Actinomycetes from Sediments in the Trondheim Fjord, Norway: Diversity and Biological Activity

The marine environment represents a largely untapped source for isolation of new microorganisms with potential to produce biologically active secondary metabolites. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest, since they are known to produce chemically diverse compounds with a wide range of biological activities. We have set out to isolate and characterize actinomycete bacteria from the sediments in one of the largest Norwegian fjords, the Trondheim fjord, with respect to diversity and antibiotic-producing potential. Approximately 3,200 actinomycete bacteria were isolated using four different agar media from the sediment samples collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first according to the morphology followed by characterization of isolates chosen as group representatives by molecular taxonomy revealed that Micromonospora was the dominating actinomycete genus isolated from the sediments. The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water samples. Nine percent of the isolates clearly required sea water for normal growth, suggesting that these strains represent obligate marine organisms. Extensive screening of the extracts from all collected isolates for antibacterial and antifungal activities revealed strong antibiotic-producing potential among them. The latter implies that actinomycetes from marine sediments in Norwegian fjords can be potential sources for the discovery of novel anti-infective agents

Humanized Ovarian Cancer Patient-Derived Xenografts for Improved Preclinical Evaluation of Immunotherapies

High-grade serous ovarian cancer (HGSOC) has poor prognosis and new treatment modalities are needed. Immunotherapy, with checkpoint inhibitors, have demonstrated limited impact. To evaluate the suitability for immunotherapeutics, contextualized preclinical models are required to secure meaningful clinical translation. Therefore, we developed and characterized humanized patient-derived xenograft (hu PDX) murine models of HGSOC, which were established by orthotopic implantation of tumor cell suspensions and intravenous injection of CD34+ cells isolated from umbilical cord blood samples. The developing human immune system in NSG and NSGS mice was followed longitudinally by flow cytometry and characterized by mass cytometry with a panel of 34 surface markers. Molecular imaging of tumor burden, survival analysis, and characterization of tumor-infiltrating immune cells was performed to assess the treatment response to anti-PD-1 (nivolumab) monotherapy. Successful generation of hu PDX models was achieved. Mice treated with nivolumab showed a decrease in tumor burden, however no significant survival benefit was identified when compared to untreated controls. No correlation was seen between PD-L1 expression and CD8 T cell infiltration and response parameters. As the characterization showed an immune infiltration of predominantly myeloid cells, similar to what is observed in HGSOC patients, the models may have the potential to evaluate the importance of myeloid cell immunomodulation as well.publishedVersio

Biosynthesis of Macrolactam BE-14106 Involves Two Distinct PKS Systems and Amino Acid Processing Enzymes for Generation of the Aminoacyl Starter Unit

SummaryBE-14106 is a macrocyclic lactam with an acyl side chain previously identified in a marine-derived Streptomyces sp. The gene cluster for BE-14106 biosynthesis was cloned from a Streptomyces strain newly isolated from marine sediments collected in the Trondheimsfjord (Norway). Bioinformatics and experimental analyses of the genes in the cluster suggested an unusual mechanism for assembly of the molecule. Biosynthesis of the aminoacyl starter apparently involves the concerted action of a distinct polyketide synthase (PKS) system and several enzymes that activate and process an amino acid. The resulting starter unit is loaded onto a second PKS complex, which completes the synthesis of the macrolactam ring. Gene inactivation experiments, enzyme assays with heterologously expressed proteins, and feeding studies supported the proposed model for the biosynthesis and provided new insights into the assembly of macrolactams with acyl side chain

Live attenuated influenza vaccine in children induces b-cell responses in tonsils

Background. Tonsils play a key role in eliciting immune responses against respiratory pathogens. Little is known about how tonsils contribute to the local immune response after intranasal vaccination. Here, we uniquely report the mucosal humoral responses in tonsils and saliva after intranasal live attenuated influenza vaccine (LAIV) vaccination in children. Methods. Blood, saliva, and tonsils samples were collected from 39 children before and after LAIV vaccination and from 16 agematched, nonvaccinated controls. Serum antibody responses were determined by a hemagglutination inhibition (HI) assay. The salivary immunoglobulin A (IgA) level was measured by an enzyme-linked immunosorbent assay. Antibody-secreting cell (ASC) and memory B-cell (MBC) responses were enumerated in tonsils and blood. Results. Significant increases were observed in levels of serum antibodies and salivary IgA to influenza A(H3N2) and influenza B virus strains as early as 14 days after vaccination but not to influenza A(H1N1). Influenza virus-specific salivary IgA levels correlated with serum HI responses, making this a new possible indicator of vaccine immunogenicity in children. LAIV augmented influenza virus-specific B-cell responses in tonsils and blood. Tonsillar MBC responses correlated with systemic MBC and serological responses. Naive children showed significant increases in MBC counts after LAIV vaccination. Conclusions. This is the first study to demonstrate that LAIV elicits humoral B-cell responses in tonsils of young children. Furthermore, salivary IgA analysis represents an easy method for measuring immunogenicity after vaccination

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80–99 years, n = 89) and younger adults (23–77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1–89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72–0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72–76%) older adults respond after two vaccinations to alpha and delta, but only 58–62% to beta and gamma, compared to 96–97% of younger vaccinees and 68–76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.publishedVersio

SARS-CoV-2 specific immune responses in overweight and obese COVID-19 patients

Obesity is a known risk factor for severe respiratory tract infections. In this prospective study, we assessed the impact of being obese or overweight on longitudinal SARS-CoV-2 humoral and cellular responses up to 18 months after infection. 274 patients provided blood samples at regular time intervals up to 18 months including obese (BMI ≥30, n=32), overweight (BMI 25-29.9, n=103) and normal body weight (BMI 18.5-24.9, n=134) SARS-CoV-2 patients. We determined SARS-CoV-2 spike-specific IgG, IgA, IgM levels by ELISA and neutralising antibody titres by neutralisation assay. RBD- and spike-specific memory B cells were investigated by ELISpot, spike- and non-spike-specific IFN-γ, IL-2 and IFN-γ/IL-2 secreting T cells by FluoroSpot and T cell receptor (TCR) sequencing was performed. Higher BMI correlated with increased COVID-19 severity. Humoral and cellular responses were stronger in overweight and obese patients than normal weight patients and associated with higher spike-specific IgG binding titres relative to neutralising antibody titres. Linear regression models demonstrated that BMI, age and COVID-19 severity correlated independently with higher SARS-CoV-2 immune responses. We found an increased proportion of unique SARS-CoV-2 specific T cell clonotypes after infection in overweight and obese patients. COVID-19 vaccination boosted humoral and cellular responses irrespective of BMI, although stronger immune boosting was observed in normal weight patients. Overall, our results highlight more severe disease and an over-reactivity of the immune system in overweight and obese patients after SARS-CoV-2 infection, underscoring the importance of recognizing overweight/obese individuals as a risk group for prioritisation for COVID-19 vaccination

Evaluation of the Sublingual Route for Administration of Influenza H5N1 Virosomes in Combination with the Bacterial Second Messenger c-di-GMP

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3′,5′)-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines

Actinomycetes from Sediments in the Trondheim Fjord, Norway: Diversity and Biological Activity

The marine environment represents a largely untapped source for isolation of new microorganisms with potential to produce biologically active secondary metabolites. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest, since they are known to produce chemically diverse compounds with a wide range of biological activities. We have set out to isolate and characterize actinomycete bacteria from the sediments in one of the largest Norwegian fjords, the Trondheim fjord, with respect to diversity and antibiotic-producing potential. Approximately 3,200 actinomycete bacteria were isolated using four different agar media from the sediment samples collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first according to the morphology followed by characterization of isolates chosen as group representatives by molecular taxonomy revealed that Micromonospora was the dominating actinomycete genus isolated from the sediments. The deep water sediments contained a higher relative amount of Micromonospora compared to the shallow water samples. Nine percent of the isolates clearly required sea water for normal growth, suggesting that these strains represent obligate marine organisms. Extensive screening of the extracts from all collected isolates for antibacterial and antifungal activities revealed strong antibiotic-producing potential among them. The latter implies that actinomycetes from marine sediments in Norwegian fjords can be potential sources for the discovery of novel anti-infective agents

Longevity of B-cell and T-cell responses after live attenuated influenza vaccination in children

Background. The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV. Methods. Thirty-eight children (3–17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4+ T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry. Results. LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccination. Strain variations were observed, in which the B strain elicited stronger responses. IFN-γ–expressing T cell counts increased significantly, and remained higher than prevaccination levels 1 year later. Expression of T-helper type 1 intracellular cytokines (interleukin 2, IFN-γ, and tumor necrosis factor α) increased after 1 dose and were boosted after the second dose. Hemagglutination inhibition titers were sustained for 1 year. Vaccine-induced memory B cell counts were significantly increased, and the response persisted for one year. Conclusions. LAIV elicited B-cell and T-cell responses that persisted for at least 1 year in children. This is a novel finding that will aid future vaccine policy