Correlates of protection against SARS-CoV-2 in rhesus macaques.

Recent studies have reported the protective efficacy of both natural1 and vaccine-induced2-7 immunity against challenge with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in rhesus macaques. However, the importance of humoral and cellular immunity for protection against infection with SARS-CoV-2 remains to be determined. Here we show that the adoptive transfer of purified IgG from convalescent rhesus macaques (Macaca mulatta) protects naive recipient macaques against challenge with SARS-CoV-2 in a dose-dependent fashion. Depletion of CD8+ T cells in convalescent macaques partially abrogated the protective efficacy of natural immunity against rechallenge with SARS-CoV-2, which suggests a role for cellular immunity in the context of waning or subprotective antibody titres. These data demonstrate that relatively low antibody titres are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may contribute to protection if antibody responses are suboptimal. We also show that higher antibody titres are required for treatment of SARS-CoV-2 infection in macaques. These findings have implications for the development of SARS-CoV-2 vaccines and immune-based therapeutic agents.

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

HAIC Combined with lenvatinib plus PD-1 versus lenvatinib Plus PD-1 in patients with high-risk advanced HCC: a real-world study.

BACKGROUND: The treatment of hepatocellular carcinoma (HCC) patients exhibiting high-risk characteristics (Vp4, and/or bile duct invasion, and/or tumor occupancy ≥ 50%) lacks standardized approaches and yields unfavorable results. This study endeavors to evaluate the safety, efficacy, and prognostic impacts of employing hepatic arterial infusion chemotherapy (HAIC), lenvatinib, and humanized programmed death receptor-1 (PD-1) in the treatment of high-risk HCC patients. METHODS: In this retrospective analysis, HCC patients with high-risk features were treated with either lenvatinib combined with PD-1 (LEN-PD1) or a combination of HAIC, lenvatinib, and PD-1 (HAIC-LEN-PD1). The study assessed the antitumor efficacy by calculating overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR). Treatment-related adverse events (TRAEs) were analyzed to assess the safety profiles. RESULTS: Between June 2019 and September 2022, a total of 61 patients were included in the LEN-PD1 group, while 103 patients were enrolled in the HAIC-LEN-PD1 group. The OS was 9.8 months in the LEN-PD1 group, whereas the HAIC-LEN-PD1 group exhibited a significantly longer median OS of 19.3 months (HR = 0.43, p < 0.001). Furthermore, PFS was notably extended in the HAIC-LEN-PD1 group compared to the LEN-PD1 group (9.6 months vs. 4.9 months, HR = 0.48, p < 0.001). Patients in the HAIC-LEN-PD1 group had a higher ORR and DCR according to the modified RECIST (76.7% vs. 23.0%, p < 0.001; 92.2% vs. 72.1%, p = 0.001). HAIC-LEN-HAIC group led to more adverse events than LEN-PD1 group, most of which were tolerable and controllable. CONCLUSION: Lenvatinib, HAIC and PD-1 showed safe and promising anti-tumor activity compared with lenvatinib alone for HCC with high-risk features.

Systemic Chemotherapy with or without Hepatic Arterial Infusion Chemotherapy for Intrahepatic Cholangiocarcinoma with Extrahepatic Oligometastasis: A Propensity Score-Matched Analysis.

PURPOSE: To compare the effectiveness of hepatic arterial infusion chemotherapy (HAIC) plus systemic chemotherapy (SYS) with that of SYS alone in patients with intrahepatic cholangiocarcinoma (ICC) with extrahepatic oligometastasis in terms of overall survival (OS) and mortality related to liver failure. MATERIALS AND METHODS: Consecutive patients diagnosed with ICC with extrahepatic oligometastasis who received either HAIC plus SYS or SYS alone between January 2019 and January 2021 were included in this retrospective cohort study. Propensity score matching (PSM) analysis was performed to address potential confounding factors. OS, progression-free survival (PFS), and intrahepatic progression-free survival (IPFS) were analyzed. The occurrence of death due to liver failure was also assessed. RESULTS: The study included a total of 179 patients, with 96 receiving SYS alone and 83 receiving HAIC plus SYS. After PSM, 83 pairs were included for further analysis. The median OS and IPFS were significantly longer in the HAIC plus SYS group compared to the SYS alone group (OS: 15.8 months vs 12.7 months; P = .023; IPFS: 9.7 vs 6.1 months; P < .001). No difference was found in PFS between the 2 groups. The HAIC plus SYS group had a significantly lower rate of mortality due to liver failure compared to the SYS alone group (42% vs 72%; P = .002). CONCLUSIONS: HAIC plus SYS is a promising treatment approach for patients with ICC and extrahepatic oligometastasis with improved OS, IPFS, and freedom from liver failure mortality compared with SYS alone.

Impact of prior Dengue immunity on Zika vaccine protection in rhesus macaques and mice.

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 -/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.

IL-33-expressing microvascular endothelial cells in human esophageal squamous cell carcinoma: Implications for pathological features and prognosis.

Accumulating evidence suggests that interleukin (IL)-33 plays a critical role in regulating angiogenesis and cancer progression. In this study, we characterized the pathological importance of IL-33 deployed by tumor microvascular endothelial cells (ECs) in human esophageal squamous cell carcinoma (ESCC). The expression of IL-33 in microvascular ECs in 80 cases of ESCC was examined with immunohistochemistry (IHC) and double immunofluorescence. IHC results showed that strong IL-33-immunoreactivity (IR) in microvessels, which were confirmed to be ECs by double immunofluorescence staining with IL-33/CD31 antibodies. Moreover, high proliferative activity was shown in IL-33-positive ECs, and the IL-33 functional receptor ST2 was expressed in microvascular ECs. Clinicopathological analysis revealed that IL-33-positive microvessel density (MVD) was positively correlated with node involvement in patients with ESCC. A log rank test showed a highly significant inverse correlation between the densities of IL-33-positive MVDs and overall survival rate, and patients with higher IL-33-positive MVDs tended to have a lower survival rate (both p < 0.05). Therefore, we concluded that IL-33 deployed by microvascular ECs correlates with advanced pathological features and the long-term survival rate, which provides new insights into the regulatory mechanisms of tumor angiogenesis in the tumor microenvironment and might serve as a promising target in patients with ESCC.

Hemodynamic Study of Stanford Type B Aortic Dissection Based on Ex Vivo Porcine Aorta Models.

BACKGROUND: In this study, we aimed to evaluate hemodynamic influence of the dissected aortic system via various ex vivo type B aortic dissection (AD) models. METHODS: Twenty-four raw porcine aortas were harvested and randomly divided into 4 groups to create various aortic models. Model A was the control group, while models B to D indicated the AD group, where models B and C presented a proximal primary entry with the false lumen (FL) lengths of 15 and 20 cm, respectively, and model D presented a 20 cm FL with a proximal primary entry and a distal reentry. All the aortic models were connected to a mock circulation loop to attain the realistic flow and pressure status. The flow distribution rate (FDR) of the aortic branches was calculated. Doppler ultrasound was applied to visualize the AD structure and to attain the velocity of flow in both the true and false lumens. Several sections of the AD were stained with hematoxylin and eosin for histologic evaluation after the experiment. RESULTS: This study demonstrated that higher pressures were found for the AD group compared with the control group. The mean systolic pressures at the inlet of models A to D were 113.34±0.81, 120.58±0.52, 117.76±0.82, and 115.87±0.42 mm Hg, respectively. The FDRs of the celiac artery in models A to D were 8.65%, 8.32%±0.15%, 7.87%±0.13%, and 8.03%±0.21%, respectively. By ultrasound visualization, the velocity of the flow at the entry to the FL in the AD group ranged in 10 to 92 cm/s. The dissection flap presented pulsatile movement, especially in the models B and C which contained 1 primary entry without distal reentries. Histological examinations indicated that AD was located between the intimal and medial layers. CONCLUSIONS: Our ex vivo models demonstrated that the configuration of the dissected aorta influenced the pressure distribution. Moreover, the dissection flap affected the FDR of the aortic branches that possibly inducing malperfusion syndrome.

Molecular characterization of a novel amalgavirus infecting lilium spp. in China.

A novel plant virus with a double-stranded (ds) RNA genome was detected in Lilium spp. in China by high-throughput sequencing and tentatively named "lily amalgavirus 2" (LAV2). The genomic RNA of LAV2 is 3432 nucleotides (nt) in length and contains two open reading frames (ORFs) that putatively encode a '1 + 2' fusion protein of 1053 amino acids (aa), generated by a '+1' programmed ribosomal frameshift (PRF). ORF1 encodes a putative 386-aa protein of unknown function, and ORF2 overlaps ORF1 by 350 nt and encodes a putative 783-aa protein with conserved RNA-dependent RNA polymerase (RdRp) motifs. The '+1' ribosomal frameshifting motif, UUU_CGN, which is highly conserved among amalgaviruses, is also found in LAV2. Sequence analysis showed that the complete genome shared 46.04%-51.59% nucleotide sequence identity with those of members of the genus Amalgavirus and had the most similarity (51.59% sequence identity) to lily amalgavirus 1 (accession no. OM782323). Phylogenetic analysis based on RdRp amino acid sequences showed that LAV2 clustered with members of the genus Amalgavirus. Overall, our data suggest that LAV2 is a new member of the genus Amalgavirus.

Reconstruction of massive bone defects after femoral tumor resection using two new-designed 3D-printed intercalary prostheses: a clinical analytic study with the cooperative utilization of multiple technologies.

BACKGROUND: To reconstruct massive bone defects of the femoral diaphysis and proximal end with limited bilateral cortical bone after joint-preserving musculoskeletal tumor resections, two novel 3D-printed customized intercalary femoral prostheses were applied. METHODS: A series of nine patients with malignancies who received these novel 3D-printed prostheses were retrospectively studied between July 2018 and November 2021. The proximal and diaphyseal femur was divided into three regions of interest (ROIs) according to anatomic landmarks, and anatomic measurements were conducted on 50 computed tomography images showing normal femurs. Based on the individual implant-involved ROIs, osteotomy level, and anatomical and biomechanical features, two alternative 3D-printed prostheses were designed. In each patient, Hounsfield Unit (HU) value thresholding and finite element analysis were conducted to identify the bone trabecula and calcar femorale and to determine the stress distribution, respectively. We described the characteristics of each prosthesis and surgical procedure and recorded the intraoperative data. All patients underwent regular postoperative follow-up, in which the clinical, functional and radiographical outcomes were evaluated. RESULTS: With the ROI division and radiographic measurements, insufficient bilateral cortical bones for anchoring the traditional stem were verified in the normal proximal femur. Therefore, two 3D-printed intercalary endoprostheses, a Type A prosthesis with a proximal curved stem and a Type B prosthesis with a proximal anchorage-slot and corresponding locking screws, were designed. Based on HU value thresholding and finite element analysis, the 3D-printed proximal stems in all prostheses maximally preserved the trabecular bone and calcar femorale and optimized the biomechanical distribution, as did the proximal screws. With the 3D-printed osteotomy guide plates and reaming guide plates, all patients underwent the operation uneventfully with a satisfactory duration (325.00 ± 62.60 min) and bleeding volume (922.22 ± 222.36 ml). In the follow-up, Harris Hip and Musculoskeletal Tumor Society scores were ameliorated after surgery (P < 0.001 and P < 0.001, respectively), reliable bone ingrowth was observed, and no major complications occurred. CONCLUSIONS: Two novel 3D-printed femoral intercalary prostheses, which achieved acceptable overall postoperative outcomes, were used as appropriate alternatives for oncologic patients with massive bone defects and limited residual bone and increased the opportunities for joint-preserving tumor resection. Several scientific methodologies utilized in this study may promote the clinical design proposals of 3D-printed implants.

A novel three-dimensional-printed patient-specific sacral implant for spinopelvic reconstruction in sacral giant cell tumour.

PURPOSE: Spinopelvic reconstruction after sacral tumour resection is one of the most demanding procedures in sacral tumour surgery. The aims of this study were to evaluate the feasibility of spinopelvic reconstruction with 3D-printed prostheses in sacral giant cell tumours and the clinical outcomes and complications at follow-up. METHODS: We retrospectively analyzed ten consecutive patients with giant cell tumors of the sacrum who underwent intralesional nerve-sparing resection with curative intent and custom implant reconstruction between 2016 and 2021. There were four males and six females with a mean age of 40.2 years (range, 25-62 years) at surgery. A computer-aided-design implant was prepared using 3D printing technology that was both matched to the bone defect and biomechanically evaluated. A 3D-printed surgical guide was used to replicate the resection procedure as planned. We analyzed operational outcomes, oncological outcomes, functional outcomes, complications, and prosthetic outcomes. Pain at rest was assessed according to a 10-cm VAS score. The results of functional improvement were evaluated using the MSTS-93 score at the final follow-up. RESULTS: All patients were observed for 26 to 61 months, with an average follow-up of 43.8 months. No deep infection or prosthetic structural failure occurred in this study. A total of 80% of patients had good neurological function and normal urinary, bowel, and ambulatory functions. The mean MSTS score was 24.1 (range, 22-26). The mean VAS score was 2 (range 0 to 2). Delayed wound healing occurred in three patients, and the wounds healed after debridement. One case had local recurrence and survived tumour-free after resection of the recurrent lesion. An aseptic loosening was found in a patient that did not require secondary surgery. By radiographical assessments, we found that 90% of implants were well osseointegrated at the final follow-up examination. CONCLUSIONS: The 3D-printed sacral implants might provide a promising strategy for spinopelvic reconstruction in sacral giant cell tumours undergoing intralesional nerve-sparing surgery with satisfactory clinical outcomes, osseointegration, and excellent durability.

Effectively predicting HIV-1 protease cleavage sites by using an ensemble learning approach.

BACKGROUND: The site information of substrates that can be cleaved by human immunodeficiency virus 1 proteases (HIV-1 PRs) is of great significance for designing effective inhibitors against HIV-1 viruses. A variety of machine learning-based algorithms have been developed to predict HIV-1 PR cleavage sites by extracting relevant features from substrate sequences. However, only relying on the sequence information is not sufficient to ensure a promising performance due to the uncertainty in the way of separating the datasets used for training and testing. Moreover, the existence of noisy data, i.e., false positive and false negative cleavage sites, could negatively influence the accuracy performance. RESULTS: In this work, an ensemble learning algorithm for predicting HIV-1 PR cleavage sites, namely EM-HIV, is proposed by training a set of weak learners, i.e., biased support vector machine classifiers, with the asymmetric bagging strategy. By doing so, the impact of data imbalance and noisy data can thus be alleviated. Besides, in order to make full use of substrate sequences, the features used by EM-HIV are collected from three different coding schemes, including amino acid identities, chemical properties and variable-length coevolutionary patterns, for the purpose of constructing more relevant feature vectors of octamers. Experiment results on three independent benchmark datasets demonstrate that EM-HIV outperforms state-of-the-art prediction algorithm in terms of several evaluation metrics. Hence, EM-HIV can be regarded as a useful tool to accurately predict HIV-1 PR cleavage sites.

Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays.

Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL-3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL-2 adapted SARS-CoV-2 pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed SΔCT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells as compared with full-length spike (S). We further compared S and SΔCT13 in terms of their sensitivity to vaccine sera, purified convalescent IgG, hACE2-mIgG, and the virus entry inhibitor BafA1. We developed a SΔCT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra- and intermediate-assay precision. Our data demonstrate that the SΔCT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge.IMPORTANCESARS-CoV-2 is the etiologic agent of the COVID-19 pandemic. The development of a high throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This SΔCT13-based pseudovirus neutralization assay should be broadly useful for the field.

Protective Efficacy of Rhesus Adenovirus COVID-19 Vaccines against Mouse-Adapted SARS-CoV-2.

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice. IMPORTANCE We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibit a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative adenovirus (Ad) vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to (i) evaluate the protective efficacy of RhAd52 vaccines and (ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate that RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

A Mock Circulation Loop for In Vitro Hemodynamic Evaluation of Aorta: Application in Aortic Dissection.

PURPOSE: Aortic dissection (AD) is a catastrophic disease with complex hemodynamic conditions, however, understandings regarding its perfusion characteristics were not sufficient. In this study, a mock circulation loop (MCL) that integrated the Windkessel element and patient-specific silicone aortic phantoms was proposed to reproduce the aortic flow environment in vitro. MATERIALS AND METHODS: Patient-specific normal and dissected aortic phantoms with 12 branching vessels were established and embedded into this MCL. Velocities for aortic branches based on 20 healthy volunteers were regarded as the standardized data for flow division. By altering boundary conditions, the proposed MCL could mimic normal resting and left-sided heart failure (LHF) conditions. Flow rates and pressure status of the aortic branches could be quantified by separate sensors. RESULTS: In normal resting condition, the simulated heart rate and systemic flow rate were 60 bpm and 4.85 L/minute, respectively. For the LHF condition, the systolic and diastolic blood pressures were 75.94±0.77 mmHg and 57.65±0.35 mmHg, respectively. By tuning the vascular compliance and peripheral resistance, the flow distribution ratio (FDR) of each aortic branch was validated by the standardized data in the normal aortic phantom (mean difference 2.4%±1.70%). By comparing between the normal and dissected aortic models under resting condition, our results indicated that the AD model presented higher systolic (117.82±0.60 vs 108.75±2.26 mmHg) and diastolic (72.38±0.58 vs 70.46±2.33 mmHg) pressures, the time-average velocity in the true lumen (TL; 36.95 cm/s) was higher than that in the false lumen (FL; 22.95 cm/s), and the blood transport direction between the TL and FL varied in different re-entries. CONCLUSIONS: The proposed MCL could be applied as a research tool for in vitro hemodynamic analysis of the aorta diseases under various physical conditions.

Vaccine protection against Zika virus from Brazil.

Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable.

Nanobodies for Accurate Recognition of Iso-tenuazonic Acid and Development of Sensitive Immunoassay for Contaminant Detection in Foods.

The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.

Sortase-Mediated Phage Decoration for Analytical Applications.

Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL-1, respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.

Differential Outcomes following Optimization of Simian-Human Immunodeficiency Viruses from Clades AE, B, and C.

Simian-human immunodeficiency virus (SHIV) infection of rhesus monkeys is an important preclinical model for human immunodeficiency virus type 1 (HIV-1) vaccines, therapeutics, and cure strategies. SHIVs have been optimized by incorporating HIV-1 Env residue 375 mutations that mimic the bulky or hydrophobic residues typically found in simian immunodeficiency virus (SIV) Env to improve rhesus CD4 binding. We applied this strategy to three SHIV challenge stocks (SHIV-SF162p3, SHIV-AE16, and SHIV-325c) and observed three distinct outcomes. We constructed six Env375 variants (M, H, W, Y, F, and S) for each SHIV, and we performed a pool competition study in rhesus monkeys to define the optimal variant for each SHIV prior to generating large-scale challenge stocks. We identified SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH as the optimal variants. SHIV-SF162p3S could not be improved, as it already contained the optimal Env375 residue. SHIV-AE16W exhibited a similar replicative capacity to the parental SHIV-AE16 stock. In contrast, SHIV-325cH demonstrated a 2.6-log higher peak and 1.6-log higher setpoint viral loads than the parental SHIV-325c stock. These data demonstrate the diversity of potential outcomes following Env375 modification in SHIVs. Moreover, the clade C SHIV-325cH challenge stock may prove useful for evaluating prophylactic or therapeutic interventions against clade C HIV-1.IMPORTANCE We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

Alpha-defensin 5 differentially modulates adenovirus vaccine vectors from different serotypes in vivo.

Adenoviral vectors have shown significant promise as vaccine delivery vectors due to their ability to elicit both innate and adaptive immune responses. α-defensins are effector molecules of the innate immune response and have been shown to modulate natural infection with adenoviruses, but the majority of α-defensin-adenovirus interactions studied to date have only been analyzed in vitro. In this study, we evaluated the role of α-defensin 5 (HD5) in modulating adenovirus vaccine immunogenicity using various serotype adenovirus vectors in mice. We screened a panel of human adenoviruses including Ad5 (species C), Ad26 (species D), Ad35 (species B), Ad48 (species D) and a chimeric Ad5HVR48 for HD5 sensitivity. HD5 inhibited transgene expression from Ad5 and Ad35 but augmented transgene expression from Ad26, Ad48, and Ad5HVR48. HD5 similarly suppressed antigen-specific IgG and CD8+ T cell responses elicited by Ad5 vectors in mice, but augmented IgG and CD8+ T cell responses and innate cytokine responses elicited by Ad26 vectors in mice. Moreover, HD5 suppressed the protective efficacy of Ad5 vectors but enhanced the protective efficacy of Ad26 vectors expressing SIINFEKL against a surrogate Listeria-OVA challenge in mice. These data demonstrate that HD5 differentially modulates adenovirus vaccine delivery vectors in a species-specific manner in vivo.