Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

African Swine Fever Virus L83L Negatively Regulates the cGAS-STING-Mediated IFN-I Pathway by Recruiting Tollip To Promote STING Autophagic Degradation.

African swine fever (ASF) is a devastating infectious disease of pigs caused by the African swine fever virus (ASFV), which poses a great danger to the global pig industry. Many viral proteins can suppress with interferon signaling to evade the host's innate immune responses. Therefore, the development of an effective vaccine against ASFV has been dampened. Recent studies have suggested that the L83L gene may be integrated into the host genome, weakening the host immune system, but the underlying mechanism is unknown. Our study found that L83L negatively regulates the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. Overexpression of L83L inhibited IFN-ß promoter and ISRE activity, and knockdown of L83L induced higher transcriptional levels of interferon-stimulated genes (ISGs) and phosphorylation levels of IRF3 in primary porcine alveolar macrophages. Mechanistically, L83L interacted with cGAS and STING to promote autophagy-lysosomal degradation of STING by recruiting Tollip, thereby blocking the phosphorylation of the downstream signaling molecules TBK1, IRF3, and IκBα and reducing IFN-I production. Altogether, our study reveals a negative regulatory mechanism involving the L83L-cGAS-STING-IFN-I axis and provides insights into an evasion strategy involving autophagy and innate signaling pathways employed by ASFV. IMPORTANCE African swine fever virus (ASFV) is a large double-stranded DNA virus that primarily infects porcine macrophages. The ASFV genome encodes a large number of immunosuppressive proteins. Current options for the prevention and control of this pathogen remain pretty limited. Our study showed that overexpression of L83L inhibited the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. In contrast, the knockdown of L83L during ASFV infection enhanced IFN-I production in porcine alveolar macrophages. Additional analysis revealed that L83L protein downregulated IFN-I signaling by recruiting Tollip to promote STING autophagic degradation. Although L83L deletion has been reported to have little effect on viral replication, its immune evade mechanism has not been elucidated. The present study extends our understanding of the functions of ASFV-encoded pL83L and its immune evasion strategy, which may provide a new basis for developing a live attenuated vaccine for ASF.

Lycium barbarum polysaccharide promotes the immunoprotective effects of a recombinant Lactobacillus plantarum vaccine expressing the Trichinella spiralis cathepsin F-like protease 1 gene.

Trichinellosis caused by Trichinella spiralis (T. spiralis) is a zoonotic disease that poses a substantial risk to human health. At present, vaccines used to prevent trichinellosis are effective, but the production of antibody levels and immunogenicity are low. Adjuvants can increase antibody levels and vaccine immunogenicity. As a result, it is critical to develop an effective adjuvant for the T. spiralis vaccine. Recent research has shown that traditional Chinese medicine polysaccharides with low-toxicity and biodegradability can act as adjuvants in vaccines. In this study, BALB/c mice were orally inoculated with a recombinant Lactobacillus plantarum (L. plantarum) vaccine expressing the T. spiralis cathepsin F-like protease 1 gene (rTs-CPF1), which was given three times at 10-day intervals. Lycium barbarum polysaccharide (LBP) was administered orally for 37 days. At 37 days after the first immunization, mice were infected with 350 T. spiralis muscle larvae (ML). Specific IgG and sIgA antibody levels against the T. spiralis CPF1 protein were increased in mice immunized with rTs-CPF1+LBP compared to those immunized with rTs-CPF1 alone. Furthermore, LBP increased IFN-γ and IL-4 expression levels, and the number of intestinal and intramuscular worms was significantly reduced in the rTs-CPF1+LBP group compared to that in the rTs-CPF1 group. In the rTs-CPF1+LBP group, the reduction rates of adult worms and muscle larvae were 47.31 % and 68.88 %, respectively. To summarize, LBP promotes the immunoprotective effects of the T. spiralis vaccine and may be considered as a novel adjuvant in parasitic vaccines.

NLRP3 Plays a Key Role in Antihelminth Immunity in the Enteral and Parenteral Stages of Trichinella spiralis-Infected Mice.

Trichinellosis is an important foodborne zoonosis, and no effective treatments are yet available. Nod-like receptor (NLR) plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis infection. The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, the role that NLRP3 plays during T. spiralis infection of mice was evaluated using enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, histopathological evaluation, bone marrow-derived macrophage (BMDM) stimulation, and immunofluorescence. The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult and migrating stages and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (interferon gamma [IFN-γ]) and suppressed the release of anti-inflammatory factors (interleukin 4 [IL-4]). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1ß and IL-18 release. This study revealed that NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis.

Lactobacillus Plantarum NC8 and its metabolite acetate alleviate type 1 diabetes via inhibiting NLRP3.

A healthy organism is the result of host-microbiome co-evolution. Microbial metabolites can also stimulate immune cells to reduce intestinal inflammation and permeability. Gut dysbiosis will lead to a variety of autoimmune diseases, such as Type 1 diabetes (T1D). Most of probiotics, such as Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium bifidium, and Streptococcus thermophiles, can improve the intestinal flora structure of the host, reduce intestinal permeability, and relieve symptoms of T1D patients if ingested above probiotics in sufficient amounts. Lactobacillus Plantarum NC8, a kind of Lactobacillus, whether it has an effect on T1D, and the mechanism of it regulating T1D is still unclear. As a member of the inflammatory family, NLRP3 inflammasome can enhance inflammatory responses by promoting the production and secretion of proinflammatory cytokines. Many previous studies had shown that NLRP3 also plays an important role in the development of T1D. When the NLRP3 gene is deleted, the disease progression of T1D will be delayed. Therefore, this study investigated whether Lactobacillus Plantarum NC8 can alleviate T1D by regulating NLRP3. The results demonstrated that Lactobacillus Plantarum NC8 and its metabolites acetate play a role in T1D by co-modulating NLRP3. Lactobacillus Plantarum NC8 and acetate can reduce the damage of T1D in the model mice, even if orally administered them in the early stage of T1D. The number of Th1/Th17 cells in the spleen and pancreatic lymph nodes (PLNs) of T1D mice were significantly reduced by oral Lactobacillus Plantarum NC8 or acetate. The expression of NLRP3 in the pancreas of T1D mice or murine macrophages of inflammatory model were significantly inhibited by treatment with Lactobacillus Plantarum NC8 or acetate. In addition, the number of macrophages in the pancreas were significantly reduced by the treatment with Lactobacillus Plantarum NC8 or acetate. In summary, this study indicated that the regulatory mechanism of Lactobacillus Plantarum NC8 and its metabolite acetate to T1D maybe via inhibiting NLRP3 and provides a novel insights into the mechanism of the alleviated role of probiotics to T1D.

The global prevalence of highly pathogenic avian influenza A (H5N8) infection in birds: A systematic review and meta-analysis.

The zoonotic pathogen avian influenza A H5N8 causes enormous economic losses in the poultry industry and poses a serious threat to the public health. Here, we report the first systematic review and meta-analysis of the worldwide prevalence of birds. We filtered 45 eligible articles from seven databases. A random-effects model was used to analyze the prevalence of H5N8 in birds. The pooled prevalence of H5N8 in birds was 1.6%. In the regions, Africa has the highest prevalence (8.0%). Based on the source, village (8.3%) was the highest. In the sample type, the highest prevalence was organs (79.7%). In seasons, the highest prevalence was autumn (28.1%). The largest prevalence in the sampling time was during 2019 or later (7.0%). Furthermore, geographical factors also were associated with the prevalence. Therefore, we recommend site-specific prevention and control tools for this strain in birds and enhance the surveillance to reduce the spread of H5N8.

Characterization and pathogenicity of the porcine epidemic diarrhea virus isolated in China.

Piglet diarrhea caused by the porcine epidemic diarrhea virus (PEDV) is a common problem on pig farms in China associated with high morbidity and mortality rates. In this study, three PEDV isolates were successfully detected after the fourth blind passage in Vero cells. The samples were obtained from infected piglet farms in Jilin (Changchun), and Shandong (Qingdao) Provinces of China and were designated as CH/CC-1/2018, CH/CC-2/2018, and CH/QD/2018. According to the analysis of the complete S protein gene sequence, the CH/CC-1/2018 and CH/CC-2/2018 were allocated to the G2b branch, while CH/QD/2018 was located in the G1a interval and was closer to the vaccine strain CV777. Successful detection and identification of the isolated strains were carried out using electron microscopy and indirect immunofluorescence. Meanwhile, animal challenge experiments and viral RNA copies determination were used to compare the pathogenicity. The results showed that CH/CC-1/2018 in Changchun was more pathogenic than CH/QD/2018 in Qingdao. In conclusion, the discovery of these new strains is conducive to the development of vaccines to prevent the pandemic of PEDV, especially that the CH/CC-1/2018, and CH/CC-2/2018 were not related to the classical vaccine strain CV777.

Lactobacillus rhamnosus GG Promotes Early B Lineage Development and IgA Production in the Lamina Propria in Piglets.

Gut microbes play an important role in the development of host B cells. It has been controversial whether GALT is the development site of B cells in pigs. By investigating the relationship between gut microbes and the development of B cells in the GALT of piglets, we found, to our knowledge for the first time, that early B cells exist in the gut lamina propria (LP) in pigs at different ages. We further used Lactobacillus rhamnosus GG (LGG) to treat piglets. The results showed that LGG promotes the development of the early B lineage, affects the composition of the Ig CDR3 repertoires of B cells, and promotes the production of IgA in the intestinal LP. Additionally, we found that the p40 protein derived from LGG can activate the EGFR/AKT and NF-κB signaling pathways, inducing porcine intestinal epithelial cells (IPEC-J2) to secrete a proliferation-inducing ligand (APRIL), which promotes IgA production in B cells. Finally, we identified ARF4 and DIF3 as candidates for p40 receptors on IPEC-J2 by GST pull-down, liquid chromatography-mass spectrometry/mass spectrometry analysis, and coimmunoprecipitation. In conclusion, LGG could promote early B cell differentiation and development in the intestinal LP in piglets and might contribute to promoting IgA production via secretion of p40, which interacts with the membrane receptors on IPEC-J2 and induces them to secrete APRIL. Our study will provide insight to aid in better utilization of probiotics to increase human health.

A novel "prime and pull" strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge.

Vaccination is still the most promising strategy for combating influenza virus pandemics. However, the highly variable characteristics of influenza virus make it difficult to develop antibody-based universal vaccines, until now. Lung tissue-resident memory T cells (TRM), which actively survey tissues for signs of infection and react rapidly to eliminate infected cells without the need for a systemic immune reaction, have recently drawn increasing attention towards the development of a universal influenza vaccine. We previously designed a sequential immunization strategy based on orally administered Salmonella vectored vaccine candidates. To further improve our vaccine design, in this study, we used two different dendritic cell (DC)-targeting strategies, including a single chain variable fragment (scFv) targeting the surface marker DC-CD11c and DC targeting peptide 3 (DCpep3). Oral immunization with Salmonella harboring plasmid pYL230 (S230), which displayed scFv-CD11c on the bacterial surface, induced dramatic production of spleen effector memory T cells (TEM). On the other hand, intranasal boost immunization using purified DCpep3-decorated 3M2e-ferritin nanoparticles in mice orally immunized twice with S230 (S230inDC) significantly stimulated the differentiation of lung CD11b+ DCs, increased intracellular IL-17 production in lung CD4+ T cells and elevated chemokine production in lung sections, such as CXCL13 and CXCL15, as determined by RNAseq and qRT‒PCR assays, resulting in significantly increased percentages of lung TRMs, which could provide efficient protection against influenza virus challenge. The dual DC targeting strategy, together with the sequential immunization approach described in this study, provides us with a novel "prime and pull" strategy for addressing the production of protective TRM cells in vaccine design.

African swine fever virus: A raised global upsurge and a continuous threaten to pig husbandry.

African swine fever (ASF) is a severe disease affecting pigs with high economic losses and endemicity in various parts of the world. So, it represents a serious threat to the global food safety. The disease was discovered in sub-Saharan Africa where still endemic, and first case was recorded in Kenya in 1921. It is now found all over the world; in Africa, Europe, Asia, and the Pacific it already affects more than 50 countries including Republic of Korea, China, Malaysia, Germany, Bhutan, and India. The P72 protein encoded by the B646L gene is the major protein that reveals high reactogenicity and antigenicity. While the P54 plays a significant role in virus pathogenesis especially cell apoptosis. Multiple virus proteins can suppress the apoptosis of the infected cell at an early stage. The disease spreads through contact with the diseased cases, contaminated fomites, and tick bites. Meanwhile, contaminated water sources might be an essential source of infection. The recovered animals have a significant role in disease persistence as silent carriers. Multiple factors might lead to the observed disease seasonality. Route of exposure, infectious dose, and herd immunity are the main determinants of disease severity and clinical signs. The several types of PCR are well-accepted standard tests for early diagnosis. Although commercial ELISAs were stipulated by OIE, it should be combined with some other virology inspections or serological assays. The ASFV-free countries should be protected against the virus entrance especially that all developed vaccines failed to provoke enough immunity status against the challenged virus. Moreover, it accelerates the speed of revealing clinical symptoms.

Protective effects of E. coli Nissle 1917 on chickens infected with Salmonella pullorum.

The probiotic E. coli Nissle 1917 (EcN) plays an important role in regulating the microbial components of the gut and preventing inflammation of the gastrointestinal tract. Currently, the long-term use of antibiotics for the treatment of lethal white diarrhea in chicks caused by Salmonella has led to increased morbidity and mutation rates. Therefore, we want to use EcN as an antibiotic alternative as an alternative approach to prevent Salmonella-induced white diarrhea in chickens. To date, there are no reports of EcN being used for the prevention and control of Salmonella pullorum (S. pullorum) in chickens. In vitro, pretreatment with EcN significantly decreased the cellular invasion of S. pullorum CVCC533 in a chicken fibroblast (DF-1) cell model. Then, 0-day-old egg-laying chickens were orally inoculated with EcN at a dose of 109 CFU/100 µL at either Day 1 (EcN1) or both Day 1 and Day 4 (EcN2). Then, S. pullorum CVCC533 was used to challenge the cells at a dose of 1.0 × 107 CFU/100 µL on Day 8. Next, the body weights and survival rates were recorded for 14 consecutive days, and the colonization of S. pullorum in the spleen and liver at 7 days post-challenge (dpc) was determined. Chicken feces were also collected at 2, 4, 6 and 8 dpc to evaluate the excretion of pathogenic bacteria in feces. The liver, duodenum and rectum samples were collected and analyzed by pathological histology at 7 dpc to evaluate the protective effect of EcN on the mucosa, villi and crypts of the small intestine. The spleen and bursa were collected, and the immune organ index was calculated. In addition, the contents of the cecum of chicks were collected at 7 dpc for 16S rRNA sequencing to detect the distribution of microbial communities in the intestine. The results showed that EcN was able to protect against CVCC533 challenge, as shown by decreased body weight loss, mortality and shedding of pathogenic bacteria in fecal samples in the EcN1 plus Salmonella challenge group (EcN1S) but not the EcN2 plus Salmonella challenge group (EcN2S). The pathogenic changes in the liver, duodenum and rectum also demonstrated that one dose but not two doses of EcN effectively prolonged the length of the pilus with decreased crypt depth, indicating its protective effects against S. pullorum. In addition, the 16S rRNA sequencing results suggested that EcN could enlarge the diversity of intestinal flora, decrease the abundance of pathogenic bacteria and increase the abundance of beneficial bacteria, such as Lactobacillus. In conclusion, EcN has shown moderate protection against S. pullorum challenge in chickens.

African swine fever virus MGF360-11L negatively regulates cGAS-STING-mediated inhibition of type I interferon production.

The type I interferon (IFN-I) signaling pathway is an important part of the innate immune response and plays a vital role in controlling and eliminating pathogens. African swine fever virus (ASFV) encodes various proteins to evade the host's natural immunity. However, the molecular mechanism by which the ASFV-encoded proteins inhibit interferon production remains poorly understood. In the present study, ASFV MGF360-11L inhibited cGAS, STING, TBK1, IKKε, IRF7 and IRF3-5D mediated activation of the IFN-ß and ISRE promoters, accompanied by decreases in IFN-ß, ISG15 and ISG56 mRNA expression. ASFV MGF360-11L interacted with TBK1 and IRF7, degrading TBK1 and IRF7 through the cysteine, ubiquitin-proteasome and autophagy pathways. Moreover, ASFV MGF360-11L also inhibited the phosphorylation of TBK1 and IRF3 stimulated by cGAS-STING overexpression. Truncation mutation analysis revealed that aa 167-353 of ASFV MGF360-11L could inhibit cGAS-STING-mediated activation of the IFN-ß and ISRE promoters. Finally, the results indicated that ASFV MGF360-11L plays a significant role in inhibiting IL-1ß, IL-6 and IFN-ß production in PAM cells (PAMs) infected with ASFV. In short, these results demonstrated that ASFV MGF360-11L was involved in regulating IFN-I expression by negatively regulating the cGAS signaling pathway. In summary, this study preliminarily clarified the molecular mechanism by which the ASFV MGF360-11L protein antagonizes IFN-I-mediated antiviral activity, which will help to provide new strategies for the treatment and prevention of ASF.

Trichinella spiralis infection ameliorates the severity of Citrobacter rodentium-induced experimental colitis in mice.

Trichinellosis is a food-borne zoonotic parasitic disease that causes serious harm to human health and the pig breeding industry. However, there are reports that Trichinella spiralis (T. spiralis) infection can treat autoimmune diseases, including enteritis and experimental autoimmune encephalitis (EAE). However, research on the mechanism of T. spiralis infection in infectious enteritis has not been fully elucidated. Therefore, this experiment used Citrobacter rodentium (C. rodentium) to induce colitis in mouse models and explored its underlying mechanisms. In this experiment, a total of 72 C57BL/6 mice were randomly divided into four groups. Experimental mice in the TS and TS + CR groups were orally inoculated with individual T. spiralis larvae. At 21 days postinfection (dpi) with T. spiralis, experimental animals in the CR and TS + CR groups were inoculated by orogastric gavage with C. rodentium. The control group received PBS only. The results indicated that the weight loss and macroscopic and microscopic colon damage of mice in the TS + CR group were significantly decreased compared with those observed in the CR group. The results of flow cytometry showed that the expression levels of IL-4, IL-10 and CD4+CD25+Foxp3+ Tregs were increased (P < 0.05), while the expression levels of IFN-γ, IL-12 and IL-17 were decreased in the spleens and MLNs of the TS + CR experimental mice compared with the colitis model mice. ELISA results revealed that the TS + CR group not only elicited a strong IgG1 response (P < 0.01) but also a low level of IgG2a response (P < 0.05) relative to the CR group. The above results demonstrated that prior exposure of mice to T. spiralis infection ameliorated the severity of C. rodentium-induced infectious colitis.

Improved pathogenicity of H9N2 subtype of avian influenza virus induced by mutations occurred after serial adaptations in mice.

H9N2 subtype, a low pathogenic avian influenza virus, is emerging as a major causative agent circulating poultry workplaces across China and other Asian countries. Increasing case number of interspecies transmissions to mammals reported recently provoked a great concern about its risks inducing global pandemics. In an attempt to understand the underlying mechanism of how the H9N2 virus disrupts the interspecies segregation to transmit to mammals. A mutant H9N2 strain was obtained by passaging the wildtype H9N2 A/chicken/Hong Kong/G9/1997 eight times from lung to lung in BALB/c mice. Our finding revealed that mice manifested severe clinical symptoms including losses of body weight, pathological damages in pulmonary sites and all died within two weeks after infected with the mutated H9N2, whereas all mice survived upon infected with wildtype strain in comparison, which suggested increased pathogenicity of the mutant strain. In addition, mice showed enhanced levels of proinflammatory cytokines in sera, including IL-6, TNF-α and IL-1ß compared to those subjected to wildtype viral infections. Sequence analysis showed that five amino acid substitutions occurred at PB2627, HA87, HA234, NP387 and M156, and a deletion mutation happened in the M gene (M157). Of these mutations, PB2 E627K played key roles in modulating lethality in mice. Taken together, the mutant H9N2 strain obtained by serial passaging of its wildtype in mice significantly increased its virulence leading to death of mice, which might be associated the accumulated mutations occurred on its genome.

Bacillus subtilis BSH has a protective effect on Salmonella infection by regulating the intestinal flora structure in chickens.

Salmonellosis is a worldwide zoonotic disease that poses a serious threat to the reproduction of livestock and poultry and the health of young animals. Probiotics including Bacillus species, have received increasing attention as a substitute for antibiotics. In this study, chicks infected with Salmonella were fed feed supplemented with the BSH to observe the pathological changes in the liver, detect the number of viable bacteria in the liver and spleen, and record the death of the chicks. The results showed that BSH could reduce the pathological changes in the liver and the invasion of Salmonella into the liver and spleen of chicks. In addition, the survival rate of chicks in the BSH experimental group was 60%, while that in the infected control group was 26%, indicating that BSH had a protective effect on chicks infected with Salmonella. Finally, the fecal microflora of 9-day-old chicks was analyzed by 16S rRNA high-throughput sequencing. The results showed that Salmonella infection could cause intestinal flora changes, while BSH could alleviate this change. In addition, BSH also promoted the proliferation of Lactobacillus salivarius in the cecum of chick. This study emphasized that BSH has anti- Salmonella infection effects in chickens and can be used as a candidate microecological preparation strain.

Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein.

Some protozoa (Plasmodium falciparum, Toxoplasma gondii, etc) are used to treat cancer because they can improve tumour-induced immunosuppression. This study aims to evaluate the antitumour effect of Eimeria stiedae oocyst soluble protein (ESSP). ESSP was extracted, and mice were injected with 5 × 105 CT26 cells in the right axilla, and then, 50 µg of ESSP was intraperitoneally injected for 5 continuous days. The effect of ESSP on tumour immunity was detected by flow cytometry 25 days after the CT26 inoculation. The results showed that ESSP can inhibit the growth of CT26 subcutaneous tumours; significantly increase the expression of MHC I, MHC II, CD80 and CD86 on the surface of splenic dendritic cells; and enhance the level of IL-12 secretion. ESSP induced an increase in the number of NK cells in the mouse spleen, and the levels of IFN-γ and CD107 were upregulated in the NK cells and CD8+ T cells. The number of metastatic nodules in the lung tumours in the mice was significantly reduced, and the number of tubes, area of the loops and total length of the tubes were significantly reduced. ESSP enhances the antitumour immune response and inhibits tumour growth, metastasis and angiogenesis.

Recombinant Lactobacillus plantarum NC8 strain expressing porcine rotavirus VP7 induces specific antibodies in BALB/c mice.

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.

Dendritic Cells Targeting Lactobacillus plantarum Strain NC8 with a Surface-Displayed Single-Chain Variable Fragment of CD11c Induce an Antigen-Specific Protective Cellular Immune Response.

Influenza A virus (H1N1) is an acute, highly contagious respiratory virus. The use of lactic acid bacteria (LAB) to deliver mucosal vaccines against influenza virus infection is a research hot spot. In this study, two recombinant Lactobacillus plantarum strains expressing hemagglutinin (HA) alone or coexpressing aCD11c-HA to target HA protein to dendritic cells (DCs) by fusion to an anti-CD11c single-chain antibody (aCD11c) were constructed. The activation of bone marrow dendritic cells (BMDCs) by recombinant strains and the interaction of activated BMDCs and sorted CD4+ or CD8+ T cells were evaluated through flow cytometry in vitro, and cellular supernatants were assessed by using an enzyme-linked immunosorbent assay kit. The results demonstrated that, compared to the HA strain, the aCD11c-HA strain significantly increased the activation of BMDCs and increased the production of CD4+ gamma interferon-positive (IFN-γ+) T cells, CD8+ IFN-γ+ T cells, and IFN-γ in the cell culture supernatant in vitro Consistent with these results, the aCD11c-HA strain clearly increased the activation and maturation of DCs, the HA-specific responses of CD4+ IFN-γ+ T cells, CD8+ IFN-γ+ T cells, and CD8+ CD107a+ T cells, and the proliferation of T cells in the spleen, finally increasing the levels of specific antibodies and neutralizing antibodies in mice. In addition, the protection of immunized mice was observed after viral infection, as evidenced by improved weight loss, survival, and lung pathology. The adoptive transfer of CD8+ T cells from the aCD11c-HA mice to NOD/Lt-SCID mice resulted in a certain level of protection after influenza virus infection, highlighting the efficacy of the aCD11c targeting strategy.

Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens.

BACKGROUND: Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry. RESULTS: In this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA') from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge. CONCLUSIONS: The novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.

A potential vaccine candidate towards chicken coccidiosis mediated by recombinant Lactobacillus plantarum with surface displayed EtMIC2 protein.

Eimeria tenella (E. tenella) has caused severe economic loss in chicken production, especially after the forbidden use of antibiotics in feed. Considering the drug resistant problem caused by misuse of chemoprophylaxis and live oocyst vaccines can affect the productivity of chickens, also it has the risk to reversion of virulence, the development of efficacious, convenient and safe vaccines is still deeply needed. In this study, the EtMic2 protein of E. tenella was anchored on the surface of Lactobacillus plantarum (L. plantarum) NC8 strain. The newly constructed strain was then used to immunize chickens, followed by E. tenella challenge. The results demonstrated that the recombinant strain could provide efficient protection against E. tenella, shown by increased relative body weight gains, percentages of CD4+ and CD8+ T cells, humoral immune response and inflammatory cytokines. In addition, decreased cecum lesion scores and fecal oocyst shedding were also observed during the experiment. In conclusion, this study proves the possibility to use L. plantarum as a vessel to deliver protective antigen to protect chickens against coccidiosis.