Immunization with an mRNA DTP vaccine protects against pertussis in rats.

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo. We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models.

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.

Susceptibility of the cultured Amazonian fish, Colossoma macropomum, to experimental infection with Aeromonas species from ornamental fish.

The global ornamental fish trade carries important risk factors for spreading pathogens between different countries and regions, not only for ornamental fish but also for cultured fish and even other animal species. In the current study, we reported the capacity of Aeromonas veronii and A. hydrophila isolated from ornamental fish to experimentally infect the reared Amazonian fish Colossoma macropomum. For this, those bacteria were identified, and a primary characterization was performed. Fish were inoculated with 0.1 mL of increasing concentrations of A. hydrophila or A. veronii (C1 = 1 × 102; C2 = 1.8 × 104; C3 = 2.1 × 106; C4 = 2.4 × 108 bacterial cells per mL) in the coelomic cavity. In the control group, fish received the same volume of sterile saline solution (0.9 %). Fish presented petechiae, skin suffusions, and mortality rates up to 100 % according to the inoculum concentration. Histopathologically, fish presented necrosis with karyolysis, loss of the cytoplasmic delimitation of cells of the renal tubules and hepatocytes, hemorrhage, cellular edema, and the presence of bacterial cells. The LD50-96h of A. veronii on C. macropomum was estimated at 2.4 × 106 CFU mL-1 and of A. hydrophila at 1.408 × 105 CFU mL-1. The results demonstrated that it is possible that Aeromonas species isolated from ornamental fish affect C. macropomum, causing similar clinical signs and lesions. This shows the importance of promoting risk control measures worldwide regarding the trade of ornamental fish.

Resistance of Nile tilapia fed with Padina boergesenii extract to Pseudomonas putida infection.

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1ß), beta-defensin (ß-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection.

Silencing of the immune gene BmPGRP-L4 in the midgut affects the growth of silkworm (Bombyx mori) larvae.

Peptidoglycan recognition proteins (PGRPs) are one of the receptors in insects' immune pathways, essential for insects to recognize the exogenous pathogens in order to activate the Toll and immune deficiency (IMD) pathway. In the silkworm Bombyx mori, previous studies focused on the short PGRPs and less is known about the long PGRPs. In this study, a long PGRP in silkworm BmPGRP-L4 was cloned and its expression and function were analysed. The results showed that BmPGRP-L4 contains a transmembrane region, a conserved PGRP domain, and an amidase-2 domain. The expression profile demonstrated that BmPGRP-L4 existed in diverse tissues including epidermis, fat body, midgut, and silk glands, with remarkably high expression in the midgut in the 5th instar. Oral infection with Escherichia coli and Staphylococcus aureus significantly induced BmPGRP-L4 in the midgut and epidermis, as well as in the fat body and silk glands. Peptidoglycan also induced the expression of BmPGRP-L4 in midgut tissue ex vivo and BmN4 cells in vitro. RNAi of BmPGRP-L4 was effective in the midgut and epidermis, while the efficiency in the fat body was transient. RNAi-mediated knock-down of BmPGRP-L4 reduced the weight and growth of the silkworm, possibly due to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

The role of Nrf2 signaling pathway in the mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

The transcription factor Nrf2 plays vital roles in detoxification and antioxidant enzymes against oxidative stress. However, the function of Nrf2 in crustaceans is not well studied. In this study, a novel Nrf2 gene from the mud crab (Sp-Nrf2) was identified. It was encoded 245 amino acids. Sp-Nrf2 expression was ubiquitously expressed in all tested tissues, with the highest expression level in the gill. Sp-Nrf2 protein was mainly located in the nucleus. The expression levels of Sp-Nrf2, and antioxidant-related genes (HO-1 and NQO-1) were induced after Vibrio parahaemolyticus infection, indicating that Nrf2 signaling pathway was involved in the responses to bacterial infection. Over-expression of Sp-Nrf2 could improve cell viability after H2O2 exposure, indicating that Sp-Nrf2 might relieve oxidative stress. Silencing of Sp-Nrf2 in vivo decreased HO-1 and NQO-1 expression. Moreover, knocking down Sp-Nrf2 in vivo can increase malondialdehyde content and the mortality of mud crabs after V. parahaemolyticus infection. Our results indicated that Nrf2 signaling pathway played a significant role in immune response against bacterial infection.

Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

Genome-wide identification, characterization and expression profiling of TRAF family genes in Sebastes schlegelii.

Tumor necrosis factor receptor-associated factors (TRAFs) are signaling mediators for Toll-like receptor (TLR) and tumor necrosis factor (TNFR) superfamily that play important roles in organism immune response. However, reports on systematic identification of TRAF gene family in teleost fish and the function of TRAFs in innate immunity of black rockfish (Sebastes schlegelii) are lacked. In our study, eight TRAF genes were identified and characterized, namely, SsTRAF2a, SsTRAF2a-like, SsTRAF2b, SsTRAF3, SsTRAF4, SsTRAF5, SsTRAF6 and SsTRAF7 in S. schegelii. Furthermore, we analyzed their sequences, conserved domains, gene structures, motif compositions, phylogeny, tissue expression patterns in healthy and Vibro. anguillarum challenged individuals. All the SsTRAFs contained typical conserved domain, including C-terminal MATH domain and N-terminal RING finger domain. Analyses of gene structures and motifs showed the distribution of exon-intron and conserved motifs in S. schegelii and serval other teleost fish. We also analyzed the expression file of SsTRAFs in five immune-relate organs, liver, spleen, kidney, gill and intestine in healthy and bacterial challenged fish. The results indicated that all SsTRAF member were widely involved in immune response after pathogenic bacteria infection. In summary, the analyses of TRAFs in S. schegelii will be helpful to better understand the diverse roles of TRAF genes in the innate immune response to bacterial challenge.

Identification and characterization of MKK6 and AP-1 in Anodonta woodiana reveal their potential roles in the host defense response against bacterial challenge.

Mitogen-activated protein kinase kinase 6 (MKK6) and activator protein-1 (AP-1) are two of the essential regulatory proteins in the p38 mitogen-activated protein kinase (MAPK) pathway, which participates in the innate immune response to bacterial infections. In this study, molluscan MKK6 (AwMKK6) and AP-1 (AwAP-1) genes were cloned and identified from Anodonta woodiana. The open reading frame (ORF) of AwMKK6 encodes for a putative polypeptide sequence of 345 amino acids containing a conserved serine/threonine protein kinase (S_TKc) domain, a SVAKT motif and a DVD domain. AwAP-1 consists of 294 amino acids including a typical nuclear localization signal (NLS), a Jun domain and a basic region leucine zipper (BRLZ) domain. Quantitative real-time PCR analysis showed that both AwMKK6 and AwAP-1 were widely expressed in all selected tissues of A. woodiana and their transcript levels in hemocytes were significantly upregulated when challenged with Aeromonas hydrophila and lipopolysaccharide (LPS). Additionally, the signaling molecules of the AwMKK6/AwAP-1 pathway including AwTLR4, AwMyD88, AwTRAF6, AwMEKK1, AwMEKK4, AwASK1, AwTAK1 and Awp38 mRNA expression showed a stronger responsiveness to LPS challenge in hemocytes of A. woodiana. RNA interference (RNAi) experiments indicated that the silencing of AwMKK6 or AwAP-1 could decrease the mRNA expression levels of immune effectors (AwTNF, AwLYZ and AwDefense). Subcellular localization studies suggested that AwMKK6 and AwAP-1 were distributed throughout the cells and nucleus, respectively, and their overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. These findings suggest that MKK6 and AP-1 play a major role in the host defense response to bacterial injection, which may make contributions to a better understanding of the immune function of the p38 MAPK pathway in mollusks.