or in Aotus immunized with particulate formulations [4]. Because peptides NRP and RP contain epitopes, which are main targets of Th1 responses and the protection is associated with strong Th1 responses [4], analysis of T-cell responses in immunized Aotus was limited to the study of IFN-g production by specific T cells.…”

“…In contrast, no IFN-g response was detected in control animals or in lymphocytes from immunized animals in response to non-infected Anopheles salivary glands, used as controls. Based on previous studies [2,3,10], strong IFN-g responses to the native protein are considered to be of good predictive value for protection against pre-erythrocytic stages of P. falciparum. …”