Structural and componential design: new strategies regulating the behavior of lipid-based nanoparticles in vivo.

Lipid-based nanoparticles have made a breakthrough in clinical disease as delivery systems due to their biocompatibility, thermal and long-term stability, high loading ability, simplicity of preparation, inexpensive production costs, and scalable manufacturing production. In particular, during the COVID-19 pandemic, this delivery system served as a vital vaccine component for virus confrontation. To obtain effective drug delivery, lipid-based nanoparticles should reach the desired sites with high efficiency, enter target cells, and release drugs. The structures and compositions of lipid-based nanoparticles can be modified to regulate these behaviors in vivo to enhance the therapeutic effects. Herein, we briefly review the development of lipid-based nanoparticles, from simple self-assembled nanovesicle-structured liposomes to multifunctional lipid nanoparticles. Subsequently, we summarize the strategies that regulate their tissue distribution, cell internalization, and drug release, highlighting the importance of the structural and componential design. We conclude with insights for further research to advance lipid-based nanotechnology.

Low energy nebulization preserves integrity of SARS-CoV-2 mRNA vaccines for respiratory delivery.

Nebulization of mRNA therapeutics can be used to directly target the respiratory tract. A promising prospect is that mucosal administration of lipid nanoparticle (LNP)-based mRNA vaccines may lead to a more efficient protection against respiratory viruses. However, the nebulization process can rupture the LNP vehicles and degrade the mRNA molecules inside. Here we present a novel nebulization method able to preserve substantially the integrity of vaccines, as tested with two SARS-CoV-2 mRNA vaccines. We compare the new method with well-known nebulization methods used for medical respiratory applications. We find that a lower energy level in generating LNP droplets using the new nebulization method helps safeguard the integrity of the LNP and vaccine. By comparing nebulization techniques with different energy dissipation levels we find that LNPs and mRNAs can be kept largely intact if the energy dissipation remains below a threshold value, for LNP integrity 5-10 J/g and for mRNA integrity 10-20 J/g for both vaccines.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 µg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

High-Precision Synthesis of RNA-Loaded Lipid Nanoparticles for Biomedical Applications.

The recent development of RNA-based therapeutics in delivering nucleic acids for gene editing and regulating protein translation has led to the effective treatment of various diseases including cancer, inflammatory and genetic disorder, as well as infectious diseases. Among these, lipid nanoparticles (LNP) have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA and thereby enhancing the therapeutic potency. These LNP consisting of ionizable lipid, helper lipid, cholesterol, and poly(ethylene glycol)-anchored lipid can stably enclose RNA and help them release into the cells' cytosol. Herein, the significant progress made in LNP research starting from the LNP constituents, formulation, and their diverse applications is summarized first. Moreover, the microfluidic methodologies which allow precise assembly of these newly developed constituents to achieve LNP with controllable composition and size, high encapsulation efficiency as well as scalable production are highlighted. Furthermore, a short discussion on current challenges as well as an outlook will be given on emerging approaches to resolving these issues.

Recent Trends in Lateral Flow Immunoassays with Optical Nanoparticles.

Rapid, accurate, and convenient diagnosis is essential for effective disease management. Various detection methods, such as enzyme-linked immunosorbent assay, have been extensively used, with lateral flow immunoassay (LFIA) recently emerging as a major diagnostic tool. Nanoparticles (NPs) with characteristic optical properties are used as probes for LFIA, and researchers have presented various types of optical NPs with modified optical properties. Herein, we review the literature on LFIA with optical NPs for the detection of specific targets in the context of diagnostics.

Elastin-targeted nanoparticles delivering doxycycline mitigate cytokine storm and reduce immune cell infiltration in LPS-mediated lung inflammation.

BACKGROUND AND PURPOSE: Cytokine storm invoked during acute and chronic lung injury promotes alveolar damage and remodeling. The current study shows that degraded elastin-targeted nanoparticles releasing doxycycline (Doxy NPs) are potent in mitigating cytokines storm, migration of immune cells in the lungs, and inhibiting inflammasome pathways in the LPS mouse model. EXPERIMENTAL APPROACH: Cytokine storm and lung injury were induced using LPS and elastase in C57BL/6 mice (rodent model for emphysema). The mice were then treated with I.V. Doxy NPs, blank NPs, or Doxy a day before LPS administration. Cytokine levels, immune cell population, and MMP activity were analyzed in broncheo-alveolar lavage fluid (BALF) 4 hours after LPS administration. Additionally, gene expression of IL-6, IL-1beta, MCP-1, NLRP3, Caspase 1 and MMPs were investigated in alveolar cells on day 3 after LPS administration. KEY RESULTS: Doxycycline NPs but not Doxycycline significantly decreased IL-6, TNF-α, IL-23 and were significantly more effective in decreasing the percentage of immune cells in the BALF. This is the first in-vivo study to demonstrate that Doxycycline can effectively inhibit inflammasome pathways in the lungs. CONCLUSION AND IMPLICATIONS: IV administration of elastin antibody conjugated Doxycycline-loaded albumin NPs can effectively modulate the local immune environment in the lungs, which is not achieved by IV Doxycycline even at 100-fold higher dose. This novel method of drug delivery can effectively lead to the repurposing of traditional Doxycycline as a potential adjunct treatment for managing the cytokine storm in the lungs in COPD and viral infections.

Biotechnology: Overcoming biological barriers to nucleic acid delivery using lipid nanoparticles.

The promise of therapeutic nucleic acids has long been tempered by difficulty in overcoming biological barriers to their delivery. The past two decades have seen the development of ionizable lipid nanoparticles as a vehicle for nucleic acid delivery and their translation to the clinic.

Optimizing the synthesis of yeast Beta-glucan via response surface methodology for nanotechnology application.

BACKGROUND: The production of biopolymers from waste resources is a growing trend, especially in high-population countries like Egypt. Beta-glucan (ß-glucan) belongs to natural polysaccharides that are derived from plant and microbial origins. In this study, following increasing demands for ß-glucan owing to its bioactive properties, a statistical model to enhance microbial ß-glucan production was evaluated for its usefulness to the food and pharmaceutical industries. In addition, a trial to convert ß-glucan polymer to nanostructure form was done to increase its bioactivity. RESULTS: Ingredients of low-cost media based on agro-industrial wastes were described using Plackett-Burman and central composite design of response surface methodology for optimizing yeast ß-glucan. Minerals and vitamin concentrations significantly influenced ß-glucan yield for Kluyveromyces lactis and nitrogen and phosphate sources for Meyerozyma guilliermondii. The maximum predicted yields of ß-glucan recovered from K. lactis and M. guilliermondii after optimizing the medium ingredients were 407 and 1188 mg/100 ml; respectively. For the first time, yeast ß-glucan nanoparticles (ßGN) were synthesized from the ß-glucan polymer using N-dimethylformamide as a stabilizer and characterized using UV-vis spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The average size of ßGN was about 300 nm as determined by DLS. The quantitative variation of functional groups between ß-glucan polymer and ßGN was evaluated by FT-IR for explaining the difference in their biological activity against Normal Homo sapiens-Hela contaminant and Hepatic cancer cell lines. CONCLUSIONS: Enriching the low-cost media based on agro-industrial wastes with nutritional ingredients improves the yield of yeast ß-glucan. The present study succeeds to form ß-glucan nanoparticles by a simple method.

A budding new approach strengthens an important message.

In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.

Antiviral Activity of Zinc Oxide Nanoparticles against SARS-CoV-2.

The highly contagious SARS-CoV-2 virus is primarily transmitted through respiratory droplets, aerosols, and contaminated surfaces. In addition to antiviral drugs, the decontamination of surfaces and personal protective equipment (PPE) is crucial to mitigate the spread of infection. Conventional approaches, including ultraviolet radiation, vaporized hydrogen peroxide, heat and liquid chemicals, can damage materials or lack comprehensive, effective disinfection. Consequently, alternative material-compatible and sustainable methods, such as nanomaterial coatings, are needed. Therefore, the antiviral activity of two novel zinc-oxide nanoparticles (ZnO-NP) against SARS-CoV-2 was investigated in vitro. Each nanoparticle was produced by applying highly efficient "green" synthesis techniques, which are free of fossil derivatives and use nitrate, chlorate and sulfonate salts as starting materials and whey as chelating agents. The two "green" nanomaterials differ in size distribution, with ZnO-NP-45 consisting of particles ranging from 30 nm to 60 nm and ZnO-NP-76 from 60 nm to 92 nm. Human lung epithelial cells (Calu-3) were infected with SARS-CoV-2, pre-treated in suspensions with increasing ZnO-NP concentrations up to 20 mg/mL. Both "green" materials were compared to commercially available ZnO-NP as a reference. While all three materials were active against both virus variants at concentrations of 10-20 mg/mL, ZnO-NP-45 was found to be more active than ZnO-NP-76 and the reference material, resulting in the inactivation of the Delta and Omicron SARS-CoV-2 variants by a factor of more than 106. This effect could be due to its greater total reactive surface, as evidenced by transmission electron microscopy and dynamic light scattering. Higher variations in virus inactivation were found for the latter two nanomaterials, ZnO-NP-76 and ZnO-NP-ref, which putatively may be due to secondary infections upon incomplete inactivation inside infected cells caused by insufficient NP loading of the virions. Taken together, inactivation with 20 mg/mL ZnO-NP-45 seems to have the greatest effect on both SARS-CoV-2 variants tested. Prospective ZnO-NP applications include an antiviral coating of filters or PPE to enhance user protection.

Lysosomal nanotoxicity: Impact of nanomedicines on lysosomal function.

Although several nanomedicines got clinical approval over the past two decades, the clinical translation rate is relatively small so far. There are many post-surveillance withdrawals of nanomedicines caused by various safety issues. For successful clinical advancement of nanotechnology, it is of unmet need to realize cellular and molecular foundation of nanotoxicity. Current data suggest that lysosomal dysfunction caused by nanoparticles is emerging as the most common intracellular trigger of nanotoxicity. This review analyzes prospect mechanisms of lysosomal dysfunction-mediated toxicity induced by nanoparticles. We summarized and critically assessed adverse drug reactions of current clinically approved nanomedicines. Importantly, we show that physicochemical properties have great impact on nanoparticles interaction with cells, excretion route and kinetics, and subsequently on toxicity. We analyzed literature on adverse reactions of current nanomedicines and hypothesized that adverse reactions might be linked with lysosomal dysfunction caused by nanomedicines. Finally, from our analysis it becomes clear that it is unjustifiable to generalize safety and toxicity of nanoparticles, since different particles possess distinct toxicological properties. We propose that the biological mechanism of the disease progression and treatment should be central in the optimization of nanoparticle design.

Antiviral and antibacterial potential of electrosprayed PVA/PLGA nanoparticles loaded with chlorogenic acid for the management of coronavirus and Pseudomonas aeruginosa lung infection.

Respiratory tract infections are a common cause of morbidity and mortality globally. The current paper aims to treat this respiratory disorder. Therefore, we elucidated the phytochemical profile of Euphorbia milii flowers and isolated chlorogenic acid (CGA) for the first time. The electrospraying technique was utilized to prepare CGA nanoparticles in polyvinyl alcohol (PVA)/PLGA polymeric matrix. Complete in vitro characterizations were performed to determine particle size, polydispersity index (PDI), zeta potential, loading efficiency (LE), scanning electron microscopy and in vitro release study. The optimum formula (F2) with a particle size (454.36 ± 36.74 nm), a surface charge (-4.56 ± 0.84 mV), % of LE (80.23 ± 5.74), an initial burst (29.46 ± 4.79) and % cumulative release (97.42 ± 4.72) were chosen for further activities. In the murine lung infection model, PVA/PLGA NPs loaded with CGA (F2) demonstrated in vivo antibacterial activity against Pseudomonas aeruginosa. Using a plaque assay, the in vitro antiviral activity was investigated. The F2 exhibited antiviral activity against coronavirus (HCoV-229E) and (Middle East respiratory syndrome coronavirus (MERS-CoV), NRCEHKU270). The IC50 of F2 against HCoV-229E and MERS-CoV was 170 ± 1.1 and 223 ± 0.88 µg/mL, respectively. The values of IC50 of F2 were significantly lower (p < .05) than that of free CGA. Therefore, the encapsulation of CGA into electrospray PVA/PLGA NPs would be a promising tool as an antimicrobial agent.

An Efficacy and Mechanism Driven Study on the Impact of Hypoxia on Lipid Nanoparticle Mediated mRNA Delivery.

Hypoxia is a common hallmark of human disease that is characterized by abnormally low oxygen levels in the body. While the effects of hypoxia on many small molecule-based drugs are known, its effects on several classes of next-generation medications including messenger RNA therapies warrant further study. Here, we provide an efficacy- and mechanism-driven study that details how hypoxia impacts the cellular response to mRNA therapies delivered using 4 different chemistries of lipid nanoparticles (LNPs, the frontrunner class of drug delivery vehicles for translational mRNA therapy utilized in the Moderna and Pfizer/BioNTech COVID-19 vaccines). Specifically, our work provides a comparative analysis as to how various states of oxygenation impact LNP-delivered mRNA expression, cellular association, endosomal escape, and intracellular ATP concentrations following treatment with 4 different LNPs across 3 different cell lines. In brief, we first identify that hypoxic cells express less LNP-delivered mRNA into protein than normoxic cells. Next, we identify generalizable cellular reoxygenation protocols that can reverse the negative effects that hypoxia imparts on LNP-delivered mRNA expression. Finally, mechanistic studies that utilize fluorescence-activated cell sorting, confocal microscopy, and enzyme inhibition reveal that decreases in mRNA expression correlate with decreases in intracellular ATP (rather than with differences in mRNA LNP uptake pathways). In presenting this data, we hope that our work provides a comprehensive efficacy and mechanism-driven study that explores the impact of differential oxygenation on LNP-delivered mRNA expression while simultaneously establishing fundamental criteria that may one day be useful for the development of mRNA drugs to treat hypoxia-associated disease.

Mitochondria-Targeted Fluorescent Nanoparticles with Large Stokes Shift for Long-Term BioImaging.

Mitochondria (MITO) play a significant role in various physiological processes and are a key organelle associated with different human diseases including cancer, diabetes mellitus, atherosclerosis, Alzheimer's disease, etc. Thus, detecting the activity of MITO in real time is becoming more and more important. Herein, a novel class of amphiphilic aggregation-induced emission (AIE) active probe fluorescence (AC-QC nanoparticles) based on a quinoxalinone scaffold was developed for imaging MITO. AC-QC nanoparticles possess an excellent ability to monitor MITO in real-time. This probe demonstrated the following advantages: (1) lower cytotoxicity; (2) superior photostability; and (3) good performance in long-term imaging in vitro. Each result of these indicates that self-assembled AC-QC nanoparticles can be used as effective and promising MITO-targeted fluorescent probes.

PEGylated Lipid Nanoparticle Formulations: Immunological Safety and Efficiency Perspective.

Lipid nanoparticles (LNPs) have been recognized as efficient vehicles to transport a large variety of therapeutics. Currently in the spotlight as important constituents of the COVID-19 mRNA vaccines, LNPs play a significant role in protecting and transporting mRNA to cells. As one of their key constituents, polyethylene glycol (PEG)-lipid conjugates are important in defining LNP physicochemical characteristics and biological activity. PEGylation has proven particularly efficient in conferring longer systemic circulation of LNPs, thus greatly improving their pharmacokinetics and efficiency. Along with revealing the benefits of PEG conjugates, studies have revealed unexpected immune reactions against PEGylated nanocarriers such as accelerated blood clearance (ABC), involving the production of anti-PEG antibodies at initial injection, which initiates accelerated blood clearance upon subsequent injections, as well as a hypersensitivity reaction referred to as complement activation-related pseudoallergy (CARPA). Further, data have been accumulated indicating consistent yet sometimes controversial correlations between various structural parameters of the PEG-lipids, the properties of the PEGylated LNPs, and the magnitude of the observed adverse effects. Detailed knowledge and comprehension of such correlations are of foremost importance in the efforts to diminish and eliminate the undesirable immune reactions and improve the safety and efficiency of the PEGylated medicines. Here, we present an overview based on analysis of data from the CAS Content Collection regarding the PEGylated LNP immunogenicity and overall safety concerns. A comprehensive summary has been compiled outlining how various structural parameters of the PEG-lipids affect the immune responses and activities of the LNPs, with regards to their efficiency in drug delivery. This Review is thus intended to serve as a helpful resource in understanding the current knowledge in the field, in an effort to further solve the remaining challenges and to achieve full potential.

Rational design of anti-inflammatory lipid nanoparticles for mRNA delivery.

Lipid nanoparticles (LNPs) play a crucial role in delivering messenger RNA (mRNA) therapeutics for clinical applications, including COVID-19 mRNA vaccines. While mRNA can be chemically modified to become immune-silent and increase protein expression, LNPs can still trigger innate immune responses and cause inflammation-related adverse effects. Inflammation can in turn suppress mRNA translation and reduce the therapeutic effect. Dexamethasone (Dex) is a widely used anti-inflammatory corticosteroid medication that is structurally similar to cholesterol, a key component of LNPs. Here, we developed LNP formulations with anti-inflammatory properties by partially substituting cholesterol with Dex as a means to reduce inflammation. We demonstrated that Dex-incorporated LNPs effectively abrogated the induction of tumor necrosis factor alpha (TNF-ɑ) in vitro and significantly reduced its expression in vivo. Reduction of inflammation using this strategy improved in vivo mRNA expression in mice by 1.5-fold. Thus, we envision that our Dex-incorporated LNPs could potentially be used to broadly to reduce the inflammatory responses of LNPs and enhance protein expression of a range of mRNA therapeutics.

Lipid nanoparticle-mediated drug delivery to the brain.

Lipid nanoparticles (LNPs) have revolutionized the field of drug delivery through their applications in siRNA delivery to the liver (Onpattro) and their use in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While LNPs have been extensively studied for the delivery of RNA drugs to muscle and liver targets, their potential to deliver drugs to challenging tissue targets such as the brain remains underexplored. Multiple brain disorders currently lack safe and effective therapies and therefore repurposing LNPs could potentially be a game changer for improving drug delivery to cellular targets both at and across the blood-brain barrier (BBB). In this review, we will discuss (1) the rationale and factors involved in optimizing LNPs for brain delivery, (2) ionic liquid-coated LNPs as a potential approach for increasing LNP accumulation in the brain tissue and (3) considerations, open questions and potential opportunities in the development of LNPs for delivery to the brain.

Lipids and Lipid Derivatives for RNA Delivery.

RNA-based therapeutics have shown great promise in treating a broad spectrum of diseases through various mechanisms including knockdown of pathological genes, expression of therapeutic proteins, and programmed gene editing. Due to the inherent instability and negative-charges of RNA molecules, RNA-based therapeutics can make the most use of delivery systems to overcome biological barriers and to release the RNA payload into the cytosol. Among different types of delivery systems, lipid-based RNA delivery systems, particularly lipid nanoparticles (LNPs), have been extensively studied due to their unique properties, such as simple chemical synthesis of lipid components, scalable manufacturing processes of LNPs, and wide packaging capability. LNPs represent the most widely used delivery systems for RNA-based therapeutics, as evidenced by the clinical approvals of three LNP-RNA formulations, patisiran, BNT162b2, and mRNA-1273. This review covers recent advances of lipids, lipid derivatives, and lipid-derived macromolecules used in RNA delivery over the past several decades. We focus mainly on their chemical structures, synthetic routes, characterization, formulation methods, and structure-activity relationships. We also briefly describe the current status of representative preclinical studies and clinical trials and highlight future opportunities and challenges.

Improvement and characterization of oral absorption behavior of clofazimine by SNEDDS: Quantitative evaluation of extensive lymphatic transport.

Clofazimine, an anti-leprosy drug, has been anticipated for a candidate to treat tuberculosis, cryptosporidiosis, and coronavirus infection, but its low oral bioavailability is considered a reason for its limited activity. In the current study, we have tried to improve the oral bioavailability of clofazimine by several SNEDDS formulations and characterized the absorption behavior from various aspects. Among four SNEDDS formulations prepared, SNEDDS A, prepared with castor oil as an oil component, provided the highest bioavailability (around 61%) and SNEDDS D, prepared with Capryol 90, gave the second highest bioavailability. SNEDDS A formed the finest nanoparticles, which were maintained under gastric and intestinal luminal conditions. The comparison in oral bioavailability between the SNEDDS formulation and its corresponding preformed nanoemulsion suggested that SNEDDS A would efficiently form nanoemulsion in the gastrointestinal tract after oral administration. AUC of mesenteric lymph node concentration was the highest for SNEDDS A, which would be one of the reasons for SNEDDS A to reveal the highest oral bioavailability. A cycloheximide-treated oral absorption study and single-pass perfusion study by utilizing a vascular-luminal perfused small intestine-liver preparation clearly indicated that over 90% of clofazimine absorbed to systemic circulation should be derived from lymphatic transport for both SNEDDS A and D. Furthermore, the fraction of dose absorbed was around 65% for SNEDDS D, but SNEDDS A achieved around 94%, indicating the excellent performance of SNEDDS A.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates.

While the rapid development of COVID-19 vaccines has been a scientific triumph, the need remains for a globally available vaccine that provides longer-lasting immunity against present and future SARS-CoV-2 variants of concern (VOCs). Here, we describe DCFHP, a ferritin-based, protein-nanoparticle vaccine candidate that, when formulated with aluminum hydroxide as the sole adjuvant (DCFHP-alum), elicits potent and durable neutralizing antisera in non-human primates against known VOCs, including Omicron BQ.1, as well as against SARS-CoV-1. Following a booster ~one year after the initial immunization, DCFHP-alum elicits a robust anamnestic response. To enable global accessibility, we generated a cell line that can enable production of thousands of vaccine doses per liter of cell culture and show that DCFHP-alum maintains potency for at least 14 days at temperatures exceeding standard room temperature. DCFHP-alum has potential as a once-yearly (or less frequent) booster vaccine, and as a primary vaccine for pediatric use including in infants.