Boosting compromised SARS-CoV-2-specific immunity with mRNA vaccination in liver transplant recipients.

BACKGROUND & AIMS: Liver transplant recipients (LTRs) demonstrate a reduced response to COVID-19 mRNA vaccination; however, a detailed understanding of the interplay between humoral and cellular immunity, especially after a third (and fourth) vaccine dose, is lacking. METHODS: We longitudinally compared the humoral, as well as CD4+ and CD8+ T-cell, responses between LTRs (n = 24) and healthy controls (n = 19) after three (LTRs: n = 9 to 16; healthy controls: n = 9 to 14 per experiment) to four (LTRs: n = 4; healthy controls: n = 4) vaccine doses, including in-depth phenotypical and functional characterization. RESULTS: Compared to healthy controls, development of high antibody titers required a third vaccine dose in most LTRs, while spike-specific CD8+ T cells with robust recall capacity plateaued after the second vaccine dose, albeit with a reduced frequency and epitope repertoire compared to healthy controls. This overall attenuated vaccine response was linked to a reduced frequency of spike-reactive follicular T helper cells in LTRs. CONCLUSION: Three doses of a COVID-19 mRNA vaccine induce an overall robust humoral and cellular memory response in most LTRs. Decisions regarding additional booster doses may thus be based on individual vaccine responses as well as evolution of novel variants of concern. IMPACT AND IMPLICATIONS: Due to immunosuppressive medication, liver transplant recipients (LTR) display reduced antibody titers upon COVID-19 mRNA vaccination, but the impact on long-term immune memory is not clear. Herein, we demonstrate that after three vaccine doses, the majority of LTRs not only exhibit substantial antibody titers, but also a robust memory T-cell response. Additional booster vaccine doses may be of special benefit for a small subset of LTRs with inferior vaccine response and may provide superior protection against evolving novel viral variants. These findings will help physicians to guide LTRs regarding the benefit of booster vaccinations.

COVID-19 mRNA booster vaccine induces transient CD8+ T effector cell responses while conserving the memory pool for subsequent reactivation.

Immunization with two mRNA vaccine doses elicits robust spike-specific CD8+ T cell responses, but reports of waning immunity after COVID-19 vaccination prompt the introduction of booster vaccination campaigns. However, the effect of mRNA booster vaccination on the spike-specific CD8+ T cell response remains unclear. Here we show that spike-specific CD8+ T cells are activated and expanded in all analyzed individuals receiving the 3rd and 4th mRNA vaccine shots. This CD8+ T cell boost response is followed by a contraction phase and lasts only for about 30-60 days. The spike-specific CD8+ T memory stem cell pool is not affected by the 3rd vaccination. Both 4th vaccination and breakthrough infections with Delta and Omicron rapidly reactivate CD8+ T memory cells. In contrast, neutralizing antibody responses display little boost effect towards Omicron. Thus, COVID-19 mRNA booster vaccination elicits a transient T effector cell response while long-term spike-specific CD8+ T cell immunity is conserved to mount robust memory recall targeting emerging variants of concern.

SARS-CoV-2-specific T-cell epitope repertoire in convalescent and mRNA-vaccinated individuals.

Continuously emerging variants of concern (VOCs) sustain the SARS-CoV-2 pandemic. The SARS-CoV-2 Omicron/B.1.1.529 VOC harbours multiple mutations in the spike protein associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. By performing in-depth comparisons of the SARS-CoV-2-specific T-cell epitope repertoire after infection and messenger RNA vaccination, we demonstrate that spike-derived epitopes were not dominantly targeted in convalescent individuals compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T-cell response compared to convalescent individuals. Booster vaccination increased the breadth of the spike-specific T-cell response in convalescent individuals but not in vaccinees with complete initial vaccination. In convalescent individuals and vaccinees, the targeted T-cell epitopes were broadly conserved between wild-type SARS-CoV-2 variant B and Omicron/B.1.1.529. Hence, our data emphasize the relevance of vaccine-induced spike-specific CD8+ T-cell responses in combating VOCs including Omicron/B.1.1.529 and support the benefit of boosting convalescent individuals with mRNA vaccines.

Apolipoprotein E genotype affects tissue metallothionein levels: studies in targeted gene replacement mice.

The apolipoprotein E (APOE) genotype is an important risk factor for ageing and age-related diseases. The APOE4 genotype (in contrast to APOE3) has been shown to be associated with oxidative stress and chronic inflammation. Metallothioneins (MT) exhibit antioxidant and anti-inflammatory activity, and MT overexpression has been shown to increase lifespan in mice. Interactions between APOE and MT, however, are largely unknown. Hence, we determined the effect of the APOE4 versus APOE3 genotype on MT levels in targeted gene replacement mice. APOE4 versus APOE3 mice exhibited significantly lower hepatic MT1 and MT2 mRNA as well as lower MT protein levels. The decrease in hepatic MT protein levels in APOE4 as compared to APOE3 mice was accompanied by lower nuclear Nrf1, a protein partly controlling MT gene expression. Cell culture experiments using hepatocytes identified allyl-isothiocyanate (AITC) as a potent MT inductor in vitro. Therefore, we supplemented APOE3 and APOE4 mice with AITC. However, AITC (15 mg/kg b.w.) could only partly correct for decreased MT1 and MT2 gene expression in APOE4 mice in vivo. Furthermore, cholesterol significantly decreased both Nrf1 and MT mRNA levels in Huh7 cells indicating that differences in MT gene expression between the two genotypes could be related to differences in hepatic cholesterol concentrations. Overall, present data suggest that the APOE genotype is an important determinant of tissue MT levels in mice and that MT gene expression may be impaired by the APOE4 genotype.

Allyl isothiocyanate as a potential inducer of paraoxonase-1--studies in cultured hepatocytes and in mice.

In this study, we tested the ability of structure-related isothiocyanates to induce the antiatherogenic enzyme paraoxonase-1 (PON1) in cultured hepatocytes. Allyl isothiocyanate (AITC), phenylethyl isothiocyanate (PEITC), and sulforaphane (SFN), but not butyl isothiocyanate (BITC) resulted in dose-dependent induction of PON1 transactivation in Huh7 cells in vitro. Induction of PON1 due to AITC was inhibited by the selective peroxisome proliferator-activated receptor γ-antagonist T0070907. AITC was used in a subsequent in vivo study in mice (n = 10 per group, Western-type diet) to test its PON1 inducing activity. Unlike in cultured hepatocytes, AITC supplementation (15 mg/kg body weight) did not increase hepatic PON1 mRNA and protein levels in mice. Thus, it is suggested that AITC may be a potent inducer of PON1 in vitro, but not in mouse liver in vivo.

Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.

Effect of quercetin on paraoxonase 2 levels in RAW264.7 macrophages and in human monocytes--role of quercetin metabolism.

There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 micromol/L) resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.

Synergistic chondroprotective effect of alpha-tocopherol, ascorbic acid, and selenium as well as glucosamine and chondroitin on oxidant induced cell death and inhibition of matrix metalloproteinase-3--studies in cultured chondrocytes.

Overproduction of reactive oxygen species and impaired antioxidant defence accompanied by chronic inflammatory processes may impair joint health. Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulate the expression of metalloproteinases which degrade the extracellular matrix. Little is known regarding the potential synergistic effects of natural compounds such as alpha-tocopherol (alpha-toc), ascorbic acid (AA) and selenium (Se) on oxidant induced cell death. Furthermore studies regarding the metalloproteinase-3 inhibitory activity of glucosamine sulfate (GS) and chondroitin sulfate (CS) are scarce. Therefore we have studied the effect of alpha-toc (0.1-2.5 micromol/L), AA (10-50 micromol/L) and Se (1-50 nmol/L) on t-butyl hydroperoxide (t-BHP, 100-500 micromol/L)-induced cell death in SW1353 chondrocytes. Furthermore we have determined the effect of GS and CS alone (100-500 micromol/L each) and in combination on MMP3 mRNA levels and MMP3 secretion in IL-1beta stimulated chondrocytes. A combination of alpha-toc, AA, and Se was more potent in counteracting t-BHP-induced cytotoxicity as compared to the single compounds. Similarly a combination of CS and GS was more effective in inhibiting MMP3 gene expression and secretion than the single components. The inhibition of MMP3 secretion due to GS plus CS was accompanied by a decrease in TNF-alpha production. Combining natural compounds such as alpha-toc, AA, and Se as well as GS and CS seems to be a promising strategy to combat oxidative stress and cytokine induced matrix degradation in chondrocytes.