Fewer NK cells from FIV-infected cats produce perforin.

<p>Whole cell suspensions from lymph nodes were cultured overnight with IL-2. Cells were then co-cultured for 4 h with target cells at an effector:target ratio of 50∶1, with monensin added after the first hour of incubation. Cells were stained for cell surface receptors CD3 and CD56 and for intracellular perforin, IFN-γ and granzyme A. Samples were immediately analyzed by flow cytometry. Columns represent mean ± SEM. FIV-infected (black columns) and SPF-control (white columns). Statistical significance was determined between FIV-infected and SPF-control cats. Statistical analysis was performed using Mann-Whitney U test. * indicates <i>P</i><0.05. SPF-control cats n = 6, FIV-infected cats n = 12.</p

Chronic FIV infection is associated with decreased lymphocyte numbers in Lm challenged LN.

<p>Chronically FIV-infected and SPF-control cats were challenged with 2.5×10⁵ cfu <i>Listeria monocytogenes</i> subcutaneously proximal to either the right metatarsal or metacarpal footpad. After 3 days, the local draining lymph node and the contralateral control nodes were removed, processed into a single cell suspension, assessed by trypan blue dye exclusion, and the total number of cells per lymph node was determined. The absolute cell number for lymphocyte subpopulations was calculated based on the percent of gated lymphocytes determined by flow cytometric analysis and total number of lymphocytes from the LN. (A) Total cell number, (B) Absolute number of NK cells (CD3⁻CD56⁺), (C) Absolute number of NKT cells (CD3⁺CD56⁺), (D) Absolute number of CD4⁺ T cells (CD3⁺CD4⁺), (E) Absolute number of CD8⁺ T cells (CD3⁺CD8⁺), and (F) Absolute number of Langerhans cells (CD1a⁺). Columns represent mean ± standard error of the mean (SEM). FIV-infected (black columns) and SPF-control animals (white columns). Statistical significance was determined between control and Lm challenged LN. Statistical analysis was performed using Wilcoxon Signed-Rank test. * indicates <i>P</i><0.05, ** indicates <i>P</i><0.01. SPF-control cats n = 8, FIV-infected cats n = 13.</p

Effect of FIV infection on PBMC subpopulations.

<p>Whole blood was collected at the time of lymph node biopsy 3 days after the Lm challenge. The absolute cell number for lymphocyte subpopulations was calculated based on the percent of gated lymphocytes determined by flow cytometric analysis multiplied by lymphocyte absolute number. (A) Absolute number of CD4⁺ T cells, (B) Absolute number of CD8⁺ T cells, (C) Absolute number of PBMC, (D) Absolute number of NK cells. Columns represent mean ± SEM. FIV-infected (black columns) and SPF-control animals (white columns). Statistical significance was determined between control and Lm challenged LN. Statistical analysis was performed using Mann-Whitney U test. ** indicates <i>P</i><0.01. SPF-control cats n = 8, FIV-infected cats n = 13.</p

Flow cytometry gating strategy and representative plots.

<p>Flow cytometry gating strategy and representative plots from control lymph node cells of a SPF-control cat (CD4⁺ and CD8⁺ T cell and NK cell gatings) and from control lymph node of a FIV-infected cat (CD4⁺CD25⁺ and FOXP3 gatings). (A) The lymphocyte population was gated based on side and forward scatter (inner gate). Lymphocyte subpopulations were gated based on expression of CD3, CD4, CD8, CD56, CD25 and FOXP3. A broader gating strategy of the total cell population (outer gate) was used to identify the CD1a⁺ subpopulation. (B) Representative dot plots are shown for AnnexinV⁺ cells within total, CD4⁺ and CD8⁺ T cells, and NK cells. Representative contour plots of BrdU⁺ and Ki67⁺ cells within total, CD4⁺ and CD8⁺ T cells, and NK cells from a FIV-infected cat are shown.</p

Effect of FIV infection on peripheral blood mononuclear cell (PBMC) proliferation.

<p>Whole blood was collected at the time of lymph node biopsy 3 days after the Lm challenge. Cell proliferation was assessed by BrdU incorporation and expression of the nuclear antigen Ki-67. (A) Absolute number of proliferating NK cells, (B) Absolute number of proliferating CD4⁺ T cells, (C) Absolute number of proliferating CD8⁺ T cells, (D) Absolute number of proliferating cells. Columns represent mean ± SEM. FIV-infected (black columns) and SPF-control animals (white columns). Statistical significance was determined between FIV-infected and SPF-control cats. Statistical analysis was performed using Mann-Whitney U test. * indicates <i>P</i><0.05. SPF-control cats n = 8, FIV-infected cats n = 13.</p

Effect of FIV infection on lymph node cell proliferation.

<p>Cell proliferation was assessed by BrdU incorporation and expression of the nuclear antigen Ki-67. The absolute number of proliferating lymphocyte subpopulations was calculated based on the percent of gated lymphocytes that either incorporated BrdU or expressed Ki-67, determined by flow cytometric analysis, and the total number of lymphocytes from the LN. (A) Absolute number of proliferating NK cells, (B) Absolute number of proliferating CD4⁺ T cells, (C) Absolute number of proliferating CD8⁺ T cells, (D) Absolute number of proliferating cells. Columns represent mean ± SEM. FIV-infected (black columns) and SPF-control animals (white columns). Statistical significance was determined between control and challenged LN, and between FIV-infected and SPF-control cats. Statistical analysis was performed using Wilcoxon Signed-Rank test and Mann-Whitney U test. * indicates <i>P</i><0.05, ** indicates <i>P</i><0.01, *** indicates <i>P</i><0.001. SPF-control cats n = 8, FIV-infected cats n = 13.</p

Mucosal Immunogenicity of Genetically Modified <i>Lactobacillus acidophilus</i> Expressing an HIV-1 Epitope within the Surface Layer Protein

<div><p>Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified <i>Lactobacillus acidophilus</i> strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the <i>Lactobacillus</i> S-layer protein for development of oral vaccines targeting specific peptides.</p></div

Induction of MPER-specific antibody production by long-term immunization.

<p>Mice received GAD19 orally every 2 weeks for 14 weeks. (a) Diluted serum (1/100) was analyzed by ELISA at each time point. Arrows represent timing of the gavage. (b) Endpoint titers (or absorbance at 450 nm) of MPER-specific serum IgG, cecal IgA, vaginal IgA, and vaginal IgG. Each symbol represents an individual mouse.</p

Validation of genetically modified <i>L</i>. <i>acidophilus</i> producing MPER-displaying S-layer proteins.

<p>The <i>L</i>. <i>acidophilus slpA</i> gene in NCK1909 was replaced with the modified <i>slpA</i> gene including MPER-encoding sequences by homologous recombination in NCK2208. (a) The gene replacement of <i>slpA</i> with the modified <i>slpA</i> was confirmed by PCR. L, DNA ladder marker. Amplified DNA fragments using primers, AK_62 and AK_65 (lane 1 and 4), AK_62 and AK_57 (lane 2 and 5), or AK_56 and AK_65 (lane 3 and 6). (b) Detection of the MPER epitope in S-layer (SlpA) protein using 2F5 mAb. Total cell proteins and purified S-layer proteins of NCK1909 and NCK2208 were separated by SDS-PAGE. The gels were stained with CBB or blotted onto PVDF membrane followed by western blot analysis using 2F5 (anti-MPER monoclonal human IgG). (c) The exposed MPER epitope was detected by flow cytometry. The <i>L</i>. <i>acidophilus</i> strains labeled with 2F5 and Alexa Fluor 488-conjugated anti-human IgG were analyzed. Relative fluorescence intensity of each strain was shown as histogram plot.</p

Typing of classes and subclasses of MPER-specific antibodies.

<p>Sera from GAD19-immunized mice were analyzed by ELISA. Each value plus SD (standard deviation) was shown.</p