An antibody-free evaluation of an mRNA COVID-19 vaccine.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, ß-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Translocation does not influence amphibian chytrid fungus prevalence among wild eastern hellbenders Cryptobranchus alleganiensis.

Disease monitoring is an essential step in translocation projects, specifically in amphibians where emerging pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd) are linked to population declines. The eastern hellbender Cryptobranchus alleganiensis is a large, fully aquatic salamander experiencing precipitous range-wide population declines; however, the role Bd plays in these declines is unclear. To augment declining hellbender populations and determine effects of translocation on Bd prevalence, we conducted a translocation study of wild adult hellbenders from 2 source streams with abundant hellbender populations to 2 streams with declining populations in east Tennessee, USA. In 2018, we implanted radio transmitters into 30 hellbenders and sampled them periodically for Bd until 17 of the 30 hellbenders were translocated in 2019. We attempted to recapture translocated hellbenders approximately every 45 d for 3 mo to determine Bd prevalence post-release. We used qPCR to detect Bd and quantify zoospore loads on positive samples. Hellbenders had a pre-translocation Bd prevalence of 50% (15 of 30), which decreased to 10% (1 of 10) post-translocation. The average zoospore load for positive samples was 73.63 ± 30.82, and no hellbenders showed signs of chytridiomycosis throughout the study. Although we detected no significant effect of translocation on Bd prevalence, we observed a reduction in Bd prevalence post-release. Our results indicate that translocation did not lead to an increase in pathogen prevalence in translocated wild adult hellbenders, suggesting that chytrid did not impact the success of short-term translocations of eastern hellbenders in the Blue Ridge ecoregion.

Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

Heartland Virus in Lone Star Ticks, Alabama, USA.

We detected Heartland virus (HRTV) in lone star nymphs collected in 2018 in northern Alabama, USA. Real-time reverse transcription PCR selective for the small segment of the HRTV genome and confirmatory sequencing of positive samples showed high identity with HRTV strains sequenced from Tennessee and Missouri.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

A standardized method for the construction of a tick drag/flag sampling approach and evaluation of sampling efficacy.

Drag sampling and flagging are two of the most effective and widely applied techniques to monitor tick populations. Despite the importance of this sampling strategy, there is a lack of standardized protocols for the construction of an inexpensive tick drag/flag. To this end, we provide a step-by-step protocol that details the construction of a tick drag/flag. We provide evidence of efficacy by comparing results obtained over 3-months at 108 locations within the William B. Bankhead National Forest, Alabama, USA. Overall, our drag/flag sampling approach yielded 1127 larvae, 460 nymphs, and 53 adults for a total of 1640 ticks representing three species. We detected significant patterns in Amblyomma americanum abundance for nymphs and adults with greater counts in June (ß = 0.91 ± 0.36, 95% CI 0.55-1.27; ß = 2.44 ± 0.63, 95% CI 1.81-3.07, respectively) and July (ß = 0.73 ± 0.36, 95% CI 0.37-1.09; ß = 1.65 ± 0.66, 95% CI 0.99-2.31, respectively) as compared to August. We also detected a significant difference in tick captures by tick drag/flag fabric type with greater captures when muslin was used as compared to flannel (ß = 1.07 ± 0.06, 95% CI 1.01-1.13). Our goal is to provide instructions to assemble a highly effective tick drag/flag using minimal supplies. Evaluation and improvements of sampling techniques is essential to understand impacts of landscape management and larger stressors, such as climate change on tick populations but also for enhancing detection of invasive non-native species.

Achieving Potent Autologous Neutralizing Antibody Responses against Tier 2 HIV-1 Viruses by Strategic Selection of Envelope Immunogens.

Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.

Continuous Flow Perfused Cadaver Model for Endovascular Training, Research, and Development.

BACKGROUND: Endovascular simulation employing computer, animal, and static models are common and useful adjuncts for teaching endovascular procedures and developing novel, complex endovascular techniques. Unfortunately, these models lack realistic haptic feedback and thus do not faithfully replicate many of the technical challenges associated with clinical endovascular procedures (e.g., arterial calcification, rigidity, and stenosis). We sought to develop a realistic and reproducible perfused cadaver model for endovascular training, device development, and research. METHODS: Fresh frozen, elderly (age 50-80 years) male cadavers were thawed and prepared for open dissection. The entire arterial tree (ascending aorta to femoral arteries) was dissected free and major branch vessels exposed. Sheaths were placed to allow outflow from selected vessels. A Dacron conduit was sewn to the ascending aorta to generate arterial inflow, which was provided by a centrifugal pump. Aortic aneurysms were created in the descending thoracic and abdominal aorta. Digital subtraction arteriography and various endovascular interventions were performed, including stent grafts and EndoAnchors deployment. RESULTS: Continuous antegrade flow was achieved in the thoracic, abdominal, iliac, and femoral segments. Open and percutaneous access at the femoral region was obtained with realistic back-bleeding and tactile feedback. Adequate, fluoroscopically documented flow was observed in both cannulated major and noncannulated smaller branches. We performed angiography with standard techniques via a pigtail catheter and contrast injector throughout the arterial system. Abdominal and thoracic endografts were deployed with appropriate angiographic guidance and realistic haptic feedback for both guidewire and stent grafts. Additional applications, including selective cannulation, aorto-iliac occlusive disease interventions, and anchor placement, were also successfully simulated. Finally, the model was used as a platform to test investigational devices. CONCLUSIONS: Our pressurized cadaver flow model successfully replicated multiple aspects of advanced endovascular procedures with haptic feedback. This novel human cadaver model allows for training and device development under clinically realistic conditions.

Ranavirus could facilitate local extinction of rare amphibian species.

There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We estimated the susceptibility of two amphibian species of conservation concern (the dusky gopher frog [Lithobates sevosus] and boreal toad [Anaxyrus boreas boreas]) to an emerging pathogen (ranavirus) using laboratory challenge experiments, and combined these data with published demographic parameter estimates to simulate the potential effects of ranavirus exposure on extinction risk. We included effects of life stage during pathogen exposure, pathogen exposure interval, hydroperiod of breeding habitat, population carrying capacity, and immigration in simulations. We found that both species were highly susceptible to ranavirus when exposed to the pathogen in water at environmentally relevant concentrations. Dusky gopher frogs experienced 100 % mortality in four of six life stages tested. Boreal toads experienced 100 % mortality when exposed as tadpoles or metamorphs, which were the only life stages tested. Simulations showed population declines, greater extinction probability, and faster times to extinction with ranavirus exposure. These effects were more evident with more frequent pathogen exposure intervals and lower carrying capacity. Immigration at natural rates did little to mitigate effects of ranavirus exposure unless immigration occurred every 2 years. Our results demonstrate that disease-induced extinction by emerging pathogens, such as ranavirus, is possible, and that threat may be especially high for species with small population sizes. For the species in this study, conservation organizations should incorporate ranavirus surveillance into monitoring programs and devise intervention strategies in the event that disease outbreaks occur.

Envelope variants circulating as initial neutralization breadth developed in two HIV-infected subjects stimulate multiclade neutralizing antibodies in rabbits.

UNLABELLED: Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE: Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested thus far have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral envelope population and tested this concept using multiple envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV envelopes circulating during the early development and maturation of breadth generated more-potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying envelope immunogens that may be more effective in generating protective antibodies in humans.

Neutralizing polyclonal IgG present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus SHIVSF162P3-infected infant rhesus macaques.

Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.

Vaccine delivery to the oral cavity using coated microneedles induces systemic and mucosal immunity.

PURPOSE: The objective of this study is to evaluate the feasibility of using coated microneedles to deliver vaccines into the oral cavity to induce systemic and mucosal immune responses. METHOD: Microneedles were coated with sulforhodamine, ovalbumin and two HIV antigens. Coated microneedles were inserted into the inner lower lip and dorsal surface of the tongue of rabbits. Histology was used to confirm microneedle insertion, and systemic and mucosal immune responses were characterized by measuring antigen-specific immunoglobulin G (IgG) in serum and immunoglobulin A (IgA) in saliva, respectively. RESULTS: Histological evaluation of tissues shows that coated microneedles can penetrate the lip and tongue to deliver coatings. Using ovalbumin as a model antigen it was found that the lip and the tongue are equally immunogenic sites for vaccination. Importantly, both sites also induced a significant (p < 0.05) secretory IgA in saliva compared to pre-immune saliva. Microneedle-based oral cavity vaccination was also compared to the intramuscular route using two HIV antigens, a virus-like particle and a DNA vaccine. Microneedle-based delivery to the oral cavity and the intramuscular route exhibited similar (p > 0.05) yet significant (p < 0.05) levels of antigen-specific IgG in serum. However, only the microneedle-based oral cavity vaccination group stimulated a significantly higher (p < 0.05) antigen-specific IgA response in saliva, but not intramuscular injection. CONCLUSION: In conclusion, this study provides a novel method using microneedles to induce systemic IgG and secretory IgA in saliva, and could offer a versatile technique for oral mucosal vaccination.

Snake co-occurrence patterns are best explained by habitat and hypothesized effects of interspecific interactions.

Snakes often occur in species-rich assemblages, and sympatry is thought to be facilitated primarily by low diet overlap, not interspecific interactions. We selected, a priori, three species pairs consisting of species that are morphologically and taxonomically similar and may therefore be likely to engage in interspecific, consumptive competition. We then examined a large-scale database of snake detection/nondetection data and used occupancy modelling to determine whether these species occur together more or less frequently than expected by chance while accounting for variation in detection probability among species and incorporating important habitat categories in the models. For some snakes, we obtained evidence that the probabilities that habitat patches are used are influenced by the presence of potentially competing congeneric species. Specifically, timber rattlesnakes (Crotalus horridus) were less likely than expected by chance to use areas that also contained eastern diamond-backed rattlesnakes (Crotalus adamanteus) when the proportion of evergreen forest was relatively high. Otherwise, they occurred together more often than expected by chance. Complex relationships were revealed between habitat use, detection probabilities and occupancy probabilities of North American racers (Coluber constrictor) and coachwhips (Coluber flagellum) that indicated the probability of competitive exclusion increased with increasing area of grassland habitat, although there was some model uncertainty. Cornsnakes (Pantherophis guttatus or Pantherophis slowinskii) and ratsnakes (Pantherophis alleghaniensis, Pantherophis spiloides, or Pantherophis obsoletus) exhibited differences in habitat selection, but we obtained no evidence that patterns of use for this species pair were influenced by current interspecific interactions. Overall, our results are consistent with the hypothesis that competitive interactions influence snake assemblage composition; the strength of these effects was affected by landscape-scale habitat features. Furthermore, we suggest that current interspecific interactions may influence snake occupancy, challenging the paradigm that contemporary patterns of snake co-occurrence are largely a function of diet partitioning that arose over evolutionary time.

High susceptibility of the endangered dusky gopher frog to ranavirus.

Amphibians are one of the most imperiled vertebrate groups, with pathogens playing a role in the decline of some species. Rare species are particularly vulnerable to extinction because populations are often isolated and exist at low abundance. The potential impact of pathogens on rare amphibian species has seldom been investigated. The dusky gopher frog Lithobates sevosus is one of the most endangered amphibian species in North America, with 100-200 individuals remaining in the wild. Our goal was to determine whether adult L. sevosus were susceptible to ranavirus, a pathogen responsible for amphibian die-offs worldwide. We tested the relative susceptibility of adult L. sevosus to ranavirus (103 plaque-forming units) isolated from a morbid bullfrog via 3 routes of exposure: intra-coelomic (IC) injection, oral (OR) inoculation, and water bath (WB) exposure. We observed 100% mortality of adult L. sevosus in the IC and WB treatments after 10 and 19 d, respectively. Ninety-five percent mortality occurred in the OR treatment over the 28 d evaluation period. No mortality was observed in the control treatment after 28 d. Our results indicate that L. sevosus is susceptible to ranavirus, and if adults in the wild are exposed to this pathogen, significant mortality could occur. Additionally, our study demonstrates that some adult amphibian species can be very susceptible to ranavirus, which has been often overlooked in North American studies. We recommend that conservation planners consider testing the susceptibility of rare amphibian species to ranavirus and that the adult age class is included in future challenge experiments.

Evaluating translocation success of wild eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Blue Ridge Ecoregion streams using pre- and post-translocation home range sizes and movement metrics.

Translocations of freshwater species have become a widespread conservation strategy to mitigate the impacts of habitat fragmentation, yet they are not often rigorously monitored using animal movement data to determine their success. We demonstrate the value of monitoring pre- and post-translocation movements and home-range sizes of a fully-aquatic, benthic stream salamander, the eastern hellbender (Cryptobranchus a. alleganiensis) to determine translocation success. We studied the home range sizes, movements, and habitat use of individuals (n = 27) in two self-sustaining populations (S1 & S2) for one year, and then subsequently collected similar data from a subset of these individuals (n = 17) that were translocated into two nearby streams (T1 & T2) with dam-isolated, declining populations in the Blue Ridge Ecoregion of Tennessee. We collected 1,571 location data points (869 pre-translocation and 715 post-translocation) from four study sites, and evaluated effects of mass, sex, and pre-translocation home range size/sedentariness, as well as habitat covariates on home range size and movements. Hellbender home range sizes increased from pre-translocation estimates at both sites, but response depended primarily on physical characteristics of release sites. Home range and fine-scale movement metrics indicated that hellbenders translocated from S1 to T1 settled in more quickly, had greater site fidelity, and smaller home ranges than hellbenders translocated from S2 to T2. Hellbender movements were influenced by cover rock size and density rather than individual characteristics. Study-long survival rates of translocated hellbenders increased from S1 to T1 (80% to 100%) and decreased from S2 to T2 (76% to 33%). Monitoring pre- and post-translocation movements was a valuable method for evaluating short-term translocation success in a freshwater environment. For future hellbender translocations, managers should prioritize selecting suitable release sites with contiguous boulder-dense areas (1-2 per m2), adequate prey (crayfish) densities (>1/m2), and habitats with low risk of predation.

Prevalence and Molecular Analysis of Hellbender (Cryptobranchus alleganiensis) Trypanosomes in Tennessee.

Populations of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have been declining for multiple decades because of a variety of stressors associated with anthropogenic habitat disturbance followed by riparian sedimentation. The influence of parasite-associated morbidity and mortality on wild hellbender populations is poorly understood. Research has detected widespread trypanosome infection in hellbenders in Virginia, US, with no other reported detections within the eastern hellbender's extensive range. In our study, trypanosomes mostly closely resembling Trypanosoma cryptobranchi were observed in a blood smear of a hellbender from Tennessee during a population survey. Banked whole blood from this hellbender along with 51 other hellbenders was molecularly tested for the 18s rRNA gene of amphibian trypanosomes using newly designed PCR primers. In total, 3/52 (5.8%) hellbenders were PCR- and sequence-positive for trypanosomes. This is the first report of the partial 18s rRNA sequence from hellbender trypanosomes from North America. Further research into trypanosome epidemiology and hellbender health implications is warranted.

Quantification of SARS-CoV-2 spike protein expression from mRNA vaccines using isotope dilution mass spectrometry.

The advent of mRNA vaccine technology has been vital in rapidly creating and manufacturing COVID-19 vaccines at an industrial scale. To continue to accelerate this leading vaccine technology, an accurate method is needed to quantify antigens produced by the transfection of cells with a mRNA vaccine product. This will allow monitoring of protein expression during mRNA vaccine development and provide information on how changes to vaccine components affects the expression of the desired antigen. Developing novel approaches that allow for high-throughput screening of vaccines to detect changes in antigen production in cell culture prior to in vivo studies could aid vaccine development. We have developed and optimized an isotope dilution mass spectrometry method to detect and quantify the spike protein expressed after transfection of baby hamster kidney cells with expired COVID-19 mRNA vaccines. Five peptides of the spike protein are simultaneously quantified and provide assurance that protein digestion in the region of the target peptides is complete since results between the five peptides had a relative standard deviation of less than 15 %. In addition, two housekeeping proteins, actin and GAPDH, are quantified in the same analytical run to account for any variation in cell growth within the experiment. IDMS allows a precise and accurate means to quantify protein expression by mammalian cells transfected with an mRNA vaccine.

Coccidiosis in Wild Eastern Newts (Notophthalmus viridescens): An Unexpected Finding in Consecutive Mortality Events in Tennessee, USA.

Wildlife diseases are a major threat for species conservation, and there is a growing need to implement more comprehensive disease response programs to better identify these diseases of concern. During March 2017, we observed moribund and dead eastern newts, Notophthalmus viridescens, in a single pond in middle Tennessee. All moribund individuals were emaciated. We euthanized and processed all individuals immediately on-site and later performed histopathology and quantitative PCR for ranavirus, the protist Perkinsea, and chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans. One newt was positive for ranavirus. Histopathology showed no evidence of ranavirosis but did reveal overwhelming coccidiosis. Overlapping partial sequences of coccidian 18S subunit DNA showed a 96.4% match with Eimeria steinhausi, suggesting that lesions were due to a previously undescribed Eimeria sp. In 2019, two more moribund newts were encountered at the same pond. Histopathology revealed the same suspicious parasitic organisms, and one individual was positive for B. dendrobatidis. Further research on how seasonal and other environmental parameters may influence coccidia-associated morbidity and mortality is warranted. These events highlight the importance of histopathologic evaluation of mortality events and provide guidance for investigation of future outbreaks.