Mucosal Gene Expression in Response to SARS-CoV-2 Is Associated with Viral Load.

Little is known about the relationships between symptomatic early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and upper airway mucosal gene expression and immune response. To examine the association of symptomatic SARS-CoV-2 early viral load with upper airway mucosal gene expression, we profiled the host mucosal transcriptome from nasopharyngeal swab samples from 68 adults with symptomatic, mild-to-moderate coronavirus disease 19 (COVID-19). We measured SARS-CoV-2 viral load using reverse transcription-quantitative PCR (RT-qPCR). We then examined the association of SARS-CoV-2 viral load with upper airway mucosal immune response. We detected SARS-CoV-2 in all samples and recovered >80% of the genome from 95% of the samples from symptomatic COVID-19 adults. The respiratory virome was dominated by SARS-CoV-2, with limited codetection of other respiratory viruses, with the human Rhinovirus C being identified in 4 (6%) samples. This limited codetection of other respiratory viral pathogens may be due to the implementation of public health measures, like social distancing and masking practices. We observed a significant positive correlation between SARS-CoV-2 viral load and interferon signaling (OAS2, OAS3, IFIT1, UPS18, ISG15, ISG20, IFITM1, and OASL), chemokine signaling (CXCL10 and CXCL11), and adaptive immune system (IFITM1, CD300E, and SIGLEC1) genes in symptomatic, mild-to-moderate COVID-19 adults, when adjusting for age, sex, and race. Interestingly, the expression levels of most of these genes plateaued at a cycle threshold (CT) value of ~25. Overall, our data show that the early nasal mucosal immune response to SARS-CoV-2 infection is viral load dependent, potentially modifying COVID-19 outcomes. IMPORTANCE Several prior studies have shown that SARS-CoV-2 viral load can predict the likelihood of disease spread and severity. A higher detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity. However, the relationship between SARS-CoV-2 viral load, airway mucosal gene expression, and immune response remains elusive. We profiled the nasal mucosal transcriptome from nasal samples collected from adults infected with SARS-CoV-2 during spring 2020 with mild-to-moderate symptoms using a comprehensive metatranscriptomics method. We observed a positive correlation between SARS-CoV-2 viral load, interferon signaling, chemokine signaling, and adaptive immune system in adults with COVID-19. Our data suggest that early nasal mucosal immune response to SARS-CoV-2 infection was viral load dependent and may modify COVID-19 outcomes.

Recombinant L. lactis vaccine LL-plSAM-WAE targeting four virulence factors provides mucosal immunity against H. pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.

Effect of vaccination route (intradermal vs. intramuscular) against porcine reproductive and respiratory syndrome using a modified live vaccine on systemic and mucosal immune response and virus transmission in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease with worldwide distribution and an enormous economic impact. To control PRRS virus (PRRSV) infection, modified live vaccines (MLVs) are widely used in the field, mainly administered via an intramuscular (IM) route. Currently, some MLVs are authorized for intradermal (ID) administration, which has many practical and welfare advantages. The objectives of the study were to compare the immune responses (systemic in blood and mucosal in lungs) and vaccine efficacy in preventing challenge strain transmission after IM or needle-free ID immunization of piglets with an MLV against PRRSV-1 (MLV1). METHODS: Groups of sixteen 5-week-old specific pathogen-free piglets were vaccinated with Porcilis PRRS® (MSD) either by an IM (V+ IM) or ID route (V+ ID) using an IDAL®3G device or kept unvaccinated (V-). Four weeks after vaccination, in each group, 8 out of the 16 piglets were challenged intranasally with a PRRSV-1 field strain, and one day later, the inoculated pigs were mingled by direct contact with the remaining 8 sentinel noninoculated pigs to evaluate PRRSV transmission. Thus, after the challenge, each group (V+ IM, V+ ID or V-) included 8 inoculated and 8 contact piglets. During the postvaccination and postchallenge phases, PRRSV replication (RT-PCR), PRRSV-specific antibodies (ELISA IgG and IgA, virus neutralization tests) and cell-mediated immunity (ELISPOT Interferon gamma) were monitored in blood and bronchoalveolar lavages (BALs). RESULTS: Postvaccination, vaccine viremia was lower in V+ ID pigs than in V+ IM pigs, whereas the cell-mediated immune response was detected earlier in the V+ ID group at 2 weeks postvaccination. In the BAL fluid, a very low mucosal immune response (humoral and cellular) was detected. Postchallenge, the vaccine efficacy was similar in inoculated animals with partial control of PRRSV viremia in V+ ID and V+ IM animals. In vaccinated sentinel pigs, vaccination drastically reduced PRRSV transmission with similar estimated transmission rates and latency durations for the V+ IM and V+ ID groups. CONCLUSIONS: Our results show that the tested MLV1 induced a faster cell-mediated immune response after ID immunization two weeks after vaccination but was equally efficacious after IM or ID immunization towards a challenge four weeks later. Considering the practical and welfare benefits of ID vaccination, these data further support the use of this route for PRRS MLVs.

Mucosal Priming with a Recombinant Influenza A Virus-Vectored Vaccine Elicits T-Cell and Antibody Responses to HIV-1 in Mice.

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Structural characteristics and mucosal immune response of the interbranchial lymphoid tissue in the gills of flounder (Paralichthys olivaceus).

A specialized lymphoepithelial tissue termed the interbranchial lymphoid tissue (ILT) is recently identified in several fish species. However, the structural variation and mucosal immune functions of the ILT remain largely unknown. In this study, the anti-Zap-70 MAb was firstly determined to specifically recognize ZAP-70 protein, and CD4-1+, CD4-2+ and CD8ß+ T-cells, but not IgM+ B cells, in peripheral blood leucocytes of flounder (Paralichthys olivaceus). Then we found that aggregates of Zap-70+ cells were located in the epithelium covering the bottom of the interbranchial cleft and along the afferent and efferent edges of the filaments in a cross view, where a meshwork of epithelial cells containing diffused lymphoid cells was exhibited, confirming these structures as the ILT; In a sagittal view, Zap-70+ cells were situated at the base of the filaments (here named as proximal ILT, pILT) and in the interlamellar epithelium (named as distal ILT, dILT). Also, a few IgM+ B cells were distributed at these sites. The lymphoepithelium within pILT and dILT was very thin with a low number of Zap-70+ cells in premetamorphosis and postclimax larvae of flounder, and got thicker containing much more Zap-70+ cells in juvenile and adult individuals. The aggregates of CD4-1+/Zap-70+, CD4-2+/Zap-70+, and CD8ß+/Zap-70+ T-cell subsets were identified in the ILT. Post bath vaccination with inactivated Edwardsiella tarda and then intraperitoneal injection of EdU, the amounts of EdU+ and Zap-70+ cells obviously increased at 3 d and 7 d, and co-localization of EdU+/Zap-70+ cells identified the presence of proliferative T cells; meanwhile, MHC class II-expressing cells were increased. These findings indicated that the ILT in gills of flounder was an important site for the induction of local T cell-mediated immunity, which would lead to a better understanding of mucosal immunity and defense mechanisms of teleost fish.

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19.

BACKGROUND: Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly being used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear. OBJECTIVES: Our aim was to evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases. METHODS: Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific IgA and IgG in sera and mucosal fluids of 2 cohorts, including SARS-CoV-2 PCR-positive patients (n = 64) and PCR-positive and PCR-negtive health care workers (n = 109). RESULTS: SARS-CoV-2-specific serum IgA titers in patients with mild COVID-19 were often transiently positive, whereas serum IgG titers remained negative or became positive 12 to 14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA were correlated with severe acute respiratory distress syndrome. Interestingly, some health care workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids were inversely correlated with age. CONCLUSIONS: Systemic antibody production against SARS-CoV-2 develops mainly in patients with severe COVID-19, with very high IgA titers seen in patients with severe acute respiratory distress syndrome, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but may stimulate mucosal SARS-CoV-2-specific IgA secretion.

Nanoparticles of conformation-stabilized canine distemper virus hemagglutinin are highly immunogenic and induce robust immunity.

BACKGROUND: Canine distemper virus (CDV) infection of ferrets, dogs, and giant pandas causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system. In this study, we tested a new candidate CDV vaccine-CDV nanoparticles-based on hemagglutinin protein. METHODS: The nanoparticles were generated from conformation-stabilized CDV hemagglutinin tetramers. Immune responses against CDV were evaluated in mice. Immunization was initiated 6 weeks after birth and boosted two times with 4-week intervals. The blood and mucosal samples were collected 2 weeks after each immunization. RESULTS: Vaccination with CDV nanoparticles elicited high levels of IgG antibody titers in mice (approximately sevenfold to eightfold higher than that obtained with soluble CDV H protein) and mucosal immune responses and developed increased CDV-specific neutralizing antibody. The mice that received nanoparticles showed significantly higher IFN-γ- and IL-4-secreting cell population in the spleen and lymph node compared with mice immunized with soluble H protein. The co-stimulatory molecular expression of CD80 and CD86 on the surface of DCs was also upregulated. CONCLUSION: The results demonstrate that self-assembly into nanoparticles can increase the immunogenicity of vaccine antigens, and nanoparticles assembled from conformation-stabilized CDV H protein can serve as a new CDV vaccine.

Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

Enterotoxigenic Escherichia coli (ETEC) remains a massive burden in developing countries with increasing morbidity and mortality rates; it is also an important pathogen in the farming industry and is a leading cause of bacterial diarrhea. Our previous study showed that nanometer-sized inclusion bodies (IBs) of the fimbrial adhesin subunit protein (FaeG), mutation heat-stable enterotoxin a (mSTa), heat-labile enterotoxin b (LTb), and STb (nontargeting) fusion protein as an oral vaccine induced both systemic and mucosal immune responses. In this study, to enhance the protective efficacy to ETEC, we used Yersinia enterocolitica adhesive and M-cell-targeting peptides to analyze high-efficiency antigen-specific immune presentation in the gut. Here, we showed that immunization with the IBs of ETEC-FaeG-mSTa-LTb-STb-induced a specific systemic and mucosal immune response in the gut, whereas the combination of both targeting peptides resulted in the highest titer, protective immune response against ETEC. A lymphocyte proliferation assay has shown that the IBs induced immunologic memory. The specific antibody of the targeting groups could effectively neutralize toxins, thereby protecting the cells of the small intestine and reducing the level of cAMP and cGMP, and the groups with double targeting showed the best effect. The most important finding was that the targeting peptides stimulate the T helper (Th) cells through Th17 and Th1 and that Th1 cells dominated the cellular immune response. We found that the targeting peptide could also activate CD11c+ on lymphoid dendritic cells, which processed and presented antigens to T cells through Th1-mediated IFN-γ and IL-12, thereby enhancing the antibody titers. The double-targeting peptide had a better effect on stimulating the immune cells to enhance the antibody titers.-Jiang, X., Xia, S., He, X., Ma, H., Feng, Y., Liu, Z., Wang, W., Tian, M., Chen, H., Peng, F., Wang, L., Zhao, P., Ge, J., Liu, D. Targeting peptide-enhanced antibody and CD11c+ dendritic cells to inclusion bodies expressing protective antigen against ETEC in mice.

10-Hydroxydecanoic Acid Potentially Elicits Antigen-Specific IgA Responses.

The effective antigen (Ag) uptake by microfold cells (M-cells) is important for the induction of an efficient mucosal immune responses. Here, we show that 10-hydroxydecanoic acid (10-HDAA) from royal jelly (RJ) potentially supports M-cell differentiation and induces effective antigen-specific mucosal immune responses in cynomolgus macaques. 10-HDAA increases the expression level of receptor activator of nuclear factor-kappaB (NF-κB) (RANK) in Caco-2 cells, which suggests that 10-HDAA potentially prompts the differentiation of Caco-2 cells into M-cells and increased transcytosis efficiency. This idea is supported by the following observations. Intranasal administration of 10-HDAA increased the number of M-cells in the epithelium overlying nasopharynx-associated lymphoid tissue (NALT) in macaques. Oral administration of 10-HDAA increased the number of M-cells in the follicle-associated epithelium (FAE) covering Peyer's patches (PPs) and significantly increased the antigen-specific immunoglobulin A (IgA) level in macaques. These findings suggest that the exogenous honeybee-derived medium-chain fatty acid 10-HDAA may effectively enhance antigen-specific immune responses.

Analysis of caecal mucosal inflammation and immune modulation during Anoplocephala perfoliata infection of horses.

Anoplocephala perfoliata is the commonest equine tapeworm, the adult parasites are attached in groups close to the ileocaecal valve causing marked inflammatory pathology. This work aimed to characterize the nature of the in vivo mucosal immune response to A perfoliata, and to investigate the role of A perfoliata excretory-secretory components in modulating in vitro immune responses. Real-time PCR detected elevation of IL13 and TGFß transcription in early-stage A perfoliata infection. In late-stage infection, IL-13, IL4 and Ifn transcripts were reduced while the regulatory cytokines, TGFß, IL10 and the transcription factor FOXP3 were increased in tissue close to the site of A perfoliata attachment; indicating downregulation of T-cell responses to A perfoliata. In vitro, A perfoliata excretory-secretory products induced apoptosis of the Jurkat T-cell line and premature cell death of ConA stimulated equine peripheral blood leucocytes. Analysis of cytokine transcription patterns in the leucocyte cultures showed a marked inhibition of IL-1 and IL-2 suggesting that a lack of T-cell growth factor transcription underlies the mechanism of the induced equine T-cell death. These preliminary findings suggest A perfoliata may have the ability to down-regulate host T-cell responses.

Plant extract-based diets differently modulate immune responses and resistance to bacterial infection in striped catfish (Pangasianodon hypophthalmus).

A feeding trial was performed to compare the effects of five ethanol herbal extracts (bhumi amla, Phyllanthus amarus Schum and Thonn [Pa]; guava, Psidium guajava L. [Pg]; sensitive plant, Mimosa pudica L. [Mp]; neem, Azadirachta indica A. Juss [Ai] and asthma plant, Euphorbia hirta L. [Eh]) on the immune response and disease resistance against Edwardsiella ictaluri infection of striped catfish (Pangasianodon hypophthalmus). Fish were fed diets supplemented with two doses of each plant extract (0% [basal diet], 0.4% Eh [Eh0.4], 2.0% Eh [Eh2.0], 0.2% Pa [Pa0.2], 1.0% Pa [Pa1.0], 0.2% Pg [Pg0.2], 1.0% Pg [Pg1.0], 0.4% Mp [Mp0.4], 2.0% Mp [Mp2.0], 0.4% Ai [Ai0.4], 2.0% Ai [Ai2.0]) for 8 weeks. Results showed that hematological parameters (total red blood cells, white blood cells, lymphocytes, monocytes, and neutrophils) of fish fed extract-based diets were significantly higher than in those fed the control diet (p < 0.05) after 4 and 8 weeks. Plasma lysozyme activity increased in fish whose diets contained both doses of Eh (p < 0.05) in week 4 (W4), whereas lysozyme activity increased in fish fed 0.2% Pa and Pg, and 2.0% Ai and Eh (p < 0.05) in week 8 (W8). The lysozyme levels in skin mucus did not significantly differ between treatments (p > 0.05) in W4 and after the bacterial challenge test. At the end of the feeding trial, levels of ACH50 significantly increased in most of extract groups compared to the control group (p < 0.05). Total immunoglobulin increased considerably in both the plasma and skin mucus of fish fed extract-supplemented diets after 8 weeks. In addition, dietary supplementation with Pg, Mp, Pa0.2, Eh2.0, and Ai0.4 for 8 weeks considerably reduced the cumulative mortality against E. ictaluri infection in striped catfish. The results suggest that plant extracts possibly modulate the striped catfish immune response in a time and dose dependent manner. Specifically, diets enriched with extracts of P. guajava at 0.2 and 1.0%, or M. pudica at 2.0% for 8 weeks, have great potential for improving striped catfish health by enhancing the immune system and reducing mortality against bacterial challenges.

In Vivo Multivesicular Body and Exosome Secretion in the Intestinal Epithelial Cells of Turtles During Hibernation.

The present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many "heterogeneous" MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome-MVB-exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.

Dietary supplementation of selenium nanoparticles modulated systemic and mucosal immune status and stress resistance of red sea bream (Pagrus major).

Dietary supplementation of selenium nanoparticles (Se-NPs) at different levels (0, 0.5, 1, and 2 mg kg-1 diet) was evaluated to find out the effects on serum and skin immune responses as well as stress resistance in the red sea bream (Pagrus major). After 45 days of experimental trial, serum and mucosal immune responses were significantly high in fish fed 1 mg Se-NPs kg-1 diet (P < 0.05). In this group, alternative complement pathway, total serum protein, antioxidant activity of catalase enzyme, serum bactericidal activity, serum lysozyme activity, and amounts of skin mucus secretions as well as stress resistance against low salinity stress increased significantly, when compared to fish fed Se-NP-free diet (P < 0.05). Furthermore, fish fed Se-NPs at 2 mg kg-1 diet exhibited higher alternative complement pathway, total serum protein, mucus lysozyme activity, serum and mucus peroxidases, amount of mucus secreted, and tolerance against low salinity stress than the fish fed Se-NP-free diet (P < 0.05). Interestingly, the nitro blue tetrazolium activity in all groups fed with diets supplemented with Se-NPs are significantly higher than Se-NP-free diet (P < 0.05). The present results demonstrate that the dietary supplementation with Se-NPs (mainly from 1 to 2 mg kg-1 level) could be useful for maintaining the overall health status of red sea bream.

Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus.

The highly infectious porcine transmissible gastroenteritis virus (TGEV), which belongs to the coronaviruses (CoVs), causes diarrhea and high mortality rates in piglets, resulting in severe economic losses in the pork industry worldwide. In this study, we used Lactobacillus plantarum (L. plantarum) to anchor the expression of TGEV antigen (S) to dendritic cells (DCs) via dendritic cell-targeting peptides (DCpep). The results show that S antigen could be detected on the surface of L. plantarum by different detection methods. Furthermore, flow cytometry and ELISA techniques were used to measure the cellular, mucosal, and humoral immune responses of the different orally gavaged mouse groups. The obtained results demonstrated the significant effect of the constructed L. plantarum expressing S-DCpep fusion proteins in inducing high expression levels of B7 molecules on DCs, as well as high levels of IgG, secretory IgA, and IFN-γ and IL-4 cytokines compared with the other groups. Accordingly, surface expression of DC-targeted antigens successfully induced cellular, mucosal, and humoral immunity in mice and could be used as a vaccine.

Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. RESULTS: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. CONCLUSIONS: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract - Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

High Smad7 sustains inflammatory cytokine response in refractory coeliac disease.

Refractory coeliac disease (RCD) is a form of coeliac disease (CD) resistant to gluten-free diet and associated with elevated risk of complications. Many effector cytokines over-produced in the gut of patients with RCD are supposed to amplify the tissue-destructive immune response, but it remains unclear if the RCD-associated mucosal inflammation is sustained by defects in counter-regulatory mechanisms. The aim of the present study was to determine whether RCD-related inflammation is marked by high Smad7, an intracellular inhibitor of transforming growth factor-ß1 (TGF-ß1 ) activity. Smad7 was evaluated in duodenal biopsy samples of patients with RCD, patients with active CD, patients with inactive CD and healthy controls by Western blotting, immunohistochemistry and real-time PCR. In the same samples, TGF-ß1 and phosphorylated (p)-Smad2/3 were evaluated by ELISA and immunohistochemistry, respectively. Pro-inflammatory cytokine expression was evaluated in RCD samples cultured with Smad7 sense or antisense oligonucleotide. Smad7 protein, but not RNA, expression was increased in RCD compared with active and inactive CD patients and healthy controls and this was associated with defective TGF-ß1 signalling, as marked by diminished p-Smad2/3 expression. TGF-ß1 protein content did not differ among groups. Knockdown of Smad7 in RCD biopsy samples reduced interleukin-6 and tumour necrosis factor-α expression. In conclusion, in RCD, high Smad7 associates with defective TGF-ß1 signalling and sustains inflammatory cytokine production. These results indicate a novel mechanism by which the mucosal cytokine response is amplified in RCD and suggest that targeting Smad7 can be therapeutically useful in RCD.

Enrichment of common carp (Cyprinus carpio) diet with medlar (Mespilus germanica) leaf extract: Effects on skin mucosal immunity and growth performance.

A feeding trial was performed to assess the effects of dietary Medlar (Mespilus germanica) leaf extract (MLE) on the growth performance, skin mucus non-specific immune parameters as well as mRNA levels of immune and antioxidant related genes in the skin of common carp (Cyprinus carpio) fingerlings. Fish were fed diets supplemented with graded levels (0, 0.25, 0.50, and 1.00%) of MLE for 49 days. The results revealed an improvement to the growth performance and feed conversion ratio in MLE fed carps (P < 0.05), regardless of the inclusion level. The immunoglobulin levels and interleukin 8 levels in the skin mucous and skin, respectively, revealed significant increment in fish fed 1% MLE (P < 0.05) in comparison with the other MLE treatments and control group. Also, feeding on 0.25% and 0.50% MLE remarkably increased skin mucus lysozyme activity (P < 0.05). However, there were no significant difference between MLE treated groups and control (P > 0.05) in case protease activity in the skin mucous or tumor necrosis factor alpha and interleukin 1 beta gene expression in the skin of carps (P > 0.05). The expression of genes encoding glutathione reductase and glutathione S-transferase alpha were remarkably increased in MLE fed carps compared to the control group (P < 0.05) while carp fed 0.50% or 1.00% MLE had significantly increased glutathione peroxidase expression in their skin (P < 0.05). The present results revealed the potentially beneficial effects of MLE on the mucosal immune system and growth performance in common carp fingerlings.

The Human Milk Oligosaccharide 2'-Fucosyllactose Quenches Campylobacter jejuni-Induced Inflammation in Human Epithelial Cells HEp-2 and HT-29 and in Mouse Intestinal Mucosa.

BACKGROUND: Campylobacter jejuni causes diarrhea worldwide; young children are most susceptible. Binding of virulent C. jejuni to the intestinal mucosa is inhibited ex vivo by α1,2-fucosylated carbohydrate moieties, including human milk oligosaccharides (HMOSs). OBJECTIVE: The simplest α1,2-fucosylated HMOS structure, 2'-fucosyllactose (2'-FL), can be predominant at ≤5 g/L milk. Although 2'-FL inhibits C. jejuni binding ex vivo and in vivo, the effects of 2'FL on the cell invasion central to C. jejuni pathogenesis have not been tested. Clinical isolates of C. jejuni infect humans, birds, and ferrets, limiting studies on its mammalian pathobiology. METHODS: Human epithelial cells HEp-2 and HT-29 infected with the virulent C. jejuni strain 81-176 human isolate were treated with 5 g 2'-FL/L, and the degree of infection and inflammatory response was measured. Four-week-old male wild-type C57BL/6 mice were fed antibiotics to reduce their intestinal microbiota and were inoculated with C. jejuni strain 81-176. The sensitivity of the resulting acute transient enteric infection and immune response to inhibition by 2'-FL ingestion was tested. RESULTS: In HEp-2 and HT-29 cells, 2'-FL attenuated 80% of C. jejuni invasion (P < 0.05) and suppressed the release of mucosal proinflammatory signals of interleukin (IL) 8 by 60-70%, IL-1ß by 80-90%, and the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2) by 50% (P < 0.05). Ingestion of 2'-FL by mice reduced C. jejuni colonization by 80%, weight loss by 5%, histologic features of intestinal inflammation by 50-70%, and induction of inflammatory signaling molecules of the acute-phase mucosal immune response by 50-60% (P < 0.05). This acute model did not induce IL-17 (adaptive T cell response), a chronic response. CONCLUSIONS: In human cells in vitro (HEp-2, HT-29) and in a mouse infection model that recapitulated key pathologic features of C. jejuni clinical disease, 2'-FL inhibited pathogenesis and its sequelae. These data strongly support the hypothesis that 2'-FL represents a new class of oral agent for prevention, and potentially for treatment, of specific enteric infectious diseases.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3'-, 4-, and 6'-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo.

BACKGROUND: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known. OBJECTIVE: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS. METHODS: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo. RESULTS: The 3 galactosyllactoses (3'-GL, 4-GL, and 6'-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α- and IL-1ß-induced expression of IL-8, MIP-3α, and MCP-1 to 48-51% and pathogen-induced IL-8 and MCP-1 to 26-30% of positive controls (P < 0.001). GOS reduced TNF-α- and IL-1ß-induced inflammatory responses to 25-26% and pathogen-induced IL-8 and MCP-1 to 36-39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01). CONCLUSION: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

Transciptomic study of mucosal immune, antioxidant and growth related genes and non-specific immune response of common carp (Cyprinus carpio) fed dietary Ferula (Ferula assafoetida).

A 8-weeks feeding trial was conducted to examine the effects of different levels (0, 0.5, 1 and 2%) of dietary Ferula (Ferula assafoetida) on expression of antioxidant enzymes (GSR, GPX and GSTA), immune (TNF-alpha, IL1B, IL- 8 and LYZ) and growth (GH, IGF1 and Ghrl) genes as well as cutaneous mucus and serum non-specific immune response in common carp. The results revealed Ferula significantly increased antioxidant gene expression (GSR and GSTA) in a dose dependent manner (P < 0.05). The expression of immune growth related genes were significantly higher in Ferula fed fish compared control group (P < 0.05). The effects of Ferula on expression of genes was more pronounced in higher doses. Feeding on Ferula supplemented diet remarkably increased skin mucus lysozyme activity (P < 0.05). However, evaluation of mucus total Ig and protease activity revealed no significant difference between control and treated groups (P > 0.05). Regarding non-specific humoral response, serum total Ig, lysozyme and ACH50 showed no remarkable variation between Ferula fed carps and control group (P > 0.05). These results indicated up-regulation of growth and health related genes in Ferula fed common carp. Further studies using pathogen or stress challenge is required to conclude that transcriptional modulation is beneficial in common carp.