Co-Administration of a Plasmid Encoding CD40 or CD63 Enhances the Immune Responses to a DNA Vaccine Against Bovine Viral Diarrhea Virus in a Mouse Model

Bovine viral diarrhea virus (BVDV) causes substantial economic losses in the livestock industry worldwide. Plasmids encoding the BVDV E2 protein are potential DNA vaccines against BVDV, but their immunogenicity has been insufficient. Here, we investigated the adjuvant effect of CD40 and CD63 on the immune responses to a BVDV E2 DNA vaccine in a mouse model. We constructed pUMVC4a-based plasmids encoding the BVDV E2 protein (pE2), mouse CD40 (pCD40), or mouse CD63 (pCD63). Protein expression by each plasmid was confirmed through Western blot analysis and immunofluorescence staining of cultured cell lines. BALB/c mice were immunized intradermally twice with pE2 in combination with, or without, pCD40 or pCD63, with 3 weeks between the two doses. pE2 with pCD40 induced significantly higher neutralizing antibody titers against BVDV than pE2 alone. Furthermore, pE2 with pCD40 or pCD63 induced significantly increased lymphocyte proliferation and IFN-γ production in response to BVDV ex vivo, compared with E2 alone. These results suggest that a plasmid encoding CD40 or CD63 can be used as an adjuvant to enhance immune responses to DNA vaccines against BVDV.

Peptide-Based Capture of Chikungunya Virus E2 Protein Using Porous Silicon Biosensor

The detection of pathogens presents specific challenges in ensuring that biosensors remain operable despite exposure to elevated temperatures or other extreme conditions. The most vulnerable component of a biosensor is typically the bioreceptor. Accordingly, the robustness of peptides as bioreceptors offers improved stability and reliability toward harsh environments compared to monoclonal antibodies that may lose their ability to bind target molecules after such exposures. Here, we demonstrate peptide-based capture of the Chikungunya virus E2 protein in a porous silicon microcavity biosensor at room temperature and after exposure of the peptide-functionalized biosensor to high temperature. Contact angle measurements, attenuated total reflectance—Fourier transform infrared spectra, and optical reflectance measurements confirm peptide functionalization and selective E2 protein capture. This work opens the door for other pathogenic biomarker detection using peptide-based capture agents on porous silicon and other surface-based sensor platforms.

In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.

Development and application of a high sensitivity immunochromatographic test strip for detecting classical swine fever virus antibody

Classical swine fever (CSF) is caused by classical swine fever virus (CSFV) and has led to huge ecnomic losses for the pig industry worldwide. Although vaccination and other control measures have been carried out, it is essential to establish a rapid and valid method for CSF vaccination monitoring and clinical diagnosis. CSFV E2 protein has been well-known as a major antigen for antibody detection. It is significant to improve affinity between E2 protein and CSFV antibody for a better performance of detection method. In this study, a recombinant E2 extracellular protein (aa 1-331), which has a native homodimer conformation and has a high affinity with anti-CSFV-E2 monoclonal antibody WH303, was expressed using Bac-to-Bac baculovirus expression system. A novel immunochromatographic test strip based on the recombinant CSFV E2 protein was developed for CSFV antibody detection. The sensitivity of this strip for detecting CSFV standard positive serum was 1:102400, 4 times higher than that of the previously developed CnC2 test strip. No cross reaction with antibodies of other swine viruses was observed. The detection of clinical swine serum samples (n=138) demonstrated that the agreements of this E2 test strip with three commercial ELISA kits were 88.40% (122/138), 86.23% (119/138), and 96.38% (133/138), respectively. Our data indicated that a novel E2 test strip with higher sensitivity has been developed and can be applied for clinical sample detections, providing a new powerful and simple approach for CSFV antibody monitoring.

Regulation of the Human Papillomavirus Lifecyle through Post-Translational Modifications of the Viral E2 Protein

The human papillomavirus (HPV) is a DNA tumor virus that infects cutaneous and mucosal epithelia where high-risk (HR) HPV infections lead to cervical, oropharyngeal, and anogenital cancers. Worldwide, nearly 5% of all cancers are caused by HR HPV. The viral E2 protein is essential for episomal replication throughout the viral lifecycle. The E2 protein is regulated by phosphorylation, acetylation, sumoylation, and ubiquitination. In this mini-review, we summarize the recent advancements made to identify post translational modifications within E2 and their ability to control viral replication.

Recent Advances on the Bovine Viral Diarrhea Virus Molecular Pathogenesis, Immune Response, and Vaccines Development

The bovine viral diarrhea virus (BVDV) consists of two species and various subspecies of closely related viruses of varying antigenicity, cytopathology, and virulence-induced pathogenesis. Despite the great ongoing efforts to control and prevent BVDV outbreaks and the emergence of new variants, outbreaks still reported throughout the world. In this review, we are focusing on the molecular biology of BVDV, its molecular pathogenesis, and the immune response of the host against the viral infection. Special attention was paid to discuss some immune evasion strategies adopted by the BVDV to hijack the host immune system to ensure the success of virus replication. Vaccination is one of the main strategies for prophylaxis and contributes to the control and eradication of many viral diseases including BVDV. We discussed the recent advances of various types of currently available classical and modern BVDV vaccines. However, with the emergence of new strains and variants of the virus, it is urgent to find some other novel targets for BVDV vaccines that may overcome the drawbacks of some of the currently used vaccines. Effective vaccination strategy mainly based on the preparation of vaccines from the homologous circulating strains. The BVDV-E2 protein plays important role in viral infection and pathogenesis. We mapped some important potential neutralizing epitopes among some BVDV genomes especially the E2 protein. These novel epitopes could be promising targets against the currently circulating strains of BVDV. More research is needed to further explore the actual roles of these epitopes as novel targets for the development of novel vaccines against BVDV. These potential vaccines may contribute to the global eradication campaign of the BVDV.

Development and Application of a High Sensitivity Immunochromatographic Test Strip for Detecting Classical Swine Fever Virus Antibody

Background: Classical swine fever (CSF) is caused by classical swine fever virus (CSFV) and has lead huge losses to the pig industry worldwide. Although vaccination and other control measures have been carried out, it is essential to establish a rapid valid method for CSF vaccination monitoring and clinical diagnosis. CSFV E2 protein has been well known as a major antigen for antibody detection. It is significant to improve affinity between E2 protein and CSFV antibody to result in a better performance of detection method. Results: In this study, a recombinant E2 extracellular protein (aa 1-331), which was with the native homodimer conformation and had a high affinity with anti-CSFV-E2 monoclonal antibody WH303, was expressed using Bac-to-Bac baculovirus expression system. A novel immunochromatographic test strip based on the recombinant CSFV E2 protein was developed for CSFV antibody detection. The sensitivity of this strip for detecting CSFV standard positive serum was 1:102400, 4 times higher than that of the former developed CnC2 test strip. No cross reaction with other swine virus antibodies was observed. The detection of clinical swine serum samples (n=138) demonstrated that the agreement of the new E2 test strip with three commercial ELISA kits was 88.40% (122/138), 86.23% (119/138), and 96.38% (133/138), respectively. Conclusion: Our data indicate that a novel E2 test strip with higher sensitivity has been developed and can be applied for clinical sample detections, providing a new powerful and simple approach for future monitoring CSFV antibodies.