Immunity by AS03ation: The natural adjuvantage.

Humans do not respond equally to vaccination. To investigate why, Mulè et al. developed a multimodal framework and found that high responders after unadjuvanted influenza vaccination exist in a naturally adjuvanted state, mimicking innate immunophenotypes following AS03-adjuvanted vaccination. This highlights biological factors that set apart high-antibody responders and how adjuvants can boost innate immune cues to improve humoral immunity.

Human anti-N1 monoclonal antibodies elicited by pandemic H1N1 virus infection broadly inhibit HxN1 viruses in vitro and in vivo.

Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies1-7. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-28-10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived11-13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

Purifying Photon Indistinguishability through Quantum Interference.

Indistinguishability between photons is a key requirement for scalable photonic quantum technologies. We experimentally demonstrate that partly distinguishable single photons can be purified to reach near-unity indistinguishability by the process of quantum interference with ancillary photons followed by heralded detection of a subset of them. We report on the indistinguishability of the purified photons by interfering two purified photons and show improvements in the photon indistinguishability of 2.774(3)% in the low-noise regime, and as high as 10.2(5)% in the high-noise regime.

Endothelial Sphingosine-1-Phosphate Receptor 4 Regulates Blood-Brain Barrier Permeability and Promotes a Homeostatic Endothelial Phenotype.

The precise regulation of blood-brain barrier (BBB) permeability for immune cells and blood-borne substances is essential to maintain brain homeostasis. Sphingosine-1-phosphate (S1P), a lipid signaling molecule enriched in plasma, is known to affect BBB permeability. Previous studies focused on endothelial S1P receptors 1 and 2, reporting a barrier-protective effect of S1P1 and a barrier-disruptive effect of S1P2. Here, we present novel data characterizing the expression, localization, and function of the S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). Hitherto, the receptor was deemed to be exclusively immune cell associated. We detected a robust expression of S1P4 in homeostatic murine BMECs (MBMECs), bovine BMECs (BBMECs), and porcine BMECs (PBMECs) and pinpointed its localization to abluminal endothelial membranes via immunoblotting of fractionated brain endothelial membrane fragments. Apical S1P treatment of BMECs tightened the endothelial barrier in vitro, whereas basolateral S1P treatment led to an increased permeability that correlated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels (MBMVs) after stroke, a neurologic disease associated with BBB impairment. RNA sequencing and qPCR analysis of BMECs suggested the involvement of S1P4 in endothelial homeostasis and barrier function. Using S1P4 knock-out (KO) mice and S1P4 siRNA as well as pharmacological agonists and antagonists of S1P4 both in vitro and in vivo, we demonstrate an overall barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic potential in CNS diseases associated with BBB dysfunction.SIGNIFICANCE STATEMENT Many neurologic diseases including multiple sclerosis and stroke are associated with blood-brain barrier (BBB) impairment and disturbed brain homeostasis. Sphingosine-1-phosphate receptors (S1PRs) are potent regulators of endothelial permeability and pharmacological S1PR modulators are already in clinical use. However, the precise role of S1P for BBB permeability regulation and the function of receptors other than S1P1 and S1P2 therein are still unclear. Our study shows both barrier-disruptive and barrier-protective effects of S1P at the BBB that depend on receptor polarization. We demonstrate the expression and novel barrier-protective function of S1P4 in brain endothelial cells and pinpoint its localization to abluminal membranes. Our work may contribute to the development of novel specific S1PR modulators for the treatment of neurologic diseases associated with BBB impairment.

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption.

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine-1-phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context-dependent manner. We utilized lipopolysaccharide (LPS)-injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood-brain barrier (BBB) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability in vitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down-regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS-treated mice lead to a breakdown of the barrier compared to serum from vehicle-treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161.

Programmable multiphoton quantum interference in a single spatial mode.

The interference of nonclassical states of light enables quantum-enhanced applications reaching from metrology to computation. Most commonly, the polarization or spatial location of single photons are used as addressable degrees of freedom for turning these applications into praxis. However, the scale-up for the processing of a large number of photons of these architectures is very resource-demanding due to the rapidly increasing number of components, such as optical elements, photon sources, and detectors. Here, we demonstrate a resource-efficient architecture for multiphoton processing based on time-bin encoding in a single spatial mode. We use an efficient quantum dot single-photon source and a fast programmable time-bin interferometer to observe the interference of up to eight photons in 16 modes, all recorded only with one detector, thus considerably reducing the physical overhead previously needed for achieving equivalent tasks. Our results can form the basis for a future universal photonics quantum processor operating in a single spatial mode.

Risk assessment and antibody responses to SARS-CoV-2 in healthcare workers.

Background: Preventing infection in healthcare workers (HCWs) is crucial for protecting healthcare systems during the COVID-19 pandemic. Here, we investigated the seroepidemiology of SARS-CoV-2 in HCWs in Norway with low-transmission settings. Methods: From March 2020, we recruited HCWs at four medical centres. We determined infection by SARS-CoV-2 RT-PCR and serological testing and evaluated the association between infection and exposure variables, comparing our findings with global data in a meta-analysis. Anti-spike IgG antibodies were measured after infection and/or vaccination in a longitudinal cohort until June 2021. Results: We identified a prevalence of 10.5% (95% confidence interval, CI: 8.8-12.3) in 2020 and an incidence rate of 15.0 cases per 100 person-years (95% CI: 12.5-17.8) among 1,214 HCWs with 848 person-years of follow-up time. Following infection, HCWs (n = 63) mounted durable anti-spike IgG antibodies with a half-life of 4.3 months since their seropositivity. HCWs infected with SARS-CoV-2 in 2020 (n = 46) had higher anti-spike IgG titres than naive HCWs (n = 186) throughout the 5 months after vaccination with BNT162b2 and/or ChAdOx1-S COVID-19 vaccines in 2021. In a meta-analysis including 20 studies, the odds ratio (OR) for SARS-CoV-2 seropositivity was significantly higher with household contact (OR 12.6; 95% CI: 4.5-35.1) and occupational exposure (OR 2.2; 95% CI: 1.4-3.2). Conclusion: We found high and modest risks of SARS-CoV-2 infection with household and occupational exposure, respectively, in HCWs, suggesting the need to strengthen infection prevention strategies within households and medical centres. Infection generated long-lasting antibodies in most HCWs; therefore, we support delaying COVID-19 vaccination in primed HCWs, prioritising the non-infected high-risk HCWs amid vaccine shortage.

Vaccination prevents severe COVID-19 outcome in patients with neutralizing type 1 interferon autoantibodies.

A hallmark of patients with autoimmune polyendocrine syndrome type 1 (APS-1) is serological neutralizing autoantibodies against type 1 interferons (IFN-I). The presence of these antibodies has been associated with severe course of COVID-19. The aims of this study were to investigate SARS-CoV-2 vaccine tolerability and immune responses in a large cohort of patients with APS-1 (N = 33) and how these vaccinated patients coped with subsequent infections. We report that adult patients with APS-1 were able to mount adequate SARS-CoV-2 spike-specific antibody responses after vaccination and observed no signs of decreased tolerability. Compared with age- and gender-matched healthy controls, patients with APS-1 had considerably lower peak antibody responses resembling elderly persons, but antibody decline was more rapid in the elderly. We demonstrate that vaccination protected patients with APS-1 from severe illness when infected with SARS-CoV-2 virus, overriding the systemic danger of IFN-I autoantibodies observed in previous studies.

Durable immune responses after BNT162b2 vaccination in home-dwelling old adults.

Objectives: Elderly are an understudied, high-risk group vulnerable to severe COVID-19. We comprehensively analyzed the durability of humoral and cellular immune responses after BNT162b2 vaccination and SARS-CoV-2 infection in elderly and younger adults. Methods: Home-dwelling old (n = 100, median 86 years) and younger adults (n = 449, median 38 years) were vaccinated with two doses of BNT162b2 vaccine at 3-week intervals and followed for 9-months. Vaccine-induced responses were compared to home-isolated COVID-19 patients (n = 183, median 47 years). Our analysis included neutralizing antibodies, spike-specific IgG, memory B-cells, IFN-γ and IL-2 secreting T-cells and sequencing of the T-cell receptor (TCR) repertoire. Results: Spike-specific breadth and depth of the CD4+ and CD8+ TCR repertoires were significantly lower in the elderly after one and two vaccinations. Both vaccinations boosted IFN-γ and IL-2 secreting spike-specific T-cells responses, with 96 % of the elderly and 100 % of the younger adults responding after the second dose, although responses were not maintained at 9-months. In contrast, T-cell responses persisted up to 12-months in infected patients. Spike-specific memory B-cells were induced after the first dose in 87 % of the younger adults compared to 38 % of the elderly, which increased to 83 % after the second dose. Memory B-cells were maintained at 9-months post-vaccination in both vaccination groups. Neutralizing antibody titers were estimated to last for 1-year in younger adults but only 6-months in the older vaccinees. Interestingly, infected older patients (n = 15, median 75 years) had more durable neutralizing titers estimated to last 14-months, 8-months longer than the older vaccinees. Conclusions: Vaccine-induced spike-specific IgG and neutralizing antibodies were consistently lower in the older than younger vaccinees. Overall, our data provide valuable insights into the kinetics of the humoral and cellular immune response in the elderly after SARS-CoV-2 vaccination or infection, highlighting the need for two doses, which can guide future vaccine design.Clinical trials.gov; NCT04706390.

Optimized Fmoc-Removal Strategy to Suppress the Traceless and Conventional Diketopiperazine Formation in Solid-Phase Peptide Synthesis.

DKP (diketopiperazine) formation is a ubiquitous side reaction in SPPS (solid-phase peptide synthesis) that is highly sequence-dependent. Secondary amino acids are extremely prone to host such a side reaction. DKP formation is predominantly induced at the Fmoc (fluorenylmethyloxycarbonyl)-removal step mediated by a secondary amine, which conventionally employs piperidine/DMF (dimethylformamide). In this study, alternative Fmoc-removal solution 2% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene)/5% piperazine/NMP (N-methyl-2-pyrrolidone) led to drastic DKP reduction relative to 20% piperidine/DMF.

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern.

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80-99 years, n = 89) and younger adults (23-77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1-89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72-0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72-76%) older adults respond after two vaccinations to alpha and delta, but only 58-62% to beta and gamma, compared to 96-97% of younger vaccinees and 68-76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.

Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5µg, 7.5µg, or 30µg), or a non-adjuvanted vaccine (30µg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5µg and 30µg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30µg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

Repeated Influenza Vaccination Boosts and Maintains H1N1pdm09 Neuraminidase Antibody Titers.

Antibodies to influenza surface protein neuraminidase (NA) have been found to reduce disease severity and may be an independent correlate of protection. Despite this, current influenza vaccines have no regulatory requirements for the quality or quantity of the NA antigen and are not optimized for induction of NA-specific antibodies. Here we investigate the induction and durability of NA-specific antibody titers after pandemic AS03-adjuvanted monovalent H1N1 vaccination and subsequent annual vaccination in health care workers in a five-year longitudinal study. NA-specific antibodies were measured by endpoint ELISA and functional antibodies measured by enzyme-linked lectin assay (ELLA) and plaque reduction naturalisation assay. We found robust induction of NA inhibition (NAI) titers with a 53% seroconversion rate (>4-fold) after pandemic vaccination in 2009. Furthermore, the endpoint and NAI geometric mean titers persisted above pre-vaccination levels up to five years after vaccination in HCWs that only received the pandemic vaccine, which demonstrates considerable durability. Vaccination with non-adjuvanted trivalent influenza vaccines (TIV) in subsequent influenza seasons 2010/2011 - 2013/2014 further boosted NA-specific antibody responses. We found that each subsequent vaccination increased durable endpoint titers and contributed to maintaining the durability of functional antibody titers. Although the trivalent influenza vaccines boosted NA-specific antibodies, the magnitude of fold-increase at day 21 declined with repeated vaccination, particularly for functional antibody titers. High levels of pre-existing antibodies were associated with lower fold-induction in repeatedly vaccinated HCWs. In summary, our results show that durable NA-specific antibody responses can be induced by an adjuvanted influenza vaccine, which can be maintained and further boosted by TIVs. Although NA-specific antibody responses are boosted by annual influenza vaccines, high pre-existing titers may negatively affect the magnitude of fold-increase in repeatedly vaccinated individuals. Our results support continued development and standardization of the NA antigen to supplement current influenza vaccines and reduce the burden of morbidity and mortality.

Gene Expression Dynamics at the Neurovascular Unit During Early Regeneration After Cerebral Ischemia/Reperfusion Injury in Mice.

With increasing distribution of endovascular stroke therapies, transient middle cerebral artery occlusion (tMCAO) in mice now more than ever depicts a relevant patient population with recanalized M1 occlusion. In this case, the desired therapeutic effect of blood flow restauration is accompanied by breakdown of the blood-brain barrier (BBB) and secondary reperfusion injury. The aim of this study was to elucidate short and intermediate-term transcriptional patterns and the involved pathways covering the different cellular players at the neurovascular unit after transient large vessel occlusion. To achieve this, male C57Bl/6J mice were treated according to an intensive post-stroke care protocol after 60 min occlusion of the middle cerebral artery or sham surgery to allow a high survival rate. After 24 h or 7 days, RNA from microvessel fragments from the ipsilateral and the contralateral hemispheres was isolated and used for mRNA sequencing. Bioinformatic analyses allowed us to depict gene expression changes at two timepoints of neurovascular post-stroke injury and regeneration. We validated our dataset by quantitative real time PCR of BBB-associated targets with well-characterized post-stroke dynamics. Hence, this study provides a well-controlled transcriptome dataset of a translationally relevant mouse model 24 h and 7 days after stroke which might help to discover future therapeutic targets in cerebral ischemia/reperfusion injury.

SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans.

Infection or vaccination induces a population of long-lived bone marrow plasma cells (BMPCs) that are a persistent and essential source of protective antibodies1-5. Whether this population is induced in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unknown. Recent reports have suggested that SARS-CoV-2 convalescent patients experience a rapid decay in their antigen-specific serum antibodies, raising concerns that humoral immunity against this virus may be short-lived6-8. Here we show that in patients who experienced mild infections (n=73), serum anti-SARS-CoV-2 spike (S) antibodies indeed decline rapidly in the first 3 to 4 months after infection. However, this is followed by a more stable phase between 4- and 8-months after infection with a slower serum anti-S antibody decay rate. The level of serum antibodies correlated with the frequency of S-specific long-lived BMPCs obtained from 18 SARS-CoV-2 convalescent patients 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy subjects with no history of SARS-CoV-2 infection. Comparable frequencies of BMPCs specific to contemporary influenza virus antigens or tetanus and diphtheria vaccine antigens were present in aspirates in both groups. Circulating memory B cells (MBCs) directed against the S protein were detected in the SARS-CoV-2 convalescent patients but not in uninfected controls, whereas both groups had MBCs against influenza virus hemagglutinin. Overall, we show that robust antigen specific long-lived BMPCs and MBCs are induced after mild SARS-CoV-2 infection of humans.

Upregulated PDK4 expression is a sensitive marker of increased fatty acid oxidation.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly three-fold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation.