Explore the vast range of titles available.

We describe the current Good Manufacturing Practice (cGMP) production and subsequent characterization of eOD-GT8 60mer, a glycosylated self-assembling nanoparticle HIV-1 vaccine candidate and germline targeting priming immunogen. Production was carried out via transient expression in the human embryonic kidney 293 (HEK293) cell line followed by a combination of purification techniques. A large-scale cGMP (200 L) production run yielded 354 mg of the purified eOD-GT8 60mer drug product material, which was formulated at 1 mg/mL in 10% sucrose in phosphate-buffered saline (PBS) at pH 7.2. The clinical trial material was comprehensively characterized for purity, antigenicity, glycan composition, amino acid sequence, and aggregation and by several safety-related tests during cGMP lot release. A comparison of the purified products produced at the 1 L scale and 200 L cGMP scale demonstrated the consistency and robustness of the transient transfection upstream process and the downstream purification strategies. The cGMP clinical trial material was tested in a Phase 1 clinical trial (NCT03547245), is currently being stored at −80 °C, and is on a stability testing program as per regulatory guidelines. The methods described here illustrate the utility of transient transfection for cGMP production of complex products such as glycosylated self-assembling nanoparticles.

•Self-adjuvanting, self-assembling protein nanoparticle to prevent malaria is made.•Amounts of selected domains of flagellin per nanoparticle are optimized.•Immune responses and protective efficacy of the vaccine are determined. To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1β, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.

Despite advances in vaccine development, influenza remains a persistent global health threat and the search for a broad-spectrum recombinant vaccine against influenza continues. The extracellular domain of the transmembrane protein M2 (M2e) of the influenza A virus is highly conserved and can be used to develop a universal vaccine. M2e is a poor immunogen by itself, but it becomes highly immunogenic when linked to an appropriate carrier. Here, we report the transient expression of a recombinant protein comprising four tandem copies of M2e fused to an artificial self-assembling peptide (SAP) in plants. The hybrid protein was efficiently expressed in Nicotiana benthamiana using the self-replicating potato virus X-based vector pEff. The protein was purified using metal affinity chromatography under denaturing conditions. The hybrid protein was capable of self-assembly in vitro into spherical particles 15–30 nm in size. The subcutaneous immunization of mice with M2e-carrying nanoparticles induced high levels of M2e-specific IgG antibodies in serum and mucosal secretions. Immunization provided mice with protection against a lethal influenza A virus challenge. SAP-based nanoparticles displaying M2e peptides can be further used to develop a recombinant “universal” vaccine against influenza A produced in plants.

Malaria infection remains a significant threat worldwide, with the development of a vaccine that induces long-term sterile immunity proving difficult due to the complexity of the parasite, Plasmodium. The identification of conserved and human leukocyte antigen (HLA)-promiscuous, protection-associated epitopes within target antigens offer promise of developing effective vaccines. We previously identified immunodominant T cell regions within selected P. falciparum (Pf) antigens using samples from semi-immune individuals in Southern Ghana. The aim was to design, produce and assess immunogenicity of Pf-antigen-specific vaccines containing conserved and promiscuous HLA class I-restricted epitopes using the self-assembling protein nanoparticle (SAPN) delivery platform. We produced five SAPN vaccines based on epitopes identified in our earlier studies from Pf antigens (PfCSP, PfAMA1 and PfTRAP) and presented by HLA supertypes HLA A*02 and HLA A*03. The vaccines were used to immunize transgenic HLA A*02 and A*03 mice at 3, 7 and 10 μg doses, and the levels of cellular responses and antibodies to sporozoites assessed by IFN-γ/IL2 FluoroSpot and immunofluorescence antibody assays (IFA), respectively. Transgenic mice vaccinated with the CSP A*02 SAPN vaccine elicited increased IFN-γ T-cell responses against the inserted epitopes, NANPNANPNV and AILSVSSFLF and significantly higher antibody responses against Pf sporozoites assessed by IFA (median titer = 10,240 in vaccinated group vs 10 in adjuvant group, p value = 0.0003).The AMA1 SAPN vaccine containing the A*02 epitope YMGNPWTEYM elicited a median IFN-γ sfc/m of 259 in the 10 μg dose group while the adjuvant only response was 47 sfc/m (p value = 0.03). The AMA1 A*03 epitope NSTCRFFVCK elicited a median IFN-γ sfc/m of 180 and 217 in the 10 and 3 μg doses respectively, while the adjuvant only response was 0 sfc/m for both doses (p value = 0.0079). Our approach demonstrates feasibility of developing a novel potentially efficacious epitope-based vaccine and affirms the potential of the SAPN technology as an effective delivery platform.

Adjuvant is required for boosting the immune responses for subunit vaccine. An emerging category of vaccine adjuvant is the self-assembling peptide that forms fibril and stimulates both humoral and cellular immunities. Based on our previous finding that a stabilized self-assembled protein nanoparticle (PNP), also called Vaccine Delivery system X (VADEX), assembled from a fusion protein composed of an amphipathic helical peptide and a superfolder green fluorescent protein can stimulate long lasting immune responses to an inserted peptide. In this report, we further introduced split-GFP technology into VADEX and evaluated the role of the amphipathic helical peptide integrity, thermal stability of PNP and the effect of self-adjuvant in antibody affinity maturation of this new platform, VADEX-pro. Our result shows the significance of amphipathic helical peptide sequence integrity in PNP assembly and the thermal stability. The immunological results provide the first evidence that the VADEX-pro PNP possess a self-adjuvant activity that is superior to a clinical stage adjuvant when evaluating the antibody binding affinity. Application of VADEX-pro based protein nanoparticle in vaccine and therapeutic antibody development will likely improve the quality of humoral immune responses.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new influenza A strains. Efficient control of infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A combination of several such antigens, including the conserved region of the second subunit of the hemagglutinin (HA2), the extracellular domain of the M2 protein (M2e), and epitopes of nucleoprotein (NP), which together can elicit an antibody- and cell-mediated immune response, would be preferred for vaccine development. In this study, we obtained recombinant virus-like particles formed by an artificial self-assembling peptide (SAP) carrying two epitopes from NP, tandem copies of M2e and HA2 peptides, along with a T helper Pan DR-binding epitope (PADRE). Fusion proteins expressed in Escherichia coli self-assembled in vitro into spherical particles with a size of 15–35 nm. Immunization of mice with these particles induced strong humoral immune response against M2e and the entire virus, and lead to the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with the influenza A virus. Our results show that SAP-based nanoparticles carrying conserved peptides from M2, HA, and NP proteins of the influenza A virus, as well as T helper epitope PADRE, can be used for the development of universal flu vaccines.

Dendritic cell (DC) targeted antigen delivery is a promising strategy to enhance vaccine efficacy and delivery of therapeutics. Self-assembling peptide-based nanoparticles and virus-like particles (VLPs) have attracted extensive interest as non-replicating vectors for nanovaccine design, based on their unique properties, including molecular specificity, biodegradability and biocompatibility. DCs are specialized antigen-presenting cells involved in antigen capture, processing, and presentation to initiate adaptive immune responses. Using DC-specific ligands for targeted delivery of antigens to DCs may be utilized as a promising strategy to drive efficient and strong immune responses. In this study, several candidates for DC-binding peptides (DCbps) were individually integrated into C-terminal of porcine circovirus type 2 (PCV2) Cap, a viral protein that could self-assemble into icosahedral VLPs with 60 subunits. The immunostimulatory adjuvant activity of DC-targeted VLPs was further evaluated in a vaccine model of PCV2 Cap. With transmission electron microscopy (TEM), expressed Cap-DCbp fusion proteins were observed self-assembled into highly ordered VLPs. Further, in dynamic light scattering (DLS) analysis, chimeric VLPs exhibited similar particle size uniformity and narrow size distribution as compared to wild type Cap VLPs. With a distinctly higher targeting efficiency, DCbp3 integrated Cap VLPs (Cap-DCbp3) displayed enhanced antigen uptake and increased elicitation of antigen presentation-related factors in BM-DCs. Mice subcutaneously immunized with Cap-DCbp3 VLPs exhibited significantly higher levels of Cap-specific antibodies, neutralizing antibodies and intracellular cytokines than those with other DCbp integrated or wild type Cap VLPs without any DCbp. Interestingly, Cap-DCbp3 VLPs vaccine induces robust cellular immune response profile, including the efficient production of IFN-γ, IL-2 and IL-10. Meanwhile, the improved proliferation index in lymphocytes with Cap-DCbp3 was also detected as compared to other VLPs. This study described the potential of DC-binding peptides for further improved antigen delivery and vaccine efficacy, explainning nanovaccine optimization in relation to a range of emerging and circulating infectious pathogens.

Background A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg- Pb / Pf CSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes ( Pf CSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. Methods Inbred mice were vaccinated with Pf CSP-SAPN in PBS. Serum antibodies were harvested and effects on P . falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. Results The results of this work demonstrate that Pf CSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P . falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, Pf CSP-SAPN-induced antibodies are able to inhibit wild-type P . falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that Pf CSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous fashion via transporter associated with antigen processing (TAP) recruitment to the early endosome (EE), and have partially delayed processing through the endoplasmic reticulum, has the potential to induce the long-lived, effector memory CD8 + T-cells as described previously. Conclusion This paper describes the examination of humoral and cellular immune mechanisms induced by Pf CSP-SAPN vaccination which result in sterile host protection against a transgenic P . berghei malaria sporozoite expressing the P . falciparum CSP, and which significantly inhibits native P . falciparum sporozoites from invading and developing within cultured human hepatocytes. These results may indicate the type and mode of action of protective antibodies needed to control P . falciparum sporozoites from infecting humans as well as a potential mechanism of induction of protective long-lived effector memory CD8 + T-cells.

Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations. One promising area of research involves self-assembled protein nanoparticles (SAPNs), which have shown potential for enhancing antigen-presenting cell uptake, B-cell activation, and lymph node trafficking. Numerous nanovaccines have been utilized in veterinary medicine, including natural self-assembled protein nanoparticles, rationally designed self-assembled protein nanoparticles, animal virus-derived nanoparticles, bacteriophage-derived nanoparticles, and plant-derived nanoparticles, which will be discussed in this review. SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases. This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine. Furthermore, this paper highlights their disadvantages and methods for improving their immunogenicity.