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Neurovirulence
Neurovirulence studies may be required for novel live virus or viral vectored vaccines if
the wild-type virus on which the vaccine is based is neurotropic or the vaccine has been
passaged through neural tissue (WHO, 2005, 2013, 2018). Much effort has been put forth
to develop small animal models of neurovirulence (Rubin, 2005; Monath, 2005) or nonanimal
models (Rubin, 2011). While these alternatives may be acceptable in some regions (Rubin,
2011) the most widely accepted model is the monkey neurovirulence test (MNVT). In the
MNVT, animals are administered a single dose of the vaccine or appropriate controls via
intrathecal and/or intraspinal routes and observed for a period of time for clinical signs of
neurotoxicity followed by histopathological evaluation of brain and spinal cord for evidence
of neurovirulence (Mire et al., 2012). Use of the NHP for this purpose must be carefully
considered as this model is not always appropriate (i.e., the wild type virus does not cause
disease in the NHP and therefore the study may not be properly controlled) and there are
ethical concerns with its use. In fact, given the limitations of the MNVT, it is no longer
required for well-characterized live virus vaccines (IABS, 2006). Data from these studies
are also often difficult to interpret given the injection itself may result in alterations in brain
tissue. Despite the limitations of and concerns surrounding the MNVT, if it is determined a
study in NHPs is needed, it is best practice to include a secondary assessment to determine
whether replicating virus is present at the site of any lesions identified in the brain and/or
spinal cord (Mire et al., 2012). As with the toxicity studies, care should be taken to ensure
only experienced individuals are responsible for study conduct and data interpretation. Dis-
cussions with HAs may be appropriate to gain concurrence on the study design or proposals
for alternate models as well as other approaches such a s conducting a biodistribution study to
evaluate biodistribution to the brain, which may be helpful in determining the need for a neu-
rovirulence test.
Conclusion
Overall, nonclinical toxicity studies are an integral part of vaccine development and are
required to support clinical trials and registration. As described above, the type and duration
of studies varies based on vaccine type, indication, components (e.g., adjuvants), etc. and are
based on specific clinical plans. To aid in the design of the development programs, world-
wide regulatory guidelines are available and provide specific guidance to vaccine manufac-
turers. While this guidance is helpful, interactions with HAs are also suggested to propose
any alternative approaches and/or ensure all regulatory expectations are met, particularly
since requirements may vary between regions.
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7. Regulatory aspects of nonclinical vaccine development238
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8
Special considerations for the
nonclinical safety assessment of
vaccines
Jayanthi J. Wolf
Regulatory Affairs and Quality Assurance, Atea Pharmaceuticals Inc., Boston,
MA, Unit ed States
OUTLINE
Introduction 240
De-risking strategies for vaccines 240
General de-risking strategies for vaccines 242
De-risking strategies for specific vaccine
types 244
Vaccines with adjuvants 245
Therapeutic vaccines 245
Pharmacokinetics and pharmacodynamics
assessments 246
Assays 246
Ligand-binding assays 247
Cell-mediated immune response assays 247
Functional assays 248
Differences in the nonclinical safety
assessment of vaccines and
biopharmaceutical drugs 248
Summary 250
References 250
CHAPTER
239
Nonclinical Development of Biologics, Vaccines and
Specialty Biologics, Second Edition
https://doi.org/10.1016/B978-0-443-13344-2.00007-9
© 2025 Elsevier Inc. All rights are reserved, including those for text
and data mining, AI training, and similar technologies.
Introduction
Vaccines are one of the most important public health advancements over the last century.
Prophylactic vaccines for infectious diseases have prevented severe disease and morbidity
throughout the world. The contribution of vaccines in reducing morbidity has been recently
evidenced during the COVID-19 pandemic. After the global emergence of SARS-CoV-2 virus
and COVID-19, there was an urgent need to develop vaccines at an unprecedented rate and
scale. Within a year of the discovery of the SARS-CoV-2 virus, several safe and effective
COVID-19 vaccines were authorized and used for mass vaccination campaigns. The positive
public health impact of COVID-19 vaccines has been clearly demonstrated because it is esti-
mated that vaccinations prevented 14e20 million deaths from COVID-19 globally between
Dec. 2020 and Dec. 2021 (Watson et al., 2022).
The safety of vaccines is carefully assessed through well-designed nonclinical and clinical
studies (see Chapter 8). The nonclinical toxicology evaluation of vaccines became a require-
ment more than 25 years ago and is a valuable tool in assessing the safety of vaccines in an-
imals prior to, and concurrent with, human clinical trials. Nonclinical toxicology studies can
provide information about the potential target organs of toxicity, which can then be used to
inform human subjects about potential risks. Nonclinical toxicology studies can also establish
a no-observable-adverse-effect level (NOAEL) and assist with determining the safe starting
dose for clinical studies. This is particularly important for prophylactic vaccines, which are
intended for healthy human subjects including infants. Details of the studies required for
nonclinical safety evaluation of vaccines have been described in other reviews and Chapter
8(Wolf et al., 2010, 2013; Green and Al-Humadi, 2017). Therefore this chapter will focus
on a discussion of de-risking strategies for vaccines and considerations for specific vaccine
types. It should be noted that due to limitations in animal models, rare adverse effects that
might occur in specific human subpopulations (e.g., autoimmunity) can only be properly
addressed in clinical studies and in postmarketing surveillance programs. Nevertheless,
nonclinical studies can provide some assurance of the general tolerability of a vacci ne and
provide a foundation for an assessment of the safety profile, which is further evaluated dur-
ing clinical trials. Nonclinical safety assessment studies allow the identification of potential
toxicities to be monitored in human trials, and could eliminate vaccine candidates that
have unacceptable risks for human testing (Wolf et al., 2010, 2013).
De-risking strategies for vaccines
Vaccines and other novel biopharmaceuticals are similar in that de-risking strategies often
involve a case-by-case approach, based on the availabi lity of animal models that might
address specific theoretical or actual concerns. However, there are some differences in the
de-risking strategies between these modalities.
Unlike other novel biopharmaceuticals, which are composed of highly purified proteins,
vaccines consist of many different types of components; including synthetic peptides, puri-
fied/recombinant proteins, peptide conjugates, protein complexes, virus-like particles, live
virus or bacteria, attenuated/inactivated virus or bacteria, viral vectors, DNA and mRNA.
To enhance the immune response of the vaccine, adjuvants are often added to the
8. Special considerations for the nonclinical safety assessment of vaccines240
II. Vaccines
formulation, which include compounds such as aluminum salts, CpG oligonucleotides,
monophosphorylated lipid A, saponins, and oil-in-water emulsions (Dey and Srivastava,
2011). Each component of the vaccine formulation needs to be carefully considered when
developing the nonclinical safety program, and novel excipients (i.e., chemicals that have
not been included in marketed products or evaluated nonclinically and/or clinically to pro-
vide some assurance of safety) might require additional evaluation from a toxicological
perspective. These considerations are unique to vaccines, since the formulation is typically
more complex than a novel monoclonal antibody or therapeutic protein formulation, which
only contains purified proteins and buffer components to stabilize the protein.
Theoretical concerns have been raised about increased toxicities with adjuvant-
containing vaccine formulations due to potential synergies between adjuvant-induced
responses and vaccine-induced responses (WHO, 2014). The safety profile of an adjuvant
would be impacted by its interactions with vaccine antigens, therefore toxicological
assessments of adjuvants are performed in the context of the full vaccine formulation
(WHO, 2014).
Vaccine toxicology studies are conducted with a formulation that is representative of the
final clinical formulation (WHO, 2014). Ideally, the clinical lot should be used, but in practice
this might not be feasible, since clinical supplies are often manufactured after the start of toxi-
cology studies. For vaccines that might be composed of live or attenuated viruses, the final
virus seed is used for the toxicology study to avoid the possibility of having any genetic dif-
ferences or mutations between the virus tested in toxicology studies and the virus tested in
clinical studies. Since vaccine formulations are very complex, consisting of multiple proteins
and chemical components, the analytical characterization of the vaccine formulation often has
some limitations when compared with well-characterized biopharmaceutical products. Due
to these limitations in analytical characterization for complex vaccine formulations, every
effort is made to test formulations in toxicology studies that are representative of the final
clinical formulation.
A unique consideration for vaccines is the need to maintain a delicate balance between
immunogenicity and reactogenicity. In novel biopharmaceutical development, the immuno-
genicity of the product is unwanted and hinders product development, whereas for vaccines
the immunogenic response is desired and inherent in the design of the vaccine. New tools
and technologies are needed in vaccine development to distinguish between the desired
immunogenic response, which will result in long-lasting immune protection and the un-
wanted proinflammatory response, which might result in severe injection site damage and
a fever/p yrogenic response.
Another unique de-risking strategy for vaccines is tied to the complex analytical character-
ization of the product. For live virus vaccines, genetic stability of the product is needed and
should be demonstrated prior to investment in manufacturing the vaccine for nonclinical and
clinical studies. An analytical characterization package that identifies all the complex compo-
nents included in the vaccine formulation is required, which might be a daunting task for vac-
cines containing live viral or bacterial components. Specific concerns that need to be
addressed include potential reversion of attenuated viral and bacterial vaccines, and neuro-
virulence of viral vaccines. Similar to other biopharmaceuticals, product release tests in
animals are needed to test for adventitious agents that are inherent to the manufacturing
process or raw materials.
De-risking strategies for vaccines 241
II. Vaccines
General de-risking strategies for vaccines
When developing the de-risking strategy for a new vaccine, the indication and patient
population for the vaccine needs to be considered. A thorough literature search can be per-
formed to determine whether there were any previous vaccines developed for the indication
and if there were specific adverse effects noted in the targeted patient population. In some
cases, vaccines may increase the severity of disease, such as with vaccines for respiratory syn-
cytial virus (RSV), dengue virus, and measles virus infections.
For example, for prophylactic RSV vaccines, human clinical trials u sing a formalin-
inactivated (FI) RSV vaccine were terminated when an increase in rates of hospitalization
and two deaths were observed in vaccinated infants who encountered RSV after
vaccination. The disease enhancement and pulmonary eosinophilia is believed to be due
to an exaggerated memory Th2 response (Fulginiti et al., 1969; Castilow et al., 2007;
Graham, 2011).
This event negatively impacted the course of RSV vaccine development until animal
models were developed, which replicated the disease enhancement phenomenon that was
observed after vaccination with formalin-inactivated RSV. The cotton rat is one of the
accepted animal models that can be used to evaluate RSV vaccine-associated enhanced
lung disease. Pulmonary histopatholog y of FI-RSV-enhanced lung disease in the cotton rat
is characterized by peribronchiolitis, bronchitis, alveolitis, and interstitial pneumonitis, where
the alveolitis and peribronchiolitis were similar to that observed in the two clinical FI-RSV
fatalities, and a scoring system was developed to facilitate assessment of the vaccine-
enhanced lung disease (Prince et al., 2001). This model can be used to screen RSV vaccines
in early nonclinical development and can also be used to support the final clinical candidate.
Safety data from cotton rat studies have supported the regulatory submissions of clinical RSV
vaccine candidates (Tang et al., 2008).
Autoimmune disease: A theoretical concern for some vaccines is the potential induction of
autoimmune diseases, although there have only been a few documented cases of autoim-
mune disease induced by vaccines (Ahmed et al., 2013). However, nonclinical models for pre-
dicting autoimmune disease are limited due to differences in genetics and physiology
between animals and humans, and the unknown etiology of autoimmune diseases. There
are no validated animal models currently available to address this concern, and the predic-
tivity of animal models evaluated to date is questionable. Species differences between ani-
mals and humans make it difficult to perform adequate nonclinical studies to address this
concern, and instead the current approach is to closely monitor for autoimmunity in clinical
studies.
Although the incidence of autoimmune-induced disease cannot be conclusively associated
with the administration of vaccines, there are some mechanisms that might contribute to this
phenomenon, such as molecular mimicry. Molecular mimicry results from an immune
response to epitopes that are shared between human proteins and antigenic components of
the vaccine. To assess for this potential toxicity, protein sequences are screened in silico to
evaluate the similarity between prote in structures of vaccine antigens and other known pro-
tein structures.
8. Special considerations for the nonclinical safety assessment of vaccines242
II. Vaccines
In silico evaluations are being used to predict allergenicity and toxicity of vaccine antigens,
particularly for recombinant peptide/protein vaccines where sequence homology searches
could be used to determine if the vaccine antigen has identity or homology to human pro-
teins/epitopes or allergens and may have the potential to induce autoimmunity or allergy.
To predict allergenicity, one of the tools that is used is AllergenFP 1.0 (AllergenFP, 2023).
AllergenFP 1.0 has a dataset of allergens and nonallergens, and uses auto cross-covariance to
generalize the length of all peptides. Analyzed epitopes are compared by calculating the
Tanimoto coefficient. If the epitope has a Tanimoto coefficient that is closer to the allergen
data, then the epitope is predicted to be an allergen (Dimitrov et al., 2014). To predict toxicity
of the epitopes, ToxinPred can be used (ToxinPred, 2023). ToxinPred uses a dipeptide-based
support vector machine (SVM) learning technique to obtain a toxicity prediction model. The
performance of the dipeptide base model has an accuracy of 94.5% (Gupta et al., 2013). While
in silico tools are used to screen vaccine candidates, there is currently a lack of in vivo evi-
dence that molecular mimicry is associated with toxicity or allergenicity of vaccines, although
it remains an issue of concern.
There are some aspects of safety evaluation that could be incorporated very early in
nonclinical development when multiple candidates and formulations are being tested. For
example, vaccine formulations are often tested in efficacy and immunogenicity studies that
do not require strict compliance to Good Laboratory Practices (non-GLP) using different an-
imal models to select the formulation that provides a robust and protective immune response.
In these studies, the vaccine formulations are typically administered in a repeat-dose manner,
similar to the intended regimen for the human clinical study. Antemortem parameters that
are routinely included in toxicology studies (such as clinical signs, ble eds for hematology
and serum biochemistry) may be included in these early non-GLP nonclinical studies to
obtain an early evaluation of vaccine safety. The inclusion of these parameters might assist
when there is a need to select between different vaccine candidate formulations. For example,
although all of the vaccine candidate formulations could provide equivalent immune re-
sponses against the antigen of interest, the hematology and serum biochemistry results might
differ in regards to a potential safety marker (e.g., clinical signs indicative of local reactoge-
nicity or alterations in liver enzym es or liver function tests (LFTs)).
A systems biology approach is being used in vaccinology in order to understand the mech-
anism by which vaccines stimulate protective immunity, and to try to predict the immuno-
genicity or efficacy of vaccines (Nakaya et al., 2012; Oberg et al., 2011). Progress has been
made in this field using microarray data from vaccine studies to identify genes and pathways,
which might be involved in developing more immunogenic vaccines. Similarly, a systems
biology approach is being evaluated to predict reactogenicity and potential general side ef-
fects of vaccines. The dilemma when trying to obtain a “reactogenicity” profile is that the
same genes and pathways that are involved in developing an immune response to the vac-
cine are also involved in the reactogenic response. One solution is to develop a reactogenicity
classifier in animals by testing marketed vaccines for which the clinical adverse event profile
has been well-studied and published. By using a blood gene module data analysis approach,
gene modules have been identified that could be used as classifiers to predict potential vac-
cine or adjuvant reactogenicity (Tang et al., 2008; Wang et al., 2012). The BIOVACSAFE
General de-risking strategies for vaccines 243
II. Vaccines
consortium evaluated six antiviral vaccines to generate training data-sets of pre-/postvacci-
nation vital signs, blood changes and whole-blood gene transcripts, and to identify putative
biomarkers of early inflammation/reactogenicity that could guide the design of subsequent
focused confirmatory studies (Weiner et al., 2019). Additional metagene- and pathway-
based analy tical approaches have been proposed to provide quantitative readouts with
biological relevance, which can be used to study both efficacy and safety to rank vaccine
candidates under development.
For vaccines that could potentially induce a severe febrile response, especially for vaccines
containing bacterial components, early de-risking includes testing for a fever or pyrogenic
response in appropriate animal models or in vitro systems such as the limulus amebocyte
lysate (LAL) assay. The most common animal model used for pyrogenicity testing is the rab-
bit, since rabbits are representative of the human response to endotoxin and nonendotoxin
pyrogens. A temperature rise after administration of the vaccine to rabbits is measured using
a rectal thermometer or thermistor probe. Febrile responses can also be measured in the same
nonclinical species in which the early immunogenicity or efficacy responses are evaluated, us-
ing rectal thermometers or thermal sensing microchips. The in vitro LAL assay is useful in
detecting pyrogens and may be used in place of animal models in cases where the vaccine
does not contain components likely to cause a false positive result (i.e., bacterial components).
However, one limitation of this assay is that it is only capable of detecting endotoxin pyro-
gens. Therefore, in vitro assays using human peripheral blood or human monocytic cell lines
could also be used to measure early cytokine responses and further understand the potential
pyrogenic response resulting from either endotoxin or nonendotoxin pyrogens. A monocyte
activation test (MAT) has bee n developed and validated as a replacement of the rabbit pyro-
gen test (Hoffmann et al., 2005). Internationally validated protocols for the evaluation of py-
rogenic contaminations have been developed using human whole blood or human monocytic
cell lines and quantifying the release of proinflammatory cytokines (e.g., interleukin-1 beta
(IL-1b) or interleukin-6 (IL-6)). In addition to being applied for detecting pyrogenic contam-
inations in aqueous samples and parenteral drugs, adaptations of the assay allow the assess-
ment of lipidic, toxic or immunomodulatory formulations (Stoddard et al., 2010). For
example, studies have demonstrated a correlation between production of proinflammatory
cytokines above a “safety threshold” in vitro and an increase in body temperature in rabbits
(Zaitseva et al., 2012). Human cell-based assays are an important tool for early screening of
new antigen and adjuvant formulations and may help to select safer candidates for further
development (Zaitseva et al., 2012).
De-risking strategies for specific vaccine types
De-risking is performed nonclinically for specific vaccine types based on existing concerns.
If adverse effects have been observed in previous clinical studies with a vaccine, these con-
cerns can sometimes be addressed using specific animal models of disease. Although it can
be challenging to use animal models of disease for toxicology studies, such as the lack of
adequate historical control data, which could result in difficulties interpreting the study re-
sults, there are benefits in using disease models in cases where there is a specific cause for
8. Special considerations for the nonclinical safety assessment of vaccines244
II. Vaccines
concern. Studies in disease models are typically performed in a non-GLP manner and supple-
ment the GLP-compliant studies that are performed in an animal model that is routinely used
for toxic ology studies.
Vaccines with adjuvants
Some adjuvants, such as TLR agonists, exhibit their effects on the immune system in a
species-specific manner (Vaure and Liu, 2014). Since human and murine responses to TLR
activation have some profound differences, species that are closer to humans (such as
nonhuman primates, NHPs) are probably more rele vant for the preclinical studies with
TLR agonists. Since ethical and financial considerations limit the use of NHPs for routine
nonclinical vaccine evaluation, NHPs in immunogenicity studies can be reused and ante-
mortem safety evaluations can be included in a non-GLP manner.
Therapeutic vaccines
Detailed regulatory guidelines do not currently exist for the nonclinical safety assessment
of therapeutic vaccines. Therefore, multiple guidelines such as those encompassing the devel-
opment of viral vectors, nucleic acids and gene therapy, should be consulted to design the
nonclinical progra m for therapeutic vaccines depending on the type of vaccine being
evaluated.
Oncology vaccines: There are several considerations for cancer vaccines in the absence of
specific guidelines. The expected use of the vaccine and dosing frequency needs to be taken
into account. Most cancer vaccines are designed to be dosed repeatedly in order to inhibit
cancer progression. However, because the total number of doses in patients is unknown
and depends on the tumor regression, a streamlined study evaluating a specific number of
doses in a series could be justified because the target may not exist in healthy animals
used for toxicology studies.
Alzheimer’s vaccines: transgenic mouse models of Alzheimer’s disease, which overex-
press the human b-amyloid protein, can be used to demonstrate that Alzheimer’sdisease
vaccine candidates do not cause adverse effects in the brain (Maier et al., 2005). A previous
therapeutic vaccine for the treatment of Alzheimer’s disease (AN1792, consisting of Ab1-
42 amyloid a ntigen and the QS-21 adjuvant) was tested in c linical studies with subjects
having mild-to-moderate Alzheimer’sdisease(Schenk, 2002). Symptoms of meningoen-
cephalitis were observed in 6% of trial patients, and were not predicted by routine
nonclinical safety studies. Retrospective nonclinical studies using several different trans-
genic mouse models of the disease were performed, where it was determined that the
inclusion of the QS-21 adjuvant in the AN1792 vac cine might have contributed to the
adverse response, shifting the immune response toward a Th1-type response including
significant elevations in IFNg (Cribbs et al., 2003). The nonclinical safety assessment of
novel vaccine candidates for Alzheimer’s disease incorporates studies in transgenic mouse
models to evaluate the potential for causing meningoencephalitis or microhemorrhage in
the brain.
De-risking strategies for specific vaccine types 245
II. Vaccines
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