Rational design and production of a chimeric antigen targeting Zika virus that induces neutralizing antibodies in mice.

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.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies.

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.

Assessing the Adjuvant Effect of Layered Double Hydroxides (LDH) on BALB/c Mice.

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.

Current status of mucosal vaccines against SARS-CoV2: a hope for protective immunity.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

An Algae-Made RBD from SARS-CoV-2 Is Immunogenic in Mice.

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.

Production and Purification of LTB-RBD: A Potential Antigen for Mucosal Vaccine Development against SARS-CoV-2.

Most of the current SARS-CoV-2 vaccines are based on parenteral immunization targeting the S protein. Although protective, such vaccines could be optimized by inducing effective immune responses (neutralizing IgA responses) at the mucosal surfaces, allowing them to block the virus at the earliest stage of the infectious cycle. Herein a recombinant chimeric antigen called LTB-RBD is described, which comprises the B subunit of the heat-labile enterotoxin from E. coli and a segment of the RBD from SARS-CoV-2 (aa 439-504, carrying B and T cell epitopes) from the Wuhan sequence and the variant of concern (VOC)-delta. Since LTB is a mucosal adjuvant, targeting the GM1 receptor at the surface and facilitating antigen translocation to the submucosa, this candidate will help in designing mucosal vaccines (i.e., oral or intranasal formulations). LTB-RBD was produced in E. coli and purified to homogeneity by IMAC and IMAC-anionic exchange chromatography. The yields in terms of pure LTB-RBD were 1.2 mg per liter of culture for the Wuhan sequence and 3.5 mg per liter for the delta variant. The E. coli-made LTB-RBD induced seric IgG responses and IgA responses in the mouth and feces of mice when subcutaneously administered and intestinal and mouth IgA responses when administered nasally. The expression and purification protocols developed for LTB-RBD constitute a robust system to produce vaccine candidates against SARS-CoV-2 and its variants, offering a low-cost production system with no tags and with ease of adaptation to new variants. The E. coli-made LTB-RBD will be the basis for developing mucosal vaccine candidates capable of inducing sterilizing immunity against SARS-CoV-2.

Nanoclays: Promising Materials for Vaccinology.

Clay materials and nanoclays have gained recent popularity in the vaccinology field, with biocompatibility, simple functionalization, low toxicity, and low-cost as their main attributes. As elements of nanovaccines, halloysite nanotubes (natural), layered double hydroxides and hectorite (synthetic) are the nanoclays that have advanced into the vaccinology field. Until now, only physisorption has been used to modify the surface of nanoclays with antigens, adjuvants, and/or ligands to create nanovaccines. Protocols to covalently attach these molecules have not been developed with nanoclays, only procedures to develop adsorbents based on nanoclays that could be extended to develop nanovaccine conjugates. In this review, we describe the approaches evaluated on different nanovaccine candidates reported in articles, the immunological results obtained with them and the most advanced approaches in the preclinical field, while describing the nanomaterial itself. In addition, complex systems that use nanoclays were included and described. The safety of nanoclays as carriers is an important key fact to determine their true potential as nanovaccine candidates in humans. Here, we present the evaluations reported in this field. Finally, we point out the perspectives in the development of vaccine prototypes using nanoclays as antigen carriers.

Production and characterization of chimeric SARS-CoV-2 antigens based on the capsid protein of cowpea chlorotic mottle virus.

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.

Expression of immunogenic poliovirus Sabin type 1 VP proteins in transgenic tobacco.

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.

Inducible expression of antigens in plants: a study focused on peptides related to multiple sclerosis immunotherapy.

Multiple sclerosis (MS) affects 2.3 million patients worldwide with no effective treatments available thus far. Depletion of autoreactive T-cells is considered the basis for immunotherapeutic approaches. For this purpose the peptides BV5S2, BV6S5, and BV13S1 have been identified as candidates for the development of a MS vaccine. Herein, the plant-based simultaneous production of these peptides is described as an effort to generate a new model of MS immunotherapy. A polyprotein comprising the sequence of the target peptides was designed having the picornaviral 2A sequence in between to mediate the release of the individual peptides upon translation. A codon optimized gene was cloned in vectors mediating constitutive (CaMV35S promoter) or inducible (AlcA promoter) expression. No transgenic tobacco plants were recovered from the constitutive vector suggesting toxicity of the target peptides. In contrast, several transformed lines were obtained with the inducible vector. The individual BV5S2, BV6S5, and BV13S1 peptides were detected in transformed lines upon ethanol-mediated induction and a quantitative analysis based on a OVA conjugate carrying the three peptides revealed accumulation levels up to 0.5 µg g-1 FW leaves. The plant-made peptides were able to induce humoral responses in orally immunized mice. This platform will be useful in the development of alternative immunotherapies against MS having low cost and safety as main attributes. Moreover the platform represents an attractive alternative for the expression of antigens having detrimental effects in plants.

Using carrot cells as biofactories and oral delivery vehicles of LTB-Syn: A low-cost vaccine candidate against synucleinopathies.

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.

Genetically-engineered plants yield an orally immunogenic PirA-like toxin from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the main etiological agent of human gastroenteritis by seafood consumption and some strains from this species causing the Acute Hepatopancreatic Necrosis Disease in shrimp have been recently reported. The PirA-like toxin from V. parahaemolyticus (ToxA) has been recently reported as an attractive antigen implicated in subunit vaccine development. Since plants are attractive hosts for the production and delivery of vaccines in the present study plants expressing ToxA were developed to account with a low cost platform for the production and oral delivery of ToxA. Tobacco plants were genetically engineered by Agrobacterium-mediated transformation to stably integrate the ToxA-coding gene into the nuclear genome. Transgenic lines were rescued in kanamycin-containing medium and analyzed by ELISA to determine ToxA yields observing levels up to 9 µg g-1 FW leaf tissues. Western blot analysis confirmed the presence of the ToxA protein in plant extracts. Immunogenicity assessment of the plant-made ToxA was performed in mice, comprising a 4-dose oral immunization scheme; revealing the induction of anti-ToxA humoral responses (IgG in serum and IgA in feces). This study opens the path for the development of low cost plant-based vaccines against Vibrio parahaemolyticus.

Design of a multiepitopic Zaire ebolavirus protein and its expression in plant cells.

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.

An overview of tolerogenic immunotherapies based on plant-made antigens.

INTRODUCTION: Over the last two decades, genetically engineered plants became attractive and mature platforms for producing vaccines and other relevant biopharmaceuticals. Autoimmune and inflammatory disorders demand the availability of accessible treatments, and one alternative therapy is based on therapeutic vaccines able to downregulate immune responses that favor pathology progression. AREAS COVERED: The current status of plant-made tolerogenic vaccines is presented with emphasis on the candidates under evaluation in test animals. Nowadays, this concept has been assessed in models of food and pollen allergies, autoimmune diabetes, asthma, arthritis, and prevention of blocking antibodies induction against a biopharmaceutical used in replacement therapies. EXPERT OPINION: According to the current evidence generated at the preclinical level, plant-made tolerogenic therapies are a promise to treat several immune-related conditions, and the beginning of clinical trials is envisaged for the next decade. Advantages and limitations for this technology are discussed.

Assessment of Carrot Callus as Biofactories of an Atherosclerosis Oral Vaccine Prototype.

Atherosclerosis is a pathology leading to cardiovascular diseases with high epidemiologic impact; thus, new therapies are required to fight this global health issue. Immunotherapy is a feasible approach to treat atherosclerosis and given that genetically engineered plants are attractive hosts for vaccine development; we previously proved that the plant cell is able to synthesize a chimeric protein called CTB:p210:CETPe, which is composed of the cholera toxin B subunit (CTB) as immunogenic carrier and target epitopes from the cholesteryl ester transfer protein (CETP461-476) and apolipoprotein B100 (p210). Since CTB:p210:CETPe was expressed in tobacco at sufficient levels to evoke humoral responses in mice, its expression in carrot was explored in the present study looking to develop a vaccine in a safe host amenable for oral delivery; avoiding the purification requirement. Carrot cell lines expressing CTB:p210:CETPe were developed, showing accumulation levels up to 6.1 µg/g dry weight. An immunoblot analysis revealed that the carrot-made protein is antigenic and an oral mice immunization scheme led to evidence on the immunogenic activity of this protein; revealing its capability of inducing serum IgG responses against p210 and CETP epitopes. This study represents a step forward in the development of an attractive oral low-cost vaccine to treat atherosclerosis.

LTB-Syn: a recombinant immunogen for the development of plant-made vaccines against synucleinopathies.

MAIN CONCLUSION: A recombinant antigen targeting α-synuclein was produced in the plant cell rendering an immunogenic protein capable to induce humoral responses in mice upon oral administration. Synucleinopathies are neurodegenerative diseases characterized by the abnormal accumulation of α-synuclein (α-Syn, a 140 amino acid protein that normally plays various neurophysiologic roles) aggregates. Parkinson's disease (PD) is the synucleinopathy with the highest epidemiologic impact and although its etiology remains unknown, α-Syn aggregation during disease progression pointed out α-Syn as target in the development of immunotherapies. Herein a chimeric protein, comprising the B subunit of the enterotoxin from enterotoxigenic Escherichia coli and α-Syn epitopes, was expressed in the plant cell having the potential to induce humoral responses following oral immunization. This approach will serve as the basis for the development of oral plant-based vaccines against PD with several potential advantages such as low cost, easy scale-up during production, and easy administration.

Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells.

KEY MESSAGE: An antigenic protein targeting two epitopes from the Zaire ebolavirus GP1 protein was expressed in plant cells rendering an antigen capable of inducing humoral responses in mouse when administered subcutaneously or orally. The 2014 Ebola outbreak made clear that new treatments and prophylactic strategies to fight this disease are needed. Since vaccination is an intervention that could achieve the control of this epidemic disease, exploring the production of new low-cost vaccines is a key path to consider; especially in developing countries. In this context, plants are attractive organisms for the synthesis and delivery of subunit vaccines. This study aimed at producing a chimeric protein named LTB-EBOV, based on the B subunit of the Escherichia coli heat-labile enterotoxin as an immunogenic carrier and two epitopes from the Zaire ebolavirus GP1 protein recognized by neutralizing antibodies. The LTB-EBOV protein was expressed in plant tissues at levels up to 14.7 µg/g fresh leaf tissue and proven to be immunogenic in BALB/c mice when administered by either subcutaneous or oral routes. Importantly, IgA and IgG responses were induced following the oral immunization. The potential use of the plant-made LTB-EBOV protein against EBOV is discussed.

Progress and future opportunities in the development of vaccines against atherosclerosis.

INTRODUCTION: Atherosclerosis represents a serious global health problem that demands new therapeutic and prophylactic interventions. Considering that atherosclerosis has autoimmune and inflammatory components, immunotherapy is a possible focus to treat this disease. Areas covered: Based on the analysis of the current biomedical literature, this review describes the status on the development of vaccines against atherosclerosis. Several targets have been identified including sequences of apolipoprotein B100 (ApoB100), cholesteryl ester transfer protein (CETP), heat shock proteins (HSP), extracellular matrix proteins, T cell receptor ß chain variable region 31 (TRBV31), the major outer membrane protein (MOMP), and the outer membrane protein 5 (Pomp5) from Chlamydia pneumoniae. Humoral and cellular immunities to these targets have been associated with therapeutic effects in murine models and humans. The evaluation of some candidates in clinical trials is ongoing. Expert commentary: New research paths based on the use of next generation vaccine production platforms are envisioned.

Towards the development of an oral vaccine against porcine cysticercosis: expression of the protective HP6/TSOL18 antigen in transgenic carrots cells.

MAIN CONCLUSION: The Taenia solium HP6/TSOL18 antigen was produced in carrot cells, yielding an immunogenic protein that induced significant protection in an experimental murine model against T. crassiceps cysticercosis when orally administered. This result supports the potential of HP6/TSOL18-carrot as a low-cost anti-cysticercosis vaccine candidate. Cysticercosis is a zoonosis caused by Taenia solium that can be prevented by interrupting the parasite life cycle through pig vaccination. Several injectable vaccine candidates have been reported, but the logistic difficulties and costs for its application limited its use in nationwide control programs. Oral plant-based vaccines can deal with this limitation, because of their easy administration and low cost. A stable expression of the HP6/TSOL18 anti-T. solium cysticercosis protective antigen in carrot calli transformed with an optimized transgene is herein reported. An antigen accumulation up to 14 µg g(-1) of dry-weight biomass was achieved in the generated carrot lines. Mouse immunization with one of the transformed calli induced both specific IgG and IgA anti-HP6/TSOL18 antibodies. A statistically significant reduction in the expected number of T. crassiceps cysticerci was observed in mice orally immunized with carrot-made HP6/TSOL18, in a similar extent to that obtained by subcutaneous immunization with recombinant HP6/TSOL18 protein. In this study, a new oral plant-made version of the HP6/TSOL18 anti-cysticercosis vaccine is reported. The vaccine candidate should be further tested against porcine cysticercosis.