Layered Double Hydroxides (LDH) as Delivery Vehicles of a Chimeric Protein Carrying Epitopes from the Porcine Reproductive and Respiratory Syndrome Virus.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes reproductive failure and respiratory symptoms, leading to huge economic losses for the pig farming industry. Although several vaccines against PRRSV are available in the market; they show an overall low efficacy, and several countries have the need for vaccines covering the local, circulating variants. This project aims at developing a new chimeric antigen targeting specific epitopes from PRRSV and evaluating two test adjuvants to formulate a vaccine candidate. The test antigen was called LTB-PRRSV, which was produced recombinantly in Escherichia coli and consisted of the heat labile enterotoxin B subunit from E. coli (LTB) and four epitopes from PRRSV. LTB-PRRSV was rescued as inclusion bodies and methods for its solubilization, IMAC-based purification, and refolding were standardized, leading to mean yields of 18 mg of pure protein per liter culture. Layered double hydroxides (LDH) have been used as vaccine adjuvants given their biocompatibility, low cost, and positive surface charge that allows an efficient adsorption of negatively charged biomolecules. Therefore, LDH were selected as delivery vehicles of LTB-PRRSV. Pure LTB-PRRSV was adsorbed onto LDH by incubation at different LDH:LTB-PRRSV mass ratios (1:0.25, 1:0.5, 1:1, and 1:2) and at pH 9.5. The best adsorption occurred with a 1:2 mass ratio, and in a sucrose-tween solution. The conjugates obtained had a polydispersity index of 0.26, a hydrodynamic diameter of 192 nm, and a final antigen concentration of 64.2 µg/mL. An immunogenicity assessment was performed by injecting mice with LDH:LTB-PRRSV, Alum/LTB-PRRSV, or LTB-PRRSV in a scheme comprising three immunizations at two-week intervals and two dose levels (1 and 5 µg). LTB-PRRSV was capable of inducing strong humoral responses, which lasted for a longer period when LDH was used as the delivery vehicle/adjuvant. The potential of LDH to serve as an attractive carrier for veterinary vaccines is discussed.

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.

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.