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.

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.

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.

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.

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.

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.

The Ability of Respiratory Commensal Bacteria to Beneficially Modulate the Lung Innate Immune Response Is a Strain Dependent Characteristic.

We investigated whether the ability of commensal respiratory bacteria to modulate the innate immune response against bacterial and viral pathogens was a shared or strain-specific characteristic. Bacterial strains belonging to the Corynebacterium pseudodiphtheriticum and Dolosigranulum pigrum species were compared by studying their influence in the Toll-like receptor (TLR)-2- and TLR3-triggered immune responses in the respiratory tract, as well as in the resistance to Respiratory Syncytial Virus (RSV) and Streptococcus pneumoniae infections. We demonstrated that nasally administered C. pseudodiphteriticum 090104 or D. pigrum 040417 were able to modulate respiratory immunity and increase the resistance against pathogens, while other strains of the same species did not influence the respiratory immune responses, demonstrating a clear strain-dependent immunomodulatory effect of respiratory commensal bacteria. We also reported here that bacterium-like particles (BLP) and cell walls derived from immunomodulatory respiratory commensal bacteria are an interesting alternative for the modulation of the respiratory immune system. Our study is a step forward in the positioning of certain strains of respiratory commensal bacteria as next-generation probiotics for the respiratory tract.

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.

A Novel In-House Enzyme-Linked Immunosorbent Assay for Genotype 3 Hepatitis E Virus Reveals High Seroprevalence in Blood Donors in Northern Argentina.

The Hepatitis E virus (HEV) is an emergent virus that causes acute hepatitis in immunocompetent hosts and chronic hepatitis in immunocompromised hosts. In Latin America, the main circulating genotype HEV-3 is usually of zoonotic origin. Diagnosis and seroprevalence studies mainly rely on the detection of specific antibodies. There are scarce data on the seroprevalence of HEV infection in Latin America mainly due to the lack of awareness of HEV circulation. Furthermore, in some countries, like Argentina, HEV testing is not included in routine assays. In order to provide tools to deepen the knowledge on HEV epidemiology in South America, we designed a new in-house ELISA based on the native recombinant protein ORF2 aa112-608 and demonstrated its potential for detecting anti-HEV immunoglobulin G (IgG) in human serum samples. The following conditions were determined: an optimal antigen concentration of 0.25 µg/ml, a serum dilution of 1:80, gelatin as a blocking agent, and a secondary antibody dilution of 1:2000. A relative sensitivity of 93.33% (95% CI: 77.9-99.2%) and a relative specificity of 99.4% (95% CI: 96.7-100%) were determined using a panel of previously characterized sera and a gold standard (HEV IgG ELISA, DIA.PRO, Italy). Further, we obtained a very good agreement (κ index = 0.94, 95% CI: 0.87-1.00) with the gold standard. We screened 813 blood donor samples with this newly developed ELISA and found a seroprevalence of 9.23% (95% confidence interval, 7.33-11.43%). We show for the first time evidence of past HEV infection in Tucuman, the most populated city in northern Argentina. We expect that this study will raise the interest of health decision makers who should intercede to include indirect testing of HEV in regular diagnostic protocols. In conclusion, the in-house ELISA developed in this work shows a very good agreement with an already licensed commercial HEV IgG ELISA (DIA.PRO, ITALY), which can be used as an epidemiologic tool for HEV surveillance.

Draft Genome Sequence of Immunobiotic Lactobacillus rhamnosus Strain IBL027, a Potential Adjuvant for Mucosal Vaccine Development.

The genome sequence of the immunomodulatory strain Lactobacillus rhamnosus strain IBL027 is described here. The reads were assembled into contigs with a total size 2,898,501 bp. The genome information will be useful for further specific genetic studies of this strain to evaluate its immunomodulatory and biotechnological properties as a vaccine adjuvant.

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.