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.

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.

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 Vaccinology Approaches for the Development of Adjuvanted Vaccines Targeted to Distinct Vulnerable Populations.

Infection persists as one of the leading global causes of morbidity and mortality, with particular burden at the extremes of age and in populations who are immunocompromised or suffer chronic co-morbid diseases. By focusing discovery and innovation efforts to better understand the phenotypic and mechanistic differences in the immune systems of diverse vulnerable populations, emerging research in precision vaccine discovery and development has explored how to optimize immunizations across the lifespan. Here, we focus on two key elements of precision vaccinology, as applied to epidemic/pandemic response and preparedness, including (a) selecting robust combinations of adjuvants and antigens, and (b) coupling these platforms with appropriate formulation systems. In this context, several considerations exist, including the intended goals of immunization (e.g., achieving immunogenicity versus lessening transmission), reducing the likelihood of adverse reactogenicity, and optimizing the route of administration. Each of these considerations is accompanied by several key challenges. On-going innovation in precision vaccinology will expand and target the arsenal of vaccine components for protection of vulnerable populations.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

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 COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines. Objective: 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: We 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. Results: Mice 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. Conclusion: We 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 summary: Obesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.

Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination.

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 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 TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant 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.

mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant.

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.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

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 (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.

Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells.

Motivation: Vaccines 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 on in vitro screening of human primary leukocytes. Summary: We 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, IFNg 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 potency in vitro and evaluated the top hit, vinblastine sulfate, for adjuvanticity in 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 PBMCs in vitro that yielded a lead compound with in vivo adjuvanticity.

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.

Waning effectiveness of SARS-CoV-2 mRNA vaccines in older adults: a rapid review.

The U.S. Centers for Disease Control and Prevention (CDC) and other health agencies have recently recommended a booster dose of COVID-19 vaccines for specific vulnerable groups including adults 65 years and older. There is limited evidence whether vaccine effectiveness (VE) in older adults decreases over time, especially against severe COVID-19. We performed a rapid review of published studies available through 4 November 2021 that provide effectiveness data on messenger RNA (mRNA) vaccines approved/licensed in the United States and identified eight eligible studies which evaluated VE in older adults. There is evidence of a decline in VE against both severe acute respiratory syndrome coronavirus 2 infection and severe COVID-19 in older adults among studies which analyzed data up to July-October 2021. Our findings suggest that VE diminishes in older adults, which supports the current recommendation for a booster dose in this population.

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.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although 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 required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

The mRNA vaccine BNT162b2 demonstrates impaired TH1 immunogenicity in human elders in vitro and aged mice in vivo.

mRNA vaccines have been key to addressing the SARS-CoV-2 pandemic but have impaired immunogenicity and durability in vulnerable older populations. We evaluated the mRNA vaccine BNT162b2 in human in vitro whole blood assays with supernatants from adult (18-50 years) and elder (≥60 years) participants measured by mass spectrometry and proximity extension assay proteomics. BNT162b2 induced increased expression of soluble proteins in adult blood (e.g., C1S, PSMC6, CPN1), but demonstrated reduced proteins in elder blood (e.g., TPM4, APOF, APOC2, CPN1, and PI16), including 30-85% lower induction of TH1-polarizing cytokines and chemokines (e.g., IFNγ, and CXCL10). Elder TH1 impairment was validated in mice in vivo and associated with impaired humoral and cellular immunogenicity. Our study demonstrates the utility of a human in vitro platform to model age-specific mRNA vaccine activity, highlights impaired TH1 immunogenicity in older adults, and provides rationale for developing enhanced mRNA vaccines with greater immunogenicity in vulnerable populations.

Overlapping Features in Kawasaki Disease-Related Arthritis and Systemic-Onset Juvenile Idiopathic Arthritis: A Nationwide Study in Japan.

Background: Arthritis may occur after the diagnosis of Kawasaki disease (KD). Most cases are self-limiting; however, some patients require prolonged treatment. Method: To characterize KD-related arthritis, 14 patients who required arthritis treatment within 30 days after the diagnosis of KD were recruited from the 23rd KD survey in Japan. Twenty-six additional patients were included from our tertiary center and literature review cohorts. Results: The estimated prevalence of KD-related arthritis in Japan was 48 per 100,000 KD patients. Patients with KD-related arthritis had an older age at onset (52 vs. 28 months, P = 0.002) and higher rate of intravenous immunoglobulin (IVIG) resistance in comparison to those without arthritis (86 vs. 17%, P < 0.001). Among 40 patients, 18 had arthritis in the acute phase KD (continued fever-onset type) and 22 did in the convalescent phase (interval fever-onset type). Both showed a similar rate of complete KD or IVIG response. Interval-type patients required biologics for arthritis control less frequently (5 vs. 39%, P = 0.02) and had a higher 2-year off-treatment rate (100 vs. 43%, P = 0.009) than continued-type ones. Interval-types showed lower serum ferritin and interleukin-18 levels than continued-types. When continued-types were grouped according to whether or not they required biologics (n = 7 and n = 11, respectively), the former subgroup had higher ferritin and interleukin-18 levels (P = 0.01 and 0.02, respectively). A canonical discriminant analysis differentiated interval-type from continued-type with the combination of age, time to arthritis, and the ferritin and matrix metalloproteinase-3 levels. Conclusion: Arthritis requiring treatment is a rare complication of KD. KD-associated arthritis includes interval-type (KD-reactive) and continued-type (true systemic-onset juvenile idiopathic arthritis [JIA] requiring biologics), and overlapping arthritis, suggesting the pathophysiological continuity of autoinflammation between KD and JIA.

The immunoregulatory function of peripheral blood CD71+ erythroid cells in systemic-onset juvenile idiopathic arthritis.

CD71+ erythroid cells (CECs) are recognized to have an immunoregulatory function via direct cell-cell interaction and soluble mediators. Circulating CECs appear in newborns or patients with hemolytic and cardiopulmonary disorders. To assess the biological role of CECs in systemic inflammation, we studied the gene expression and function in systemic-onset juvenile idiopathic arthritis (SoJIA). Peripheral blood mononuclear cells of SoJIA patients expressed upregulated erythropoiesis-related genes. It represented the largest expansion of CECs during active phase SoJIA among other inflammatory diseases. Despite the opposing roles of erythropoietin and hepcidin in erythropoiesis, both serum levels were in concert with the amounts of SoJIA-driven CECs. Circulating CECs counts in inflammatory diseases were positively correlated with the levels of C-reactive protein, IL-6, IL-18, or soluble TNF receptors. Co-culture with active SoJIA-driven CECs suppressed secretions of IL-1ß, IL-6, and IL-8 from healthy donor monocytes. The top upregulated gene in SoJIA-driven CECs was ARG2 compared with CECs from cord blood controls, although cytokine production from monocytes was suppressed by co-culture, even with an arginase inhibitor. CECs are driven to the periphery during the acute phase of SoJIA at higher levels than other inflammatory diseases. Circulating CECs may control excessive inflammation via the immunoregulatory pathways, partly involving arginase-2.