Lambda Phage-Based Antibody-Stimulating Platform Targeting EGFRvIII.

The nucleocapsid (N) protein of the severe acute respiratory syndrome coronavirus (SARSCoV) is the most abundant structural protein in virus-infected cells. Its primary function is to pack ~30 kb positive-sense viral RNA genome that includes a 5' cap into a ribonucleoprotein (RNP) complex known as capsid. Among the 4 major structural proteins of SARS-CoV, the N protein exhibits the highest expression levels in the host cells. We produced a recombinant SARS-CoV N protein (SARSN1) and used it to detect specific antibodies in mice vaccinated with a live probiotic vaccine containing the coronavirus nucleocapsid protein.

No influence of cadmium on the production of specific antibodies in mice

Rotavirus is the major enteric pathogen that is responsible for more than 500,000 deaths of children worldwide annually. The use of live-attenuated oral rotavirus vaccines has significantly reduced child mortality rate in developed countries. However, the situation still remains unacceptably worse in most developing countries due to low efficacy of the vaccines in the region. The vaccines often do not reach the developing countries due to financial and logistic challenges. A high burden of rotavirus disease and the unresolved challenges with the current rotavirus vaccines, particularly in developing countries, have ignited efforts to develop next-generation low-cost, effective and safe vaccine candidates. Virus-like particles (VLPs) have come into focus for their promising application in vaccination because of their unique and attractive set of properties including safety, self-adjuvanticity, developability, economy and the ability to be formulated for stability without a cold chain. Their impact on human health is already evident through commercialization of VLP vaccines against hepatitis B virus infection, human papillomavirus-induced cervical cancer and hepatitis E virus infection. Rotavirus-like particles (RLPs) with proven preclinical immunogenicity and protective efficacy are considered as safe and effective vaccine candidates. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising approaches in reducing process complexity and cost. Depending on the physicochemical properties, size and /or surface density of modules, modularization may affect production of stable VLP forming subunits, termed capsomeres, and prevent VLP assembly. This thesis demonstrates a multipronged approach for a low-cost production of stable bacterially-produced modular murine polyomavirus capsomeres and in vitro assembled VLPs presenting a rotavirus highly conserved peptide epitope (RV10) and a conformational and a virus neutralizing 18 kDa antigen (VP8*), separately. The experimental works in this thesis were carried out to address challenges associated with the adverse effects of inserted module’s physicochemical properties, size and density on the production of modular capsomeres and VLPs. Particularly, the expression and purification of stable modular capsomeres for in vitro modular VLP assembly and the enhancement of module-specific immune response were investigated. The outcomes of the work in this thesis are: (i) a rapid and simple high-throughput screening method based on dynamic light scattering measurements was developed to identify additives for enhanced stability of modular capsomeres; (ii) using synthetic biology designs, the hydrophobicity of RV10 modules was engineered for enhanced stability of modular capsomeres; (iii) in vitro assembled modular VLPs displaying RV10 modules were obtained via a module titration approach using Escherichia coli protein co-expression strategy; and (iv) highly stable and immunogenic modular VLPs displaying conformational VP8* were produced using a combination of Escherichia coli protein co-expression, simplified modular capsomere purification steps and cell-free in vitro VLP assembly. The outcomes of this thesis expand the potential and plasticity of the murine polyomavirus VLP platform for presentation of antigenic modules regardless of their adverse physicochemical properties and large size. This work introduces, to the best of our knowledge, the first bacterially-produced capsomeres and in vitro assembled VLPs displaying either hydrophobic RV10 peptide modules or 18 kDa rotavirus VP8* large antigens. The modular capsomeres (CapVP8*) and modular VLPs (VLP-VP8*) induced high levels of VP8*-specific antibodies in mice. The high immunogenicity of CapVP8* and VLP-VP8*likely indicates protective efficacy and makes them a more viable vaccine candidate for further development to prevent rotavirus in the developing world at affordable cost. The strategies developed in this thesis potentially provides a cost-effective production route for modular capsomere and VLPs presenting other antigenic modules from rotavirus and other target pathogens, highly suitable to manufacturing economics for the developing world.

Trypanosoma cruzi: Immune response in mice immunized with parasite antigens

Enhancement of antigen-specific antibody production by extracellular slime products from slime-forming Lactococcus lactis subspecies cremoris SBT 0495 in mice

BackgroundA vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination.MethodsA conserved gene in Plasmodium, the Plasmodium berghei gene (PBANKA_020570) encoding a 51 kDa protein (pb51 gene), was identified through search of the PlasmoDB database using a combination of expression and protein localization criteria. A partial domain of the Pb51 protein was expressed in a prokaryotic expression system (rPb51) and used for immunization in mice. The protein expression profile and localization were studied by Western blot and indirect immunofluorescence assay (IFA), respectively. The inhibitory effect of the anti-rPb51 antibodies on parasite proliferation was evaluated in erythrocytes in vivo. The transmission-blocking activity of the immune sera was determined by in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA).ResultsThe rPb51 elicited specific antibodies in mice. Western blot confirmed Pb51 expression in schizonts, gametocytes and ookinetes. IFA showed localization of Pb51 on the outer membranes of schizonts, gametocytes, zygotes, retorts, ookinetes and sporozoites of P. berghei. Mice immunized with the rPb51 protein significantly reduced parasite proliferation and gametocyte conversion in vivo. Moreover, the rPb51 antisera also significantly reduced the in vitro ookinete conversion when added into the ookinete culture medium. In DFA, mice immunized with the rPb51 reduced the prevalence of mosquito infection by 21.3% and oocyst density by 54.8%.ConclusionsIn P. berghei, P51 was expressed in both asexual erythrocytic and sexual stages and localized on the surface of these stages with the exception of the ring stage. The anti-rPb51 antibodies inhibited both P. berghei proliferation in mice and transmission of the parasite to mosquitoes.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009–10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention.

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand’s cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors

The development of cancer immunotherapies is progressing rapidly with a variety of technological approaches. They consist of "cancer vaccines", which are based on the idea of vaccination, "effector cell therapy", classified as passive immunotherapy, and "inhibition of immunosuppression", which intends to break immunological tolerance to autoantigens or immunosuppressive environments characterizing antitumor immune responses. Recent reports showing clinical evidence of efficacy of immune checkpoint inhibitors and adoptive immunotherapies with tumor-infiltrating lymphocytes and tumor-specific receptor gene-modified T cells indicate the beginning of a new era for cancer immunotherapy. This guidance summarizes ideas that will be helpful to those who plan to develop cancer immunotherapy. The aims of this guidance are to discuss and offer important points in early phase clinical studies of innovative cancer immunotherapy, with future progress in this field, and to contribute to the effective development of cancer immunotherapy aligned with the scope of regulatory science. This guidance covers cancer vaccines, effector cell therapy, and inhibition of immunosuppression, including immune checkpoint inhibitors.

Comparative reactogenicity and immunogenicity of 23 valent pneumococcal vaccine administered by intramuscular or subcutaneous injection in elderly adults

Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the desired antibodies selected from the libraries are packaged within the phage particles, linking genotype and phenotype. Here, we describe the application of this technology to the construction of a phage-displayed single-domain antibody (sdAb) library based on the heavy chain antibody repertoire of a llama, the panning of the library against a peptide antigen and the expression, purification, and characterization of sdAbs isolated by panning.

Hydrogels, a class of materials with a 3Dnetwork structure, are widely used in various applications of therapeutic delivery, particularly cancer therapy. Micro and nanogels as miniaturized structures of the bioengineered hydrogels may provide extensive benefits over the common hydrogels in encapsulation and controlled release of small molecular drugs, macromolecular therapeutics, and even cells. Cancer immunotherapy is rapidly developing, and micro/nanostructured hydrogels have gained wide attention regarding their engineered payload release properties that enhance systemic anticancer immunity. Additionally, they are a great candidate due to their local administration properties with a focus on local immune cell manipulation in favor of active and passive immunotherapies. Although applied locally, such micro/nanostructured can also activate systemic antitumor immune responses by releasing nanovaccines safely and effectively inhibiting tumor metastasis and recurrence. However, such hydrogels are mostly used as locally administered carriers to stimulate the immune cells by releasing tumor lysate, drugs, or nanovaccines. In this review, the latest developments in cancer immunotherapy are summarized using micro/nanostructured hydrogels with a particular emphasis on their function depending on the administration route. Moreover, the potential for clinical translation of these hydrogel-based cancer immunotherapies is also discussed.

Hiroshi Shiku, MD, PhD: In Memoriam (1943–2022)