Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain

It is now well accepted that the structural protein Pr55Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development

Virus-Like Particles of SARS-Like Coronavirus Formed by Membrane Proteins from Different Origins Demonstrate Stimulating Activity in Human Dendritic Cells

The pathogenesis of SARS coronavirus (CoV) remains poorly understood. In the current study, two recombinant baculovirus were generated to express the spike (S) protein of SARS-like coronavirus (SL-CoV) isolated from bats (vAcBS) and the envelope (E) and membrane (M) proteins of SARS-CoV, respectively. Co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (BVLPs) as demonstrated by electron microscopy. Incorporation of S protein of vAcBS (BS) into VLPs was confirmed by western blot and immunogold labeling. Such BVLPs up-regulated the level of CD40, CD80, CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-α in immature dendritic cells (DCs). Immune responses were compared in immature DCs inoculated with BVLPs or with VLPs formed by S, E and M proteins of human SARS-CoV. BVLPs showed a stronger ability to stimulate DCs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of IL-6 and TNF-α. Further study indicated that IFN-γ+ and IL-4+ populations in CD4+ T cells increased upon co-cultivation with DCs pre-exposed with BVLPs or SARS-CoV VLPs. The observed difference in DC-stimulating activity between BVLPs and SARS CoV VLPs was very likely due to the S protein. In agreement, SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than SARS-CoV S DNA in mice. Our data have demonstrated for the first time that SL-CoV VLPs formed by membrane proteins of different origins, one from SL-CoV isolated from bats (BS) and the other two from human SARS-CoV (E and M), activated immature DCs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. Finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of SARS-like CoV

VLPs and particle strategies for cancer vaccines

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IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

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Comparison of lymphokine secretion and responsiveness of human T cell clones isolated in IL-4 and in IL-2

Interleukin (IL)-4 has been shown to be secreted simultaneously with IL-2 and interferon (IFN)-gamma by the majority of CD4+ human T cell clones isolated and cultured using IL-2 as a growth factor. Moreover, IL-4 was found to be as efficient as IL-2 to promote the outgrowth of human T cell clones. In this study we have investigated the pattern of lymphokine production by human T cell clones isolated and cultured in IL-4. Most of the CD4+ T cell clones isolated in IL-4 were found to have the ability to simultaneously secrete IL-2, IL-4, and IFN-gamma upon activation. The T cell clones isolated in IL-4 produced, in general, more IL-4 and less IL-2 than the clones isolated and cultured in IL-2. This tendency did not appear to be a stable feature inasmuch as when representative CD4+ T cell clones were split and cultured in either IL-2 or IL-4, the clones in IL-2 secreted more IL-2 and less IL-4 than the same cells cultured in IL-4. These results indicate that the isolation and culture of human CD4+ T cells in IL-4 did not lead to an "irreversible" development of these cells into Th-1- or Th-2-like cells. Clones isolated in IL-4 responded better to IL-4 than they did to IL-2. On the other hand, T cell clones from the same donor isolated in IL-2 showed the reverse pattern since these latter cells were found to respond better to IL-2 than to IL-4. Furthermore, "nonresponsiveness" of a T cell clones in a [3H]TdR assay to either IL-2 or IL-4 is not a stable feature since clones, unresponsive to a particular lymphokine, could be adapted to become responsiv

Antigen-specific, but not natural killer, activity of T cell receptor-gamma delta cytotoxic T lymphocyte clones involves secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and influx of Ca2+ ions

Analysis of Ag specificity of TRC-gamma delta+ T cells in humans has been hampered by the fact that cloned lines of these cells expanded in IL-2 generally display high NK-like cytotoxic activity. A TCR-gamma delta+ CTL clone, isolated in IL-4, strongly lysed a specific stimulator cell, the EBV-transformed cell line JY, but failed to lyse K562 and other target cells sensitive for NK cell activity. Subsequent culture of this clone (CD124) in IL-2 induced high cytotoxic activity against the NK sensitive target cells. K562 cells were unable to induce the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester [(BLT)-serine esterase] or influx of Ca2+ ions in clone CD124 cultured in either IL-4 or IL-2. In contrast, JY cells induced high BLT-serine esterase secretion and an increase of cytosolic Ca2+ levels. By using a combination of a 51Cr-release assay and a BLT-serine esterase secretion assay, the reactivity of clone CD124 against a limited number of target cells was analyzed. CD124 which expresses HLA-A2 and -B7, recognized an Ag shared by JY (HLA-A2; B7; C blank; DR4,6) and one haplotype expressed by the cell line SPS (HLA-A1; B14; Cw6; DR4). The only specificity shared by SPS and JY was HLA-DR4. However, clone CD124 failed to lyse 5 other HLA-DR4+ target cells. The cytotoxic activity of clone CD124 was inhibited by the class I MHC specific mAb W6/32 and the anti-beta 2m mAb A88, but not, or only marginally, by the anti HLA-DQ mAb SPV-L3 or the anti-HLA-DR mAb 135. These data strongly suggest that clone CD124 recognizes a class I MHC Ag different from HLA-A, -B, or -