E2 Antibody Research Articles

Development of an indirect ELISA for the immunoprotection evaluation of E2 antibodies against classical swine fever virus

The Chinese government's reclassification of Classical Swine Fever (CSF) from a class Ⅰ to a class Ⅱ animal infectious disease, now also including CSF under the disease eradication program, reflects the significant progress made through extensive immunization with CSF vaccines. In light of this advancement, there is an imperative need for an expedient and accurate method to assess the levels of immunoprotection against classical swine fever virus (CSFV) in vaccinated pigs, a critical component in the campaign to eradicate the disease. This study develops an indirect enzyme-linked immunosorbent assay (iELISA) based on a highly glycosylated E2 protein stable expressed in CHO-K1 mammalian cells. Statistical analysis revealed strong positive correlations between the iELISA and VNT results (r = 0.9063, p < 0.0001) that were much greater than those between the IDEXX ELISA and VNT results (r = 0.8126, p < 0.0001). Taking the VNT data as the standard, the consistency of the iELISA (κ =0.880) was greater than that of the IDEXX ELISA (κ =0.699). In summary, the iELISA provides a more efficient and precise method for assessing CSFV immunity in pigs. Its reliable detection of immunoprotection levels against CSFV makes it an essential tool for optimizing CSF vaccination strategies. Consequently, its application can significantly support the ongoing efforts to eradicate CSF.

Phase II trial assessing safety, efficacy and immune correlates of heterologous prime-boost with pBI-11 (IM) and TA-HPV (IM) plus pembrolizumab for advanced, PD-L1 CPS≥1, hrHPV+ oropharyngeal cancer.

TPS6118 Background: HPV-related OPSCC occurs in younger patients, has a significantly better prognosis, and is most often caused by HPV subtype 16. There is a 90% overall survival for HPV+/p16+ OPSCC (40% for HPV-/p16- OPSCC), which can be cured with multimodality care. For patients with R/M disease, treatment with pembrolizumab (P) +/- chemotherapy is palliative, with a median OS of approximately 12 months for patients with PD-L1 combined positive score (cps) &gt;1 treated with P alone. Heterologous prime boost with DNA priming followed by vaccinia-based boosting against HPV16 viral antigens will be studied using pBI-11 [(DNA vaccine pNGVL4a-SigE7 (detox)/HSP70, HPV16 E7(detox)/ HPV18 E7(detox), HPV16 E6(detox), and HPV18 E6(detox)] followed by TA-HPV (recombinant vaccinia-human papillomavirus (denoted TA-HPV) derived from the Wyeth strain of vaccinia which carries modified E6 and E7 genes from HPV types 16 and 18). This study is approved by the Vanderbilt University IRB and is open accrual (NCT NCT05799144). Methods: Patients with R/M, HPV positive OPSCC without prior therapy for R/M disease, without contraindications to immunotherapy and with a PD-L1 cps &gt; 1 will be screened. There is a 6 patient safety run-in. Treatment with pBI-11 vaccine (2 IM injections on weeks 1 and 4) plus one IM administration of TA-HPV on week 7 will be given in combination with P IV on weeks 1, 4, and 7). Following restaging, all patients will continue (including those with PD) with 3 more cycles of P followed by restaging. The primary clinical outcome is RR to addition of vaccine in the 50% of predicted P non-responders (NR) who proceed to Part II (ie conversion to responders). All responders will continue to receive P until progression. Approximately 54 patients with be enrolled. Two research tumor biopsies and blood draws will be obtained pre- and on treatment (week7-9). Tumor tissue cores will be utilized for immunohistochemical and molecular tests (e.g., expression of immune cell and viral markers, TCR sequencing and total transcripts). Blood with be tested for HPV16/18 E6, E7 antibodies and vaccinia virus neutralizing assays; proliferative responses of peripheral blood lymphocytes to stimulation by HPV16/18 E6 and E7; HPV16/18 E6- and E7-Specific T cells; and HPV DNA in plasma. Data from the 1st four patients enrolled will be presented. Clinical trial information: NCT05799144 .

A liposome encapsulated methylene blue-mediated electrochemical and UV–visible dual mode split-type immunoassay for the detection of 17β-estradiol

Herein, we fabricated an electrochemical (EC) and UV–visible absorption (UV–vis) dual mode split-type immunoassay for the detection of 17β-estradiol (E2), which was mediated by liposome encapsulated methylene blue (MB@lip). MB molecule acted as the probe in the EC and UV–vis absorption dual mode detections, and its release was controlled by liposome. The competitive immune recognition was conducted between the E2 in the sample and E2 conjugated bovine serum protein (E2-BSA) adsorbed on the 96-wells plate in combining with E2 antibody labeled with MB@lip (E2-Ab/MB@lip). MB molecule could be released from the resulting immune composite of E2-BSA/E2-Ab/MB@lip in the presence of Triton X-100, and quantified by UV–vis and EC methods. The three-dimensional cross-linked reduced graphene oxide/Ti3C2 (3D-rGO/Ti3C2) aerogel was prepared through hydrothermal method, then complexed with the electroactive anthraquinone (AQ) and used as the electrode modified material. The AQ/3D-rGO/Ti3C2 composite had high surface area and provided abundant adsorption sites for MB, and the displacement/competitive behavior between AQ and MB could dexterously achieve the ratiometric EC detection of E2. In addition, the inherent blue color of MB allowed it to be analyzed by UV–vis absorption method. The proposed dual mode detection method exhibited broad linear ranges of 0.1 pg mL−1 to 50 ng mL−1 (by UV–vis) and 0.03 pg mL−1 to 50 ng mL−1 (by EC) for E2 detection, and the detection limits were 0.023 pg mL−1 (S/N = 3) and 8.0 fg mL−1 (S/N = 3), respectively. Moreover, the proposed immunoassay exhibited good practicability and was applied to monitor E2 in milk and serum successfully.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

A single-point mutation in the rubella virus E1 glycoprotein promotes rescue of recombinant vesicular stomatitis virus.

Rubella virus (RuV) is an enveloped plus-sense RNA virus and a member of the Rubivirus genus. RuV infection in pregnant women can lead to miscarriage or an array of severe birth defects known as congenital rubella syndrome. Novel rubiviruses were recently discovered in various mammals, highlighting the spillover potential of other rubiviruses to humans. Many features of the rubivirus infection cycle remain unexplored. To promote the study of rubivirus biology, here, we generated replication-competent recombinant VSV-RuV (rVSV-RuV) encoding the RuV transmembrane glycoproteins E2 and E1. Sequencing of rVSV-RuV showed that the RuV glycoproteins acquired a single-point mutation W448R in the E1 transmembrane domain. The E1 W448R mutation did not detectably alter the intracellular expression, processing, glycosylation, colocalization, or dimerization of the E2 and E1 glycoproteins. Nonetheless, the mutation enhanced the incorporation of RuV E2/E1 into VSV particles, which bud from the plasma membrane rather than the RuV budding site in the Golgi. Neutralization by E1 antibodies, calcium dependence, and cell tropism were comparable between WT-RuV and either rVSV-RuV or RuV containing the E1 W448R mutation. However, the E1 W448R mutation strongly shifted the threshold for the acid pH-triggered virus fusion reaction, from pH 6.2 for the WT RuV to pH 5.5 for the mutant. These results suggest that the increased resistance of the mutant RuV E1 to acidic pH promotes the ability of viral envelope proteins to generate infectious rVSV and provide insights into the regulation of RuV fusion during virus entry and exit.IMPORTANCERubella virus (RuV) infection in pregnant women can cause miscarriage or severe fetal birth defects. While a highly effective vaccine has been developed, RuV cases are still a significant problem in areas with inadequate vaccine coverage. In addition, related viruses have recently been discovered in mammals, such as bats and mice, leading to concerns about potential virus spillover to humans. To facilitate studies of RuV biology, here, we generated and characterized a replication-competent vesicular stomatitis virus encoding the RuV glycoproteins (rVSV-RuV). Sequence analysis of rVSV-RuV identified a single-point mutation in the transmembrane region of the E1 glycoprotein. While the overall properties of rVSV-RuV are similar to those of WT-RuV, the mutation caused a marked shift in the pH dependence of virus membrane fusion. Together, our studies of rVSV-RuV and the identified W448R mutation expand our understanding of rubivirus biology and provide new tools for its study.

Establishment and application of an indirect ELISA for Getah virus E2 antibody detection

Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.

Detection of serum biomarkers of HPV-16 driven oropharynx and oral cavity cancer in Brazil

BackgroundHPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. MethodsHPV was genotyped by PCR-hybridization. All HPV DNA positive and ∼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. ResultsThe prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. ConclusionsOur findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.

Helicobacter pylori-induced fibroblast-derived Serpin E1 promotes gastric cancer growth and peritoneal dissemination through p38 MAPK/VEGFA-mediated angiogenesis

BackgroundFibroblasts, especially cancer-associated fibroblasts (CAFs), represent the predominant stromal cell population in the tumor microenvironment and have an important function in tumorigenesis by interacting with tumor cells. However, their interaction remains elusive in an inflammatory tumor microenvironment induced by Helicobacter pylori (H. pylori).MethodsThe expression of Serpin family E member 1 (Serpin E1) was measured in fibroblasts with or without H. pylori infection, and primary gastric cancer (GC) cells. Serpin E1 knockdown and overexpression fibroblasts were generated using Serpin E1 siRNA or lentivirus carrying Serpin E1. Co-culture models of fibroblasts and GC cells or human umbilical vein endothelial cells (HUVECs) were established with direct contact or the Transwell system. In vitro functional experiments and in vivo tumorigenesis assay were employed to study the malignant behaviors of GC cells interacting with fibroblasts. ELISA was used for quantifying the levels of Serpin E1 and VEGFA in the culture supernatant. The tube formation capacity of HUVECs was assessed using a tube formation assay. Recombinant human Serpin E1 (recSerpin E1), anti-Serpin E1 antibody, and a MAPK pathway inhibitor were utilized to treat HUVECs for elucidating the underlying molecular mechanisms.ResultsSerpin E1 was predominantly expressed in gastric CAFs. H. pylori infection significantly enhanced the expression and secretion of Serpin E1 by CAFs. Both fibroblast-derived Serpin E1 and recSerpin E1 enhanced the growth, invasion, and migration of GC cells, along with increased VEGFA expression and tube formation in HUVECs. Furthermore, the co-inoculation of GC cells and fibroblasts overexpressing Serpin E1 triggered the expression of Serpin E1 in cancer cells, which facilitated together xenograft tumor growth and peritoneal dissemination of GC cells in nude mice, with an increased expression of Ki67, Serpin E1, CD31 and/or VEGFA. These processes may be mediated by Serpin E1-induced migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs.ConclusionH. pylori infection induces Serpin E1 expression in fibroblasts, subsequently triggering its expression in GC cells through their interaction. Serpin E1 derived from these cells promotes the migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs, thereby facilitating GC growth and peritoneal metastasis. Targeting Serpin E1 signaling is a potential therapy strategy for H. pylori-induced GC.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Elucidation of novel SNPs affecting immune response to classical swine fever vaccination in pigs using immunogenomics approach.

The host genetic makeup plays a significant role in causing the within-breed variation among individuals after vaccination. The present study was undertaken to elucidate the genetic basis of differential immune response between high and low responder Landlly (Landrace X Ghurrah) piglets vis-à-vis CSF vaccination. For the purpose, E2 antibody response against CSF vaccination was estimated in sampled animals on the day of vaccination and 21-day post-vaccination as a measure of humoral immune response. Double-digestion restriction associated DNA (ddRAD) sequencing was undertaken on 96 randomly chosen Landlly piglets using Illumina HiSeq platform. SNP markers were called using standard methodology. Genome-wide association study (GWAS) was undertaken in PLINK program to identify the informative SNP markers significantly associated with differential immune response. The results revealed significant SNPs associated with E2 antibody response against CSF vaccination. The genome-wide informative SNPs for the humoral immune response against CSF vaccination were located on SSC10, SSC17, SSC9, SSC2, SSC3 and SSC6. The overlapping and flanking genes (500Kb upstream and downstream) of significant SNPs were CYB5R1, PCMTD2, WT1, IL9R, CD101, TMEM64, TLR6, PIGG, ADIPOR1, PRSS37, EIF3M, and DNAJC24. Functional enrichment and annotation analysis were undertaken for these genes in order to gain maximum insights into the association of these genes with immune system functionality in pigs. The genetic makeup was associated with differential immune response against CSF vaccination in Landlly piglets while the identified informative SNPs may be used as suitable markers for determining variation in host immune response against CSF vaccination in pigs.

Safety and Immunogenicity of Chimeric Pestivirus KD26_E2LOM in Piglets and Calves.

A chimeric pestivirus (KD26_E2LOM) was prepared by inserting the E2 gene of the classical swine fever virus (CSFV) LOM strain into the backbone of the bovine viral diarrhea virus (BVDV) KD26 strain. KD26_E2LOM was obtained by transfecting the cDNA pACKD26_E2LOM into PK-15 cells. KD26_E2LOM chimeric pestivirus proliferated to titers of 106.5 TCID50/mL and 108.0 TCID50/mL at 96 h post-inoculation into PK-15 cells or MDBK cells, respectively. It also reacted with antibodies specific for CSFV E2 and BVDV Erns, but not with an anti-BVDV E2 antibody. Piglets (55-60 days old) inoculated with a high dose (107.0 TCID50/mL) of KD26_E2LOM produced high levels of CSFV E2 antibodies. In addition, no co-habiting pigs were infected with KD26_E2LOM; however, some inoculated pigs excreted the virus, and the virus was detected in some organs. When pregnant sows were inoculated during the first trimester (55-60 days) with a high dose (107.0 TCID50/mL) of KD26_E2LOM, anti-CSFV E2 antibodies were produced at high levels; chimeric pestivirus was detected in one fetus and in the ileum of one sow. When 5-day-old calves that did not consume colostrum received a high dose (107.0 TCID50/mL) of KD26_E2LOM, one calf secreted the virus in both feces and nasal fluid on Day 2. A high dose of KD26_E2LOM does not induce specific clinical signs in most animals, does not spread from animal to animal, and generates CSFV E2 antibodies with DVIA functions. Therefore, chimeric pestivirus KD26_E2LOM is a potential CSFV live marker vaccine.

B-017 The Effect of Temperature on the Performance of Streptavidin-coated Paramagnetic Particles in Immunoassay Applications

Abstract Background Streptavidin-coated paramagnetic particles (streptavidin particles) are widely used in clinical immunoassays. Streptavidin has the highest affinity with biotin among all known non-covalent interaction we know today. With biotinylated molecules, be they small or large, strong binding of such molecules can be brought to streptavidin in a reliable and robust manner. In addition, streptavidin itself is a stable molecule, more resistant to heat, proteolysis, and chemical denaturation than a typical protein (1). Many publications illustrate the binding sites, binding forces, and configurations of streptavidin with or without biotin (2, 3). From our experience working with streptavidin particles in immunoassays, we know that the challenges to their applications are mainly with baseline signal, biotin interference, and thermal stability. This abstract describes our findings on the thermal stability of assay reagents made from streptavidin particles. Methods Intrigued by a recent publication that reports the effect of temperature on the affinity and stoichiometry of streptavidin with biotin (1), suggesting streptavidin’s binding capacity as a function of temperature in the range of 4°C to 40°C, we coupled streptavidin particles (Dynabeads T1 type) with biotinylated thyroxine hormone (T4), estradiol (E2), folate binding protein (FBP), and anti NT-proBNP antibody at varied temperatures between 4°C and 40°C, and then evaluated their coating capacity and thermal stability at 30°C or 37°C. The detection was alkaline phosphatase conjugates with anti T4 antibody, folate analogue, anti E2 antibody, and anti NT-proBNP antibody, respectively. The evaluation used Mindray’s automated chemiluminescent analyzer, CL6000i. Results Our data indicate that temperature plays a critical role in enhancing the performances of streptavidin particles in these immunoassays. From pre-coupling treatment of the particle to coupling with biotinylated ligands and subsequently post-coupling blocking and aging, temperature shows great impacts on the stability and tolerance to heat of the coupled particles at elevated temperatures. In contrast to the effectiveness of pre-coupling washing at 50°C or 55°C, coupling of streptavidin particles with biotinylated ligands favors a lower temperature, for example, ambient temperature and 4°C. Coupling at 37°C to 40°C results in slightly lower RLU signals in all four assays. Coupling at lower temperatures improves signal level and, more importantly, enhances thermal resistance of coupled particles. Post-coupling aging and optimization of diluent compositions can additionally improve accelerated stability data obtained at 37°C. Conclusion There are two mechanisms that negatively impact the stability performance of streptavidin particles coupled with ligands: One is gradual loss of loosely bound streptavidin molecules and the other streptavidin’s configurational and structural changes triggered by temperature changes. Both mechanisms can be countered by proper control of temperature in the multiple steps of making the solid phase reagents. Our findings can help immunoassay developers to design more robust assay reagents involving streptavidin particles.

Immune protection induced by E2 recombinant glycoprotein of bovine viral diarrhea virus in a murine model.

Bovine viral diarrhea virus (BVDV) is considered the most important viral pathogen in ruminants worldwide due to the broad range of clinical manifestations displayed by infected animals. Therefore, infection with BVDV leads to severe economic losses in several countries' beef and dairy industries. Vaccination prevents reproductive failure and gastrointestinal and respiratory disorders caused by BVDV infection. However, considering their limitations, conventional vaccines such as live, attenuated, and killed viruses have been applied. Hence, different studies have described subunit vaccines as an effective and safe alternative for BVDV protection. Therefore, in this study, the ectodomain of E2 (E2e) glycoprotein from NADL BVDV strain was expressed in mammalian cells and used in two vaccine formulations to evaluate immunogenicity and protection against BVDV conferred in a murine model. Formulations consisted of solo E2e glycoprotein and E2e glycoprotein emulsified in adjuvant ISA 61 VG. Five groups of 6 mice of 6-to-8-week-old were immunized thrice on days 1, 15, and 30 by intraperitoneal injection with the mentioned formulations and controls. To evaluate the conferred protection against BVDV, mice were challenged six weeks after the third immunization. In addition, the humoral immune response was evaluated after vaccination and challenge. Mice groups inoculated with solo E2e and the E2e + ISA 61 VG displayed neutralizing titers; however, the E2 antibody titers in the E2e + ISA 61 VG group were significantly higher than the mice group immunized with the solo E2e glycoprotein. In addition, immunization using E2e + ISA 61 VG prevents animals from developing severe lesions in surveyed tissues. Moreover, this group acquired protection against the BVDV challenge, evidenced by a significant reduction of positive staining for BVDV antigen in the lungs, liver, and brain between the experimental groups. Our findings demonstrated that using E2e + ISA 61 VG induces greater BVDV protection by an early humoral response and reduced histopathological lesions and BVDV antigen detection in affected organs, indicating that E2e + ISA 61 VG subunit formulation can be considered as a putative vaccine candidate against BVDV. The efficacy and safety of this vaccine candidate in cattle requires further investigation.

Cross-reactivities and cross-neutralization of different envelope glycoproteins E2 antibodies against different genotypes of classical swine fever virus.

Classical swine fever (CSF) is a highly contagious swine disease caused by the classical swine fever virus (CSFV), wreaking havoc on global swine production. The virus is divided into three genotypes, each comprising 4-7 sub-genotypes. The major envelope glycoprotein E2 of CSFV plays an essential role in cell attachment, eliciting immune responses, and vaccine development. In this study, to study the cross-reaction and cross-neutralizing activities of antibodies against different genotypes (G) of E2 glycoproteins, ectodomains of G1.1, G2.1, G2.1d, and G3.4 CSFV E2 glycoproteins from a mammalian cell expression system were generated. The cross-reactivities of a panel of immunofluorescence assay-characterized serum derived from pigs with/without a commercial live attenuated G1.1 vaccination against different genotypes of E2 glycoproteins were detected by ELISA. Our result showed that serum against the LPCV cross-reacted with all genotypes of E2 glycoproteins. To evaluate cross-neutralizing activities, hyperimmune serum from different CSFV E2 glycoprotein-immunized mice was also generated. The result showed that mice anti-E2 hyperimmune serum exhibited better neutralizing abilities against homologous CSFV than heterogeneous viruses. In conclusion, the results provide information on the cross-reactivity of antibodies against different genogroups of CSFV E2 glycoproteins and suggest the importance of developing multi-covalent subunit vaccines for the complete protection of CSF.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1–338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Prevalence of oral and blood oncogenic human papillomavirus biomarkers among an enriched screening population: Baseline results of the MOUTH study.

Human papillomavirus (HPV)-related oropharyngeal cancer screening is being explored in research studies, but strategies to identify an appropriate population are not established. The authors evaluated whether a screening population could be enriched for participants with oncogenic HPV biomarkers using risk factors for oral HPV. Participants were enrolled at Johns Hopkins Hospitals and Mount Sinai Icahn School of Medicine. Eligible participants were either men aged 30 years or older who had two or more lifetime oral sex partners and a personal history of anogenital dysplasia/cancer or partners of patients who had HPV-related cancer. Oral rinse and serum samples were tested for oncogenic HPV DNA, RNA, and E6 or E7 antibodies, respectively. Participants with any biomarker were considered at-risk. Of 1108 individuals, 7.3% had any oncogenic oral HPV DNA, and 22.9% had serum antibodies for oncogenic HPV E6 or E7. Seventeen participants (1.5%) had both oral and blood biomarkers. HPV type 16 (HPV16) biomarkers were rarer, detected in 3.7% of participants, including 20 with oral HPV16 DNA and 22 with HPV16 E6 serum antibodies (n=1 had both). In adjusted analysis, living with HIV (adjusted odds ratio, 2.65; 95% CI, 1.60-4.40) and older age (66-86 vs. 24-45years; adjusted odds ratio, 1.70; 95% CI, 1.07-2.70) were significant predictors of being at risk. Compared with the general population, the prevalence of oral HPV16 (1.8% vs. 0.9%), any oncogenic oral HPV DNA (7.3% vs. 3.5%), and HPV16 E6 antibodies (2.2% vs. 0.3%) was significantly elevated. Enrichment by the eligibility criteria successfully identified a population with higher biomarker prevalence, including HPV16 biomarkers, that may be considered for screening trials. Most in this group are still expected to have a low risk of oropharyngeal cancer.

Dual-Mode Electrochemical Competitive Immunosensor Based on Cd2+/Au/Polydopamine/Ti3C2 Composite and Copper-Based Metal-Organic Framework for 17β-Estradiol Detection.

Herein, a dual-mode electrochemical competitive immunosensor was constructed for the detection of 17β-estradiol (E2) based on differential pulse voltammetry (DPV) and chronoamperometry (i-t). During the immune recognition process, the E2 antibody (E2-Ab) was immobilized on the Cd2+/Au/polydopamine/Ti3C2 (Cd2+/Au/pDA/Ti3C2) composite-modified electrode; then, the E2-conjugated bovine serum albumin (E2-BSA) was labeled with a copper-based metal-organic framework (Cu-MOF) and competed with E2 in combining the E2-Ab. The Cu-MOF was not only an electroactive species but also possessed good electrocatalytic activity toward H2O2. Thus, E2 could be quantified according to the peak current change of the Cu-MOF in DPV curve or the variation of H2O2 reduction current. For DPV quantification, Cd2+ was introduced as an internal reference in this case, and a highly reproducible ratio readout was obtained. The as-prepared dual-mode E2 electrochemical immunosensor showed good linear relationship in the ranges of 1 pg mL-1-10 ng mL-1 (DPV) and 10 pg mL-1-10 ng mL-1 (i-t), and the detection limits were 0.47 and 5.4 pg mL-1 (S/N = 3), respectively. Furthermore, the dual-mode electrochemical immunosensor exhibited good practicability in real sample analysis.

Comparative efficacy evaluation of different CSF vaccines in pigs with CSF maternally derived antibodies

Classical swine fever (CSF) is a highly contagious and important swine disease in China. Sporadic outbreaks with mild clinical signs are still being reported despite massive vaccination with the CSF C-strain vaccine. One possible reason for vaccine failure could be interference from maternally derived antibodies (MDAs) during vaccination in the field. The aim of this study was to evaluate the efficacy of different CSF vaccines in the presence of MDAs and to assess the different vaccination schemes in the field. The results demonstrated that vaccination with a single dose of C-strain-PK vaccine protected pigs against severe clinical signs and significantly reduced viremia. The impact of MDAs was negligible. The interference was also mild during a prime and boost vaccination scheme using the C-strain-ST vaccine. In contrast, a significant influence of MDAs on the efficacy of the subunit E2 vaccine in a one-dose vaccination scheme was observed, with pigs showing severe clinical signs, CSF-associated death, typical pathological lesions and a high level of viremia after challenge, despite robust E2 antibody induction. A field vaccination and challenge study further confirmed the superior effectiveness of a single dose of C-strain-PK vaccine in the presence of MDAs in comparison to a routine prime and boost vaccination scheme applied in the field, with pigs having fever, chronic signs, significant viremia and shedding after challenge. Delaying the vaccination time from the age of 28 days to 45 days, when MDA was low, was beneficial for improving the clinical protection and immunity induced by vaccines. Altogether, the results presented here emphasize that a high-quality vaccine and a scientific design of the vaccination scheme based on serological surveillance are essential pillars to control and eliminate CSF in China.

Usefulness of high-risk HPV early oncoprotein (E6 and E7) serological markers in the detection of cervical cancer: A systematic review and meta-analysis.

We reviewed the literature on the importance of selected anti-high-risk human papillomavirus (HR-HPV)antibodies (namely, 16/18 and early oncoproteinsE6 and E7) as potential serological markers for early detection of individuals at high risk of cervical cancer. We searched for studies in PubMed and Embase databases published from 2010 to 2020 on antibodies against HR-HPV E6 and E7 early proteins and cervical cancer. Pooled sensitivity and specificity for HPV16 and HPV18 antibodies were calculated using a bivariate hierarchical random-effects model. A total of 69 articles were identified; we included three studies with 1550 participants. For the three HPV16/18 E6 and E7 antibody tests, enzyme-linked immunosorbent assay-based assays had a sensitivity of 18% for detecting CIN2+ (95% confidence interval [CI]: 15-21) and a specificity of 96% (95% CI: 92-98), for slot-blot, sensitivity was 28.9% (95% CI: 23.3-35.1) and specificity was 72% (95% CI: 66.6-77.0) for detecting CIN2+, and for multiplex HPV serology assay based on a glutathione S-transferase, sensitivity was 16% (95% CI: 8.45-28.6) and specificity was 98% (95% CI: 97-99) for detecting invasive cervical cancer. HR-HPV16/18E6 and E7 serological markers showed high specificity, but sensitivity was suboptimal for the detection of cervical cancer in either population screening settings or as point-of-care screening tests.

Combination treatment with 17β-estradiol and anti-PD-L1 suppresses MC38 tumor growth by reducing PD-L1 expression and enhancing M1 macrophage population in MC38 colon tumor model

17β-estradiol (E2) is known to have a protective effect in colorectal cancer (CRC); thus, E2 may be effective for cancer immunotherapy in CRC. The aim of this study is to evaluate the effect of combination therapy with E2 and anti-programmed cell death receptor-1 ligand (PD-L1) antibodies, and the effects of sex and estrogen on colon tumor growth, PD-L1 expression, and tumor-associated cell populations in an MC38 colon tumor model. Male mice showed increased MC38 colon tumor growth and PD-L1 expression in tumor sections as well as higher proportion of cancer-associated fibroblasts (CD45−CD31−CD140a+), PD-L1-expressing tumor cells (CD45−CD274+) and tumor-associated macrophages (TAMs) (CD11b+F4/80+CD274+) compared to female mice. E2 treatment prior to MC38 injection significantly reduced these phenomena in male mice. Furthermore, co-treatment with E2 and anti-PD-L1 antibodies significantly inhibited MC38 tumor growth and reduced PD-L1-expressing cells in male mice compared to treatment with either E2 or anti-PD-L1 antibodies alone. Combination treatment with E2 and anti-PD-L1 decreased TAM population (CD11b+F4/80+) in the tumor mass while increasing M1 TMAs (CD11b+F4/80+CD86+). These results suggest that estrogen inhibits MC38 tumor growth by downregulating PD-L1 expression and regulating tumor-associated cell populations. Furthermore, estrogen boosted the effect of anti-PD-L1 antibody in the MC38 tumor model.