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Antimicrobial Agents and Chemotherapy, April 2009, p. 1679-1682, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01334-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Radioimmunotherapy Is Effective against High-Inoculum Cryptococcus neoformans Infection in Mice and Does Not Select for Radiation-Resistant Cryptococcal Cells{triangledown}

Ruth A. Bryan,1 Zewei Jiang,1 Xianchun Huang,1 Alfred Morgenstern,2 Frank Bruchertseifer,2 Rani Sellers,3 Arturo Casadevall,4,5,# and Ekaterina Dadachova1,4*,#

Departments of Nuclear Medicine,1 Pathology,3 Microbiology and Immunology,4 Medicine, Albert Einstein College of Medicine, Bronx, New York,5 European Commission, Joint Research Centre, Institute for Transuranium Elements, Karlsruhe, Germany2

Received 5 October 2008/ Returned for modification 15 November 2008/ Accepted 31 December 2008


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ABSTRACT
 
We investigated the utility of radioimmunotherapy (RIT) in the treatment of experimental cryptococcal infection with high-inoculum and the possibility of RIT treatment selecting for fungal cells with radiation-resistant phenotypes. RIT reduced mortality in high-burden infections, and we found no evidence for the development of radiation-resistant cells.


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INTRODUCTION
 
In response to the need for novel treatments for infectious diseases, our laboratory has been developing a radioimmunotherapy (RIT) approach (reviewed in reference 4). Cryptococcus neoformans, our model organism, has well-characterized antibody reagents and animal models. We previously reported that the survival of A/JCr mice systemically infected with 105 C. neoformans cells was significantly prolonged by treatment with beta emitter 188-rhenium (188Re)- or alpha emitter 213-bismuth (213Bi)-labeled monoclonal antibody (MAb) 18B7, which recognizes the polysaccharide capsule of C. neoformans (5). Clinically, patients present at different stages of infection, some with high microbial burdens for which the efficacy of RIT is unknown. Another question is whether RIT selects for radiation-resistant fungal cells, which would interfere with follow-up RIT.

We hypothesized that 188Re, which has a 16.9-h physical half-life, would be more likely than 213Bi (46-min half-life) (1) to deliver radioactivity carried by MAb 18B7 (3) to 106 C. neoformans cells (strain 24067; ATCC, Manassas, VA). Our animal experiments followed the guidelines of the Albert Einstein College of Medicine Institute for Animal Studies. Groups of five A/JCr mice (NCI; Bethesda, MD) were infected i.v. with 106 C. neoformans cells and treated intraperitoneally 24 h later with 100 to 200 µCi of 188Re-18B7 (30 µg MAb per mouse) or 30 µg of unlabeled 18B7. A/JCr mice were used because they are highly susceptible to i.v. infection, possibly due to a partial complement deficiency (9). Infection with 106 C. neoformans cells delivers a high inoculum that translates into a high organism burden and increased levels of glycoronoxylomannan (GXM), as would be expected in an established infection. In fact, even in infections with 105 cells, the levels of GXM in the blood of A/JCr mice are equal to those in patients with cryptococcosis (5).

Kaplan-Meyer plots (Fig. 1a) showed that all doses of 188Re-18B7 significantly (P < 0.05) prolonged survival; 125 and 150 µCi were most effective, and 200 µCi was least effective. These doses should deliver radiation to any C. neoformans cells in the host that can be accessed by a labeled antibody. There would be 8 x 109 C. neoformans cells 24 h after infection with 106 cells; 100 µCi 188Re contain 3.2 x 1011 atoms, at least 50 radioactive atoms per C. neoformans cell. This study with mice systemically infected with 106 C. neoformans cells demonstrates that RIT can reduce mortality even with high fungal burdens. Previously, we reported decreased fungal burdens in lungs and brains following treatment with 188Re (5), where the survival rate of mice infected with 105 C. neoformans cells was the highest in the group treated with 100 µCi, while the organ fungal burden was the lowest for those treated with 200 µCi. There is no linear dose response in RIT in general (reviewed in reference 8), and with the increased infection burden the therapeutic window seems to narrow. Hematologic toxicity at the high end of the dose curve seems to outweigh the therapeutic benefit of reduction of the fungal burden by high doses (7).


Figure 1
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  		FIG. 1. RIT of C. neoformans in vivo and in vitro. (a) Survival percentages for A/JCr mice infected i.v. with 106 C. neoformans cells and treated 24 h later with 100 to 200 µCi 188Re-18B7 MAb. Control mice were given matching amounts of unlabeled (cold) 18B7 MAb. (b) In vitro killing of C. neoformans with 188Re-18B7 MAb. Each sample contained 105 fungal cells. (c) In vitro killing of C. neoformans with 213Bi-18B7 MAb. Each sample contained 105 fungal cells. (d) Average percentages of body weight change in 213Bi-18B7-treated and control mice. (e) Average percentages of body weight change in 188Re-18B7-treated and control mice. (f) Median survival in days of A/JCr mice infected i.v. with 5 x 104 C. neoformans cells and treated 24 h later with 150 µCi 188Re-18B7 or 125 µCi 213Bi-18B7 MAb. Untreat., untreated; ReRIT/CN naive, mice infected with CNnaive cells and treated with 188Re-18B7; Bi RIT/CN naive, mice infected with CNnaive cells and treated with 213Bi-18B7; ReRIT/CN ReRIT, mice infected with CNRe RIT cells and treated with 188Re-18B7; Bi RIT/CN BiRIT, mice infected with CNBi RIT cells and treated with 213Bi-18B7. (g) Kaplan-Meyer curves showing survival of A/JCr mice infected i.v. with 5 x 104 C. neoformans cells and treated 24 h later with 150 µCi 188Re-18B7 MAb.

A second goal was to evaluate the retention of RIT sensitivity in C. neoformans cells isolated from RIT-treated mice. The emergence of radiation-resistant cells would be a concern for subsequent RIT and the therapeutic outcome. To generate RIT-treated C. neoformans cells, A/JCr mice were infected i.v. with 5 x 104 cells and treated 24 h later with either 150 µCi 188Re-18B7 or 125 µCi 213Bi-18B7 or were left untreated. The surviving mice were sacrificed, and their lungs were homogenized and plated on Sabouraud's agar. Isolated colonies were grown overnight in Sabouraud's broth. To assess the radiosensitivity of the cells in vitro, C. neoformans cells from ATCC (CNnaive cells), C. neoformans cells recovered from untreated A/JCr mice (CNpassaged cells), and C. neoformans cells recovered from mice given 188Re-18B7 MAb (CNRe RIT cells) or 213Bi-18B7 MAb (CNBi RIT cells) were treated with 188Re-18B7 or 213Bi-18B7 MAb as previously described (2). Naive, passaged, or RIT-pretreated cells were equally radiosensitive to both 188Re and 213Bi (Fig. 1b and c).

To evaluate the possibility that RIT might select for C. neoformans cells resistant to radiation in vivo, we infected A/JCr mice with CNRe RIT, CNBi RIT, and CNnaive cells. Infected mice were treated with 150 µCi 188Re-18B7 or 125 µCi 213Bi-18B7 24 h post-i.v. infection and then monitored for survival and weight loss. We detected no differences in weight loss for mice infected with CNnaïve cells and mice infected with CNRe RIT or CNBi RIT cells. All groups lost weight after infection (Fig. 1d and e); however, mice receiving RIT with 213Bi-18B7 lost significantly less weight at the nadir (27 to 30 days) than untreated controls (P < 0.007 by Student's t test) (Fig. 1d). By contrast, the trend for groups treated with 188Re-18B7 was to lose more weight than untreated groups (P = 0.06) (Fig. 1e). RIT with 188Re-18B7 was more radiotoxic in mice with chronic C. neoformans lung infection than RIT with 213Bi-18B7 (7); the longer range of 188Re emissions in tissue may damage healthy tissues. Lethality in mice infected with CNRe RIT or CNBi RIT cells was the same as in mice infected with CNnaïve cells (P > 0.05) (Fig. 1f). The survival of mice treated with 213Bi-18B7 MAb was longer (P = 0.04) than of those treated with 188Re-18B7 (Fig. 1g), probably due to the higher killing power of alpha particles from 213Bi than of electrons from 188Re.

At 130 days postinfection, the lungs and brains from selected mice from each group were plated for CFU or analyzed histologically for signs of inflammation, possible radiation scarring (by using hematoxylin and eosin stain), and the presence of C. neoformans cells (by using Gomori methenamine-silver nitrate stain [GMS]). No striking difference between the groups was evident. The pathology in these chronically infected mice was generally focal and circumscribed, consisting of areas of lymphocytic and histiocytic infiltrates in areas containing cryptococcal cells (Fig. 2). Organ cultures from some mice from each treatment group had no CFU, consistent with the clearance of infection in both the brain and lung. Radiation fibrosis in the lungs was nonexistent (Fig. 2), consistent with previous observations (7).


Figure 2
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  		FIG. 2. Histology of brains and lungs from A/JCr mice infected i.v. with 5 x 104 C. neoformans cells and treated after 24 h with 125 µCi 213Bi-18B7 MAb. Mice were sacrificed 3 months posttreatment. (a, c, e, g, and h) Hematoxylin and eosin staining. (b, d, and f) GMS staining. (a and b) Lung from a 213Bi-18B7-treated CNnaive mouse, showing scattered alveolar macrophages with GMS-positive material within the cytoplasm (x200 magnification). The insert represents expansion of the boxed region. (c and d) Brain from a 213Bi-18B7-treated CNBi RIT mouse showing lymphohistiocytic meningitis at the base of the brain, with intralesional cryptococci (x200 magnification). The insert represents expansion of the boxed region. (e and f) Lungs from the same mouse as in panels c and d, showing a focal granuloma with central foamy macrophages which are encircled by lymphocytes. Macrophages contain GMS-positive organisms (x400 magnification). (g and h) Overview of the lungs (x25 magnification). (g) Lung from mouse infected with CNnaive and treated with 213Bi-18B7. (h) Lung from mouse infected with CNBi RIT and treated with 213Bi-18B7. All magnifications are original.

Treatment of C. neoformans with particulate radiation leads to the loss of clonogenicity (6, 2), which would explain the absence of radiation-resistant phenotypes after RIT. The residual cells which replicate after RIT most likely were protected from radiolabeled antibodies by a biofilm, an abscess, or a host cell. Like other antifungal therapies, RIT reduces the cryptococcal burden but does not eradicate infection. The efficacy of RIT might be enhanced by combining it with antifungal drugs or by repeated fractionated treatments. RIT provides a novel approach to antifungal therapy, potentially applicable to a wide spectrum of human mycoses.


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ACKNOWLEDGMENTS
 
E. Dadachova is a Sylvia and Robert S. Olnick Faculty Scholar in Cancer Research and is supported by NIH grant AI60507. A. Casadevall is supported by the following NIH grants: AI33774-11, HL59842-07, AI33142-11, AI52733-02, and GM 07142-01. A. Morgenstern and F. Bruchertseifer are supported by the European Commission.

Part of the results of this study were presented at the 108th ASM General Meeting, June 2008, Boston, MA.


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FOOTNOTES
 
* Corresponding author. Mailing address: Albert Einstein College of Medicine, Department of Nuclear Medicine, 1695A Eastchester Rd., Bronx, NY 10461. Phone: (718) 405-8485. Fax: (718) 405-8457. E-mail: edadacho{at}aecom.yu.edu Back

{triangledown} Published ahead of print on 12 January 2009. Back

# E. Dadachova and A. Casadevall share the senior authorship of the paper. Back


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Antimicrobial Agents and Chemotherapy, April 2009, p. 1679-1682, Vol. 53, No. 4
0066-4804/09/$08.00+0     doi:10.1128/AAC.01334-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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