Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.
Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) VH (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the “effector Ab,” that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer—one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH—was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.
Background. A dramatic increase in morbidity and mortality from Clostridium difficile infection (CDI) due to the recent emergence of virulent, antibiotic-resistant strains has led to a search for alternatives to antibiotics, including vaccines and immune-based therapy that target the 2 key toxins—TcdA and TcdB.
Methods. We investigated the efficacy of specific human monoclonal antibodies (HuMab) and alpaca polyclonal antibodies against each toxin separately and in combination in the gnotobiotic piglet model of CDI. Additionally, the HuMab and polyclonal antibodies were exploited to investigate the precise contribution of each toxin to systemic and/or gastrointestinal (GI) tract disease.
Results. Our results indicate that TcdB is an important virulence factor associated with GI and systemic pathology. Administration of anti-TcdB antibody alone or with anti-TcdA protected 100% of piglets from development of systemic CDI and minimized GI lesions. Conversely, 100% of piglets administered only anti-TcdA developed severe GI and systemic disease, with 67%–83% fatality, faring worse than placebo-treated control animals.
Conclusions. These results highlight the importance of TcdB in the pathogenesis of CDI and the effectiveness of TcdB-specific antibody in treating CDI. However, the results raise new questions regarding the nature of TcdA interaction with therapeutic antibodies.
Clostridium difficile; anti-toxin antibodies; TcdA; TcdB; systemic disease
Botulinum neurotoxin (BoNT) is responsible for causing botulism, a potentially fatal disease characterized by paralysis of skeletal muscle. Existing specific treatments include polyclonal antisera derived from immunized humans or horses. Both preparations have similar drawbacks, including limited supply, risk of adverse effects and batch to batch variation. Here, we describe a panel of six highly protective sheep monoclonal antibodies (SMAbs) derived from sheep immunized with BoNT/A1 toxoid (SMAbs 2G11, 4F7) or BoNT/A1 heavy chain C-terminus (HcC) (SMAbs 1G4, 5E2, 5F7, 16F9) with or without subsequent challenge immunization with BoNT/A1 toxin. Although each SMAb bound BoNT/A1 toxin, differences in specificity for native and recombinant constituents of BoNT/A1 were observed. Structural differences were suggested by pI (5E2 = 8.2; 2G11 = 7.1; 4F7 = 8.8; 1G4 = 7.4; 5F7 = 8.0; 16F9 = 5.1). SMAb protective efficacy vs. 10,000 LD50 BoNT/A1 was evaluated using the mouse lethality assay. Although not protective alone, divalent and trivalent combinations of SMabs, IG4, 5F7 and/or 16F9 were highly protective. Divalent combinations containing 0.5–4 μg/SMAb (1–8 μg total SMAb) were 100% protective against death with only mild signs of botulism observed; relative efficacy of each combination was 1G4 + 5F7 > 1G4 + 16F9 >> 5F7 + 16F9. The trivalent combination of 1G4 + 5F7 + 16F9 at 0.25 μg/SMAb (0.75 μg total SMAb) was 100% protective against clinical signs and death. These results reflect levels of protective potency not reported previously.
botulinum neurotoxin; botulinum toxin; BoNT; BoNT/A1; BoNT/A2; monoclonal antibodies; sheep monoclonal antibodies; immunotherapy; passive immunization; botulism
Hemolytic uremic syndrome (HUS) leading to acute kidney failure, is a condition linked to the production of primarily Shiga toxin 2 (Stx2) by some E. coli serotypes. We have previously shown that Stx2 A subunit-specific human monoclonal antibody (HuMAb) 5C12, and B subunit-specific HuMAb 5H8 inhibit cultured cell death, and protect mice and piglets from fatal Stx2-intoxication. We have also shown that 5H8 blocks binding of Stx2 to its cell-surface receptor globotriaosyl ceramide (Gb3), whereas Stx2 when complexed with 5C12 binds Gb3 with higher affinity than Stx2. The mechanism by which 5C12 neutralizes Stx2 in vitro involves trapping of Stx2 in the recycling endosomes and releasing it into the extracellular environment. Because of the clinical implications associated with the formation of Stx2/antibody complexes and the potential for trapping and clearance through a severely damaged kidney associated with HUS, we investigated the likely site(s) of Stx2/antibody localization and clearance in intoxicated mice treated with antibody or placebo.
Mice were injected with radiolabeled Stx2 (125I-Stx2) 4 hours after administration of 5C12, 5H8, or phosphate buffered saline (PBS) and the sites of localization of labeled Stx2, were investigated 3, 24 and 48 hours later. The liver recorded statistically much higher concentrations of labeled Stx2 for groups receiving 5C12 and 5H8 antibodies after 3, 24 and 48 hours, as compared with the PBS group. In contrast, highest levels of labeled Stx2 were detected in the kidneys of the PBS group at all 3 sampling times. Mice receiving either of the two HuMAbs were fully protected against the lethal effect of Stx2, as compared with the fatal outcome of the control group.
The results suggest that HuMAbs 5C12 and 5H8 promoted hepatic accumulation and presumably clearance of toxin/antibody complexes, significantly diverting Stx2 localization in the kidneys, the target of Stx2 and the cause of HUS. This is in contrast to the fatal outcome of the control group receiving PBS. The results also confirm earlier observations that both HuMAbs are highly and equally protective against Stx2 intoxication in mice.
Shiga toxin; Radiolabel; Antibody; Toxin elimination; Toxin concentration; Pharmacokinetic; Human monoclonal antibody
Upon binding to intestinal epithelial cells, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector translocated intimin receptor (Tir), which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium intimin are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.
Citrobacter rodentium; intimin; enterohemorrhagic Escherichia coli; invasin; enteropathogenic Escherichia coli
Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.
Upon intestinal colonization, enterohemorrhagic Escherichia coli (EHEC) induces epithelial cells to generate actin “pedestals” beneath bound bacteria, lesions that promote colonization. To induce pedestals, EHEC utilizes a type III secretion system to translocate into the mammalian cell bacterial effectors such as translocated intimin receptor (Tir), which localizes in the mammalian cell membrane and functions as a receptor for the bacterial outer membrane protein intimin. Whereas EHEC triggers efficient pedestal formation during mammalian infection, EHEC cultured in vitro induces pedestals on cell monolayers with relatively low efficiency. To determine whether growth within the mammalian host enhances EHEC pedestal formation, we compared in vitro-cultivated bacteria with EHEC directly isolated from infected piglets. Mammalian adaptation by EHEC was associated with a dramatic increase in the efficiency of cell attachment and pedestal formation. The amounts of intimin and Tir were significantly higher in host-adapted than in in vitro-cultivated bacteria, but increasing intimin or Tir expression, or artificially increasing the level of bacterial attachment to mammalian cells, did not enhance pedestal formation by in vitro-cultivated EHEC. Instead, a functional assay suggested that host-adapted EHEC translocate Tir much more efficiently than does in vitro-cultivated bacteria. These data suggest that adaptation of EHEC to the mammalian intestine enhances bacterial cell attachment, expression of intimin and Tir, and translocation of effectors that promote actin signaling.
host adaptation; actin assembly; translocation; EHEC; intimin; Tir
Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD50) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD50 of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.
Infection of children with Shiga toxin (Stx)-producing Escherichia coli (STEC) can lead to hemolytic-uremic syndrome (HUS) in 5 to 10% of patients. Stx2, one of two toxins liberated by the bacterium, is directly linked with HUS. We have previously shown that Stx-specific human monoclonal antibodies protect STEC-infected animals from fatal systemic complications. The present study defines the protective antibody dose in relation to the time of treatment after the onset of diarrhea in infected gnotobiotic piglets. Using the mouse toxicity model, we selected 5C12, an antibody specific for the A subunit, as the most effective Stx2 antibody for further characterization in the piglet model in which piglets developed diarrhea 16 to 40 h after bacterial challenge, followed by fatal neurological symptoms at 48 to 96 h. Seven groups of piglets received doses of 5C12 ranging from 6.0 mg/kg to 0.05 mg/kg of body weight, administered parenterally 48 h after bacterial challenge. The minimum fully protective antibody dose was 0.4 mg/kg, and the corresponding serum antibody concentration in these piglets was 0.7 μg (±0.5)/ml, measured 7 to 14 days after administration. Of 40 infected animals which received Stx2 antibody treatment of ≥0.4 mg/kg, 34 (85%) survived, while only 1 (2.5%) of 39 placebo-treated animals survived. We conclude that the administration of the Stx2-specific antibody was protective against fatal systemic complications even when it was administered well after the onset of diarrhea. These findings suggest that children treated with this antibody, even after the onset of bloody diarrhea, may be equally protected against the risk of developing HUS.
Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(κ) [IgM(κ)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(κ) elements specific for subunit B) were isolated. Two IgM(κ) Hu-MAbs (2D9 and 15G9) and three IgG1(κ) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.
Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and κ light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1κ A-subunit-specific and 3 IgG1κ and 1 IgG3κ B-subunit-specific antibodies. Six IgG1κ A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1κ B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of >95% activity of 1 ng of Stx2 in the presence of 0.04 μg of HuMAb in vitro and significant prolongation of survival of mice given 50 μg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.
Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene (URE1) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1, and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.
The antibody response to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) in BALB/c mice frequently expresses the 2H1 idiotype (Id) and is restricted in variable gene usage. This study examined the immunogenicity of GXM-protein conjugates, V (variable)-region usage, and 2H1 Id expression in seven mouse strains: BALB/c, C57BL/6, A/J, C3H, NZB, NZW, and (NZB × NZW)F1 (NZB/W). All mouse strains responded to vaccination with GXM conjugated to tetanus toxoid (TT), the relative magnitude of the antibody response being BALB/c ∼ C3H > C57BL/6 ∼ NZB ∼ NZW ∼ NZB/W > A/J. Analysis of serum antibody responses to GXM with polyclonal and monoclonal antibodies to the 2H1 Id revealed significant inter- and intrastrain differences in idiotype expression. Thirteen monoclonal antibodies (MAbs) (two immunoglobulin M [IgM], three IgG3, one IgG1, three IgG2a, two IgG2b, and two IgA) to GXM were generated from one NZB/W mouse and one C3H/He mouse. The MAbs from the NZB/W mouse were all 2H1 Id positive (Id+) and structurally similar to those previously generated in BALB/c mice, including the usage of a VH from the 7183 family and Vκ5.1. Administration of both 2H1 Id+ and Id− MAbs from NZB/W and C3H/H3 mice prolonged survival in a mouse model of cryptococcosis. Our results demonstrate (i) that V-region restriction as indicated by the 2H1 Id is a feature of both primary and secondary responses of several mouse strains; and (ii) that there is conservation of V-region usage and length of the third complementarity-determining region in antibodies from three mouse strains. The results suggest that V-region restriction is a result of antibody structural requirements necessary for binding an immunodominant antigen in GXM.
Hemolytic-uremic syndrome (HUS) is a serious disease in children, attributable in the majority of cases to infection with Shiga toxin (Stx)-producing Escherichia coli. Using gnotobiotic piglets orally infected with E. coli O157:H7, which develop Stx-related cerebellar lesions and fatal neurological symptoms, we show that administration of Stx2-specific antiserum well after challenge protected, in a dose-response fashion, against these symptoms for at least 24 h after bacterial challenge. Twenty-six of 30 piglets given Stx2 antiserum survived the challenge, compared to only 4 of 16 animals given control serum or saline. Given our observations in piglets, Stx antibody of human origin may likewise prevent HUS in children.
The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(κ)] is in preclinical development for treatment of Cryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.
Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.