Frank Camacho

Early embryonic loss is a major cause of reproductive inefficiency in cattle, primarily due to premature luteolysis. Interferon tau (IFN-τ), secreted by the trophoblast, plays a critical role in maternal recognition of pregnancy by maintaining corpus luteum function. However, its practical application has been limited by its rapid degradation and short half-life in vivo. Here, we developed a novel formulation of recombinant bovine IFN-τ, combining chitosan-based microencapsulation with starch–chitosan hydrogel delivery, enabling sustained intrauterine release. This dual-delivery strategy offers a significant improvement over conventional IFN-τ administration methods that rely on repeated intrauterine infusions of soluble protein. The rbIFN-τ was expressed in Pichia pastoris, purified to 90.1% homogeneity, and structurally validated via homology modeling and molecular docking, confirming its interaction with type I interferon receptors. The encapsulated formulation retained antiviral activity, stimulated transcription of interferon-stimulated genes (PKR, OAS1, OAS2), and showed sustained release in vitro for up to 26 days. In vivo evaluation demonstrated safety and biological efficacy, with treated cattle showing inhibited luteolysis, sustained serum progesterone levels, and preserved corpus luteum integrity. This formulation represents a promising biotechnological approach to improve reproductive efficiency through a long-acting, species-specific IFN-τ delivery system.

Mucosal vaccines represent a transformative approach to immunization by stimulating immune responses at mucosal surfaces—the primary entry points for many infectious agents. These vaccines can induce localized mucosal immunity, which is characterized by secretory IgA (SIgA) and robust systemic immune memory. Despite these advantages, mucosal vaccine development faces significant barriers, including antigen degradation, low mucosal absorption, and insufficient immunogenicity. Recent advances in adjuvant technology, antigen delivery platforms, and recombinant expression systems offer promising solutions to overcome these limitations. This review provides a comprehensive overview of current mucosal vaccine strategies, highlights state-of-the-art innovations in formulation and delivery, and identifies critical challenges and future research directions necessary to accelerate clinical translation and global implementation.

Early embryonic loss is a significant cause of reproductive inefficiency and economic loss in cattle, largely due to inadequate maternal recognition of pregnancy and premature luteolysis. Interferon tau (IFN-τ), a trophoblast-derived signaling molecule, is essential for maintaining corpus luteum function and embryo survival in ruminants, but its rapid degradation and short half-life have limited its practical use. This study developed a novel delivery system for bovine recombinant IFN-τ (brIFN-τ) produced in Pichia pastoris, utilizing chitosan-based microencapsulation and starch-chitosan hydrogels for sustained intrauterine release. The recombinant protein was successfully expressed, purified, and structurally validated, confirming its interaction with bovine interferon receptors. Encapsulated IFN-τ showed controlled, prolonged in vitro release and retained biological activity, demonstrated by increased viability and antiviral response in bovine cells exposed to Mengo virus. In vivo, the microencapsulated formulation was safe and effectively inhibited luteolysis, as shown by sustained progesterone levels and reduced corpus luteum regression compared to controls. These results indicate that hydrogel-delivered, microencapsulated brIFN-τ is a promising biotechnological strategy to modulate luteolysis, enhance maternal recognition of pregnancy, and reduce early embryonic loss in cattle, potentially improving reproductive efficiency and supporting more sustainable livestock management.

Effective drug delivery to the central nervous system (CNS) remains a challenge due to the blood-brain barrier (BBB). Macromolecules such as proteins and peptides are unable to cross BBB and have poor therapeutic efficacy due to little or no drug distribution. A promising alternative is the conjugation of a drug to a shuttle molecule that can reach the CNS via receptor-mediated transcytosis (RMT). Several receptors have been described for RMT, such as low-density lipoprotein receptor-related protein 1 (LRP1). We used phage display technology combined with an in vitro BBB model to identify LRP1 ligands. A single domain antibody (dAb) library was used to enrich for species that selectively bind to immobilised LRP1 ligand. We obtained a novel nanobody, dAb D11, that selectively binds to LRP1 receptor and mediates in vitro internalisation of phage particles in brain endothelial cells, with a dissociation constant Kd of 183.1 ± 85.8 nM. The high permeability of D11 was demonstrated by an in vivo biodistribution assay in mice. We discovered D11, the first LRP1 binding dAb with BBB permeability. Our findings will contribute to the development of RMT-based drugs for the treatment of CNS diseases.

Hantavirus Pulmonary Syndrome (HPS), caused by the Andes virus (ANDV), is a significant public health concern in South America, with no available vaccines or specific treatments. Neutralizing antibodies targeting ANDV glycoproteins Gn and Gc have shown protective effects in animal models, making them promising vaccine candidates. This study reports the recombinant expression, purification, and immunogenicity evaluation of sGn and sGc glycoproteins produced in the methylotrophic yeast Pichia pastoris. Initially, sGn was expressed as a secretable protein, but it formed aggregates once accumulated in the culture medium. To address this, the same strain was transformed with a vector expressing Gc, based on the hypothesis that Gn folding depends on Gc presence. However, co-expression of Gn and Gc in Pichia resulted in both proteins becoming trapped as insoluble aggregates inside the yeast, resembling inclusion bodies seen in E. coli. The recombinant proteins were solubilized and purified using metal ion affinity chromatography, followed by a refolding step before being used for immunization. The purified sGn and sGc antigens, formulated with aluminum hydroxide (AlOH), were tested in Syrian hamsters. The immunization results showed that the sGn-sGc formulation induced a strong Th1-biased immune response, characterized by high levels of IFN-γ, IL-12, and IL-6 cytokines, along with a specific antibody response against both Gn and Gc, with titers exceeding 1:10,000. These findings suggest the potential of these antigens as promising candidates for a subunit vaccine against HPS.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that primarily infects cattle but has a wide range of hosts. It represents a global health problem affecting the livestock industry tremendously, with economic losses of about 3 billion annually. Dairy cattle produce a decline of 10% in terms of liters of milk produced and 5% in the meat industry because of live weight loss and seized carcasses in abattoirs. The core of the current control measures implemented in most countries against bTB is based on the diagnosis with tuberculin skin test (TST) and culling of infected animals. Unfortunately, control programs have failed to eradicate the disease since no vaccine protects cattle from infection. Moreover, the strain of M. bovis Bacillus Calmette Guerin, BCG used as a vaccine for human tuberculosis, interferes with surveillance tools. Nowadays, most researchers have been working on improving the efficacy of BCG through a prime-boost strategy that involves a first immunization with BCG and a booster with different types of vaccines. A less explored approach by experts has been the development of a new vaccine that only includes some protective antigens of M. bovis that should be absent ot in low representation in TST. On the other hand, because TST precludes implementation of Bacille Calmette-Guérin (BCG) vaccine–based control programs, several investigations have been carried out to replace the TST with a DIVA test that allows to differentiation infected from vaccinated animals. In this review, most of the studies cited agree that without an effective vaccine and a compatible diagnosis, no program for eradication would be successful against tuberculosis in cattle. Keywords: bovine tuberculosis; bTB; Mycobacterium bovis; diagnosis; tuberculin skin test, TST, bPPD; vaccines, DIVA.

The epidermal growth factor (EGF) is one of the most critical ligands of the EGF receptor (EGFR), a well-known oncogene frequently overexpressed in cancerous cells and an important therapeutic target in cancer. The EGF is the target of a therapeutic vaccine aimed at inducing an anti-EGF antibody response to sequester this molecule from serum. However, strikingly, very few investigations have focused on EGF immunotargeting. Since the use of nanobodies (Nbs) for EGF neutralization may be an effective therapeutic strategy in several types of cancer, in this study, we decided to generate anti-EGF Nbs from a recently constructed, phage-displaying synthetic nanobody library. To our knowledge, this is the first attempt to obtain anti-EGF Nbs from a synthetic library. By applying a selection strategy that uses four different sequential elution steps along with three rounds of selection, we obtained four different EGF-specific Nb clones, and also tested their binding capabilities as recombinant proteins. The obtained results are very encouraging and demonstrate the feasibility of selecting nanobodies against small antigens, such as the EGF, from synthetic libraries.

Nanobodies (Nbs) are single domain antibody fragments derived from heavy-chain antibodies found in members of the Camelidae family. They have become a relevant class of biomolecules for many different applications because of several important advantages such as their small size, high solubility and stability, and low production costs. On the other hand, synthetic Nb libraries are emerging as an attractive alternative to animal immunization for the selection of antigen-specific Nbs. Here, we present the design and construction of a new synthetic nanobody library using the phage display technology, following a structure-based approach in which the three hypervariable loops were subjected to position-specific randomization schemes. The constructed library has a clonal diversity of 10⁸ and an amino acid variability that matches the codon distribution set by design at each randomized position. We have explored the capabilities of the new library by selecting nanobodies specific for three antigens: vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF) and the glycoprotein complex (GnGc) of Andes virus. To test the potential of the library to yield a variety of antigen-specific Nbs, we introduced a biopanning strategy consisting of a single selection round using stringent conditions. Using this approach, we obtained several binders for each of the target antigens. The constructed library represents a promising nanobody source for different applications.

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS–PAGE and Western blot. The K_D values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high K_D values (10⁻⁸M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.