Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides.

Extraction of nucleic acids (NAs) is critical for many methods in molecular biology and bioanalytical chemistry. NA extraction has been extensively studied and optimized for a wide range of applications and its importance to society has significantly increased. The COVID-19 pandemic highlighted the importance of early and efficient NA testing, for which NA extraction is a critical analytical step prior to the detection by methods like polymerase chain reaction.

An Approach for Antigen-Agnostic Identification of Virus-Like Particle-Displayed Epitopes that Engage Specific Antibody V Gene Regions.

Antibody complementarity determining regions (CDRs) participate in antigen recognition, but not all participate equally in antigen binding. Here we describe a technique for discovering strong, specific binding partners to defined motifs within the CDRs of chimeric, engineered antibodies using affinity selection and counter-selection of epitopes displayed on bacteriophage MS2-based virus-like particles (VLPs). As an example, we show how this technique can be used to identify families of VLPs that interact with antibodies displaying the CDRs encoded by the germline precursor of a broadly neutralizing monoclonal antibody against HIV-1.

The Immunogenicity of a VLP-based Malaria Vaccine Targeting CSP in Pregnant and Neonatal Mice.

Maternal antibodies are passively transferred to the fetus via the placenta during gestation and can play an important role in protecting the newborn from infection. For example, in malaria-endemic regions, maternal antibodies likely provide substantial protection against malaria in the first 6 months of life. However, circulating maternal antibodies can also interfere with vaccine efficacy.

A method for mapping the linear epitopes targeted by the natural antibody response to Zika virus infection using a VLP platform technology.

Zika virus (ZIKV), a mosquito-borne pathogen, is associated with neurological complications in adults and congenital abnormalities in newborns. There are no vaccines or treatments for ZIKV infection. Understanding the specificity of natural antibody responses to ZIKV could help inform vaccine efforts.

Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site.

Expansion and Refinement of Deep Sequence-Coupled Biopanning Technology for Epitope-Specific Antibody Responses in Human Serum.

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses.

Antibodies to Variable Domain 4 Linear Epitopes of the Major Outer Membrane Protein Are Not Associated with Chlamydia Resolution or Reinfection in Women.

is an obligate intracellular bacterium. infection is the most prevalent bacterial sexually transmitted infection and can lead to pelvic inflammatory disease and infertility in women. There is no licensed vaccine for prevention, in part due to gaps in our knowledge of -specific immune responses elicited during human infections.

Vaccination with VLPs Presenting a Linear Neutralizing Domain of Hla Elicits Protective Immunity.

The pore-forming cytotoxin α-hemolysin, or Hla, is a critical virulence factor that promotes infection by causing tissue damage, excessive inflammation, and lysis of both innate and adaptive immune cells, among other cellular targets. In this study, we asked whether a virus-like particle (VLP)-based vaccine targeting Hla could attenuate Hla-mediated pathogenesis. VLPs are versatile vaccine platforms that can be used to display target antigens in a multivalent array, typically resulting in the induction of high titer, long-lasting antibody responses.

Factors That Govern the Induction of Long-Lived Antibody Responses.

The induction of long-lasting, high-titer antibody responses is critical to the efficacy of many vaccines. The ability to produce durable antibody responses is governed by the generation of the terminally differentiated antibody-secreting B cells known as long-lived plasma cells (LLPCs). Once induced, LLPCs likely persist for decades, providing long-term protection against infection.

Assessing Antibody Specificity in Human Serum Using Deep Sequence-Coupled Biopanning.

Affinity selection using phage-display technologies is a powerful tool for identifying the peptide epitopes of monoclonal antibodies. Coupling affinity selection with deep sequencing technologies allows for the broad assessment of selectant populations. Here, we describe a method for using a phage-display platform to assess antibody specificity in human serum.

Qß Virus-like particle-based vaccine induces robust immunity and protects against tauopathy.

Tauopathies, including frontotemporal dementia (FTD) and Alzheimer's disease (AD) are progressive neurodegenerative diseases clinically characterized by cognitive decline and could be caused by the aggregation of hyperphosphorylated pathological tau (pTau) as neurofibrillary tangles (NFTs) inside neurons. There is currently no FDA-approved treatment that cures, slows or prevents tauopathies. Current immunotherapy strategies targeting pTau have generated encouraging data but may pose concerns about scalability, affordability, and efficacy.

Identification and Immunological Characterization of the Ligand Domain of Plasmodium vivax Reticulocyte Binding Protein 1a.

Background: Erythrocyte invasion by malaria parasites is essential for blood-stage development. Consequently, parasite proteins critically involved in erythrocyte invasion, such as the Plasmodium vivax reticulocyte binding proteins (RBPs) that mediate preferential invasion of reticulocytes, are considered potential vaccine targets. Thus, targeting the RBPs could prevent blood-stage infection and disease.

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection.

Display of single-chain variable fragments on bacteriophage MS2 virus-like particles.

Background: Virus-like particles (VLPs) of the RNA bacteriophage MS2 have many potential applications in biotechnology. MS2 VLPs provide a platform for peptide display and affinity selection (i.e.

Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses.

Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection.

Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein.

Background: Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) . The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.

Engineering virus-like particles as vaccine platforms.

Virus-like particles (VLPs) have been utilized as vaccine platforms to increase the immunogenicity of heterologous antigens. A variety of diverse VLP types can serve as vaccine platforms, and research has focused on engineering VLPs to improve their efficacy as vaccines, enhance their stability, and allow for more versatile display of antigens. Here, we review selected VLP vaccine platforms, highlight efforts to improve these platforms through structure-informed rational design, and point to areas of future research that will assist in these efforts.

Optimized Formulation of a Thermostable Spray-Dried Virus-Like Particle Vaccine against Human Papillomavirus.

Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP)-based vaccine for HPV infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multicomponent excipient system and by optimizing the spray-drying parameters using a half-factorial design approach.

Identification of Anti-CA125 Antibody Responses in Ovarian Cancer Patients by a Novel Deep Sequence-Coupled Biopanning Platform.

High-grade epithelial ovarian cancer kills more women than any other gynecologic cancer and is rarely diagnosed at an early stage. We sought to identify tumor-associated antigens (TAA) as candidate diagnostic and/or immunotherapeutic targets by taking advantage of tumor autoantibody responses in individuals with ovarian cancer. Plasma-derived IgG from a pool of five patients with advanced ovarian cancer was subjected to iterative biopanning using a library of bacteriophage MS2 virus-like particles (MS2-VLPs) displaying diverse short random peptides.

Epitope-Specific Anti-hCG Vaccines on a Virus Like Particle Platform.

The possibility of a contraceptive vaccine targeting human chorionic gonadotropin has long been recognized, but never fully realized. Here we describe an epitope-specific approach based on immunogenic display of hCG-derived peptides on virus-like particles of RNA bacteriophage. A number of recombinant VLPs were constructed, each displaying a different hCG-derived peptide.

Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display.

We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens.

Preclinical refinements of a broadly protective VLP-based HPV vaccine targeting the minor capsid protein, L2.

An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types.

Affinity selection of epitope-based vaccines using a bacteriophage virus-like particle platform.

Display of epitopes on virus-like particles (VLPs) is a highly effective technique for enhancing the immunogenicity of antigens that are poorly immunogenic in their native context. VLP-based vaccines can be used to elicit long-lasting, high-titer antibody responses against diverse target antigens, even self-antigens. Most VLP platform-based vaccines are rationally engineered; specific target epitopes or domains are arrayed so that they are displayed at high-valency on the surface of VLPs.

Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP) technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs.