An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including coronavirus disease 2019. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines. OBJECTIVE: We sought to establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. METHODS: A murine model of diet-induced obesity and insulin resistance was used to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. RESULTS: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet, HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T-cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in mice fed a normal diet but not in HFD mice. CONCLUSIONS: The study demonstrated impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases.

Precision Vaccine Adjuvants for Older Adults: A Scoping Review.

Older adults, defined as those ≥60 years of age, are a growing population vulnerable to infections including severe acute respiratory syndrome coronavirus 2. Although immunization is a key to protecting this population, immunosenescence can impair responses to vaccines. Adjuvants can increase the immunogenicity of vaccine antigens but have not been systematically compared in older adults. We conducted a scoping review to assess the comparative effectiveness of adjuvants in aged populations. Adjuvants AS01, MF59, AS03, and CpG-oligodeoxynucleotide, included in licensed vaccines, are effective in older human adults. A growing menu of investigational adjuvants, such as Matrix-M and CpG plus alum, showed promising results in early phase clinical trials and preclinical studies. Most studies assessed only 1 or 2 adjuvants and no study has directly compared >3 adjuvants among older adults. Enhanced preclinical approaches enabling direct comparison of multiple adjuvants including human in vitro modeling and age-specific animal models may derisk and accelerate vaccine development for older adults.

Detailed analysis of Japanese patients with adenosine deaminase 2 deficiency reveals characteristic elevation of type II interferon signature and STAT1 hyperactivation.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive inflammatory disease caused by loss-of-function mutations in both alleles of the ADA2 gene. Most patients with DADA2 exhibit systemic vasculopathy consistent with polyarteritis nodosa, but large phenotypic variability has been reported, and the pathogenesis of DADA2 remains unclear. OBJECTIVES: This study sought to assess the clinical and genetic characteristics of Japanese patients with DADA2 and to gain insight into the pathogenesis of DADA2 by multi-omics analysis. METHODS: Clinical and genetic data were collected from 8 Japanese patients with DADA2 diagnosed between 2016 and 2019. ADA2 variants in this cohort were functionally analyzed by in vitro overexpression analysis. PBMCs from 4 patients with DADA2 were subjected to transcriptome and proteome analyses. Patient samples were collected before and after introduction of anti- TNF-α therapies. Transcriptome data were compared with those of normal controls and patients with other autoinflammatory diseases. RESULTS: Five novel ADA2 variants were identified in these 8 patients and were confirmed pathogenic by in vitro analysis. Anti-TNF-α therapy controlled inflammation in all 8 patients. Transcriptome and proteome analyses showed that upregulation of type II interferon signaling was characteristic of DADA2. Network analysis identified STAT1 as a key regulator and a hub molecule in DADA2 pathogenesis, a finding supported by the hyperactivation of STAT1 in patients' monocytes and B cells after IFN-γ stimulation. CONCLUSIONS: Type II interferon signaling and STAT1 are associated with the pathogenesis of DADA2.

Delayed Development of Coronary Artery Aneurysm in Patients with Kawasaki Disease Who Were Clinically Responsive to Immunoglobulin.

OBJECTIVE: To clarify the frequency and characteristics of discrepant outcomes of intravenous immunoglobulin (IVIG) between fever and coronary artery aneurysms (CAAs) in patients with Kawasaki disease. STUDY DESIGN: This study included 325 patients who responded to oral aspirin and IVIG alone. The main outcome was CAA 4 weeks after disease onset. CAA was defined as ≥2.5 of maximum z score (Zmax) representing the highest value of 4 coronary artery branches. Immunoglobulin dosage and sequential changes in Zmax were reviewed to investigate the effects on fever and timing of CAA development. Logistic regression analyses with receiver operating characteristic curves using clinical and laboratory variables including the initial Zmax were performed to identify predictors of CAA at 4 weeks. RESULTS: CAAs were either persistent or appeared de novo 4 weeks after diagnosis in 13 of 325 patients who responded to a single or repeated IVIG. Four single-dose IVIG-responders developed CAA although they had pretreatment Zmax of <2.0. The 2 single-dose IVIG responders with the greatest pretreatment Zmax (>4.5) developed persistent CAA. Receiver operating characteristic analysis demonstrated Zmax of 2.57 as the cut-off for predicting CAA. Multivariable analyses identified >2.5 Zmax (OR 9.08, 95% CI 1.26-65.3, P = .028, 50% sensitivity, 91% specificity) as the sole risk factor for CAA at 4 weeks in single-dose IVIG responders. CONCLUSIONS: Delayed development and persistence of CAA in single-dose IVIG responders indicate that some factors other than those responsible for systemic inflammation may contribute to vasculitis in CAA. Baseline Zmax 2.5 aids in predicting CAAs.

Toward precision adjuvants: optimizing science and safety.

PURPOSE OF REVIEW: The gradual replacement of inactivated whole cell and live attenuated vaccines with subunit vaccines has generally reduced reactogenicity but in many cases also immunogenicity. Although only used when necessary, adjuvants can be key to vaccine dose/antigen-sparing, broadening immune responses to variable antigens, and enhancing immunogenicity in vulnerable populations with distinct immunity. Licensed vaccines contain an increasing variety of adjuvants, with a growing pipeline of adjuvanted vaccines under development. RECENT FINDINGS: Most adjuvants, including Alum, Toll-like receptor agonists and oil-in-water emulsions, activate innate immunity thereby altering the quantity and quality of an adaptive immune response. Adjuvants activate leukocytes, and induce mediators (e.g., cytokines, chemokines, and prostaglandin-E2) some of which are biomarkers for reactogenicity, that is, induction of local/systemic side effects. Although there have been safety concerns regarding a hypothetical risk of adjuvants inducing auto-immunity, such associations have not been established. As immune responses vary by population (e.g., age and sex), adjuvant research now incorporates principles of precision medicine. Innovations in adjuvant research include use of human in vitro models, immuno-engineering, novel delivery systems, and systems biology to identify biomarkers of safety and adjuvanticity. SUMMARY: Adjuvants enhance vaccine immunogenicity and can be associated with reactogenicity. Novel multidisciplinary approaches hold promise to accelerate and de-risk targeted adjuvant discovery and development. VIDEO ABSTRACT: http://links.lww.com/MOP/A53.

Previous antibiotic use and the development of Kawasaki disease: a matched pair case-control study.

BACKGROUND: Kawasaki disease (KD) is an acute febrile illness with systemic vasculitides, mostly affecting infants and young children. The etiology of KD is still unclear; however, altered gut microbiota have been recently implicated as a contributing factor for the development of vasculitis. METHODS: We conducted an age- and gender-matched case-control study on 50 patients and 200 control subjects to search for potential factors leading to intestinal dysbiosis associated with KD. Data were analyzed using conditional multivariable logistic regression. RESULTS: Previous antibiotic administration was associated with the patients who developed KD (odds ratio [OR] 11.7, 95% confidence interval [CI] 4.7-29.1, P < 0.0001), but not other variables, including breastfeeding and group nursery. In subgroup analyses, cesarean birth was indicated as an associated factor in addition to previous antibiotic administration in infants under 12 months of age (OR: 8.0, 95% CI: 1.8-34.4, P = 0.005), but not in older children. CONCLUSIONS: The association between previous antibiotic administration and the onset of KD was demonstrated. Antibiotics may contribute to the development of KD by affecting the intestinal microbiota in infants and young children.

A Nationwide Survey of Pediatric-onset Japanese Encephalitis in Japan.

BACKGROUND: Japanese encephalitis (JE) is the leading cause of viral encephalitis with high mortality and morbidity in Asia. In Japan, however, the active recommendation of JE vaccine was retracted in 2005 because of the potential risk of acute disseminated encephalomyelitis. We aimed to determine the recent incidence of childhood-onset JE after the domestic change of vaccination policy in Japan, and to analyze the clinical features of affected children. METHODS: A retrospective nationwide survey was conducted for pediatric patients with JE in Japan from 1995 to 2015. The national surveillance system was used to identify the pediatric patients with JE. Follow-up questionnaires were sent to analyze their clinical and neuroimaging profiles. RESULTS: Among a total of 109 patients registered to the national surveillance, 10 (9%) were less than age 15 years. The annual incidence rate of childhood-onset JE was higher during 2005-15 than that during 1995-2004 (4.3 × 10-3 vs 1.1 × 10-3 per 100000, respectively; P = .04). Endemic regions overlapped with prefectures that farmed pigs harboring antibodies against JE virus with high prevalence. Detailed clinical data were collected from 9 patients. None of them died, but 5 of 9 patients (56%) had neurological sequelae after recovery. One patient who was partially vaccinated with 2 doses of JE vaccine fully recovered from a coma. The age of 3 years or less was associated with unfavorable neurological prognosis. CONCLUSIONS: Our data provide evidence for the importance and prophylactic effect of the JE vaccine in young children in the endemic area.

Clinical Utility of Highly Purified 10% Liquid Intravenous Immunoglobulin in Kawasaki Disease.

Compared with a 5% intravenous immunoglobulin, a 10% intravenous immunoglobulin as the first-line treatment of Kawasaki disease significantly reduced the fever duration (10 vs 13 hours, P = .022) among the responders, and the interval to adjunctive therapy for nonresponders (47 vs 49 hours, P = .035). There were no severe adverse events.

Procalcitonin levels predicting the infliximab response of immunoglobulin resistant Kawasaki disease.

OBJECTIVE: To search the predictive factors of infliximab resistance in intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD) patients. STUDY DESIGN: Twenty-seven patients with KD who received infliximab after 4-5 g/kg of IVIG therapy from 2013 to 2015 were consecutively recruited in this study. They were divided into two groups: patients who responded to infliximab (infliximab-responsive group, n = 15) and patients who required additional therapy for the disease control (infliximab-resistant group, n = 12). We analyzed the clinical and laboratory parameters just before the infliximab treatment including serum levels of procalcitonin and cytokines with respect to the infliximab response. RESULTS: Serum procalcitonin concentration (P = 0.017), neutrophils to lymphocytes ratio (P = 0.013), and % neutrophils (P = 0.004) were higher, and serum sodium concentration (P = 0.017) was lower in infliximab-resistant group than those of infliximab-responsive group, respectively. Multivariate logistic regression analyses indicated that higher procalcitonin concentration (odds ratio [OR] 1.48, 95% confidence interval [CI] 1.00-5.00, P = 0.046) and lower sodium levels (OR 0.64, 95% CI 0.32-1.00, P = 0.047), but not other variables, were associated with infliximab-resistance. Serum procalcitonin concentrations positively correlated with the serum levels of interleukin-6, soluble tumor necrosis factor receptor type 1 and type 2, respectively. Analyses of the receiver operating characteristic (ROC) curve showed that the cut-off value of procalcitonin 2.0 ng/ml had 58.3% of sensitivity and 93.3% of specificity. ROC analysis yielded an area under the curve (AUC) of 0.739 to predict infliximab-resistance. CONCLUSION: Serum procalcitonin might be an effective biomarker to predict infliximab resistance in severe KD patients who are refractory to IVIG treatment.

Activation of Nod1 Signaling Induces Fetal Growth Restriction and Death through Fetal and Maternal Vasculopathy.

Intrauterine fetal growth restriction (IUGR) and death (IUFD) are both serious problems in the perinatal medicine. Fetal vasculopathy is currently considered to account for a pathogenic mechanism of IUGR and IUFD. We previously demonstrated that an innate immune receptor, the nucleotide-binding oligomerization domain-1 (Nod1), contributed to the development of vascular inflammations in mice at postnatal stages. However, little is known about the deleterious effects of activated Nod1 signaling on embryonic growth and development. We report that administration of FK565, one of the Nod1 ligands, to pregnant C57BL/6 mice induced IUGR and IUFD. Mass spectrometry analysis revealed that maternally injected FK565 was distributed to the fetal tissues across placenta. In addition, maternal injection of FK565 induced robust increases in the amounts of CCL2, IL-6, and TNF proteins as well as NO in maternal, placental and fetal tissues. Nod1 was highly expressed in fetal vascular tissues, where significantly higher levels of CCL2 and IL-6 mRNAs were induced with maternal injection of FK565 than those in other tissues. Using Nod1-knockout mice, we verified that both maternal and fetal tissues were involved in the development of IUGR and IUFD. Furthermore, FK565 induced upregulation of genes associated with immune response, inflammation, and apoptosis in fetal vascular tissues. Our data thus provided new evidence for the pathogenic role of Nod1 in the development of IUGR and IUFD at the maternal-fetal interface.

Memory B-Cell Pools Predict the Immune Response to Pneumococcal Conjugate Vaccine in Immunocompromised Children.

BACKGROUND: The immune responses to pneumococcal conjugate vaccine (PCV) are low in immunocompromised hosts. The effect of memory B cells on the immune response to PCV remains elusive. METHODS: In this prospective study, 53 children who received 7-valent PCV were enrolled. Antipneumococcal immunoglobulin G (IgG) levels and opsonization index (OI) titers, along with lymphocyte subsets, were investigated in immunocompromised and immunocompetent hosts. Immunocompromised patients comprised 8 hematopoietic stem cell transplant recipients (group A) and 9 immunosuppressive therapy recipients (group B), and controls consisted of 14 children aged >1 year (group C) and 22 infants (group D). RESULTS: Serotype-specific IgG concentrations and OIs in group A were lower than those in group C. These did not differ among groups B, C, and D. The rates of achieving immunity (defined as an IgG level of 1.0 µg/mL and an OI of 8) in group A were also lower than in group C. Despite the sustained numbers of total T cells and B cells, CD27(+) B-cell and CD4(+) T-cell counts in group A were lower than those in group C. In group B, the immunoglobulin D-expressing CD27(-) B-cell count was only lower than that in group C. CONCLUSIONS: Circulating numbers of CD27(+) B cells, rather than CD4(+) T cells, may predict the effective PCV responses in immunocompromised children.

Polymyxin-B immobilized column-direct hemoperfusion for adolescent toxic shock syndrome.

Toxic shock syndrome (TSS) is a critical illness associated with toxin from Staphylococcus aureus. Despite recent advances in critical care, mortality remains high and additional effective therapy is required. We report an adolescent case of TSS successfully treated with direct hemoperfusion using polymyxin-B immobilized fiber (PMX-DHP). The patient with spina bifida also had ischial pressure ulcer, and developed TSS associated with methicillin-resistant S. aureus. Despite conventional treatment, the patient developed refractory shock, which was immediately improved with PMX-DHP. PMX-DHP has been widely used for the treatment of sepsis to remove circulating endotoxins produced by Gram-negative bacteria, but beneficial effects have also been reported for Gram-positive bacterial infection. To our knowledge, this is the first report on PMX-DHP for TSS in an adolescent patient, and we propose that PMX-DHP could be a new treatment strategy for severe TSS in children as well.

Clinical and genetic investigation of 17 Japanese patients with hyperekplexia.

AIM: The aim of the study was to determine clinical and genetic characteristics of Japanese patients with hyperekplexia. METHOD: Clinical courses, responses to antiepileptic drugs, outcomes, and genetic testing were investigated in 17 Japanese patients (nine males, eight females, median age 1y, range birth-45y) with hyperekplexia. RESULTS: In all patients, muscle stiffness and startle responses appeared soon after birth. Only seven patients were diagnosed with hyperekplexia before 1 year of age. Seven patients had been misdiagnosed with other disorders such as epilepsy and adult-onset anxiety neurosis. Umbilical/inguinal hernias were seen in 10 patients. Life-threatening events were noted in four patients. Clonazepam was the most effective drug. Muscle stiffness completely disappeared in 12 patients before 5 years of age, whereas startle responses resolved in only three patients. Mutations in the GLRA1 and GLRB genes were identified in 16 patients and one patient respectively. In 14 patients, the mutation showed autosomal dominant inheritance; in the other three, inheritance was autosomal recessive. p.R271Q of GLRA1 was the most frequent mutation, found in 10 patients. Novel mutations, p.A272P and p.A384P of GLRA1, were detected. Clinical severity and outcome varied even in the same family. INTERPRETATION: Early correct diagnosis is essential for prevention of accidental injuries and to provide appropriate treatments for hyperekplexia. Clonazepam is effective, although the time taken for startle responses to resolve varied.

The uselessness of procalcitonin in the diagnosis of focal bacterial central nervous system infection.

We investigated the utility of procalcitonin in early diagnosis of bacterial central nervous system (CNS) infection. Serum procalcitonin level was markedly elevated in the patients with systemic meningitis but not in the patients with brain abscess and subdural empyema. Procalcitonin may be useless to diagnose focal bacterial CNS infection.

The utility of biomarkers in differentiating bacterial from non-bacterial lower respiratory tract infection in hospitalized children: difference of the diagnostic performance between acute pneumonia and bronchitis.

The aim of this study is to investigate the utility of several biomarkers in differentiating bacterial community-acquired lower respiratory tract infection (CA-LRTI) from non-bacterial CA-LRTI in children and the difference of their diagnostic performance between pneumonia and bronchitis. A retrospective cohort study composed of 108 pediatric patients hospitalized for CA-LRTI was performed during 2010-2013. Based on the findings of chest X-ray and sputum samples, patients were divided into 4 categories, group of bacterial pneumonia or bronchitis, and non-bacterial (viral or etiology-unknown) pneumonia or bronchitis. Peripheral white blood cell and neutrophil counts, and serum C-reactive protein (CRP) and procalcitonin (PCT) levels were compared among the 4 groups. Finally, 54 patients were the subject of this study. In the patients with pneumonia, serum CRP and PCT levels were significantly elevated in the group of bacterial pneumonia (CRP: p = 0.02, PCT: p = 0.0008). The area under the receiver operating characteristic curve for PCT for distinguishing between bacterial and non-bacterial pneumonia was the largest, and sensitivity, specificity, positive predictive value and negative predictive value of PCT were best among 4 markers. On the other hand, in the patients with bronchitis, neutrophil count was significantly decreased in non-bacterial bronchitis whereas no significant differences of WBC count, CRP level or PCT level were seen. In conclusion, PCT was the most useful marker to differentiate bacterial pneumonia whereas neutrophil count contributed most to the discrimination of bacterial bronchitis. The diagnostic performance of biomarkers may be different between pneumonia and bronchitis.

Staphylococcal endocarditis as the first manifestation of heritable protein S deficiency in childhood.

A 12-year-old Japanese girl developed infective endocarditis and central nervous system disease. The previously healthy girl showed altered consciousness and abnormal behaviors along with the classical signs of septic emboli. Staphylococcus aureus was isolated from peripheral blood, but not, the pleocytotic cerebrospinal fluid. Diagnostic imaging studies revealed a vegetative structure in the morphologically normal heart, and multiple thromboembolisms in the brain and spleen. Low plasma activity of protein S (12%) and thrombophilic family history allowed the genetic study, demonstrating that she carried a heterozygous mutation of PROS1 (exon 13; 1689C > T, p.R474C). Surgical intervention of the thrombotic fibrous organization and subsequent anticoagulant therapy successfully managed the disease. There are no reports of infective endocarditis in childhood occurring as the first presentation of heritable thrombophilia. Protein S deficiency might be a risk factor for the development or exacerbation of infective endocarditis in children having no pre-existing heart disease.

Adjuvantation of a SARS-CoV-2 mRNA vaccine with controlled tissue-specific expression of an mRNA encoding IL-12p70.

Messenger RNA (mRNA) vaccines were pivotal in reducing severe acute respiratory syndrome 2 (SARS-CoV-2) infection burden, yet they have not demonstrated robust durability, especially in older adults. Here, we describe a molecular adjuvant comprising a lipid nanoparticle (LNP)-encapsulated mRNA encoding interleukin-12p70 (IL-12p70). The bioactive adjuvant was engineered with a multiorgan protection (MOP) sequence to restrict transcript expression to the intramuscular injection site. Admixing IL-12-MOP (CTX-1796) with the BNT162b2 SARS-CoV-2 vaccine increased spike protein-specific immune responses in mice. Specifically, the benefits of IL-12-MOP adjuvantation included amplified humoral and cellular immunity and increased immune durability for 1 year after vaccination in mice. An additional benefit included the restoration of immunity in aged mice to amounts comparable to those achieved in young adult animals, alongside amplification with a single immunization. Associated enhanced dendritic cell and germinal center responses were observed. Together, these data demonstrate that an LNP-encapsulated IL-12-MOP mRNA-encoded adjuvant can amplify immunogenicity independent of age, demonstrating translational potential to benefit vulnerable populations.

From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.