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Background The opioid epidemic worsened during the coronavirus disease 2019 (COVID-19) pandemic. Synthetic opioids (primarily fentanyl) comprise the most common drugs involved in overdose (OD) death. A vaccine that blocks fentanyl from reaching the brain to prevent OD is under development, and insight is needed into its acceptability. Methods Using a semi-structured interview guide, persons with opioid use disorder (OUD), family, professionals, and the public were interviewed about attitudes and concerns regarding a fentanyl vaccine. Reactions to fictional clinical vignettes of persons at risk of OUD because of pain and/or substance use histories were collected, analyzed, and quantified for favorability. Interviews were transcribed, coded, and analyzed thematically. Results Among N = 64 participants, (70.3% female, average age 32.4 years), attitudes were favorable toward a fentanyl vaccine, with preference for lifelong durability (76% of n = 55 asked). Perceived benefits centered on the potential for a life-saving intervention, suffering averted, healthcare dollars saved, and the utility of a passive harm reduction strategy. Concerns centered on uncertainty regarding vaccine safety, questions about efficacy, worry about implications for future pain management, stigma, and need for supportive counseling and guidance to personalize decision making. Reactions to vignettes revealed complex attitudes toward fentanyl vaccination when considering recipient age, health history, and future risks for addiction and pain. Conclusions Positive responses to a fentanyl vaccine were found along with appreciation for the complexity of a vaccine strategy to prevent OD in the setting of pain and uncertain durability. Further research is needed to elucidate operational, ethical, and communications strategies to advance the model.

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.

BackgroundObesity and Type 2 Diabetes Mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines.ObjectiveTo 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.MethodsWe utilized a murine model of diet-induced obesity and insulin resistance to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection.ResultsMice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet (ND), 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 ND mice but not in HFD mice.ConclusionWe demonstrate 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.Capsule summaryObesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.

Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant (TLR7/8A) based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized a) the formulation delivery system, b) structure, and c) immunologic activity of novel small molecule imidazoquinoline TLR7/8As towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8A overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.

SummaryWe describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ∼2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNγ and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers—an 8.6% hit rate—we downselected to five scaffolds that demonstrated robust efficacy and potencyin vitroand evaluated the top hit, vinblastine sulfate, for adjuvanticityin vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCsin vitrothat yielded a lead compound within vivoadjuvanticity.MotivationVaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based onin vitroscreening of human primary leukocytes.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specificin vitromodelling, we identified the combination of a small molecule STING agonist (2′3′-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive synergistic activation of neonatal dendritic cells and precision CD4 Th cell expansion via the IL-12/IFNγ axis. We further demonstrate that vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating-cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust T helper (Th)1 bias, high frequency of T follicular helper (TFH) cells and germinal center B (GC B) cells along with IgG2c-skewed humoral responsein vivo. Dual loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PSs and CL075-PSsin vivo. These data validate an optimally designed adjuvantation system, via age-selected small molecule synergy and a multi-component nanocarrier formulation, as an effective approach to induce type 1 immune responses in early life.

Abstract Infection is the predominant cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, very young infants fail to respond optimally to most vaccines currently in use, especially neonates. In 2005, Stanley Plotkin proposed that new delivery systems would spur a new revolution in pediatric vaccinology, just as attenuation, inactivation, cell culture of viruses, genetic engineering, and adjuvantation had done in preceding decades. Recent advances in the field of immunoengineering, which is evolving alongside vaccinology, have begun to increasingly influence vaccine formulation design. Historically, the particulate nature of materials used in many vaccine formulations was empiric, often because of the need to stabilize antigens or reduce endotoxin levels. However, present vaccine delivery systems are rationally engineered to mimic the size, shape, and surface chemistry of pathogens, and are therefore often referred to as “pathogen-like particles”. More than a decade from his original assessment, we re-assess Plotkin’s prediction. In addition, we highlight how immunoengineering and advanced delivery systems may be uniquely capable of enhancing vaccine responses in vulnerable populations, such as infants. Impact Immunoengineering and advanced delivery systems are leading to new developments in pediatric vaccinology. Summarizes delivery systems currently in use and development, and prospects for the future. Broad overview of immunoengineering’s impact on vaccinology, catering to Pediatric Clinicians and Immunologists.

The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused significant mortality, especially among older adults whose distinct immune system reflects immunosenescence. Multiple SARS-CoV-2 vaccines have received emergency use authorization and/or licensure from the US Food and Drug Administration and throughout the world. However, their deployment has heighted significant limitations, such by age-dependent immunogenicity, requirements for multiple vaccine doses, refrigeration infrastructure that is not universally available, as well as waning immunity. Thus, there was, and continues to be a need for continued innovation during the pandemic given the desire for dose-sparing, formulations stable at more readily achievable temperatures, need for robust immunogenicity in vulnerable populations, and development of safe and effective pediatric vaccines. In this context, optimal SARS-CoV-2 vaccines may ultimately rely on inclusion of adjuvants as they can potentially enhance protection of vulnerable populations and provide dose-sparing effects enabling single shot protection.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (T

SUMMARYDevelopment of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.One Sentence SummaryAlum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

Other than clean drinking water, vaccines have been the most effective public health intervention in human history, yet their full potential is still untapped. To date, vaccine development has been largely limited to empirical approaches focused on infectious diseases and has targeted entire populations, potentially disregarding distinct immunity in vulnerable populations such as infants, elders, and the immunocompromised. Over the past few decades innovations in genetic engineering, adjuvant discovery, formulation science, and systems biology have fueled rapid advances in vaccine research poised to consider demographic factors (e.g., age, sex, genetics, and epigenetics) in vaccine discovery and development. Current efforts are focused on leveraging novel approaches to vaccine discovery and development to optimize vaccinal antigen and, as needed, adjuvant systems to enhance vaccine immunogenicity while maintaining safety. These approaches are ushering in an era of precision vaccinology aimed at tailoring immunization for vulnerable populations with distinct immunity. To foster collaboration among leading vaccinologists, government, policy makers, industry partners, and funders from around the world, the Precision Vaccines Program at Boston Children’s Hospital hosted the 2nd International Precision Vaccines Conference (IPVC) at Harvard Medical School on the 17th–18th October 2019. The conference convened experts in vaccinology, including vaccine formulation and adjuvantation, immunology, cell signaling, systems biology, biostatistics, bioinformatics, as well as vaccines for non-infectious indications such as cancer and opioid use disorder. Herein we review highlights from the 2nd IPVC and discuss key concepts in the field of precision vaccines.

Current acellular-pertussis (aP) vaccines appear inadequate for long-term pertussis control because of short-lived efficacy and the increasing prevalence of pertactin-negative isolates which may negatively impact vaccine efficacy. In this study, we added fimbriae (FIM)2 and FIM3 protein to licensed 2-, 3- or 5-component aP vaccines (Pentavac(®), Boostrix(®), Adacel(®), respectively) to assess whether an aP vaccine with enhanced FIM content demonstrates enhanced efficacy. Vaccine-induced protection was assessed in an intranasal mouse challenge model. In addition, potential reactogenicity was measured by biomarkers in a human whole blood assay (WBA) in vitro and benchmarked the responses against licensed whole cell pertussis (wP) and aP vaccines including Easyfive(®), Pentavac(®) and Pentacel(®). The results show that commercial vaccines demonstrated reduced efficacy against pertactin-negative versus pertactin-positive strains. However, addition of higher amounts of FIM2/3 to aP vaccines reduced lung colonization and increased vaccine efficacy against a pertactin-negative strain in a dose- dependent manner. Improvements in efficacy were similar for FIM2 and FIM3-expressing strains. Increasing the amount of FIM2/3 proteins in aP formulations did not alter vaccine- induced biomarkers of potential reactogenicity including prostaglandin E(2), cytokines and chemokines in human newborn cord and adult peripheral blood tested in vitro. These results suggest that increasing the quantity of FIM proteins in current pertussis vaccine formulations may further enhance vaccine efficacy against B. pertussis infection without increasing the reactogenicity of the vaccine.

Vaccines have been traditionally developed with the presumption that they exert identical immunogenicity regardless of target population and that they provide protection solely against their target pathogen. However, it is increasingly appreciated that vaccines can have off-target effects and that vaccine immunogenicity can vary substantially with demographic factors such as age and sex. Bacille Calmette-Guerin (BCG), the live attenuated Mycobacterium bovis vaccine against tuberculosis (TB), represents a key example of these concepts. BCG vaccines are manufactured under different conditions across the globe generating divergent formulations. Epidemiologic studies have linked early life immunization with certain BCG formulations to an unanticipated reduction (∼50%) in all-cause mortality, especially in low birthweight males, greatly exceeding that attributable to TB prevention. This mortality benefit has been related to prevention of sepsis and respiratory infections suggesting that BCG induces "heterologous" protection against unrelated pathogens. Proposed mechanisms for heterologous protection include vaccine-induced immunometabolic shifts, epigenetic reprogramming of innate cell populations, and modulation of hematopoietic stem cell progenitors resulting in altered responses to subsequent stimuli, a phenomenon termed "trained immunity." In addition to genetic differences, licensed BCG formulations differ markedly in content of viable mycobacteria key for innate immune activation, potentially contributing to differences in the ability of these diverse formulations to induce TB-specific and heterologous protection. BCG immunomodulatory properties have also sparked interest in its potential use to prevent or alleviate autoimmune and inflammatory diseases, including type 1 diabetes mellitus and multiple sclerosis. BCG can also serve as a model: nanoparticle vaccine formulations incorporating Toll-like receptor 8 agonists can mimic some of BCG's innate immune activation, suggesting that aspects of BCG's effects can be induced with non-replicating stimuli. Overall, BCG represents a paradigm for precision vaccinology, lessons from which will help inform next generation vaccines.

Intranasal mucosal vaccines are an attractive approach to induce protective mucosal immune responses. Activation of lung antigen presenting cells (APCs), a phenotypically and functionally heterogeneous cell population located at distinct mucosal sites, may be key to the immunogenicity of such vaccines. Understanding responsiveness of newborn lung APCs to adjuvants may the inform design of efficacious intranasal vaccines for early life, when most infections occur. Here, we characterized and phenotyped APCs from neonatal (7 days of life) and adult (6–8 weeks of age) mice. Neonatal mice demonstrated a relatively high abundance of alveolar macrophages (AMs), with lower percentages of plasmacytoid dendritic cells (pDCs), CD103+ (cDC1), and CD11b+ (cDC2) DCs. Furthermore, neonatal CD103+ and CD11b+ DC subsets demonstrated a significantly lower expression of maturation markers (CD40, CD80, and CD86) as compared to adult mice. Upon stimulation of lung APC subsets with a panel of pattern recognition receptor (PRR) agonists, including those engaging TLRs or STING, CD11c+ enriched cells from neonatal and adult mice lungs demonstrated distinct maturation profiles. Of the agonists tested, the TLR5 ligand, flagellin, was most effective at activating neonatal lung APCs, inducing significantly higher expression of maturation markers on CD103+ (cDC1) and CD11b+ (cDC2) subsets. Intranasal administration of flagellin induced a distinct migration of CD103+ and CD11b+ DC subsets to the mediastinal lymph nodes (mLNs) of neonatal mice. Overall, these findings highlight age-specific differences in the maturation and responsiveness of lung APC subsets to different PRR agonists. The unique efficacy of flagellin in enhancing lung APC activity suggests that it may serve as an effective adjuvant for early life mucosal vaccines.

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.

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.

Infections are most frequent at the extremes of life, especially among newborns, reflecting age-specific differences in immunity. Monocytes maintain tissue-homeostasis and defence-readiness by escaping circulation in the absence of inflammation to become tissue-resident antigen presenting cells in vivo. Despite equivalent circulating levels, neonates demonstrate lower presence of monocytes inside peripheral tissues as compared to adults. To study the ability of monocytes to undergo autonomous transendothelial extravasation under biologically accurate circumstances we engineered a three-dimensional human vascular-interstitial model including collagen, fibronectin, primary endothelial cells and autologous untreated plasma. This microphysiological tissue construct enabled age-specific autonomous extravasation of monocytes through a confluent human endothelium in the absence of exogenous chemokines and activation. Both CD16− and CD16+ newborn monocytes demonstrated lower adherence and extravasation as compared to adults. In contrast, pre-activated tissue constructs were colonized by newborn monocytes at the same frequency than adult monocytes, suggesting that neonatal monocytes are capable of colonizing inflamed tissues. The presence of autologous plasma neither improved newborn homeostatic extravasation nor shaped age-specific differences in endothelial cytokines that could account for this impairment. Newborn monocytes demonstrated significantly lower surface expression of CD31 and CD11b, and mechanistic experiments using blocking antibodies confirmed a functional role for CD31 and CD54 in neonatal homeostatic extravasation. Our data suggests that newborn monocytes are intrinsically impaired in extravasation through quiescent endothelia, a phenomenon that could contribute to the divergent immune responsiveness to vaccines and susceptibility to infection observed during early life.

Intranasal mucosal vaccines are of interest in that they may induce protective mucosal immune responses. Activation of lung antigen presenting cells (APCs), a phenotyoically and functionally heterogeneous cell population located at distinct mucosal sites, may be key to the immunogenicity of such vaccines. Characterizing responsiveness of newborn lung APCs to adjuvants may inform design of efficacious intranasal vaccines for early life, when most infections occur. We characterized APCs from neonatal (+ (cDC1) and CD11b+ (cDC2) DCs. Furthermore, neonatal CD103+ and CD11b+ DC subsets demonstrated an inverse expression of maturation markers as compared to adult mice. Upon stimulation of lung APC subsets with a panel of pattern recognition receptor (PRR), including TLR and STING, agonists, CD11c+ enriched cells from neonatal and adult mice lungs demonstrated distinct maturation profiles. The TLR5 ligand, flagellin, was most effective at activating neonatal lung APCs, inducing significantly higher expression of maturation markers on CD103+ (cDC1) and CD11b+ (cDC2) subsets. Intranasal administration of flagellin induced a distinct migration of CD103+ and CD11b+ DC subsets to the mediastinal lymph nodes (mLNs) of neonatal mice. Overall, these findings highlight age specific differences in the maturation and responsiveness of lung APC subsets to different PRR agonists. The unique efficacy of flagellin in enhancing lung APC activity suggests that it may serve as an effective adjuvant for early life mucosal vaccines.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate long-lasting adaptive immunity. While it is known that PRRs detect unique chemical patterns associated with invading microorganism, if and how the physical properties of PRR ligands influence development of the immune response is largely overlooked. Through the study of fungal mannans we present data that put the physical form of PRR ligands at the center of the process that determines the outcome of 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 periphery and dLN. When combined with SARS-CoV-2 Spike, this formulation elicits neutralizing anti-SARS-CoV-2 antibodies that cross-react with pathogenic coronaviruses. Thus, the physical properties of fungal ligands can be harnessed for rational adjuvant design and vaccine development.

Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1β) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood. Newborn cord and adult blood were treated with PTX (50–400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR. Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1β with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation. PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Due to functionally distinct cell-mediated immunity, newborns and infants are highly susceptible to infection with intracellular pathogens. Indeed, neonatal Ag-presenting dendritic cells (DCs) demonstrate impaired Th1 responses to many candidate adjuvants, including most TLR agonists (TLRAs). Combination adjuvantation systems may provide enhanced immune activation but have typically been developed without regard to the age of the target population. We posited that distinct combinations of TLRAs and C-type lectin receptor agonists may enhance Th1 responses of newborn DCs. TLRA/C-type lectin receptor agonist combinations were screened for enhancement of TNF production by human newborn and adult monocyte-derived DCs cultured in 10% autologous plasma or in newborn cord, infant, adult, and elderly whole blood. Monocyte-derived DC activation was characterized by targeted gene expression analysis, caspase-1 and NF-κB studies, cytokine multiplex and naive autologous CD4+ T cell activation. Dual activation of newborn DCs via the C-type lectin receptor, macrophage-inducible C-type lectin (trehalose-6,6-dibehenate), and TLR7/8 (R848) greatly enhanced caspase-1 and NF-κB activation, Th1 polarizing cytokine production and autologous Th1 polarization. Combined activation via TLR4 (glycopyranosyl lipid adjuvant aqueous formulation) and Dectin-1 (β-glucan peptide) acted synergistically in newborns and adults, but to a lesser extent. The degree of synergy varied dramatically with age, and was the greatest in newborns and infants with less synergy in adults and elders. Overall, combination adjuvant systems demonstrate markedly different immune activation with age, with combined DC activation via Macrophage-inducible C-type lectin and TLR7/8 representing a novel approach to enhance the efficacy of early-life vaccines.

Most infections occur in early life, prompting development of novel adjuvanted vaccines to protect newborns and infants. Several Toll-like receptor (TLR) agonists (TLRAs) are components of licensed vaccine formulations or are in development as candidate adjuvants. However, the type and magnitude of immune responses to TLRAs may vary with the TLR activated as well as age and geographic location. Most notably, in newborns, as compared to adults, the immune response to TLRAs is polarized with lower Th1 cytokine production and robust Th2 and anti-inflammatory cytokine production. The ontogeny of TLR-mediated cytokine responses in international cohorts has been reported, but no study has compared cytokine responses to TLRAs between U.S. neonates and infants at the age of 6months. Both are critical age groups for the currently pediatric vaccine schedule. In this study, we report quantitative differences in the production of a panel of 14 cytokines and chemokines after in vitro stimulation of newborn cord blood and infant and adult peripheral blood with agonists of TLR4, including monophosphoryl lipid A (MPLA) and glucopyranosyl lipid Adjuvant aqueous formulation (GLA-AF), as well as agonists of TLR7/8 (R848) and TLR9 (CpG). Both TLR4 agonists, MPLA and GLA-AF, induced greater concentrations of Th1 cytokines CXCL10, TNF and Interleukin (IL)-12p70 in infant and adult blood compared to newborn blood. All the tested TLRAs induced greater infant IFN-α2 production compared to newborn and adult blood. In contrast, CpG induced greater IFN-γ, IL-1β, IL-4, IL-12p40, IL-10 and CXCL8 in newborn than in infant and adult blood. Overall, to the extent that these in vitro studies mirror responses in vivo, our study demonstrates distinct age-specific effects of TLRAs that may inform their development as candidate adjuvants for early life vaccines.

Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles, paralleling responses to adjuvant-containing vaccines in vivo. Age-specific in vitro modeling coupled with proteomics may provide fresh insight into the ontogeny of adjuvant action thereby informing targeted adjuvanted vaccine development for distinct age groups.

Infection is a significant cause of mortality and immunization is the most promising biomedical intervention to reduce this burden. However, multiple booster doses are often required to protect those with distinct immunity such as the young, elders, and immunocompromised individuals. Adjuvants are molecules that potentiate vaccine responses. There is a need for a larger toolbox of adjuvants, since Alum is the only adjuvant currently incorporated into licensed vaccines in the pediatric setting. We utilized a unique in vitro approach to discover new adjuvants, screening a ~10k small molecule library via a no-wash 384-well quantitative TNF immunoassay in peripheral blood mononuclear cells (PBMCs) from 3 adult healthy human donors. Preliminary hits were based on induced TNF production in at least two of the three donors screened. We identified a novel imidazopyrimidine (IMP) scaffold named PVP-037 which demonstrated efficacy, potency, and adjuvanticity. PVP-037 demonstrated broad age-independent immune-stimulatory ability in human PBMCs and dendritic cells, including key Th-polarizing cytokines such as IL-12, and significant enhancement murine humoral responses to recombinant hemagglutinin (rHA) proteins in the quadrivalent influenza. PVP-037 further demonstrated limited activity in several common cell lines (THP-1) and murine/porcine primary leukocytes. Structure activity relationship (SAR) studies identified the analog PVP-144-3 with greater in vitro efficacy across species (porcine, non-human primates) and murine in vivo adjuvanticity. Overall, we discovered an IMP adjuvant via high throughput screening of human mononuclear cells in vitro which correlated with enhanced influenza immunization in mice.

Introduction Neonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production. Methods Whole newborn and adult blood was treated with PTX (50–200 μM), DEX (10−10–10−7 M), or AZI (2.5–20 μM), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity. Results PTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation. PTX preferentially inhibited pro-inflammatory cytokines especially TNF. DEX inhibited IL-10 in newborn, and TNF, IL-1β, IL-6 and interferon-α in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1β, IL-6 and IL-10, and LPS-induced IL-1β and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1β, and IL-6, and R848-induced IL-1β and interferon-α, while (PTX+AZI) synergistically decreased induction of TNF, IL-1β, and IL-6. Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA. Conclusions Age, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.

The goal of immunization is the induction of an immune response to protect from infection or disease (1). Until the end of the 20th century, adjuvantation science was limited to the use aluminum salts (alum) (2). Over the past 20 years there has been explosive growth of information regarding pattern recognition receptors (PRRs) that can activate leukocytes and thereby enhance immune responses (3). In parallel, a growing menu of adjuvants, agents that boost responses to vaccinal antigens following immunization, is now becoming available to immunologists and vaccinologists. However, immunity over life is not static but rather changes with age (4). Here, we will summarize the adjuvants currently in clinical use, new adjuvants in development and potential uses and benefits of a new “adjuvant toolbox” for developing novel early life vaccines (5).

Immunization is key to preventing infectious diseases, a leading cause of death early in life. However, due to age-specific immunity, vaccines often demonstrate reduced efficacy in newborns and young infants as compared to adults. Here, we combined in vitro and in vivo approaches to identify adjuvant candidates for early life immunization. We employed newborn and adult bone marrow-derived dendritic cells (BMDCs) to perform a screening of pattern recognition receptor agonists and found that the stimulator of interferon genes ligand 2′3′-cGAMP (hereafter cGAMP) induces a comparable expression of surface maturation markers in newborn and adult BMDCs. Then, we utilized the trivalent recombinant hemagglutinin (rHA) influenza vaccine, Flublok, as a model antigen to investigate the role of cGAMP in adult and early life immunization. cGAMP adjuvantation alone could increase rHA-specific antibody titers in adult but not newborn mice. Remarkably, as compared to alum or cGAMP alone, immunization with cGAMP formulated with alum (Alhydrogel) enhanced newborn rHA-specific IgG2a/c titers ~400-fold, an antibody subclass associated with the development of IFNγ-driven type 1 immunity in vivo and endowed with higher effector functions, by 42 days of life. Highlighting the amenability for successful vaccine formulation and delivery, we next confirmed that cGAMP adsorbs onto alum in vitro. Accordingly, immunization early in life with (cGAMP+alum) promoted IFNγ production by CD4+ T cells and increased the proportions and absolute numbers of CD4+ CXCR5+ PD-1+ T follicular helper and germinal center (GC) GL-7+ CD138+ B cells, suggesting an enhancement of the GC reaction. Adjuvantation effects were apparently specific for IgG2a/c isotype switching without effect on antibody affinity maturation, as there was no effect on rHA-specific IgG avidity. Overall, our studies suggest that cGAMP when formulated with alum may represent an effective adjuvantation system to foster humoral and cellular aspects of type 1 immunity for early life immunization.

Immunization is key to protecting term and preterm infants from a heightened risk of infection. However, preterm immunity is distinct from that of the term, limiting its ability to effectively respond to vaccines routinely given at birth, such as hepatitis B vaccine (HBV). As part of the Expanded Program on Immunization, HBV is often given together with the live-attenuated vaccine Bacille Calmette–Guérin (BCG), known to activate multiple pattern-recognition receptors. Of note, some clinical studies suggest BCG can enhance efficacy of other vaccines in term newborns. However, little is known about whether BCG can shape Th-polarizing cytokine responses to HBV nor the age-dependency of such effects, including whether they may extend to the preterm. To characterize the effects of BCG on HBV immunogenicity, we studied individual and combined administration of these vaccines to cord newborn and adult human whole blood and mononuclear cells in vitro and to neonatal and adult mice in vivo. Compared to either BCG or HBV alone, (BCG + HBV) synergistically enhanced in vitro whole blood production of IL-1β, while (BCG + HBV) also promoted production of several cytokines/chemokines in all age groups, age-specific enhancement included IL-12p70 in the preterm and GM-CSF in the preterm and term. In human mononuclear cells, (BCG + HBV) enhanced mRNA expression of several genes including CSF2, which contributed to clustering of genes by vaccine treatment via principle component analysis. To assess the impact of BCG on HBV immunization, mice of three different age groups were immunized subcutaneously with, BCG, HBV, (BCG + HBV) into the same site; or BCG and HBV injected into separate sites. Whether injected into a separate site or at the same site, co-administration of BCG with HBV significantly enhanced anti-HBV IgG titers in mice immunized on day of life-0 or -7, respectively, but not in adult mice. In summary, our data demonstrate that innate and adaptive vaccine responses of preterm and term newborns are immunologically distinct. Furthermore, BCG or “BCG-like” adjuvants should be further studied as a promising adjuvantation approach to enhance immunogenicity of vaccines to protect these vulnerable populations.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro . PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4 + T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution.

The human immune system is comprised of cellular and molecular components designed to coordinately prevent infection while avoiding potentially harmful inflammation and auto-immunity. Immunity varies with age, reflecting unique age-dependent challenges including fetal gestation, the neonatal phase and infancy. Herein, we review novel mechanistic insights into early life immunity, with emphasis on emerging models of human immune ontogeny, which may inform age-specific translational development of novel anti-infectives, immunomodulators and vaccines.

Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6−/− mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6−/− independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.

Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

Background Group B Neisseria meningitidis, an endotoxin-producing Gram-negative bacterium, causes the highest incidence of group B meningococcus (MenB) disease in the first year of life. The Bexsero vaccine is indicated in Europe from 8 weeks of age. Endotoxin components of outer membrane vesicles (OMVs) or soluble lipopolysaccharide (LPS) represent a potential source of inflammation and residual reactogenicity. The purpose of this study was to compare novel candidate MenB vaccine formulations with licensed vaccines, including Bexsero, using age-specific human in vitro culture systems. Methods OMVs from wild type- and inactivated lpxL1 gene mutant-N. meningitidis strains were characterized in human neonatal and adult in vitro whole blood assays and dendritic cell (DC) arrays. OMVs were benchmarked against licensed vaccines, including Bexsero and whole cell pertussis formulations, with respect to Th-polarizing cytokine and prostaglandin E2 production, as well as cell surface activation markers (HLA-DR, CD86, and CCR7). OMV immunogenicity was assessed in mice. Results ΔlpxLI native OMVs (nOMVs) demonstrated significantly less cytokine induction in human blood and DCs than Bexsero and most of the other pediatric vaccines (e.g., PedvaxHib, EasyFive, and bacillus Calmette–Guérin) tested. Despite a much lower inflammatory profile in vitro than Bexsero, ΔlpxLI nOMVs still had moderate DC maturing ability and induced robust anti-N. meningitidis antibody responses after murine immunization. Conclusion A meningococcal vaccine comprised of attenuated LPS-based OMVs with a limited inflammatory profile in vitro induces robust antigen-specific immunogenicity in vivo.

Infection is the most common cause of mortality early in life, in large measure due to suboptimal early life vaccination strategies as compared to older age groups. New adjuvants are absolutely cardinal to further optimize current immunization approaches. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is now urgent because of the resurgence of pertussis in many countries, contemporaneous and likely related to the switch from whole cell to acellular vaccines. In this context, age-specific adjuvant strategies may be key to enhance early life immunogenicity by creating a vaccine formulation that induces both robust and persistent immunity (i.e., overcome waning immunity) to B. pertussis. In the present study, we firstly optimized the a) formulation delivery system, b) stability, and c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes. Next, in both adult and neonatal mouse models, we demonstrate that this TLR7/8 adjuvant can overcome neonatal hyporesponsiveness to acellular pertussis vaccination by driving Th1 favoring responses to a licensed acellular vaccine (DTaP). This potent immunization strategy may be of fundamental importance in vaccine development and could represent a new paradigm for effective pertussis immunization in early life.

Dendritic cells (DCs) are professional antigen-presenting cells that play a vital role in shaping adaptive immunity. DC maturation begins when exogenous danger signals bind to the appropriate toll-like receptor (TLR) and initiate expression of cell surface markers and the secretion of cytokines. This process occurs through defined mitogen-activated protein kinase (MAPK) signalling pathways. Of the 13 known mammalian TLRs, lipopolysaccharide (LPS), which activates TLR4, is the most commonly used ligand for the maturation of DCs in vitro. This comprehensive study measures cytokine secretion and cell surface marker expression in murine bone-marrow-derived DCs following maturation with LPS compared to DCs matured with a panel of other TLR-ligands (zymosan A (TLR2/6), PGN (TLR2), poly(I:C) (TLR3), flagellin (TLR5) and CpG-ODN1826 (TLR9)). The role of MAPK signalling pathways in the maturation process was also examined. Results demonstrate that zymosan A and CpG induce comparable cytokine and cell surface marker profiles to LPS. The remaining ligands differed significantly for cytokine and CD40 expression, but not for CD80 and CD86 expression. While there were differences for MAPK signalling pathways for all ligands, the effect of the inhibitors were broadly similar. These findings broaden our knowledge of TLR ligand-matured DCs.

Item does not contain fulltext Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B-cell proliferation and Ab production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naive B-cell class-switch recombination (CSR) and cytokine production. Gene expression levels of most TLRs was similar between newborn and adult B cells, except that newborn naive B cells expressed more TLR9 than adult naive B cells. Neonatal naive B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn naive B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn naive B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early-life immunization.

Background Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization. Objective Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. Methods Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist–encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow–derived DCs enabled benchmarking of the TLR8 agonist–encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25–loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. Results Although alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited TH-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist–adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4+ T-cell numbers. Conclusion TLR8 agonist–encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines., Graphical abstract