Repeated-Dose Toxicity, Biodistribution, and Shedding Assessments With a ChAd155 Respiratory Syncytial Virus Vaccine Candidate Evaluated in Rabbits and Rats.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections (LRTI) in infants, and toddlers and vaccines are not yet available. A pediatric RSV vaccine (ChAd155-RSV) is being developed to protect infants against RSV disease. The ChAd155-RSV vaccine consists of a recombinant replication-deficient chimpanzee-derived adenovirus (ChAd) group C vector engineered to express the RSV antigens F, N, and M2-1. The local and systemic effects of three bi-weekly intramuscular injections of the ChAd155-RSV vaccine was tested in a repeated-dose toxicity study in rabbits. After three intramuscular doses, the ChAd155-RSV vaccine was considered well-tolerated. Changes due to the vaccine-elicited inflammatory reaction/immune response were observed along with transient decreases in platelet count without physiological consequences, already reported for other adenovirus-based vaccines. In addition, the biodistribution and shedding of ChAd155-RSV were also characterized in two studies in rats. The distribution and persistence of the ChAd155-RSV vaccine candidate was consistent with other similar adenovector-based vaccines, with quantifiable levels of ChAd155-RSV observed at the injection site (muscle) and the draining lymph nodes up to 69 days post administration. The shedding results demonstrated that ChAd155-RSV was generally not detectable in any secretions or excreta samples. In conclusion, the ChAd155-RSV vaccine was well-tolerated locally and systemically.

Lack of effects on female fertility or pre- and postnatal development of offspring in rats after exposure to AS03-adjuvanted recombinant plant-derived virus-like particle vaccine candidate for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted tens of millions of people in a worldwide pandemic. A recently developed recombinant Plant-Derived Virus-Like Particle Vaccine candidate for COVID-19 (CoVLP) formulated with AS03 has been shown to be well-tolerated and highly immunogenic in healthy adults. Since the target population for the vaccine includes women of childbearing potential, the objective of the study was to evaluate any untoward prenatal and postnatal effects of AS03-adjuvanted CoVLP administered intramuscularly to Sprague-Dawley female rats before cohabitation for mating (22 and 8 days prior) and during gestation (Gestation Days [GD] 6 and 19). The embryo-fetal development (EFD) cohort was subjected to cesarean on GD 21 and the pre/post-natal (PPN) cohort was allowed to naturally deliver. Effects of AS03-adjuvanted CoVLP was evaluated on pregnant rats, embryo-fetal development (EFD), during parturition, lactation and the development of the F1 offspring up to weaning Vaccination with AS03-adjuvanted CoVLP induced an antibody response in F0 females and anti-SARS-CoV-2 spike-specific maternal antibodies were detected in the offspring at the end of the gestation and lactation periods. Overall, there was no evidence of untoward effects of AS03-adjuvanted CoVLP on the fertility or reproductive performance of the vaccinated F0 females. There was no evidence of untoward effects on embryo-fetal development (including teratogenicity), or early (pre-weaning) development of the F1 offspring. These results support the acceptable safety profile of the AS03-adjuvanted CoVLP vaccine for administration to women of childbearing potential.

Chimeric hemagglutinin supra-seasonal universal influenza vaccine candidates administered sequentially by the intramuscular route are locally and systemically well-tolerated in rabbits.

Sequential intramuscular immunization with chimeric hemagglutinins (cHA) composed of the same conserved HA stalk domain and distinct HA heads is a proposed strategy to produce a supra-seasonal universal influenza vaccine. To evaluate the local tolerance and the local and systemic effects of this strategy, two studies were performed in rabbits. In the first study, two different split virion monovalent cHA vaccines, containing cH5/1N1 and cH8/1N1, with or without AS01 or AS03, were injected at a two-week interval. In the second study, animals were given these vaccines and two weeks later an additional dose of split virion monovalent cHA vaccine containing cH11/1N1, with or without AS01 or AS03. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation, and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed three days after the last dose and after a treatment-free recovery period. The treatment-related changes included body weight loss and food consumption decrease, increases in neutrophil count, C-reactive protein and fibrinogen levels. Microscopic signs of inflammation at the injection sites and immune stimulation of the draining lymph nodes and spleen were also noticed. Most post-injection findings could be linked to the transient inflammation due to the establishment of the desired vaccine-elicited immune response, and were mainly observed in the adjuvanted groups. In conclusion, the sequential administration of different cHA vaccines was locally and systemically well-tolerated in rabbits.

Nonclinical safety assessment of repeated administration and biodistribution of ChAd3-EBO-Z Ebola candidate vaccine.

ChAd3-EBO-Z is an investigational adenovirus-based vaccine for the prevention of Ebola virus disease. Two nonclinical studies were performed to evaluate the biodistribution, local tolerance and potential local and systemic toxic effects of this vaccine. In the biodistribution study, rats received a single intramuscular injection of either ChAd3-EBO-Z or saline. Enlargement of the draining lymph nodes, starting on day 2, was noticed in ChAd3-EBO-Z-treated rats, indicating that an immune response had taken place. Viral DNA was mainly found at the injection sites and in the draining lymph nodes, from where it progressively disappeared during the observation period, while it was found only transiently and occasionally in other organs. In the repeated-dose toxicity study, either ChAd3-EBO-Z or saline was administered intramuscularly to rabbits on two occasions with a 2-week interval. General health status, rectal temperature, local tolerance, ophthalmology, hematology, coagulation and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed. Treatment-related changes included a transient increase in neutrophil count, C-reactive protein and fibrinogen levels, and a transient decrease in platelet count. As expected, microscopic observations 3 days after the second injection were related to the elicited inflammatory reaction, and these inflammatory responses had almost completely disappeared 29 days after the second immunization. In conclusion, the vaccine was locally and systemically well-tolerated and the viral vector was partially or totally cleared from the organs where it disseminated, supporting the clinical development of the vaccine.

Oral Vaccination with Replication-Competent Adenovirus in Mice Reveals Dissemination of the Viral Vaccine beyond the Gastrointestinal Tract.

Since the 1970s, replication-competent human adenoviruses 4 and 7 have been used as oral vaccines to protect U.S. soldiers against the severe respiratory diseases caused by these viruses. These vaccines are thought to establish a digestive tract infection conferring protection against respiratory challenge through antibodies. The success of these vaccines makes replication-competent adenoviruses attractive candidates for use as oral vaccine vectors. However, the inability of human adenoviruses to replicate efficiently in laboratory animals has hampered the study of such vectors. Here, we used mouse adenovirus type 1 (MAV-1) in mice to study oral replication-competent adenovirus-based vaccines. We show that MAV-1 oral administration provides protection that recapitulates the protection against homologous respiratory challenge observed with adenovirus 4 and 7 vaccines. Moreover, live oral MAV-1 vaccine better protected against a respiratory challenge than inactivated vaccines. This protection was linked not only with the presence of MAV-1-specific antibodies but also with a better recruitment of effector CD8 T cells. However, unexpectedly, we found that such oral replication-competent vaccine systemically spread all over the body. Our results therefore support the use of MAV-1 to study replication-competent oral adenovirus-based vaccines but also highlight the fact that those vaccines can disseminate widely in the body.IMPORTANCE Replication-competent adenoviruses appear to be promising vectors for the development of oral vaccines in humans. However, the study and development of these vaccines suffer from the lack of any reliable animal model. In this study, mouse adenovirus type 1 was used to develop a small-animal model for oral replication-competent adenovirus vaccines. While this model reproduced in mice what is observed with human adenovirus oral vaccines, it also highlighted that oral immunization with such a replication-competent vaccine is associated with the systemic spread of the virus. This study is therefore of major importance for the future development of such vaccine platforms and their use in large human populations.

Signal management in pharmacovigilance and human risk assessment of CpG 7909, integrating embryo-fetal and post-natal developmental toxicity studies in rats and rabbits.

The potential reproductive and developmental toxicity of the synthetic oligodeoxynucleotide (ODN) CpG 7909, a component of GSK's AS15 immunostimulant, was examined in rat and rabbit studies following intermittent intramuscular injections. Previous studies using subcutaneous and intraperitoneal injections in mice, rats and rabbits revealed that CpG ODNs induced developmental effects. To analyze the safety signal, GSK conducted additional animal studies using the intended clinical route of administration. CpG 7909 injections were administered intramuscularly to rats or rabbits 28 and 14days before pairing, on 4 or 5 occasions during gestation, and on lactation day 7. The No Observed Adverse Effect Level for female fertility, embryo-fetal and pre- and post-natal development was 4.2mg/kg in both species, approximately 500-fold higher than the anticipated human dose. In conclusion, the anticipated risk to humans is considered low for sporadic intramuscular exposure to CpG 7909.

Intramuscularly administered herpes zoster subunit vaccine has no effects on fertility, pre- and post-natal development in Sprague-Dawley rats.

The herpes zoster subunit vaccine (HZ/su) is an investigational vaccine for the prevention of shingles, a disease caused by the varicella zoster virus (VZV). It is composed of recombinant VZV glycoprotein E (gE) and AS01. We assessed the potential toxic effects of gE/AS01 and AS01 alone on female and male fertility, and on embryo-fetal, pre- and post-natal development in Sprague-Dawley rats. Females were immunized before pairing and during gestation. Half of the pregnant rats were used for embryo-fetal investigations. The ones that gave birth were immunized during lactation and offspring were analysed. In a male fertility study, rats were immunized before pairing. After mating, the untreated females were sacrificed and the fetuses examined. In addition, male fertility parameters were evaluated. Results indicated that female mating performance and fertility, pre- and post-natal survival and offspring development, male mating performance and fertility were unaffected by intramuscular administration of the zoster candidate vaccine gE/AS01.

Evaluation of the potential effects of AS03-adjuvanted A(H1N1)pdm09 vaccine administration on the central nervous system of non-primed and A(H1N1)pdm09-primed cotton rats.

An increased risk of narcolepsy following administration of an AS03-adjuvanted A(H1N1)pdm09 pandemic influenza vaccine (Pandemrix™) was described in children and adolescents in certain European countries. We investigated the potential effects of administration of the AS03-adjuvanted vaccine, non-adjuvanted vaccine antigen and AS03 Adjuvant System alone, on the central nervous system (CNS) in one-month-old cotton rats. Naïve or A(H1N1)pdm09 virus-primed animals received 2 or 3 intramuscular injections, respectively, of test article or saline at 2-week intervals. Parameters related to systemic inflammation (hematology, serum IL-6/IFN-γ/TNF-α) were assessed. Potential effects on the CNS were investigated by histopathological evaluation of brain sections stained with hematoxylin-and-eosin, or by immunohistochemical staining of microglia, using Iba1 and CD68 as markers for microglia identification/activation, albumin as indicator of vascular leakage, and hypocretin. We also determined cerebrospinal fluid (CSF) hypocretin levels and hemagglutination-inhibiting antibody titers. Immunogenicity of the AS03-adjuvanted A(H1N1)pdm09 pandemic influenza vaccine was confirmed by the induction of hemagglutination-inhibiting antibodies. Both AS03-adjuvanted vaccine and AS03 alone activated transient innate (neutrophils/eosinophils) immune responses. No serum cytokines were detected. CNS analyses revealed neither microglia activation nor inflammatory cellular infiltrates in the brain. No differences between treatment groups were detected for albumin extravascular leakage, CSF hypocretin levels, numbers of hypocretin-positive neuronal bodies or distributions of hypocretin-positive axonal/dendritic projections. Consequently, there was no evidence that intramuscular administration of the test articles promoted inflammation or damage in the CNS, or blood-brain barrier disruption, in this model.

Non-clinical safety assessment of single and repeated administration of gE/AS01 zoster vaccine in rabbits.

HZ/su is an investigational recombinant subunit vaccine for the prevention of shingles, a disease resulting from the reactivation of varicella zoster virus. The vaccine is composed of recombinant varicella zoster virus glycoprotein E (gE), and liposome-based Adjuvant System AS01. To evaluate the potential local and systemic effects of this vaccine, three studies were performed in rabbits. In the first two studies, rabbits received a single intramuscular (IM; study 1) or subcutaneous (SC; study 2) dose of gE/AS01, AS01 alone (in study 2 only) or saline, and the local tolerance was evaluated up to 3 days after administration. Under these conditions, only local inflammatory reactions at the injection sites were detected by microscopic evaluation. In the third study, gE/AS01, AS01 alone or saline, were injected SC or IM on four occasions at 2 week intervals. General health status, local tolerance, ophthalmology, haematology and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed after termination of the study. The only treatment-related changes included a transient increase in neutrophils, C-reactive protein and fibrinogen levels and microscopic signs of inflammation at the injection sites, which are expected observations related to the elicited inflammatory reaction. The SC and IM routes of administration produced similar systemic effects. However, microscopic findings at the injection sites differed. One month after the last injection, recovery was complete in all groups. In conclusion, the single and repeated SC and IM administration of the gE/AS01 vaccine were locally and systemically well-tolerated in rabbits and support the clinical development of the vaccine. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Adjuvant Systems on the cardiovascular and respiratory functions in telemetered conscious dogs and anaesthetised rats.

Adjuvants Systems (AS) containing immunostimulant combinations are used in human vaccines. Safety pharmacology studies evaluated the cardiorespiratory effects of AS in conscious telemetered dogs and in anaesthetised rats. Sixteen telemetered beagle dogs (4/group) received intramuscular injections of saline at Day 0, and one clinical dose of AS01, AS03, AS04 or AS15 at Day 7 (7× the equivalent human dose on a bodyweight basis). Bodyweights were measured through Day 14 and cardiorespiratory parameters and body temperature through 72 h post-treatment. Anaesthetised rats (4/group) received one intravenous injection of AS01, AS03 or AS15 at 1 mL/kg bodyweight (140× the equivalent human dosages), or saline. Cardiorespiratory parameters were measured for 120 min post-dose. In dogs, food consumption and mean bodyweight decreased with AS03, and mean body temperature slightly increased with AS01, AS03 and AS15, but were not considered to be adverse. Cardiovascular effects (a slight, reversible increase in mean heart rate and shortened mean RR/PR/QT-intervals) were observed with AS15. No relevant clinical effects or effects on QRS-complex/QTc-interval durations, arterial pressure or respiratory parameters were observed. In rats, there were no consistent treatment-related effects. Collectively, this suggests that AS01, AS03, AS04 and AS15 are not associated with potentially deleterious effects on ventricular repolarisation, atrio/intra-ventricular conductivities or respiratory functions.

Non-clinical safety and biodistribution of AS03-adjuvanted inactivated pandemic influenza vaccines.

Pandemic-influenza vaccines containing split-inactivated-virus antigen have been formulated with the immunostimulatory Adjuvant System AS03 to enhance the antigen immunogenicity and reduce antigen content per dose. AS03 is an oil-in-water emulsion containing α-tocopherol, squalene and polysorbate 80. To support the clinical development of AS03-adjuvanted pandemic-influenza vaccines, the local and systemic toxicity of test articles containing split-influenza A(H5N1) and/or AS03 were evaluated after 3-4 intramuscular (i.m.) injections in rabbits. Treatment-related effects were restricted to mild inflammatory responses and were induced primarily by the test articles containing AS03. The injection-site inflammation was mild at 3 days, and minimal at 4 weeks after the last injection; and was reflected by signs of activation in the draining lymph nodes and by systemic effects in the blood including a transient increase of neutrophils. In addition, a study in mice explored the biodistribution of A(H5N1) vaccines or AS03 through radiolabelling the antigen or constituents of AS03 prior to injection. In this evaluation, 57-73% of AS03's principal constituents had cleared from the injection site 3 days after injection, and their different clearance kinetics were suggestive of AS03's dissociation. All these AS03 constituents entered into the draining lymph nodes within 30 min after injection. In conclusion, the administration of repeated doses of the H5N1/AS03 vaccine was well tolerated in the rabbit, and was primarily associated with transient mild inflammation at the injection site and draining lymph nodes. The biodistribution kinetics of AS03 constituents in the mouse were consistent with AS03 inducing this pattern of inflammation.

Non-clinical safety assessment of single and repeated intramuscular administration of a human papillomavirus-16/18 vaccine in rabbits and rats.

The human papillomavirus (HPV)-16/18 vaccine (Cervarix®) is a prophylactic vaccine for the prevention of cervical cancer. The vaccine contains recombinant virus-like particles assembled from the L1 major capsid proteins of the cervical cancer-causing viral types HPV-16 and HPV-18, and Adjuvant System 04 (AS04), which contains the immunostimulant MPL and aluminium salt. To evaluate potential local and systemic toxic effects of the HPV-16/18 vaccine or AS04 alone, three repeated-dose studies were performed in rabbits and rats. One rabbit study also included a single-dose evaluation. In rabbits (~2.5 kg), the full human dose (HD) of the vaccine was evaluated (0.5 ml per injection site), and in rats (~250 g), 1/5 HD of vaccine was evaluated, corresponding to ≥ 12 times the dosage in humans relative to body weight. In both animal models, the treatment-related changes included a slight transient increase in the number of circulating neutrophils as well as a local inflammatory reaction at the injection site. These treatment-related changes were less pronounced after four doses of AS04 alone than after four doses of the HPV-16/18 vaccine. Additional treatment-related changes in the rat included lower albumin/globulin ratios and microscopic signs of inflammation in the popliteal lymph nodes. In both animal models, 13 weeks after the fourth dose, recovery was nearly complete, although at the injection site in some animals there were signs of discoloration, muscle-fibre regeneration and focal points of macrophage infiltration. Therefore, in these non-clinical models, the single and repeated dose administrations of the HPV-16/18 vaccine or AS04 alone were safe and well tolerated.

Local tolerance and systemic toxicity of single and repeated intramuscular administrations of two different formulations of the RTS,S malaria candidate vaccine in rabbits.

RTS,S malaria antigen is weakly immunogenic as such and needs to be formulated with an adjuvant to improve the magnitude and duration of the immune responses to RTS,S. Two Adjuvant Systems, AS01 and AS02 were evaluated during the development of the RTS,S vaccine. The evaluation included non-clinical studies in rabbits to evaluate the local intramuscular tolerance following administration on a single occasion, and the local and systemic effects following repeated administrations of RTS,S/AS01 or RTS,S/AS02 formulations. In the first study, rabbits were injected on one occasion with RTS,S/AS01, RTS,S/AS02 or controls, and the local intramuscular tolerance was evaluated up to 3 days after injection. In the second study, the different formulations were injected on Days 0, 14, 28 and 42. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Macroscopic and microscopic evaluations were made after termination of the study. No sign of toxicity was detected following single or repeated administrations of the adjuvanted RTS,S formulations. Changes in haematology or clinical chemistry parameters were indicative of a developing immune response in the groups receiving either RTS,S formulation. All examined parameters returned to normal within 28 days after the last injection. The absence of toxicological effects following the injection of RTS,S/AS01 or RTS,S/AS02 in rabbits was supportive of further clinical evaluation of these two formulations.

Non-clinical safety evaluation of single and repeated intramuscular administrations of MAGE-A3 Cancer Immunotherapeutic in rabbits and cynomolgus monkeys.

The MAGE-A3 recombinant protein combined with AS15 immunostimulant (MAGE-A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non-clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE-A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE-A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post-injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3-month treatment-free period (2/5 per gender per group). Local and systemic reactions and MAGE-A3-specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post-mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post-injection), but returned to normal after 1-8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment-related macroscopic findings were recorded after the treatment-free period. MAGE-A3-specific antibody and T-cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE-A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys.

Narcolepsy and A(H1N1)pdm09 vaccination: shaping the research on the observed signal.

Epidemiological data from several European countries suggested an increased risk of the chronic sleep disorder narcolepsy following vaccination with Pandemrix(™), an AS03-adjuvanted, pandemic A(H1N1)pdm09 influenza vaccine. Further research to investigate potential associations between Pandemrix™ vaccination, A(H1N1)pdm09 influenza infection and narcolepsy is required. Narcolepsy is most commonly caused by a reduction or absence of hypocretin produced by hypocretin-secreting neurons in the hypothalamus, and is tightly associated with HLA-II DQB1*06:02. Consequently, research focusing on CD4(+) T-cell responses, building on the hypothesis that for disease development, T cells specific for antigen(s) from hypocretin neurons must be activated or reactivated, is considered essential. Therefore, the following key areas of research can be identified, (1) characterization of hypothetical narcolepsy-specific auto-immune CD4(+) T cells, (2) mapping epitopes of such T cells, and (3) evaluating potential mechanisms that would enable such cells to gain access to the hypothalamus. Addressing these questions could further our understanding of the potential links between narcolepsy and A(H1N1)pdm09 vaccination and/or infection. Of particular interest is that any evidence of a mimicry-based mechanism could also explain the association between narcolepsy and A(H1N1)pdm09 influenza infection.

Evaluation of C-reactive protein as an inflammatory biomarker in rabbits for vaccine nonclinical safety studies.

INTRODUCTION: Inflammatory reactions are one of the potential safety concerns that are evaluated in the framework of vaccine safety testing. In nonclinical studies, the assessment of the inflammation relies notably on the measurement of biomarkers. C-reactive protein (CRP) is an acute-phase plasma protein of hepatic origin that could be used for that purpose in toxicity studies with rabbits. METHODS: To evaluate the utility of CRP as an additional inflammatory biomarker in adjuvant or vaccine toxicity studies, rabbits were injected on Day 0 with saline, aluminium phosphate, aluminium hydroxide, Adjuvant System (AS)01, AS03, AS15, or diphtheria-tetanus-whole cell pertussis-hepatitis B vaccine (DTPw-HB). Body weights, haematology parameters, CRP and fibrinogen levels were measured daily up to Day 7. Macroscopic changes at the injection site were also evaluated up to Day 7. At Day 7, a histopathological examination of the injection site was performed. RESULTS: Like fibrinogen, CRP levels rapidly increased after the injection of Adjuvant Systems or DTPw-HB, peaking at Day 1, and returning to baseline in less than a week. The magnitude of the CRP increase was consistently higher than that of fibrinogen with a larger fold increase from background, providing a more sensitive evaluation. The number of circulating heterophils was also increased on Day 1 after the injection of Adjuvant Systems or DTPw-HB. The highest increases in CRP levels were observed after the injection of DTPw-HB or AS03, and were also associated with the persistence of mixed inflammatory cell infiltrates (including heterophils) at the injection sites on Day 7. No increases in CRP levels and in circulating heterophils were observed after injection of the aluminium salt adjuvants. DISCUSSION: Our study supports the use of CRP as an accurate biomarker of acute inflammation in rabbits for vaccine toxicity studies and highlights an association between increased CRP levels and the recruitment of heterophils.