University of Groningen
In vitro approaches for the evaluation of human vaccines
Signorazzi, Aurora
DOI:
10.33612/diss.166150822
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2021
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Signorazzi, A. (2021). In vitro approaches for the evaluation of human vaccines. University of Groningen.
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Chapter 6
Summarizing discussion
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Background
As highlighted in Chapter 1, the development of vaccines is a complex process,
traditionally dominated by trial and error [1]. Long and costly investigations, often
including animal studies, are required. Many vaccine candidates fail to reach the final
stages of development because the results obtained in animals are not translatable
to humans [2]. In addition to their use in research towards new vaccines, in vivo
methods are also heavily employed during batch surveillance of commercially
available vaccines [3]. However, animal tests – despite being the standard evaluation
method for many vaccines – suffer from large variability in the results [4]. The
development of alternative methods is therefore necessary to obtain more consistent
results and to decrease the ethical and economical burdens of animal studies [5,6].
Different types of in vitro assays can be used for the refinement, reduction or
replacement of animal tests during vaccine development and production [7].
Analytical methods study the physico- and immunochemical properties of the
vaccine candidate and its components, such as the antigens’ characteristics and
functionality [8]. Cellular assays can instead be used to characterize the reaction of
immune cells to the formulation [9]. In this thesis, both analytical and cell-based
assays were used to evaluate different properties of two model viral vaccines for
human use, tick-borne encephalitis (TBE) vaccine and influenza vaccine.
Characterizing TBE vaccine-specific immune responses
In Chapters 2-4, we aimed to study the interaction of human immune cells with the
TBE vaccine. Additionally, we harnessed the knowledge gained in these studies to
develop a cell-based assay that could contribute to the replacement of mandatory
in vivo potency testing of TBE vaccine batches. The findings of these chapters are
summarized in Figure 1.
The knowledge on the immune responses to the TBE vaccine is currently limited to
the induction of envelope protein-directed antibodies and of TBEV-specific CD4
+T
cells [10–12]. Innate immune responses, that provide useful information on the
mechanism of action of a vaccine and that can be easily evaluated for assessing the
quality of the formulation, have not been characterized yet. In Chapter 2, we aimed
to investigate the early responses of human immune cells to the TBE vaccine. Primary
cells such as peripheral blood mononuclear cells (PBMCs), despite presenting
donor-dependent variability [13], are a versatile platform for the characterization of vaccine-
or pathogen-associated responses [14–16].
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Figure 1. Graphical summary on the development, assessment and application of an in
vitro system for the evaluation of the TBE vaccine. ❶ Using cell-based assays, we found
that freshly isolated or cryopreserved PBMCs can respond consistently and specifically to I-TBEV, but not to the adjuvanted TBE vaccine. ❷ THP-1-derived cells, instead, do not respond specifically to either formulation. ❸ In freshly isolated PBMCs, the surface protein and the viral genome (sensed via Toll-like receptor 7, red endosomal receptor in the figure) of I-TBEV stimulate IFN-producing plasmacytoid dendritic cells. IFN production induces differentiation of plasmablasts and increase in total antibodies production. ❹ Cryopreserved PBMCs can distinguish high- from low-quality I-TBEV batches, as they induce distinct expression levels of IFN-stimulated genes (ISGs). The expression of ISGs correlates with the percentage of high-quality formulation in a mixed sample. ❺ Using the same platform, we characterized the innate responses in vitro to inactivated or live TBEV; both induce significant upregulation in the transcription of ISGs and of RIG-I-like receptor (RLR) genes, and a limited inflammatory response. In this platform, I-TBEV sensing involves RLRs (blue cytosolic receptor), while the live virus is sensed by Toll-like as well as RIG-I-like receptors. Image created with BioRender.
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In this study, freshly isolated PBMCs were stimulated with the adjuvanted TBE vaccine
and its primary component, formalin-inactivated TBEV (I-TBEV). The cellular
responses were then studied in several cell subsets.
Characterization of molecular markers and proteins expressed and produced by
PBMCs revealed that I-TBEV induced type I interferon (IFN) responses, important
mediators in immunity against live TBEV [17–19]. The I-TBEV-induced IFN-α
production was found to be critical for the differentiation of B cells to plasmablasts.
Additionally, stimulation with I-TBEV resulted in increased production of total IgG
and IgM as compared to untreated cells. The adjuvanted vaccine formulation, on the
other hand, was not able to induce upregulated expression or production of IFN and
interferon-stimulated genes, nor increased plasmablast differentiation or total
antibody production. Aluminum hydroxide, present in the adjuvanted vaccine as well
as in the excipient, had an inhibitory effect on the induction of I-TBEV-associated
responses and caused a decrease in cell viability. Indeed, Al(OH)
3is known to
interfere with readouts in cellular assays, due to both its in vitro toxicity and
antigen-aggregating mechanism [20–23] – notwithstanding its proven safety and adjuvanting
potency in humans [24,25]. Thus, the characterization of the cell responses to the TBE
vaccine was further performed using the vaccine antigen-carrying component,
I-TBEV.
After identifying the induction of type I interferon as characteristic of the response
to I-TBEV, we aimed to pinpoint the cell population(s) involved in the signaling.
Plasmacytoid dendritic cells (pDCs) were found to be in large part responsible for the
I-TBEV-induced IFN-α production. Additionally, I-TBEV promoted the differentiation
of a subpopulation of pDCs, P1-pDCs, previously shown to be highly specialized in
interferon production following virus stimulation [26].
The immunostimulatory mechanism of I-TBEV was shown to require a functional
surface protein, needed for cell binding and entry, and an intact viral genome, sensed
upon internalization of the pathogen. In fact, antibody-mediated virus neutralization,
denaturation of the glycoprotein or inhibition of the RNA-sensing Toll-like receptor
7 (TLR7) all resulted in the abrogation of I-TBEV-induced production of IFN, cytokines
and antibodies, and in the inhibition of cell differentiation. The important role of TLR7
in flavivirus sensing was brought to light by previous in vivo studies, which found a
link between TLR7 deficiency in animal models and increased replication of Langat
virus (a naturally attenuated flavivirus in the TBEV serogroup) as well as of Japanese
encephalitis virus (a mosquito-borne neurotropic flavivirus) [27,28]. Triggering of
TLR7 in TBEV sensing has not been previously proven, but only postulated [18]; here,
we show that this receptor is involved in the response of freshly isolated PBMCs to
inactivated TBEV. Thus, the stimulating properties of the genomic RNA appear to be
retained upon virus inactivation by formaldehyde.
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These results highlight that the functional integrity of the E glycoprotein and of the
viral genome are preserved during manufacturing of the TBE vaccine and are
required for the induction of type I interferon, an important link between innate and
adaptive immunity [29,30].
Establishing an in vitro evaluation system for the TBE
vaccine
As for other vaccines, the potency of the TBE vaccine is evaluated for each newly
produced batch using a lethal challenge test on immunized mice [31]. Recently,
several in vitro assays have been developed as alternatives to animal-based tests for
the quality control of vaccines [9,15,32–36]. A strategy that could reduce the need of
in vivo tests for vaccine batch release is the consistency approach: newly produced
batches of a vaccine are compared to reference ones (of proven in vivo potency) to
verify their conformity with respect to a series of quality attributes assessed in vitro
[8]. In Chapter 3, we applied the knowledge gained in Chapter 2 to develop a
cell-based method for assessing the conformity of TBE vaccine batches with respect to
the induction of innate immune responses. Together with other in vitro assays, this
method could contribute to the reduction (or replacement) of in vivo potency tests
for lot release [3]. The cellular platform for such an assay should display distinctive
responses to high- and low-quality vaccine batches and, to be applicable in a quality
control environment, should respond in a reproducible manner.
Antigen-presenting cells (APCs), as the sentinels of the immune system and main
targets of TBEV in the first stages of infection [37], are an interesting candidate
platform for measuring quality-related biomarkers. We assessed two different
APC-like platforms: one cell line-based and one primary cell-based. Cell lines are
advantageous as a consistent source of cells not subject to donor-dependent
variation. In this study we used THP-1 cells, long used as a monocyte, macrophage
and dendritic cell model [38–40]. Primary cells, on the other hand, are more closely
representative of in vivo responses than cell lines [41], and were identified in Chapter
2 as able to display I-TBEV-induced responses. Here, we used frozen-thawed PBMCs,
which would be more convenient than freshly isolated cells in an industry setting.
Using RT-qPCR as readout, we showed that the THP-1-derived platforms were
unsuitable for our purposes, as the cells showed no vaccine-specific activation. Yet,
THP-1 cells were able to generate an antiviral response after stimulation with live
TBEV. Indeed, THP-1 cells are permissive to TBEV infection [42], but produce lower
amounts of TBEV-induced chemokines than other cell lines [43]. The responses of
THP-1 cells to I-TBEV, a less potent stimulus than the live virus, might therefore have
been too low to reach the limit of detection.
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Primary cell-based platforms, in contrast, were able to respond specifically to I-TBEV
through increased transcription of interferon-stimulated genes (ISGs), in line with the
results from our previous study. Unsurprisingly, also in this platform the presence of
alum in the adjuvanted vaccine induced cytotoxicity; thus, we focused on assessing
quality parameters in I-TBEV.
Using the inactivated virus to stimulate PBMCs, we found that, in multiple donors,
the expression of several ISGs was similarly induced by different (conforming) vaccine
batches and differentially regulated in response to high- and low-quality batches.
Additionally, we evaluated the sensitivity of the assay, and showed that it could
distinguish between I-TBEV batches with differences in the formulation quality as low
as 20%.
These results support the suitability of a cell- (and specifically PBMC-) based platform
for batch conformity testing of the non-adjuvanted TBE vaccine. Such a platform
could be used for in-process control testing of vaccine batches, as well as for
screening vaccine candidates during development [14,15,44–46].
Applying in vitro evaluation systems: analysis of TBE
vaccine-associated pathways
Applying the platform presented in Chapter 3, in Chapter 4 we aimed to further
study the functions and pathways induced by I-TBEV. Additionally, we set out to
compare differences in the response to replicating and inactivated virus, to analyze
possible effects of the inactivation process on the immunogenicity of the vaccine.
Using RNA sequencing, we assessed the innate immune responses of PBMCs to live
and inactivated TBEV by analyzing the cells’ transcriptome. The results show that
both stimuli induced a distinct interferon-dominated signature in PBMCs, confirming
our previous findings on the important role of type I IFN in TBEV-associated
responses and in line with data on common early immune signatures induced by
several vaccines [17,47].
Inflammatory responses, on the other hand, were selectively regulated. After
treatment of the PBMCs with (I-)TBEV, the JAK-STAT pathway was upregulated, while
the expression of genes involved in interleukin signaling, antigen presentation and
lymphoid–non-lymphoid cell interaction was downregulated. These results are in
accordance with our earlier findings [48], but appear characteristic of the pathogen.
Indeed, other whole inactivated vaccines (WIVs), such as those against influenza virus
and respiratory syncytial virus, induce upregulation of some of the genes we found
downregulated in (I-)TBEV-treated cells [44,49–51].
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After characterizing the downstream response of (I-)TBEV-stimulated PBMCs, we
next focused on identifying the pattern recognition receptors (PRRs) involved in the
transduction of the signal. RNA-Seq had identified, next to the characteristic IFN
signature, an upregulation of cytoplasmic RIG-I-like receptors (RLRs) in cells
stimulated with live or inactivated TBEV. Thus, we analyzed the involvement in (I-)
TBEV sensing of RLRs – as well as of TLRs, prompted by the findings in Chapter 2.
Using pathway-specific inhibitors and reporter cell lines, we demonstrated that RLRs
are involved in the initiation of the antiviral response to I-TBEV and live TBEV. Indeed,
activation of RLRs is known as a predominant mechanism in sensing flaviviruses
[52,53]. TLR pathways were implicated in the induction of the antiviral response
against live TBEV, but did not appear to be involved in the sensing of the inactivated
virus in this study. This result is in apparent contrast with the TLR7 involvement
identified in Chapter 2 in freshly isolated cells. However, the use of frozen-thawed
PBMCs in Chapter 4 might explain these differences.
Indeed, cryopreservation of PBMCs has been shown to affect the pDC population
[54], identified in Chapter 2 as the primary source of I-TBEV-induced IFN-α. Further
investigation corroborated the hypothesis of a detrimental effect of cryopreservation
on pDC functionality, as a reduction by 75% of the I-TBEV-induced IFN-α was
observed in PBMCs that have undergone the freezing and thawing process (Etna et
al., unpublished results). Our working theory is therefore that pDCs, in which the
RIG-I pathway is dispensable for an RIG-IFN response [17], would preferably sense and
respond to I-TBEV through the TLR7 pathway. Other myeloid cells, less affected by
the cryopreservation process [55], could sense I-TBEV mainly through RLRs instead.
The predominant role of RIG-I in I-TBEV sensing by myeloid cells other than pDCs
would also explain the induction of the type I interferon response and of the
JAK-STAT pathways [56] and the unsuccessful activation of the THP-1 cells in Chapter 3,
as these monocytic tumor cells show a much lower expression of this receptor
compared to primary cells [57] and other cell lines [58].
Overall, Chapter 2 and 4 highlight the importance of PRR stimulation by the viral
RNA for the induction of an immune response by the TBE vaccine. The
immuno-stimulating properties conferred by the presence of the viral genome in a vaccine are
already known; for instance, influenza vaccines containing the viral RNA are more
immunogenic than formulations lacking it [44,49,59]. In the absence of the viral
genome (as in vaccines based on virus-like particles), TLR7 or RIG-I agonists have
been proposed as adjuvants to enhance poorly immunogenic vaccine candidates
[60–64]. While such an adjuvanting strategy is attractive from an immunogenic point
of view, its implementation has been hindered by safety concerns [65]. The
preservation of the viral genome during the production of whole inactivated vaccines
is therefore paramount, as this feature provides important self-adjuvanting
properties to the formulation.
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Collectively, the data presented in Chapters 2-4 brings new insights into the innate
response elicited and the signaling pathways engaged by live TBEV and by the
inactivated virus – the primary constituent of the TBE vaccine. Additionally, the results
obtained prove the applicability of the designed PBMC-based system for a variety of
purposes, from mechanistic immunological studies (Chapter 2 and 4) to batch
release testing (Chapter 3).
Applying in vitro evaluation systems: analysis of
inactivation-associated effects
The data presented in the previous chapters underscored the importance of
maintaining the structure and functionality of the viral particles and their
components during the preparation of inactivated vaccines. The integrity of the viral
proteins and genome can be affected by the inactivation process through the
introduction of nicks or cross-links [66,67]. In Chapter 5, using immunochemical and
cell-based methods, we assessed different inactivation procedures for their effect on
the virions’ integrity and functionality – here with influenza as a model.
In the case of influenza vaccines, all formulations derived from whole viral particles
(with the exception of live-attenuated vaccines) share a virus inactivation step [68].
The concept behind the inactivation procedure is that the process must inhibit the
replication of the virus without impacting on its antigenicity (for example by altering
the vaccine antigens) [69–71]. Influenza inactivation is usually achieved by exposure
of the live virus to β-propiolactone (BPL) or formaldehyde (FA), two common
inactivating chemicals [72]. Pharmacopoeias specify the maximal concentration of
these compounds to be used during the inactivation procedure; yet, the
standardization of all other parameters is left to the manufacturers. Studies have
shown that excessive inactivation with FA or BPL may cause alterations to the
antigens that result in diminished functionality of the viral particles in the vaccine
[73–78]. The effects of FA on the physicochemical properties of proteins are known
[79], while BPL is traditionally thought to mainly affect nucleic acids [67,80]. However,
recent studies have highlighted that BPL could have an effect on proteins as well
[68,81].
In this study, we assessed the impact of the two inactivating agents on the
immunochemical properties of the virus particles in several influenza virus strains, to
provide insights for the improvement and harmonization of influenza vaccine
production. Our results show that both BPL and FA caused alterations in the
properties of the virions, as summarized in Table 1. Additionally, the magnitude of
the effects caused by the treatments was found to be strain-dependent.
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Table 1. Impact of different chemical inactivation methods on influenza virus infectivity, binding, fusion and TLR7 stimulation capacity. These properties were assessed in live and
inactivated virus preparations and compared across several influenza virus strains. The number of symbols indicates the degree of efficacy (green checks) or of the detrimental effects on the virions (red arrows) of the inactivation treatments.
Generally, BPL was more efficient in inactivating the viral particles than formaldehyde,
as assessed by measuring the residual infectivity. This result was not unexpected, as
low temperature FA-treatment was previously found to cause incomplete
inactivation [82]. Following the particles’ interaction with cells, we then assessed the
ability of the inactivated virus to bind to cell membranes (measured using the
hemagglutinating properties of influenza virus [83]), to fuse in conditions of low pH
[84] and to activate TLR7, a receptor present in the endosomal compartment,
indicating successful cell entry [85]. Both BPL and FA impacted on the binding and
fusion capacity of the viral particles, in accordance with previous studies [81,86,87].
In our head-to-head comparison, BPL caused greater loss of these properties than
FA. The stimulation of a TLR7 reporter cell line was also affected by the inactivation
treatments. In this case, the inactivation with FA impacted more on influenza-induced
TLR7 activation than the BPL treatment. While we did not investigate the direct effect
of the inactivating agents on the viral RNA, other studies showed that the FA
inactivation procedure could affect the integrity of the genome [87–89]. However,
the effects of an inactivating agent on the RNA-TLR7 interaction alone are difficult
to estimate. Stimulation of an endosomal receptor presupposes binding and
internalization of the virus, processes that can be affected by inactivation-induced
protein alterations.
In summary, this study shows that chemical inactivation impacts on various
properties of influenza virus in a strain-dependent way, and that many other factors
beyond the concentration of the inactivating agent itself – most prominently the
temperature used during the inactivation process [72,82,90,91] – have to be
considered. While subtle discrepancies in differently-inactivated viruses do not
necessarily translate to significantly distinct safety and potency of the vaccine in vivo
[92], it should be noted that a thorough investigation has not yet been presented.
For such an analysis, this study describes relatively simple but highly informative
methods that could be used in an in vitro vs in vivo evaluation to assess and
ultimately select better inactivation strategies.
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In vitro vaccine evaluation systems: challenges and
opportunities
This thesis has presented a number of in vitro vaccine evaluation systems; a relevant
question that remains to be addressed is for which applications can these methods
actually be used, and at what stages of the vaccine development process. In vitro
evaluation systems can in general be applied for different purposes:
•
Research on vaccines and vaccine-induced responses.
In vitro methods can be used to study antigen conformation, alterations,
vaccines’ mechanisms of action and relevant pathways. For analyses focused on
the properties of the vaccine itself – such as antigen assembly, quantification and
adsorption to the adjuvant – immunochemical methods are already extensively
used as they provide a level of detail not achievable with animal studies [93–96].
The evaluation of vaccine-induced immune responses in vitro presents a
different level of complexity, as the immune system responds to extraneous
agents through dynamic interactions between several cell types and
environments. Innate immune responses are relatively easily measurable, since
they can be generated by individual cell types – in particular APCs. Cellular assays
focused on the interaction of APCs with vaccines have identified selected
pathogen-specific vaccine-induced responses [9,44,46,97,98]. Omics approaches
have pushed this even further, allowing us to obtain a complete picture of the in
vitro innate responses to a particular vaccine (as was done in Chapter 4) and to
draw more general conclusions regarding vaccine-associated signatures [99–
103]. For what concerns the evaluation of adaptive responses (the induction of
which is the actual goal of vaccination), the use of in vitro methods can prove
more complicated. Adaptive responses necessitate cell interactions;
antigen-specific activation of B or T cell lymphocytes requires the presence of unique
cells with a particular B- or T-cell receptor molecule [104,105]. Nevertheless,
novel in vitro platforms for the evaluation of B and T cell responses are being
developed [14,106], and will eventually allow prediction of adaptive responses
upon vaccination, particularly when using systems based on several modules
[107]. Thus, basic research on vaccines and vaccine-induced immune responses
remains one of the major applications of immunochemical and cell-based in vitro
methods.
•
Selection and comparison of promising vaccine candidates.
Apart from their application in vaccine research, in vitro methods offer
interesting possibilities for the pre-clinical development stage. In this phase,
cellular methods are already in use – however mainly for toxicity assessments
rather than efficacy determination, for which animal studies remain the go-to
approach [108].
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The use of animal models has resulted in the identification of many vaccine
candidates. Yet, a number of them did not perform adequately in subsequent
clinical trials, calling into question the relevance of such animal models for the
prediction of responses in humans [109,110]. Most notably, research into HIV
vaccines has faced many setbacks, as more than 90 vaccine candidates that were
successful in animal studies failed in humans [111]. This result is especially
disappointing considering that for HIV vaccines the animal models often
included non-human primates, expected to generate highly translational results.
In hindsight, this turned out to be a dangerous assumption, as it led researchers
to dismiss unsupportive in vitro data [112,113]. Therefore, the use of alternative
methods for the determination of vaccine candidates’ efficacy should not be
secondary but parallel – or even prior – to in vivo studies. The establishment of
panels of assays could further improve the translational value of the in vitro
results, by offering a comprehensive overview of the vaccine candidates and of
the associated responses. The use of high-throughput techniques could further
speed up this selection process [114]. In addition to their application in
pre-clinical development, in vitro methods can support the selection of the most
promising vaccine candidates in the clinical phases through head-to-head
comparisons of several candidates.
•
Assessment of the quality of commercial vaccines.
As abundantly discussed, one of the most extensive uses of animals in the
vaccine development and production environment lies in lot release testing
[115]. Despite the fact that these in vivo tests have been used in quality control
for several years, intrinsic issues pertaining the reproducibility of results from
animal experiments can cause high variations [116]. In adherence to the
consistency approach, in vitro testing systems can be used to replace, reduce or
refine animal tests – thereby addressing scientific, economical and ethical
concerns. In vitro assay can evaluate the vaccine quality and batch-to-batch
consistency using different strategies. Immunochemical binding assays, allowing
antigen quantification as a measure of vaccine efficacy, are in use in parallel to
(or proposed as replacement of) in vivo assays for some human as well as
veterinary vaccines [33,117–120]. While still not commonly used for the quality
control of vaccines, several types of cell-based in vitro methods can assess the
biological activity as relevant indication of the potency in vivo. Cellular assays
developed to evaluate the quality or conformity of the formulation do so by
measuring the production of vaccine-encoded genes, the cell responses to the
vaccine or the vaccine-conferred in vitro protection [9,15,121–123]. A number of
these alternative methods are in the process of being accepted and implemented
by pharmacopeias, as the use of in vitro assays for the purpose of batch release
testing of vaccines is being promoted by the scientific, industrial and regulatory
communities alike [124].
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Keeping these purposes in mind, the in vitro methods – and especially the cell-based
assays – presented in this thesis can be re-evaluated with respect to their potential
application. A summary of the conclusions drawn is shown in Table 2.
For basic research on human vaccines, in vitro studies offer the possibility of testing
the effect of the formulations on the target species’ cells – which, as previously
discussed, can respond quite differently than what results from animal models might
suggest. As shown in Chapters 2 and 4, the cell-based methods described allow the
investigation of vaccine-associated innate responses. At the research stage of vaccine
development, in vitro systems using primary cell platforms may provide more
representative data than that obtained with cell lines. As shown in Chapter 3,
immortalized cells might present different permissiveness and responsiveness to the
pathogen in its live or inactivated form.
Since basic research is often performed across several institutions, the methods and
platforms need to be relatively universally available, simple and inexpensive. Such
was the case for the assays employed in this thesis to study cytokine production,
expression of genes and surface markers, and functionality of the virions. An example
of basic research in which the PBMC-based assay described would be valuable is the
in vitro investigation of the immunological basis of vaccine failure. Unsuccessful
immunization following vaccination occurs most frequently in the elderly [125], and
has been observed for viral vaccines against TBE, influenza, herpes zoster, hepatitis
B, mumps and measles [126–131]. Analyzing immune cells from subjects in which the
vaccine did not provide protection and comparing the induction of
vaccine-associated biomarkers between donors from different age groups may help unravel
the mechanisms of immunosenescence, and could lead to improved immunization
efficacy.
Similarly to their application in vaccine research, the cell-based methods described
can be employed in the pre-clinical and clinical phases. In parallel – or prior – to the
in vivo studies performed to evaluate potency and safety, in vitro methods such as
those presented in this thesis could systematically assess several formulations and
expedite the selection of the most promising vaccine candidates. For assays
monitoring the activation of immune cells, an initial investigation into the cellular
responses occurring in the animal models following immunization should be
performed to confirm the relevance of the potential biomarkers analyzed in vitro.
Besides supporting the pre-clinical development phase, primary cell-based methods
(such as those used in Chapters 2 and 3) can also be applied during the clinical stage:
in particular, ex vivo analyses of the responses in immune cells from the vaccinated
subjects can offer an in-depth characterization of the vaccines’ function in humans.
These screenings are indeed becoming increasingly common during early clinical
studies of new vaccines [132–135], as they provide a better understanding of the
underlying immunization mechanism.
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Table 2. Applicability of cellular platforms at the different stages of vaccine research, development and production. 3 green checks indicate that the platforms are already suitable
and applicable. 2 or 1 checks indicate platforms that are not as suitable or that require further optimization (superscripts). A red cross indicates a platform unsuitable for the scope.
The most difficult stage for the implementation of primary cell-based methods is
arguably the batch release testing for commercial vaccines. In this phase more than
in all the others, the system(s) used (be it animal models, cellular platforms or
analytical methods) must be practical - due to the high frequency of testing – and as
consistent as possible, to allow comparisons across multiple batches. So far,
cell-based assays are used only for toxicity assays, while immunochemical methods are
accepted as alternatives for potency assessments [136,137]. As the interest in
replacing (or at least reducing) animal testing is now increasing in all sectors involved
in the vaccine industry, more and more cell-based assays are being developed for
potency assessments [124]. The implementation of such methods for the evaluation
of vaccine batches is therefore a question of when, not if.
Yet, some cellular platforms present more challenges than others; primary cells are
obviously less easily retrievable than cell lines, and, as shown in Chapter 3, can
exhibit donor-specific responses – at the very least quantitatively. These hurdles can
be overcome by bulk processing of PBMCs, which could be isolated, screened and
selected all at once, thereby allowing consecutive use of consistent aliquots.
Additionally, pooling of PBMCs from multiple donors could further increase their
applicability by mitigating variations in responses [13,138]. PBMC pooling, however,
would only be applicable for assessing the innate immune responses to a vaccine
after short-term stimulation. Longer incubation might lead to allogeneic responses
in cells from mixed donors [139], which would affect the assay readouts.
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The solutions proposed can minimize the intrinsic problematics derived from using
primary cells. However, for the purpose of ensuring batch-to-batch conformity,
the consistency approach prescribes the use of in vitro assays not necessarily for
predicting the potency in vivo, but for analyzing the consistency of vaccine batches
based on quality attributes [8], which – once identified – could be assessed in the
most convenient platform. Thus, the choice of platform does not have to favor the
cells that preserve the most in vivo properties (such as PBMCs), but rather those that
present the least complexity of usage and the highest reproducibility. Consequently,
cell lines remain the most homogeneous platform for the purpose of vaccine batch
assessment, and were our initial choice for the assay described in Chapter 3. If a cell
line responsive to the vaccine is identified, the development and adoption of a cell
line-based vaccine testing system would prove more feasible than the
standardization of a primary cell platform.
For the identification of such a cell line (and of potential readouts)
,primary cell-based
assays can nevertheless be of help: once the pathways and receptors involved in the
specific response to the vaccine are characterized (as was done in Chapters 2 and
4), the candidate platforms could be efficiently screened and selected. In the case of
the TBE vaccine, for example, a potential platform (other than the PBMC-based one)
should possess functional RIG-I-like receptors and potent interferon responses.
Some commercial cell lines with these characteristics are available and even include
reporter constructs, which would allow fast and simple readouts [140,141]. If these
commercial platforms were to present low sensitivity or non-responsiveness,
transfecting the receptor of interest to create a custom-made stable cell line (an
approach already taken for some toxicological assays [142]) could be considered.
In vitro vaccine evaluation systems: future perspectives
With the continuous advancements in bioinformatics and the steady decrease in cost
of omics approaches, it is safe to predict that multiparametric analyses will become
more and more common in the vaccinology field [143]. For basic research purposes,
a more in-depth analysis into the cell-specific responses to a vaccine could be
obtained through single-cell sequencing of in vitro vaccine-stimulated cells [144].
This method is similar to that described in Chapter 4 but offers the possibility of
zooming in on the responses of individual cells to identify cell type-specific functions
and pathways induced. Deconvolution methods, which can associate observations in
bulk transcriptomics data (from unfractionated PBMCs) to specific immune subsets
[145], can already partially address this task by inferring cell type proportions
[146,147]. However, the profiling of cellular immune responses in individual subsets
enables a highly detailed characterization of the vaccine-elicited cellular immunity,
especially if combined with proteomics or metabolomics analysis [102]. Furthermore,
single-cell sequencing could potentially suggest the most appropriate cell types for
the development of cellular platforms to be used in subsequent batch release testing.
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Besides advanced readout methods, several promising and increasingly
sophisticated cellular platforms are being developed. These cell-based models try to
replicate the complexity of the immune system, which exerts its response through an
extensive range of organs, tissues and cell types with specialized functions. Complex
systems (usually) present the disadvantage of being expensive and difficult to
standardize across different laboratories. Yet, they may also provide an opportunity
to overcome the necessarily reductionist approaches of conventional in vitro
methods to the investigation of immune responses.
The MIMIC® (modular immune in vitro construct) system represents one such an
example. This system consists of several modules, that aim to predict the migration
of APCs in peripheral tissues or the induction of naïve and recall B and T cell
responses in lymphoid tissues using isolated PBMCs and tissue constructs [148]. An
advantage of the MIMIC® system is that it offers the possibility to automate the
testing process and evaluate the responses of a large number of donors
simultaneously, features that favor its use in a pre-clinical development setting. A
comparison of the immune responses in vivo, ex vivo and in the MIMIC®-based assay
showed the suitability of the system to support vaccine development also during
clinical studies [149]. In this study, the platform was in fact able to reflect
age-associated differences in vaccination-induced immunity, showing reduced
influenza-specific B and T cell responses in elderly donors that paralleled the in vivo antibody
responses.
Adding another layer of complexity are the three-dimensional systems using
micro-organoids and organ- or tissue-on-a-chip models [150,151]. Many of these platforms
are being developed for cancer immunology research [152–154], but could very well
be applied for the investigation of vaccine-associated responses. A recent work by
Wagar et al. describes a functional organotypic system that recapitulates key
germinal center features, including the production of antigen-specific antibodies,
somatic hypermutation, plasmablast differentiation and class-switch recombination,
allowing the investigation of adaptive immune responses upon vaccination in vitro
using human cells [155].
These novel platforms and technologies have the potential of further improving our
understanding of vaccine immunology. For the purpose of lot-to-lot testing of
vaccines, however, they might prove difficult to harmonize across multiple vaccine
manufacturing sites and contract research organizations, and complex to validate for
the approval by health agencies. Simpler methods, such as those described in this
thesis – not attempting to fully mimic the in vivo immune response, but evaluating
(several) key aspects of vaccine quality – could in the near future advance the
implementation of the consistency approach, eventually leading to a vaccine
development and testing environment that is both more effective for humans and
less painful to animals.
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