Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP-38) is a multifunctional neuropeptide known to influence key physiological processes in teleost fish, including somatic growth and immune regulation. Building upon this foundation, our study further characterizes the contribution of PACAP-38 to antibacterial defenses in Nile tilapia. Following intraperitoneal administration of the synthetic peptide, we observed a significant increase in the bacterial growth inhibitory activity of skin and gill mucus against Edwardsiella tarda and Pseudomonas aeruginosa, concomitant with the modulation of Oreochromicins gene expression. Furthermore, PACAP-38 administration, both alone and in combination with a P. aeruginosa bacterin, significantly improved survival rates following an experimental challenge with this pathogen. In a complementary experiment, oral delivery of PACAP-38 via modified feed not only enhanced fish welfare but also reduced the bacterial load of P. aeruginosa in gill mucus post-infection and increased Oreochromicins expression in mucosal tissues. Collectively, these findings demonstrate that PACAP-38 enhances mucosal antibacterial defenses in vivo, solidifying its role as an effective immunomodulatory peptide in teleost. With further development, PACAP-38 presents a promising alternative to conventional antibiotics or inactivated vaccines in aquaculture.

Aquaculture has been established as a sustainable alternative to traditional fisheries, which face challenges such as overexploitation and environmental degradation. However, disease outbreaks, often caused by poor farming conditions, pollution, and environmental stress, remain a major concern, leading to economic losses and increasing the risk of antibiotic resistance due to the overuse of antibiotics. Therefore, it is crucial to seek new strategies that improve fish health and well-being, preventing drug resistance and promoting sustainable practices. GHRP-6, a synthetic growth hormone-releasing peptide that mimics ghrelin, has shown potential immunostimulatory properties and feed efficiency in fish. In this study, we evaluated the effects of orally administered GHRP-6 in an oil-based formulation on juvenile tilapia (Oreochromis sp.) challenged or unchallenged with Pseudomonas aeruginosa. We assessed its influence on immune gene expression and digestive enzyme activity. The results demonstrated that GHRP-6 treatment significantly enhanced growth performance (weight and length), reduced in vivo bacterial load after infection, and modulated key genes related to innate and adaptive immunity in the gills, intestine and head kidney. In addition, our results demonstrated, for the first time, a direct link between a growth hormone secretagogue in fish and the modulation of specific enzyme activity in the gut following a bacterial challenge. These findings highlight the potential of GHRP-6 as a dietary immunomodulator and growth promoter in fish farming, offering a promising strategy to reduce antibiotic usage and promote more sustainable aquaculture practices.

Disease outbreaks in aquaculture are a major challenge, often leading to the widespread use of antibiotics for prevention and treatment. However, excessive and improper antibiotic use leads to antimicrobial resistance, posing risks to both aquatic species and human health. Therefore, researchers are looking into new alternatives such as antimicrobial peptides. Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with antimicrobial and immunomodulatory properties, has shown potential applications in fish and shellfish aquaculture. Nevertheless, few studies have been conducted to date in shellfish species. Given the importance of crustacean aquaculture, the current study aimed to assess the effect of synthetic Clarias gariepinus PACAP-38 and a PACAP modified sequence in shrimp L. vannamei during a Vibrio parahaemolyticus infection. Shrimp were treated for one week with four injections of each peptide administrated every 48 h. After the last treatment, animals were inoculated with a sublethal dose of V. parahaemolyticus, and samples were collected at 0-, 20-, and 68-h post-inoculation. The immunological status of the shrimp was assessed by measuring hemolymph immunological indicators and RT-qPCR. The results suggest that the modified form of PACAP exhibited an immunomodulatory effect, mostly maintaining stable levels of immune parameters throughout the challenge, unlike the native PACAP-38 and control groups, which exhibited more fluctuating responses. This modified variant of PACAP also demonstrated a strong antibacterial effect against V. parahaemolyticus. Overall, our findings provide more insights regarding the role of PACAP in crustacean immunology and its potential use as an alternative to antibiotics in aquaculture.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide with multiple biological functions including immunomodulation. While well-studied in vertebrates, its effects in invertebrates like Litopenaeus vannamei remain largely unexplored. Given the shrimp industry's global economic importance and losses due to disease, understanding PACAP's role in shrimp immunity offers a promising avenue for enhancing aquaculture sustainability and productivity. The present study evaluates the immune response to a single PACAP injection, investigating whether an individual administration is able to trigger a significant immune response, and the potential detrimental effects related to the administration route. Thus, effects of native PACAP-38 (PACAP1) and its scrambled version (PACAP5), as well as saline solution (vehicle) injection, over time were evaluated on shrimp immune responses. Samples were collected at 0-, 6-, 24-, 48-, and 72-hours posttreatment (hpt). Immune parameters such as the total number of circulating hemocytes (THC), the concentration of proteins, and hemagglutination and phenoloxidase activities, in the hemolymph were analyzed. Reverse transcription-quantitative PCR (RT-qPCR) was conducted on hemocyte, gill, and hepatopancreas samples to evaluate the differential expression of immune-related genes. Results showed that the treatments triggered an early inflammatory response marked by reduced circulating hemocytes during the first 24 hpt. PACAP-treated groups recovered baseline hemocyte levels by 48 hpt, unlike the saline control, suggesting an immunomodulatory role for PACAP. Additionally, PACAP influenced the tissue-specific expression of key immune genes such as crustin, HSP70 and superoxide dismutase. Overall, both the injection procedure and/or the administration of saline negatively impacted the shrimp's immune status, an effect that was partially mitigated by PACAP treatment. Thus, further research needs to explore new routes of administration such as dietary inclusion or immersion baths, to optimize PACAP’s immunomodulatory potential in shrimp health management.

IntroductionSubunit vaccines represent a safer alternative to live attenuated formulations. However, they often require potent adjuvants and delivery systems to elicit robust immunity, particularly against highly contagious diseases such as Classical Swine Fever (CSF).MethodsIn this study, we investigated the immunogenicity and protective efficacy of a novel mucosal subunit vaccine comprising the chimeric E2-CD154 protein, co-administered with the mucosal adjuvant c-di-AMP, in domestic pigs. Optimal dosing and immunization schedules for sublingual immunization were determined, followed by a challenge experiment using a highly virulent CSF virus (CSFV) strain.ResultsOur results showed that sublingual co-administration of E2-CD154 and the STING agonist c-di-AMP conferred robust clinical protection, effectively prevented viral replication, and restricted the dissemination of infectious virus. This combination induced strong systemic IgG and IgA responses and neutralizing antibodies against multiple CSFV strains, achieving outcomes comparable with the commercial Porvac® vaccine, administered intramuscularly. Importantly, virus isolation from tonsils confirmed the absence of infectious virus in pigs immunized with E2-CD154 and c-di-AMP, unlike those receiving E2-CD154 or the adjuvant alone. Moreover, immunized animals exhibited minimal IFN-α serum levels post-challenge, indicating reduced innate activation and viral replication.DiscussionThese findings provide evidence, in a large mammalian host such as the pig, that c-di-AMP functions as an adjuvant for a recombinant E2-CD154 protein delivered sublingually, enhancing immune responses consistent with protection against viral replication. Together, these results offer insights into the development of non-replicating, DIVA-compatible platforms against CSFV and support the rational design of next-generation subunit vaccines targeting viral pathogens relevant to both veterinary and human medicine.

IntroductionSea lice are crustacean ectoparasites affecting Atlantic salmon production worldwide and impediments to industry growth. Chemical treatment has been the method of choice to control infestation with increasing resistance. Vaccination is an environmentally friendly alternative for sea lice control; however, obtaining high levels of lice reduction through active immunization has proven difficult. This study aimed to explore the efficacy of two sea lice vaccine prototypes under laboratory-controlled conditions.MethodsTherein, fish were vaccinated with two chimeric antigens, TT-P0 or P0-my32, using oil-adjuvanted vaccine formulations and a prime-boost vaccination protocol. Fish were experimentally challenged with copepodids at 2, 5, and 11 months post-prime vaccination.Results and discussionTT-P0 vaccinated fish had a significantly lower lice number at all three challenges, 88, 90, and 20%, respectively, compared to controls. The P0-my32 vaccine gave high protection at early time points post-vaccination, with 91 and 75.4% reduction at 3 and 6 months, respectively, fading off at 12 months (4.2% reduction vs. control). The TT-P0 group had a significantly lower lice number than controls at the 11-month challenge. A higher degree of protection coincided with higher circulating antibody levels against homologous antigens. This proof of concept study encourage the use of vaccination as a tool to reduce the lice burden in salmon, and preclinical and clinical testing at a large scale is needed to document the level of protection attained under field conditions.

Porcine respiratory diseases have a huge economic impact on pig production. The highest incidence of these diseases is commonly linked to Streptococcus suis, Pasteurella multocida and Bordetella bronchiseptica, some of which are zoonotic posing a risk to human health. The inappropriate and excessive use of conventional antibiotics, as usual procedure for treating respiratory diseases in pigs, has generated the emergence of antimicrobial resistance (AMR), which urgently requires the development of alternative approaches to current antimicrobials. Antimicrobial peptides (AMPs) have rapidly garnered interest as novel therapeutic candidates. Oreochromicin-2 (Oreoch-2), an AMP previously isolated from Oreochromis niloticus gills, has shown broad antibacterial properties against several species. However, studies about its effect on porcine respiratory pathogens and its potential use for the treatment of swine respiratory diseases are not available. In this work we determined the in vitro antimicrobial activity of the peptide against S. suis by a broth microdilution method. Oreoch-2 showed a MIC of 3.13 mu M against this pathogen. For in vivo experiments, Yorkshire x Landrace crossbred (LYxL35) weaning pigs aged 30-33 days were allocated in pens containing ten pigs each. To study the influence of the peptide on health status, a total clinical score was determined. The administration of Oreoch-2 improved the clinical behavior of the animals, similar to the conventional antibiotic shotapen, respect to the placebo group. A reduction of bacterial loads in the respiratory tract and lungs was observed in Oreoch-2-treated animals as compared to the placebo group. It was shown that peptide treated-piglets displayed significantly higher serum IgG concentration compared to the control group. These results demonstrated Oreoch-2 potential as an antimicrobial and immunostimulant drug candidate against respiratory diseases in pigs.

Ghrelin, a peptide hormone originally known for its role in growth hormone secretion and appetite regulation, is a key immunomodulator in vertebrates, including fish. This hormone and its receptor (GHS-R) are widely expressed in immune cells—T cells, B cells, neutrophils, and macrophages—and tissues. In fish, Ghrelin modulates physiological processes (e.g., reproduction or energy balance) and improves immune defense mechanisms. Studies have demonstrated its ability to promote phagocytic activity, regulate cytokine production, and stimulate antimicrobial peptide production, enhancing resistance to pathogenic infections. Likewise, the development of ghrelin mimetics, like GHRP-6, with a wide range of functionalities, further increases the potential of their use in aquaculture, showing promising results in improving growth and immune responses in fish. Ghrelin and its analogs represent a promising alternative for boosting fish's immune response against pathogens. Thus, understanding Ghrelin's regulatory mechanisms in fish immunity may reveal practical applications for aquaculture practices, helping achieve fish welfare and sustainability. Keywords: Ghrelin; immune system; fish; growth hormone secretagogue; cytokines; antimicrobial activity

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.

Understanding the intricate ecological interactions within the microbiome of arthropod vectors is crucial for elucidating disease transmission dynamics and developing effective control strategies. In this study, we investigated the ecological roles of Coxiella-like endosymbiont (CLE) and Anaplasma marginale across larval, nymphal, and adult stages of Rhipicephalus microplus. We hypothesized that CLE would show a stable, nested pattern reflecting co-evolution with the tick host, while A. marginale would exhibit a more dynamic, non-nested pattern influenced by environmental factors and host immune responses. Our findings revealed a stable, nested pattern characteristic of co-evolutionary mutualism for CLE, occurring in all developmental stages of the tick. Conversely, A. marginale exhibited variable occurrence but exerted significant influence on microbial community structure, challenging our initial hypotheses of its non-nested dynamics. Furthermore, in silico removal of both microbes from the co-occurrence networks altered network topology, underscoring their central roles in the R. microplus microbiome. Notably, competitive interactions between CLE and A. marginale were observed in nymphal network, potentially reflecting the impact of CLE on the pathogen transstadial-transmission. These findings shed light on the complex ecological dynamics within tick microbiomes and have implications for disease management strategies.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved multifunctional neuropeptide belonging to the secretin/glucagon/vasoactive intestinal peptide superfamily. In recent years, the role of PACAP in immunoregulation has been partially elucidated in mammals and teleost fishes, demonstrating that PACAP modulates both innate and acquired immunity. However, in invertebrates, little is known about its immunostimulant and antimicrobial properties. In previous research, studies performed in Litopenaeus (Penaeus) vannamei have revealed that Clarias gariepinus PACAP-38 improves immune functions like lectins, superoxide dismutase and nitric oxide synthase derived metabolites in postlarvae shrimp. In this study, the immunomodulatory effects of C. gariepinus PACAP-38 and four PACAP sequence modified peptides in juvenile L. vannamei were evaluated. The effect of several administrations of PACAP and its variants on shrimp immune effectors, as well as the effect of different doses was examined. Results revealed that PACAP can influence hemolymph immune parameters, and immune-related gene mRNA levels in the hemocytes, gill and hepatopancreas with different effects depending on the modification of the peptide sequence, the dose and the tissue assessed. Altogether, these findings provide valuable insights regarding the role of PACAP in crustacean innate immunity and highlight the need for further research into its potential application as an alternative to antibiotics in aquaculture.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1β and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1β protein from previously accumulated transcripts or the cleavage of accumulated IL-1β precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Recent work has identified pituitary adenylate cyclase activating polypeptide (PACAP) as a potential antimicrobial and immune stimulating agent which may be suitable for use in aquaculture. However, its effects on teleost immunity are not well studied and may be significantly different than what has been observed in mammals. In this study we examined the effects of PACAP on the Atlantic salmon macrophage cell line SHK-1. PACAP was able to increase the expression of LPS-induced il-1β in at concentrations of 1 uM when administered 24h prior to LPS stimulation. Furthermore, concentrations as low as 40nM had an effect when administered both 24h prior and in tandem with LPS. PACAP was also capable of increasing the expression of il-1β and tnf-α in SHK-1 cells challenged with a low dose of heat-killed Flavobacterium columnare. We attempted to get a better understanding of the mechanism underlying this enhancement of il-1β expression by manipulating downstream signaling of PACAP with inhibitors of phosphodiesterase and phospholipase C activity. We found that inducing cAMP accumulation with phosphodiesterase inhibitors failed to recapitulate the effect of PACAP administration on LPS-mediated il-1β expression by PACAP, while use of a phospholipase C inhibitor caused a PACAP-like enhancement in LPS-mediated il-1β expression. Interestingly, the VPAC1 receptor inhibitor PG97-269, but not the PAC1 inhibitor max.d.4, also was capable of causing a PACAP-like enhancement in LPS-mediated il-1β expression. This suggests that fish do not utilize the PACAP receptors in the same manner as mammals, but that it still exerts an immunostimulatory effect that make it a good immunostimulant for use in aquaculture.

Aquaculture is an essential economic activity worldwide, supplying the requirements of various nations. The larval stage is one of the most critical periods during the production cycle in aquaculture. To contribute to amplifying the knowledge related to viable methods to enhance this activity, the present study investigated the effects of prebiotic fructooligosaccharide (FOS) and the synthetic peptide GHRP-6 in the growth of fish, as well as its repercussions on a series of digestive enzyme activities and immune response parameters. Tilapias (Oreochromis niloticus) larvae were divided into three groups: group 1, fed with pre-formulated feed; group 2, fed with feed + FOS; group 3, fed with feed + GHRP-6. We measured the gain in size and weight at 72 and 87 days after starting the diets. At the end of treatment, we continue feeding the animals with a standard diet using the pre-formulated feed. We measured immune system parameters and digestive enzyme activities on larvae homogenate, focusing on lysozyme and antiprotease activity and α-amylase, trypsin and esterase activity, respectively. Results show a significant increment in size and weight in those fish fed with feed + FOS. Meanwhile, the primary enzyme activity was detected in those fish fed with feed + GHRP-6. On rough terms, both food combinations stimulated the immune system. Our study suggests that FOS and GHRP-6 could be desirable additives to incorporate into the tilapia larvae feeding and increase production efficiency. Keywords: aquaculture, digestive enzymes activity, FOS, GHRP-6, immune system, larvae

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.

Porvac is a classical swine fever (CSF) subunit vaccine. It is safe and induces a robust neutralizing antibody response, sterilizing immunity, and early protection, and it prevents vertical transmission in pregnant sows. The methodology to approve Porvac batches is a challenging experiment in pigs with a virulent CSF virus strain. However, there is an ethical reason to reduce, at minimum, the use of animals in these lethal experiments. The knowledge indicates that neutralizing antibody titers in the blood could be a good correlate of protection. The results of 22 challenge experiments involving 116 Porvac vaccinated and 38 unvaccinated animals were analyzed. All vaccinated animals remained free from CSF clinical signs and pathological lesions and were negative for viral isolation after the challenge. In contrast, all unvaccinated pigs developed clinical and pathological signs of the disease and had to be euthanized eight days post-challenge. All vaccinated pigs exhibited high neutralizing antibody titers, with a geometric mean value of 1: 5153. The lower titer registered was 1: 800. A complete correspondence between neutralizing antibody titers and protection was demonstrated. These results support substituting the viral challenge test for the neutralizing peroxidase-linked assay in the release of Porvac® batches. Keywords. Classical swine fever; virus; subunit vaccine; viral challenge; neutralizing antibodies

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.