Protective efficacy of subsp. <i>novicida</i> vaccination against subsequent subsp. <i>tularensis</i> challenge in Fischer 344 rats.

<p>Groups of Fischer 344 rats (n = 6) were vaccinated intratracheally with 10² or 10⁵ CFU of subsp. <i>novicida</i> in PBS or mock vaccinated with PBS alone. (A) Thirty days later, rats were challenged intratracheally with subsp. <i>tularensis</i> (10⁴ CFU) and monitored daily for morbidity and mortality. *<i>P</i> = 0.182, **<i>P</i><0.05 (B) Fourteen days after vaccination with 10⁵ CFU of subsp. <i>novicida</i> or PBS alone, rats were sacrificed, cervical lymph nodes removed, and whole cell populations were recalled with 10⁵ CFU of UV-inactivated subsp. <i>novicida</i>, media alone, or the unrelated antigen HEL for 72 hr. Culture supernatants were analyzed for antigen-specific IFN-γ production. *<i>P</i><0.001 (C) Thirty days after vaccination with 10⁵ CFU of subsp. <i>novicida</i> or PBS alone, blood was collected and sera were prepared. Sera were analyzed for antigen-specific total antibody (Ig H & L), IgG1, and IgG2a, as well as reaction to the unrelated antigen HEL by ELISA. Results are represented as 50% binding titers. Results are representative of two separate experiments. *<i>P</i><0.01, **<i>P</i> = 0.118.</p

Intracellular replication of <i>Francisella</i> strains within Fischer 344 hepatocytes.

<p>Livers were collected from Fischer 344 rats and hepatocytes were isolated. Cells were seeded in 96 well plates (2×10⁵ cells/well) and allowed to adhere. Cells were infected with 10 MOI of (A) subsp. <i>novicida</i>, (B) LVS, (C) subsp. <i>holarctica</i> or (D) subsp. <i>tularensis</i> for 2 hr, gentamicin treated for 1 hr, and incubated at 37°C for 72 hr. At the indicated time points after infection (3, 24, 48 and 72 hr), cells were lysed and serial dilutions were plated to quantify intracellular bacteria. Results are representative of three separate experiments. *<i>P</i> = 0.02, **<i>P</i><0.01.</p

Intratracheal LD₅₀ Doses of <i>Francisella</i> Strains in Fischer 344 Rats.

<p>Groups of Fischer 344 rats (n = 6) were challenged intratracheally with increasing doses (10² to 10⁷ CFU) of subsp. <i>novicida</i> strain U112, subsp. <i>holarctica</i> strain LVS, subsp. <i>holarctica</i> strain OR96-0246, or subsp. <i>tularensis</i> strain SCHU S4 and monitored daily for morbidity and mortality and the LD₅₀ and mean time to death (MTD) calculated. Results for each strain are representative of at least two separate experiments.</p

Contribution of Fischer 344 rat BMDM nitric oxide production on intracellular replication of <i>Francisella</i> strains.

<p>(A) Supernatants from intracellular replication assays (3, 24, 48 and 72 h) were analyzed for the presence of nitric oxide. Concentrations of nitric oxide following infection of BMDM with (i) subsp. <i>novicida</i>, (ii) LVS, (iii) subsp. <i>holarctica</i>, and (iv) subsp. <i>tularensis</i>. Results are representative of three separate experiments. (B) Fischer 344 BMDM were seeded in 96 well plates (2×10⁵ cells/well) and allowed to adhere. Cells were infected with 10 MOI of (i) subsp. <i>novicida</i> or (ii) subsp. <i>tularensis</i> for 2 hr, with or without N-methyl-L-arginine acetate salt as a nitric oxide inhibitor, treated for 1 hr with gentamicin, and incubated at 37°C for 72 hr. At the indicated time points (3, 24, 48 and 72 h) after challenge, cells were lysed and serial dilutions were plated to quantify intracellular bacteria. Results are representative of two separate experiments. <b>*</b><i>P</i><0.001, <b>**</b><i>P</i><0.05.</p

Uptake and replication of <i>Francisella</i> strains within Fischer 344 rat BMDM.

<p>Bone marrow derived macrophages from Fischer 344 rats were seeded in 96 well plates (2×10⁵ cells/well) and allowed to adhere. Cells were infected (10 or 100 MOI) for two hr with subsp. <i>novicida</i>, LVS, subsp. <i>holarctica</i>, or subsp. <i>tularensis</i>. (A) Serial dilutions of culture supernatants (extracellular bacteria) and cell lysates (intracellular bacteria) at (i) 10 MOI and (ii) 100 MOI were plated to determine ability of macrophages to take up bacteria. Results are representative of two separate experiments. (B) Following a 2 hr infection period, cells were treated with gentamicin for 1 hr, and then incubated at 37°C for 72 hr. At the indicated time points (3, 24, 48 and 72 h), cells were lysed and serial dilutions of lysates were plated to quantify intracellular bacteria. Intracellular replication profiles of (i) subsp. <i>novicida</i>, (ii) LVS, (iii) subsp. <i>holarctica</i>, or (iv) subsp. <i>tularensis</i>. Results are representative of at least three separate experiments.</p

Comparison of basal phagocytic ability of BMDM from Fischer 344 rats and BALB/c mice.

<p>Bone marrow derived macrophages from (A) Fischer 344 rats and (B) BALB/c mice were seeded in 6 well plates (1×10⁶ cells/well) and allowed to adhere. Cells were incubated with fluorescent microspheres (10 or 100 beads/cell) for 1 hr to allow for phagocytosis. Cells were collected into 5 ml polystyrene tubes and stained with either mouse anti-rat CD11b AF647 or rat anti-mouse CD11b APC to confirm the macrophage population. The numbers of intracelllular beads were quantified by flow cytometry (P5 = 1 bead, P6 = 2 beads, P7≥3 beads). Results are representative of two separate experiments.</p

Effect of naïve rat serum on uptake and replication of <i>Francisella</i> strains within Fischer 344 rat BMDM.

<p>Bone marrow derived macrophages were seeded in 96 well plates (2×10⁵ cells/well) and allowed to adhere. Cells were infected (10 MOI) with (A) LVS or (B) subsp. <i>tularensis</i> for 2 hr with or without 10% naïve rat serum as an opsonin, treated for 1 hr with gentamicin, and incubated at 37°C for 72 hr. At the indicated time points (3, 24, 48, or 72 h) cells were lysed and serial dilutions of lysates were plated to quantify intracellular bacteria. Results are representative of two separate experiments.</p

Nomenclature and construction of heavy-chain hybrids of mAb F26G3.

<p>The CH1, CH2 and CH3 domains of IgG3 mAb were replaced with the respective domains of the IgG2b subclass switch variant of mAb F26G3.</p

Binding behavior of parental and heavy-chain domain hybrid mAbs.

<p>A – Scatchard plots for determination of affinities from equilibrium binding by SPR. B – Dissociation constants (K_D in nM) for each mAb calculated from Panel A. C - Binding by ELISA. Results are reported as the concentration of each mAb (ng/ml) that produced an OD₄₅₀ = 2.0 in a standard ELISA format.</p

Assessment of binding of subclass switch families of mAbs F24F2 and F26G3 by fluorescence perturbation.

<p>Changes in mAb fluorescence (excitation wavelength = 284 nm; emission wavelength = 341 nm) are reported for parental F24F2 or F26G3 IgG3 and subclass switch variants upon addition of increasing amounts of synthetic (γ-d-Glu)₅. The solid lines are hyperbolic fits of the data to the equation: y = (ax^b)/(c^b+x^b), where c is the apparent dissociation constant (K_D) in µM.</p