Muscles, Deltoid

A recombinant uncleaved clade C HIV-1 envelope gp140 protein (CN54gp140) produced by Polymun Scientific (Klosterneuburg, Austria) to GMP specification, which has previously been reported to be immunogenic in a number of preclinical and clinical studies (26 (link), 27 (link)), was used for the X001 clinical trial. The vaccine Ag CN54gp140 was administered intramuscularly into the deltoid muscle of the upper arm at a dosage of 100 µg CN54gp140 formulated with 5 µg GLA-AF [Infectious Disease Research Institute, Seattle, WA, USA (28 (link))] in a total volume of 0.4 ml. GLA has been shown to enhance antibody responses in mice, non-human primates, and humans without causing adverse reactions (29 (link), 30 (link)). Good adjuvanticity was observed in a previous influenza clinical trial where they used 2.5 µg GLA and a clinical trial of a leishmaniasis vaccine using 2 or 5 µg GLA in an oil-in-water emulsion formulation (GLA-SE), which was safe and well tolerated (30 (link), 31 (link)). Since much of the reactogenicity of GLA-SE is believed to be associated with its oil-in-water form, the aqueous formulation GLA-AF was assumed to be less reactogenic. Subsequent animal studies revealed that GLA-AF has broadly equivalent adjuvant activity to GLA-SE and based on these findings for the current trial a 5-µg dose of GLA was selected (32 (link)). Further, good tolerability of GLA-AF at a dose of 5 µg in combination with the CN54gp140 Ag has been demonstrated in previous clinical trials conducted by us (27 (link), 33 (link)).

The muscle activations estimated from the musculoskeletal model during the upper-extremity isometric force task were used to calculate synergies. The muscle activations from all force directions were combined into an m × t matrix, V, where m was the number of muscles (i.e., 30) and t was the number of force directions (i.e., 1000). The activations for each muscle were normalized to unit-variance to ensure that the synergies were not biased toward high-variance muscles (Roh et al., 2012 (link)). NNMF was used to calculate synergies (Lee and Seung, 1999 (link); Tresch et al., 1999 (link), 2006 (link)) such that V = W^*C where W is the m × n matrix with n synergies and C is the n × t matrix of synergy activation coefficients. Thus, each column of W represents the weights of each muscle for one synergy, and each row of C represents how much the corresponding synergy was activated or used to generate force in each direction. The number of synergies, n, was set at four to compare to the prior experimental study. The NNMF algorithm was implemented within an iterative optimization which tested random initial estimates of W and C and selected the muscle weights and activation timings that minimized the sum of squared error between V and the muscle activations.
To demonstrate that our simulation was consistent with experimental observation, we first compared the synergies estimated from the musculoskeletal model to the synergies from the experimental protocol reported by Roh et al. (2012 (link)). The experimental protocol included EMG from eight muscles: the brachioradialis, biceps brachii, triceps brachii (long and lateral heads), deltoid (anterior, medial, and posterior fibers), and pectoralis major (clavicular fibers). Thus, for this comparison, we used the activations from the musculoskeletal model for the eight muscles with EMG to calculate synergies using NNMF. We compared the synergies from the musculoskeletal model to the experimental synergies from eight unimpaired subjects. We calculated the similarity of the synergies as the average correlation coefficient. To evaluate if the synergies from the simulation were within the inter-subject variability, we compared the synergies from the musculoskeletal model to the experimental synergies of each subject. We calculated the similarity of the experimental synergies from each subject to one another to evaluate the inter-subject variability. Each subject's synergies were then compared to the simulated synergies to evaluate the similarity between the experimental and simulated synergies. We used an equivalence test to determine if the similarity of the experimental and simulated synergies were within the inter-subject similarity with a significance level of 0.05. For both the inter-subject similarity and similarity between experimental and simulated, we report the 95% confidence intervals.

Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and were conducted with approved animal protocols from the Institutional Animal Care and Use Committee (IACUC) at the research facilities. NHP studies were conducted at the University of Louisiana at Lafayette New Iberia Research Center.
Two cohorts of vaccinated NHPs received a booster immunization after randomizing each group within a cohort based on their baseline characteristics (Fig. 1).
In the mRNA-primed cohort, six adult male and six adult female Mauritius cynomolgus macaques (Macaca fascicularis) aged 4–10 years, selected based on their responses to the primary vaccination, were randomly allocated to three groups of four animals according to their baseline characteristics.
In the subunit-primed cohort, 15 adult male Indian rhesus macaques (Macaca mulatta) aged 4–7 years were randomly allocated to three groups of five animals. In the priming phase, animals received two immunizations of either Sanofi’s mRNA COVID (ancestral D614) experimental candidate vaccines or CoV2 preS dTM-AS03 (ancestral D614) vaccine through the intramuscular route in the deltoid at day 0 and day 21. Seven months after the primary immunization, both cohorts were immunized with CoV2 preS dTM (ancestral)–AS03, CoV2 preS dTM (Beta)–AS03, and a bivalent CoV2 preS dTM (ancestral + Beta)–AS03. All groups received a total dose of 5 µg of CoV2 preS dTM antigen. All immunologic analyses were performed blinded on serum collected at 7, 14, 21, 28, 56, 84 days, and 6 months post-boost injection for D614G and Beta seroneutralizations; on D14, 56, 84, and 6 months for Delta; on D14 and 6 months for Omicron (BA.1), Omicron BA.4/5, and SARS-CoV1. Animal studies were conducted in compliance with all relevant local, state, and federal regulations, and were approved by the New Iberia Research Center.

The groups consisted of three female and three male Indian rhesus macaques (Macaca mulatta), 2–3 years of age, weighing 3–6 kg. Prior to vaccination or bleedings, macaques were ketamine-anesthetized or with a mixture of ketamine/dexmedetomidine with atipamezole reversal and monitored regularly until fully recovered from anesthesia. In brief, macaques were injected IM into the deltoid muscle with 500 µL of mRNA or YF-17D vaccines. mRNA vaccine groups consisted of YF prM-E mRNA-LNP (10 or 20 µg), YF NS1 mRNA-LNP (10 µg), and a bivalent prM-E/NS1 vaccine (10 µg of each mRNA-LNP). The positive control group was vaccinated with a full human dose of the YF-17D licensed vaccine (17D-204, Stamaril^®, Sanofi Pasteur). One animal from the positive control group (animal r18015) presented diarrhea and Campylobacter coli infection and was treated with azithromycin for 5 days before the first vaccination. All mRNA vaccine groups were administered twice at a 4-week interval while the YF-17D vaccine was injected once. After vaccination, the site of injection was monitored for an inflammatory response. Blood draws were performed before the vaccination (weeks –2 and 0), post first vaccination (weeks 1, 3, and 4), post second vaccination (weeks 5, 6, 8, 12, 16, and 24) to collect serum for serology, and for the measurement of cytokines and chemokines before and 16 h after the first and second immunization (Supplementary Fig. 1d).

All clavicles were scanned with IQon Spectral Computed Tomography (CT) (Philips Healthcare, Netherlands) at the University Hospital of Miyazaki (Miyazaki, Japan). A 3D model of the clavicles was reconstructed from CT data using the application software MIMICS 23.0 (Materialise, Leuven, Belgium). Using these data, the bone surface configuration concerning the insertion area of the muscles and the non-attachment area from the model were evaluated.
Generally, a medical device is used to fix clavicle fractures. We selected two types of plates: the anterior plate (VA-LCP Anterior Clavicle Plate, 10 holes, 101 mm, Synthes^®, Tokyo, Japan) and the superior plate (LCP Superior Clavicle Plate, 7 holes, 110 mm, Synthes^®, Tokyo, Japan), the lengths of which were sufficient to cover three or more holes in the proximal and distal parts. Three-dimensional templating was performed on both the superior and anterior clavicle plates using CT data (Fig. 1B). The area of coverage by the anterior and superior plates on the sternocleidomastoid, trapezius, pectoralis major, and deltoid muscles were measured using Materialise 3-matic 15.0 software (Materialise, Leuven, Belgium). The areas covered by these muscles were measured using the following formula: medial length × lateral length and were evaluated statistically.