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.

Aerosol delivery of SARS-CoV-2 human monoclonal antibodies in macaques limits viral replication and lung pathology.

Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.

Broad host susceptibility of North American amphibian species to Batrachochytrium salamandrivorans suggests high invasion potential and biodiversity risk.

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen of amphibians that is emerging in Europe and could be introduced to North America through international trade or other pathways. To evaluate the risk of Bsal invasion to amphibian biodiversity, we performed dose-response experiments on 35 North American species from 10 families, including larvae from five species. We discovered that Bsal caused infection in 74% and mortality in 35% of species tested. Both salamanders and frogs became infected and developed Bsal chytridiomycosis. Based on our host susceptibility results, environmental suitability conditions for Bsal, and geographic ranges of salamanders in the United States, predicted biodiversity loss is expected to be greatest in the Appalachian Region and along the West Coast. Indices of infection and disease susceptibility suggest that North American amphibian species span a spectrum of vulnerability to Bsal chytridiomycosis and most amphibian communities will include an assemblage of resistant, carrier, and amplification species. Predicted salamander losses could exceed 80 species in the United States and 140 species in North America.

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.

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.

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.

Efficacy of two reduction methods in conjunction with 3-D-printed patient-specific pin guides for aligning simulated comminuted tibial fractures in cadaveric dogs.

OBJECTIVE: To assess the feasibility and accuracy of using 2 methods for reduction and alignment of simulated comminuted diaphyseal tibial fractures in conjunction with 3-D-printed patient-specific pin guides. SAMPLE: Paired pelvic limbs from 8 skeletally mature dogs weighing 20 to 35 kg. METHODS: CT images of both tibiae were obtained, and 3-D reconstructions of the tibiae were used to create proximal and distal patient-specific pin guides. These guides were printed and used to facilitate fracture reduction and alignment in conjunction with either a 3-D-printed reduction guide or a linear type 1A external fixator. Postreduction CT images were used to assess the accuracy of pin guide placement and the accuracy of fracture reduction and alignment. RESULTS: The 3-D-printed guides were applied with acceptable ease. Guides for both groups were placed with minor but detectable deviations from the planned location (P = .01), but deviations were not significantly different between groups. Fracture reduction resulted in similar minor but detectable morphological differences from the intact tibiae (P = .01). In both groups, fracture reduction and alignment were within clinically acceptable parameters for fracture stabilization by means of minimally invasive plate osteosynthesis. CLINICAL RELEVANCE: Virtual surgical planning and fabrication of patient-specific 3-D-printed pin guides have the potential to facilitate fracture reduction and alignment during use of minimally invasive plate osteosynthesis for fracture stabilization.

Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis.

Increasingly, antibodies are being used to treat and prevent viral infections. In the context of HIV, efficacy is primarily attributed to dose-dependent neutralization potency and to a lesser extent Fc-mediated effector functions. It remains unclear whether augmenting effector functions of broadly neutralizing antibodies (bNAbs) may improve their clinical potential. Here, we use bNAb 10E8v4 targeting the membrane external proximal region (MPER) to examine the role of antibody-mediated effector and complement (C') activity when administered prophylactically against SHIV challenge in rhesus macaques. With sub-protective dosing, we find a 78-88% reduction in post-acute viremia that is associated with 10E8v4-mediated phagocytosis acting at the time of challenge. Neither plasma nor tissue viremic outcomes in vivo is improved with an Fc-modified variant of 10E8v4 enhanced for C' functions as determined in vitro. These results suggest that effector functions inherent to unmodified 10E8v4 contribute to efficacy against SHIVSF162P3 in the absence of plasma neutralizing titers, while C' functions are dispensable in this setting, informing design of bNAb modifications for improving protective efficacy.

CD4+ T Cells Are Dispensable for Induction of Broad Heterologous HIV Neutralizing Antibodies in Rhesus Macaques.

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies.

Quantification of SARS-CoV-2 spike and nucleocapsid proteins using isotope dilution tandem mass spectrometry.

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.

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.

ABSENCE OF BATRACHOCHYTRIUM SALAMANDRIVORANS IN A GLOBAL HOTSPOT FOR SALAMANDER BIODIVERSITY.

Batrachochytrium salamandrivorans (Bsal) is an emerging fungal pathogen that affects salamander and newt populations in Asia and Europe. In the Western Hemisphere, Bsal represents a major threat to endemic amphibian populations, which have not evolved resistance to infection, and which could experience local extinction events such as those observed in European fire salamanders (Salamandra salamandra). We report findings of a survey focusing specifically on wild lungless salamanders in the southeastern US, the most biodiverse location for salamander species globally. Between May 2016 and July 2018, we conducted 25 surveys at 10 sites across three ecoregions in Tennessee, US. Using quantitative (q)PCR, we screened water samples and skin swabs from 137 salamanders in five plethodontid genera. Although single replicates of six samples amplified during qPCR cycling, no samples could be confirmed as positive for the presence of Bsal with 28S rRNA PCR and independent laboratory screening. It is probable that we found false positive results, as reported by other researchers using the same assay. We offer recommendations for future monitoring efforts.

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.

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.

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.

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.

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA.

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (ß=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

Prevalence of Ophidiomyces ophiodiicola, the Causative Agent of Snake Fungal Disease, in the Interior Plateau Ecoregion of Tennessee, USA.

The fungal pathogen Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, has been implicated in declines of North American snake populations since 2006 and the geographic range of this pathogen is still not fully known. In Tennessee, US, O. ophiodiicola has been detected since 2012, but large portions of the state have not been surveyed for this pathogen. Our primary objective was to monitor the prevalence of O. ophiodiicola in the Interior Plateau ecoregion of Tennessee by swabbing all snakes that were encountered during road cruising survey efforts in 2017 and 2018. Eleven snakes of four species, copperhead (Agkistrodon contortrix), common water snake (Nerodia sipedon), black kingsnake (Lampropeltis nigra), and smooth earthsnake (Virginia valeriae), tested positive for the presence of O. ophiodiicola. Overall, 9.2% (11/120) of snakes sampled tested positive for the presence of O. ophiodiiola, and we further observed a seasonal trend in detections with summer months having the greatest frequency of detections. Our results extend the known geographic range of O. ophiodiicola in Tennessee by adding four previously unconfirmed O. ophiodiicola-positive counties. Further sampling will need to be conducted across west Tennessee because this is the most data-deficient region of the state. Our results offer additional evidence of the presence of this pathogen in Tennessee and will help researchers further understand the geographic distribution and host range.

Adenosine deaminase-1 enhances germinal center formation and functional antibody responses to HIV-1 Envelope DNA and protein vaccines.

Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (TFH) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the TFH-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of TFH cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only these mice had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.