Spore Dark Injection 8 Download
Hercules Montero
Human infections caused by the toxin-producing, anaerobic and spore-forming bacterium Paeniclostridium sordellii are associated with a treatment-refractory toxic shock syndrome (TSS). Reproductive-age women are at increased risk for P. sordellii infection (PSI) because this organism can cause intrauterine infection following childbirth, stillbirth, or abortion. PSI-induced TSS in this setting is nearly 100% fatal, and there are no effective treatments. TcsL, or lethal toxin, is the primary virulence factor in PSI and shares 70% sequence identity with Clostridioides difficile toxin B (TcdB). We therefore reasoned that a neutralizing monoclonal antibody (mAB) against TcdB might also provide protection against TcsL and PSI. We characterized two anti-TcdB mABs: PA41, which binds and prevents translocation of the TcdB glucosyltransferase domain into the cell, and CDB1, a biosimilar of bezlotoxumab, which prevents TcdB binding to a cell surface receptor. Both mABs could neutralize the cytotoxic activity of recombinant TcsL on Vero cells. To determine the efficacy of PA41 and CDB1 in vivo, we developed a transcervical inoculation method for modeling uterine PSI in mice. In the process, we discovered that the stage of the mouse reproductive cycle was a key variable in establishing symptoms of disease. By synchronizing the mice in diestrus with progesterone prior to transcervical inoculation with TcsL or vegetative P. sordellii, we observed highly reproducible intoxication and infection dynamics. PA41 showed efficacy in protecting against toxin in our transcervical in vivo model, but CDB1 did not. Furthermore, PA41 could provide protection following P. sordellii bacterial and spore infections, suggesting a path for further optimization and clinical translation in the effort to advance treatment options for PSI infection.
P. sordellii infection (PSI) in humans is rare but typically fatal. It is most frequently observed as a uterine infection in postpartum women following childbirth, stillbirth, or abortion. Once identified, antibiotics can be used to eradicate the bacteria, but these are not effective at neutralizing the secreted TcsL lethal toxin that can cause a treatment-refractory toxic shock syndrome. A neutralizing antibody against TcsL would address this problem in concept but has never been directly tested. TcsL shares high sequence identity with C. difficile TcdB, the primary virulence factor in C. difficile infection (CDI). We characterized two C. difficile TcdB antibodies, CDB1, a biosimilar to a clinically available drug, and PA41, in the neutralization capabilities of TcsL. In the process, we established a non-surgical, uterine infection model and made the discovery that disease symptoms varied with the reproductive cycle of the animals. This opens the door for new research questions at the interface of bacterial spore and toxin biology with reproductive health. While CDB1 did not provide protection against PSI in our animal model, PA41 did show protection. If developed for CDI, this antibody could have an added therapeutic utility in the life-threatening instances of human PSI.
Copyright: 2022 Bernard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Human infections caused by the toxin-producing, anaerobic and spore-forming bacterium Paeniclostridium sordellii are associated with a treatment-refractory toxic shock syndrome (TSS) and are typically lethal [1]. Reproductive-age women are at increased risk for P. sordellii infection (PSI) because this organism can cause intrauterine infection following childbirth or abortion [1]. Clinical indications of disease include a marked leukemoid reaction, i.e., a vast increase in white blood cells, increased vascular permeability, hemoconcentration, and, in most cases, the absence of a fever [1]. When women present with PSI-TSS, very little is known on how to treat the patient [1]. In most cases, a hysterectomy is performed along with definitive antibiotic therapy. However, even if antibiotics are successful in killing the bacteria, there are bacterial toxins that can continue circulating the body to cause disease.
P. sordellii secretes two cytotoxins that are similar in structure and function to toxins generated by the pathogen Clostridioides difficile: lethal toxin (TcsL), similar to C. difficile TcdB, sharing 76% sequence identity, and hemorrhagic toxin (TcsH), similar to C. difficile TcdA, sharing 78% sequence identity. Both TcsL and TcsH, like the C. difficile toxins, are glucosyltransferases that inactivate host GTPases. Some TcsL-positive isolates lack the gene encoding TcsH and are rapidly lethal in an animal model, indicating that TcsH is not essential for virulence [2]. Genetically derived TcsL-mutant strains were nonlethal in a mouse infection model giving evidence that TcsL is an essential virulence factor responsible for disease in PSI [3]. Neutralizing the cytopathic effect of TcsL might protect humans against toxic shock caused by TcsL-expressing P. sordellii.
Developing effective interventions against PSI (and TSS) is stymied by a lack of animal models and an incomplete understanding of how P. sordellii induces disease. Some investigators have used intraperitoneal injection of toxin [8,9], but this model is not optimal in terms of physiological relevance. To increase relevancy, a uterine mouse model was established to study PSI-associated TSS [10]. This intrauterine infection involves survival surgery to allow for ligation at the cervical junction and direct introduction of bacteria into the uterine lumen [10]. This model, however, provides additional pain and stress to the animals and increases the risk of infecting the blood stream directly. To address this problem, we developed an innovative mouse model system in which to study PSI using a transcervical (TC) inoculation method. Such a model allows for a non-surgical transfer of inoculum through the vaginal orifice, past the cervix, and directly into the uterus. This method eliminates the need for intensive survival surgery and produces a disease that more closely represents the nature of PSI in postnatal and post-abortive women.
To assess the effects of anti-TcdB antibodies PA41 and CDB1 on TcsL in vitro, cell neutralization assays were performed. First, Vero cells were treated with serial dilutions of TcsL in the presence and absence of 100 nM concentrations of the mABs for 72 hours and then assayed for ATP levels as an indicator of viability (Fig 1A). In panel B of Fig 1, we display the same data as a bar graph, indicating the concentrations of TcsL where we see a statistically significant difference. At 100 fM TcsL, PA41, but not CDB1, provided a statistically significant improvement in viability, although TcsL is not very toxic at this concentration. The cells, however, were very sensitive to 1 pM TcsL, and PA41 was able to completely neutralize the cytotoxic activity of the toxin. CDB1, also, was able to show statistically significant neutralization at this TcsL concentration, though to a lesser extent than PA41. Vero cells were not viable following intoxication with 10 pM and 100 pM TcsL alone. In the presence of PA41, cells were completely or partially viable when intoxicated with 10 pM or 100 pM TcsL, respectively. CDB1 was not able to protect at 10 pM or 100pM TcsL. Altogether, both antibodies neutralized the cytotoxicity of TcsL on Vero cells, though PA41 appeared more effective than CDB1 in all conditions. We also performed a dose titration of both antibodies at a cytotoxic dose of TcsL (1 pM) to get an understanding of their relative potencies (Fig 1C and 1D). Strong neutralization of TcsL was observed with 100 pM PA41. Following a titration of CDB1, we report a sharp reduction of antibody potency below 100 nM.
To assess the effects of PA41 and CDB1 on TcsL in vivo, it was first necessary to determine the lowest lethal intraperitoneal (IP) dose of TcsL. Female, 9-12-week-old, C57BL/6J mice were IP injected with 1 ng and 2.5 ng TcsL in 100 uL PBS (Fig 2A). In this study, 2.5 ng TcsL was the lowest lethal dose administered, with all animals succumbing to intoxication prior to 24 hours post administration. Consistent with previous findings [9], all mice intoxicated with this amount and higher of TcsL had a buildup of fluid in the thoracic and peritoneal cavities (S1 Fig). Mice intoxicated with 1 ng TcsL survived the study and showed no signs of disease.
Next, we wanted to perform a survival study with 2.5 ng TcsL IP alone or in the presence of PA41 or CDB1 (Fig 2B). Following our in vitro assays, we determined a 10000-fold excess (0.75mg/kg) of antibody to lethal TcsL (2.5ng) would be a reasonable dose to use in our in vivo IP intoxications. We found that PA41 was able to effectively neutralize TcsL, and all the animals survived the study with no signs of disease. CDB1 was not able to completely neutralize TcsL when given at 0.75 mg/kg, resulting in a survival of 66%. This result led us to increase the antibody dose ten-fold to 7.5 mg/kg to be used for all in vivo studies moving forward to give both antibodies the best opportunity for neutralization. To better understand how long the antibody circulates in the bloodstream following a single administration of PA41, we performed a three-day study where on Day 0, three animals were administered 7.5 mg/kg PA41. On days 1, 2, and 3, an animal was euthanized and the whole blood was collected. From western blot analysis of the serum using an anti-human Fab antibody, we could consistently observe PA41 in serum each day for up to three days without showing signs of depletion (S2 Fig).
From these in vivo studies, both C. difficile monoclonal antibodies were able to neutralize 2.5 ng TcsL following IP injection, with PA41 appearing more effective than CDB1. In addition, antibody administration protected mice from TcsL-induced pleural effusion.
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