The Anti-SARS-CoV-2 IgG1 and IgG3 Antibody Isotypes with Limited Neutralizing Capacity against Omicron Elicited in a Latin Population a Switch toward IgG4 after Multiple Doses with the mRNA Pfizer-BioNTech Vaccine.

The aim of this study was to analyze the profiles of IgG subclasses in COVID-19 convalescent Puerto Rican subjects and compare these profiles with those of non-infected immunocompetent or immunocompromised subjects that received two or more doses of an mRNA vaccine. The most notable findings from this study are as follows: (1) Convalescent subjects that were not hospitalized developed high and long-lasting antibody responses. (2) Both IgG1 and IgG3 subclasses were more prevalent in the SARS-CoV-2-infected population, whereas IgG1 was more prevalent after vaccination. (3) Individuals that were infected and then later received two doses of an mRNA vaccine exhibited a more robust neutralizing capacity against Omicron than those that were never infected and received two doses of an mRNA vaccine. (4) A class switch toward the "anti-inflammatory" antibody isotype IgG4 was induced a few weeks after the third dose, which peaked abruptly and remained at high levels for a long period. Moreover, the high levels of IgG4 were concurrent with high neutralizing percentages against various VOCs including Omicron. (5) Subjects with IBD also produced IgG4 antibodies after the third dose, although these antibody levels had a limited effect on the neutralizing capacity. Knowing that the mRNA vaccines do not prevent infections, the Omicron subvariants have been shown to be less pathogenic, and IgG4 levels have been associated with immunotolerance and numerous negative effects, the recommendations for the successive administration of booster vaccinations to people should be revised.

Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli-lipopolysaccharide, whereas it modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

IMPORTANCE: Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cell's activation via bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by TLR4. For this reason, scientists have aimed to develop antagonists able to block TLR4 and, thereby the cytokine storm. We report here that a mixture of mu-class isoforms from the F. hepatica GST protein family administered intraperitoneally 1 h prior to a lethal LPS injection can modulate the dynamics and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice while significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock. These results suggest that native F. hepatica glutathione S-transferase is a promising candidate for drug development against endotoxemia and other inflammatory diseases.

Inflammatory cytokines and a diverse cervicovaginal microbiota associate with cervical dysplasia in a cohort of Hispanics living in Puerto Rico.

Cervical cancer (CC) is women's fourth most common cancer worldwide. A worrying increase in CC rates in Hispanics suggests that besides Human papillomavirus infections, there may be other cofactors included in the epithelial microenvironment that could play a role in promoting the disease. We hypothesized that the cervical microbiome and the epithelial microenvironment favoring inflammation is conducive to disease progression in a group of Hispanics attending gynecology clinics in Puerto Rico. Few studies have focused on the joint microbiota and cytokine profile response in Hispanics outside the US, especially regarding the development of precancerous lesions. We aimed to investigate the relationship between the cervicovaginal microbiome and inflammation in Hispanic women living in PR while considering cervical dysplasia and HPV genotype risk. Cervical samples collected from 91 participants coming to gynecology clinics in San Juan, underwent 16S rRNA genes (V4 region) profiling, and cytokines were measured using Luminex MAGPIX technology. Cytokines were grouped as inflammatory (IL-1ß, TNFα, IFNγ, IL-6), anti-inflammatory (IL- 4, IL-10, TGFß1), and traffic-associated (IL-8, MIP1a, MCP1, IP10). They were related to microbes via an inflammation scoring index based on the quartile and tercile distribution of the cytokine's concentration. We found significant differences in the diversity and composition of the microbiota according to HPV type according to carcinogenic risk, cervical disease, and cytokine abundance. Community State Types (CSTs) represents a profile of microbial communities observed within the vaginal microbiome ecological niche, and Lactobacillus-depleted CST IV had ~ 90% dominance in participants with high-grade squamous intraepithelial lesions and high-risk HPV. The increasing concentration of pro-inflammatory cytokines was associated with a decrease in L. crispatus. In contrast, dysbiosis-associated bacteria such as Gardnerella, Prevotella, Atopobium concomitantly increased with pro-inflammatory cytokines. Our study highlights that the cervical microbiota of Hispanics living in Puerto Rico is composed mostly of diverse CST profiles with decreased Lactobacillus and is associated with a higher pro-inflammatory environment. The joint host-microbe interaction analyses via cytokine and microbiota profiling have very good translational potential.

Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli -lipopolysaccharide whereas modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

The helminth Fasciola hepatica is known as a master of immunomodulation. It suppresses antigen specific Th1 responses in concurrent bacterial infections while promoting the Th2/Treg regulatory responses, thus demonstrating its anti-inflammatory ability in vivo . We have recently demonstrated that a single intraperitoneal injection with native F. hepatica Glutathione S -Transferase (nFhGST), mostly comprised of mu-class isoforms, can suppresses the cytokine storm and increasing the survival rate in a mouse model of septic shock (1). Knowing that the peritoneal macrophages in response to microbial stimuli play essential roles in the defense, tissue repairment, and maintenance of homeostasis, the present study aimed to determine whether nFhGST could modulate the amount and dynamic of these cells concurrently to the suppression of pro-inflammatory cytokines. The remarkable findings described in this article are, (i) nFhGST suppresses serum IL-12, TNF-α, and IFN-γ in BALB/c mice challenged with a lethal dose of LPS, (ii) Although nFhGST does not elicit IL-10, it was able to significantly suppress the high levels of LPS-induced IL-10, which is considered a key cytokine in the pathophysiology of sepsis (2). iii) nFhGST prevent the disappearance of large peritoneal macrophages (LPM) whereas significantly increasing this population in the peritoneal cavity (PerC) of LPS treated animals, (iv) nFhGST promotes the alternative activation of macrophages whereas suppress the classical activation of macrophages in vitro by expressing high levels of Ym-1, a typical M2-type marker, secreting the production of IL-37, and preventing the production of TNF-α, iNOS2 and nitric oxide, which are typical markers of M1-type macrophages, (v) nFhGST suppress the bacterial phagocytosis of macrophages, a role that plays both, M1-and M2-macrophages, thus partially affecting the capacity of macrophages in destroying microbial pathogens. These findings present the first evidence that nFhGST is an excellent modulator of the PerC content in vivo, reinforcing the capacity of nFhGST as an anti-inflammatory drug against sepsis in animal models. Importance: Sepsis is an infection that can lead to a life-threatening complication. Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cells' activation by bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by toll-like receptor 4 (TLR4). For this reason, scientists aimed to develop antagonists able to block the cytokine storm by blocking TLR4. We report here that a mixture of mu-class isoforms from the F. hepatica glutathione S-transferase (nFhGST) protein family administered intraperitoneally 1 h after a lethal LPS injection, is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock whereas modulate the dynamic and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice. These results suggest that nFhGST is a prominent candidate for drug development against endotoxemia and other inflammatory diseases.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Limited Impact of Delta Variant's Mutations on the Effectiveness of Neutralization Conferred by Natural Infection or COVID-19 Vaccines in a Latino Population.

The SARS-CoV-2 pandemic has impacted public health systems all over the world. The Delta variant seems to possess enhanced transmissibility, but no clear evidence suggests it has increased virulence. Our data show that pre-exposed individuals had similar neutralizing activity against the authentic COVID-19 strain and the Delta and Epsilon variants. After only one vaccine dose, the neutralization capacity expanded to all tested variants in pre-exposed individuals. Healthy vaccinated individuals showed a limited breadth of neutralization. One vaccine dose did induce similar neutralizing antibodies against the Delta as against the authentic strain. However, even after two doses, this capacity only expanded to the Epsilon variant.

Limited impact of Delta variant’s mutations in the effectiveness of neutralization conferred by natural infection or COVID-19 vaccines in a Latino population.

The SARS-CoV-2 pandemic has impacted public health systems all over the world. The Delta variant seems to possess enhanced transmissibility, but no clear evidence suggests it has increased virulence. Our data shows that pre-exposed individuals had similar neutralizing activity against the authentic COVID-19 strain and the Delta and Epsilon variants. After one vaccine dose, the neutralization capacity expands to all tested variants. Healthy vaccinated individuals showed a limited breadth of neutralization. One vaccine dose induced similar neutralizing antibodies against the Delta compared to the authentic strain. However, even after two doses, this capacity only expanded to the Epsilon variant.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells.

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1ß, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

Function Is More Reliable than Quantity to Follow Up the Humoral Response to the Receptor-Binding Domain of SARS-CoV-2-Spike Protein after Natural Infection or COVID-19 Vaccination.

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.

Early immunologic response to mRNA COVID-19 vaccine in patients receiving biologics and/or immunomodulators.

Patients with immune conditions and immune-modifying therapies were excluded from the Covid-19 vaccine trials. Studies have shown conflicting response to different vaccines in persons receiving immune suppressors or biologics. The aim of this study is to evaluate humoral and cellular response to Covid-19 vaccines in patients with Inflammatory Bowel Disease (IBD) using biologic and/or immunomodulatory (IMM) therapies. Methods: Participants are adults with IBD receiving biologics or IMM planning to receive a Covid 19 vaccine. Cellular immunity (CD4+ and CD8+ T cell levels) with flow cytometry are measured at baseline and 2 weeks after each vaccine dose. Humoral immunity (antibody titers and neutralizing capacity,VNT%) is analyzed by ELISA at baseline, 2 weeks after each dose, and 6 and 12 months after vaccine. We present the early results of the first 19 subjects. The study is approved by the IRB. Results: 19 subjects (18 in biologics and 1 in IMM) who received 2 doses of the Pfizer-BioNTech vaccine are included. Total IgG antibodies increased 21.13 times after the first dose and 90 times after the second dose. VTN% increased 11.92 times after the first dose and 53.79 times after the second dose. When compared with a healthy control cohort, total IgG antibodies and VTN% were lower in the subjects after the first dose. After the second dose, IgG antibodies increased but remained lower than controls, but VTN% were similar to controls. CD4 and CD8 mean levels had an upward trend after vaccination. Conclusions: Neutralizing capacity response to the vaccine in subjects was similar to a healthy cohort in spite of lower increases in total IgG antibodies. The CD4 and CD8 results observed may support the capacity to mount an effective cellular response in patients on biologics. Larger studies are needed to determine vaccine efficacy in these patients.

Personalized health and the coronavirus vaccines-Do individual genetics matter?

Vaccines represent preventative interventions amenable to immunogenetic prediction of how human variability will influence their safety and efficacy. The genetic polymorphism among individuals within any population can render possible that the immunity elicited by a vaccine is variable in length and strength. The same immune challenge (virus and/or vaccine) could provoke partial, complete or even failed protection for some individuals treated under the same conditions. We review genetic variants and mechanistic relationships among chemokines, chemokine receptors, interleukins, interferons, interferon receptors, toll-like receptors, histocompatibility antigens, various immunoglobulins and major histocompatibility complex antigens. These are the targets for variation among macrophages, dendritic cells, natural killer cells, T- and B-lymphocytes, and complement. The technology platforms (mRNA, viral vectors, proteins) utilized to produce vaccines against SARS-CoV-2 infections may each trigger genetically distinct immune reactogenic profiles. With DNA biobanking and immunoprofiling of recipients, global COVID-19 vaccinations could launch a new era of personalized healthcare.

Function is more reliable than quantity to follow up the humoral response to the Receptor Binding Domain of SARS- CoV-2 Spike protein after natural infection or COVID-19 vaccination.

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. We also report that serum neutralization capacity correlates with IgG titers, wherein IgG1 was the predominant isotype (62.71%), followed by IgG4 (15.25%), IgG3 (13.56%), and IgG2 (8.47%) at the earliest tested timepoint. IgA titers were detectable in just 28.81% of subjects, and only 62.71% of subjects had detectable IgM in the first sample despite confirmation of infection by a molecular diagnostic assay. Our data suggests that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from 10 out of the 59 subjects which had received an initial first dose of mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for induction of neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern seen after natural infection, after the second vaccine dose, the total anti-S antibodies titers declined, however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. The decline in anti-S antibody titer, however, was significantly less in pre-exposed individuals, highlighting the potential for natural infection to prime a more robust immune response to the vaccine. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. However, this difference was significant only when neutralizing antibody titers were compared among the two groups. No differences were observed between naturally infected and vaccinated individuals when total anti-S antibodies and IgG titers were measured. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that a functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies. In this context, our results also support standardizing methods of assessing the humoral response to SARS-CoV-2 when determining vaccine efficacy and describing the immune correlates of protection for SARS-CoV-2.

Fasciola hepatica GST downregulates NF-κB pathway effectors and inflammatory cytokines while promoting survival in a mouse septic shock model.

Parasitic helminths and helminth-derived molecules have demonstrated to possess powerful anti-inflammatory properties and confirmed therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress specific Th1 specific immune responses induced by concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. In this study, we demonstrate that native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, majorly comprised of Mu-class GST isoforms, significantly suppresses the LPS-induced TNFα and IL1ß of mouse bone-marrow derived macrophages in vitro and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice exposed to lethal doses of LPS increasing their survival rate by more than 85%. Using THP1-Blue CD14 cells, a human monocyte cell line, we also demonstrate that nFhGST suppresses NF-κB activation in response to multiple TLR-ligands, including whole bacteria clinical isolates and this suppression was found to be dose-dependent and independent of the timing of exposure. Moreover, the suppressive effect of nFhGST on NF-κB activation was shown to be independent of enzyme activity or secondary structure of protein. As part of its anti-inflammatory effect nFhGST target multiple proteins of the canonic and non-canonic NF-κB signaling pathway as well as also JAK/STAT pathway. Overall, our results demonstrate the potent anti-inflammatory properties of nFhGST and its therapeutic potential as an anti-inflammatory agent.

Outlining key inflammation-associated parameters during early phase of an experimental gram-negative sepsis model in rhesus macaques (Macaca mulatta).

The aim of this study was to identify inflammation-associated markers during the early phase of sepsis in rhesus macaque. Four rhesus macaques were given an intravenous dose of 1010 CFU/kg of E. coli. Blood samples were collected before, or 30 minutes, 2, 4, 6 and 8 hours after E. coli infusion. Physiological parameters, bacteremia, endotoxemia, C-reactive protein (CRP), procalcitonin (PCT), and plasma cytokines/chemokines were determined for each animal. Bacteremia was present in all animals from 30 minutes to 3 hours after E. coli infusion whereas endotoxin was detected during the full-time course. CRP and PCT levels remained at detectable levels during the whole experimental window suggesting an ongoing inflammatory process. Signature cytokines and chemokines such as TNF-α, MIP-1α, and MIP-1ß peaked about 2 hours after E. coli infusion and decreased thereafter. Plasma IL-6, IL-12p40, IFN-γ, and IL-1Ra, as well as I-TAC, MIG, IP-10 and MCP-1, remained at detectable levels after 4 hours of E. coli infusion. This nonhuman primate model could be useful for the assessment of new therapeutics aiming to suppress key inflammatory markers throughout sepsis early phases.

Fh15 Blocks the Lipopolysaccharide-Induced Cytokine Storm While Modulating Peritoneal Macrophage Migration and CD38 Expression within Spleen Macrophages in a Mouse Model of Septic Shock.

Sepsis caused by Gram-negative bacteria is the consequence of an unrestrained infection that continuously releases lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to multiorgan failure and death. After scrutinizing the immune modulation exerted by a recombinant Fasciola hepatica fatty acid binding protein termed Fh15, our group demonstrated that addition of Fh15 to murine macrophages 1 h prior to LPS stimulation significantly suppresses the expression of proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL1-ß). The present study aimed to demonstrate that Fh15 could exert a similar anti-inflammatory effect in vivo using a mouse model of septic shock. Among the novel findings reported in this article, (i) Fh15 suppressed numerous serum proinflammatory cytokines/chemokines when injected intraperitoneally 1 h after exposure of animals to lethal doses of LPS, (ii) concurrently, Fh15 increased the population of large peritoneal macrophages (LPMs) in the peritoneal cavity (PerC) of LPS-injected animals, and (iii) Fh15 downregulated the expression on spleen macrophages of CD38, a cell surface ectoenzyme with a critical role during inflammation. These findings present the first evidence that the recombinant parasitic antigen Fh15 is an excellent modulator of the PerC cell content and in vivo macrophage activation, endorsing Fh15's potential as a drug candidate against sepsis-related inflammatory response.IMPORTANCE Sepsis is a potentially life-threatening complication of an infection. Sepsis is mostly the consequence of systemic bacterial infections leading to exacerbated activation of immune cells by bacterial products, resulting in enhanced release of inflammatory mediators. Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a critical factor in the pathogenesis of sepsis, which is sensed by Toll-like receptor 4 (TLR4). The scientific community highly pursues the development of antagonists capable of blocking the cytokine storm by blocking TLR4. We report here that a recombinant molecule of 14.5 kDa belonging to the Fasciola hepatica fatty acid binding protein (Fh15) is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock when administered by the intraperitoneal route 1 h after a lethal LPS injection. These results suggest that Fh15 is an excellent candidate for drug development against endotoxemia.

Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Assessment of Fasciola hepatica glutathione S-transferase as an antigen for serodiagnosis of human chronic fascioliasis.

Due to the unsatisfactory performance of parasitological diagnosis of human fascioliasis; the use of immunodiagnosis based on the detection of anti-Fasciola antibodies is traditionally used as a diagnostic alternative using total or purified parasite excretory-secretory products (ESPs). Glutathione S-transferase (GST) protein, one of the F. hepatica ESP components, possesses well-known roles in the detoxification of xenobiotic and endogenously derived toxins within the host bile environment. GST has shown to be a good target for vaccine or drug development against fascioliasis. The current study aimed to evaluate the potential of GST protein purified from a soluble crude extract of adult flukes as an antigen for serodiagnosis of chronic human fascioliasis by indirect ELISA. The study included a panel of 116 serum samples collected from individuals with confirmed fascioliasis, individuals carrying heterologous parasitic infections and healthy subjects. The parasitological examination was used as gold standard and a previously optimized ESP-ELISA was used to compare the performance of the GST-ELISA method. Results demonstrated that GST-ELISA is 94.3% sensitive, 80.2% specific and exhibits a moderate positive correlation (r = 0.555) and substantial agreement (k = 0.786) with the results obtained with the ESP-ELISA method. Moreover, because no sera from patients with early F. hepatica infection were available, GST-ELISA was then tested with sera from rabbits experimentally infected with F. hepatica metacercariae. The assay was able to detect anti-Fasciola antibodies as early as the 3rd week of infection (p < 0.0001) with peaks at 4th and 10th week post-infection.

Fasciola hepatica ESPs Could Indistinctly Activate or Block Multiple Toll-Like Receptors in a Human Monocyte Cell Line.

Fasciola hepatica is a parasitic helminth that induces Th2/Treg responses in its mammalian host. Some reports have suggested that ESPs achieve these polarized immune responses by delaying the activation of dendritic cells and macrophages during the early stages of innate immunity, a process that is mediated by TLR4. The present study aimed to investigate whether TLRs other than TLR4 could also be targeted by F. hepatica ESPs. To achieve this aim a screening system was optimized using THP1-Blue CD14 cells. ESPs were first separated based on their molecular weight and according their net charge by ion exchange chromatography (IEC). Results demonstrated that F. hepatica ESPs mainly cathepsin, serpin and endopin are capable of activating TLR2, TLR4, TLR8 and likely also TLR5 and TLR6. In contrast, fatty acid binding protein strongly suppressed the stimulation induced by various TLR-ligands. Further studies are needed to understand how these apparent contradictory effects of molecules of the same protein mix complement each other in the context of an active infection resulting in the polarized Th2-immune response that characterize F. hepatica infections.

Recombinant Fasciola hepatica fatty acid binding protein suppresses toll-like receptor stimulation in response to multiple bacterial ligands.

Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1ß and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.