Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene. As expected, the persistence of the HD vector was markedly higher than that of the FG vector. For both vectors, the majority of the vector DNA remained extrachromosomal and predominantly in the form of episomal monomers. However, using a quantitative gel-purification-based integration assay, a portion of the detectable vector was found to be associated with high molecular weight (HMW) genomic DNA, indicating potential integration with a frequency of up to ~44 and 7000 integration events per µg cellular genomic DNA (or ~0.0003 and 0.05 integrations per cell, respectively) for the FG and HD Ad5 vectors, respectively, following intravenous injection of 1 × 1011 virus particles. To confirm integration occurred (versus residual episomal vector DNA co-purifying with genomic DNA), we characterized nine independent integration events using Repeat-Anchored Integration Capture (RAIC) PCR. Sequencing of the insertion sites suggests that both of the vectors integrate randomly, but within short segments of homology between the vector breakpoint and the insertion site. Eight of the nine integrations were in intergenic DNA and one was within an intron. These findings represent the first quantitative assessment and characterization of Ad5 vector integration following intravenous administration in vivo in wild-type mice.

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].

Evaluation of a 9-valent HPV vaccine in Sprague-Dawley rats: Nonclinical studies assessing general, reproductive, and developmental toxicity.

GARDASIL®9, a 9-valent vaccine against human papillomavirus (9vHPV), was developed to prevent diseases mediated by HPV types 6/11/16/18/31/33/45/52/58. During the development of the vaccine, three nonclinical safety studies were conducted to evaluate repeat-dose toxicity and prenatal and postnatal developmental toxicity in Sprague-Dawley rats. In all studies, the vaccine was administered via intramuscular injections of 0.5 mL (the human dose) divided equally into each quadriceps muscle. In the repeat-dose toxicity study, potential local and systemic toxic effects of the 9vHPV vaccine were evaluated after 4 doses given 21 days apart and after a 21-day recovery period. In the prenatal study, virgin females were dosed at 5 and 2 weeks prior to mating and on Gestation Day [GD] 6 (3 total doses). Potential postnatal developmental toxicity of the vaccine formulation was evaluated after 4 total doses (premating to lactation). There were no treatment-related unscheduled deaths in any studies. In the 3-month repeat-dose toxicity study, no adverse effects in male or female rats were observed. Anticipated systemic effects representing immunological responses and local inflammatory reactions at the injection sites were noted in the vaccine-treated groups, with a trend toward recovery by the end of the 21-day recovery period. In the prenatal developmental toxicity study, there was no evidence of toxicity in females given the vaccine. There were no effects on fertility or reproductive performance of the parental females and no evidence of developmental toxicity. In the postnatal study, there was no evidence of toxicity in vaccine-treated females and no evidence of developmental toxicity based on standard postnatal parameters, including behavioral testing and reproductive performance. The vaccine induced antibody responses in all studies and vaccine-specific antibodies were detected in offspring in the developmental toxicity studies. These results support the favorable safety profile of GARDASIL®9.

Current strategies in the non-clinical safety assessment of biologics: New targets, new molecules, new challenges.

Nonclinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these innovative and often complex drugs. Emerging topics in this field were discussed recently at the 2016 Annual US BioSafe General Membership meeting. The presentations and subsequent discussions from the main sessions are summarized. The topics covered included: (i) specialty biologics (oncolytic virus, gene therapy, and gene editing-based technologies), (ii) the value of non-human primates (NHPs) for safety assessment, (iii) challenges in the safety assessment of immuno-oncology drugs (T cell-dependent bispecifics, checkpoint inhibitors, and costimulatory agonists), (iv) emerging therapeutic approaches and modalities focused on microbiome, oligonucleotide, messenger ribonucleic acid (mRNA) therapeutics, (v) first in human (FIH) dose selection and the minimum anticipated biological effect level (MABEL), (vi) an update on current regulatory guidelines, International Council for Harmonization (ICH) S1, S3a, S5, S9 and S11 and (vii) breakout sessions that focused on bioanalytical and PK/PD challenges with bispecific antibodies, cytokine release in nonclinical studies, determining adversity and NOAEL for biologics, the value of second species for toxicology assessment and what to do if there is no relevant toxicology species.

Clinical development of a recombinant Ebola vaccine in the midst of an unprecedented epidemic.

The 2014-2016 Ebola outbreak caused over 28,000 cases and 11,000 deaths. Merck & Co. Inc., Kenilworth, NJ USA and NewLink Genetics are working with private and public partners to develop and license an Ebola vaccine that was evaluated extensively during the outbreak. The vaccine referred to as V920 is a recombinant vesicular stomatitis virus (rVSV) in which the VSV-G envelope glycoprotein (GP) is completely replaced by the Zaire ebolavirus GP (rVSVΔG-ZEBOV-GP). Eight Phase I and four Phase II/III clinical trials enrolling approximately 17,000 subjects were conducted in parallel to the outbreak to assess the safety, immunogenicity, and/or efficacy of V920. Immunogenicity data demonstrate that anti-GP antibodies are generally detectable by ELISA by 14days postvaccination with up to 100% seroconversion observed by 28days post dose. In addition, the results of a ring vaccination trial conducted by the WHO and their partners in Guinea suggest robust vaccine efficacy within 10days of receipt of a single dose of vaccine. The vaccine is generally well-tolerated when administered to healthy, non-pregnant adults. The development of this vaccine candidate in the context of this unprecedented epidemic has involved the close cooperation of large number of international partners and highlights what we as a public health community can accomplish when working together towards a common goal. Study identification: V920-001 to V920-012. CLINICALTRIALS.GOV identifiers: NCT02269423; NCT02280408; NCT02374385; NCT02314923; NCT02287480; NCT02283099; NCT02296983; NCT02344407; NCT02378753; NCT02503202.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

An Evaluation of the Impact of PD-1 Pathway Blockade on Reproductive Safety of Therapeutic PD-1 Inhibitors.

This report discusses the principles of reproductive toxicity risk assessment for biopharmaceuticals blocking the PD-1/programmed cell death ligand 1 (PD-L1) pathway, which have been developed for the treatment of patients with advanced malignancies. The PD-1/PD-L1 pathway is a T-cell co-inhibitory pathway that normally maintains immune tolerance to self. Its role in pregnancy is to maintain immune tolerance to the fetal allograft. In cancer patients, this signaling pathway is hijacked by some neoplasms to avoid immune destruction. PD-1/PD-L1-blocking agents enhance functional activity of the target lymphocytes to eventually cause immune rejection of the tumor. A therapeutic blockade of PD-1/PD-L1 pathway that occurs at full target engagement provides a unique challenge to address the risk to pregnancy because disruption of the same pathway may also reduce or abrogate maternal immune tolerance to the fetal alloantigens inherited through the father. Typically, nonclinical reproductive and developmental toxicity (DART) studies in animals (rats and rabbits) with clinical drug candidates are conducted to identify potential risk in humans and to determine exposure margin for the effects on reproduction as part of the risk assessment. However, for biopharmaceuticals for which the desired mechanism of action cannot be separated from potential deleterious effects to the fetus and when the only relevant toxicology species is nonhuman primate (NHP), the risk to reproduction can be predicted by a mechanism-based assessment using data generated from murine surrogate models as supportive information without conducting DART in NHPs. Such an approach has been used in the evaluation of pregnancy risk of anti-PD-1 agent, pembrolizumab, and has been demonstrated as an important alternative to performing DART studies in NHPs.

Lack of effects on male fertility from a quadrivalent HPV vaccine in Sprague-Dawley rats.

BACKGROUND: Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases in both men and women. A recently developed quadrivalent HPV vaccine, Gardasil, has been shown to be highly effective in the prevention of several HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on male fertility including reproductive performance, sperm evaluations, and histology of the testes. In addition, anti-HPV antibodies were measured during the study. METHODS: Group 1 (30 male rats) received the full human dose of vaccine (0.5 mL, ∼200-fold excess based on body weight) by intramuscular injection at 6 weeks, 3 weeks, and 3 days prior to cohabitation. Group 2 males received only 1 dose at 3 days prior to cohabitation. Additional groups (20 male rats each) were administered PBS or Merck Aluminum Adjuvant similarly to Group 1. Ten males in the vaccine-treated groups were bled for immunogenicity assays after each dose. Twenty males per group were mated to untreated female rats. Cesarean sections were performed on Gestation Day 15 or 16. Cohabited males were necropsied and sperm count and motility were evaluated. RESULTS: There were no unscheduled deaths during the study and no evidence of toxicity in vaccine-treated male rats. The vaccine induced a specific antibody response to the 4 HPV types after each injection. There were no effects on the cesarean-section parameters of females or reproductive parameters of the cohabited male rats, including histomorphology of testes and epididymis, sperm count, and sperm motility. CONCLUSIONS: These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects on routine measures of male fertility in rats.

Lack of effects on fertility and developmental toxicity of a quadrivalent HPV vaccine in Sprague-Dawley rats.

Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases, with approximately half of the HPV-infected people being adolescents and young adults. A recently developed quadrivalent HPV vaccine, GARDASIL((R)), has been shown to be highly effective in the prevention of a number of HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on female fertility and F1 development, growth, behavior, and reproductive performance. In addition, anti-HPV antibodies in the F0 females and F1 offspring were measured during the study. Two groups of 65 virgin Sprague-Dawley rats were administered two or four intramuscular injections of the vaccine (full human dose of 0.5 mL at 5 and 2 weeks prior to mating, on Gestation Day [GD] 6, and Lactation Day [LD] 7; or GD 6 and LD 7 only). Additional groups of rats were administered phosphate-buffered saline or Merck Aluminum Adjuvant (MAA) at the same four times. All females were mated to males of the same stock. Cesarean sections were performed on 22/group on GD 21, 22/group were allowed to deliver, and remaining females used for blood collections or replacements. F0 female fertility parameters were evaluated. An extensive number of prenatal, perinatal, and postnatal parameters were evaluated in the F1 generation. There were no unscheduled deaths during the study. There was no evidence of toxicity in the F0 females given either MAA or vaccine. There were no effects on the fertility or reproductive performance of the F0 females. There was no evidence of developmental toxicity to the F1 generation, including fetal body weight and morphology, postnatal growth and development, behavior, and reproductive performance. The quadrivalent vaccine induced a specific antibody response to the four HPV types in the F0 female rats following one or multiple injections. Antibodies against all four HPV types were transferred to the F1 generation during gestation and/or lactation, likely via the placenta and milk, respectively. The passively transferred antibodies persisted up to Postnatal Day 77 when they were last measured. These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects in either the treated F0 female rats or the F1 generation.

Quantifying the titer and quality of adenovirus stocks.

The broad application of recombinant adenoviruses to the development of vaccines and gene therapy vectors has encouraged the development of molecular assays for the facile quantitation of adenoviral particles and the assignment of their infectious potency. The Genome Quantitation Assay (GQA) and the QPCR-Based Potency Assay (QPA) developed for adenoviruses offer the attributes of precision, rapidity, and high throughput either performed manually or facilitated by simple automated liquid handling systems. These assay attributes allow for accelerated process development support and product characterization and release. The assays for adenovirus could offer the additional advantage in that their quantitation is based on viral replication independent of cytopathology permitting quantitation of serotypes that cause minimal cytopathic effect (CPE) in 293 cells and specificity that allows the components of multivalent vaccines to be discriminated and quantitated for release.

Using QPCR to assign infectious potencies to adenovirus based vaccines and vectors for gene therapy: toward a universal method for the facile quantitation of virus and vector potency.

The assignment of infectious potency to test articles of adenovirus has been conducted mainly using classical end-point dilution methods, which rely on virus induced cytopathology to reveal the presence of infectious virus. These assays suffer the disadvantages of labor intensity, duration, throughput restriction and variability. In the course of our development of an Ad5 based HIV vaccine for clinical evaluation, we sought a facile method for the assignment of potency to the numerous test articles generated during the development of bioprocesses for bulk manufacture, downstream purification and formulation. In this paper we describe a quantitative PCR based potency assay (QPA) which uses QPCR to quantitate adenovirus genomes replicated 24h after the inoculation of a test article on 293 cell monolayers, and then relates that mass to potency by interpolation to a standard curve of replicated adenovirus genomes constructed with a reference adenovirus standard to which infectious potency has been previously assigned in the classical end-point dilution assay. The QPA assay for adenovirus is simple and rapid, with a throughput capacity adequate to the potency assay demands of bioprocess development, and with a precision expressed as a root variability of 16.8% R.S.D., allowing for close discriminations of the products of alternative process configurations. The adenovirus QPA principle can be applied to the quantitation of infectious potency of both RNA and DNA viruses and we report briefly on the development of QPA assays for measles and mumps. QPA assays owing to their simplicity and easy automation, rapidity, capacity and precision hold promise to become widely practiced methods for the quantitation of the potency of live virus vaccines and other recombinant virus vectors.

Production and formulation of adenovirus vectors.

Adenovirus vectors have attracted considerable interest over the past decade, with ongoing clinical development programs for applications ranging from replacement therapy for protein deficiencies to cancer therapeutics to prophylactic vaccines. Consequently, considerable product, process, analytical, and formulation development has been undertaken to support these programs. For example, "gutless" vectors have been developed in order to improve gene transfer capacity and durability of expression; new cell lines have been developed to minimize recombination events; production conditions have been optimized to improve volumetric productivities; analytical techniques and scaleable purification processes have advanced towards the goal of purified adenovirus becoming a "well-characterized biological"; and liquid formulations have been developed which maintain virus infectivity at 2-8 degrees C for over 18 months. These and other advances in the production of adenovirus vectors are discussed in detail in this review. In addition, the needs for the next decade are highlighted.

Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d.

For members of the CD1 family of beta(2)-microglobulin-associated lipid-presenting molecules, tyrosine-based motifs in the cytoplasmic tail and invariant chain (Ii) govern glycoprotein trafficking through endosomal compartments. Little is known about the intracellular pathways of CD1 trafficking and antigen presentation. However, in vitro studies with cells transfected with mutant CD1 that had a truncated cytoplasmic tail have suggested a role for these tyrosine motifs in some, but not all, antigenic systems. By introducing a deletion of the tyrosine motif into the germ line, and through homologous recombination in embryonic stem cells, we now describe knock-in mice with the CD1d cytoplasmic tail deleted. Despite adequate surface CD1d expression and the presence of Ii, these mutant mice showed multiple and selective abnormalities in intracellular trafficking, antigen presentation and T cell development, demonstrating the critical functions of the CD1d cytoplasmic tail motif in vivo.