Multiantigen pan-sarbecovirus DNA vaccines generate protective T cell immune responses.

SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell-based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2-transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness.

A third vaccination with a single T cell epitope confers protection in a murine model of SARS-CoV-2 infection.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Pyroptosis-inducing active caspase-1 as a genetic adjuvant in anti-cancer DNA vaccination.

Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.

Dominant Antiviral CD8+ T Cell Responses Empower Prophylactic Antibody-Eliciting Vaccines Against Cytomegalovirus.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

A poly-neoantigen DNA vaccine synergizes with PD-1 blockade to induce T cell-mediated tumor control.

The combination of immune-stimulating strategies has the potency to improve immunotherapy of cancer. Vaccination against neoepitopes derived from patient tumor material can generate tumor-specific T cell immunity, which could reinforce the efficacy of checkpoint inhibitor therapies such as anti-PD-1 treatment. DNA vaccination is a versatile platform that allows the inclusion of multiple neoantigen-coding sequences in a single formulation and therefore represents an ideal platform for neoantigen vaccination. We developed an anti-tumor vaccine based on a synthetic DNA vector designed to contain multiple cancer-specific epitopes in tandem. The DNA vector encoded a fusion gene consisting of three neoepitopes derived from the mouse colorectal tumor MC38 and their natural flanking sequences as 40 amino acid stretches. In addition, we incorporated as reporter epitopes the helper and CTL epitope sequences of ovalbumin. The poly-neoantigen DNA vaccine elicited T cell responses to all three neoantigens and induced functional CD8 and CD4 T cell responses to the reporter antigen ovalbumin after intradermal injection in mice. The DNA vaccine was effective in preventing outgrowth of B16 melanoma expressing ovalbumin in a prophylactic setting. Moreover, the combination of therapeutic DNA vaccination and anti-PD-1 treatment was synergistic in controlling MC38 tumor growth whereas individual treatments did not succeed. These data demonstrate the potential of DNA vaccination to target multiple neoepitopes in a single formulation and highlight the cooperation between vaccine-based and checkpoint blockade immunotherapies for the successful eradication of established tumors.

A novel nutritional supplement prevents muscle loss and accelerates muscle mass recovery in caloric-restricted mice.

BACKGROUND: Muscle atrophy is defined as decreased muscle mass, associated with aging as well as with various chronic diseases and is a fundamental cause of frailty, functional decline and disability. Frailty represents a huge potential public health issue worldwide with high impact on healthcare costs. A major clinical issue is therefore to devise new strategies preventing muscle atrophy. In this study, we tested the efficacy of Vital01, a novel oral nutritional supplement (ONS), on body weight and muscle mass using a caloric restriction-induced mouse model for muscle atrophy. METHODS: Mice were calorically restricted for 2 weeks to induce muscle atrophy: one control group received 60% kcal of the normal chow diet and one intervention group received 30% kcal chow and 30 kcal% Vital01. The effects on body weight, lean body mass, muscle histology and transcriptome were assessed. In addition, the effects of Vital01, in mice with established muscle atrophy, were assessed and compared to a standard ONS. To this end, mice were first calorically restricted on a 60% kcal chow diet and then refed with either 100 kcal% chow, a mix of Vital01 (receiving 60% kcal chow and 40 kcal% Vital01) or with a mix of standard, widely prescribed ONS (receiving 60 kcal% chow and 40 kcal% Fortisip Compact). RESULTS: Vital01 attenuated weight loss (-15% weight loss for Vital01 vs. -25% for control group, p < 0.01) and loss of muscle mass (Vital01 with -13%, -12% and -18%, respectively, for gastrocnemius, quadriceps and tibialis vs. 25%, -23% and -28%, respectively, for control group, all p < 0.05) and also restored body weight, fat and muscle mass more efficiently when compared to Fortisip Compact. As assessed by transcriptome analysis and Western blotting of key proteins (e.g. phospoAKT, mTOR and S6K), Vital01 attenuated the catabolic and anabolic signaling pathways induced by caloric restriction and modulated inflammatory and mitochondrial pathways. In addition, Vital01 affected pathways related to matrix proteins/collagens homeostasis and tended to reduce caloric restriction-induced collagen fiber density in the quadriceps (with -27%, p = 0.051). CONCLUSIONS: We demonstrate that Vital01 preserves muscle mass in a calorically restricted mouse model for muscle atrophy. Vital01 had preventive effects when administered during development of muscle atrophy. Furthermore, when administered in a therapeutic setting to mice with established muscle atrophy, Vital01 rapidly restored body weight and accelerated the recurrence of fat and lean body mass more efficiently than Fortisip Compact. Bioinformatics analysis of gene expression data identified regulatory pathways that were specifically influenced by Vital01 in muscle.

MARK-AGE biomarkers of ageing.

Many candidate biomarkers of human ageing have been proposed in the scientific literature but in all cases their variability in cross-sectional studies is considerable, and therefore no single measurement has proven to serve a useful marker to determine, on its own, biological age. A plausible reason for this is the intrinsic multi-causal and multi-system nature of the ageing process. The recently completed MARK-AGE study was a large-scale integrated project supported by the European Commission. The major aim of this project was to conduct a population study comprising about 3200 subjects in order to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any marker in isolation.

Substitutions near the receptor binding site determine major antigenic change during influenza virus evolution.

The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.

Accelerated age-related cognitive decline and neurodegeneration, caused by deficient DNA repair.

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage of Ercc1(Δ/-) mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an age-dependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.

Pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice.

BACKGROUND: Pomegranate seed oil has been shown to protect against diet induced obesity and insulin resistance. OBJECTIVE: To characterize the metabolic effects of punicic acid on high fat diet induced obesity and insulin resistance. DESIGN: High-fat diet or high-fat diet with 1% Pomegranate seed oil (PUA) was fed for 12 weeks to induce obesity and insulin resistance. We assessed body weight and composition (pSABRE DEXA-scan), energy expenditure (Columbus Instruments) and insulin sensitivity at the end of the 12 weeks. RESULTS: PSO intake resulted in a lower body weight, 30.5±2.9 vs 33.8±3.2 g PSO vs HFD respectively, p=0.02, without affecting food intake or energy expenditure. The lower body weight was fully explained by a decreased body fat mass, 3.3±2.3 vs 6.7±2.7 g for PSO and HFD fed mice, respectively, p=0.02. Insulin clamps showed that PSO did not affect liver insulin sensitivity but clearly improved peripheral insulin sensitivity, 164±52% vs 92±24% for PSO and HFD fed mice respectively, p=0.01. CONCLUSIONS: We conclude that dietary PSO ameliorates high-fat diet induced obesity and insulin resistance in mice, independent of changes in food intake or energy expenditure.

CE-MS for metabolic profiling of volume-limited urine samples: application to accelerated aging TTD mice.

Metabolic profiling of biological samples is increasingly used to obtain more insight into the pathophysiology of diseases. For translational studies, biological samples from animal models are explored; however, the volume of these samples can be a limiting factor for metabolic profiling studies. For instance, only a few microliters of urine is often available from small animals like mice. Hence, there is a need for a tailor-made analytical method for metabolic profiling of volume-limited samples. In the present study, the feasibility of capillary electrophoresis time-of-flight mass spectrometry (CE-ToF-MS) for metabolic profiling of urine from mice is evaluated. Special attention is paid to the analytical workflow; that is, such aspects as sample preparation, analysis, and data treatment are discussed from the metabolomics viewpoint. We show that metabolites belonging to several chemical families can be analyzed in mouse urine with the CE-ToF-MS method using minimal sample pretreatment and an in-capillary preconcentration procedure. This exemplifies the advantages of CE-ToF-MS for metabolic profiling of volume-limited samples as loss of material is minimized. The feasibility of the CE-ToF-MS-based workflow for metabolic profiling is illustrated by the analysis of urine samples from wild-type as well as from TTD mutant mice, which are a model for the accelerated aging, with osteoporosis being one of the main hallmarks.

Age-related motor neuron degeneration in DNA repair-deficient Ercc1 mice.

Degeneration of motor neurons contributes to senescence-associated loss of muscle function and underlies human neurodegenerative conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors contributing to motor neuron vulnerability and degenerative phenotypes in vivo are therefore important for our understanding of the neuromuscular system in health and disease. Here, we analyzed neurodegenerative abnormalities in the spinal cord of progeroid Ercc1(Delta/-) mice that are impaired in several DNA repair systems, i.e. nucleotide excision repair, interstrand crosslink repair, and double strand break repair. Ercc1(Delta/-) mice develop age-dependent motor abnormalities, and have a shortened life span of 6-7 months. Pathologically, Ercc1(Delta/-) mice develop widespread astrocytosis and microgliosis, and motor neuron loss and denervation of skeletal muscle fibers. Degenerating motor neurons in many occasions expressed genotoxic-responsive transcription factors p53 or ATF3, and in addition, displayed a range of Golgi apparatus abnormalities. Furthermore, Ercc1(Delta/-) motor neurons developed perikaryal and axonal intermediate filament abnormalities reminiscent of cytoskeletal pathology observed in aging spinal cord. Our findings support the notion that accumulation of DNA damage and genotoxic stress may contribute to neuronal aging and motor neuron vulnerability in human neuromuscular disorders.

Metabolic profiling of accelerated aging ERCC1 d/- mice.

Aging is a fundamental biological process for which the mechanism is still largely unknown due to its complex and multifactorial nature. Animal models allow us to simplify this complexity and to study individual factors separately. As there are many causative links between DNA repair deficiency and aging, we studied the ERCC1(d/-) mouse, which has a modified ERCC1 gene, involved in the Nucleotide Excision Repair, and as a result has a premature aging phenotype. Profiling of these mice on different levels can give an insight into the mechanisms underlying the aging phenotype. In the current study, we have performed metabolic profiling of serum and urine of these mice in comparison to wild type and in relation to aging by (1)H NMR spectroscopy. Analysis of metabolic trajectories of animals from 8 to 20 weeks suggested that wild type and ERCC1(d/-) mutants have similar age-related patterns of changes; however, the difference between genotypes becomes more prominent with age. The main differences between these two genetically diverse groups of mice were found to be associated with altered lipid and energy metabolism, transition to ketosis, and attenuated functions of the liver and kidney.

Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes.

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin.