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.

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.

Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding.

INTRODUCTION: Formalin fixation and paraffin embedding (FFPE) is a standard method for tissue sample storage and preservation in pathology archives. The Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) is a useful method for gene expression analysis, but its sensitivity is significantly decreased in FFPE tissue due to the fixation process. This process results in chemical modifications of RNA, cross-links proteins to RNA, and degrades RNA in these archived samples, hindering the reverse transcription step of the conventional RT-pPCR method and preventing generation of a cDNA that is long enough for the subsequent quantitative PCR step. METHODS: In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides. RESULTS: Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3months to 4years, and was found to be comparable to results obtained with frozen liver tissues. DISCUSSION: Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue.

A multi-species assay for siRNA-mediated mRNA knockdown analysis without the need for RNA purification.

INTRODUCTION: Various animal models are routinely used to evaluate the efficacy and toxicity of small interfering RNA (siRNA) therapeutics. Given that the most common measure of efficacy with siRNA therapeutics is mRNA knockdown, the development of a single assay for quantification of siRNA-mediated mRNA knockdown in multiple species would provide significant time and cost-savings during preclinical development. METHODS AND RESULTS: We have developed an assay targeting short consensus sequences of a particular mRNA in multiple species using the principles of a recently-reported stem-loop RT-qPCR method (Chen et al., 2005). The multi-species RT-qPCR assay is highly sensitive, reproducible, has a dynamic range of seven orders of magnitude, and it can be used to quantify a specific mRNA in crude tissue homogenates without the need for RNA purification. Compared to the limitations of conventional RT-qPCR assays, this assay provides a simple and robust tool for mRNA quantification to evaluate siRNA-mediated mRNA knockdown. DISCUSSION: This assay can potentially become a routine method for mRNA quantification to evaluate siRNA-mediated mRNA knockdown.

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.

Nonclinical safety assessment of vaccines and adjuvants.

To ensure the safe administration of vaccines to humans, vaccines (just like any new chemical entity) are evaluated in a series of nonclinical safety assessment studies that aim at identifying the potential toxicities associated with their administration. The nonclinical safety assessment of vaccines, however, is only part of a testing battery performed prior to human administration, which includes (1) the evaluation of the vaccine in efficacy and immunogenicity studies in animal models, (2) a quality control testing program, and (3) toxicology (nonclinical safety assessment) testing in relevant animal models. Although each of these evaluations plays a critical role in ensuring vaccine safety, the nonclinical safety assessment is the most relevant to the evaluation in human clinical trials, as it allows the identification of potential toxicities to be monitored in human trials, and in some cases, eliminates candidates that have unacceptable risks for human testing. This review summarizes the requirements for the nonclinical testing of vaccines and adjuvants needed in support of all phases of human clinical trials.

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.

Application of PCR technology in vaccine product development.

The complex process of vaccine product development needs to be tightly controlled and closely monitored to ensure vaccine quality and consistency. Since its inception, PCR has been widely used in all stages of vaccine product development as a tool to assist in the evaluation of vaccine quality, safety and efficacy. In this review, the general principles of conventional and real-time quantitative PCR (Q-PCR) technology and its application in vaccine product development for quantitation of vaccine dose (genome quantitation assay), infectivity (Q-PCR-based potency assay), process residuals, stability, adventitious agents, safety assessment and clinical studies are described. The future outlook and the advantages and disadvantages of this technology are also discussed.

Ensuring the quality, potency and safety of vaccines during preclinical development.

There is an abundance of vaccines currently in development, with most of them exploring novel mechanisms, adjuvants and/or delivery systems not only for traditional prophylactic use, but also for therapeutic uses. As vaccines are generally administered to healthy individuals, ensuring their quality, potency and safety becomes crucial, especially prior to evaluation in humans. To ensure these key attributes, vaccine developers need to incorporate them as early in the development program as possible, starting in basic research and continuing through preclinical, clinical and postmarketing development. Fortunately for vaccine developers, ample guidance is available from various regulatory agencies to enlighten the long and arduous path of vaccine development. This review will highlight these regulatory expectations, and provide some clarity as to why they are in place.

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.