Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer.

Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines.

Natural killer cells in human pregnancy.

Natural killer (NK) cells account for 70% of the leukocytes in the mucosal lining of the uterus (the decidua) in the first trimester of pregnancy. They are CD56(superbright) granulated cells expressing a repertoire of Killer-cell Immunoglobulin-like Receptors (KIR) skewed towards recognising HLA-C, which is the only classical class I MHC found on placental trophoblast cells. The function of decidual NK cells is not yet known, but there is evidence to suggest that they are involved in mediating trophoblast invasion into the decidua and modifying maternal spiral arteries to increase blood flow to the placenta. In order to characterise decidual NK cells and to understand their interactions with other cells at the maternal-foetal interface, it is useful to be able to isolate these cells. Here, we describe methods for the isolation and culture of decidual NK cells, decidual stromal cells and trophoblast cells from human first trimester tissue samples.

Allelic expression patterns of KIR3DS1 and 3DL1 using the Z27 and DX9 antibodies.

KIR3DL1 is one of the best-characterised inhibitory NK cell receptors. Unusually, one common allele at the 3DL1 locus encodes an activating receptor known as 3DS1. There is genetic evidence for a protective role of 3DS1 in certain viral diseases, but there has been uncertainty about expression of the 3DS1 protein. Using transfection, we show that surface expression of 3DS1 is reliant on the adaptor protein DNAX-activating protein 12 (DAP12). KIR3DS1 was recognised by the antibody Z27, a reagent that also detects KIR3DL1 but no other killer immunoglobulin-like receptor (KIR) molecule. Z27 stained 3DS1 on the surface of fresh circulating NK cells from 3DS1/3DS1 homozygotes. By double-staining with Z27 and DX9, an antibody specific for 3DL1, we obtained evidence that in 3DS1/3DL1 heterozygous donors significant numbers of NK cells express 3DS1 without co-expressing 3DL1 and that NK cells expressing both alleles are difficult to detect.

Methods for isolation of cells from the human fetal-maternal interface.

During human pregnancy, fetal placental cells known as trophoblast invade into the uterine mucosal lining, coming into contact with maternal cells including a specialized population of leukocytes. In order to understand the interaction of maternal cells with trophoblast, it is useful to be able to isolate the various cell types from the fetal-maternal interface so that they may be characterized and co-cultured in vitro. This chapter details the methods we use in our laboratory for enrichment and/or purification of trophoblast cells, decidual leukocytes, decidual natural killer cells, decidual stromal cells, and decidual glandular epithelial cells from human first-trimester tissue samples.

Molecular characterization of KIR3DL3.

Killer-cell immunoglobulin-like receptors (KIRs) are a structurally and functionally diverse family of molecules expressed by natural killer (NK) cells and T-cell subsets. The most centromeric gene in the human KIR cluster is KIR3DL3, a framework gene that is present in all haplotypes. KIR3DL3 has only one immunoreceptor tyrosine-based inhibitory motif and lacks the exon encoding the stem between the Immunoglobulin domains and the transmembrane region. We have investigated expression of KIR3DL3 in blood and decidual NK cells by reverse transcriptase polymerase chain reaction (RT-PCR) and protein analysis using a KIR3DL3-specific monoclonal antibody, CH21. KIR3DL3 mRNA was only detected in the CD56(bright) subset in cells from peripheral blood and in CD56(bright) decidual NK cells. The CD56(bright) NK92 cell line was also positive. Quantitative RT-PCR indicated a trend for higher expression of KIR3DL3 in female peripheral blood mononuclear cells compared to that in male. Using a bisulphite conversion method, we found that the promoter of KIR3DL3 was strongly methylated. Surface protein expression was detectable after demethylation. Like other KIRs, KIR3DL3 is highly polymorphic, and we detected 14 variants in 25 unrelated individuals. Nucleotide substitutions were scattered throughout the sequence, with a cluster of alleles at the start of the transmembrane region at the site where the remnant of the linking stem present in other KIR is found. We conclude that the KIR3DL3 gene is not a pseudogene but encodes a protein that is not expressed in healthy individuals. Protein expression might be induced under certain developmental or pathological situations.