Epitope-coated polymer particles elicit neutralising antibodies against Plasmodium falciparum sporozoites

The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.

Detection of Antibodies Against the SARS-CoV-2 Spike Protein and Analysis of the Peripheral Blood Mononuclear Cell Transcriptomic Profile, 15 Years After Recovery From SARS

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shows a high degree of homology with SARS-CoV. They share genes, protein sequences, clinical manifestations, and cellular entry patterns. Thus, SARS research may serve helpful in gaining a better understanding of the current coronavirus disease 2019 (COVID-19) pandemic. Serum antibodies from convalescent patients with SARS collected in 2018 were used to target the recombinant SARS-CoV-2 spike protein via a chemiluminescence microsphere immunoassay. Antibodies of convalescent patients with SARS exhibited serous immune cross-reactivity with the SARS-CoV-2 spike protein. The serous antibodies, excluding S22 of convalescent patients with SARS, did not competitively inhibit the binding of SARS-CoV-2 spike protein to ACE2. T cellular immunity research was conducted in vitro using peripheral blood mononuclear cells (PBMCs) stimulated by pooled peptide epitopes 15 years post-infection. Interferon gamma was detected and the PBMC transcriptomic profile was obtained. The heatmap of the transcriptomic profile showed that mRNAs and circRNAs of the SARS group clustered together after being stimulated by the peptide epitope pool. Differentially expressed mRNAs were most significantly enriched in immunity and signal transduction (P < 0.01). SARS elicits cytokine and chemokine responses, partially consistent with previously published data about COVID-19. Overall, our results indicate that antibodies from convalescent patients with SARS persisted for 15 years and displayed immune cross-reactivity with the SARS-CoV-2 spike protein. The immune status of patients with SARS 15 years post-infection may provide a better understanding of the future immune status of patients with COVID-19.

Peptide-Based Vaccines for Neurodegenerative Diseases: Recent Endeavors and Future Perspectives

The development of peptide-based vaccines for treating human neurodegenerative diseases has been the eventual aim of many research endeavors, although no active immunotherapies have been approved for clinical use till now. A typical example of such endeavors is the effort to develop vaccines for Alzheimer’s disease based on the beta-amyloid peptide, which continues to be intensively investigated despite previous setbacks. In this paper, recent developments in peptide-based vaccines which target beta-amyloid as well as tau protein and α-synuclein are presented. Particular focus has been directed toward peptide epitopes and formulation systems selected/developed and employed to enhance vaccine efficacy and safety. Results from both, human clinical trials and animal preclinical studies conducted mainly in transgenic mice have been included. Future perspectives on the topic are also briefly discussed.

Virtual Screening to Identify the Protein Network Interaction of Berberine with Red Complex Pathogens

Introduction: Periodontal disease is an infection of the tissues that hold your teeth in place. It's typically caused by poor brushing and flossing habits that allow plaque, a sticky film of bacteria, to build up on the teeth and harden. Elimination of these pathogens from the site of infection remains a perplexing task, which demands the use of antibiotics. The emergence of drug resistant forms has spurred interest into identifying novel therapeutic targets against these pathogens. Aim: The present study employs virtual screening method to identify the protein network interaction of berberine with red complex pathogens. Materials and Methods: Computational tools were used to identify the targets, assess their functional role and virulence property. Further, the peptide epitopes present in the virulence factors were identified using the BepiPred tool. The subcellular location of the virulence proteins was also elucidated using PSORTb. Results: Berberine was found to target vital protein transporters such as TetR family transcriptional regulator and MerR family transcriptional regulator, which is known to play a crucial role in the survival of bacterial cells. Conclusion: Hence the present study provides preliminary data on the protein targets of berberine against red complex pathogens. However, in vitro studies using the compound is warranted to further confirm the efficacy of the compound.

FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.

In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles

Simultaneous delivery of mRNA to multiple populations of antigen (Ag)-specific CD8+ T cells is challenging given the diversity of peptide epitopes and polymorphism of class I major histocompatibility complexes (MHCI). We developed Ag-presenting nanoparticles (APNs) for mRNA delivery using pMHCI molecules that were refolded with photocleavable peptides to allow rapid ligand exchange by UV light and site-specifically conjugated with a lipid tail for post-insertion into preformed mRNA lipid nanoparticles. Across different TCR transgenic mouse models (P14, OT-1, Pmel), UV-exchanged APNs bound and transfected their cognate Ag-specific CD8+ T cells equivalent to APNs produced using conventionally refolded pMHCI molecules. In mice infected with PR8 influenza, multiplexed delivery of UV-exchanged APNs against three immunodominant epitopes led to ~50% transfection of a VHH mRNA reporter in cognate Ag-specific CD8+ T cells. Our data shows that UV-mediated peptide exchange can be used to rapidly produce APNs for mRNA delivery to multiple populations of Ag-specific T cells in vivo.

A New Workflow to Generate Monoclonal Antibodies against Microorganisms

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.

HER2/neu-Based Peptide Vaccination-Pulsed with B-Cell Epitope Induced Efficient Prophylactic and Therapeutic Antitumor Activities in TUBO Breast Cancer Mice Model

Breast cancer is the most common invasive cancer diagnosed among women. A cancer vaccine has been recognized as a form of immunotherapy with a prominent position in the prevention and treatment of breast cancer. The majority of current breast cancer vaccination strategies aim to stimulate antitumor T-cell responses of the HER2/neu oncogene, which is abnormally expressed in breast cancer cells. However, the role of the B-cell humoral response is often underappreciated in the cancer vaccine design. We have advanced this idea by elucidating the role of B-cells in cancer vaccination by designing a chimeric antigenic peptide possessing both cytotoxic T lymphocytes (GP2) and B-cell (P4) peptide epitopes derived from HER2/neu. The chimeric peptide (GP2–P4) was further conjugated to a carrier protein (KLH), forming a KLH–GP2–P4 conjugate. The immunogenicity of KLH–GP2–P4 was compared with KLH–GP2 (lacking the B-cell epitope) in BALB/c mice. Mice immunized with KLH–GP2–P4 elicited more potent antigen-specific neutralizing antibodies against syngeneic TUBO cells (cancer cell line overexpressing HER2/neu) that was governed by a balanced Th1/Th2 polarization in comparison to KLH–GP2. Subsequently, these immune responses led to greater inhibition of tumor growth and longer survival in TUBO tumor-bearing mice in both prophylactic and therapeutic challenge experiments. Overall, our data demonstrated that the B-cell epitope has a profound effect in orchestrating an efficacious antitumor immunity. Thus, a multi-epitope peptide vaccine encompassing cytotoxic T-lymphocytes, T-helper and B-cell epitopes represents a promising strategy in developing cancer vaccines with a preventive and therapeutic modality for the effective management of breast cancer.

Poor CD4+ T Cell Immunogenicity Limits Humoral Immunity to P. falciparum Transmission-Blocking Candidate Pfs25 in Humans

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission.