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

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 and Parkinson 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 β protein in Alzheimer 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 suggested that further evaluation of gene based vaccines and immunotherapies against neurodegenerative diseases are warranted to determine their potential clinical utility.

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 by 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 2 most common neurodegenerative diseases, Alzheimer disease (AD) and Parkinson disease (PD), which are characterized by progressive dementia and motor system disturbances respectively.^6-17

Conventional active and passive immunotherapies against Alzheimer and Parkinson 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 β amyloid (Aβ) 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 Aβ. Promising results in these experiments were obtained including a lowering of brain Aβ 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 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 Aβ T 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, Aβ 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-Aβ monoclonal antibodies (mAb), demonstrated some efficacy in appropriate Tg mouse models²¹ 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, to mediate biological activity, is determined and the potential adverse effects eliminated. As such, further clinical trials are planned.²³ As well, in addition to Aβ, 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 α-synuclein (α−syn). Specifically, the aggregated form of this disordered protein is hypothesized be a major contributor to the PD pathogenesis.²⁶ Both α−syn peptide or recombinant protein vaccines as well as passive immunotherapeutic strategies targeting α−syn against PD have been evaluated.^8,9,27 In α−syn expressing Tg mice or in a rat model expressing α−syn genes, delivered by an adeno-associated virus 9 (AAV-9) vector, the use of α−syn peptide, recombinant protein, peptide pulsed dendritic cell (DC) vaccines or passive delivery of anti-α−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 and Parkinson 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 which target 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 immunogen can result in long term expression of the vaccine antigen as opposed to the typical necessity for repeated immunizations with conventional 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. Irrespective of the generation of side effects, viral vectors typically induce both antibody and T cell immune responses, which are typically 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. As suggested however, the generation of T cell responses (i.e. cytotoxic T lymphocytes) by this strategy, in the context of 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 do not have significant toxicity. 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 Aβ expressing AAV vector decreased Aβ burden and cognitive deficits in murine Tg AD models.³⁵ As well, Hara and colleagues reported the development of an oral Aβ vaccine, delivered by an AAV vector, which mediated a decrease in Aβ 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 α−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 y.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 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 by 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, the development of adverse effects after peptide vaccination has occurred, due likely to the untoward generation of T cell responses against Aβ in addition to suggested safety concerns with of the co-administered adjuvant. To that end, naked DNA based immunization was initially evaluated against Aβ for AD in the mid 2000s.⁴⁴ In those provocative studies a DNA vaccine expressing the Aβ peptide, delivered by gene gun technology, resulted in a significant bias for the generation of Th2 immune responses. Other studies have indicated that Aβ DNA vaccines decreased Th1 cell proliferation coupled with a decrease in the potentially deleterious pro-inflammatory cytokines IFN-γ and IL-17, as compared to Aβ peptide vaccines.⁴⁵ Similar results were noted in a Aβ peptide prime: Aβ DNA boost vaccination strategy.⁴⁶ These findings suggested that Aβ 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 Aβ is the ability to overcome 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 Aβ vaccine consisting of Aβ DNA + Aβ peptide, administered concomitantly, resulted in very high levels of anti-Aβ antibodies, presumably due to the ability of this vaccination method to overcome the immune hypo-responsiveness of Aβ, 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 Aβ DNA vaccine conjugated to a non-self T cell epitope, PADRE.³⁸ Also, studies evaluating the role of polymorphic MHC genes on responses to Aβ 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

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 α−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- α−syn antibodies as well as an increase and decrease in levels of IL-4 and IFN-γ, respectively. This finding indicates a potentially protective effect of DNA vaccination in this model and warrants further evaluation. As well, α−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 a DNA vaccine could assist in overcoming α−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 Aβ 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 and Parkinson disease

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 putatively minimally effective levels, this method may result in a better safety profile than 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-α−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 is 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. Currently, although this strategy has not been specifically applied to biologically active anti- Aβ or anti-α−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 that 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 α−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 generate the higher expression levels that were 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 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 investigated in terms of vaccination potential. In addition, some investigators have suggested that both Aβ and tau should be targeted concomitantly by conventional drugs and/or immune-based interventions. As such, these types of studies are being pursued. Likewise in PD the aggregated pathologic form of the protein α−syn is a reasonable molecule to target for vaccination and immunotherapy. In addition to conventional vaccination and passive immunotherapeutic approaches being evaluated against these neurodegenevative diseases, 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. As summary, Table 1 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.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Table 1.

Active and passive Immunotherapeutic strategies against Alzheimer and Parkinson diseases

	References
active peptide/recombinant protein vaccines	Aβ: 3–5,10–13,15,18,19,20α-syn: 6,8,9
active AAV-based vaccines	Aβ: 35,36α-syn: none to date
active naked DNA plasmid vaccines	Aβ: 38,39,44,45,46,48,49α-syn: 37
conventional passive antibody immunotherapies	Aβ: 4,5,21–23,55α-syn: 9,27,28,29,30
gene-based passive antibody immunotherapies	Aβ: none to dateα-syn: none to date

AD = Alzheimer disease; PD = Parkinson disease; Aβ = β amyloid; α-syn = α synuclein; AAV = adeno-associated virus.