FluoroSpot Analysis of TLR-Activated Monocytes Reveals Several Distinct Cytokine-Secreting Subpopulations.

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine-secreting cells on the single-cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL-1ß, IL-6, TNF-α and MIP-1ß were secreted by larger populations of responding cells (25.9-39.2%) compared with the smaller populations of GM-CSF (9.1%), IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore, when studying co-secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL-1ß and/or IL-6 and those secreting TNF-α, MIP-1ß, GM-CSF, IL-10 and IL-12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation, several subpopulations with distinct cytokine-secreting profiles could be identified.

Amplified antigen-specific immune responses in HIV-1 infected individuals in a double blind DNA immunization and therapy interruption trial.

Immunotherapy in patients with HIV-1 infection aims to restore and broaden immunological competence, reduce viral load and thereby permit longer periods without combined antiretroviral treatment (cART). Twelve HIV-1-infected patients on cART were immunized on the skin with DNA plasmids containing genes of several HIV-1 subtypes with or without the addition of hydroxyurea (HU), or with placebo. The mean net gain of HIV-specific CD8+ T cell responses were higher and broader in the HIV DNA vaccine groups compared to non-vaccinated individuals (p<0.05). The vaccine-induced immune responses per se had no direct effect on viral replication. In all patients combined, including placebo, the viral set point after a final structured therapy interruption (STI) was lower than prior to initiation of cART (p=0.003). Nadir CD4 levels appeared to strongly influence the post-STI viral titers. After the sixth immunization or placebo, patients could stay off cART for a median time of 15 months. The study shows that HIV DNA immunization induces broader and higher magnitudes of HIV-specific immune responses compared to structured therapy interruptions alone. Although compromised by small numbers of patients, the study also demonstrates that well-monitored STI may safely function as an immunological read out of HIV vaccine efficacy.

Cross-clade immune responses to Gag p24 in patients infected with different HIV-1 subtypes and correlation with HLA class I and II alleles.

Individuals infected with different subtypes of HIV-1 (A, B, C, D, CRF01_AE and CRF02_AG) were analyzed for their antigen-specific immune response with respect to their HLA genetics. The p24 Gag protein was selected for analysis, since previous studies of the same cohort of patients had shown that almost 80% of these individuals responded to Gag peptides of subtypes A, B and/or C. A large number of Gag antigen-specific responses were recorded. Both previously recognized as well as new epitopes were identified, assumed to bind HLA classes I and/or II. Fifteen individuals showed class I cellular responses to T cell epitopes irrespective of the infecting virus subtype. For five individuals infected with subtypes A, B, D and CRF02_AG, new T cell epitopes are described. Responses related to the patient's class I alleles are frequent, and several new putative class II responses were found.

Pre-clinical evaluation of a CEA DNA prime/protein boost vaccination strategy against colorectal cancer.

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.

The rationale behind a vaccine based on multiple HIV antigens.

The viral diversity of HIV-1 is likely to require a vaccine strategy that induces broad cellular and humoral anti-HIV-1 immunity. Our strategy is based on multiple HIV-1 DNA immunogens together with adjuvant recombinant granulocyte-macrophage stimulating factor. This article describes pre-clinical and clinical work preceding the initiation of clinical HIV-1 phase I/II trials.