Freeze-dried Group Research Articles

Equivalence of freeze-dried and liquid-frozen formulations of MVA-BN as smallpox and mpox vaccine

ABSTRACT Modified Vaccinia Ankara Bavarian Nordic (MVA-BN) as a smallpox and mpox vaccine has been approved in its liquid-frozen (LF) formulation in the US, Canada, and EU. A freeze-dried (FD) formulation may offer additional benefits, such as a longer shelf life and reduced dependence on cold chain storage and transport. In a phase 2 clinical trial, 651 vaccinia-naïve participants were vaccinated with two doses of MVA-BN LF or FD, 4 weeks apart. The objectives were to compare MVA-BN FD with LF in terms of vaccine-induced immune responses, safety, and reactogenicity. Non-inferiority of the immune response was assessed by the 95% CI of the geometric mean ratios. Both formulations induced robust vaccinia-specific humoral and cellular immune responses. At peak humoral responses (Week 6), geometric means of total antibody titers were 1096 (95% CI 1013, 1186) from the FD group and 877 (95% CI 804, 956) from the LF group, achieving the primary endpoint of non-inferiority of MVA-BN FD compared to MVA-BN LF. At peak cellular responses (Week 2), geometric means of T cell spot forming units were 449 (95% CI 341, 590) from the FD group and 316 (95% CI 234, 427) from the LF group. Both formulations of MVA-BN were well tolerated, with similar unsolicited AEs and solicited systemic reactions in both groups but slightly more local reactions in the FD group. No vaccine-related serious adverse events (SAEs) or vaccine-related AE of special interest were reported. The FD formulation of MVA-BN was shown to be equivalent to MVA-BN LF.

Real-time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus

Whether it is better to use viable or non-viable probionts in aquaculture is still a matter of debate. In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated. Three forms of this probiont: (1) heat-killed (HK), (2) live spray (LI) and (3) freeze-dried (FD) were incorporated into a basal (control) diet for rainbow trout O. mykiss. The LI and FD diets are referred to as viable diets. A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments. The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF-β), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real-time polymerase chain reaction. The tested immune genes were up-regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene. The TNF gene was found to be highly (P<0.05) up-regulated (5000-fold) in groups fed both viable forms (LI, FD). With regard to the TGF-β gene, the spleen of the HK and FD groups showed significant up-regulation of 20- and 30-folds respectively. The IFN gene was up-regulated (P<0.05) in all treatments, but more in the viable diet treatments. Kidney and spleen tissues showed similar expression patterns, i.e. all of these genes were up-regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet.

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or −196 °C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1 h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 °C (11%) and +25 °C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (≥10 spikes/h) was higher in the non-dried control group (79%; P < 0.05) than in the FD groups (58, 55, and 54% for storage at −196, +4, and +25 °C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12 h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P < 0.05) resumed meiosis than those injected with +25, +4 and −196 °C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P < 0.05) than those in all the FD groups (34, 27, and 28% for storage at −196, +4, and +25 °C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or −196 °C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.

321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P &lt; 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs