BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis.

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Toxicologic Pathology Forum: mRNA Vaccine Safety-Separating Fact From Fiction.

SARS-CoV-2 spread rapidly across the globe, contributing to the death of millions of individuals from 2019 to 2023, and has continued to be a major cause of morbidity and mortality after the pandemic. At the start of the pandemic, no vaccines or anti-viral treatments were available to reduce the burden of disease associated with this virus, as it was a novel SARS coronavirus. Because of the tremendous need, the development of vaccines to protect against COVID-19 was critically important. The flexibility and ease of manufacture of nucleic acid-based vaccines, specifically mRNA-based products, allowed the accelerated development of COVID-19 vaccines. Although mRNA-based vaccines and therapeutics had been in clinical trials for over a decade, there were no licensed mRNA vaccines on the market at the start of the pandemic. The rapid development of mRNA-based COVID-19 vaccines reduced serious complications and death from the virus but also engendered significant public concerns, which continue now, years after emergency-use authorization and subsequent licensure of these vaccines. This article summarizes and addresses some of the safety concerns that continue to be expressed about these vaccines and their underlying technology.

Toxicologic Pathology Forum: Tissue Evaluation in Nonclinical Toxicity Studies for Prophylactic Vaccines.

Nonclinical toxicity testing (GLP) of prophylactic vaccines to support human clinical trials is outlined in the World Health Organization nonclinical vaccine-development guidelines, which are followed by most regulatory agencies globally. Vaccine GLP toxicity studies include at least two groups: a buffer control (often phosphate-buffered saline) group and a highest anticipated clinical dose formulation group. However, studies may include additional groups, including lower-dose formulation groups and adjuvant-containing formulation control groups. World Health Organization guidelines touch upon expectations for dose group and tissue selection for microscopic evaluation, but there is variation in the interpretation of this aspect of these guidelines between vaccine developers. This opinion piece proposes a scientifically based approach for defining appropriate groups to evaluate in the dosing and recovery phases in nonclinical vaccine toxicity studies, as well as suggestions on selecting tissues for microscopic evaluation at the recovery phase of studies to promote alignment between vaccine manufacturers.

Modeling SARS-CoV-2: Comparative Pathology in Rhesus Macaque and Golden Syrian Hamster Models.

Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.

Review of Current Vaccine Development Strategies to Prevent Coronavirus Disease 2019 (COVID-19).

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak that started in Wuhan, China, in 2019 resulted in a pandemic not seen for a century, and there is an urgent need to develop safe and efficacious vaccines. The scientific community has made tremendous efforts to understand the disease, and unparalleled efforts are ongoing to develop vaccines and treatments. Toxicologists and pathologists are involved in these efforts to test the efficacy and safety of vaccine candidates. Presently, there are several SARS-CoV-2 vaccines in clinical trials, and the pace of vaccine development has been highly accelerated to meet the urgent need. By 2021, efficacy and safety data from clinical trials are expected, and potentially a vaccine will be available for those most at risk. This review focuses on the ongoing SARS-CoV-2 vaccine development efforts with emphasis on the nonclinical safety assessment and discusses emerging preliminary data from nonclinical and clinical studies. It also provides a brief overview on vaccines for other coronaviruses, since experience gained from these can be useful in the development of SARS-CoV-2 vaccines. This review will also explain why, despite this unprecedented pace of vaccine development, rigorous standards are in place to ensure nonclinical and clinical safety and efficacy. [Box: see text].

Peripheral Nerve Microscopic Changes Related to Study Procedures: Two Nonclinical Case Studies.

Peripheral nerves are routinely examined microscopically during the nonclinical safety assessment of therapeutics. In addition to test article-related on- or off-target changes, microscopic changes in peripheral nerves may also be caused by study procedures, such as parenteral test article administration and blood or tissue sampling. We present 2 nonclinical case studies in which nonstandard peripheral nerves had study procedure-related histologic changes. The first case study describes mouse trigeminal nerve changes as a result of blood sampling via retro-orbital sinus puncture. These changes included minimal-to-mild nerve fiber (axonal) degeneration associated with macrophage infiltration. The second case study presents rat brachial plexus changes associated with animal handling and blood sampling. Brachial plexus changes included minimal-to-moderate inflammation, focal hemorrhage, and nerve fiber degeneration. In both cases, the histological changes were morphologically indistinguishable from those that might be due to test article. Therefore, careful consideration of the incidence and severity across groups and a review of study procedures to rule out handling-related nerve damage are essential before identifying a test article-related effect on peripheral nerves. Study design considerations to avoid such procedure-related changes will be discussed, as well as sampling strategies to help distinguish these from test article-related effects.

Evaluation of Rat Acute Phase Proteins as Inflammatory Biomarkers for Vaccine Nonclinical Safety Studies.

The objectives were to characterize the kinetics of acute phase proteins (APPs) α-2 macroglobulin (A2M), α-1 acid glycoprotein (A1AGP), and fibrinogen (FIB), and injection site macroscopic and microscopic findings following intramuscular administration of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (TDaP; Adacel); adjuvants (aluminum phosphate [AlPO4]; aluminum hydroxide, Al[OH]3; CpG/Al[OH]3; or Quillaja saponaria 21 [QS-21]); or saline to female Wistar Han rats. Intravascular lipopolysaccharide (LPS) was a positive control. Injection sites and lymph nodes were evaluated microscopically, using hematoxylin and eosin (H&E) stained sections, 48 hours postdose (HPD) and compared with APP concentrations; A2M and A1AGP were measured using Meso Scale Discovery analyzer. Fibrinogen was measured on STA Compact analyzer. In a time-course study, APP peaked at 24 or 48 HPD. In a subsequent study at 48 HPD, injection site microscopic changes included inflammation and muscle degeneration/necrosis, which was different in severity/nature between groups. The APPs were not increased in rats administered saline, Al(OH)3, or AlPO4. Fibrinogen and A1AGP increased in rats administered CpG/Al(OH)3, QS-21, or TDaP; and A2M increased in rats administered QS-21. Fibrinogen, A2M, and A1AGP increased after LPS administration. Acute phase proteins can be used to monitor inflammatory responses to adjuvants; however, some adjuvants may induce inflammation without higher APPs.

Scientific and Regulatory Policy Committee Points to Consider*: Approaches to the Conduct and Interpretation of Vaccine Safety Studies for Clinical and Anatomic Pathologists.

The design and execution of toxicology studies supporting vaccine development have some unique considerations relative to those supporting traditional small molecules and biologics. A working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee conducted a review of the scientific, technical, and regulatory considerations for veterinary pathologists and toxicologists related to the design and evaluation of regulatory toxicology studies supporting vaccine clinical trials. Much of the information in this document focuses on the development of prophylactic vaccines for infectious agents. Many of these considerations also apply to therapeutic vaccine development (such as vaccines directed against cancer epitopes); important differences will be identified in various sections as appropriate. The topics addressed in this Points to Consider article include regulatory guidelines for nonclinical vaccine studies, study design (including species selection), technical considerations in dosing and injection site collection, study end point evaluation, and data interpretation. The intent of this publication is to share learnings related to nonclinical studies to support vaccine development to help others as they move into this therapeutic area. [Box: see text].

Phenotypic Modulation of Skeletal Muscle Fibers in LPIN1-Deficient Lipodystrophic ( fld) Mice.

Lipin-1 ( Lpin1)-deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the ß-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1-/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid-Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1-/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1-/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

Whole chromosome aneuploidy in the brain of Bub1bH/H and Ercc1-/Δ7 mice.

High levels of aneuploidy have been observed in disease-free tissues, including post-mitotic tissues such as the brain. Using a quantitative interphase-fluorescence in situ hybridization approach, we previously reported a chromosome-specific, age-related increase in aneuploidy in the mouse cerebral cortex. Increased aneuploidy has been associated with defects in DNA repair and the spindle assembly checkpoint, which in turn can lead to premature aging. Here, we quantified the frequency of aneuploidy of three autosomes in the cerebral cortex and cerebellum of adult and developing brain of Bub1b(H/H) mice, which have a faulty mitotic checkpoint, and Ercc1(-/Δ7) mice, defective in nucleotide excision repair and inter-strand cross-link repair. Surprisingly, the level of aneuploidy in the brain of these murine models of accelerated aging remains as low as in the young adult brains from control animals, i.e. <1% in the cerebral cortex and ∼0.1% in the cerebellum. Therefore, based on aneuploidy, these adult mice with reduced life span and accelerated progeroid features are indistinguishable from age-matched, normal controls. Yet, during embryonic development, we found that Bub1b(H/H), but not Ercc1(-/Δ7) mice, have a significantly higher frequency of aneuploid nuclei relative to wild-type controls in the cerebral cortex, reaching a frequency as high as 40.3% for each chromosome tested. Aneuploid cells in these mutant mice are likely eliminated early in development through apoptosis and/or immune-mediated clearance mechanisms, which would explain the low levels of aneuploidy during adulthood in the cerebral cortex of Bub1b(H/H) mice. These results shed light on the mechanisms of removal of aneuploidy cells in vivo.

Translating Mouse Models.

Mice and humans branched from a common ancestor approximately 80 million years ago. Despite this, mice are routinely utilized as animal models of human disease and in drug development because they are inexpensive, easy to handle, and relatively straightforward to genetically manipulate. While this has led to breakthroughs in the understanding of genotype-phenotype relationships and in the identification of therapeutic targets, translation of beneficial responses to therapeutics from mice to humans has not always been successful. In a large part, these differences may be attributed to variations in the alignment of protein expression and signaling in the immune systems between mice and humans. Well-established inbred strains of "The Laboratory Mouse" vary in their immune response patterns as a result of genetic mutations and polymorphisms arising from intentional selection for research relevant traits, and even closely related substrains vary in their immune response patterns as a result of genetic mutations and polymorphisms arising from genetic drift. This article reviews some of the differences between the mouse and human immune system and between inbred mouse strains and shares examples of how these differences can impact the usefulness of mouse models of disease.

Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes.

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.

Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.

Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.

Platelet-activating factor contributes to Bacillus anthracis lethal toxin-associated damage.

The lethal toxin (LeTx) of Bacillus anthracis plays a central role in the pathogenesis of anthrax-associated shock. Platelet-activating factor (PAF) is a potent lipid mediator that has been implicated in endotoxin-associated shock. In this study, we examined the contribution of PAF to the manifestations of lethal toxin challenge in WT mice. LeTx challenge resulted in transient increase in serum PAF levels and a concurrent decrease in PAF acetylhydrolase activity. Inhibition of PAF activity using PAF antagonists or toxin challenge of PAF receptor negative mice reversed or ameliorated many of the pathologic features of LeTx-induced damage, including changes in vascular permeability, hepatic necrosis, and cellular apoptosis. In contrast, PAF inhibition had minimal effects on cytokine levels. Findings from these studies support the continued study of PAF antagonists as potential adjunctive agents in the treatment of anthrax-associated shock.

Helios induces epigenetic silencing of IL2 gene expression in regulatory T cells.

Regulatory T cells (Tregs) play a critical role in maintaining immune tolerance and preventing autoimmune disease. Tregs express the transcription factor Foxp3, which acts as a master regulator of their differentiation and controls their capacity to suppress T cell responses. Tregs have an intrinsically anergic phenotype and do not produce IL-2 or proliferate upon stimulation ex vivo. Recent studies identified that Helios, a member of the Ikaros family of transcription factors, is expressed in Tregs. However, its specific function is not fully understood. In this study, we show that Helios regulates IL-2 production in Tregs by suppressing Il2 gene transcription. Loss of Helios in Tregs breaks their anergic phenotype and results in derepression of the Il2 locus, allowing Tregs to display increased baseline proliferation and to produce IL-2 following stimulation. Conversely, forced expression of Helios in CD4(+)Foxp3(-) T cells results in a loss of their normal ability to produce IL-2. Helios acts by binding to the Il2 promoter and inducing epigenetic modifications that include histone deacetylation. We also show that loss of Helios in Tregs results in decreased Foxp3 binding to the Il2 promoter, indicating that Helios promotes binding of Foxp3 to the Il2 promoter. Interestingly, the loss of Helios in Tregs also causes a decrease in suppressive capacity. Our results identify Helios as a key regulator of Il2 expression in Tregs, contributing to the maintenance of the anergic phenotype.

ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3(-/-) mice.

Reactive oxygen species (ROS) participate in normal intracellular signalling and in many diseases including cancer and aging, although the associated mechanisms are not fully understood. Forkhead Box O (FoxO) 3 transcription factor regulates levels of ROS concentrations, and is essential for maintenance of hematopoietic stem cells. Here, we show that loss of Foxo3 causes a myeloproliferative syndrome with splenomegaly and increased hematopoietic progenitors (HPs) that are hypersensitive to cytokines. These mutant HPs contain increased ROS, overactive intracellular signalling through the AKT/mammalian target of rapamycin signalling pathway and relative deficiency of Lnk, a negative regulator of cytokine receptor signalling. In vivo treatment with ROS scavenger N-acetyl-cysteine corrects these biochemical abnormalities and relieves the myeloproliferation. Moreover, enforced expression of Lnk by retroviral transfer corrects the abnormal expansion of Foxo3(-/-) HPs in vivo. Our combined results show that loss of Foxo3 causes increased ROS accumulation in HPs. In turn, this inhibits Lnk expression that contributes to exaggerated cytokine responses that lead to myeloproliferation. Our findings could explain the mechanisms by which mutations that alter Foxo3 function induce malignancy. More generally, the work illustrates how deregulated ROS may contribute to malignant progression.

The colon: from banal to brilliant.

The colon serves as the habitat for trillions of microbes, which it must maintain, regulate, and sequester. This is managed by what is termed the mucosal barrier. The mucosal barrier separates the gut flora from the host tissues; regulates the absorption of water, electrolytes, minerals, and vitamins; and facilitates host-flora interactions. Colonic homeostasis depends on a complex interaction between the microflora and the mucosal epithelium, immune system, vasculature, stroma, and nervous system. Disruptions in the colonic microenvironment such as changes in microbial composition, epithelial cell function/proliferation/differentiation, mucus production/makeup, immune function, diet, motility, or blood flow may have substantial local and systemic consequences. Understanding the complex activities of the colon in health and disease is important in drug development, as xenobiotics can impact all segments of the colon. Direct and indirect effects of pharmaceuticals on intestinal function can produce adverse findings in laboratory animals and humans and can negatively impact drug development. This review will discuss normal colon homeostasis with examples, where applicable, of xenobiotics that disrupt normal function.

Next-Generation Contraceptive Intravaginal Ring: Comparison of Etonogestrel and Ethinyl Estradiol In Vitro and In Vivo Release from 3D-Printed Intravaginal Ring and NuvaRing.

Intravaginal rings (IVRs) represent a well-established, woman-controlled and sustained vaginal drug delivery system suitable for a wide range of applications. Here, we sought to investigate the differences in etonogestrel (ENG) and ethinyl estradiol (EE) release from a 3D-printed IVR utilizing continuous liquid interface production (CLIP™) (referred to as CLIPLOW for low drug loading and CLIPHIGH IVRs for high drug loading) and NuvaRing, a commercially available injection molded IVR. We conducted in vitro release studies in simulated vaginal fluid to compare the release of ENG and EE from CLIPLOW IVRs and NuvaRing. CLIPLOW IVRs had a similar hormone dose to NuvaRing and exhibited slightly slower ENG release and greater EE release in vitro compared to NuvaRing. When administered to female sheep, NuvaRing demonstrated greater ENG/EE levels in plasma, vaginal tissue and vaginal fluids compared to CLIPLOW IVR despite similar drug loadings. Leveraging observed hormones levels in sheep from NuvaRing as an effective contraceptive benchmark, we developed a long-acting CLIPHIGH IVR with increased ENG and EE doses that demonstrated systemic and local hormone levels greater than the NuvaRing for 90 days in sheep. No signs of toxicity were noted regarding general health, colposcopy, or histological analysis in sheep after CLIPHIGH IVR administration. Our results provided (1) a comparison of ENG and EE release between a 3D-printed IVR and NuvaRing in vitro and in vivo, (2) a preclinical pharmacokinetic benchmark for vaginally delivered ENG and EE and (3) the generation of a 90-day CLIP IVR that will be utilized in future work to support the development of a long-acting ENG/EE IVR combined with an antiretroviral for the prevention of HIV and unplanned pregnancy.