Background and Aims

Hemorrhagic fever with renal syndrome (HFRS) is a viral zoonotic disease primarily caused by the Hantaan virus (HTNV). This illness is prevalent in over 70 countries worldwide, posing a significant public health challenge due to its high endemicity. The glycoprotein (GP) of HTNV is a key structural protein that is crucial for triggering both humoral and cellular immune responses, making GP-specific immunoprophylaxis a promising therapeutic approach for HFRS.

Methods

Lysosome-associated membrane protein 1 (LAMP1) has the ability to target antigens to lysosomes and endosomes, thereby enhancing the immunogenicity of nucleic acid vaccines. In this study, we developed a recombinant DNA vaccine for HTNV GP that utilizes LAMP1 to direct the vaccine to the major histocompatibility complex (MHC) class II compartment. We conducted thorough computational analyses to evaluate the properties of the vaccine molecule and its potential immune responses in the body. Initial animal studies confirmed the effectiveness of GP-derived Th epitopes and multi-epitope vaccines.

Results

We designed two vaccines based on Gnc molecules and optimized them using a LAMP-targeting approach. Following three immunizations, mice receiving the pVAX-LAMP/Gnc vaccine exhibited increased splenocyte-specific IFN-γ secretion and higher serum antibody titers, particularly in terms of neutralizing activity. Furthermore, the efficacy of these molecular therapies was supported by preliminary in silico findings and laboratory animal experiments. By facilitating lysosomal trafficking and antigen presentation, the LAMP1 targeting strategy significantly enhanced both humoral and cellular immune responses specific to EBOV-GP.

Conclusions

Our research expands the strategic framework for improving DNA vaccine design and presents a promising candidate for HFRS prevention, establishing a foundation for future antiviral vaccine strategies.

Introduction: Breast cancer is the most diagnosed type of cancer in women and the leading cause of death by cancer worldwide. In recent years, active immunotherapy using vaccines has been raised as a novel approach to conventional treatments. Peptide-based vaccines are developed using overexpressed proteins in the tumor, commonly known as tumor antigens, that can stimulate the humoral immune response. Objective: To evaluate in Balb/c mice the production of antibodies induced by inoculation with doses of 30, 50, and 100 µg of the multi-peptide-based vaccine prototype derived from tumoral antigens. Material and methods: The vaccine prototype included the peptides derived from the following proteins: mammaglobin-α, NY-ESO-1, PLAC-1, Syntenin-1, and MAGE-A3. These were selected by in silicio analysis. Peptides were mixed with Freund incomplete adjuvant and inoculated subcutaneously during days 1, 15, 45, and 60 in twelve Balb/C mice classified into four experimental groups (three experimental groups and a placebo group). The mice were bled during days 0, 40, and 80, and serum samples were used to detect the presence of antibodies (IgM, IgG1, IgG2a, IgG2b, and IgG3) and the recognition of each individual peptide by assays of Indirect ELISA and Dot blot. Results: We observed the production of IgM and subclasses of IgG in the three experimental groups (30, 50, and 100 µg), mainly the subclasses IgG2a and IgG2b. In the Dot blot, we observed that immunized mice produce antibodies against all the peptides; particularly, the peptides derived from Syntenin-1, PRAME, mammaglobin-α, and PLAC-1 were the most immunogenic. Conclusion: The multi-peptide-based vaccine prototype induced antibody production against each peptide. The results can contribute to the development of future in vivo experiments focused on the effect of the administration of peptides on avoiding tumoral growth.

Neoplasms and reduced productivity in chickens caused by avian leukosis viruses (ALVs) have led to significant economic losses in the poultry industry. Due to the frequent genomic mutations that give rise to new viral variants, traditional whole-virus vaccines have demonstrated inadequate protection. With the high prevalence of ALV in farms in China, it is urgent to develop novel vaccines to control ALV. The capsid protein p27 is highly conserved across all current ALV subtypes, and studies have shown that p27-specifc antibodies in ALV-infected chickens were able to neutralize ALV, making it a promising target for vaccine design. We screened anti-p27 monoclonal antibodies using phage display technology from antibodies 111C and 86C that could neutralize ALVs. To identify neutralizing epitopes, antibody–antigen docking simulation was first used to predict epitopes that might interact with neutralizing antibodies. Then, the predicted peptides were generated, which were then identified by ELISA and Western blot to bind to neutralizing monoclonal antibodies. The affinities of the neutralizing antibodies in binding to the identified epitopes were as high as 10⁻⁹ M, validating the neutralizing epitopes were correct. These epitopes were found to be highly conserved among most ALV variants and could be displayed on virus-like particles (VLPs) to form vaccine candidates. These vaccines will be tested in chickens to assess both their immunogenicity and protection against ALVs. The reverse vaccinology used in this study provides an excellent paradigm for the development of protective vaccines against ALV, with broader implications for future vaccine research.

Respiratory infections caused by Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa remain life-threatening to Cystic Fibrosis (CF) patients. Immunotherapies are attractive alternatives to protect CF patients against these infections. In order to identify new targets for the development of immunoprotective therapies, we used a surfomics approach to find putative surface-exposed proteins. This methodology combined with immunoinformatics tools allowed the identification of surface-exposed proteins containing B-cell epitopes [1]. The OmpA-like protein BCAL2645 was chosen and demonstrated by Western blotting and ELISA assays to be immunoreactive against sera from CF patients with a record of Bcc infections. The protein was characterized as multifunctional and important in the infection process. An anti-BCAL2645 polyclonal antibody was produced and was found to decrease the number of adhered and invading B.cenocepacia bacteria to human cells in vitro by more than 70% [2]. A cross-effect against P.aeruginosa and B.multivorans using this antibody was also observed, strongly decreasing the adhesion and invasion of these species to the human bronchial epithelial cell line CFBE41o-[3]. Using the animal model Galleria mellonella, the antibody was found to confer protection against these infections. These results highlight the potential of anti-BCAL2645 antibodies for passive immunization therapies to prevent infections against two of the most problematic bacterial species infecting CF patients. Preliminary results from passive immunization strategies under study using anti-BCAL2645 antibodies will be presented.

[1] Seixas, AMM; etal. Surface-Exposed Protein Moieties of Burkholderia cenocepacia J2315 in Microaerophilic and Aerobic Conditions. Vaccines 2024,12,398. https://doi.org/10.3390/vaccines12040398

[2] Seixas, AMM; etal.. A Polyclonal Antibody Raised against the Burkholderia cenocepacia OmpA-like Protein BCAL2645 Impairs the Bacterium Adhesion and Invasion of Human Epithelial Cells In Vitro. Biomedicines 2021,9,1788. https://doi.org/10.3390/biomedicines9121788

[3] Seixas, AMM; etal. A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B.multivorans Virulence. Vaccines 2024,12,207. https://doi.org/10.3390/vaccines12020207

Oropouche virus (OROV) is the pathogen that causes Oropouche fever, an endemic disease in Amazonian regions, whose symptomatology could include complications in the nervous and blood systems, and for which there are no specific treatments or vaccines. The development of vaccines has been supported by advances in areas such as immunoinformatics and the introduction of new vaccination platforms such as viral vectors for the delivery of pathogen antigens. The aim of the present work was to design a vaccine against the Oropouche virus based on a multi-epitope protein and using immunoinformatics. A consensus proteome of OROV was generated from the alignment of sequences stored in databases. Based on this proteome, CD8+ T cell, CD4+ T cell and B cell epitope prediction programs were used to identify peptides with epitope potential. The epitopes to be included in the vaccine were then selected based on the following criteria: high promiscuity towards HLA-I and HLA-II alleles that are prevalent in the countries most affected by the virus, conservation of the peptide among the different OROV strains, non-homology of the peptide with the human proteome, and the absence of toxicity and allergenicity of the peptide. After the filtering and selection process, eleven T cell epitopes, which allowed for a total coverage of the most frequent HLA alleles among OROV-exposed countries, and three B cell epitopes were selected. The chosen epitopes were concatenated by linkers to generate a multi-epitope protein, which also contains the sequence of the β-defensin 3 as an adjuvant. The multi-epitope protein will be evaluated by molecular docking analysis against TLR4 to identify its potential to activate the immune system. This study proposes a vaccine candidate against the Oropouche virus that will be tested in preclinical models in the next phase of this project.

Background: Lymphatic filariasis is a neglected tropical disease (NTD) affecting more than 863 million people in 47 countries across the world. A multi-epitope prophylactic/therapeutic vaccination targeting filarial defense proteins would be invaluable in achieving the current goal of LF elimination.

Method: In this study, a combination of immunomics and immune-informatics was applied to construct a multi-epitope vaccine candidate. The antigenic proteins were identified by immune blotting against different categories of Wuchereria bancrofti-infected LF sera.

Result: The major antigenic proteins were heat shock protein 70, Tubulin beta chain, Enolase, Galectin, and 14-3-3 zeta. The five antigens were combined together to construct a multi-epitope vaccine after predicting the linear B-cell and T-cell epitopes of individual antigens. A three-dimensional model of the candidate vaccine was predicted, followed by refinement, and was validated using RAMPAGE and PROCHECK servers. A Toll-like receptor (TLR) agonist, a 50S ribosomal subunit of Mycobacterium tuberculosis, was included in the candidate vaccine to enhance vaccine immunogenicity. The docking of the chimeric peptide vaccine against the TLR5 resulted in high binding efficiency for the docked complex. The in silico immune simulation provided a significant increase in CD4⁺ T-cell and CD8⁺ T-cell populations.

Conclusion: In summary, the recombinant putative vaccine showed high immunogenicity which could be experimentally validated in the future for the development of a potent LF vaccine. Furthermore, by employing multi-epitope structures and constructing a cocktail vaccine for LF, this study has the potential to represent an important milestone in the development of an anti-filarial vaccine.

Influenza remains a persistent global threat, with the rapid mutation of its surface proteins, particularly hemagglutinin (HA), challenging the effectiveness of seasonal vaccines. Universal vaccination seems to be a promising way of combatting the influenza virus's rapid mutation rate. The concept of a universal vaccine aims to provide broad protection against diverse strains by targeting conserved viral epitopes. In this review, I explored some of the major hurdles in achieving this goal, focusing on antigen selection, immunodominance, and the challenges posed by nanoparticle-based and T-cell-mediated vaccine platforms. The pursuit of a universal influenza vaccine faces numerous challenges despite advancements in the identification of conserved antigens and innovative technologies. Different types of vaccines have been put into clinical trials, such as viral vector and mRNA-based vaccines, non-replicating viral vector vaccines, and broadly neutralized antibodies (bnAbs), which have been proven to meet the research demands of the universal influenza vaccine. While promising, these approaches face significant barriers, including issues of antigen stability, delivery, and struggles of eliciting durable cross-reactive immune responses in mucosal tissues. Novel platforms like mRNA vaccines and viral vector vaccines show promise in providing broad protection against virus mutations. Breakthroughs in vaccine design continue to be made, aiming to enhance cross-protective efficacy and defense against potential pandemics. The results highlight that although significant progress has been made, overcoming the scientific, technical, and regulatory challenges is essential for achieving a truly universal influenza vaccine. In conclusion, ongoing global collaboration and innovative research are crucial for overcoming these barriers. However, the mutation rate of the influenza virus presents a significant obstacle in the path towards a commercially available universal vaccine.

Malaria is a major public health problem. Estimates suggestthat there were over 249 million cases of malaria in the year 2022. Most of the severe malaria cases and almost all the malaria-related deaths are caused by malaria parasite Plasmodium falciparum. The malaria parasite P. falciparum has unique cytoadherence properties through which it sequesters in the host's microvascular endothelial cells. The P. falciparum-infected erythrocytes can bind to the host's microvascular endothelial cells, platelets, uninfected erythrocytes and other immune cells, such as dendritic cells, and sequester in the host's blood vasculature. The cytoadherence of P. falciparum-infected erythrocytes in the brain and placenta has been associated with severe malaria disease. Cytoadherence to microvascular endothelial cells causes obstruction in blood flow, endothelial cell activation, and the release of proinflammatory cytokines. Studies have also shown that the gC1qR-mediated cytoadherence of P. falciparum-infected erythrocytes to dendritic cells can lead to the inhibition of the dendritic cells and therefore may contribute to the immune suppression of the innate immune system of the host. Cytoadherence is mediated by specific receptor–ligand interactions. Earlier, we identified a novel receptor gC1qR for cytoadherence to brain microvascular endothelial cells and platelets. The P. falciparum-infected erythrocytes can bind to dendritic cells, platelets and brain microvascular endothelial cells via gC1qR as a receptor. Cytoadherence to gC1qR has been implicated in the pathogenesis of severe malaria by several studies. We have also mapped the interacting domain of PfEMP1 interacting with gC1qR. Since gC1qR has an important role in severe malaria pathogenesis and the inhibition of dendritic cells have a role in innate and adaptive immunity, a vaccine targeting gC1qR and its PfEMP1 ligand interaction will likely have a broader protection, eliciting a longer-lasting immunity against P. falciparum infections.

An improved vaccine against tuberculosis (TB) is urgently needed to enhance the impact of TB immunisation offered by BCG, the only licensed TB vaccine, which is currently unable to substantially reduce the number of TB cases globally. Most new TB vaccine candidates use the parenteral and invasive route of immunisation without showing significant superiority to BCG. Wide-ranging vaccination strategies should be considered to improve the chances of finding an ideal TB vaccine. Here, we explore the potential of treating TB with a non-invasive immunisation route, specifically looking at the oral route of vaccination as a potential strategy for developing a new TB vaccine, which is arguably the most preferred route of vaccination due to its ease of administration at a lower cost compared to injectable vaccines. The downstream hostile gut environment poses a major challenge to oral vaccine development. Several strategies for overcoming this challenge have been studied, such as using live-attenuated vectors and nanoparticles. Our group is at an early stage of developing an oral TB vaccine candidate based on a chimeric secretory IgA-Ag85B subunit vaccine constructed to withstand the gut environment and reach the mucosal immune cells to initiate immune responses. We are currently evaluating the immunogenicity of the vaccine candidate as a booster to BCG in a mouse model to further assess its potential. BALB/c mice primed with BCG were given the vaccine candidate orally, with subsequent collection of samples from the euthanised mice 4 weeks post-booster to determine the T-cell responses in the spleen using flow cytometry and the anti-Ag85B IgG levels in serum and the anti-Ag85B IgA levels in bronchoalveolar lavage using ELISA. The findings from this study can offer new knowledge regarding oral immunisation using a recombinant secretory IgA construct that could potentially become a candidate vaccine for future TB vaccine development.

Fowl adenovirus serotype 4 (FAdV-4), leading to Hepatitis-hydropericardium syndrome (HHS) in chickens, has caused huge economic losses to the poultry industry in China since 2015. Therefore, effective therapeutic approaches to prevent FAdV-4 is critical for HHS control. In this study, we identified two monoclonal antibodies, 27A-5 and 42A-10, to specifically bind to and neutralize FAdV-4 viruses in vitro. Pre-incubation of both antibodies with FAdV-4 hindered the viruses from infecting LMH cells, with the complete blocking concentrations being 33.3 μg/mL for 27A-5 and 16.6 μg/mL for 42A-10. In addition, whether these antibodies pose viral neutralization abilities in vivo was determined, as was whether they could be used as therapeutic antibodies in the future. In chickens, the survival rates were 100% after administration with both antibodies (10 mg/kg), while the fatality rates of untreated chickens were 100%. In addition, a low dose (5mg/kg) of the 27A-5 antibody could save all animals. Moreover, pathologic changes such as pericardial sac liquid and liver damages in survivors were remarkably reduced. Further analysis demonstrated that inflammation caused by FAdV-4 infection was decreased in antibody treated animals. More importantly, the viral loads in treated chickens were almost cleared, indicating that the antibodies were effectively neutralizing the FAdV-4 viruses in vivo. In conclusion, the two antibodies could neutralize FAdV-4 in vitro and in vivo, which means that they have the potential to be therapeutic antibodies for HHS treatment. This study lays a foundation for the development of safe and effective therapeutic methods against FAdV-4.