In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

Serologic evidence of the circulation of the hepatitis E virus and the prevalence of antibodies against hepatitis A in an indigenous population in northern Argentina.

In 2005 a universal vaccination program against hepatitis A was introduced in Argentina. Nevertheless, there are still some unvaccinated marginal population groups. There are no data about the seroprevalence of hepatitis E in the northern region of Argentina mainly because of lack of awareness of this emergent pathogen. We aimed to determine the seroprevalence of hepatitis A, and hepatitis E in an indigenous population in northern Argentina. One hundred and twenty six (126) donor serum samples collected near San Salvador de Jujuy were analyzed for anti-HAV IgG and HEV IgG and IgM, alkaline phosphatase and transaminase values. Volunteers were interviewed about their living conditions, animal farming, consumption of tap water or river water, and level of education. Seroprevalence of specific anti-HAV antibodies was high (80.2%, 95% confidence interval, 72.1-86.7%) in children under 5 years of age, indicating early infection in life. Seroprevalence of anti-HEV antibodies was 5.6% (95% CI: 2.3-11.2%), being slightly higher than the values found in healthy patients from other regions of the country. Although we could not characterize the genotype of the circulating HEV strain, the clear epidemiological difference between seroprevalence of HAV and HEV in a community with poor sanitary conditions suggest that the circulating HEV strains spread through a different transmission route than HAV. Furthermore a significant correlation between anti-HEV IgG and swine farming was found (p<0.05), which supports a zoonotic transmission path. We reassessed the epidemiological pattern of HAV infection and reported evidence of HEV infection for the first-time in a community belonging to the Guarani ethnic group, highlighting the need to include hepatitis E testing in routine diagnostics in the region.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

OBJECTIVE: To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. METHODS: Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. RESULTS: Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. CONCLUSIONS: Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells.

BACKGROUND: Previous findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized. RESULTS: We aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer's patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN-α and -ß in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1ß, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs. CONCLUSIONS: It was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge.

The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 possesses immunomodulatory activities in the gastrointestinal and respiratory tracts when administered orally. Its adhesion to the intestinal mucosa does not condition its beneficial effects. The intranasal administration of L. rhamnosus CRL1505 is more effective than the oral route at modulating immunity in the respiratory tract. Nonetheless, it has not yet been established whether the adherence of the CRL1505 strain to the respiratory mucosa is needed to provide the immune benefits to the host. In this study, we evaluated the role of adhesion to the respiratory mucosa of the mucus-binding factor (mbf) knock-out L. rhamnosus CRL1505 mutant (Δmbf CRL1505) in the context of a Toll-like receptor 3 (TLR3)-triggered innate immunity response. In vitro adhesion studies in porcine bronchial epitheliocytes (PBE cells) indicated that L. rhamnosus Δmbf CRL1505 adhered weakly compared to the wild-type strain. However, in vivo studies in mice demonstrated that the Δmbf CRL1505 also reduced lung damage and modulated cytokine production in the respiratory tract after the activation of TLR3 to a similar extent as the wild-type strain. In addition, the mutant and the wild-type strains modulated the production of cytokines and antiviral factors by alveolar macrophages in the same way. These results suggest that the Mbf protein is partially involved in the ability of L. rhamnosus CRL1505 to adhere to the respiratory epithelium, but the protein is not necessary for the CRL1505 strain to exert its immunomodulatory beneficial effects. These findings are a step forward in the understanding of molecular interactions that mediate the beneficial effects of nasally administered probiotics.

Bacterium-like Particles from Corynebacterium pseudodiphtheriticum as Mucosal Adjuvant for the Development of Pneumococcal Vaccines.

Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

Unraveling swine hepatitis E in the central region of Argentina through ELISA development and epidemiological insights.

Hepatitis E virus (HEV) is a public health concern globally, causing acute viral hepatitis in humans. Genotype-3 HEV (HEV-3), the most frequently genotype detected in South America, is zoonotic and the main reservoirs are the domestic pig and wild boar. Circulation of HEV-3 in Argentina has been confirmed in humans as well as in pig herds, wild boar and environmental waters. However, data are scarce mainly due to the inaccessibility of serological assays in this country. In order to provide insights in the epidemiology of HEV in swine in Argentina, we developed an indirect ELISA based on the native recombinant protein ORF2 and conducted a serological survey to determine the prevalence of seropositive swine in small-scale pig farms in the central region of Argentina. The method was evaluated in a panel of 157 serum samples, resulting in relative sensitivity of 98.6 % (95 % CI 95 %-100 %) and relative specificity of 97.7 % (95 % CI 94 %-100 %) compared to a commercial test. An almost perfect agreement was obtained between the two tests (Kappa index of 0.961). A survey on 294 samples from 49 small-scale farms resulted in a seropositivity rate of 54 %. Seropositive animals were found in 34 out of 49 (69.4 %) farms. Most of the farms (70.6 %) had over 50 % of seropositive animals. The wide spreading of HEV in the swine population of Tandil, Argentina, underscore the need to better understand the epidemiology of HEV in the region, enabling the implementation of targeted interventions to mitigate the impact of this virus on public health.

A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus.

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Exploring hepatitis E virus seroprevalence and associated risk factors among the human population in Tandil, Buenos Aires, Argentina.

Background: Hepatitis E virus (HEV) infection is a common cause of acute clinical hepatitis worldwide and is emerging as a disease in Argentina. It is primarily transmitted through contaminated water and food, following the fecal-oral route. Furthermore, is a zoonotic disease with swine as the primary reservoir. Prevalence of HEV infection in humans in several regions of Argentina remains unknown. Objectives: (i) Determine the seroprevalence of HEV among the human population in Tandil, Buenos Aires, Argentina; (ii) Evaluate its association with demographic, socioeconomic and other risk exposures variables, and (iii) Describe and analyze spatial patterns related to HEV infection. Methods: From August 2020 to July 2021, serum samples were collected from 969 individuals aged 1-80 years. Seroprevalence and 95% Confidence Interval was determined. To assess the factors associated with the presence of anti-HEV antibodies, associations between the variables and seropositivity were evaluated through bivariate and multivariate analysis. Spatial scanning for clusters of positivity was carried out. Factors associated with these clusters were also assessed. Results: Anti-HEV antibodies were detected in 4.64% (IC 95% 3.27-6.02) of samples. Dark urine was associated with seropositivity (p = 0.02). Seropositivity was linked with the presence of natural water courses near their households (p = 0.02); the age (p = 0.04); and previous travel to Europe (p = 0.04). A spatial cluster of low rates of HEV seropositivity was detected, with greater distance of the households to water courses associated to the cluster, and male sex inversely associated to it. Discussion and conclusion: This study is the first study to investigate the prevalence of HEV in the population from Tandil, Buenos Aires, Argentina. Considering HEV infection in the differential diagnosis in individuals presenting acute hepatitis is highlighted. The incorporation of HEV testing into blood screening policies should be mandatory. Factors related to the infection and spatial patterns of high and low risk were determined, and should be considered when implementing specific preventive measures.

Systematic screening for novel, serologically reactive Hepatitis E Virus epitopes.

BACKGROUND: The National Institutes of Health classified Hepatitis E as an emerging disease since Hepatitis E Virus (HEV) is the major cause of acute hepatitis in developing countries. Interestingly, an increasing number of sporadic cases of HEV infections are described in industrialized countries as zoonosis from domestic livestock. Despite the increasing relevance of this pathogen in clinical virology, commercial antibody assays are mainly based on fragments of HEV open reading frame (ORF) 2 and ORF3. The largest ORF1 (poly-)protein, however, is not part of current testing formats. METHODS: From a synthesized full length HEV genotype 1 cDNA-bank we constructed a complete HEV gene library consisting of 15 respective HEV ORF domains. After bacterial expression and purification of nine recombinant HEV proteins under denaturating conditions serum profiling experiments using 55 sera from patients with known infection status were performed in microarray format. SPSS software assessed the antigenic potential of these nine ORF domains in comparison to seven commercial HEV antigens (genotype 1 and 3) by performing receiver operator characteristics, logistic regression and correlation analysis. RESULTS: HEV antigens produced with our method for serum profiling experiments exhibit the same quality and characteristics as commercial antigens. Serum profiling experiments detected Y, V and X domains as ORF1-antigens with potentially comparable diagnostic significance as the well established epitopes of ORF2 and ORF3. However no obvious additional increase in sensitivity or specificity was achieved in diagnostic testing as revealed by bioinformatic analysis. Additionally we found that the C-terminal domain of the potential transmembrane protein ORF3 is responsible for IgG and IgM seroreactivity. Data suggest that there might be a genotype specific seroreactivity of homologous ORF2-antigens. CONCLUSIONS: The diagnostic value of identified ORF1 epitopes might not necessarily improve sensitivity and specificity, but broaden the overall quality of existing test systems. ORF2 and ORF3-antigens are still commonly used in diagnostic assays and possibly hold the potential to serologically differentiate between genotype 1 and 3 infections. Our systematic approach is a suitable method to investigate HEV domains for their serologic antigenicity. Epitope screening of native viral domains could be a preferable tool in developing new serologic test components.

Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

Lactiplantibacillus plantarum as a Potential Adjuvant and Delivery System for the Development of SARS-CoV-2 Oral Vaccines.

The most important characteristics regarding the mucosal infection and immune responses against the Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) as well as the current vaccines against coronavirus disease 2019 (COVID-19) in development or use are revised to emphasize the opportunity for lactic acid bacteria (LAB)-based vaccines to offer a valid alternative in the fight against this disease. In addition, this article revises the knowledge on: (a) the cellular and molecular mechanisms involved in the improvement of mucosal antiviral defenses by beneficial Lactiplantibacillus plantarum strains, (b) the systems for the expression of heterologous proteins in L. plantarum and (c) the successful expressions of viral antigens in L. plantarum that were capable of inducing protective immune responses in the gut and the respiratory tract after their oral administration. The ability of L. plantarum to express viral antigens, including the spike protein of SARS-CoV-2 and its capacity to differentially modulate the innate and adaptive immune responses in both the intestinal and respiratory mucosa after its oral administration, indicates the potential of this LAB to be used in the development of a mucosal COVID-19 vaccine.

Complete Genome Sequence of Burkholderia gladioli Phage Maja.

Burkholderia gladioli is a Gram-negative bacterium associated with cystic fibrosis infections. Here, we describe the genome sequence of B. gladioli phage Maja. Maja is most related to another Burkholderia phage, BcepF1, and may be a temperate phage, despite the absence of repressor or integrase homologs in its genome sequence.

Complete Genome Sequence of Burkholderia gladioli Myophage Mana.

Burkholderia gladioli is known to cause respiratory tract infections in cystic fibrosis patients. Here, we describe the annotation of the 38,038-bp genome sequence of Mana, a P2-like phage of B. gladioli Understanding the genomic characteristics of phages infecting pathogens like B. gladioli can lead to advancements in phage therapy.

Complete Genome Sequence of Burkholderia cenocepacia Phage Mica.

Burkholderia cenocepacia is a multidrug-resistant Gram-negative pathogen known to colonize patients with chronic granulomatous disease and cystic fibrosis. Here, we describe Burkholderia phage Mica, which is predicted to be a lysogenic myophage based on the similarity of its structural proteins to Enterobacteria phage P2 and Burkholderia phage KL3.

Immunobiotic Lactobacilli Improve Resistance of Respiratory Epithelial Cells to SARS-CoV-2 Infection.

Previously, we reported that immunomodulatory lactobacilli, nasally administered, beneficially regulated the lung antiviral innate immune response induced by Toll-like receptor 3 (TLR3) activation and improved protection against the respiratory pathogens, influenza virus and respiratory syncytial virus in mice. Here, we assessed the immunomodulatory effects of viable and non-viable Lactiplantibacillus plantarum strains in human respiratory epithelial cells (Calu-3 cells) and the capacity of these immunobiotic lactobacilli to reduce their susceptibility to the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immunobiotic L. plantarum MPL16 and CRL1506 differentially modulated IFN-ß, IL-6, CXCL8, CCL5 and CXCL10 production and IFNAR2, DDX58, Mx1 and OAS1 expression in Calu-3 cells stimulated with the TLR3 agonist poly(I:C). Furthermore, the MPL16 and CRL1506 strains increased the resistance of Calu-3 cells to the challenge with SARS-CoV-2. L. plantarum MPL16 induced these beneficial effects more efficiently than the CRL1506 strain. Of note, neither non-viable MPL16 and CRL1506 strains nor the non-immunomodulatory strains L. plantarum CRL1905 and MPL18 could modify the resistance of Calu-3 cells to SARS-CoV-2 infection or the immune response to poly(I:C) challenge. To date, the potential beneficial effects of immunomodulatory probiotics on SARS-CoV-2 infection and COVID-19 outcome have been extrapolated from studies carried out in the context of other viral pathogens. To the best of our knowledge, this is the first demonstration of the ability of immunomodulatory lactobacilli to positively influence the replication of the new coronavirus. Further mechanistic studies and in vivo experiments in animal models of SARS-CoV-2 infection are necessary to identify specific strains of beneficial immunobiotic lactobacilli like L. plantarum MPL16 or CRL1506 for the prevention or treatment of the COVID-19.

Early detection of Varicella-Zoster virus (VZV)-specific T-cells before seroconversion in primary varicella infection: case report.

Here we report the case of a 54-year old, immunocompetent German patient with primary varicella whose Varicella-Zoster Virus (VZV)-specific T-cell responses could be detected early in infection and before the onset of seroconversion. This case demonstrates that the detection of VZV-specific T-cells may under certain circumstances support the diagnosis of a primary varicella infection, as for example in cases of atypical or subclinical varicella or in the absence of detectable VZV DNA in plasma.

Immunomodulatory Properties of Bacterium-Like Particles Obtained From Immunobiotic Lactobacilli: Prospects for Their Use as Mucosal Adjuvants.

Non-viable lactic acid bacteria (LAB) have been proposed as antigen delivery platforms called bacterium-like particles (BLPs). Most studies have been performed with Lactococcus lactis-derived BLPs where multiple antigens were attached to the peptidoglycan surface and used to successfully induce specific immune responses. It is well-established that the immunomodulatory properties of LAB are strain dependent and therefore, the BLPs derived from each individual strain could have different adjuvant capacities. In this work, we obtained BLPs from immunomodulatory (immunobiotics) and non-immunomodulatory Lactobacillus rhamnosus and Lactobacillus plantarum strains and comparatively evaluated their ability to improve the intestinal and systemic immune responses elicited by an attenuated rotavirus vaccine. Results demonstrated that orally administered BLPs from non-immunomodulatory strains did not induce significant changes in the immune response triggered by rotavirus vaccine in mice. On the contrary, BLPs derived from immunobiotic lactobacilli were able to improve the levels of anti-rotavirus intestinal IgA and serum IgG, the numbers of CD24+B220+ B and CD4+ T cells in Peyer's patches and spleen as well as the production of IFN-γ by immune cells. Interestingly, among immunobiotics-derived BLPs, those obtained from L. rhamnosus CRL1505 and L. rhamnosus IBL027 enhanced more efficiently the intestinal and systemic humoral immune responses when compared to BLPs from other immunobiotic bacteria. The findings of this work indicate that it is necessary to perform an appropriate selection of BLPs in order to find those with the most efficient adjuvant properties. We propose the term Immunobiotic-like particles (IBLPs) for the BLPs derived from CRL1505 and IBL027 strains that are an excellent alternative for the development of mucosal vaccines.

Nasal priming with immunobiotic lactobacilli improves the adaptive immune response against influenza virus.

The nasal priming with Lactobacillus rhamnosus CRL1505 modulates the respiratory antiviral innate immune response and improves protection against influenza virus (IFV) challenge in mice. However, the potential beneficial effect of the CRL1505 strain on the adaptive immune response triggered by IFV infection or vaccination was not evaluated before. In this work, we demonstrated that nasally administered L. rhamnosus CRL1505 is able to improve both the humoral and cellular adaptive immune responses induced by IFV infection or vaccination. Higher levels of IFV-specific IgA and IgG as well as IFN-γ were found in the serum and the respiratory tract of CRL1505-treated mice after IFV challenge. Lactobacilli treated mice also showed reduced concentrations of IL-17 and improved levels of IL-10 during IFV infection. The differential balance of inflammatory and regulatory cytokines induced by L. rhamnosus CRL1505 contributed to the protection against IFV by favoring an effective effector immune response without inducing inflammatory-mediated lung damage. The optimal immunomodulatory effect of the CRL1505 strain was achieved with viable bacteria. However, non-viable L. rhamnosus CRL1505 was also efficient in improving the adaptive immune responses generated by IFV challenges and therefore, emerged as an interesting alternative for vaccination of immunocompromised hosts. Similar to other immunomodulatory properties of lactobacilli, it was shown here that the adjuvant effect in the context of IFV vaccination was a strain dependent ability, since differences were found when L. rhamnosus CRL1505 and the immunomodulatory strain L. rhamnosus IBL027 were compared. This investigation represents a thorough exploration of the role of immunobiotic lactobacilli in improving humoral and cellular adaptive immune responses against IFV in the context of both infection and vaccination.

Lipoteichoic Acid Is Involved in the Ability of the Immunobiotic Strain Lactobacillus plantarum CRL1506 to Modulate the Intestinal Antiviral Innate Immunity Triggered by TLR3 Activation.

Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein (dltD) knockout CRL1506 strain (L. plantarumΔdltD) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 (L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-ß, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarumΔdltD or L. plantarum WT showed higher levels of IFN-ß while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-ß and IL-10 and reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarumΔdltD-treated mice showed enhanced levels of IFN-ß after poly(I:C) challenge. However, treatment of mice with L. plantarumΔdltD was not able to increase IL-10 or reduce CD3+NK1.1+CD8αα+ cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation.