Serum antibody profiling identifies vaccine-induced correlates of protection against aerosolized ricin toxin in rhesus macaques.

Inhalation of the biothreat agent, ricin toxin (RT), provokes a localized inflammatory response associated with pulmonary congestion, edema, neutrophil infiltration, and severe acute respiratory distress. The extreme toxicity of RT is the result of the toxin's B chain (RTB) promoting rapid uptake into alveolar macrophages and lung epithelial cells, coupled with the A chain's (RTA) potent ribosome-inactivating properties. We previously reported that intramuscular vaccination of rhesus macaques with a lyophilized, alum-adsorbed recombinant RTA subunit vaccine (RiVax®) was sufficient to confer protection against a lethal dose of aerosolized RT. That study implicated RT-specific serum IgG, toxin-neutralizing activity (TNA), and epitope-specific responses as being associated with immunity. However, it was not possible to define actual correlates of protection (COP) because all vaccinated animals survived the RT challenge. We addressed the issue of COP in the current study, by vaccinating groups of rhesus macaques with RiVax® following the previously determined protective regimen (100 µg on study days 0, 30 and 60) or one of two anticipated suboptimal regimens (100 µg on study days 30 and 60; 35 µg on study days 0, 30, and 60). Two unvaccinated animals served as controls. The animals were challenged with ~5 × LD50s of aerosolized RT on study day 110. We report that all vaccinated animals seroconverted prior to RT challenge, with the majority also having measurable TNA, although neither antibody levels nor TNA reached statistical significance with regard to a correlation with protection. By contrast, survival correlated with pre-challenge, epitope-specific serum IgG levels, derived from a competitive sandwich ELISA using a panel of toxin-neutralizing monoclonal antibodies directed against distinct epitopes on RiVax®. The identification of a species-neutral, competitive ELISA that correlates with vaccine-induced protection against RT in nonhuman represents an important advance in the development of medical countermeasures (MCM) against a persistent biothreat.

Utility of Newborn Dried Blood Spots to Ascertain Seroprevalence of SARS-CoV-2 Antibodies Among Individuals Giving Birth in New York State, November 2019 to November 2021.

Importance: Serosurveys can be used to monitor population-level dynamics of COVID-19 and vaccination. Dried blood spots (DBSs) collected from infants contain maternal IgG antibodies and are useful for serosurveys of individuals recently giving birth. Objectives: To examine SARS-CoV-2 antibody prevalence in pregnant individuals in New York State, identify associations between SARS-CoV-2 antibody status and maternal and infant characteristics, and detect COVID-19 vaccination among this population. Design, Setting, and Participants: A population-based, repeated cross-sectional study was conducted to detect SARS-CoV-2 nucleocapsid (N) and spike (S) IgG antibodies. Deidentified DBS samples and data submitted to the New York State Newborn Screening Program between November 1, 2019, and November 30, 2021, were analyzed. Exposures: Prenatal exposure to SARS-CoV-2 antibodies. Main Outcomes and Measures: The presence of IgG antibodies to SARS-CoV-2 N and S antigens was measured using a microsphere immunoassay. Data were analyzed by geographic region and compared with reported COVID-19 cases and vaccinations among reproductive-aged females (15-44 years of age). Data were stratified by infant birth weight, gestational age, maternal age, and multiple birth status. Results: Dried blood spot samples from 415 293 infants (median [IQR] age, 1.04 [1.00-1.20] days; 210 805 [51.1%] male) were analyzed for SARS-CoV-2 antibodies. The first known antibody-positive infant in New York State was born on March 29, 2020. SARS-CoV-2 seroprevalence reflected statewide and regional COVID-19 cases among reproductive-aged females in the prevaccine period. From February through November 2021, S seroprevalence was strongly correlated with cumulative vaccinations in each New York State region and in the state overall (rs = 0.92-1.00, P ≤ .001). S and N seroprevalences were significantly lower in newborns with very low birth weight (720 [14.8%] for S and 138 [2.8%] for N, P < .001) and low birth weight (5160 [19.3%] for S and 1233 [4.6%] for N, P = .009) compared with newborns with normal birth weight (77 116 [20.1%] for S and 19 872 [5.2%] for N). Lower N and higher S seroprevalences were observed in multiple births (odds ratio [OR], 0.84; 95% CI, 0.75-0.94; P = .002 for N and OR, 1.24; 95% CI, 1.18-1.31; P < .001 for S) vs single births and for maternal age older than 30 years (OR, 0.87; 95% CI, 0.80-0.94; P < .001 for N and OR, 1.17; 95% CI, 1.11-1.23; P < .001 for S) vs younger than 20 years. Conclusions and Relevance: In this study, seroprevalence in newborn DBS samples reflected COVID-19 case fluctuations and vaccinations among reproductive-aged women during the study period. These results demonstrate the utility of using newborn DBS testing to estimate SARS-CoV-2 seroprevalence in pregnant individuals.

Selenoproteins and the senescence-associated epitranscriptome.

Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.

Serological Analysis Identifies Consequential B Cell Epitopes on the Flexible Linker and C-Terminus of Decorin Binding Protein A (DbpA) from Borrelia burgdorferi.

Decorin binding protein A (DbpA) is a surface adhesin of Borrelia burgdorferi, the causative agent of Lyme disease. While DbpA is one of the most immunogenic of B. burgdorferi's nearly 100 lipoproteins, the B cell epitopes on DbpA recognized by humans following B. burgdorferi infection have not been fully elucidated. In this report we profiled ~270 B. burgdorferi-seropositive human serum samples for IgM and IgG reactivity with a tiled DbpA 18-mer peptide array derived from B. burgdorferi sensu stricto strains B31 and 297. Using enzyme-linked immunosorbent assays (ELISA) and multiplex immunoassays (MIA), we identified 12 DbpA-derived peptides whose antibody reactivities were significantly elevated (generally <10-fold) in B. burgdorferi-seropositive sera, compared to those measured in a healthy cohort. The most reactive peptide (>80-fold IgG, 10-fold IgM) corresponded to residues 64 to 81, which map to an exposed flexible loop between DbpA's α-helix 1 and α-helix 2. This loop, whose sequence is identical between strains B31 and 297, overhangs DbpA's substrate binding pocket. A second strongly reactive antibody target (>80-fold IgG, 3 to 5-fold IgM) mapped to DbpA's C-terminus, a lysine rich tail implicated in attachment to glycosaminoglycans. We postulate that antibody responses against these two targets on DbpA could limit B.burgdorferi's ability to attach to and colonize distal tissues during the early stages of infection. IMPORTANCE The bacterium, Borrelia burgdorferi, is the causative agent of Lyme disease, the most reported tick-borne illness in the United States. In humans, clinical manifestations of Lyme disease are complex and can persist for months, even in the face of a robust antibody response directed against numerous B. burgdorferi surface proteins, including decorin binding protein A (DbpA), which is involved in the early stages of infection. In this study we employed ~270 serum samples from B. burgdorferi-seropositive individuals to better understand human antibody reactivity to specific regions (called epitopes) of DbpA and how such antibodies may function in limiting B. burgdorferi dissemination and tissue colonization.

Durable Immunity to Ricin Toxin Elicited by Intranasally Administered Monoclonal Antibody-Based Immune Complexes.

Inhalation of ricin toxin (RT) elicits profuse inflammation and cell death within the upper and lower airways, ultimately culminating in acute respiratory distress syndrome. We previously reported that the effects of pulmonary RT exposure in mice are nullified by intranasal administration of an mAb mixture consisting of PB10, directed against ricin's enzymatic subunit (RTA), and SylH3, directed against ricin's binding subunit (RTB). We now report that delivery of PB10 and SylH3 as an RT-mAb immune complex (RIC) to mice by the intranasal or i.p. routes stimulates the rapid onset of RT-specific serum IgG that persists for months. RIC administration also induced high-titer, toxin-neutralizing Abs. Moreover, RIC-treated mice were immune to a subsequent 5 × LD50 RT challenge on days 30 or 90. Intranasal RIC administration was more effective than i.p. delivery at rendering mice immune to intranasal RT exposure. Finally, we found that the onset of RT-specific serum IgG following RIC delivery was independent of FcγR engagement, as revealed through FcγR knockout mice and RICs generated with PB10/SylH3 LALA (leucine to alanine) derivatives. In conclusion, a single dose of RICs given intranasally to mice was sufficient to stimulate durable protective immunity to RT by an FcγR-independent pathway.

Vaccine Strain and Wild-Type Clades of Varicella-Zoster Virus in Central Nervous System and Non-CNS Disease, New York State, 2004-2019.

Since the introduction of the varicella-zoster virus (VZV) vaccine in the United States in 1995, there has been a dramatic decrease in both the number and severity of varicella cases. However, VZV surveillance data and information on the VZV clade distribution in central nervous system (CNS) disease and non-CNS disease in New York State is not available. To investigate this, cerebrospinal fluid (CSF) samples from patients with encephalitis or meningitis and non-CSF samples from patients with non-CNS disease manifestations consistent with VZV, collected from 2004 to 2019, were tested with molecular VZV assays. A total of 341 CSF and 1,398 non-CSF samples that tested positive by a VZV-specific real-time PCR assay were further characterized as wild-type or vaccine strain by 3 biallelic real-time PCR assays targeting single nucleotide polymorphism (SNP) markers in open reading frame (ORF) 62. Genotyping was then performed on wild-type strains by conventional PCR and sequencing of 500-bp regions in ORFs 21, 22, and 50. Sequence analysis identified clades 1 to 5 in both sample types with a virtually identical clade distribution between CSF and non-CSF samples. In addition, 19 clade 6 and 13 clade 9 samples were detected in non-CSF samples after implementation of an expanded genotyping scheme, including ORF 29, 38, and 67. These clades were not detected in any CSF samples. Finally, a total of 28 vaccine strains were detected, 25 in the non-CSF samples and 3 in the CSF samples. All three cases of vaccine strain with CNS involvement experienced relatively minor symptoms of aseptic meningitis and fully recovered. These results support the evidence that while the VZV vaccine is capable of causing CNS disease, it is still a rare event and symptoms are typically less severe than those caused by wild-type infection.

Durable Immunity to Ricin Toxin Elicited by a Thermostable, Lyophilized Subunit Vaccine.

The development of vaccines against biothreat toxins like ricin (RT) is considered an integral component of the U.S. national security efforts. RiVax is a thermostable, lyophilized RT subunit vaccine adsorbed to aluminum salt adjuvant intended for use by military personnel and first responders. Phase 1 studies indicated that RiVax is safe and immunogenic, while a three-dose intramuscular vaccination regimen in nonhuman primates elicited protection against lethal dose RT challenge by aerosol. Here, we investigated, in a mouse model, the durability of RiVax-induced antibody responses and corresponding immunity to lethal dose RT challenge. Groups of mice were subcutaneously administered 3 or 1 µg of RiVax on days 0 and 21 and challenged with 10× 50% lethal dose (LD50) RT by injection at six different intervals over the course of 12 months. Serum antibody titers and epitope-specific competition assays were determined prior to each challenge. We report that the two-dose, 3-µg regimen conferred near-complete protection against RT challenge on day 35 and complete protection thereafter (challenge days 65, 95, 125, 245, and 365). The two-dose, 3-µg regimen was superior to the 1-µg regimen as revealed by slight differences in survival and morbidity scores (e.g., hypoglycemia, weight loss) on challenge days 35 and 365. In separate experiments, a single 3-µg RiVax vaccination proved only marginally effective at eliciting protective immunity to RT, underscoring the necessity of a prime-boost regimen to achieve full and long-lasting protection against RT. IMPORTANCE Ricin toxin (RT) is a notorious biothreat, as exposure to even trace amounts via injection or inhalation can induce organ failure and death within a matter of hours. In this study, we advance the preclinical testing of a candidate RT vaccine known as RiVax. RiVax is a recombinant nontoxic derivative of RT's enzymatic subunit that has been evaluated for safety in phase I clinical trials and efficacy in a variety of animal models. We demonstrate that two doses of RiVax are sufficient to protect mice from lethal dose RT challenge for up to 1 year. We describe kinetics and other immune parameters of the antibody response to RiVax and discuss how these immune factors may translate to humans.

Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

Evaluation of Specimen Types and Saliva Stabilization Solutions for SARS-CoV-2 Testing.

Identifying SARS-CoV-2 infections through aggressive diagnostic testing remains critical to tracking and curbing the spread of the COVID-19 pandemic. Collection of nasopharyngeal swabs (NPS), the preferred sample type for SARS-CoV-2 detection, has become difficult due to the dramatic increase in testing and consequent supply strain. Therefore, alternative specimen types have been investigated that provide similar detection sensitivity with reduced health care exposure and the potential for self-collection. In this study, the detection sensitivity of SARS-CoV-2 in nasal swabs (NS) and saliva was compared to that of NPS using matched specimens from two outpatient cohorts in New York State (total n = 463). The first cohort showed only a 5.4% positivity, but the second cohort (n = 227) had a positivity rate of 41%, with sensitivity in NPS, NS, and saliva of 97.9%, 87.1%, and 87.1%, respectively. Whether the reduced sensitivity of NS or saliva is acceptable must be assessed in the settings where they are used. However, we sought to improve on it by validating a method to mix the two sample types, as the combination of nasal swab and saliva resulted in 94.6% SARS-CoV-2 detection sensitivity. Spiking experiments showed that combining them did not adversely affect the detection sensitivity in either. Virus stability in saliva was also investigated, with and without the addition of commercially available stabilizing solutions. The virus was stable in saliva at both 4°C and room temperature for up to 7 days. The addition of stabilizing solutions did not enhance stability and, in some situations, reduced detectable virus levels.

Sensitization of Airway Epithelial Cells to Toxin-Induced Death by TNF Superfamily Cytokines.

The TNF superfamily of proinflammatory and proapoptotic cytokines influence tissue-wide responses to molecular insults such as small molecules, toxins, and viral infections that perturb cellular homeostasis at the level of DNA replication, transcription, and translation. In the context of acute lung injury, for example, TNF superfamily members like TNF-α and TRAIL can severely exacerbate disease pathophysiology. This chapter describes a systematic approach to optimization of mammalian cell viability assays and transcriptional profiling through nCounter® Technology to permit a detailed examination of how TNF-α and TRAIL modulate programmed cell death pathways in concert with ricin toxin, a ribosome-inactivating protein (RIP) and a potent inducer of acute respiratory distress. We compare two widely used luciferase- and colorimetric-based cell viability assays and provide optimization protocols for adherent and non-adherent cell lines. We provide a computational workflow to facilitate downstream analysis of datasets generated from nCounter® gene expression panels. While combined treatment with ricin toxin and TRAIL serves as the exemplar, the methodologies are applicable to any TNF superfamily member in combination with any biological agent of interest.

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin.

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD50 of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration's "Animal Rule."

A Humanized Monoclonal Antibody Cocktail to Prevent Pulmonary Ricin Intoxication.

PB10 IgG1, a monoclonal antibody (MAb) directed against an immunodominant epitope on the enzymatic subunit (RTA) of ricin toxin (RT), has been shown to passively protect mice and non-human primates from an aerosolized lethal-dose RT challenge. However, it was recently demonstrated that the therapeutic efficacy of PB10 IgG1 is significantly improved when co-administered with a second MAb, SylH3, targeting RT's binding subunit (RTB). Here we report that the PB10/SylH3 cocktail is also superior to PB10 alone when used as a pre-exposure prophylactic (PrEP) in a mouse model of intranasal RT challenge. The benefit of the PB10/SylH3 cocktail prompted us to engineer a humanized IgG1 version of SylH3 (huSylH3). The huPB10/huSylH3 cocktail proved highly efficacious in the mouse model, thereby opening the door to future testing in non-human primates.

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin.

Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by pro-inflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin's B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin's enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.

An intranasally administered monoclonal antibody cocktail abrogates ricin toxin-induced pulmonary tissue damage and inflammation.

Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal inflammatory response in the airways following inhalation. Uptake of ricin by alveolar macrophages (AM) and other pulmonary cell types occurs via two parallel pathways: one mediated by ricin's B subunit (RTB), a galactose-specific lectin, and one mediated by the mannose receptor (MR;CD206). Ricin's A subunit (RTA) is a ribosome-inactivating protein that triggers apoptosis in mammalian cells. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA and administered intravenously, was able to rescue Rhesus macaques from lethal aerosol dose of ricin. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of lung macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of lung macrophages, and a significant dampening of PMN recruitment into the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB- and MR-dependent uptake pathways.

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin-Ab complexes.

Ricin toxin is a plant-derived, ribosome-inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)-with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a "lactose-sensitive" pathway mediated by ricin's galactose/N-acetylgalactosamine-specific lectin subunit (RTB), and a "mannose-sensitive" pathway mediated by the mannose receptor (MR; CD206) or other C-type lectins capable of recognizing the mannose-side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin-specific mouse MAb and camelid single-domain (VH H) antibodies to protect KCs and LSECs from ricin-induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VH Hs afforded little (<40%) or even no protection to LSECs against ricin-induced death. Complete protection of LSECs was only achieved with MAb or VH H cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose-sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab-based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.

Rescue of rhesus macaques from the lethality of aerosolized ricin toxin.

Ricin toxin (RT) ranks at the top of the list of bioweapons of concern to civilian and military personnel alike, due to its high potential for morbidity and mortality after inhalation. In nonhuman primates, aerosolized ricin triggers severe acute respiratory distress characterized by perivascular and alveolar edema, neutrophilic infiltration, and severe necrotizing bronchiolitis and alveolitis. There are currently no approved countermeasures for ricin intoxication. Here, we report the therapeutic potential of a humanized mAb against an immunodominant epitope on ricin's enzymatic A chain (RTA). Rhesus macaques that received i.v. huPB10 4 hours after a lethal dose of ricin aerosol exposure survived toxin challenge, whereas control animals succumbed to ricin intoxication within 30 hours. Antibody intervention at 12 hours resulted in the survival of 1 of 5 monkeys. Changes in proinflammatory cytokine, chemokine, and growth factor profiles in bronchial alveolar lavage fluids before and after toxin challenge successfully clustered animals by treatment group and survival, indicating a relationship between local tissue damage and experimental outcome. This study represents the first demonstration, to our knowledge, in nonhuman primates that the lethal effects of inhalational ricin exposure can be negated by a drug candidate, and it opens up a path forward for product development.

TRAIL (CD253) Sensitizes Human Airway Epithelial Cells to Toxin-Induced Cell Death.

Inhalation of ricin toxin is associated with the onset of acute respiratory distress syndrome (ARDS), characterized by hemorrhage, inflammatory exudates, and tissue edema, as well as the nearly complete destruction of the lung epithelium. Here we report that the Calu-3 human airway epithelial cell line is relatively impervious to the effects of ricin, with little evidence of cell death even upon exposure to microgram amounts of toxin. However, the addition of exogenous soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL; CD253) dramatically sensitized Calu-3 cells to ricin-induced apoptosis. Calu-3 cell killing in response to ricin and TRAIL exposure was partially inhibited by caspase-8 and caspase-3/7 inhibitors, consistent with involvement of extrinsic apoptotic pathways in cell death. We employed nCounter Technology to define the transcriptional response of Calu-3 cells to ricin, TRAIL, and the combination of ricin plus TRAIL. An array of genes associated with inflammation and cell death were significantly upregulated upon treatment with ricin toxin and were further amplified upon addition of TRAIL. Of particular note was interleukin-6 (IL-6), whose expression in Calu-3 cells increased 300-fold upon ricin treatment and more than 750-fold upon ricin and TRAIL treatment. IL-6 secretion by Calu-3 cells was confirmed by cytometric bead array analysis. On the basis of these finding, we speculate that the severe airway epithelial cell damage observed in animal models following ricin exposure is a result of a positive-feedback loop driven by proinflammatory cytokines such as TRAIL and IL-6.IMPORTANCE Ricin toxin is a biothreat agent that is particularly damaging to lung tissue following inhalation. A hallmark of ricin exposure is widespread inflammation and concomitant destruction of the airway epithelium. In this study, we investigated the possible interaction between ricin and known proinflammatory cytokines associated with lung tissue. Using an established human airway epithelial cell line, we demonstrate that epithelial cell killing by ricin is significantly enhanced in the presence of the proinflammatory cytokine known as TRAIL (CD253). Moreover, epithelial cells that are simultaneously exposed to ricin and TRAIL produced large amounts of secondary proinflammatory signals, including IL-6, which in the context of the lung would be expected to exacerbate toxin-induced tissue damage. Our results suggest that therapies designed to neutralize proinflammatory cytokines such as TRAIL and IL-6 may limit the bystander damage associated with ricin exposure.

Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine.

Biodefense vaccine are destined to be stockpiled for periods of time and deployed in the event of a public health emergency. In this report, we compared the potency of liquid and lyophilized (thermostabilized) formulations of a candidate ricin toxin subunit vaccine, RiVax, adsorbed to aluminum salts adjuvant, over a 12-month period. The liquid and lyophilized formulations were stored at stressed (40 °C) and unstressed (4 °C) conditions and evaluated at 3, 6 and 12-month time points for potency in a mouse model of lethal dose ricin challenge. At the same time points, the vaccine formulations were interrogated in vitro by competition ELISA for conformational integrity using a panel of three monoclonal antibodies (mAbs), PB10, WECB2, and SyH7, directed against known immunodominant toxin-neutralizing epitopes on RiVax. We found that the liquid vaccine under stress conditions declined precipitously within the first three months, as evidenced by a reduction in in vivo potency and concomitant loss of mAb recognition in vitro. In contrast, the lyophilized RiVax vaccine retained in vivo potency and conformational integrity for up to one year at 4 °C and 40 °C. We discuss the utility of monitoring the integrity of one or more toxin-neutralizing epitopes on RiVax as a possible supplement to animal studies to assess vaccine potency.

Differential Effects of Temperature and Mosquito Genetics Determine Transmissibility of Arboviruses by Aedes aegypti in Argentina.

Aedes aegypti (L.) (Diptera: Culicidae) have a global distribution and are the primary vector of a number of mosquito-borne viruses responsible for epidemics throughout the Americas. As in much of South America, the threat from pathogens including dengue virus (DENV; Flaviviridae, Flavivirus) and chikungunya virus (CHIKV; Togaviridae, Alphavirus) has increased in Argentina in recent years. The complexity of transmission cycles makes predicting the occurrence and intensity of arbovirus outbreaks difficult. To gain a better understanding of the risk of DENV and CHIKV in Argentina and the factors influencing this risk, we evaluated the role of population and temperature in the vector competence and vectorial capacity (VC) of Ae. aegypti from geographically and ecologically distinct locations. Our results demonstrate that intrinsic and extrinsic factors including mosquito population, viral species, and temperature significantly influence both vector competence and overall VC of Ae. aegypti in Argentina, yet also that the magnitude of these influences is highly variable. Specifically, results suggest that CHIKV competence is more dependent on mosquito genetics than is DENV competence, whereas temperature has a greater effect on DENV transmission. In addition, although there is an overall positive correlation between temperature and competence for both viruses, there are exceptions to this for individual virus-population combinations. Together, these data establish large variability in VC for these pathogens among distinct Ae. aegypti populations in Argentina and demonstrate that accurate assessment of arbovirus risk will require nuanced models that fully consider the complexity of interactions between virus, temperature, mosquito genetics, and hosts.

Fine-Specificity Epitope Analysis Identifies Contact Points on Ricin Toxin Recognized by Protective Monoclonal Antibodies.

Ricin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report, we describe five new mouse mAbs directed against an immunodominant region, so-called epitope cluster II, on the surface of ricin's ribosome-inactivating enzymatic subunit A (RTA). The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10× LD50 ricin challenge by injection. Only three of the mAbs (LE4, PH12, and TB12) afforded protection over the 7-d study period. Neither binding affinity nor in vitro toxin-neutralizing activity could fully account for LE4, PH12, and TB12's potent in vivo activity relative to the other six mAbs. However, epitope mapping studies by hydrogen exchange-mass spectrometry revealed that LE4, PH12, and TB12 shared common contact points on RTA corresponding to RTA α-helices D and E and ß-strands d and e located on the back side of RTA relative to the active site. The other six mAbs recognized overlapping epitopes on RTA, but none shared the same hydrogen exchange-mass spectrometry profile as LE4, PH12, and TB12. A high-density competition ELISA with a panel of ricin-specific, single-domain camelid Abs indicated that even though LE4, PH12, and TB12 make contact with similar secondary motifs, they likely approach RTA from different angles. These results underscore how subtle differences in epitope specificity can significantly impact Ab functionality in vivo. ImmunoHorizons, 2018, 2: 262-273.