Bohemian Rhapsody of Future Drug Delivery Systems: Rational Changes Necessary for the Next Revolution.

Controlled drug delivery technology has matured for more than 70 years, starting from a twice-a-day oral formulation to 6 month long-acting injectable formulations. Further technological advances require superior formulations to treat various diseases more efficiently. Developing future formulations with practical innovations for treating existing and new diseases necessitates our continued efforts to overcome at least three main hurdles. They include (i) drug delivery with reduced side effects, (ii) long-term treatment of chronic diseases, and (iii) the overcoming of biological barriers. Such efforts start with the improved ability to accurately test drug delivery efficacy using proper controls. Future development can be aided by artificial intelligence if used properly. The next revolution of drug delivery systems will be augmented if implementation is given equal weight as discovery. Such a process can be accelerated with the systemic revamp of the research funding structure and cultivating a new generation of scientists who can think differently.

Treatment efficacy of 532-nm diode laser glottoplasty in patients with sulcus vocalis: a prospective study.

PURPOSE: This study prospectively assessed the efficacy and safety of 532-nm diode laser glottoplasty in patients with sulcus vocalis. METHODS: A prospective human trial was performed from August 2016 to September 2021. 532-nm diode laser glottoplasty was performed in 30 consecutive patients with sulcus vocalis who suffered from voice problems. Patients underwent acoustic aerodynamic, perceptual, stroboscopic, and Voice Handicap Index-10 (VHI-10) evaluations before and 1, 6, and 12 months after laser glottoplasty. RESULTS: Most subjective parameters showed significant improvement (P < 0.05) at 6 months after laser glottoplasty and remained stable at 12 months. Most objective parameters showed significant improvement (P < 0.05) at 12 months after laser glottoplasty. Complications during follow-up included mild vocal fold vibration reduction in 3.3% of patients (1/30) and persistent vocal fold edema in 3.3% of patients (1/30). CONCLUSIONS: Statistically significant voice improvement at 12 months after 532-nm diode laser glottoplasty was achieved without serious complications.

The Impact of Post-Processing Temperature on PLGA Microparticle Properties.

PURPOSE: Biodegradable poly(lactide-co-glycolide) (PLGA) microparticles loaded with either risperidone or naltrexone were prepared from an emulsification homogenization process. The objective of this study was to determine the impact the post-treatment temperature has on the properties and subsequent performance of the microparticles. METHODS: The post-treatment temperature of an ethanolic solution was characterized from 10 ~ 35ºC for the naltrexone and risperidone micropartilces. RESULTS: The wash temperature resulted in a typical triphasic in vitro release pattern at low wash temperatures or a biphasic pattern consisting of an elevated release rate at higher post-treatment temperatures. The post-treatment temperature largely influences the particle morphology, residual solvent levels, glass transition temperature, and drug loading and is molecule dependent, whereby these characteristics subsequently influence the drug release rate. CONCLUSION: The study highlights the importance of both the post-treatment process and control during manufacturing to obtain a formulation within the desired product profile.

Multiphysics Simulation of Local Transport and Absorption Coupled with Pharmacokinetic Modeling of Systemic Exposure of Subcutaneously Injected Drug Solution.

INTRODUCTION: Subcutaneous (SC) injectables have become more acceptable and feasible for administration of biologics and small molecules. However, efficient development of these products is limited to costly and time-consuming techniques, partially because absorption mechanisms and kinetics at the local site of injection remain poorly understood. OBJECTIVE: To bridge formulation critical quality attributes (CQA) of injectables with local physiological conditions to predict systemic exposure of these products. METHODOLOGY: We have previously developed a multiscale, multiphysics computational model to simulate lymphatic absorption and whole-body pharmacokinetics of monoclonal antibodies. The same simulation framework was applied in this study to compute the capillary absorption of solubilized small molecule drugs that are injected subcutaneously. Sensitivity analyses were conducted to probe the impact by key simulation parameters on the local and systemic exposures. RESULTS: This framework was capable of determining which parameters had the biggest impact on small molecule absorption in the SC. Particularly, membrane permeability of a drug was found to have the biggest impact on drug absorption kinetics, followed by capillary density and drug diffusivity. CONCLUSION: Our modelling framework proved feasible in predicting local transport and systemic absorption from the injection site of small molecules. Understanding the effect of these properties and how to model them may help to greatly expedite the development process.

Scanning Analysis of Sequential Semisolvent Vapor Impact To Study Naltrexone Release from Poly(lactide-co-glycolide) Microparticles.

Poly(lactide-co-glycolide) (PLGA)-based microparticle formulations have been a mainstay of long-acting injectable drug delivery applications for decades. Despite a long history of use, tools and techniques to analyze and understand these formulations are still under development. Recently, a new characterization method was introduced known as the surface analysis after sequential semisolvent impact using sequential semisolvent vapors. The vapor-based technique is named, for convenience, surface analysis of (semisolvent) vapor impact (SAVI). In the SAVI method, discretely controlled quantities of selected organic semisolvents in the vapor phase were applied to PLGA microparticles to track particle morphological changes by laser scanning confocal microscopy. Subsequently, the morphological images were analyzed to calculate mean peak height (Sa), core height (Sk), kurtosis (Sku), dale void volume (Vvv), the density of peaks (Spd), maximum height (Hm), and the shape ratio (Rs). Here, the SAVI method was applied to naltrexone-loaded microparticles manufactured internally and Vivitrol, a commercial formulation. SAVI analysis of these microparticles indicated that the two primary mechanisms controlling the naltrexone release were the formation of discrete, self-crystallized portions of naltrexone within the PLGA structure and the degradation of PLGA chains through nucleophilic substitution. The relatively higher amounts of naltrexone crystals resulted in prolonged release than lower amounts of crystals. Data from gel permeation chromatography, differential scanning calorimetry, and in vitro release measurements all point to the importance of naltrexone crystal formation. This study highlights the utility of SAVI for gaining further insights into the microstructure of PLGA formulations and using SAVI data to support research, product development, and quality control applications for microparticle formulations of pharmaceuticals.

Potential Roles of the Glass Transition Temperature of PLGA Microparticles in Drug Release Kinetics.

Poly(lactic-co-glycolic acid) (PLGA) has been used for long-acting injectable drug delivery systems for more than 30 years. The factors affecting the properties of PLGA formulations are still not clearly understood. The drug release kinetics of PLGA microparticles are influenced by many parameters associated with the formulation composition, manufacturing process, and post-treatments. Since the drug release kinetics have not been explainable using the measurable properties, formulating PLGA microparticles with desired drug release kinetics has been extremely difficult. Of the various properties, the glass transition temperature, Tg, of PLGA formulations is able to explain various aspects of drug release kinetics. This allows examination of parameters that affect the Tg of PLGA formulations, and thus, affecting the drug release kinetics. The impacts of the terminal sterilization on the Tg and drug release kinetics were also examined. The analysis of drug release kinetics in relation to the Tg of PLGA formulations provides a basis for further understanding of the factors controlling drug release.

Engineering Quick- and Long-acting Naloxone Delivery Systems for Treating Opioid Overdose.

PURPOSE: Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick- and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. METHODS: The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 µm and 50 µm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with ß-arrestin recruitment assays. RESULTS: The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 µm to 23 µm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 µm to 23 µm helped eliminate the lag phase and obtain the steady-state drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a µ-opioid receptor, to recruit ß-arrestin 2. CONCLUSIONS: Naloxone released from the three selected formulations could inhibit DAMGO-induced ß-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration.

Prevention of Opioid Abuse and Treatment of Opioid Addiction: Current Status and Future Possibilities.

Prescription opioid medications have seen a dramatic rise in misuse and abuse, leading regulators and scientists to develop policies and abuse-deterrent technologies to combat the current opioid epidemic. These abuse-deterrent formulations (ADFs) are intended to deter physical and chemical tampering of opioid-based products, while still providing safe and effective delivery for therapeutic purposes. Even though formulations with varying abuse-deterrent technologies have been approved, questions remain about their effectiveness. While these formulations provide a single means to combat the epidemic, a greater emphasis should be placed on formulations for treatment of addiction and overdose to help those struggling with opioid dependence. This article analyzes various ADFs currently in clinical use and explores potential novel systems for treatment of addiction and prevention of overdose.

Insights and Lessons from a Scientific Conference on Non-Invasive Delivery of Macromolecules.

A growing share of the pharmaceutical development pipeline is occupied by macromolecule drugs, which are primarily administered by injection. Despite decades of attempts, non-invasive delivery of macromolecules has seen only a few success stories. Potential benefits of non-invasive administration include better patient acceptance and adherence and potentially better efficacy and safety. Greater inter-disciplinary dialogue and collaboration are integral to realizing these benefits.

A Fast and Sensitive Method for the Detection of Leuprolide Acetate: A High-Throughput Approach for the In Vitro Evaluation of Liquid Crystal Formulations.

The suitability of using fluorescence spectroscopy to rapidly assay drug release by quantifying the time-dependent increase in total intrinsic protein fluorescence was assessed. Leuprolide acetate, a synthetic nonapeptide analogue of gonadotropin-releasing hormone (GnRH or LHRH), is the active pharmaceutical ingredient used to treat a wide range of sex hormone-related disorders, including advanced prostatic cancer, endometriosis, and precocious puberty. During the in vitro evaluation of drug delivery technologies for leuprolide acetate, one of the most time-consuming steps is the detection and accurate quantification of leuprolide release from formulation candidates. Thus far, the dominant means for leuprolide detection involves conventional multistep high-performance liquid chromatography (HPLC) methods, requiring sampling, dilutions, sample filtration, and chromatography, which can take up to 40 min for each sample. With the increasing demand for assay adaptation to high-throughput format, here we sought to exploit fluorescence spectroscopy as a tool to develop a novel method to rapidly assay the in vitro release of leuprolide acetate. By utilizing the intrinsic fluorescence of the tryptophan (Trp) and tyrosine (Tyr) amino acid residues present in the leuprolide nonapeptide, the in vitro release from liquid crystal formulations was accurately quantified as a function of fluorescence intensity. Here, we demonstrate that assaying leuprolide release using intrinsic protein fluorescence in a 96-well format requiring volumes as low as 100 µL is a cost-effective, rapid, and highly sensitive alternative to conventional HPLC methods. Furthermore, the high signal-to-noise ratios and robust Z'-factors of >0.8 indicate high sensitivity, precision, and feasibility for miniaturization, high-throughput format adaptation, and automation.

Drug Delivery Research: The Invention Cycle.

Controlled drug delivery systems have been successful in introducing improved formulations for better use of existing drugs and novel delivery of biologicals. The initial success of producing many oral products and some injectable depot formulations, however, reached a plateau, and the progress over the past three decades has been slow. This is likely due to the difficulties of formulating hydrophilic, high molecular weight drugs, such as proteins and nucleic acids, for targeting specific cells, month-long sustained delivery, and pulsatile release. Since the approaches that have served well for delivery of small molecules are not applicable to large molecules, it is time to develop new methods for biologicals. The process of developing future drug delivery systems, termed as the invention cycle, is proposed, and it starts with clearly defining the problems for developing certain formulations. Once the problems are well-defined, creative imagination examines all potential options and selects the best answer and alternatives. Then, innovation takes over to generate unique solutions for developing new formulations that resolve the previously identified problems. Ultimately, the new delivery systems will have to go through a translational process to produce the final formulations for clinical use. The invention cycle also emphasizes examining the reasons for success of certain formulations, not just the reasons for failure of many systems. Implementation of the new invention cycle requires new mechanisms of funding the younger generation of scientists and a new way of identifying their achievements, thereby releasing them from the burden of short-termism.

Effects of the Microparticle Shape on Cellular Uptake.

Physical forms of microparticles and nanoparticles, such as the size, charge, and shape, are known to affect endocytosis. Improving the physical designs of the drug carriers can increase the drug uptake efficiency and the subsequent drug efficacy. Simple shapes, such as sphere and cylinder, have been studied for their ability for endocytosis. To have a better understanding of the shape effect on cellular uptake, different particle shapes were prepared, using the keyboard character shapes, and their impacts on cellular uptake were examined. The results showed that shapes with higher aspect ratios and sharper angular features have a higher chance of adhering to the cells and become internalized by the cancer cells. The local interaction between the cell membrane and the part of the microparticle in contact with the cell membrane also plays a crucial role in determining the outcome.

Silymarin-Loaded Nanoparticles Based on Stearic Acid-Modified Bletilla striata Polysaccharide for Hepatic Targeting.

Silymarin has been widely used as a hepatoprotective drug in the treatment of various liver diseases, yet its effectiveness is affected by its poor water solubility and low bioavailability after oral administration, and there is a need for the development of intravenous products, especially for liver-targeting purposes. In this study, silymarin was encapsulated in self-assembled nanoparticles of Bletilla striata polysaccharide (BSP) conjugates modified with stearic acid and the physicochemical properties of the obtained nanoparticles were characterized. The silymarin-loaded micelles appeared as spherical particles with a mean diameter of 200 nm under TEM. The encapsulation of drug molecules was confirmed by DSC thermograms and XRD diffractograms, respectively. The nanoparticles exhibited a sustained-release profile for nearly 1 week with no obvious initial burst. Compared to drug solutions, the drug-loaded nanoparticles showed a lower viability and higher uptake intensity on HepG2 cell lines. After intravenous administration of nanoparticle formulation for 30 min to mice, the liver became the most significant organ enriched with the fluorescent probe. These results suggest that BSP derivative nanoparticles possess hepatic targeting capability and are promising nanocarriers for delivering silymarin to the liver.

Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer.

A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis-aconitic anhydride (CA, a pH-sensitive linker) to obtain PEI-CA-DOX conjugates. The PEI-CA-DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI-CA-DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI-CA-DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma-bearing mice, the PEI-CA-DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long-term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI-CA-DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.

Smart Nanoparticles for Drug Delivery: Boundaries and Opportunities.

Various pharmaceutical particles have been used in developing different drug delivery systems ranging from traditional tablets to state-of-the-art nanoparticle formulations. Nanoparticle formulations are unique in that the small size with huge surface area sometimes provides unique properties that larger particles and bulk materials do not have. Nanoparticle formulations have been used in improving the bioavailability of various drugs, in particular, poorly soluble drugs. Nanoparticle drug delivery systems have found their unique applications in targeted drug delivery to tumors. While nanoparticle formulations have been successful in small animal xenograft models, their translation to clinical applications has been very rare. Developing nanoparticle systems designed for targeted drug delivery, e.g., treating tumors in humans, requires clear understanding of the uniqueness of nanoparticles, as well as limitations and causes of failures in clinical applications. It also requires designing novel smart nanoparticle delivery systems that can increase the drug bioavailability and at the same time reduce the drug's side effects.

Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug.

The importance and advantages of three-dimensional (3D) cell cultures have been well-recognized. Tumor cells cultured in a 3D culture system as multicellular tumor spheroids (MTS) can bridge the gap between in vitro and in vivo anticancer drug evaluations. An in vitro 3D tumor model capable of providing close predictions of in vivo drug efficacy will enhance our understanding, design, and development of better drug delivery systems. Here, we developed an in vitro 3D tumor model by adapting the hydrogel template strategy to culture uniformly sized spheroids in a hydrogel scaffold containing microwells. The in vitro 3D tumor model was to closely simulate an in vivo solid tumor and its microenvironment for evaluation of anticancer drug delivery systems. MTS cultured in the hydrogel scaffold are used to examine the effect of culture conditions on the drug responses. Free MTS released from the scaffold are transferred to a microfluidic channel to simulate a dynamic in vivo microenvironment. The in vitro 3D tumor model that mimics biologically relevant parameters of in vivo microenvironments such as cell-cell and cell-ECM interactions, and a dynamic environment would be a valuable device to examine efficiency of anticancer drug and targeting specificity. These models have potential to provide in vivo correlated information to improve and optimize drug delivery systems for an effective chemotherapy.

FRET imaging reveals different cellular entry routes of self-assembled and disulfide bonded polymeric micelles.

Although nanocarriers hold promise for cancer chemotherapy, their intracellular drug delivery pathways are not fully understood. In particular, the influence of nanocarrier stability on cellular uptake is still uncertain. By physically loading hydrophobic FRET probes, we revealed different intracellular drug delivery routes of self-assembled and disulfide bonded micelles. The self-assembled micelles were structurally dissociated by micelle-membrane interactions, and the hydrophobic probes were distributed on the plasma membrane. Alternatively, intact disulfide bonded micelles carrying hydrophobic probes were internalized into cancer cells via multiple endocytic pathways. Following internalization, disulfide bonded micelles were decomposed in early endosomes by glutathione-mediated disulfide bond reduction, exposing the probes to intracellular organelles.

A Novel Intervention That Prevents Vocal Fold Scarring.

OBJECTIVES: We evaluated the preventive efficacy of stromal vascular fraction (SVF) for vocal fold scar in a rabbit model. STUDY DESIGN: Animal model. METHODS: The study included 40 male New Zealand white rabbits: 20 received vocal fold scar surgery served as normal controls (control group). The other 20 received the same vocal fold scar surgery with SVF injection (SVF group) Histological and high-speed video analyses of vocal fold vibration were performed 4 weeks after scar surgery and SVF injection. The maximum amplitude of vocal fold vibration was used to assess vocal fold vibration. A real-time PCR study was also performed to evaluate the scar regeneration and remodeling including TGF-ß1, IL-6, procollagen-1, MMP-2, 9, and HAS-2, 3. RESULTS: Vocal fold vibration analyses indicated that the maximum amplitude differences in the vibration of the SVF group were significantly higher than the control group. The histological findings showed that the collagen density ratio were significantly lower in the SVF group compared to the control group. Real-time Polymerase Chain Reaction (PCR) study showed significant increases of MMP-2, 9 and HAS-2, 3, and a decrease of TGF-ß1, IL-6, procollagen-1 in the SVF group compared to the control group. CONCLUSIONS: Based on the vocal fold vibration study, histological findings, and real-time PCR study, SVF injection showed preventive activity and improvement of vocal fold vibration for vocal fold scar in a rabbit model.

Preparation and evaluation of injectable microsphere formulation for longer sustained release of donepezil.

In this study, donepezil-loaded PLGA and PLA microspheres (Dp-PLGA-M/Dp-PLA-M) and Dp-PLA-M wrapped in a polyethylene glycol-b-polycaprolactone (PC) hydrogel (Dp-PLA-M/PC) were prepared to reduce the dosing frequency of injections to treat Alzheimer's disease patients. Dp-PLGA-M and Dp-PLA-M with a uniform particle size distribution were repeatably fabricated in nearly quantitative yield and with high encapsulated Dp yields using an ultrasonic atomizer. The injectability and in vitro and in vivo Dp release, biodegradation, and inflammatory response elicited by the Dp-PLGA-M, Dp-PLA-M, and Dp-PLA-M/PC formulations were then compared. All injectable formulations showed good injectability with ease of injection, even flow, and no clogging using a syringe needle under 21-G. The injections required a force of <1 N. According to the biodegradation rate of micro-CT, GPC and NMR analyses, the biodegradation of Dp-PLA-M was slower than that of Dp-PLGA-M, and the biodegradation rate of Dp-PLA-M/PC was also slower. In the Dp release experiment, Dp-PLA-M sustained Dp for longer compared with Dp-PLGA-M. Dp-PLA-M/PC exhibited a longer sustained release pattern of two months. In vivo bioavailability of Dp-PLA-M/PC was almost 1.4 times higher than that of Dp-PLA-M and 1.9 times higher than that of Dp-PLGA-M. The variations in the Dp release patterns of Dp-PLGA-M and Dp-PLA-M were explained by differences in the degradation rates of PLGA and PLA. The sustained release of Dp by Dp-PLA-M/PC was attributed to the fact that the PC hydrogel served as a wrapping matrix for Dp-PLA-M, which could slow down the biodegradation of PLA-M, thus delaying the release of Dp from Dp-PLA-M. Dp-PLGA-M induced a higher inflammatory response compared to Dp-PLA-M/PC, suggesting that the rapid degradation of PLGA triggered a strong inflammatory response. In conclusion, Dp-PLA-M/PC is a promising injectable Dp formulation that could be used to reduce the dosing frequency of Dp injections.

Comparison of humoral and cellular immune responses between ChAd-BNT heterologous vaccination and BNT-BNT homologous vaccination following the third BNT dose: A prospective cohort study.

Introduction: The differential immune responses after two additional BNT162b2 (BNT) booster doses between ChAdOx1 nCoV-10 (ChAd)-primed and BNT-primed groups have not been elucidated. The aim of this study was to compare vaccine-induced humoral and cellular immune responses and evaluate breakthrough infection between the two vaccination strategies. Methods: In 221 healthy subjects (111 in the ChAd group), longitudinal immune responses were monitored at 3, 4, and 6 months after the 2nd dose and 1, 3, and 6 months after the 3rd dose. Humoral immunity was measured by two fully automated chemiluminescent immunoassays (Elecsys and Abbott) and a surrogate virus neutralization test (sVNT). Cellular immunity was assessed by two interferon-γ (IFN-γ) release assays (QuantiFERON SARS-CoV-2 and Covi-FERON). Results: After the 2nd dose of BNT vaccination, total antibody levels were higher in the ChAd group, but IgG antibody and sVNT results were higher in the BNT group. Following the 3rd dose vaccination, binding antibody titers were significantly elevated in both groups (ChAD-BNT; 15.4 to 17.8-fold, BNT-BNT; 22.2 to 24.6-fold), and the neutralizing capacity was increased by 1.3-fold in both cohorts. The ChAd-BNT group had lower omicron neutralization positivity than the BNT-BNT group (P = 0.001) at 6 months after the 3rd dose. Cellular responses to the spike antigen also showed 1.7 to 3.0-fold increases after the 3rd dose, which gradually declined to the levels equivalent to before the 3rd vaccination. The ChAd cohort tended to have higher IFN-γ level than the BNT cohort for 3-6 months after the 2nd and 3rd doses. The frequency of breakthrough infection was higher in the ChAd group (44.8%) than in the BNT group (28.1%) (P = 0.0219). Breakthrough infection induced increased humoral responses in both groups, and increase of cellular response was significant in the ChAd group. Discussion: Our study showed differential humoral and cellular immune responses between ChAd-BNT-BNT heterologous and BNT-BNT-BNT homologous vaccination cohorts. The occurrence of low antibody levels in the ChAd-primed cohort in the humoral immune response may be associated with an increased incidence of breakthrough infections. Further studies are needed on the benefits of enhanced cellular immunity in ChAd-primed cohorts.