We compared the kinetics of amphotericin B (AMB) lung accumulation and fungal clearance by liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) in a neutropenic murine model of invasive pulmonary mucormycosis (IPM).Immunosuppressed BALB/c mice were inoculated with 1 ؋ 10 6 Rhizopus oryzae spores and administered L-AMB or ABLC at daily intravenous doses of 1, 5, or 10 mg/kg of body weight for 5 days starting 12 h after infection. At a dose of 10 mg/kg/day, both L-AMB and ABLC were effective at reducing the R. oryzae lung fungal burden and achieved lung tissue concentrations exceeding the isolate mean fungicidal concentration (MFC) of 8 g/ml by 72 h. When ABLC was dosed at 5 mg/kg/day, the ABLC-treated animals had significantly higher AMB lung concentrations than the L-AMB treated animals at 24 h (6.64 and 1.44 g/g, respectively; P ‫؍‬ 0.013) and 72 h (7.49 and 1.03 g/g, respectively; P ‫؍‬ 0.005), and these higher concentrations were associated with improved fungal clearance, as determined by quantitative real-time PCR (mean conidial equivalent of R. oryzae DNA per lung, 4.44 ؎ 0.44 and 6.57 ؎ 0.74 log 10 , respectively; P < 0.001). Analysis of the AMB tissue concentration-response relationships revealed that the suppression of R. oryzae growth in the lung required tissue concentrations that approached the MFC for the infecting isolate (50% effective concentration, 8.19 g/g [95% confidence interval, 2.81 to 18.1 g/g]). The rates of survival were similar in the animals treated with L-AMB and ABLC at 10 mg/kg/day. These data suggest that higher initial doses may be required during L-AMB treatment than during ABLC treatment of experimental IPM.Invasive pulmonary mucormycosis (IPM) is an uncommon but frequently fatal angioinvasive mold infection that has increased in incidence over the last decade, especially in patients with hematological malignancies and recipients of hematopoietic stem cell transplantation (HSCT) (23). In a recent multicenter, prospective observational study of invasive fungal infections in HSCT recipients, mucormycosis was the third most common invasive fungal infection (7.2%), behind invasive aspergillosis (59.2%) and invasive candidiasis (24.8%) (21). Data from the Centers for Disease Control and Prevention Transplant Associated Infection Surveillance Network (TRANSNET) reported that the incidence of mucormycosis in U.S. transplant centers increased nearly sixfold from 2001 to 2004, with Rhizopus being the most frequently isolated genus (22).Although new diagnostic and treatment options have improved the survival rates in patients with invasive pulmonary aspergillosis (IPA) over the last decade, the prognosis for patients with IPM remains poor, as only one-third of the patients survive beyond 12 weeks after the diagnosis (13, 21, 23). The outcome of IPM is heavily dependent on a timely diagnosis, as the initial clinical manifestations and radiographic appearance of IPM are often indistinguishable from those of IPA, and the first-line antifungals used to treat aspergillosis, ...

The reformulation of amphotericin B (AMB) into a lipid complex (AMB lipid complex [ABLC]) or liposomal carrier (liposomal AMB [L-AMB])changes the rate and extent of drug distribution to the lung. The importance of pharmacokinetic differences among the various lipid AMB formulations in the treatment of invasive pulmonary aspergillosis (IPA) remains unknown. We compared the kinetics of AMB lung accumulation and fungal clearance of ABLC-and L-AMB-treated mice with acute IPA. BALB/c mice were immunosuppressed with cyclophosphamide and cortisone before intranasal inoculation with 1.5 ؋ 10 6 Aspergillus fumigatus 293 conidia. ABLC or L-AMB was administered in daily intravenous doses (1, 5, or 10 mg/kg of body weight), starting 12 h after infection and continuing until day 5. At predetermined times (0, 24, 72, and 120 h), mice were euthanized, and lungs were harvested for determinations of lung fungal burdens (quantitative PCR) and total AMB lung tissue concentrations. Both ABLC and L-AMB were effective at reducing lung fungal burdens at doses of >5 mg/kg/day. Clearance of A. fumigatus during the first 24 h was associated with AMB tissue concentrations of >4 g/g. At 5 mg/kg/day, ABLC produced a more rapid fungal clearance than did L-AMB, but at the end of therapy, fungal burden reductions were similar for both formulations and were not improved with higher dosages. These data suggest that ABLC delivers active AMB to the lung more rapidly than does L-AMB, resulting in faster Aspergillus clearance in an experimental model of IPA. However, pharmacodynamic differences between the two formulations were less apparent when mice were dosed at 10 mg/kg/day.Despite the availability of new treatment options, lipid formulations of amphotericin B (AMB) continue to play a central role in the management of invasive pulmonary aspergillosis (IPA) due to their broad spectrum and low potential for cross-resistance with other antifungals (5). Currently, the following three lipid formulations are approved for the treatment of IPA in patients who have failed or are intolerant to other therapies: AMB colloidal dispersion, AMB lipid complex (ABLC), and liposomal AMB (L-AMB). All three AMB formulations differ in terms of their lipid composition and particle size, resulting in different pharmacokinetic characteristics when the drugs are administered in vivo. For example, L-AMB consists of small unilamellar particles (60 to 70 nm) that avoid uptake by cells of the reticuloendothelial system (RES) (7,8,14,25). Hence, intravenous administration of L-AMB results in sustained, high concentrations of encapsulated AMB in the serum, with a somewhat delayed distribution of free drug to tissues. Conversely, intravenous administration of ABLC results in relatively low serum AMB concentrations due to the rapid RES cell uptake of the large lipid complex (1,600 to 11,000 nm) (1). Extensive RES cell uptake of ABLC is thought to account for the more rapid distribution of ABLC to certain organs, such as the lungs, than that of other formulations (18,19). The cl...

Failure to induce synthesis of neutralizing antibodies to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to antibodies in their germline variable (V) region configuration expressed as B cell receptors, insufficient adaptive mutations in antibody variable (V) regions and conformational instability of gp120. We employed peptide analogs of gp120 residues 421-433 (CD4BDcore) to identify antibodies produced without prior exposure to HIV (constitutive antibodies). The CD4BDcore peptide was recognized by single chain Fv (scFv) fragments from non-infected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework (FR) segments of a VH4-family scFv with the corresponding VH3-family FRs from scFv JL427 improved the CD4BDcore peptide binding activity, suggesting a CD4BDcore binding site outside the pocket formed by the complementarity determining regions. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BDcore-specific serum IgAs from non-infected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BDcore-specific, HIV neutralizing murine IgM with heavy and light chain V regions (VH and VL regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BDcore peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive antibodies as an innate, germline-encoded activity directed to the superantigenic CD4BDcore epitope that is available for amplification for vaccination against HIV.

Mucormycosis is an uncommon fungal infection that has been increasingly reported in severely immunocompromised patients receiving Aspergillus-active antifungals. Although clinical studies and pre-clinical animal models have suggested a unique predisposition for breakthrough mucormycoses in patients receiving voriconazole, no study has specifically evaluated the selection dynamics of various Aspergillus -active antifungal classes in vivo. We utilized an Aspergillus fumigatus:Rhizopus oryzae (10:1) model of mixed fungal pneumonia in corticosteroid-immunosuppressed mice to compare the selection dynamics of daily liposomal-amphotericin B (L-AMB), micafungin (MCFG) and voriconazole (VRC) therapy. A. fumigatus and R. oryzae lung fungal burden were serially monitored in parallel using non-cross-amplifying quantitative real-time PCR assays for each fungal genus. Additionally, experiments were performed where the R. oryzae component of the mixed inoculum was serially-passed on VRC-containing agar before animal infection. We found prior exposure to voriconazole in vitro, consistently resulted in a 1.5-2 log 10 increase in R. oryzae fungal burden by day +5 in vivo relative to animals infected with the non-VRC preexposed inoculum, irrespective of the antifungal-treatment administered in mice (P ≤ 0.02 all treatment groups). Mice infected with the VRC-preexposed inoculum and subsequently treated with saline or VRC had the highest mortality rates (82-86%), followed by MCFG (55%) then L-AMB (39%, P = 0.04 vs. control). However, in vivo treatment alone with voriconazole alone did not consistently increase the virulence of non- voriconazole preexposed R. oryzae versus controls. We conclude that exposure of R. oryzae sporangiospores to voriconazole in vitro modulates the subsequent growth rate and/or virulence of the fungus in vivo, which reduces effectiveness of Mucorales-active antifungals. The mechanisms underlying this phenotypic change are unknown.

Despite a lack of appreciable antifungal activity in vitro, both anidulafungin and caspofungin were still modestly effective in vivo against a laboratory-generated A. fumigatus mutant harbouring the Ser678Pro mutation in Fks1p. This persistent activity, combined with impaired fitness of the isolate in vivo, could partially explain why microbiologically documented echinocandin-resistance in Aspergillus species remains a rare clinical occurrence.