Loss of Allergen 1 Confers a Hypervirulent Phenotype That Resembles Mucoid Switch Variants of Cryptococcus neoformans

ABSTRACT Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Δ gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Δ cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Δ mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.

R5X4 Viruses Are Evolutionary, Functional, and Antigenic Intermediates in the Pathway of a Simian-Human Immunodeficiency Virus Coreceptor Switch

ABSTRACT To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIVSF162P3N-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIVSF162P3N and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.

Induction of tumor cell death by anti-GD2 monoclonal antibodies (MoAb): Requirement of antibody Fc and a long residence time (slow koff)

13507 Background: Anti-GD2 MoAbs have shown clinical activities against neuroblastoma (NB) and other neuroectodermal tumors. Besides complement mediated (CMC) and antibody-dependent cell mediated cytotoxicities (ADCC), they can directly induce cell death. However, MoAb and their genetic constructs differ substantially in death-inducing efficiency. Although multivalent binding is important for these functions, the roles of association on rate (kon), dissociation off rate (koff), valency and Fc have not been carefully defined. Methods: We tested a panel of purified anti-GD2 MoAbs, MoAb fragment, and single chain variable fragment (scFv) fusion protein. The kinetics of in vitro binding was measured by surface plasmon resonance (SPR) using BIACORE 2000. NB cell lines (LAN1, NMB7, BE(2)C, SKNER, SKNJD and SKNLP) were treated with MoAb in vitro, their survivals were studied by the chromogenic assay and IC50 derived using Sigmaplot. Results: The most efficient cell kill against NB was 3F8 (mIgG3) with an average IC50 of 4.7 ug/ml. In contrast, IC50 was >30 ug/ml for 14.G2a (mIgG2a), ME361 (mIgG2a) and S220–51 (mIgG3). It was >100 ug/ml for 3F8 class-switch variant 3G6 (mIgM), 3F8-F(ab’)2(fragment), and 5F11scFv-streptavidin (tetramer). By SPR, the apparent affinity Kd (equilibrium dissociation constant) of 3F8 [1.1x10-8M] was higher than that of 3F8-F(ab’)2 [3.3x10-8M], 14.G2a [9.0x10- 8M], ME361 [4.6x10-7M], and S220–51 [4.6x10-6 M], but lower than that of decameric IgM 3G6 [6.5x10- 9 M] and tetrameric 5F11scFv-streptavidin [2.6x10-9M]. The affinity advantage of 3F8 over other bivalent MoAbs was primarily due to its slower koff [3.0x10-4 sec-1] when compared to other MoAbs [1.1x10- 3-7.3x10-2 sec-1]. Conclusions: Different structural forms of anti-GD2 MoAb have distinct efficiency in inducing NB cell death as well as binding affinity to GD2. Since these properties can affect CMC and ADCC, they may in turn influence the clinical efficacy of immunotherapy. Our findings suggest the importance of an intact IgG3 molecule (the presence of Fc), as well as a long residence time (slow koff). No significant financial relationships to disclose.

Passive Intranasal Monoclonal Antibody Prophylaxis against Murine Pneumocystis carinii Pneumonia

ABSTRACT Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.

High Pathogenic Potential of Low-Affinity Autoantibodies in Experimental Autoimmune Hemolytic Anemia

To assess the potency of low-affinity anti–red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti–mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a–injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.