The aggregation and spread of alpha‐synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the ...establishment of a safe and effective disease‐modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB‐Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat‐labile Escherichia coli enterotoxin LTB as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB‐Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB‐Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 μg/mL. Furthermore, the purified soluble rLTB‐Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB‐Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies.
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•Implications of bioinformatics on the development of epitope-based vaccines are described.•A compilation of useful bioinformatics tools for vaccine development is ...presented.•Implications on developing vaccines against hypervariable viruses are described.•Perspectives for the field are identified.
Exploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned.
Around 1.7 million people die annually due to enteric infections, which are mainly caused by ETEC (Enterotoxigenic
E. coli
),
V. cholera
,
V. parahaemolyticus
,
and Salmonella
. There are currently ...licensed vaccines against cholera and salmonella, whose distribution is chain-dependent making difficult to efficiently distribute them in poor countries. In this context plants are attractive hosts for the synthesis and delivery of subunit vaccines that could be produced at very low costs and widely distributed ensuring vaccination coverage. Subunit vaccines often demand the use of adjuvants to reach proper immunogenicity. Several bacterial toxins have been used as mucosal and systemic adjuvants and a potential molecule for this purpose is the PirA-like toxin (ToxA) from
V. parahaemolyticus
, which was deemed highly immunogenic in some species. In this study a protein named
ToxAentero
was expressed in tobacco plants to initiate the development of accessible vaccines against enteric diseases.
ToxAentero
is based on ToxA, as adjuvant carrier, and epitopes from ETEC,
V. cholerae
,
V. parahaemolyticus
, and
S. typhimurium
. The production yields reached up to 5.46 µg g
− 1
fresh leaf tissue. The plant-made
ToxAentero
was found immunogenic in mice immunized by oral or subcutaneous routes in terms of the induction of IgG (sera) and IgA (feces) humoral responses against most of the target epitopes from the enteric pathogens. This study opens the path for the development of a promising oral plant-based multiepitopic vaccine candidate in the fight against enteric diseases.
Key message
A multiepitopic protein targeting several enteric pathogens, was expressed in plants and characterized in mice; revealing that it is immunogenic and thus is proposed as an attractive vaccine candidate.
The current vaccines used to fight against COVID-19 are effective, however the induction of protective immunity is a pending goal required to prevent viral transmission, prevent the generation of new ...variants, and ultimately eradicate SARS-CoV-2. Mucosal immunization stands as a promising approach to achieve protective immunity against SARS-CoV-2; therefore, it is imperative to innovate the current vaccines by developing mucosal candidates, focusing not only on their ability to prevent severe COVID-19 but to neutralize the virus before invasion of the respiratory system and other mucosal compartments.
This review covers the current advances on the development of anti-COVID-19 mucosal vaccines. Biomedical literature, including PubMed and clinicaltrials.gov website, was analyzed to identify the state of the art for this field. The achievements in preclinical and clinical evaluations are presented and critically analyzed.
There is a significant advance on the development of mucosal vaccines against SARSCoV-2, which is a promise to increase the efficacy of immunization against this pathogen. Both preclinical and clinical evaluation for several candidates have been performed. The challenges in this road (e.g. low immunogenicity, a reduced number of adjuvants available, and inaccurate dosage) are identified and also critical perspectives for the field are provided.
The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome. Currently, there is no specific ...treatment or vaccine approved to combat this disease, and thus, developing safe and effective vaccines is a relevant goal. In this study, a multi-epitope protein called rpZDIII was designed based on a series of ZIKV antigenic sequences, a bacterial carrier, and linkers. The analysis of the predicted 3D structure of the rpZDIII chimeric antigen was performed on the AlphaFold 2 server, and it was produced in E. coli and purified from inclusion bodies, followed by solubilization and refolding processes. The yield achieved for rpZDIII was 11 mg/L in terms of pure soluble recombinant protein per liter of fermentation. rpZDIII was deemed immunogenic since it induced serum IgG and IgM responses in mice upon subcutaneous immunization in a three-dose scheme. Moreover, sera from mice immunized with rpZDIII showed neutralizing activity against ZIKV. Therefore, this study reveals rpZDIII as a promising immunogen for the development of a rationally designed multi-epitope vaccine against ZIKV, and completion of its preclinical evaluation is guaranteed.
•Synuclein is a possible target for immunotherapies development.•A chimeric antigen targeting three epitopes from α-Syn was designed (LTB-Syn).•LTB-Syn was produced in carrot cell lines at 2.5 μg/g ...dry biomass.•LTB-Syn showed high stabillity in dried plant tissues for 6 months at 25 °C.•LTB-Syn served as priming agent in mice, leading to significant humoral responses.
Synucleinopathies are conditions that remain with no available effective treatments thus far. Immunotherapy is a possible path to fight against such pathologies by inducing antibodies against alpha-synuclein (α-Syn), which could induce the clearance of its pathologic form. Looking to develop a new low-cost, effective vaccine against synucleinopathies; we have designed a chimeric plant-made antigen comprising the subunit B of the enterotoxin from enterotoxigenic E. coli and three B cell epitopes from α-Syn, which is named LTB-Syn. In the present study, LTB-Syn was produced in carrot cell lines as appropriate platform for the formulation of oral vaccines not requiring purification. The development of transgenic carrot cell lines took 8 months and the LTB-Syn yield reached 2.3 μg/g dry biomass. The antigen encapsulated in lyophilized carrot cells was highly stable at room temperature over a six-month period and upon heating at 50 °C for 2 h. Moreover, LTB-Syn was able to prime immune responses that, in combination with parenteral boosting using an OVA-Syn conjugate, induced significant humoral resposes in mice. Thus the carrot-made oral LTB-Syn vaccine is a promising candidate that deserves further analyses to advance in its preclinical evaluation.
The discovery and validation of new adjuvants are critical areas for vaccinology. Mineral materials (e.g., alum microparticles) have been used for a long time as adjuvants in human vaccine ...formulations. Nonetheless, the use of nanosized materials is a promising approach to diversify the properties of adjuvants. Nanoclays are potential adjuvants proposed by some research groups. However, their adjuvant mechanisms and safety have not been fully elucidated. Herein, we aimed at expanding the knowledge on the potential adjuvanticity of layered double hydroxide (LDH) nanoparticles by reporting a detailed method for the synthesis and characterization of LDHs and the adsorption of a model antigen (bovine serum albumin, BSA). LDHs varying in diameter (from 56 to 88 nm) were obtained, and an in vitro evaluation revealed that the LDHs are not inherently toxic. BSA was passively adsorbed onto the LDHs, and the immunogenicity in mice of the conjugates obtained was compared to that of free BSA and BSA co-administered with alum (Alum-BSA). The LDH-BSA conjugates induced a higher humoral response that lasted for a longer period compared with that of free BSA and Alum-BSA, confirming that LDH exerts adjuvant effects. The 56 nm LDH particles were deemed as the more efficient carrier since they induced a higher and more balanced Th1/Th2 response than the 88 nm particles. This study is a contribution toward expanding the characterization and use of nanoclays in vaccinology and justifies further studies with pathogen-specific antigens.
Despite the current advances in global vaccination against SARS-CoV-2, boosting is still required to sustain immunity in the population, and the induction of sterilizing immunity remains as a pending ...goal. Low-cost oral immunogens could be used as the basis for the design of affordable and easy-to-administer booster vaccines. Algae stand as promising platforms to produce immunogens at low cost, and it is possible to use them as oral delivery carriers since they are edible (not requiring complex purification and formulation processes). Herein, a Chlamydomonas-made SARS-CoV-2 RBD was evaluated as an oral immunogen in mice to explore the feasibility of developing an oral algae-based vaccine. The test immunogen was stable in freeze-dried algae biomass and able to induce, by the oral route, systemic and mucosal humoral responses against the spike protein at a similar magnitude to those induced by injected antigen plus alum adjuvant. IgG subclass analysis revealed a Th2-bias response which lasted over 4 months after the last immunization. The induced antibodies showed a similar reactivity against either Delta or Omicron variants. This study represents a step forward in the development of oral vaccines that could accelerate massive immunization.
•The four VPs from Poliovirus Sabin 1 are potential targets for subunit vaccine development.•Codon-optimized genes coding for the VPs were expressed in transgenic tobacco plants.•The yields of VPs in ...leaf tissues ranged 0.31–16.85 μg/g of fresh weight.•The plant-made VPs elicited systemic and mucosal humoral responses in mice upon subcutaneous and oral immunizations.
One of the milestones of vaccinology is the depletion of the global impact of Poliomyelitis. The current vaccines to deal with Polio comprise the Sabin and Salk formulations. The main limitation of the former is the use of attenuated viruses that can revert into pathogenic forms, whereas the latter is more expensive and induces no protection in the intestinal tract; the site of virus replication. Genetically engineered plants cope with such limitations. In addition, they offer a low-cost alternative for production, storage and delivery of vaccines. This technology has been narrowly applied in the development of Polio vaccines.
Herein, we explored the ability of tobacco cells to express the immunogenic VP1, VP2, VP3, and VP4 Polio antigens, which are relevant for vaccine development. Evidence on the expression of the plant-made Polio VPs is presented and an immunogenicity assessment proved their capacity to induce local and systemic humoral responses when administered by subcutaneous and oral routes. The plant-made VPs will be useful in the development of low-cost vaccine formulations able to induce effective mucosal immunity without the risks associated to the use of attenuated viruses; therefore there is a potential for this technology to contribute toward Polio eradication.
The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for ...developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.
•Cowpea chlorotic mottle virus capsid protein (CCMV CP) is an attractive epitope carrier.•Epitope based vaccines are a possible approach to innovate vaccination against SARS-CoV-2.•CCMV CP-based chimeras targeting SARS-CoV-2 were efficiently produced in E. coli.•The three CCMV CP-based chimeras were purified at a high level by IMAC.•The three CCMV CP-based chimeras showed attractive antigenicity and immunogenicity.
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