Vaccination with poly-L-arginine as immunostimulant for peptide vaccines: induction of potent and long-lasting T-cell responses against cancer antigens.

Vaccines that induce high numbers of sustained T cell responses are urgently needed for the treatment of numerous diseases including cancer. Antigen-presenting cells (APCs), the most important of which are dendritic cells, orchestrate antigen-dependent T cell responses in that they present antigens to T cells in an appropriate environment. Here we present evidence that after vaccination with a simple mixture of the cationic poly-amino acid poly-L-arginine and tumor antigen-derived peptide antigens, large numbers of antigen-specific T cells are induced and APCs mediate the generation of T lymphocytes. We observe that after s.c. injection, MHC class II(+) cells infiltrate injection sites and are loaded with large amounts of antigen in vivo under the influence of poly-L-arginine. Consequently, numerous antigen-charged APCs can be detected in draining lymph nodes of vaccinated animals. Antigen-specific T cell responses induced are systemic and were readily detected more than 4 months after the last vaccination, the latest time point we measured. By contrast, even after repeat injections, we were consistently unable to detect antibody responses against poly-L-arginine, allowing this compound to be used for numerous booster injections. Clinical trials in cancer patients using poly-L-arginine as immunostimulant will be carried out in the near future.

The dawn of gene isolation.

Ever since it became clear through the work of Watson and Crick that the gene is a stretch of double stranded helical DNA and is understandable in chemical terms, biochemists have striven to get their hands on isolated genes. The isolation of the ribosomal genes of Xenopus laevis in 1966 provided a first instance where a purified DNA of known function could be investigated, long before the advent of gene cloning technologies. The second instance was the purification of the Lac operon from Escherichia coli. Later, but still before the gene cloning days the 5S RNA genes of X. laevis and the histone genes of the sea urchin Psammechinus miliaris were isolated by physico-chemical methods, but their isolation marked the end of an era. By 1975, gene cloning technology was well established and the isolation of genes quickly became an everyday occurrence.

The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene.

Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator". The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered. This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development. We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes. We conclude that the H2A enhancer is bipartite, is located approx. 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter. Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene. Within this structure, there is an insulator element.

The artificial antimicrobial peptide KLKLLLLLKLK induces predominantly a TH2-type immune response to co-injected antigens.

Cationic antimicrobial peptides (CAMPs) are active defence components of the innate immune system. Several artificial CAMPs have been designed as antibiotic peptide therapeutics, but none have been reported to exert adjuvant activity in animal models. Here we show for the first time that an artificial CAMP, KLKLLLLLKLK (KLKL5KLK), is a potent inducer of adaptive immunity to co-injected antigens in vivo. High levels of antigen-specific antibodies were obtained after co-injection of KLKL5KLK with the model antigen ovalbumin (OVA) or a commercially available influenza vaccine. We show that KLKL5KLK induces a sustained immune response with a prevalent TH2 profile when co-injected with proteinaceous and peptide-based antigens. Furthermore, the immuno-enhancing activity of peptide KLKL5KLK was retained when C-terminally amidated or synthesised as retro-all-D-peptide. We provide evidence that KLKL5KLK enhances the association of antigen to antigen-presenting cells and forms a depot of antigen at the site of injection, making it an interesting adjuvant for novel vaccine design.