HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood.

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n=20) or placebo (n=4) before and after vaccination. 11 cytokines were measured: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses.

Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.

Background: Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible.

Methods: Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining.

Comparison of benchtop microplate beta counters with the traditional gamma counting method for measurement of chromium-51 release in cytotoxic assays.

The most traditional method used to measure the lytic activity of cytotoxic T lymphocytes or natural killer (NK) cells is the chromium release assay (CRA). No study has been reported that systematically compares the traditional gamma counting method with various benchtop microplate scintillation formats to measure chromium release. Here we investigated the utilization of microplate beta counters in comparison with the traditional gamma counting method to quantitate antigen-specific cytolysis, lymphokine-activated killer (LAK) activity, and NK activity in the CRA.

Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles.

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance.

Simultaneous measurement of several cytokines using small volumes of biospecimens.

The role of host immunity in the development of virus-induced cancers has been difficult to elucidate, in part because of our inability to effectively measure multiple immune parameters using available amounts of biological material. The objective of the present study was to validate the use of a newly developed multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines [interleukin/(IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, and tumor necrosis factor-alpha]. Supernatants obtained from peripheral blood mononuclear cell cultures stimulated with various different mitogens and antigens (including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and media alone) were selected for study.