The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli.

The lactose operon of Escherichia coli is a paradigm system for quantitative understanding of gene regulation in prokaryotes. Yet, none of the many mathematical models built so far to study the dynamics of this system considered the fact that the Lac repressor regulates its own transcription by forming a transcriptional roadblock at the O3 operator site. Here we study the effect of autoregulation on intracellular LacI levels and also show that cAMP-CRP binding does not affect the efficiency of autoregulation. We built a mathematical model to study the role of LacI autoregulation in the lactose utilization system. Previously, it has been argued that negative autoregulation can significantly reduce noise as well as increase the speed of response. We show that the particular molecular mechanism, a transcriptional roadblock, used to achieve self-repression in the lac system does neither. Instead, LacI autoregulation balances two opposing states, one that allows quicker response to smaller pulses of external lactose, and the other that minimizes production costs in the absence of lactose.

In vivo study of targeted nanomedicine delivery into Langerhans cells by multiphoton laser scanning microscopy.

Epidermal Langerhans cells (LCs) function as professional antigen-presenting cells of the skin. We investigated the LC-targeting properties of a special mannose-moiety-coated pathogen-like synthetic nanomedicine DermaVir (DV), which is capable to express antigens to induce immune responses and kill HIV-infected cells. Our aim was to use multiphoton laser microscopy (MLM) in vivo in order to visualize the uptake of Alexa-labelled DV (AF546-DV) by LCs. Knock-in mice expressing enhanced green fluorescent protein (eGFP) under the control of the langerin gene (CD207) were used to visualize LCs. After 1 h, AF546-DV penetrated the epidermis and entered the eGFP-LCs. The AF546-DV signal was equally distributed inside the LCs. After 9 h, we observed AF546-DV signal accumulation that occurred mainly at the cell body. We demonstrated in live animals that LCs picked up and accumulated the nanoparticles in the cell body.

Specific contacts of the -35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression.

The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of 'extended -10' promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor -35 element. To investigate whether specific recognition of the -35 element would affect the regulation of P1 by GalR, we mutagenized the -35 element of P1, isolated variants of the -35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the -10 and -35 elements concomitantly. Our results suggest that promoters that lack specific -35 element recognition allow decoupling of local chromosome structure from transcription initiation.

Direct and indirect effects in the regulation of overlapping promoters.

Optimal response to environmental stimuli often requires activation of certain genes and repression of others. Dual function regulatory proteins play a key role in the differential regulation of gene expression. While repression can be achieved by any DNA binding protein through steric occlusion of RNA polymerase in the promoter region, activation often requires a surface on the regulatory protein to contact RNAP and thus facilitate transcription initiation. RNAP itself is also a DNA binding protein, therefore it can function as a transcriptional repressor. Searching the Escherichia coli promoter database we found that ∼14% of the identified 'forward' promoters overlap with a promoter oriented in the opposite direction. In this article we combine a mathematical model with experimental analysis of synthetic regulatory regions to investigate interference of overlapping promoters. We find that promoter interference depends on the characteristics of overlapping promoters. The model predicts that promoter strength and interference can be regulated separately, which provides unique opportunities for regulation. Our experimental data suggest that in principle any DNA binding protein can be used for both activation and repression of promoter transcription, depending on the context. These findings can be exploited in the construction of synthetic networks.

DermAll nanomedicine for allergen-specific immunotherapy.

Allergen-specific immunotherapy (ASIT) the only disease-modifying treatment for IgE-mediated allergies is characterized with long treatment duration and high risk of side effects. We investigated the safety, immunogenicity and efficacy of a novel ASIT, called DermAll, in an experimental allergic rhinitis model. We designed and characterized DermAll-OVA, a synthetic plasmid pDNA/PEIm nanomedicine expressing ovalbumin (OVA) as model allergen. DermAll-OVA was administered topically with DermaPrep device to target Langerhans cells. To detect the clinical efficacy of DermAll ASIT we quantified the nasal symptoms and characterized the immunomodulatory activity of DermAll ASIT by measuring cytokine secretion after OVA-stimulation of splenocytes and antibodies from the sera. In allergic mice DermAll ASIT was as safe as Placebo, balanced the allergen-induced pathogenic TH2-polarized immune responses, and decreased the clinical symptoms by 52% [32%, 70%] compared to Placebo. These studies suggest that DermAll ASIT is safe and should significantly improve the immunopathology and symptoms of allergic diseases. FROM THE CLINICAL EDITOR: A novel allergen-specific immunotherapy for IgE-mediated allergies is presented in this paper, using an experimental allergic rhinitis model and a synthetic plasmid pDNA/PEIm nanomedicine expressing ovalbumin as model allergen. Over 50% reduction of symptoms was found as the immune system's balance was favorably altered toward more TH2-polarized immune responses.

T cell immunity ameliorates COVID-19 disease severity and provides post-exposure prophylaxis after peptide-vaccination, in Syrian hamsters.

Background: The emergence of novel SARS-CoV-2 variants that resist neutralizing antibodies drew the attention to cellular immunity and calls for the development of alternative vaccination strategies to combat the pandemic. Here, we have assessed the kinetics of T cell responses and protective efficacy against severe COVID-19 in pre- and post-exposure settings, elicited by PolyPEPI-SCoV-2, a peptide based T cell vaccine. Methods: 75 Syrian hamsters were immunized subcutaneously with PolyPEPI-SCoV-2 on D0 and D14. On D42, hamsters were intranasally challenged with 102 TCID50 of the virus. To analyze immunogenicity by IFN-γ ELISPOT and antibody secretion, lymphoid tissues were collected both before (D0, D14, D28, D42) and after challenge (D44, D46, D49). To measure vaccine efficacy, lung tissue, throat swabs and nasal turbinate samples were assessed for viral load and histopathological changes. Further, body weight was monitored on D0, D28, D42 and every day after challenge. Results: The vaccine induced robust activation of T cells against all SARS-CoV-2 structural proteins that were rapidly boosted after virus challenge compared to control animals (~4-fold, p<0.05). A single dose of PolyPEPI-SCoV-2 administered one day after challenge also resulted in elevated T cell response (p<0.01). The vaccination did not induce virus-specific antibodies and viral load reduction. Still, peptide vaccination significantly reduced body weight loss (p<0.001), relative lung weight (p<0.05) and lung lesions (p<0.05), in both settings. Conclusion: Our study provides first proof of concept data on the contribution of T cell immunity on disease course and provide rationale for the use of T cell-based peptide vaccines against both novel SARS-CoV-2 variants and supports post-exposure prophylaxis as alternative vaccination strategy against COVID-19.

Dynamic features of gene expression control by small regulatory RNAs.

Small regulatory RNAs (sRNAs) in eukaryotes and bacteria play an important role in the regulation of gene expression either by binding to regulatory proteins or directly to target mRNAs. Two of the best-characterized bacterial sRNAs, Spot42 and RyhB, form a complementary pair with the ribosome binding region of their target mRNAs, thereby inhibiting translation or promoting mRNA degradation. To investigate the steady-state and dynamic potential of such sRNAs, we examine the 2 key parameters characterizing sRNA regulation: the capacity to overexpress the sRNA relative to its target mRNA and the speed at which the target mRNA is irreversibly inactivated. We demonstrate different methods to determine these 2 key parameters, for Spot42 and RyhB, which combine biochemical and genetic experiments with computational analysis. We have developed a mathematical model that describes the functional properties of sRNAs with various characteristic parameters. We observed that Spot42 and RyhB function in distinctive parameter regimes, which result in divergent mechanisms.

Safety and Activity of PolyPEPI1018 Combined with Maintenance Therapy in Metastatic Colorectal Cancer: an Open-Label, Multicenter, Phase Ib Study.

PURPOSE: Although chemotherapy is standard of care for metastatic colorectal cancer (mCRC), immunotherapy has no role in microsatellite stable (MSS) mCRC, a "cold" tumor. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine derived from 7 tumor-associated antigens (TAA) frequently expressed in mCRC. This study assessed PolyPEPI1018 combined with first-line maintenance therapy in patients with MSS mCRC. PATIENTS AND METHODS: Eleven patients with MSS mCRC received PolyPEPI1018 and Montanide ISA51VG adjuvant subcutaneously, combined with fluoropyrimidine/biologic following first-line induction with chemotherapy and a biologic (NCT03391232). In Part A of the study, 5 patients received a single dose; in Part B, 6 patients received up to three doses of PolyPEPI1018 every 12 weeks. The primary objective was safety; secondary objectives were preliminary efficacy, immunogenicity at peripheral and tumor level, and immune correlates. RESULTS: PolyPEPI1018 vaccination was safe and well tolerated. No vaccine-related serious adverse event occurred. Eighty percent of patients had CD8+ T-cell responses against ≥3 TAAs. Increased density of tumor-infiltrating lymphocytes were detected post-treatment for 3 of 4 patients' liver biopsies, combined with increased expression of immune-related gene signatures. Three patients had objective response according to RECISTv1.1, and 2 patients qualified for curative surgery. Longer median progression-free survival for patients receiving multiple doses compared with a single dose (12.5 vs. 4.6 months; P = 0.017) suggested a dose-efficacy correlation. The host HLA genotype predicted multi-antigen-specific T-cell responses (P = 0.01) indicative of clinical outcome. CONCLUSIONS: PolyPEPI1018 added to maintenance chemotherapy for patients with unresectable, MSS mCRC was safe and associated with specific immune responses and antitumor activity warranting further confirmation in a randomized, controlled setting.

In Silico Model Estimates the Clinical Trial Outcome of Cancer Vaccines.

Over 30 years after the first cancer vaccine clinical trial (CT), scientists still search the missing link between immunogenicity and clinical responses. A predictor able to estimate the outcome of cancer vaccine CTs would greatly benefit vaccine development. Published results of 94 CTs with 64 therapeutic vaccines were collected. We found that preselection of CT subjects based on a single matching HLA allele does not increase immune response rates (IRR) compared with non-preselected CTs (median 60% vs. 57%, p = 0.4490). A representative in silico model population (MP) comprising HLA-genotyped subjects was used to retrospectively calculate in silico IRRs of CTs based on the percentage of MP-subjects having epitope(s) predicted to bind ≥ 1-4 autologous HLA allele(s). We found that in vitro measured IRRs correlated with the frequency of predicted multiple autologous allele-binding epitopes (AUC 0.63-0.79). Subgroup analysis of multi-antigen targeting vaccine CTs revealed correlation between clinical response rates (CRRs) and predicted multi-epitope IRRs when HLA threshold was ≥ 3 (r = 0.7463, p = 0.0004) but not for single HLA allele-binding epitopes (r = 0.2865, p = 0.2491). Our results suggest that CRR depends on the induction of broad T-cell responses and both IRR and CRR can be predicted when epitopes binding to multiple autologous HLAs are considered.

A Peptide Vaccine Candidate Tailored to Individuals' Genetics Mimics the Multi-Targeted T Cell Immunity of COVID-19 Convalescent Subjects.

Long-term immunity to coronaviruses likely stems from T cell activity. We present here a novel approach for the selection of immunoprevalent SARS-CoV-2-derived T cell epitopes using an in silico cohort of HLA-genotyped individuals with different ethnicities. Nine 30-mer peptides derived from the four major structural proteins of SARS-CoV-2 were selected and included in a peptide vaccine candidate to recapitulate the broad virus-specific T cell responses observed in natural infection. PolyPEPI-SCoV-2-specific, polyfunctional CD8+ and CD4+ T cells were detected in each of the 17 asymptomatic/mild COVID-19 convalescents' blood against on average seven different vaccine peptides. Furthermore, convalescents' complete HLA-genotype predicted their T cell responses to SARS-CoV-2-derived peptides with 84% accuracy. Computational extrapolation of this relationship to a cohort of 16,000 HLA-genotyped individuals with 16 different ethnicities suggest that PolyPEPI-SCoV-2 vaccination will likely elicit multi-antigenic T cell responses in 98% of individuals, independent of ethnicity. PolyPEPI-SCoV-2 administered with Montanide ISA 51 VG generated robust, Th1-biased CD8+, and CD4+ T cell responses against all represented proteins, as well as binding antibodies upon subcutaneous injection into BALB/c and hCD34+ transgenic mice modeling human immune system. These results have implications for the development of global, highly immunogenic, T cell-focused vaccines against various pathogens and diseases.

Structure and function of the D-galactose network in enterobacteria.

Galactose is important for the survival and virulence of bacteria. In Escherichia coli, galactose is utilized by the Leloir pathway, which is controlled by a complex network. To shed light on the potential functions the galactose network could perform, we performed bioinformatical analysis of reference genome sequences belonging to the Enterobacteriaceae family. We found that several genomes have reduced numbers of components compared to the E. coli galactose system, suggesting that the network can be optimized for different environments. Typically, genes are removed by deletions; however, in Yersinia pestis, the galactose mutarotase (galM) gene is inactivated by a single-base-pair deletion. Lack of GalM activity indicates that the two anomers of D-galactose are used for different purposes, α-D-galactose as a carbon source and ß-D-galactose for induction of UDP-galactose synthesis for biosynthetic glycosylation. We demonstrate that activity of the galM gene can be restored by different single-base-pair insertions. During the evolution of Y. pestis to become a vector-transmitted systemic pathogen, many genes were converted to pseudogenes. It is not clear whether pseudogenes are present to maintain meiotrophism or are in the process of elimination. Our results suggest that the galM pseudogene has not been deleted because its reactivation may be beneficial in certain environments.

Identification of cohesive ends and genes encoding the terminase of phage 16-3.

Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3'-protruding complementary G/C-rich sequences. terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems.

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA.

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16-3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16-3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co-evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.

immX immunity region of rhizobium phage 16-3: two overlapping cistrons of repressor function.

16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions. We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V). The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the immX region. Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens. The X(V) part (1 kb) contained a target for X(U/L) repressor action. Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However, X(V1) could also be folded into a transcription terminator. Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.

Binding sites of different geometries for the 16-3 phage repressor.

Prokaryotic repressor-operator systems provide exemplars for the sequence-specific interactions between DNA and protein. The crucial atomic contacts of the two macromolecules are attained in a compact, geometrically defined structure of the DNA-protein complex. The pitch of the DNA interface seems an especially sensitive part of this architecture because changes in its length introduce new spacing and rotational relations in one step. We discovered a natural system that may serve as a model for investigating this problem: the repressor of the 16-3 phage of Rhizobium meliloti (helix-turn-helix class protein) possesses inherent ability to accommodate to various DNA twistings. It binds the cognate operators, which are 5'-ACAA-4 bp-TTGT-3' (O(L)) and 5'-ACAA-6 bp-TTGT-3' (O(R)) and thus differ 2 bp in length, and consequently the two half-sites will be rotated with respect to each other by 72 degrees in the idealized B-DNA (64 degrees by dinucleotide steps calculations). Furthermore, a synthetic intermediate (DNA sequence) 5'-ACAA-5 bp-TTGT-3' (O5) also binds specifically the repressor. The natural operators and bound repressors can form higher order DNA-protein complexes and perform efficient repression, whereas the synthetic operator-repressor complex cannot do either. The natural operators are bent when complexed with the repressor, whereas the O5 operator does not show bending in electrophoretic mobility assay. Possible structures of the complexes are discussed.