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Cancer is a leading cause of death worldwide. The search for innovative therapeutic approaches is a principal focus of medical research. Vaccine strategies targeting a number of tumor-associated antigens are currently being evaluated. To date, none have garnered significant success. Purportedly, an immunosuppressive tumor microenvironment and the accumulation of regulatory T cells contribute to a lack of tumor vaccine efficacy. Aspartyl/asparaginyl β-hydroxylase (ASPH), a promising therapeutic target, is overexpressed in a variety of malignant tumors but is expressed negligibly in normal tissues. Computer analysis predicted that ASPH expresses four peptide sequences (epitopes) capable of stimulating regulatory T cell activity. The abolition of these putative regulatory T cell epitopes increased the CD4 + and CD8 + effector T cell responses to monocyte-derived dendritic cells pulsed with a modified, epitope-depleted version of ASPH in an ex vivo human lymphoid tissue-equivalent coculture system while simultaneously decreasing the overall number of FoxP3 + regulatory T cells. These findings suggest that the efficacy of all new vaccine candidates would profit from screening and eliminating potential tolerogenic regulatory T cell epitopes.

Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency in functional phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine (Phe) in patients' blood and organs. Affected patients encounter severe developmental delay, neurological deficits, and behavioral abnormalities when not treated. Early diagnosis and treatment are extremely important; newborn screening programs have been implemented in most countries to ensure early identification of patients with PKU. Despite available treatment options, several challenges remain: life-long adherence to a strict diet, approval of some medications for adults only, and lack of response to these therapies in a subpopulation of patients. Therefore, there is an urgent need for treatment alternatives. An mRNA-based approach tested in PKU mice showed a fast reduction in the accumulation of Phe in serum, liver and brain, the most significant organ affected. Repeated injections of LNP-formulated mouse PAH mRNA rescued PKU mice from the disease phenotype for a prolonged period of time. An mRNA-based approach could improve the quality of life tremendously in PKU patients of all ages by replacing standard-of-care treatments., Competing Interests: CW-T and FC-T are salaried employees of CureVac AG, Tübingen, Germany. NH was salaried employee of CureVac AG, Tübingen, Germany at the beginning of the project; and is salaried employee of Atriva Therapeutics GmbH, Tübingen, Germany. The authors declare that this study received funding from CureVac AG. The funder had the following involvement in the study: study design, collection, analysis, interpretation of data, the writing of this article, and the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cacicedo, Weinl-Tenbruck, Frank, Limeres, Wirsching, Hilbert, Pasha Famian, Horscroft, Hennermann, Zepp, Chevessier-Tünnesen and Gehring.)

Childhood mortality represents a major issue with 5. 3 million worldwide deaths of children under 5 years of age in 2019. Approximately half of those deaths can be attributed to easily preventable, infectious diseases. Currently approved neonatal vaccines are typically effective only after multiple doses leaving infants especially vulnerable during the first 6 months of life. Survival rates could be improved significantly by developing new and more potent vaccines that are capable of overcoming inherently tolerogenic neonatal immune systems. TLR agonists have garnered a great deal of attention in recent years due to their extensive capacities to activate innate immunity. Herein, the superior capacity of the TLR7/8 agonist, resiquimod (R848), to activate adult and neonatal primary peripheral blood dendritic cells is demonstrated. Moreover, R848 can be conjugated to polyethylene glycol and encapsulated in ovalbumin nanocapsules to efficiently co-deliver antigen and adjuvant in vitro . This study is among the first to demonstrate the capacity of encapsulated R848 to activate neonatal dendritic cells. These findings support the potential incorporation of R848 as adjuvant in neonatal vaccines, making them more effective in eliciting a robust immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wirsching, Machtakova, Borgans, Pretsch, Fichter, Cacicedo, Thérien-Aubin, Landfester and Gehring.)

Hereditary tyrosinemia type 1 is an inborn error of amino acid metabolism characterized by deficiency of fumarylacetoacetate hydrolase (FAH). Only limited treatment options (e.g., oral nitisinone) are available. Patients must adhere to a strict diet and face a life-long risk of complications, including liver cancer and progressive neurocognitive decline. There is a tremendous need for innovative therapies that standardize metabolite levels and promise normal development. Here, we describe an mRNA-based therapeutic approach that rescues Fah -deficient mice, a well-established tyrosinemia model. Repeated intravenous or intramuscular administration of lipid nanoparticle-formulated human FAH mRNA resulted in FAH protein synthesis in deficient mouse livers, stabilized body weight, normalized pathologic increases in metabolites after nitisinone withdrawal, and prevented early death. Dose reduction and extended injection intervals proved therapeutically effective. These results provide proof of concept for an mRNA-based therapeutic approach to treating hereditary tyrosinemia type 1 that is superior to the standard of care., Competing Interests: C.W. and F.C. are salaried employees of CureVac AG, Tübingen, Germany. N.H. was salaried employee of CureVac AG, Tübingen, Germany at the beginning of the project; and is salaried employee of MRM Health, Zwijnaarde, Belgium. M.L.C., D.F., S.W., S.G., B.K.S., J.H., J.G.O., J.B.H., and F.Z. declare no competing interests., (© 2022 The Author(s).)

The first cases of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were reported by Chinese authorities at the end of 2019. The disease spread quickly and was declared a global pandemic shortly thereafter. To respond effectively to infection and prevent viral spread, it is important to delineate the factors that affect protective immunity. Herein, a cohort of convalescent healthcare workers was recruited and their immune responses were studied over a period of 3 to 9 months following the onset of symptoms. A cross-reactive T cell response to SARS-CoV-2 and endemic coronaviruses, i.e., OC43 and NL63, was demonstrated in the infected, convalescent cohort, as well as a cohort composed of unexposed individuals. The convalescent cohort, however, displayed an increased number of SARS-CoV-2-specific CD4 + T cells relative to the unexposed group. Moreover, unlike humoral immunity and quickly decreasing antibody titers, T cell immunity in convalescent individuals was maintained and stable throughout the study period. This study also suggests that, based on the higher CD4 T cell memory response against nucleocapsid antigen, future vaccine designs may include nucleocapsid as an additional antigen along with the spike protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wirsching, Harder, Heymanns, Gröndahl, Hilbert, Kowalzik, Meyer and Gehring.)

Nanocapsules are an excellent platform for the delivery of macromolecular payloads such as proteins, nucleic acids or polyprodrugs, since they can both protect the sensitive cargo and target its delivery to the desired site of action. However, the release of macromolecules from nanocapsules remains a challenge due to their restricted diffusion through the nanoshell compared to small molecule cargo. Here, we designed degradable protein nanocapsules with varying crosslinking densities of the nanoshell to control the release of model macromolecules. While the crosslinking did not influence the degradability of the capsules by natural proteases, it significantly affected the release profiles. Furthermore, the optimized protein nanocapsules were successfully used to deliver and effectively release a bioactive macromolecular vaccine adjuvant in vitro and, thus, can be used as an efficient platform for the design of potential nanovaccines.

Water-soluble low molecular weight drugs, such as the synthetic glucocorticoid dexamethasone (DXM), can easily leak out of nanocarriers after encapsulation due to their hydrophilic nature and small size. This can lead to a reduced therapeutic efficacy and therefore to unwanted adverse effects on healthy tissue. Targeting DXM to inflammatory cells of the liver like Kupffer cells or macrophages is a promising approach to minimize typical side effects. Therefore, a controlled transport to the cells of interest and selective on-site release is crucial. Aim of this study was the development of a DXM-phosphate-based polyprodrug and the encapsulation in silica nanocontainers (SiO 2 NCs) for the reduction of inflammatory responses in liver cells. DXM was copolymerized with a linker molecule introducing pH-cleavable hydrazone bonds in the backbone and obtaining polyprodrugs (PDXM). Encapsulation of PDXMs into SiO 2 NCs provided a stable confinement avoiding uncontrolled leakage. PDXMs were degraded under acidic conditions and subsequently released out of SiO 2 NCs. Biological studies showed significantly enhanced anti-inflammatory capacity of the polyprodrug nanoformulations over non-encapsulated DXM or soluble polyprodrugs. These results demonstrate the advantage of combining the polyprodrug strategy with nanocarrier-mediated delivery for enhanced control of the delivery of water-soluble low molecular weight drugs.

Immunosuppression with glucocorticoids is a common treatment for autoimmune liver diseases and after liver transplant, which is however associated with severe side-effects. Targeted delivery of glucocorticoids to inflammatory cells, e.g. liver macrophages and Kupffer cells, is a promising approach for minimizing side effects. Herein, we prepare core-shell silica nanocapsules (SiO 2 NCs) via a sol-gel process confined in nanodroplets for targeted delivery of dexamethasone (DXM) for liver immunosuppressive therapy. DXM with concentrations up to 100 mg mL -1 in olive oil are encapsulated while encapsulation efficiency remains over 95% after 15 days. Internalization of NCs by non-parenchymal murine liver cells significantly reduces the release of inflammatory cytokines, indicating an effective suppression of inflammatory response of liver macrophages. Fluorescent and magnetic labeling of the NCs allows for monitoring their intracellular trafficking and biodegradation. Controlled interaction with blood proteins and good colloidal stability in blood plasma are achieved via PEGylation of the NCs. Specific proteins responsible for stealth effect, such as apolipoprotein A-I, apolipoprotein A-IV, and clusterin, are present in large amounts on the PEGylated NCs. In vivo biodistribution investigations prove an efficient accumulation of NCs in the liver, underlining the suitability of the SiO 2 NCs as a dexamethasone carrier for treating inflammatory liver diseases.

Natural killer (NK) cells are innate lymphocytes with a strong antitumor ability. In tumor patients, such as multiple myeloma (MM) patients, an elevated number of NK cells after stem cell transplantation (SCT) has been reported to be correlated with a higher overall survival rate. With the aim of improving NK cell use for adoptive cell therapy, we also addressed the cytotoxicity of patient-derived, cytokine-stimulated NK cells against MM cells at specific time points: at diagnosis and before and after autologous stem cell transplantation. Remarkably, after cytokine stimulation, the patients' NK cells did not significantly differ from those of healthy donors. In a small cohort of MM patients, we were able to isolate autologous tumor cells, and we could demonstrate that IL-2/15 stimulated autologous NK cells were able to significantly improve their killing capacity of autologous tumor cells. With the aim to further improve the NK cell killing capacity against MM cells, we investigated the potential use of NK specific check point inhibitors with focus on NKG2A because this inhibitory NK cell receptor was upregulated following ex vivo cytokine stimulation and MM cells showed HLA-E expression that could even be increased by exposure to IFN-γ. Importantly, blocking of NKG2A resulted in a significant increase in the NK cell-mediated lysis of different MM target cells. Finally, these results let suggest that combining cytokine induced NK cell activation and the specific check point inhibition of the NKG2A-mediated pathways can be an effective strategy to optimize NK cell therapeutic approaches for treatment of multiple myeloma.

Researchers are working on key issues in regenerative medicine in many of the over 280 academic institutions in Germany. Moreover, nonuniversity research institutions such as Helmholtz-, Max-Planck-, Fraunhofer- and Leibniz-Centers provide important contributions to the field of regenerative medicine in Germany. Research on stem cells is conducted in the area of pluripotent stem cells (in particular basic research on induced pluripotent stem [iPS] cells, but also embryonic stem cells) and adult (tissue-specific) stem and progenitor cells, as well as cancer stem cells (both on basic science and translational levels).

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.

The yeast Candida albicans is a harmless member of the normal microflora on the mucosal surfaces of most healthy persons, but it can cause severe opportunistic infections in immunosuppressed patients. To become a successful human commensal and pathogen, C. albicans has evolved host adaptation mechanisms on different levels. The regulated expression of virulence and other genes in response to environmental signals allows an optimal adaptation to new host niches during the course of an infection. In addition, C. albicans is able to switch between different cell types in a reversible and apparently random fashion. Phenotypic switching involves the coordinated regulation of phase-specific genes, and the resulting generation of selected, pre-programmed cell types may represent an additional strategy to adapt to certain host environments. Finally, C. albicans produces genetically altered variants at a high rate. This microevolution ensures survival when the pathogen encounters new adverse conditions, as exemplified by the development of stable drug-resistant variants under the selection pressure caused by antimycotic therapy. Thus, rather than the possession of single dominant virulence factors, it is its remarkable versatility that makes C. albicans the most important fungal pathogen of humans.

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.

Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA(R) developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.