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.

Formulation of Nanovaccines toward an Extended Immunity against Nicotine.

Nicotine vaccines have been investigated to assist with smoking cessation. Because smoking cessation is a long process, past nicotine vaccines required multiple injections to achieve long-term efficacy. It would be of great significance if extended efficacy can be achieved with fewer injections. Here, we report the assembly of lipid-polylactic acid (PLA) and lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) based nicotine vaccines. Mice immunized with the lipid-PLGA vaccine produced higher titers of nicotine-specific antibodies than the lipid-PLA vaccine in short-term. However, the lipid-PLA vaccine was found to induce long-lasting antibodies. Three months after the immunization, only mice that received first two injections of the lipid-PLGA vaccine and a third injection of the lipid-PLA vaccine achieved a significantly lower brain nicotine concentration of 65.13 ± 20.59 ng/mg than 115.88 ± 37.62 ng/mg from the negative controls. The results indicate that not only the stability of the vaccines but also the combination of the vaccines impacted the long-term efficacy of the immunization. Lastly, both the body weight and the histopathology study suggest that the vaccines were safe to mice. These findings suggest that long-term immunity against nicotine can be realized by a rational administration of nanovaccines of different levels of stability.

Métodos alternativos como parte de las nuevas tendencias en el control de calidad de vacunas / Alternative methods as part of the new trends in vaccine quality control

El control de calidad de las vacunas resulta fundamental para las actividades de producción, liberación lote a lote y comercialización de las vacunas. Sin embargo, en la actualidad este es un proceso que por su concepción es lento y costoso debido a que se apoya en la realización de extensas pruebas en animales para demostrar la potencia y seguridad de estos productos biológicos. El desarrollo de métodos alternativos inspirados en el principio de las 3Rs (Reducción, Refinamiento y Reemplazo) constituye una tendencia que debe impactar de manera muy significativa en la reducción de los tiempos de liberación y el costo del proceso de control de calidad de vacunas en los próximos años. En particular la sustitución de las pruebas de potencia y toxicidad in vivo por procedimientos alternativos más relevantes, rápidos, exactos, reproducibles, robustos y baratos, que incluyen la serología, la cuantificación directa de antígeno, los ensayos en cultivos celulares y el enfoque a consistencia, por solo mencionar algunos; implica un cambio de paradigma, con indiscutibles repercusiones éticas, logísticas, económicas y científico-técnicas, para el aseguramiento de los parámetros de calidad de los inmunobiológicos con el mejor balance costo-beneficio: las vacunas. Los fundamentos técnicos de estos métodos alternativos, sus ventajas y nivel de implementación a nivel internacional, así como sus principales limitaciones, son abordados en este trabajo(AU)

Vaccine quality control is crucial for the manufacturing, lot release and commercialization activities worldwide. However, the current process is by-design too slow and expensive because is based on large animal assays for assuring the potency and safety of these important biological products. The development of 3Rs alternative methods (Reduction, Refinement and Replacement) is a trend able to significantly reduce the releasing times and costs of the vaccine quality control processes in the next few years. Particularly, the replacement of the animals-based potency and toxicity assays by alternative procedures more relevant, fast, accurate, reproducible and cheap, including serology, direct antigen quantification, cell culture tests and the Consistency Approach, for just mentioning some of them, implies a paradigm shift, with undisputable ethical, logistical, economic, scientific and technical repercussions for ensuring the vaccine quality parameters. Theoretical basements, advantages and implementation levels of the alternatives methods as well as their main limitations are presented in this paper(AU)

Efficacy and safety of immunological adjuvants. Where is the cut-off?

Research over the past several decades has provided insight into the mode of action of adjuvants. However, the main focus of attention has been the efficacy in the induction of protective immunogenicity, while less effort has been devoted to the study of toxicity mechanisms. Evidences suggest that several mechanisms that are responsible for the immunostimulating effects are, at the same time, responsible of the adverse effects. In this context, it is often very difficult to establish the boundaries between immunostimulation and immunotoxicity to reach the ideal balance of efficacy/safety. During decades, hundreds of adjuvants and adjuvant formulations have been proposed as immunostimulants for vaccines but very few have been used in human vaccines due to toxicity concerns. In this review, relevant aspects about immunotoxicology of adjuvants, based on clinical and experimental studies are discussed. Some effects are only observed under hyperstimulating regimens using non-approved adjuvants for human use, but these are nonetheless useful to understanding basic principles of adjuvant toxicity. The acute local and systemic reactions, during the first hours and those that can be observed after the third day of vaccination in the inoculation site and systemically are discussed.

A rationally designed peptide IA-2-P2 against type 1 diabetes in streptozotocin-induced diabetic mice.

Recent studies have investigated the potential of type 1 diabetes mellitus-related autoantigens, such as heat shock protein 60, to induce immunological tolerance or to suppress the immune response. A functional 24-residue peptide derived from heat shock protein 60 (P277) has shown anti-type 1 diabetes mellitus potential in experimental animals and in clinical studies, but it also carries a potential atherogenic effect. In this study, we have modified P277 to retain an anti-type 1 diabetes mellitus effect and minimize the atherogenic potential by replacing the P277 B epitope with another diabetes-associated autoantigen, insulinoma antigen-2 (IA-2), to create the fusion peptide IA-2-P2. In streptozotocin-induced diabetic C57BL/6J mice, the IA-2-P2 peptide displayed similar anti-diabetic effects to the control P277 peptide. Also, the IA-2-P2 peptide did not show atherogenic activity in a rabbit model. Our findings indicate the potential of IA-2-P2 as a promising vaccine against type 1 diabetes mellitus.

A novel method for deriving thresholds of toxicological concern for vaccine constituents.

Safety assessment evaluating the presence of impurities, residual materials, and contaminants in vaccines is a focus of current research. Thresholds of toxicological concern (TTCs) are mathematically modeled levels used for assessing the safety of many food and medication constituents. In this study, six algorithms are selected from the open-access ToxTree software program to derive a method for calculating TTCs for vaccine constituents: In Vivo Rodent Micronucleus assay/LD50, Benigni-Bossa/LD50, Cramer Extended/LD50, In Vivo Rodent Micronucleus assay/TDLo, Benigni-Bossa/TDLo, and the Cramer Extended/TDLo. Using an initial dataset (n = 197) taken from INCHEM, RepDose, RTECS, and TOXNET, the chemicals were divided into two families: "positive" - based on the presence of structures associated with adverse outcomes, or "negative" - no such structures or having structures that appear to be protective of health. The final validation indicated that the Benigni-Bossa/LD50 method is the most appropriate for calculating TTCs for vaccine constituents. Final TTCs were designated as 18.06 µg/person and 20.61 µg/person for the Benigni-Bossa/LD50 positive and negative structural families, respectively.

Gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides.

We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments.

Safety assessment of adjuvanted vaccines: Methodological considerations.

Adjuvants mainly interact with the innate immune response and are used to enhance the quantity and quality of the downstream adaptive immune response to vaccine antigens. Establishing the safety of a new adjuvant-antigen combination is achieved through rigorous evaluation that begins in the laboratory, and that continues throughout the vaccine life-cycle. The strategy for the evaluation of safety pre-licensure is guided by the disease profile, vaccine indication, and target population, and it is also influenced by available regulatory guidelines. In order to allow meaningful interpretation of clinical data, clinical program methodology should be optimized and standardized, making best use of all available data sources. Post-licensure safety activities are directed by field experience accumulated pre- and post-licensure clinical trial data and spontaneous adverse event reports. Continued evolution of safety evaluation processes that keep pace with advances in vaccine technology and updated communication of the benefit-risk profile is necessary to maintain public confidence in vaccines.

Historical data analyses and scientific knowledge suggest complete removal of the abnormal toxicity test as a quality control test.

In the early 1900s, the abnormal toxicity test (ATT) was developed as an auxiliary means to ensure safe and consistent antiserum production. Today, the ATT is utilized as a quality control (QC) release test according to pharmacopoeial or other regulatory requirements. The study design has not been changed since around 1940. The evidence of abnormal toxicity testing as a prediction for harmful batches is highly questionable and lacks a scientific rationale. Numerous reviews of historical ATT results have revealed that no reliable conclusions can be drawn from this QC measure. Modern pharmaceutical manufacturers have thorough control of the manufacturing process and comply with good manufacturing practice rules. Contaminants are appropriately controlled by complying with the validated manufacturing processes and strict QC batch release confirming batch-to-batch consistency. Recognizing that product safety, efficacy, and stability can be ensured with strict QC measures, nowadays most regulatory authorities do not require the ATT for most product classes. In line with the replacement, reduction, and refinement (3Rs) initiative, the test requirement has been deleted from approximately 80 monographs of the European Pharmacopoeia and for the majority of product classes in the United States. For these reasons, it is recommended that the ATT should be consistently omitted world-wide and be removed from pharmacopoeias and other regulatory requirements.

[Immunological adjuvants. Determinant factors in the efficacy-toxicity ratio of the contemporary vaccines]. / Adyuvantes inmunológicos. Determinantes en el balance eficacia-toxicidad de las vacunas contemporáneas.

To achieve effective and safe vaccines for the prevention of not yet controlled or re-emergent infectious diseases, one of the more importance aspects is to have immunological adjuvants that allow inducing a protective immune response with an appropriate safety profile. Since 1926 the aluminium compounds have been used as adjuvants for human vaccines, and only in the last 10 years have some new products been registered. Although there an enormous quantity of proposed candidates, the toxicity is the main factor that has limited their introduction into the clinic. In this work the mechanism of action are updated, and the toxicity of the immunological adjuvants are revised, especially those that have obtained clinical approval or are close to getting it.

Developmental toxicity testing of vaccines.

Preventative and therapeutic vaccines are increasingly used during pregnancy and present special considerations for developmental toxicity testing. The various components of the vaccine formulation (i.e., protein or polysaccharide antigen, adjuvants, and excipients) need to be assessed for direct effects on the developing conceptus. In addition, possible adverse influences of the induced antibodies on fetal and/or postnatal development need to be evaluated. A guidance document on the preclinical testing of preventative and therapeutic vaccines for developmental toxicity was issued by the FDA in 2006. Preclinical studies are designed to assess possible influences of vaccines on pre- and postnatal development. The choice of model animal for these experiments is influenced by species differences in the timing and extent of the transfer of the induced maternal antibodies to the fetus. The cross-placental transport of maternal immunoglobulins generally only occurs in late gestation and tends to be greater in humans and monkeys than in non-primate species. For many vaccines, the rabbit shows a greater rate of prenatal transfer of the induced antibodies than rodents. For biotechnology-derived vaccines that are not immunogenic in lower species, nonhuman primates may be the only appropriate models. It may be advisable to test new adjuvants using the ICH study designs for conventional pharmaceuticals in addition to the developmental toxicity study with the final vaccine formulation.

Regulatory assessment of reproductive toxicology data.

This chapter outlines a regulator's personal approach to the assessment of reproductive toxicology data in the context of the assessment of the overall nonclinical data package for pharmaceutical agents. Using as a framework the International Conference on Harmonisation Common Technical Document headings, guidance is provided on the expectations of regulators for the presentation and discussion of the data by the applicant to facilitate the risk assessment process. Consideration is given to the use of reproductive toxicology data in the assessment process for both clinical trial applications (CTAs) and marketing authorization applications (MAAs). Suggestions for some guiding principles in drafting of the various product information documents (for example the Investigator's Brochure (IB) for CTAs and the Nonclinical Overview and Summary of Product Characteristics for MAAs) are included.

Vaccine-related beliefs and practices of parents of children with autism spectrum disorders.

Although the assertion of a link between vaccines and autism has been scientifically rejected, the theory continues to be popular and may influence the attitudes of parents of children with autism spectrum disorders. The authors sought to assess how often parents change or discontinue their child's vaccine schedule after autism spectrum disorder diagnosis and whether beliefs about the etiology of autism affect their decision to do so. The authors surveyed 197 (43%) of 460 eligible parents of children under 18 years of age with autism spectrum disorders who were enrolled in a state-funded agency that provides services to those with developmental disabilities in western Los Angeles County. Half of the parents discontinued or changed vaccination practices, and this was associated with a belief that vaccines contributed to autism spectrum disorders, indicating a potential subset of undervaccinated children. Educational tools should be designed to assist physicians when talking to parents of children with autism spectrum disorders about vaccination.

Study designs for the nonclinical safety testing of new vaccine products.

During the development of a new vaccine, the purpose of nonclinical studies is to provide safety information to support the clinical development and licensure of the product. In this article the study designs currently accepted for the nonclinical safety testing of new vaccines are described for single dose, local tolerance, repeat dose toxicity and safety pharmacology studies; these studies together form the basis of a typical nonclinical safety evaluation dossier. The detailed design of the preclinical package must take account of the intended clinical use, patient population, route of administration, formulation, dose level and immunisation schedule. The test item that is used for these studies must be adequately representative of the intended clinical formulation. The animal model used for these studies must be selected on criteria of relevance. Single dose toxicity studies provide information on acute actions or the potential effect of accidental overdose, but this information is often available from the repeat dose toxicity study, obviating the need for the acute study. Local tolerance studies provide information on tissue reactions at the site of administration. Evaluation of the findings must distinguish between normal tissue responses to injected material and findings indicative of undesirable pathological changes. The repeated dose toxicity studies are the principal studies that support the safety profile of the vaccines. The design of these studies must take full account of the features of the vaccine in the choice of treatment regime, dose levels, pharmacodynamic monitoring and timing of investigations and sacrifice. Safety pharmacology studies are performed to evaluate the potential for undesirable secondary pharmacological actions of vaccines if there is data to suggest that such studies are needed; this evaluation is made on a case by case basis. In the absence of specific guidance the design of studies for therapeutic vaccines follows the same general principles as those for anti-infective vaccines.

Developmental and reproductive toxicity testing of vaccines.

The majority of new preventative and therapeutic vaccines are now assessed for developmental toxicity according to guidelines issued by the FDA in 2006. Despite the absence of confirmed effects in humans, vaccines are frequently suspected of having adverse side-effects on the development of children. Such suspicions are perhaps unavoidable considering the extremely widespread use of vaccines. The preclinical developmental toxicology studies are designed to assess possible influences of each component of the vaccine formulation-and the induced antibodies-on the development of the conceptus, neonate and suckling organism. Immune modulation by a vaccine or an adjuvant could, for instance, affect the outcome of pregnancy by interfering with the natural shift in immune balance of the mother during gestation. Maternal immunoglobulins are transferred from the mother to the offspring in order to confer passive immunity during early life. This maternal antibody transport is prenatal in humans and monkeys, but tends to be delayed until after birth in other species. Therefore, a suitable model species needs to be chosen for preclinical studies in order to ensure exposure of the foetus to the induced maternal antibodies following vaccination. Rabbits are the best laboratory model for prenatal immunoglobulin transfer, but rodents are more practical for the necessary postnatal investigations. Non-human primates are the only appropriate models for the testing of vaccines that are not immunogenic in lower species. It is advisable to test new adjuvants separately according to the ICH S5(R2) guidelines. Preclinical paediatric investigations are not currently required for vaccines, even though most vaccines are given to children. Other areas of regulatory concern include developmental immunotoxicity and effects on the preimplantation embryo. Because of the limitations of the available animal models for developmental toxicity testing, pharmacovigilance is essential.

Reproduction and juvenile animal toxicology studies in the rat with a new allergy vaccine adjuvanted with monophosphoryl lipid A (MPL(®)) for the treatment of grass pollen allergy.

Pollinex(®) Quattro Grass has been developed for the prevention or relief of allergic symptoms caused by pollen in both adults and children. Reproduction and juvenile animal toxicology studies have been performed. Subcutaneous injection on Day 14 prior to pairing and on Days 6 and 13 of gestation to pregnant rats at 2000SU/0.5 mL elicited no signs of maternal or embryo-foetal toxicity. Mating, fertility, fecundity and pup parameters were all unaffected by treatment. Once-weekly subcutaneous administration at ascending doses of 300, 800, 2000 and 2000SU/0.5 mL followed by a 4 week non-dose period to juvenile rats from 3 weeks of age showed no signs of obvious toxicity. As in a previously performed adult animal toxicology study with the vaccine, not unexpected, but relatively minor, immuno-stimulatory effects were seen in this study along with injection site reaction which can largely be attributed to the presence of tyrosine in the formulation.

Progress in understanding adjuvant immunotoxicity mechanisms

Over the last twenty years research has provided an important insight into the mechanisms responsible for the immunotoxicity of both local and systemic adverse reactions following the use of immunostimulating drugs and adjuvants. In this article we provide an update of the present knowledge relating to the various parameters and reactants of the immune system at the cellular as well as molecular level that are believed to play a key role in reactogenicity. We discuss evidence obtained from observations in vitro, in vivo in animal models and from clinical applications, including adjuvants used in large scale vaccination today. The data discussed are mainly taken from animal models following hyperstimulation of the immune system; either by the use of very powerful adjuvants, like Freunds that are too toxic for use in practical vaccination, by deliberate high dose application of adjuvants or by the in vivo application of cytokines. Although such hyperstimulating regimens are unlikely to find their way into practical vaccination of humans, this information is of great value as it may facilitate the understanding of the toxicity mechanisms, aid the design of standardised models for the assessment of adjuvant safety and the possible application of new adjuvants in vaccines for humans(AU)