Successive Immunization With Epitope-Decreasing Dengue Antigens Induced Conservative Anti-Dengue Immune Responses

© Copyright © 2020 Hou, Ye, Loo, Wong and Chen. Repeated homologous antigen immunization has been hypothesized to hinder antibody diversification, whereas sequential immunization with heterologous immunogens can educate B cell differentiations towards conserved residues thereby facilitating the generation of cross-reactive immunity. In this study, we developed a sequential vaccination strategy that utilized epitope-decreasing antigens to reinforce the cross-reactivity of T and B cell immune responses against all four serotypes dengue virus. The epitope-decreasing immunization was implemented by sequentially inoculating mice with antigens of decreasing domain complexity that first immunized with DENV1 live-attenuated virus, following by the Envelope protein (Env), and then Env domain III (EDIII) subunit protein. When compared to mice immunized with DENV1 live-attenuated virus three times, epitope-decreasing immunization induced higher TNF-α CD8+ T cell immune response against consensus epitopes. Epitope-decreasing immunization also significantly improved neutralizing antibody response to heterologous serotypes. Moreover, this sequential approach promoted somatic hypermutations in the immunoglobulin gene of antigen-specific memory B cells in comparison to repeated immunization. This proof-of-concept work on epitope-decreasing sequential vaccination sheds light on how successively exposing the immune system to decreasing-epitope antigens can better induce cross-reactive antibodies

Sequential immunization induces strong and broad immunity against all four dengue virus serotypes.

A major challenge in dengue vaccine development is the need to induce immunity against four dengue (DENV) serotypes. Dengvaxia®, the only licensed dengue vaccine, consists of four variant dengue antigens, one for each serotype. Three doses of immunization with the tetravalent vaccine induced only suboptimal protection against DENV1 and DENV2. Furthermore, vaccination paradoxically and adversely primes dengue naïve subjects to more severe dengue. Here, we have tested whether sequential immunization induces stronger and broader immunity against four DENV serotypes than tetravalent-formulated immunization. Mice were immunized with four DNA plasmids, each encoding the pre-membrane and envelope from one DENV serotype, either sequentially or simultaneously. The sequential immunization induced significantly higher levels of interferon (IFN)γ- or tumor necrosis factor (TNF)α-expressing CD4+ and CD8+ T cells to both serotype-specific and conserved epitopes than tetravalent immunization. Moreover, sequential immunization induced higher levels of neutralizing antibodies to all four DENV serotypes than tetravalent vaccination. Consistently, sequential immunization resulted in more diversified immunoglobulin repertoire, including increased complementarity determining region 3 (CDR3) length and more robust germinal center reactions. These results show that sequential immunization offers a simple approach to potentially overcome the current challenges encountered with tetravalent-formulated dengue vaccines

Plasmodium falciparum replication factor C subunit 1 is involved in genotoxic stress response

About half the world's population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localisation to the nuclear periphery, indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication, apoptosis and DNA repair pathways leading to cell death. Additionally, mislocalisation of the endogenously tagged protein confirmed its essential role in parasites' replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in P. falciparum.Ministry of Education (MOE)National Medical Research Council (NMRC)Accepted versionNational Medical Research Council, Grant/Award Numbers: CBRG12nov014, OFIRG17may073; Singapore Ministry of Education, Grant/Award Number: NGF‐2017‐03‐03

Dengue Mosaic Vaccines Enhance Cellular Immunity and Expand the Breadth of Neutralizing Antibody Against All Four Serotypes of Dengue Viruses in Mice

An estimated 400 million people in the world are infected with any of the four types of dengue virus (DENV) annually. The only licensed dengue vaccine cannot effectively prevent infection with all of the four DENVs, especially in those immunologically naïve at baseline. In this study, we explored a mosaic vaccine approach, which utilizes an artificial recombinant sequence for each serotype to achieve maximum coverage of variant epitopes in the four DENVs. We determined the immunogenicity and cross-reactivity of DNA plasmids encoding individual mosaic sequences or the natural sequences in mice. We show that the mosaic vaccines, particularly those targeting DENV serotype 1 and 2, improved vaccine immunogenicity by increasing the percentage of antigen-specific IFNγ- or TNFα-secreting CD4 and CD8 T cells, and titers of neutralizing antibodies. The mosaic vaccine diversified and broadened anti-dengue T cell responses and cross-reactive neutralizing antibodies against all four serotypes. The mosaic vaccines also induced higher level of antigen-specific B cell responses. These results suggest that mosaic vaccines comprising of DENV serotype 1 and 2 variant epitopes could stimulate strong and broad immune responses against all four serotypes. Keywords: dengue viruses, mosaic vaccine, T cell responses, neutralizing antibodies, cross-reactivitySingapore-MIT Alliance for Research and Technology (SMART

Engineered Lysins With Customized Lytic Activities Against Enterococci and Staphylococci

The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains – catalytic domain (CD) and cell wall binding domain (CBD) – to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing Enterococcus faecium while targeting Enterococcus faecalis and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the E. faecium cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.Ministry of Education (MOE)National Research Foundation (NRF)Published versionThis research was supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) program, through the Singapore–MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group. We acknowledge funding support from NMRC OFIRG20nov-0079. Contributions of LNL were supported by the National Research Foundation and Ministry of Education Singapore under its Research Center of Excellence Program at SCELSE. A.S. and J.L. acknowledge support from the Singapore‐MIT Alliance (SMA) Graduate Fellowship and MOE Tier 2 Grant MOE2018-T2-2-13. Proteomics work was performed in part in the MIT Center for Environmental Health Sciences Bioanalytical Core, which is supported by Center grant P30‐ES002109 from the National Institute of Environmental Health Sciences with the aid of Dr. Michael Demott

Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture

Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) for label-free purification of cultured RBCs from human hematopoietic stem cells. An advancement in sorting and closed-loop manufacturing of viable human RBCs

Hydrogen Sulfide Sensitizes Acinetobacter baumannii to Killing by Antibiotics

The production of endogenous hydrogen sulfide (H₂S) has been shown to confer antibiotic tolerance in all bacteria studied to date. Therefore, this mediator has been speculated to be a universal defense mechanism against antibiotics in bacteria. This is assuming that all bacteria produce endogenous H₂S. In this study, we established that the pathogenic bacteria Acinetobacter baumannii does not produce endogenous H₂S, giving us the opportunity to test the effect of exogenous H₂S on antibiotic tolerance in a bacterium that does not produce it. By using a H₂S-releasing compound to modulate the sulfide content in A. baumannii, we demonstrated that instead of conferring antibiotic tolerance, exogenous H₂S sensitized A. baumannii to multiple antibiotic classes, and was able to revert acquired resistance to gentamicin. Exogenous H₂S triggered a perturbation of redox and energy homeostasis that translated into hypersensitivity to antibiotic killing. We propose that H₂S could be used as an antibiotic-potentiator and resistance-reversion agent in bacteria that do not produce it

Human natural killer cells control Plasmodium falciparum infection by eliminating infected red blood cells

Immunodeficient mouse–human chimeras provide a powerful approach to study host-specific pathogens, such as Plasmodium falciparum that causes human malaria. Supplementation of immunodeficient mice with human RBCs supports infection by human Plasmodium parasites, but these mice lack the human immune system. By combining human RBC supplementation and humanized mice that are optimized for human immune cell reconstitution, we have developed RBC-supplemented, immune cell-optimized humanized (RICH) mice that support multiple cycles of P. falciparum infection. Depletion of human natural killer (NK) cells, but not macrophages, in RICH mice results in a significant increase in parasitemia. Further studies in vitro show that NK cells preferentially interact with infected RBCs (iRBCs), resulting in the activation of NK cells and the elimination of iRBCs in a contact-dependent manner. We show that the adhesion molecule lymphocyte-associated antigen 1 is required for NK cell interaction with and elimination of iRBCs. Development of RICH mice and validation of P. falciparum infection should facilitate the dissection of human immune responses to malaria parasite infection and the evaluation of therapeutics and vaccines.Singapore-MIT Alliance for Research and Technology (Interdisciplinary Research Group in Infectious Disease Research Program