Gene-based vacines and immunotherapeutic strategies against neurodegenerative diseases-Potential utility and limitations

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Gene-based vaccines and immunotherapeutic strategies
against neurodegenerative diseases: Potential utility
and limitations
Jeremy J. Kudrna
a
& Kenneth E. Ugen
a
a
Department of Molecular Medicine, Morsani College of Medicine, University of South
Florida, Tampa, FL 33612
Accepted author version posted online: 30 Jun 2015.
To cite this article: Jeremy J. Kudrna & Kenneth E. Ugen (2015): Gene-based vaccines and immunotherapeutic strategies
against neurodegenerative diseases: Potential utility and limitations, Human Vaccines & Immunotherapeutics, DOI:
10.1080/21645515.2015.1065364
To link to this article: http://dx.doi.org/10.1080/21645515.2015.1065364
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1
Gene-based vaccines and immunotherapeutic strategies against
neurodegenerative diseases: potential utility and limitations
Jeremy J. Kudrna and Kenneth E. Ugen#
Department of Molecular Medicine
Morsani College of Medicine
University of South Florida
Tampa, FL 33612
#Corresponding Author Email: kugen@health.usf.edu
Abstract
There has been a recent expansion of vaccination and immunotherapeutic strategies from
controlling infectious diseases to the targeting of non-infectious conditions including neurodegenerative
disorders. In addition to conventional vaccine and immunotherapeutic modalities, gene-based methods
that express antigens for presentation to the immune system by either live viral vectors or non-viral
naked DNA plasmids have been developed and evaluated. This mini-review/commentary summarizes the
advantages and disadvantages, as well as the research findings to date, of both of these gene-based
vaccination approaches in terms of how they can be targeted against appropriate antigens within the
Alzheimer’s and Parkinson’s disease pathogenesis processes as well as potentially against targets in other
neurodegenerative diseases. Most recently, the novel utilization of these viral vector and naked DNA
gene-based technologies includes the delivery of immunoglobulin genes from established biologically
active monoclonal antibodies. This modified passive immunotherapeutic strategy has recently been
applied to deliver passive antibody immunotherapy against the pathologically relevant amyloid beta
Downloadedby[JeremyKudrna]at12:3803July2015

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protein in Alzheimer’s disease. The advantages and disadvantages of this technological application of
gene-based immune interventions, as well as research findings to date are also summarized. In sum, it is
argued that further evaluation of gene based vaccines and immunotherapies against neurodegenerative
diseases are warranted to determine their potential clinical utility.

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Introduction
Historically, vaccines were developed and utilized for the control and prevention of a number of
infectious diseases and by virtue of this action has had a considerable role in improving public health and
increasing life expectancy 1. More recently, vaccine and immunotherapeutic strategies have been applied
to non-infectious diseases. Specifically, a number of cancers of non-infectious origin, which typically
generate altered molecules following malignant transformation, have had these putative antigens
targeted for vaccine and immunotherapeutic interventions 2. Even more recently several
neurodegenerative diseases have been targeted as well for immune-based prophylactic and
immunotherapeutic strategies 3, 4. These strategies are based on the generation of mutated or altered self-
proteins that can overcome immunological tolerance and can often function as antigens 5, 6. The majority
of vaccine research and development in this area have centered on the two most common
neurodegenerative diseases, Alzheimer’s disease (AD) and Parkinson’s disease (PD), which are
characterized by progressive dementia and motor system disturbances respectively 6-17.
Conventional active and passive immunotherapies against Alzheimer’s and Parkinson’s disease
Immune based strategies against these diseases are attractive, since, particularly in the case of AD,
there are few, if any available effective conventional pharmacological therapies 7. Initial vaccination
studies have targeted the beta amyloid (A ) protein, which is theorized to be important in AD disease
etiology and/or pathogenesis 7. These studies were performed in transgenic (Tg) mice that express
human A Promising results in these experiments were obtained including a lowering of brain A levels
as well as an amelioration of cognitive deficits in the mice 10, 11, 13, 15. The transition of these Tg mouse
vaccination studies to human trials have suggested some potential clinical efficacy, but have also produced

4
some serious adverse side effects, resulting in the cessation of the clinical trial 18, 19. It is hypothesized
that the vaccine adjuvant used in this clinical vaccine trial as well as the associated activation of
A cell epitopes (i.e. resulting in potential Th1 autoimmune responses), may have been major
mediators of the most severe side effect, aseptic meningoencephalitis 4, 20. Therefore, A peptides
devoid of T cell epitopes are currently being evaluated for safety and efficacy as vaccines 10, 11.
Likewise, a putatively safer adjuvant-free passive immunotherapy approach, using anti-A monoclonal
antibodies (mAb), demonstrated some efficacy in appropriate Tg mouse models 21 but initial human
clinical testing using this strategy, with humanized versions of the murine mAbs, failed to demonstrate
any long-term apparent significant clinical benefit in terms of preventing or slowing cognitive decline,
with also some concern about potential toxic adverse effects 22. It is hypothesized that the failure of the
passive immunotherapy approach may have been due to an uncertainty relating to the appropriate time
point in the AD pathogenesis process at which to administer the antibodies 23. Irrespective of these
findings, there continues to be some enthusiasm that such a passive immunotherapeutic strategy could
be effective if the appropriate timing of the mAb administration is determined and the potential adverse
effects eliminated. As such, further clinical trials are planned 23. As well, in addition to A the tau protein,
which is a component of neurofibrillary tangles theorized as well to be relevant in AD pathogenesis, has
also been suggested to be a potential vaccine and immunotherapy target 24. Therefore, immune-based
strategies targeting the pathologic form of tau are in pre-clinical and clinical testing 25.
In comparison to AD, vaccine and immunotherapy research and development targeting relevant
protein antigens in PD are more limited 9. The major protein theorized to be associated with the
pathologic dopaminergic neuron loss in PD is alpha-synuclein ( syn). Specifically, the aggregated form
of this disordered protein is hypothesized be a major contributor to the PD pathogenesis 26. Both syn
peptide or recombinant protein vaccines as well as passive immunotherapeutic strategies targeting
syn against PD have been evaluated 8, 9 27. In syn expressing Tg mice or in a rat model expressing

5
syn genes, delivered by an adeno-associated virus 9 (AAV-9) vector, the use of syn peptide,
recombinant protein, peptide pulsed dendritic cell (DC) vaccines or passive delivery of anti- syn
antibodies have been demonstrated to ameliorate some of the pathologic and cognitive deficits
characteristic of these models 6, 28-30. These findings indicate that immune-based interventions might
have some utility against PD as well as against AD 9.
Gene-based active and passive immunotherapeutic strategies against Alzheimer’s and Parkinson’s
disease: Adeno-associated virus and naked DNA plasmid mediated delivery
In addition to the more conventional peptide and protein vaccine and passive antibody immune-
based strategies against AD and PD that have been evaluated, gene-based vaccines and
immunotherapeutic methods have been recently examined targeting these diseases. Typically, this gene-
based vaccine and immunotherapeutic technologies have included viral vector and non-viral based (i.e.
naked DNA) delivery strategies 31, 32.
Viral vector based vaccine delivery methods have an advantage over inactivated peptide/protein
preparations or passive antibody administrations since, in principle, a single administration of the viral-
vector based vaccine can result in long term expression of the vaccine antigen as opposed to the typical
necessity for repeated immunizations with peptide or recombinant protein antigens or passive
antibodies 32, 33. However, it has also been suggested that continued long-term expression of antigens by
viral vectors might result in adverse effects, including the potential generation of autoimmunity. Because
viral vectors typically induce both humeral antibody and T cell immune responses, they are often useful
for controlling a number of infectious agents such as viruses 33. As well, such “live” preparations are less
likely to require the addition of an adjuvant to stimulate effective immune responses. However, the
generation of T cell responses (i.e. cytotoxic T lymphocytes) by this strategy, in the context of

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neurodegenerative diseases such as AD, likely increases the development of adverse effects even in
comparison to “adjuvanted” peptide or recombinant protein vaccines. Other putative disadvantages with
the use of viral vector based vaccines, both generally as well as related to applications against
neurodegenerative diseases, are the potential adverse effects elicited by stimulation of preexisting
immunity against the viral vector backbone. These responses can typically influence both safety and
efficacy. This has been particularly problematic for adenovirus vectors 32. In contrast, adeno-associated
(AAV) viral vectors do not typically induce significant untoward anti-vector backbone immune responses
32. Furthermore, these vectors are non-pathogenic and lack of toxic activity. Therefore, AAV vectors have
typically been a more attractive viral vector based delivery of DNA for gene therapy purposes as well as
for expression of vaccine antigens against infectious and non-infectious diseases. These vectors are also
attractive for use since they mediate long-term expression in a number of tissues, including in non-
dividing cells such as neurons 34. To that end, for vaccination purposes, Mouri et. al. demonstrated that an
A expressing AAV vector decreased A burden and cognitive deficits in murine Tg AD models 35. As
well, Hara and colleagues reported the development of an oral A vaccine, delivered by an AAV vector,
which mediated a decrease in A burden in the brain 36. To our knowledge, however, viral vectors such
as AAV have not been evaluated, to date, for the delivery and analysis of potential efficacy of syn
vaccines in rodent models of PD.
In addition to viral vector based strategies, non-viral naked DNA vaccines have been developed
and evaluated against a number of targets including infectious agents, cancers as well as more recently
neurodegenerative diseases such as AD and PD 37-40. This non-viral DNA vaccine strategy has been
developed and evaluated over the past 25 years 31. Initial studies were performed with intramuscular
injection of the DNA plasmid with transfection of muscles cells and antigen presenting cells such as
dendritic cells (DC), for subsequent expression, processing and presentation to the immune system 31.
DNA vaccines have several conceptual and practical advantages over other vaccine types including

7
relative ease of production, stability, as well as a favorable safety profile including a lack of anti-DNA
immune responses which has been a problematic limitation of some of aforementioned viral vector based
delivery systems 31. In terms of inducing both antibody and cellular immune responses, DNA vaccines
appear to mimic responses elicited live attenuated and viral vector based vaccines 31. However, the
practical clinical utility of the conventional naked DNA vaccine approach has been limited by the low
level of delivery of DNA into host cells 31. This inefficient delivery coupled with other factors affecting
expression, such as non-optimized delivery plasmids, have been theorized to hamper the ability of this
vaccine approach to generate biologically active immune responses of clinical significance 31, 40, 41.
Recently, various plasmid optimization and delivery enhancement methods have been evaluated, most
notably, the use of electric pulses (i.e. in vivo electroporation) to enhance the uptake of DNA plasmids, for
vaccination as well as other applications 40, 41.
This technique, designated EP, uses electric pulses to disrupt plasma membrane permeability to
create temporary pores that facilitate entry of molecules, including conventional drugs and DNA into cells
40, 41. EP is hypothesized to mediate, in the case of DNA delivery, the enhancement of expression and
ultimately the immunogenicity of the antigens generated by the administered genes 40, 41. In addition to
specific vaccination applications, EP has also been demonstrated to have immunotherapeutic potential
for the delivery and enhancement of the expression of disease modifying immunomodulatory cytokines
42, 43. To date, results from a number of clinical trials utilizing EP to deliver DNA has demonstrated an
excellent safety profile for EP with only limited and temporary side effects, coupled with some clinical
efficacy 40. Therefore, EP mediated delivery is theorized to have great potential in overcoming some of
the initial limitations of DNA vaccines which may allow this technology to be clinically useful.
The application of novel and safe vaccination strategies, such as naked DNA is particularly
relevant for AD, since, as indicated above, development of adverse effects from peptide vaccination has
occurred, due likely to the untoward generation of T cell responses against in addition to suggested
safety concerns with the use of co-administered adjuvants. To that end, naked DNA based immunization

8
was initially evaluated against A for AD in the mid 2000s 44. In those provocative studies a DNA vaccine
expressing the peptide of AD, delivered by gene gun technology, resulted in a significant bias for the
generation of Th2 immune responses. Other studies have indicated that DNA vaccines decreased Th1
cell proliferation coupled with a decrease in the potentially deleterious pro-inflammatory cytokines IFN-
and IL-17, as compared to peptide vaccines 45. Similar results were noted in a peptide prime:
DNA boost vaccination strategy 46. These findings suggested that DNA vaccines induced a
response that likely will safe and potentially effective 39, 41. In addition, as indicated, a long-standing issue
with vaccination using a “self” molecule such as is overcoming immune tolerance. It is on this issue,
as well, that a naked DNA plasmid vaccination strategy may have advantages and potential utility. In fact,
DNA vaccination has been demonstrated to break immunological tolerance to a prion protein vaccine in a
prion disease model, with the immunization mediating an amelioration of the pathology associated with
this disorder 47. In the context of AD it has been indicated that a combination vaccine consisting of
DNA + peptide, administered concomitantly, resulted in very high levels of anti- antibodies,
presumably due to the ability of this vaccination method to overcome the immune hypo-responsiveness
of which is likely associated with immunological tolerance 48. Likewise, DNA vaccine approaches
against AD have involved attempts to avoid the effects of potentially autoreactive and harmful T cell
responses. One strategy, on this issue, has used an epitope specific DNA vaccine conjugated to a non-
self T cell epitope, PADRE 38. Also, studies evaluating the role of polymorphic MHC genes on responses to
DNA vaccination have also been explored 38, 49.
Overall, based on studies to date, there appears to be logistical and practical advantages to further
explore the utilization of naked DNA vaccines against AD and perhaps other neurodegenerative diseases
where specific antigens can be targeted. As such, human clinical trial evaluations of this strategy are
planned 39.

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In terms of the application of DNA based vaccines against PD to our knowledge only one report
has been published. In that study Chen et. al. utilized an optimized DNA plasmid designated VAX1-IL-
4/SYN-B which targets the PD pathologically relevant syn protein 37. This was tested in a chemically
induced (i.e. 1-Methyl-4-phenyl-1, 2,3,6-tetrahydropyridine=MPTP) rodent PD model. The results of the
study indicated that the vaccine stimulated high levels of anti- syn antibodies as well as an increase
and decrease in levels of IL-4 and IFN- respectively. This finding indicates a potentially protective effect
of DNA vaccination in this model, which warrants further evaluation. As well, syn in PD pathogenesis,
similar to Aβ in AD, functions normally as a self-protein and can be immunologically tolerant. Therefore
an approach such as DNA vaccines could assist in overcoming syn immune tolerance, thus making it
further a potentially useful immune-based therapy or prophylaxis against PD.
As indicated previously, because of some of the disadvantages, including the development of
adverse effects following active vaccination with A in the clinical trials, there has been an impetus to
develop and evaluate passive immunotherapeutic (i.e. humanized or human mAbs) strategies against
neurodegenerative diseases such as AD and PD. However, some mAb preparations against Aβ tested in
clinical trials have been associated with some adverse effects as well, including the development of
cerebral edema and micro-hemorrhages 22, 50. In addition, the mAb preparations tested to date failed
to mediate any significant clinical effect 22, 50. This lack of efficacy, as indicated previously, might have
been due to a lack of understanding as to when to begin mAb administration in the AD pathogenesis
process, in addition to issues as to what dose to utilize as well as the number and time intervals of
administrations.
Gene-based delivery of immunoglobulin DNA sequences from monoclonal antibodies with
biological activity against antigenic targets in Alzheimer’s and Parkinson’s disease

10
Based on the use and utility of viral vector and non-viral based DNA delivery for active
immunization against neurodegenerative diseases such as AD, it has been hypothesized that, likewise,
these methods could administer established biologically active mAbs through injection of light and
heavy chain immunoglobulin expressing genes. It is reasoned that such a strategy could result in long-
term expression of mAbs, obviating the need for repeated conventional passive administrations of
antibodies that were generated through tissue culture or other in vitro methods that typically entail
costly and labor intensive purifications processes. As well, such gene-based immune therapies could
be administered very early in the putative AD or PD pathogenesis process and, as such, may overcome
some of the factors that could have limited the success of the conventional passive immunotherapy
clinical trials. As well, because levels of mAbs are continuously generated in vivo over time at
minimally effective levels, this method may result in a better safety profile that the conventional mAb
immunotherapy strategy where repeated bolus injections of usually large doses of antibodies are
required due to pharmacokinetic and pharmacodynamics considerations. Specifically, some
investigations using a gene-based strategy for in vivo mAb generation have been performed targeting
Aβ in AD. This method, designated vectored immunoprophylaxis 51, utilizes AAV vectors that express
light and heavy chain immunoglobulin or single chain antibody genes of established anti- Aβ mAbs.
The studies using this technique have demonstrated some efficacy in terms of decreasing Aβ
deposition and ameliorating cognitive deficits in rodent models of AD 52-54, 55. To date, to our
knowledge, this AAV delivery system for mAb genes has not been utilized for targeting anti- syn
mAbs against PD.
Although the approach of using AAV vectors to deliver anti- Aβ mAbs have demonstrated some
proof-of-concept efficacy, this viral vector based strategy will likely have the same concerns and
disadvantages noted in the utilization of this method for delivery of vaccine antigen expressing genes.
Therefore, it could be argued that a non-viral vector based naked DNA plasmid delivery method, as
used for active vaccination, could also be implemented for delivery of mAb expressing genes.

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Currently, although this strategy has not been specifically applied to biologically active anti- Aβ or
anti- syn mAbs, it has been evaluated using other target mAbs. The study by Tjelle et.al. demonstrated
that this DNA plasmid delivery strategy was able to produce mAbs of correct structure
and biological activity following EP mediated delivery of the antibody expressing genes 56. More recently
Muthumani and colleagues used an optimized DNA plasmid EP delivery to target a broadly neutralizing
anti-HIV mAb. In this study it was shown that after delivery of the mAb DNA, mice were able to generate
in their sera long-term expression of antibodies that were able to neutralize HIV-1 in vitro 57. These
proof-of-concept studies suggest that this non-viral DNA plasmid delivery method might have
applications in terms of targeting mAbs against syn and Aβ as well as other potential antigens. Even
though this method has a likely better safety profile than viral vector based delivery systems, further
optimization of the strategy is needed in order to allow the higher expression levels attained by AAV
delivery methods. Overall, however, this naked DNA plasmid delivery method for this novel passive
immunotherapy strategy warrants further evaluation.
Conclusions and Summary
In conclusion, vaccine and immunotherapeutic methods against neurodegenerative diseases
such as AD and PD, which have “targetable” antigens that „break” immune tolerance, are still viable
prophylactic/therapeutic strategies that continue to be evaluated. This is irrespective of the safety and
efficacy concerns indicated by Aβ peptide immune-based strategies against AD. Although historically
Aβ has been targeted for vaccine and immunotherapeutic development it can be argued that tau, a
pathologic protein in AD involved in the generation of neurofibrillary tangles, should likewise be
investigation in terms of vaccination potential. In addition, some investigators have suggested that
both Aβ and tau should be targeted concomitantly by conventional drugs as well as by immune-based
interventions. As such, these types of studies are being pursued. Likewise in PD the aggregated

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pathologic form of the protein syn is a reasonable molecule to target for vaccination and
immunotherapy. In addition to conventional vaccination and passive immunotherapeutic approaches
being evaluated in this area, more recently gene-based strategies such as viral vectors and non-viral
(i.e. naked DNA) expression plasmids have been and are currently being investigated. There are
advantages and disadvantages with both viral and non-viral based gene delivery methods, but the
naked DNA approach is attractive because of some of its logistical and safety characteristics. Methods
to enhance expression and effectiveness of naked DNA vaccine delivered antigens and
immunotherapies through various optimization methods makes this strategy viable and attractive
approach against neurodegenerative diseases such as AD and PD. The Table presented in this mini-
review lists the different vaccine and immunotherapeutic strategies that have been evaluated against
AD and PD, along with relevant references in which results from investigations on these different
approaches are reported.

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Table 1. Active and passive Immunotherapeutic strategies against Alzheimer and Parkinson’s diseases
Vaccination and Passive Immunotherapy
Strategies Against Aβ for AD and α-syn for PD
References
active peptide/recombinant protein vaccines Aβ: 3, 4, 5, 10, 11, 12, 13, 15, 18, 19, 20
α-syn: 6, 8, 9
active AAV-based vaccines Aβ: 35, 36
α-syn: none to date
active naked DNA plasmid vaccines Aβ: 38, 39, 44, 45, 46, 48, 49
α-syn: 37
conventional passive antibody immunotherapies Aβ: 4, 5, 21, 22, 23, 55
α-syn: 9, 27, 28, 29,30
gene-based passive antibody immunotherapies Aβ: none to date
α-syn: none to date
AD = Alzheimer’s disease; PD = Parkinson’s Disease; Aβ = beta amyloid; α-syn = alpha synuclein; AAV = adeno-
associated virus

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