Stepping up ELISpot: Multi-Level Analysis in FluoroSpot Assays

ELISpot is one of the most commonly used immune monitoring assays, which allows the functional assessment of the immune system at the single cell level. With its outstanding sensitivity and ease of performance, the assay has recently advanced from the mere single function cell analysis to multifunctional analysis by implementing detection reagents that are labeled with fluorophores (FluoroSpot), allowing the detection of secretion patterns of two or more analytes in a single well. However, the automated evaluation of such assays presents various challenges for image analysis. Here we dissect the technical and methodological requirements for a reliable analysis of FluoroSpot assays, introduce important quality control measures and provide advice for proper interpretation of results obtained by automated imaging systems

Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease

Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathway

Catalytic activities of the recombinant integrases.

<p>Catalytic activities of the recombinant integrases.</p

Expression of integrase variants in <i>E.coli</i> BL21(DE3).

<p>SDS-PAGE analysis of the purified consensus HIV-1 clade A integrase (IN_a, lane 1), its inactivated variant (IN_in, lane 2) and the inactivated IN variant with elvitegravir resistance mutations (IN_in_e3, lane 3) eluted from the Ni-NTA-agarose column with 500 mM imidazole, followed by staining with Coomassie Blue (<b>A</b>). Western blotting of integrase preparations (diluted 1∶50) after SDS-PAGE and transfer, using polyclonal rabbit anti-IN antibodies (<b>B</b>). Data are representative of three independent experiments.</p

Expression of IN variants in eukaryotic cells.

<p>Western blotting of lysates of HeLa cells transfected with the pVaxIN_a (lane 1), pVaxIN_a_e3 (lane 2), pVaxIN_in (lane 3), pVaxIN_in_e3 (lane 4), or empty vector pVax1 (lane 5); recombinant IN of HXB2 carrying 6His-tag (34 kDa) loaded in the amounts of 0.5, 2.5, 5 and 10 ng/well (lanes 7 to 10, respectively). Blot was stained with the rabbit polyclonal anti-IN antibodies, stripped, and re-stained with the monoclonal anti-actin antibodies. Molecular mass markers as defined by the protein ladder (Page Ruler Prestained Protein Ladder, Fermentas; lane 6) are given to the left (<b>A</b>). Average amount of the IN variants expressed per transfected human (HeLa, HEK293) or mouse (NIH3T3) cell (results of two independent runs, each done in duplicate) (<b>B</b>).</p

Average radiance at the sites of the IN/Luc-reporter genes co-injection correlates to IN-specific cytokine response.

<p>Inverse correlation of the bioluminescence represented by the average radiance (BLI) at the injection site on day 21 after the immunization to: % CD4+T cells secreting IFN-γ, IL-2, IFN-γ/IL-2, and IFN-γ/IL-2/TNF-α (<b>A</b>); % CD8+T cells secreting IFN-γ, TNF-α, IFN-γ/TNF-α and IFN-γ/IL-2/TNF-α (<b>B</b>). Correlations of BLI on days 4, 9, 15 and 21 to the triple IFN-γ/IL-2/TNF-α secretion by CD4+ T cells by day 23 (<b>C</b>). Results of the BLI and FACS analysis of the data collected in two independent experiments (2 times × 4 mice in each group) were analyzed by the Spearman rank-order test (Statistica AXA 10).</p

Integrase activities: 3′-processing and DNA strand transfer by IN variants.

<p>Products of 3′-processing and strand transfer of the synthetic DNA duplexes with ³²P-labeled B-strands (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t001" target="_blank">Table 1</a>) by the consensus HIV-1 clade A integrase (IN_a), its inactivated variant (IN_in), and the inactivated variant with elvitegravir resistance mutations (IN_in_e3) were separated by gel electrophoresis and quantified using Image-QuantTM 4.1 software. The 3′-processing assay: U5 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (lanes 2, 3, and 4, respectively) (<b>A</b>). The strand transfer reaction: U5-2 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively); T – the strand transfer products (<b>B</b>). Incubation of the non-specific DNA Ran in the absence of integrases (lane 1), and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively) (<b>C</b>). Activities of HXB2 integrase, 3′-processing (1), strand transfer (2) (<b>D</b>). Tests were performed with 100 nM integrases and 10 nM DNA. Products were separated in denaturing 20% PAAG with 7M urea (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a> for details). Data are representative of two independent experiments.</p

IFN-γ/IL-2 Fluorospot assay of the splenocytes of mice immunized with IN gene variants.

<p>The results of IFN-γ/IL-2 Fluorospot performed on splenocytes of mice immunized with plasmids encoding consensus IN (IN_a), inactivated consensus IN (IN_in), inactivated consensus IN with mutations conferring resistance to elvitegravir (IN_in_e3), or empty vector. Splenocytes were stimulated <i>in vitro</i> with a Luc-derived peptide (LUC), and individual or pooled IN-derived peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t003" target="_blank">Table 3</a>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a>. IN-specific <i>in vitro</i> secretion of IFN-γ (<b>A</b>), IL-2 (<b>B</b>), and dual secretion of IFN-γ/IL-2 (<b>C</b>). Responses represent the average number of signal-forming units (sfu) per mln cells in two independent experiment runs, each done in duplicate,+SD.</p

Sequence and structure of the synthetic IN genes.

<p>Amino acid sequences of the consensus HIV-1 clade A integrase (IN_a), its inactive variant containing mutation in the active site D64V (IN_in), and inactive variant with mutations conferring resistance to elvitegravir H51Y, E92Q, S147G, E157Q, K160Q (IN_in_e3), all with Met-Gly dipeptide on the N-terminus (<b>A</b>); Schematic representation of the structure of the synthetic genes. The following regions are indicated: IN ORF including the Met-Gly dipeptide, 5′- and 3′-end nucleotide flanks with <i>BamHI</i> and <i>EcoRI</i> restriction sites (<b>B</b>).</p