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Single domain antibodies (sdAbs) have substantial biophysical advantages over conventional antibodies and are increasingly being employed as components of immunotherapeutic agents. One particularly favorable property is the ability to link different sdAbs into heteromultimers. This feature allows production of single molecules capable of simultaneously targeting more than one antigen. In particular, cooperative binding of multiple linked sdAbs to non-overlapping epitopes on the same target can produce synergistic improvements in target affinity, variant specificity, and in vivo potencies. Here we seek to test the option of increased component sdAbs in these heteromultimers by testing different sdAb heterohexamers in which each of the six camelid sdAb components (VHHs) can neutralize one of three different Botulinum neurotoxin (BoNT) serotypes, A, B or E. Each heterohexamer bound all three targeted BoNT serotypes and protected mice from at least 100 MIPLD50 of each serotype. To test the potential of mRNA therapeutics encoding long sdAb heteromultimers, one heterohexamer was encoded as replicating RNA (repRNA), formulated with a cationic nanocarrier, and delivered to mice via intramuscular injection. Heterohexamer antitoxin serum expression levels were easily detected by 8 hours post-treatment, peaked at 5-10 nM around two days, and persisted for more than three days. Mice treated with the formulated repRNA one day post-treatment survived challenge with 100 MIPLD50 of each toxin serotype, demonstrating the function of all six component VHHs. Use of long sdAb multimers, administered as proteins or repRNA, offer the potential for substantially improved versatility in the development of antibody-based therapeutics.

Botulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.

Leptospirosis is a zoonotic disease with worldwide distribution caused by pathogenic Leptospira spp. Pathogenic Leptospira spp. are shed in urine of infected hosts and transmitted via ingestion of contaminated food or water, inoculation, inhalation of aerosolized urine, and absorption through mucous membranes. Leptospirosis is of particular concern in tropical and subtropical regions such as Barranquilla, Colombia. Recent reports indicate that in Barranquilla, rodents, dogs, and humans have a high leptospiral seroprevalence; and amongst zoo mammals, nonhuman primates have a high prevalence of Leptospira spp. infection. We therefore sought to determine whether primates in captivity at the Barranquilla Zoo were exposed to Leptospira spp. and whether there was a probable causal transmission link between the primates and peridomestic rodents. Samples were collected from 29 captive nonhuman primates, 15 free-ranging rats (Rattus rattus), and 10 free-ranging squirrels (Sciurus granatensis). Serum samples from primates, rats, and squirrels were evaluated via microagglutination test (MAT) vs 24 reference Leptospira serovars. Blood and urine from the primates and kidney tissue from the rats and squirrels were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and polymerase chain reaction (PCR) of lipL32 was performed to determine whether active infection was present. Leptospiral seroprevalence was found to be 66.7% (10/15) in rats, 60% (6/10) in squirrels, and 6.9% (2/29) in neotropical primates. Ateles hybridus and Ateles fusciceps had positive titers to serogroups Cynopteri and Ictohaemorrhagiae, respectively. Of the rodents that had antibodies against Leptospira spp., 90% of the rats and 66.7% of the squirrels corresponded to the serovar australis. Interestingly, all animals were culture and PCR negative, indicating Leptospira spp. exposure in the absence of current infection. While their status as maintenance hosts needs to be investigated further, this is the first study to show leptospiral seropositivity in red-tailed squirrels (S. granatensis).

A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications., The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A.

The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA–lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long‐lasting serum antibody titers in vivo , thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA‐mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.

Influenza A virus (IAV) is known to circulate among human and animal reservoirs, yet there are few studies that address the potential for urban rodents to carry and shed IAV. Rodents are often used as influenza models in the lab, but the few field studies that have looked for evidence of IAV in rodents have done so primarily in rural areas following outbreaks of IAV in poultry. This study sought to assess the prevalence of IAV recovered from wild Norway rats in a dense urban location (Boston). To do this, we sampled the oronasal cavity, paws, and lungs of Norway rats trapped by the City of Boston’s Inspectional Services from December 2016 to September 2018. All samples were screened by real-time, reverse transcriptase PCR targeting the conserved IAV matrix segment. A total of 163 rats were trapped, 18 of which (11.04%) were RT-PCR positive for IAV in either oronasal swabs (9), paw swabs (9), both (2), or lung homogenates (2). A generalized linear model indicated that month and geographic location were correlated with IAV-positive PCR status of rats. A seasonal trend in IAV-PCR status was observed with the highest prevalence occurring in the winter months (December-January) followed by a decline over the course of the year, reaching its lowest prevalence in September. Sex and weight of rats were not significantly associated with IAV-PCR status, suggesting that rodent demography is not a primary driver of infection. This pilot study provides evidence of the need to further investigate the role that wild rats may play as reservoirs or mechanical vectors for IAV circulation in urban environments across seasons.

The backyard chicken (BYC) movement in the USA has increased human contact with poultry and subsequently, human contact with the pathogen Salmonella. However, to date, there have been few studies assessing prevalence of Salmonella in backyard flocks, despite the known public health risk this zoonotic bacterium poses. The objective of this study was to characterize human-BYC interactions and assess the prevalence of Salmonella among BYC flocks. We interviewed 50 BYC owners using a structured questionnaire to determine flock and household characteristics that facilitate contact with BYC and that may be associated with Salmonella in the BYC environment. Composite faecal material, cloacal swabs and dust samples from 53 flocks housed on 50 residential properties in the Greater Boston, Massachusetts area were tested for Salmonella using standard culture techniques and confirmed using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometer. Microbroth dilution and whole genome sequencing were used to determine phenotypic and genotypic resistance profiles, respectively, and sequence results were used to determine multilocus sequencing type. No owners self-reported a diagnosis of salmonellosis in the household. Over 75% of a subset of owners reported that they and their children consider BYC pets. This perception is evident in how owners reported interacting with their birds. Salmonella enterica subspecies enterica serotype Kentucky ST152 (serogroup C)-a strain not commonly associated with human infection-was confirmed in one flock, or 2% of tested flocks, and demonstrated resistance to tetracycline and streptomycin. We detected Salmonella at low prevalence in BYC. Further study of the health effects of exposure to zoonotic gastrointestinal pathogens such as Salmonella among families with BYC is warranted.

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only V H domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

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 (LD 50 ) 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 LD 50 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.

Background: Inflammatory airway disease (IAD) in horses, similar to asthma in humans, is a common cause of chronic poor respiratory health and exercise intolerance due to airway inflammation and exaggerated airway constrictive responses. Human rhinovirus is an important trigger for the development of asthma; a similar role for viral respiratory disease in equine IAD has not been established yet. Methods: In a case–control study, horses with IAD (n = 24) were compared to control animals from comparable stabling environments (n = 14). Horses were classified using pulmonary function testing and bronchoalveolar lavage. PCR for equine rhinitis virus A and B (ERAV, ERBV), influenza virus (EIV), and herpesviruses 2, 4, and 5 (EHV-2, EHV-4, EHV-5) was performed on nasal swab, buffy coat from whole blood, and cells from BAL fluid (BALF), and serology were performed. Categorical variables were compared between IAD and control using Fisher’s exact test; continuous variables were compared with an independent t-test. For all analyses, a value of P

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2−/−) and the parental strain. Parental mice lived significantly longer than NOS2−/− mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2−/− and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2−/− mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2−/− mice resulted in significantly higher levels of IFN-γ, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1α than parental mice. Ab administration had different effects on infected NOS2−/− and parental mice with respect to IFN-γ, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1α levels. Ab administration increased lung levels of IFN-γ in parental mice and reduced levels in NOS2−/− mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.

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.

Epitope specificity and isotype influence mAb efficacy against Cryptococcus neoformans; however, the relative contribution of each attribute is poorly understood. To date, only mAbs that recognize two epitopes of capsular glucuronoxylomannan (GXM), defined by the IgG1 mAbs 2H1 and E1, consistently mediate protection against C. neoformans. The role of epitope specificity was further examined using six additional IgG1 mAbs and serotype D C. neoformans ATCC 24067. mAbs 3C2, 439, and 471 recognize the 2H1 epitope, whereas mAbs 339, 1255, and 302 recognize two separate epitopes. mAbs 3C2, 439, and 471 competed for GXM with the IgA mAb 18G9, a 2H1 mAb family member, whereas mAbs 302, 339, and 1255 did not. Each mAb bound GXM similarly, as determined by agglutination, direct Ag binding, Ag inhibition, and indirect capsular immunofluorescence assays. mAb apparent affinity constants for GXM ranged from 5 to 26 × 107 M−1 with mAb 1255 > 3C2 > 339 > 439 > 471 > 302. Each mAb significantly prolonged survival (p < 0.05); the average survival times of control and mice passively immunized with mAbs 3C2, 302, 339, 439, 471, and 1255 were 10.8, 36.6, 33, 25.5, 24.9, 17, and 22.6 days, respectively. Although each mAb enhanced J774.16 cell fungicidal activity, differences were observed in the ability of each mAb to facilitate attachment and ingestion of cryptococci. These results indicate 1) two additional epitope specificities associated with mAb efficacy, 2) differences in opsonic and protective efficacy for IgG1 anti-GXM mAbs, 3) an association between affinity and protective efficacy, and 4) additional support for association between an annular indirect capsular immunofluorescence pattern and mAb efficacy.

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) V H (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.

Two immunoglobulin M monoclonal antibodies (mAbs) derived from the same B cell recognize different epitopes on the capsular polysaccharide of the pathogenic yeast, Cryptococcus neoformans. Their respective epitopes are located in spatially distinct regions of the capsule. Passive administration of one mAb prolonged survival whereas the other mAb did not. The results indicate that specificity is an important determinant of antibody efficacy against C. neoformans and that somatic mutations occurring during the antibody response can affect the protective efficacy of antibodies to C. neoformans.

Clostridium difficile, an anaerobic, spore-forming, gram-positive bacillus, is the leading cause of antibiotic-associated diarrhea in developing countries. A hypervirulent, epidemic strain, designated NAP1/027/BI, has caused an increase in C. difficile infection (CDI) case and fatality rates since the early 2000s [1, 2]. The symptoms of CDI, ranging from mild diarrhea to severe disease including pseudomembranous colitis, toxic megacolon, sepsis, shock, and even death, are thought to be caused mainly by 2 toxins—toxin A (TcdA) and toxin B (TcdB). Understanding the relative roles of these 2 toxins in disease pathogenesis is of great importance. Although the toxins have been investigated using numerous methods over the past several decades, the results have been conflicting. In earlier studies using culture supernatants or purified toxins in rodent or rabbit models, TcdA and/or TcdB were administered into the gastrointestinal (GI) tract directly via gavage or intestinal loop. Results indicated that TcdA was the essential virulence factor, with TcdB unable to induce lesions when administered in the absence of TcdA [3, 4]. When highly purified toxins are administered systemically to rodents, either together or separately, both are able to induce disease and death [5]. The finding of cardiotoxicity associated with TcdB in the zebrafish model more recently highlighted the potential for systemic actions of the toxins and the importance of TcdB, in particular [6], although this has yet to apply to mammalian species. Active and passive immunity have also been used to investigate the roles of the 2 toxins. Using toxoid for immunization in hamsters, 1 study found that immunization with both TcdA and TcdB toxoids was necessary for complete protection [7], but another study found that immunization with only TcdA toxoid was necessary for protection [8]. Investigation of naturally occurring serum antitoxin antibodies in human patients placed an emphasis on the importance of anti-TcdA in preventing recurrence of CDI [9]. Human monoclonal antibodies (HuMab) have been generated against both toxins, and studies in hamsters revealed that HuMab against both toxins prevented CDI in hamsters [10]. Likewise, studies in human patients also highlighted the importance of antibodies against both toxins, not just TcdA, for protection from recurrence [11]. In the past several years, methods for genetic manipulation of C. difficile have allowed for creation of isogenic mutant strains that produce only TcdA or TcdB. One such study showed TcdB to be the essential virulence factor in the hamster model [12], but another study found that both TcdA and TcdB are important for virulence [13]. The results of these numerous studies indicate that neither toxin can be ignored in terms of the importance in disease pathogenesis or for development of novel treatments and therapeutics. However, we are still left with many unanswered questions. In this study we used HuMabs [10] and alpaca polyclonal antibodies against TcdA and/or TcdB in the piglet model of CDI [14] to further investigate the roles of the 2 toxins in pathogenesis and to provide more information on the use of antitoxin antibodies for prevention and treatment of CDI in human patients.

Background 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. Results 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. Conclusions 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.

An IgG1 monoclonal antibody generated from a mouse infected with Cryptococcus neoformans modified the course of intravenous cryptococcal infection in A/J mice by prolonging survival and reducing lung fungal burden, brain mass, and serum polysaccharide levels. The results demonstrate that C. neoformans infection can elicit useful antibodies.Key words: Cryptococcus neoformans, monoclonal antibody, antibody immunity.

Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis.

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.

Isotype switching is a process by which immunoglobulin variable gene regions initially proximal to and expressed with the mu constant region gene (C mu) can rearrange downstream to other constant region genes. Thus the same antigen binding site can be expressed with each of the other constant region isotypes and perform the full panoply of effector functions. Although isotype switching is thought to involve highly reiterated 'switch site' sequences located 5' to constant region genes, the exact role of these switch sites is unknown. It has been reported that prior to switching, the 'donor' switch site 5' of C mu occasionally undergoes deletions, but it is not known whether this is an adventitious event or one which predisposes to or prevents isotype switching. Since T cell independent (TI) immune responses are dominated by IgM while T cell dependent (TD) responses are associated with switching to IgG, we have examined the state of the mu switch site in 51 IgM-producing hybridomas isolated from a variety of TI and TD responses. Although more hybridomas from the TI responses studied exhibited S mu deletions, deletion of S mu also occurred in hybridomas isolated from TD responses. Analysis of a well-characterized clonally related subset of IgM hybridomas also revealed that mu switch region deletion can be associated with the productive allele.

The molecular characteristics of the humoral immune response to a serotype A Cryptococcus neoformans infection were compared with the response elicited by a cryptococcal glucuronoxylomannan-tetanus toxoid (GXM-TT) conjugate. Anticryptococcal monoclonal antibodies (mAbs) isolated from both responses have previously been shown to recognize the same antigenic determinant of cryptococcal GXM. Southern blot and sequence analyses indicate that the hybridomas isolated from each response arose from only a few precursor B cells. All the mAbs generated from the infected and GXM-TT conjugate-immunized mice utilize the same VH7183 family member: JH2/JH4, v kappa 5.1, and J kappa 1; mAbs generated by different B cells had complementarity-determining region 3's (CDR3s) composed of seven amino acids with a common sequence motif. Thus, the molecular analysis of these anticryptococcal mAb-producing hybridomas indicated that the response to both cryptococcal infection and conjugate immunization was oligoclonal and highly restricted with regard to immunoglobulin gene utilization. The GXM-TT conjugate primarily stimulated isotype switching and clonal proliferation, and did not result in hybridomas expressing additional immunoglobulin repertoires. The mAbs from both responses had a number of replacement mutations at the 5' end of CDR2 that appear to be the result of antigen-driven selection. Somatic mutation also resulted in altered epitope specificity for one mAb, 13F1. Passive administration of representative mAbs from different clones generated in response to the GXM-TT conjugate prolonged survival of lethally infected mice.

This study was designed to develop a simple, noninvasive method for saliva collection: a first step toward developing new diagnostic tests to survey gorillas for infectious diseases. The subjects included free-ranging mountain gorillas (Gorilla beringei beringei) in the Parc National des Volcans, Rwanda, and a group of orphan mountain and Grauer's gorillas (Gorilla heringei graueri) housed nearby in a temporary holding facility. Three collection methods were used to recover saliva from discarded forest food: swabbing, soaking, and washing. Saliva was also collected from orphan gorillas maintained in a captive setting by using dental ropes inside mesh bags. The presence of gorilla saliva in each sample was confirmed by using a salivary s-amylase assay and forensic press test paper. The recovery of gorilla DNA was verified by polymerase chain reaction by using primers specific to mountain and Grauer's gorillas. Of the three collection techniques used to recover saliva from forest food, directly swabbing plant bite marks was the most effective. Wild celery (Peucedanum linderi) provided for the most consistent saliva recovery and is eaten year round by mountain gorillas in Rwanda. This study shows that gorilla saliva can be recovered easily and noninvasively from known individual free-ranging gorillas by collecting pieces of wild celery discarded as the gorillas forage and from captive gorillas by offering them juice-soaked dental ropes inside mesh bags. Both methods can be used to recover gorilla DNA for genetic studies. Saliva collected from free-ranging and captive gorillas may prove to be a useful biologic sample for the development of new diagnostic tests and hormonal analysis.

Several murine monoclonal antibodies (MAbs) specific for the capsular glucuronoxylomannan of Cryptococcus neoformans were studied for their capacity to confer protection when passively administered to lethally infected mice. The MAb group studied recognized at least three distinct epitopes and included immunoglobulin M (IgM), IgG3, IgG1, and IgA isotypes. The protection model used A/J and BALB/c mice infected intraperitoneally with 10(8) cryptococci. The MAbs were administered either immediately preceding or, in one experiment, 24 to 48 h prior to infection. Protective efficacy was assessed by the ability of passively administered MAbs to prolong the survival of lethally infected mice. Three IgM MAbs, each of which recognized a distinct epitope, were able to prolong survival of lethally infected mice to different extents. A set of IgM, IgG3, IgG1 and IgA MAbs which utilize the same immunoglobulin gene elements and were derived from the same B-cell clone exhibited significant class differences in protective efficacy with IgA, IgG1 > IgM > IgG3. The results confirm that protective MAbs against C. neoformans capsular polysaccharide exist and strongly suggest that both epitope specificity and isotype are important determinants of protective efficacy.

The antibody responses of BALB/c mice to serotype A Cryptococcus neoformans capsular polysaccharide (CNPS) were compared after cryptococcal infection and immunization with a serotype A glucuronoxylomannan-tetanus toxoid conjugate (GXM-TT). Infection rarely resulted in a rise of serum antibody titer to CNPS. In contrast, mice immunized with GXM-TT produced serum IgM and IgG to CNPS. Six IgM and one IgG1 monoclonal antibodies (MAbs) were generated from the spleen of one infected mouse. Nine IgM, 1 IgG3, 16 IgG1, and 7 IgA MAbs were generated from the spleen of one GXM-TT-immunized mouse. All MAbs generated from both mice bound to the GXM fraction of the capsular polysaccharide. For some MAbs, de-O-acetylation of serotype A GXM abolished or greatly reduced MAb binding compared with the native GXM. All MAbs reacted with CNPS from C. neoformans serotypes A-D. MAbs generated from the infected mouse competitively inhibited the binding of MAbs generated from the GXM-TT-immunized mouse. These results indicate that some antibodies elicited by infection with C. neoformans or by immunization with GXM-TT bind to the same antigenic determinant in the GXM.

Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producingEscherichia 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.

Cryptococcus neoformans causes life-threatening infections in immunocompromised patients, including those with AIDS and hematological malignancies (28). Cryptococcal infection in individuals with impaired immunity has high mortality, and those who survive the acute infection often have chronic infections. Given that the therapy of cryptococcosis is unsatisfactory, there is interest in vaccine development (14). Control of C. neoformans infection is associated with cell-mediated immunity (28) and granuloma formation (23). However, there is strong evidence that humoral immunity can also be important. Antibody to the capsular glucuronoxylomannan (GXM) can prolong survival, reduce organ fungal burden, enhance granulomatous inflammation, and clear capsular polysaccharide antigen in infected mice (reviewed in references 3, 21, and 36). This suggests that vaccines which elicit high titers of protective antibodies may be useful for prevention of C. neoformans infection. GXM alone is unlikely to be an effective vaccine because it is poorly immunogenic and can be immunosuppressive (10, 26, 34). Conjugation of GXM to a protein carrier produces a highly immunogenic vaccine (6, 12, 13, 42). Mice immunized with a GXM-tetanus toxoid (GXM-TT) vaccine live longer and have lower CFU counts than controls when challenged with C. neoformans infection (12). Molecular and idiotypic analyses of antibodies to GXM produced in response to infection or GXM-TT immunization revealed restriction in variable gene usage (5, 8, 29). Murine monoclonal antibodies (MAbs) to GXM can be classified into five groups based on molecular and idiotypic characteristics (5). Group II MAbs include several protective antibodies and are defined by a heavy-chain V (variable)-region using a VH from the 7183 gene family, a seven-amino-acid D/N segment which results in a fixed-length third complementarity-determining (CDR3) region, a light-chain V region using Vκ5.1, and reactivity with the 2H1 idiotype (Id) (5). The 2H1 Id is expressed by MAb 2H1, which is the prototypical group II antibody to GXM (5). MAb 2H1 has been demonstrated to protect against C. neoformans in murine intravenous (33), intracerebral (31), intraperitoneal (i.p.) (32), and intratracheal (20) infection. The crystal structure of MAb 2H1 has been solved with and without peptide mimetics (43). A MAb with a structure similar to that of 2H1 is in advanced preclinical development for use as adjunctive therapy in cryptococcal infections (4). However, expression of 2H1 Id is not sufficient for protection since antibodies with the same V-region usage manifest large differences in protective efficacy based on isotype (44) and fine specificity (30). The molecular and cellular mechanisms which produce V-region-restricted responses are poorly understood. Here we studied the antibody response to a GXM conjugate vaccine in genetically different strains of mice, including three autoimmune strains. Our initial goal was to study the extent of V-region restriction in GXM-binding antibodies and generate MAbs different in specificities and molecular structure from those already available. We hypothesized that if restriction was the result of clonal deletion of self-reactive antibodies, then autoimmune mice would have a less restricted antibody response. Furthermore, we hypothesized that antibody responses in genetically different mouse strains might be quite different. This appeared to be confirmed by preliminary experiments with anti-idiotypic reagents to the 2H1 Id. However, molecular analysis of hybridomas from mice with 2H1 Id-negative (Id−) responses does not support either hypothesis since all mice studied made antibodies to GXM which are structurally similar to MAb 2H1. Instead, our findings suggest that V-region restriction in GXM-binding antibodies is a result of constrained requirements for antigen binding.

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.

The efficacy of combination therapy with 5-flucytosine and an IgG1 monoclonal antibody to Cryptococcus neoformans capsular glucuronoxylomannan was studied in vitro with J774. 16 murine macrophage-like cells and in vivo in murine cryptococcal infection. The combination of 5-flucytosine and IgG1 was more effective in reducing the numbers of C. neoformans colony-forming units in vitro and in vivo than either agent alone.

The efficacy of monoclonal antibody (MAb 2H1) against diverse strains of Cryptococcus neoformans was studied in a murine model of intravenous infection. For six of eight strains, administration of MAb prior to infection prolonged survival of mice. For two strains, 371 and SB4A, administration of MAb prior to infection did not prolong survival in multiple experiments with inocula ranging from 10(2) to 10(6) yeast cells per mouse. Mice infected with strains 371 and SB4A had fewer CFU than non-MAb-treated controls, but the CFU reduction was not sufficient to affect survival. Serum glucuronoxylomannan (GXM) levels varied for the different C. neoformans strains. For mice that did not receive MAb 2H1, there was a positive correlation between lung fungal burden and serum GXM levels. MAb 2H1-treated mice had significantly reduced serum GXM levels. The results indicate that the efficacy of MAb 2H1 administration in prolonging survival and/or reducing organ CFU varies with the C. neoformans strain.