Elicited Responses Research Articles

Simultaneous detection and characterization of antigen-specific B cells and CD4 +and CD8 +T cell responses upon natural infection and vaccination

Abstract An important lesson learned from the COVID-19 pandemic is the need for a more comprehensive assessment of the immune status upon a natural infection and vaccination. In addition to measuring antibody titers, monitoring the status of antigen-specific B and T cell must be taken into consideration to gain a more complete understanding of the elicited immune response and its durability. The objective of this study was to develop an immune monitoring assay based on the Dextramer ®technology for simultaneous monitoring of antigen-specific B and T cells (CD4 and CD8). The assay can be applied in combination with flow cytometry and in single cell RNA-seq analysis. The assay builds on Dextramer ®reagents displaying full-length spike protein (specific B cell assessment) or MHC-peptide complexes displaying virus peptides (detecting specific CD8 or CD4 T cells). The assay was evaluated by longitudinal assessment of immunity in individuals vaccinated against SARS-CoV-2 by combining the assay with flow cytometry (n=5) and the Chromium 10× RNA-seq platform (n=2). The latter allows an in-depth view of the immune status by simultaneous investigation of the transcriptome, surface proteins and receptor sequences of antigen-specific cells. Our results show that by using the virus-specific Dextramer ®assay we could monitor changes in SARS-CoV-2-specific B- and T-cell responses before and after primary vaccination and booster doses using flow cytometry and single cell RNA-seq analysis. These results show that the Dextramer ®assay can aid in understanding the elicitation and persistence of antigen-specific responses. Moreover, the assay can easily be tailored to any antigen-specificity by replacing the ligands coupled to the Dextramer ®reagents.

Nucleated red blood cells: unexpected orchestrators of fetal immunity

Abstract Red blood cells (RBCs) are not typically considered active immune mediators. This dogma stems from the fact that RBC precursors discard organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Intriguingly, nucleated RBCs (nRBCs) circulate in human fetuses and neonates. Due to evolutionary pressure for successful reproduction, circulation of nRBCs during pregnancy is likely important. However, the role of nRBCs in uteroremains unknown. To define the role of human nRBCs, we queried single cell RNA-seq data and found that transcriptomics support nRBCs as putative immune mediators. Unexpectedly, we found that nRBCs express antigen processing & presentation machinery. nRBCs constitutively express MHC II & co-stimulatory molecules, hallmarks of specialized antigen-presenting cells. We further demonstrated that nRBCs internalize and cleave antigens. Currently, we aim to determine if nRBCs present antigens to T cells and to characterize the elicited response. In addition, we found that nRBCs express pattern recognition receptors capable of detecting pathogens and initiating an antimicrobial response. We demonstrated that nRBCs respond to viral infection through upregulation of antiviral genes, interferons/interferon-stimulated genes, MHC I, and pro-inflammatory cytokines/chemokines. These data suggest a putative functionality for nRBCs in mediating antiviral immunity in utero. We are currently investigating antiviral functions of nRBCs. Together, our findings shed light on an unexpected orchestrator of fetal immunity. Knowledge of the fetal immune system has potential to help us understand health and disease during pregnancy, as a neonate, and likely every stage of life after. Supported by grants from NIH/NIDDK 5T32DK007780, NIH/NIGMS K12GM081259, Burroughs Wellcome Fund 2022 Next Gen Pregnancy Initiative Award

Abstract 661: Wnt16 Promotes Smooth Muscle Mitochondrial Function And Contractile Phenotype Via Taz (Wwtr1) Relays, Limiting Aneurysmal Dilatation Of Ascending Aorta

Wnt16 is selectively expressed in arterial vascular smooth muscle (VSM) as well as in bone and skin. Non-synonymous WNT16 cSNPs that increase risk for low bone mass also convey risk for composite cardiovascular events (stroke, MI, heart failure, CV death) in the Dallas Heart Study after adjustment for age, sex, ancestry, systolic blood pressure, and body mass index (OR = 1.25, p = 0.02) . To better understand Wnt16’s role in cardiovascular biology, we generated Wnt16-/-;LDLR-/- mice. Blood pressure and pulse wave velocity were reduced in Wnt16-/-;LDLR-/- mice vs. LDLR-/- controls. Ascending aorta (AscAo) dilatation following angiotensin-II (AngII) infusion was worsened with Wnt16 deficiency, while no differences existed at baseline. Contractile markers such as Acta2 - known to convey AscAo aneurysm risk and reduce pressure with genetic deficiency - were down-regulated in Wnt16-/- VSM, with reduction in TGFbeta-induced contraction. De-differentiation markers (C3, Sca1) were upregulated, and mitochondrial respiration (Seahorse oximetry) was significantly impaired with Wnt16 deficiency. Acta2 protein was reduced with Wnt16 deficiency as was aortic Ankrd1, a target of Yap and Taz(Wwtr1) activation via nuclear TEAD-directed transcription. Wnt16-/- VSM exhibited reduced nuclear Yap+Taz. RNAi targeting Wnt16 or Taz (not Yap1 ) phenocopied Wnt16 deficiency, inhibited Wnt16 up-regulation of contractile markers and reversed C3 suppression. Wnt16 deficiency increased the Yap-Taz inhibitory protein Ccm2 in VSM. Moreover, RNAi targeting Yap-Taz inhibitory kinases Lats1/2 or Ccm2 -- but not Ccm3 or Stk3/Stk4 -- elicited responses reciprocal to Wnt16 deficiency. Biotin proximity labeling following expression of Taz-BioID2 fusion protein identified TEAD1, IQGAP3 and specific MAP4K components of the STRIPAK complex as functionally important Wnt16-Taz signaling mediators. TEAD cognates in Acta2 and C3 promoters convey responses to Wnt16 and Taz+Tead1, and Wnt16 treatment increased Taz association with the Acta2 promoter in aortic VSM. Thus, Wnt16 promotes the contractile phenotype and mitochondrial function in VSM and limits AngII aneurysmal remodeling in AscAo. Strategies that augment the VSM Wnt16-Taz relay may mitigate aneurysmal remodeling.

CRYOPRESERVATION OF PRIMARY SPLENOCYTES FROM ANTIBODY PRODUCED MICE AGAINST Bitis arientans SNAKE VENOM

Bitis Arientans are associated with the highest number of morbidity and mortality in Nigeria.The most effective treatments against snakebite is the administration of antivenom.Antibodies that is specific for a single epitope of an antigen are obtain by isolating antibody-secreting lymphocytes. These lymphocytes are found within the Splenocytes cells which can be used for a wide variety of immunology-based applications. However, development of hybridoma cell is rare and time consuming. Therefore, valuable time and cell materials can be saved through cryopreservation. This research aimed at Cryopreservation of primary splenocytes from Antibody produced mice against Bitis arientans snake venom. The LD50 of Bitis Arientans venom toxicity was determined in mice according to World Health Organization guidelines. Antibody production was achieved using six mice after immunization for six weeks and antibody titer were determined by indirect ELISA method. Myeloma cells line X63 Ag8.653 were cultured and Mouse Splenocytes with the highest immune response were removed aseptically by mechanical method and cells viability was determined then the isolated splenocytes were cryopreserved in cryovial tube stored at -80C.The LD50 was found to be 1.8mg/kg and Elisa analysis showed mice 2 and 6 to elicited highest immune response with IgG Concentration 3.1µg/ml and 4.6µg/ml. isolated splenocytes were counted to be 6.5x10 and 1.2x10 cells for mice 2 and 6 and myeloma cells to be 1.95x105 cells.In this finding antibody against Bitis Arientans venom were produced and mouse splenocytes were isolated and cryopreserved. Thus, cryopreserved splenocytes and myeloma can be used for the generation of monoclonal antibody

Omicron BA.1-specific T-cell responses in adults vaccinated with CoronaVac or BNT162b2 in Hong Kong: an observational cohort study

The primary aim of using vaccines in public health responses to SARS-CoV-2 variants of concern is to reduce incidence of severe disease, for which T-cell responses are essential. There is a paucity of data on vaccine-induced T-cell immunity to omicron (B.1.1.529). We aimed to compare SARS-CoV-2 omicron BA.1-specific T-cell responses in adults vaccinated with CoronaVac or BNT162b2. For this observational cohort, we recruited adults (aged ≥18 years) from three vaccination centres in Hong Kong. We included participants from four cohorts (cohort 1: participants who received two doses of either BNT162b2 or CoronaVac, cohort 2: participants who received two doses and a booster, cohort 3: participants who received two doses and a booster and had a breakthrough omicron infection, and cohort 4: participants who had a previous non-omicron infection and subsequently received one dose of vaccine). People with confirmed history of COVID-19 at recruitment were excluded from cohort 1 and cohort 2. We collected blood samples before vaccination (for cohort 1 and 2), 1-month following vaccination (for all cohorts), and during convalescence for cohort 3 and 4) and determined the proportion of IFNγ+CD4+ and IFNγ+CD8+ T cells in peripheral blood against SARS-CoV-2 using flow cytometry with peptide pools of SARS-CoV-2 wild type or omicron BA.1. The primary outcome was proportion of CD4+ and CD8+ T cells against SARS-CoV-2 1 month after exposure (ie, vaccination or breakthrough infection). Overall, between May 21, 2020, and Aug 31, 2021, we recruited 659 participants (231 [35%] men and 428 [65%] women). Of these participants, 428 were included in cohort 1 (214 [50%] received BNT162b2 and 214 [50%] received CoronaVac); 127 in cohort 2 (48 [38%] received all BNT162b2, 40 [31%] received all CoronaVac, and 39 [31%] received two CoronaVac and a booster with BNT162b2); 58 in cohort 3, and 46 in cohort 4 (16 [35%] received CoronaVac and 30 [65%] received BNT162b2). Vaccine-induced T-cell responses to the wild-type and omicron BA.1 variants were generally similar in adults receiving two doses of either CoronaVac (CD4+ cells p=0·33; CD8+ cells p=0·70) or BNT162b2 (CD4+ cells p=0·28; CD8+ cells p=1·0). Using a peptide pool of all structural proteins for stimulation, BNT162b2 induced a higher median frequency of omicron-specific CD4+ T cells in adults younger than 60 years (CD4+ cells 0·012% vs 0·010%, p=0·031; CD8+ cells 0·003% vs 0·000%, p=0·055) and omicron-specific CD8+ T cells in people aged 60 years or older (CD4+ cells 0·015% vs 0·006%, p=0·0070; CD8+ cells 0·007% vs 0·000%, p=0·035). A booster dose of either BNT162b2 or CoronaVac after two doses of CoronaVac boosted waning T-cell responses, but T-cell responses did not exceed those at 1 month after the second dose (CoronaVac CD4+ p=0·41, CD8+ p=0·79; BNT162b2 CD4+ p=0·70 CD8+ p=0·80). The evidence that mRNA and inactivated vaccines based on the ancestral SARS-CoV-2 virus elicited T-cell responses to SARS-CoV-2 omicron variants might explain the high observed vaccine effectiveness against severe COVID-19 shown by both types of vaccine, despite great differences in neutralising antibody responses. The use of either vaccine can be considered if the primary aim is to reduce severity and death caused by the new omicron subvariants; however, BNT162b2 is preferable for adults older than 60 years. The Health and Medical Research Fund Commissioned Research on the Novel Coronavirus Disease and S H Ho Foundation.

Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania.

Despite decades of research devoted to finding a vaccine against leishmaniasis, we are still lacking a safe and effective vaccine for humans. Given this scenario, the search for a new prophylaxis alternative for controlling leishmaniasis should be a global priority. Inspired by leishmanization-a first generation vaccine strategy where live L. major parasites are inoculated in the skin to protect against reinfection-live-attenuated Leishmania vaccine candidates are promising alternatives due to their robust elicited protective immune response. In addition, they do not cause disease and could provide long-term protection upon challenge with a virulent strain. The discovery of a precise and easy way to perform CRISPR/Cas-based gene editing allowed the selection of safer null mutant live-attenuated Leishmania parasites obtained by gene disruption. Here, we revisited molecular targets associated with the selection of live-attenuated vaccinal strains, discussing their function, their limiting factors and the ideal candidate for the next generation of genetically engineered live-attenuated Leishmania vaccines to control leishmaniasis.

Patients’ views on health promotion and disease prevention services provided by healthcare workers in a South African tertiary hospital

BackgroundPatients’ views and experiences in healthcare institutions provide a means of assessing the quality of services patients receive from healthcare workers (HCWs). However, the views of patients on the health promotion (HP) and disease prevention (DP) services offered by HCWs and the delivery mode have not been adequately studied.AimThis study assessed the views of patients on HP and DP services provided by various categories of HCWs.SettingThe study was conducted at a tertiary hospital in the Nelson Mandela Bay Municipality, South Africa.MethodAn exploratory cross-sectional study was conducted among 500 patients. The questionnaire elicited responses from patients regarding the HP and DP services received from the different cadres of HCWs at three different admission phases: pre-admission phase (PAP), admission phase (ADP), and post-admission phase (POP). Descriptive, bivariate, and multivariate analysis was conducted.ResultsIn the PAP, most patients (83.33%, n = 5; 87.85%, n = 217; and 76.14%, n = 150) seen by the rehabilitation health workers, medical doctors, and nurses respectively were empowered to manage their health. Patients attended to by nurses were 0.45 (95% CI 0.27–0.74) times less likely than those attended to by medical doctors to receive information that that will help them address the physical and environmental needs. In the ADP, patients attended to by nurses were less likely, compared to those attended to by medical doctors to be empowered to have good control over their health. In the POP, patients attended to by nurses are more likely to have their health behaviours change for better compared to those not seen by any HCW.ConclusionPatients attending tertiary hospital received greater HP and DP services during the PAP and ADP of patient care. Greatest influence for behavioural change of patients on HP and DP were achieved from the medical doctors, nurses and rehabilitation service staff. Improving structural factors may prove beneficial in enhancing patients’ experience from all HCW groups and phases of patient care.

Collective emotions during the COVID-19 outbreak.

The COVID-19 pandemic has exposed the world's population to unprecedented health threats and changes to social life. High uncertainty about the novel disease and its social and economic consequences, together with increasingly stringent governmental measures against the spread of the virus, likely elicited strong emotional responses. We analyzed the digital traces of emotional expressions in tweets during 5 weeks after the start of outbreaks in 18 countries and six different languages. We observed an early strong upsurge of anxiety-related terms in all countries, which was related to the growth in cases and increases in the stringency of governmental measures. Anxiety expression gradually relaxed once stringent measures were in place, possibly indicating that people were reassured. Sadness terms rose and anger terms decreased with or after an increase in the stringency of measures and remained stable as long as measures were in place. Positive emotion words only decreased slightly and briefly in a few countries. Our results reveal some of the most enduring changes in emotional expression observed in long periods of social media data. Such sustained emotional expression could indicate that interactions between users led to the emergence of collective emotions. Words that frequently occurred in tweets suggest a shift in topics of conversation across all emotions, from political ones in 2019, to pandemic related issues during the outbreak, including everyday life changes, other people, and health. This kind of time-sensitive analyses of large-scale samples of emotional expression have the potential to inform risk communication. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Shatavarin-IV saponin adjuvant elicits IgG and IgG2b responses against Staphylococcus aureus bacterin in a murine model

Asparagus adscendens Roxb. also known as “safed musli” or “shatavari” is a medicinal plant commonly found in South Asian countries. Shatavari is effective for the treatment of gastric ulcers, renal stones, bronchitis, diabetes, diabetic neuropathy, irritable bowel syndrome, alcohol withdrawal and has reported immunostimulatory effects. In this study, the adjuvant potential of Shatavarin-IV saponin against Staphylococcus aureus bacterin in mice was investigated. Shatavarin-IV was evaluated for its toxicity and immunomodulatory potential against S. aureus bacterin in mice. Cellular and humoral immune responses were assessed. Shatavarin-IV was isolated from the fruit extract of Asparagus adscendens. The confirmation of the isolated molecule as Shatavarin-IV was done via TLC-based comparison with the standard molecule. Further, the structure was confirmed by using extensive spectroscopic analyses and comparing the observed data with literature reports. It was found safe up to the dose of 0.1 mg in the mice model. Shatavarin-IV adjuvant elicited IgG and IgG2b responses at the dose of 40 μg against S. aureus bacterin. However, the cell-mediated immune response was lesser as compared with the commercial Quil-A saponin . We demonstrated that Shatavarin-IV saponin adjuvant produced an optimum humoral immune response against S. aureus bacterin. These results highlight the potential of Shatavarin-IV as an adjuvant in a combination adjuvant in vaccine formulations for induction of potent immune response.

Auditory nerve responses to combined optogenetic and electrical stimulation in chronically deaf mice

Objective. Optogenetic stimulation of the auditory nerve offers the ability to overcome the limitations of cochlear implants through spatially precise stimulation, but cannot achieve the temporal precision nor temporal fidelity required for good hearing outcomes. Auditory midbrain recordings have indicated a combined (hybrid) stimulation approach may permit improvements in the temporal precision without sacrificing spatial precision by facilitating electrical activation thresholds. However, previous research has been conducted in undeafened or acutely deafened animal models, and the impact of chronic deafness remains unclear. Our study aims to compare the temporal precision of auditory nerve responses to optogenetic, electrical, and combined stimulation in acutely and chronically deafened animals. Methods. We directly compare the temporal fidelity (measured as percentage of elicited responses) and precision (i.e. stability of response size and timing) of electrical, optogenetic, and hybrid stimulation (varying sub-threshold or supra-threshold optogenetic power levels combined with electrical stimuli) through compound action potential and single-unit recordings of the auditory nerve in transgenic mice expressing the opsin ChR2-H134R in auditory neurons. Recordings were conducted immediately or 2–3 weeks following aminoglycoside deafening when there was evidence of auditory nerve degeneration. Main results. Results showed that responses to electrical stimulation had significantly greater temporal precision than optogenetic stimulation (p < 0.001 for measures of response size and timing). This temporal precision could be maintained with hybrid stimulation, but only when the optogenetic stimulation power used was below or near activation threshold and worsened with increasing optical power. Chronically deafened mice showed poorer facilitation of electrical activation thresholds with concurrent optogenetic stimulation than acutely deafened mice. Additionally, responses in chronically deafened mice showed poorer temporal fidelity, but improved temporal precision to optogenetic and hybrid stimulation compared to acutely deafened mice. Significance. These findings show that the improvement to temporal fidelity and temporal precision provided by a hybrid stimulation paradigm can also be achieved in chronically deafened animals, albeit at higher levels of concurrent optogenetic stimulation levels.

Unmasking the truth: Experimental evidence of facemask compliance in Bangladesh, Kenya, and Nigeria during the COVID-19 pandemic.

High levels of compliance with public health measures are critical to ensure a successful response to the COVID-19 pandemic and other public health emergencies. However, most data on compliance are self-reported and the tendency to overreport due to social desirability could yield biased estimates of actual compliance. A list experiment is a widely used method to estimate social desirability bias in self-reported estimates of sensitive behaviours. We estimate rates of compliance with facemask mandates in Kenya, Nigeria, and Bangladesh using data from phone surveys conducted in March-April 2021. Data on compliance were collected from two different survey modules: a self-reported compliance module (stated) and a list experiment (elicited). We find large gaps between stated and elicited rates of facemask wearing for different groups depending on specific country contexts and high levels of overreporting of facemask compliance in self-reported surveys: there was an almost 40 percentage point gap in Kenya, 30 percentage points in Nigeria, and 20 percentage points in Bangladesh. We also observe differences in rates of self-reported facemask wearing among key groups but not using the elicited responses from the list experiment, which suggest that social desirability bias may vary by demographics. Data collected from self-reported surveys may not be reliable to monitor ongoing compliance with public health measures. Moreover, elicited compliance rates indicate levels of mask wearing are likely much lower than those estimated using self-reported data.

Investigating the impact of visual perspective in a motor imagery-based brain-robot interaction: A pilot study with healthy participants.

Motor Imagery (MI)-based Brain Computer Interfaces (BCI) have raised gained attention for their use in rehabilitation therapies since they allow controlling an external device by using brain activity, in this way promoting brain plasticity mechanisms that could lead to motor recovery. Specifically, rehabilitation robotics can provide precision and consistency for movement exercises, while embodied robotics could provide sensory feedback that can help patients improve their motor skills and coordination. However, it is still not clear whether different types of visual feedback may affect the elicited brain response and hence the effectiveness of MI-BCI for rehabilitation. In this paper, we compare two visual feedback strategies based on controlling the movement of robotic arms through a MI-BCI system: 1) first-person perspective, with visual information that the user receives when they view the robot arms from their own perspective; and 2) third-person perspective, whereby the subjects observe the robot from an external perspective. We studied 10 healthy subjects over three consecutive sessions. The electroencephalographic (EEG) signals were recorded and evaluated in terms of the power of the sensorimotor rhythms, as well as their lateralization, and spatial distribution. Our results show that both feedback perspectives can elicit motor-related brain responses, but without any significant differences between them. Moreover, the evoked responses remained consistent across all sessions, showing no significant differences between the first and the last session. Overall, these results suggest that the type of perspective may not influence the brain responses during a MI- BCI task based on a robotic feedback, although, due to the limited sample size, more evidence is required. Finally, this study resulted into the production of 180 labeled MI EEG datasets, publicly available for research purposes.

Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates.

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 109CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate.

Identification of volatile compounds from fruits aroma and citrus essential oils and their effect on oviposition of Ceratitis capitata (Diptera: Tephritidae).

Ceratitis capitata (Wiedemann), also known as the Mediterranean fruit fly, is one of the most serious pests for several fresh fruit commodities causing extensive fruit losses worldwide. The response of C. capitata adults to fruit and nonfruit volatiles has been extensively studied. However, the linkage between fruit volatiles and the female ovipositional choice has not been fully elucidated. The present study focused on identifying the volatile organic compounds emitted by detached intact fresh fruits (oranges, lemons, bergamots, and apples) and citrus essential oils and evaluate their effect on Mediterranean fruit fly oviposition. There were more than 130 and 45 volatiles compounds detected in fruits odors and citrus essential oils, respectively. The volatile profile of fruits was dominated either from terpenes and terpenoids or from esters of butanoic, hexanoic, and octanoic acids while limonene was by far the most abundant compound in all citrus essential oils. Oviposition of C. capitata was strongly affected by volatiles emanated from both intact fruit and the citrus essential oils. Regarding the volatiles of the intact fruits, the odor of sweet orange elicited strong oviposition responses to females, while bergamot had the least stimulatory effect on oviposition. Bergamot oil also elicited the least oviposition stimulation compared to sweet orange and lemon essential oils. Our discussion elaborates on the role of fruit volatiles on host finding behavior and fruit susceptibility to C. capitata infestation and includes possible practical implication of the above findings.

Recent advances in antigen targeting to antigen-presenting cells in veterinary medicine.

Advances in antigen targeting in veterinary medicine have gained traction over the years as an alternative approach for diseases that remain a challenge for traditional vaccines. In addition to the nature of the immunogen, antigen-targeting success relies heavily on the chosen receptor for its direct influence on the elicited response that will ensue after antigen uptake. Different approaches using antibodies, natural or synthetic ligands, fused proteins, and DNA vaccines have been explored in various veterinary species, with pigs, cattle, sheep, and poultry as the most frequent models. Antigen-presenting cells can be targeted using a generic approach, such as broadly expressed receptors such as MHC-II, CD80/86, CD40, CD83, etc., or focused on specific cell populations such as dendritic cells or macrophages (Langerin, DC-SIGN, XCR1, DC peptides, sialoadhesin, mannose receptors, etc.) with contrasting results. Interestingly, DC peptides show high specificity to DCs, boosting activation, stimulating cellular and humoral responses, and a higher rate of clinical protection. Likewise, MHC-II targeting shows consistent results in enhancing both immune responses; an example of this strategy of targeting is the approved vaccine against the bovine viral diarrhea virus in South America. This significant milestone opens the door to continuing efforts toward antigen-targeting vaccines to benefit animal health. This review discusses the recent advances in antigen targeting to antigen-presenting cells in veterinary medicine, with a special interest in pigs, sheep, cattle, poultry, and dogs.

Distinct in vitro and in vivo neutralization profiles of monoclonal antibodies elicited by the receptor binding domain of the ancestral SARS-CoV-2.

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.

Abstract OT1-20-02: Intralesional injection of anti-PD-1 (pembrolizumab) and OX40L/IL-23/IL-36 mRNAs (mRNA-2752) results in regression of DCIS characterized by lymphocytic infiltrates

Abstract Background: High-grade DCIS with immune infiltrates may represent lesions that are able to be kept in check by the immune system, but still are at risk for progression to invasive disease. We and others have demonstrated that the presence of T cells, and in particular the spatial proximity of CD8+/PD-1+ T cells and PD-L1+ cells predicts response to chemotherapy and PD-1 inhibitors in the setting of invasive triple negative breast cancer. DCIS with high-risk features (e.g. large, palpable, high grade, HR-, or HER2+) often have T cell infiltrates. We hypothesized that it might be possible to potentiate the immune response in high-risk DCIS with immune checkpoint blockade. Since mortality for DCIS is extremely low, we proposed an intralesional pembrolizumab (pembro) treatment approach to avoid systemic adverse effects. In a phase 1 dose escalation study of single agent pembro we found 2 doses of 8 mg, administered intralesionally 2-3 wks apart, was tolerable, induced immunological changes within the DCIS lesions, but had little clinical benefit. Herein, we expanded the number of injections to 4 and also tested a combination with mRNA-2752 (Moderna), an mRNA-based therapeutic encoding a T cell co-stimulator OX40L, and pro-inflammatory cytokines IL-23 and IL-36 gamma, to determine if we could find a dose that was both tolerable and elicited anti-tumor responses. Methods Women eligible for this study had DCIS with at least 2 of the following high-risk features: age&amp;lt; 45; high grade, extensive comedonecrosis; palpable mass; hormone receptor negative [HR-]; HER2+; size &amp;gt;5 cm, or microinvasion. A dose expansion cohort was performed using 4 doses of 8 mg pembro, 2-3 wks apart (n=5). We then combined pembro with mRNA-2752 (n=8 cases), initially at 8 and 4 mg, respectively. Dose reductions were implemented based on AEs. Patients received an MRI before and after 2 injections, spaced 2-3 wks apart. A total of 4 injections were allowed. Core biopsy or surgery was conducted after the last MRI. Results We observed an increase in T cell density in the dose expansion cohort (pembro only), except when there was a paucity of T cells in the pre-treatment biopsy. However, only 1 patient in this cohort demonstrated a reduction in lesion size (clinically and on MRI). As of 22SEP22, 8 patients have received the combination of pembro and mRNA-2752; 7 are evaluable. Two patients with minimal T cell infiltrates at baseline (both HR+) failed to respond, based on imaging or pathology. 4/5 patients with moderate to high T cell infiltrates at baseline had partial (2) or complete responses (2) based on imaging (surgery pending), one of whom had absence of DCIS on post-therapy core biopsy and a clear MRI 4 months later. Correlative studies (immune cell densities, spatial proximities) will be presented at the meeting along with complete data (including post-treatment pathology) on the first 8 patients. Side effects were independent of response and included Gr1/2 fever, myalgias, and fatigue starting within 12 hours of injection (3-4 days), enlargement of regional nodes by day 2, and Gr 1/2 erythema and induration of the breast starting at 4 days and lasting 4-20 days. Earlier onset and persistence of symptoms with subsequent exposure required dose reductions for the majority of patients to maintain tolerability (avoid fevers over 39.4°C, intense erythema and swelling of the breast lasting &amp;gt; 4 days). No AEs were higher than grade 2. Conclusion: The combination of intralesional pembro and mRNA-2752 demonstrated modulation of the tumor immune microenvironment and robust antitumor activity in high-risk DCIS (typically HR- or HER2+) with existing T cell infiltrates. This is an ongoing study; the optimal Phase 2 dose for the combination will be based on the totality of evolving safety and translational data. Citation Format: Laura J. Esserman, Alexa Glencer, Kirithiga Ramalingam, Christopher J. Schwartz, Alexander Borowsky, Gillian L. Hirst, Rachel Woody, Nicole Schindler, Nola M. Hylton, Michael Campbell. Intralesional injection of anti-PD-1 (pembrolizumab) and OX40L/IL-23/IL-36 mRNAs (mRNA-2752) results in regression of DCIS characterized by lymphocytic infiltrates [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-20-02.

In-vivo time course of organ uptake and blood-brain-barrier permeation of poly(L-lactide) and poly(perfluorodecyl acrylate) nanoparticles with different surface properties in unharmed and brain-traumatized rats.

Traumatic brain injury (TBI) has a dramatic impact on mortality and quality of life and the development of effective treatment strategies is of great socio-economic relevance. A growing interest exists in using polymeric nanoparticles (NPs) as carriers across the blood-brain barrier (BBB) for potentially effective drugs in TBI. However, the effect of NP material and type of surfactant on their distribution within organs, the amount of the administrated dose that reaches the brain parenchyma in areas with intact and opened BBB after trauma, and a possible elicited inflammatory response are still to be clarified. The organ distribution, BBB permeation and eventual inflammatory activation of polysorbate-80 (Tw80) and sodiumdodecylsulfate (SDS) stabilized poly(L-lactide) (PLLA) and poly(perfluorodecyl acrylate) (PFDL) nanoparticles were evaluated in rats after intravenous administration. The NP uptake into the brain was assessed under intact conditions and after controlled cortical impact (CCI). A significantly higher NP uptake at 4 and 24 h after injection was observed in the liver and spleen, followed by the brain and kidney, with minimal concentrations in the lungs and heart for all NPs. A significant increase of NP uptake at 4 and 24 h after CCI was observed within the traumatized hemisphere, especially in the perilesional area, but NPs were still found in areas away from the injury site and the contralateral hemisphere. NPs were internalized in brain capillary endothelial cells, neurons, astrocytes, and microglia. Immunohistochemical staining against GFAP, Iba1, TNFα, and IL1β demonstrated no glial activation or neuroinflammatory changes. Tw80 and SDS coated biodegradable PLLA and non-biodegradable PFDL NPs reach the brain parenchyma with and without compromised BBB by TBI, even though a high amount of NPs are retained in the liver and spleen. No inflammatory reaction is elicited by these NPs within 24 h after injection. Thus, these NPs could be considered as potentially effective carriers or markers of newly developed drugs with low or even no BBB permeation.

Suprapontine Structures Modulate Brainstem and Spinal Networks

Several spinal motor output and essential rhythmic behaviors are controlled by supraspinal structures, although their contribution to neuronal networks for respiration and locomotion at birth still requires better characterization. As preparations of isolated brainstem and spinal networks only focus on local circuitry, we introduced the in vitro central nervous system (CNS) from neonatal rodents to simultaneously record a stable respiratory rhythm from both cervical and lumbar ventral roots (VRs).Electrical pulses supplied to multiple sites of brainstem evoked distinct VR responses with staggered onset in the rostro-caudal direction. Stimulation of ventrolateral medulla (VLM) resulted in higher events from homolateral VRs. Stimulating a lumbar dorsal root (DR) elicited responses even from cervical VRs, albeit small and delayed, confirming functional ascending pathways. Oximetric assessments detected optimal oxygen levels on brainstem and cortical surfaces, and histological analysis of internal brain structures indicated preserved neuron viability without astrogliosis. Serial ablations showed precollicular decerebration reducing respiratory burst duration and frequency and diminishing the area of lumbar DR and VR potentials elicited by DR stimulation, while pontobulbar transection increased the frequency and duration of respiratory bursts. Keeping legs attached allows for expressing a respiratory rhythm during hindlimb stimulation. Trains of pulses evoked episodes of fictive locomotion (FL) when delivered to VLM or to a DR, the latter with a slightly better FL than in isolated cords.In summary, suprapontine centers regulate spontaneous respiratory rhythms, as well as electrically evoked reflexes and spinal network activity. The current approach contributes to clarifying modulatory brain influences on the brainstem and spinal microcircuits during development.Graphical Novel preparation of the entire isolated CNS from newborn rats unveils suprapontine modulation on brainstem and spinal networks. Preparation views (A) with and without legs attached (B). Successful fictive respiration occurs with fast dissection from P0-P2 rats (C). Decerebration speeds up respiratory rhythm (D) and reduces spinal reflexes derived from both ventral and dorsal lumbar roots (E).

Influence of female reproductive state and of fishing-capture stress on the oxygen uptake rate of a viviparous elasmobranch.

In animals discarded after a fishing capture event, the elicited stress response necessary to ensure their survival is energetically costly. This energy is diverted from other important biological activities, including growth and reproduction, possibly impairing them. Given that elasmobranchs are among the most threatened vertebrate groups, estimating capture-induced energetic changes and comparing these variations to the energy requirements of pregnancy maintenance is necessary. In pregnant southern fiddler rays (Trygonorrhina dumerilii), we measured changes in oxygen uptake rate (ṀO2 ; a proxy for metabolic rate and energy usage) in response to trawling simulation and air exposure, and estimated the oxygen requirements of sustaining late-term pregnancy and embryos. ṀO2 was measured in pregnant females, before (prestress ṀO2 ) and after trawling simulation (after-capture ṀO2 ), and again after females gave birth (postpartum ṀO2 ). After-capture ṀO2 was 31.7% lower than ṀO2 measured in minimally stressed females, suggesting a reduction in energy expenditure. This reduction is likely triggered by an initially excessive energetic investment in the stress response, and is aimed at shutting down nonessential activities to redirect energy to processes fundamental for survival. Prestress ṀO2 was 78.5% higher than postpartum ṀO2 . Capture simulation decreased ṀO2 to values similar to those observed postpartum, suggesting a capture-induced reduction in oxygen and energy allocation to pregnancy and embryonic respiration, which could be associated with reproductive impairments. These data, by better estimating the impact of capture and discard on energetic requirements and reproductive fitness, may support the introduction of area and/or seasonal closures to fishing.