The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

To evaluate the immunogenicity of human immunodeficiency virus (HIV) type 1 p55^gag virus-like particles (VLPs) released by budding from yeast spheroplasts, we have analyzed the effects of yeast VLPs on monocyte-derived dendritic cells (DCs). Yeast VLPs were efficiently incorporated into DCs via both macropinocytosis and endocytosis mediated by mannose-recognizing receptors, but not the mannose receptor. The uptake of yeast VLPs induced DC maturation and enhanced cytokine production, notably, interleukin-12 p70. We showed that yeast membrane components may contribute to DC maturation partly through Toll-like receptor 2 signaling. Thus, Gag particles encapsulated by yeast membrane may have an advantage in stimulating Gag-specific immune responses. We found that yeast VLPs, but not the control yeast membrane fraction, were able to activate both CD4⁺ and CD8⁺ T cells of HIV-infected individuals. We tested the effect of cross-presentation of VLP by DCs in two subjects recruited into a long-term nonprogressor-slow progressor cohort. When yeast VLP-loaded DCs of these patients were cocultured with peripheral blood mononuclear cells for 7 days, approximately one-third of the Gag-specific CD8⁺ T cells were activated and became perforin positive. However, some of the Gag-specific CD8⁺ T cells appeared to be lost during in vitro culture, especially in a patient with a high virus load. Our results suggest that DCs loaded with yeast VLPs can activate Gag-specific memory CD8⁺ T cells to become effector cells in chronically HIV-infected individuals, but there still remain unresponsive Gag-specific T-cell populations in these patients.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against HIV. On the other hand, due to the susceptibility of DCs to HIV infection, virus replication is strongly enhanced in DC–T cell interaction via an immunological synapse formed during the antigen presentation process. When HIV-1 is isolated from individuals newly infected with the mixture of R5 and X4 variants, R5 is predominant, irrespective of the route of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced by antigen presentation, we postulated that the selective expansion of R5 may largely occur at the level of DC–T cell interaction. Thus, the immunological synapse serves as an infectious synapse through which the virus can be disseminated in vivo. We used fluorescent recombinant X4 and R5 HIV-1 consisting of a common HIV-1 genome structure with distinct envelopes, which allowed us to discriminate the HIV-1 transmitted from DCs infected with the two virus mixtures to antigen-specific CD4⁺ T cells by flow cytometry. We clearly show that the selective expansion of R5 over X4 HIV-1 did occur, which was determined at an early entry step by the activation status of the CD4⁺ T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs. Our results imply a promising strategy for the efficient control of HIV infection. The cellular tropism of HIV-1 is determined by the binding of HIV-1 envelope to chemokine coreceptors, CCR5 or CXCR4, in addition to a major entry receptor, CD4. The mystery still now is that despite the mixed infection of CCR5-utilizing (R5) and CXCR4-utilizing (X4) HIV-1 in many AIDS patients, R5 is predominantly isolated from newly infected individuals whatever the mode of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced initially by antigen-presenting DCs, we postulated that the selective expansion of R5 may largely occur at the level of antigen-dependent DC–T cell interaction, called immunological synapse. Thus, the immunological synapse serves as an infectious synapse through which the virus can be rapidly disseminated in vivo. In this study, we prepared X4 and R5 HIV-1 expressing red or green fluorescence and showed that the selective expansion of R5 over X4 did occur, depending on the activation status of CD4⁺ T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs.

Background: The differentiation of CD8⁺T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown.Methods: In order to explore the potential implication of microRNAs in CD8⁺T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8⁺T cell subsets defining the major steps of the T cell differentiation pathway.Results: We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease.Conclusions: This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8⁺T lymphocytesin vivo, which is likely to impact on specific cellular functions.