Early Diagnosis of Pathogen Infection by Cell-Based Activation Immunoassay

Figure 1. IFNγ ELISPOT response in the spleen following intranasal infection with F. tularensis. Mice were infected with a sub-lethal dose (102 cfu, 0.1 LD50) of LVS strain, or with a lethal dose of 105 cfu LVS or 102 Schu strain (100 LD50). Animals were sacrificed and splenocytes were stimulated in situ in an ELISPOT assay with UV-inactivated LVS (UViLVS, 5E7 cfu equivalent/mL) or left untreated (no antigen). Images of 106 cells/well of splenocytes from naïve (A) or sub-lethal LVS-infected mice (day 3 post infection) (B) are presented. (C) Quantification of IFNγ spots represent groups of 3 mice, collected in the indicated time-points following sub-lethal LVS infection, and individually assayed as described above. (D) Three mice were collected 3 days following lethal intranasal infection with LVS (105 cfu) or Schu strain (102 cfu), and assayed as described above. Data are representative of three (LVS infections) and two (SchuS4 infections) independently performed experiments. ** p < 0.01.

Figure 2. IFNγ ELISPOT response in the spleen following subcutaneous infection with B. anthracis and Y. pestis. Mice were infected with B. anthracis strain Sterne ΔhtrA lefMUT Δcya (A) or Y. pestis subsp EV76 (B). Four days later splenocytes were stimulated with B. anthracis core antigens and PA protein or formalin-inactivated EV76, respectively. Data are representative of three independently performed experiments. ** p < 0.01.

Figure 3. IFNγ ELISPOT response in the spleen following intranasal infection with Influenza strain A/Puerto Rico/8/1934 H1N1. Mice were infected with 20 pfu and groups of 3 spleens were collected in the indicated time points. Splenocytes were stimulated in situ in an ELISPOT assay with MHC peptide epitopes (1 µM total peptide concentration). Two immunodominant CD4 peptides epitopes (I-Ab-restricted, NP311-325 QVYSLIRPNENPAHK, designated QVHK; NA161-175 SVAWSASACHDGMGW, designated SVGW), two CD8 peptide epitopes (Db-restricted, NP366-374 ASNENMETM, designated ASTM; PA224-233 SSLENFRAYV, designated SSYV) and a mixture of the four were used for stimulation. Data are representative of three independently performed experiments. ** p < 0.01.

Figure 4. ELISPOT response in the blood following F. tularensis infection. Groups of three mice were infected intranasal with F. tularensis LVS (100 cfu) and bled 7 and 13 days post infection. PBMC from naïve and infected mice were stimulated with UViLVS as describe or left untreated. Data are representative of two independently performed experiments. * p < 0.05, ** p < 0.01.

Figure 5. Cellular and humoral response in non-human primates following B. anthracis infection. Three individual animals were infected subcutaneously with 5 × 108 spores of Sterne ΔhtrA lefMUT Δcya strain. The animals were bled in the indicated time points and their immune response was evaluated by ELISPOT (A) and ELISA (B) as described in the text. * p < 0.05, ** p < 0.01.

Figure 6. RNA-seq analysis. (A) Experimental flow chart: Splenocytes from five naive mice and five LVS-infected mice were stimulated in situ with UViLVS for 3 h. Total RNA was extracted and mRNA was sequenced. The number of genes that were significantly up-regulated (B) or down-regulated (C) in infected and naïve mice in response to antigen stimulation, as described in the text. (D) Real-time PCR validation of mmp9 and ifng genes in splenocytes from LVS-infected versus naïve mice. The expression level was normalized to the hprt gene. * p < 0.05.