Production and Immunogenicity Assessment of LTp50: An Escherichia coli-Made Chimeric Antigen Targeting S1- and S2-Epitopes from the SARS-CoV-2/BA.5 Spike Protein.

Subunit vaccines stand as a leading approach to expanding the current portfolio of vaccines to fight against COVID-19, seeking not only to lower costs but to achieve long-term immunity against variants of concern and have the main attributes that could overcome the limitations of the current vaccines. Herein a chimeric protein targeting S1 and S2 epitopes, called LTp50, was designed as a convenient approach to induce humoral responses against SARS-CoV-2. LTp50 was produced in recombinant Escherichia coli using a conventional pET vector, recovering the expected antigen in the insoluble fraction. LTp50 was purified by chromatography (purity > 90%). The solubilization and refolding stages helped to obtain a stable protein amenable for vaccine formulation. LTp50 was adsorbed onto alum, resulting in a stable formulation whose immunogenic properties were assessed in BALB/c mice. Significant humoral responses against the S protein (BA.5 variant) were detected in mice subjected to three subcutaneous doses (10 µg) of the LTp50/alum formulation. This study opens the path for the vaccine formulation optimization using additional adjuvants to advance in the development of a highly effective anti-COVID-19 vaccine directed against the antigenic regions of the S protein, which are less prone to mutations.

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.

A general photoiniferter approach to the surface functionalization of acrylic and methacrylic structures written by two-photon stereolithography.

Two-photon stereolithography (TPS) is an established additive fabrication technique allowing the voxel-by-voxel direct writing of even intricate 3D nano/microstructures via the polymerization of a photoresin. An obvious way to tune the chemical functionalities of such nano/microstructures is formulating a photoresin with the desired functional monomer(s). Unfortunately, this makes every photoresin "unique" in terms of viscosity and reactivity, thus requiring a tedious and often time-consuming optimization of its printing parameters. In this work, we describe a general approach for the chemical functionalization of TPS-written structures based on two commercial photoresins. Our strategy entailed the grafting of functional polymer layers via an innovative approach based on photoiniferter coupling to unreacted double bonds and photopolymerization. After writing woodpiles as 3D model structures, we demonstrated the viability of this approach by anchoring a photoiniferter via its photoinduced addition to the residual CC on the structure's surface triggered by green light. This in turn allowed for the blue light-mediated, surface-initiated photopolymerization of functional monomers. Molecularly imprinted polymer films were also easily synthesized by using the same approach on model honeycombs. The imprinted layers resulted in only a minimal increase in size with no effect on the geometrical features of the honeycombs. Overall, this strategy offers a general approach for the surface modification of TPS-written (meth)acrylic structures with a wide variety of functional polymers via photoiniferter polymerization.

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.

Synthesis and immunogenicity assessment of a gold nanoparticle conjugate for the delivery of a peptide from SARS-CoV-2.

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.

Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase.

Bioremediation with genetically modified microalgae is becoming an alternative to remove metalloids and metals such as cadmium, a contaminant produced in industrial processes and found in domestic waste. Its removal is important in several countries including Mexico, where the San Luis Potosi region has elevated levels of it. We generated a construct with a synthetic gene for γ-glutamylcysteine synthetase and employed it in the chloroplast transformation of Chlamydomonas reinhardtii. In dose-response kinetics with media containing from 1 to 20 mg/L of cadmium, both the transplastomic clone and the wild-type strain grew similarly, but the former removed up to 32% more cadmium. While the growth of both decreased with higher concentrations of cadmium, the transplastomic clone removed 20 ± 9% more than the wild-type strain. Compared to the wild-type strain, in the transplastomic clone the activity of glutathione S-transferase and the intracellular glutathione increased up to 2.1 and 1.9 times, respectively, in media with 2.5 and 10 mg/mL of cadmium. While 20 mg/L of cadmium inhibited the growth of both, the transplastomic clone gradually duplicated. These results confirm the expression of the synthetic gene gshA in the transformed strain as revealed in its increased removal uptake and metabolic response.

Arsenic removal using Chlamydomonas reinhardtii modified with the gene acr3 and enhancement of its performance by decreasing phosphate in the growing media.

Arsenic contamination of groundwater is a significant problem in countries like Mexico, where San Luis Potosi is among the regions registering severe levels of it. Bioremediation with microalgae capable to absorb and metabolize metals or metalloids like arsenic reduces their toxicity and is a cost-effective approach compared to physical-chemical processes. We evaluated the capability of Chlamydomonas reinhardtii to remove arsenate and compared it with an acr3-modified recombinant strain, which we produced by transforming the wild-type strain with Agrobacterium tumefaciens using the construct pARR1 including a synthetic, optimized acr3 gene from Pteris vittata, a hyper-accumulator of arsenic. We monitored the growth of both strains in media with arsenate, containing a standard or a 10-fold decreased amount of phosphate. Comparing both strains in media initially with 0.5, 1, and 1.5 mg/L of arsenate, the acr3-modified strain removed 1.5 to 3 times more arsenic than the wild-type strain. Moreover, the arsenic uptake rate increased 1.2 to 2.3 times when growing the acr3-modified strain in media with decreased phosphate, while the uptake rate for the wild-type strain scarcely changed under the same conditions. These results confirm the expression of the acr3 gene in C. reinhardtii and its potential application to remove arsenic.

K88 Fimbrial Adhesin Targeting of Microspheres Containing Gentamicin Made with Albumin Glycated with Lactose.

The formulation and characterization of gentamicin-loaded microspheres as a delivery system targeting enterotoxigenic Escherichia coli K88 (E. coli K88) was investigated. Glycated albumin with lactose (BSA-glucose-ß (4-1) galactose) was used as the microsphere matrix (MS-Lac) and gentamicin included as the transported antibiotic. The proposed target strategy was that exposed galactoses of MS-Lac could be specifically recognized by E. coli K88 adhesins, and the delivery of gentamicin would inhibit bacterial growth. Lactosylated microspheres (MS-Lac1, MS-Lac2 and MS-Lac3) were obtained using a water-in-oil emulsion, containing gentamicin, followed by crosslinking with different concentrations of glutaraldehyde. Electron microscopy displayed spherical particles with a mean size of 10-17 µm. In vitro release of gentamicin from MS-Lac was best fitted to a first order model, and the antibacterial activity of encapsulated and free gentamicin was comparable. MS-Lac treatments were recognized by plant galactose-specific lectins from Ricinus communis and Sophora japonica and by E. coli K88 adhesins. Results indicate MS-Lac1, produced with 4.2 mg/mL of crosslinker, as the best treatment and that lactosylated microsphere are promising platforms to obtain an active, targeted system against E. coli K88 infections.