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Keywords:

  • autoimmunity;
  • diabetes mellitus;
  • Jenner;
  • multiple sclerosis;
  • vaccination

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

Abstract.  Steinman L (Stanford University, Stanford, CA, USA). Inverse vaccination, the opposite of Jenner’s concept, for therapy of autoimmunity (foresight). J Intern Med 2010: 267: 441–451.

DNA-based vaccines to induce antigen-specific inhibition of immune responses in human autoimmune diseases represent the inverse of what Jenner intended when he invented vaccination. Jenner’s vaccine induced antigen-specific immunity to small pox. DNA vaccines for autoimmunity have been developed in preclinical settings, and now tested in human trials. The first two clinical trials, one in relapsing remitting multiple sclerosis, and the other in type 1 diabetes indicate that specific inhibition of antigen-specific antibody and T-cell responses is attainable in humans. Further development of this approach is ongoing. This new version of immunization termed ‘inverse vaccination’ when applied to autoimmune diseases, may allow targeted reduction of unwanted antibody and T-cell responses to autoantigens, while leaving the remainder of the immune system intact. The method of specifically reducing a pathological adaptive autoimmune response is termed inverse vaccination.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

What is meant by the inverse of Jennerian vaccination?

In 1796, Edward Jenner wrote,

I selected a healthy boy, about eight years old, for the purpose of inoculation for the cow-pox. The matter was taken from a sore on the hand of a dairymaid, who was infected by her master’s cows, and it was inserted, on the 14th of May, 1796, into the arm of the boy by means of two superficial incisions, barely penetrating the cutis, each about half an inch long [1].

He continued,

In order to ascertain whether the boy, after feeling so slight an affection of the system from the cow-pox virus, was secure from the contagion of the smallpox, he was inoculated the 1st of July following with variolous matter, immediately taken from a pustule. Several slight punctures and incisions were made on both his arms, and the matter was carefully inserted, but no disease followed. The same appearances were observable on the arms as we commonly see when a patient has had variolous matter applied, after having either the cow-pox or smallpox. Several months afterwards he was again inoculated with variolous matter, but no sensible effect was produced on the constitution.

Jenner’s powerful assertion was that ‘the cow-pox protects the human constitution from the infection of the smallpox’ [1]. These classic experiments performed in the eighteenth century established the remarkable idea that the immune system could be turned on, in a highly specific manner that afforded protection from a highly virulent and often fatal viral infection. In the past three centuries, quite surprisingly there has been very little progress, at least in man, in establishing what would be the very opposite of Jenner’s vaccination. In experimental animals there are scores of papers demonstrating that it is possible to induce control or eliminate specific immune responses, although reducing ongoing autoimmune responses has been more challenging [2]. The opposite of what Jenner intended would involve reducing or eliminating specifically a particular undesired immune response. Such an approach would with high likelihood provide enormous benefit to an individual suffering from an autoimmune disease stemming from an immune response to that component of what we call ‘self’, in the immunological sense.

Inverse Jennerian vaccination in the context of current approaches to autoimmunity

Current approaches to treatment of autoimmunity were for the most part taken from drugs that have already been approved to treat lymphoid malignancy like the anti-CD20 monoclonal Rituxan [3–5] or the anti-CD52 monoclonal antibody [6, 7], Campath, or to reduce transplant rejection, with various monoclonal anti-CD3 antibodies [8, 9]. Thus, some of the most popular approaches to treating autoimmunity include extinguishing nearly all of one’s CD20 B cells [3–5], paralysing all of one’s CD3 T cells [8–10], killing all of one’s CD52 white blood cells [6, 7], impairing ingress of all T and B cells to affected organs via integrin blockade [11–14] or impeding egress of lymphoctyes out of lymph nodes with modulators of spingosine phosphate receptors [15]. So it is instructive to emphasize Jenner’s observation:

what renders the cow-pox virus so extremely singular is that the person who has been thus affected is forever after secure from the infection of the small pox; neither exposure to the variolous effluvia, nor the insertion of the matter into the skin, producing this distemper [1].

The singularity of his approach to modulation of adaptive immunity is the essence of what my colleagues and I are attempting in human autoimmune disease: We are trying to shut down specific unwanted T- and B-cell responses in individuals already afflicted with diseases thought to be autoimmune such as multiple sclerosis (MS) and type 1 diabetes. Instead of undertaking approaches that have been translated from the fields of transplant rejection and from cancer therapy, where wide swathes of the immune response are extinguished, we are attempting a much focused approach for controlling unwanted adaptive immune responses.

Identification of targets in particular autoimmune diseases for inverse vaccination

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

In 1999 we initiated a two-pronged attack to first identify the targets of adaptive immunity in various autoimmune diseases, and then to develop a way to reduce these unwanted adaptive responses. Large-scale arrays were developed where hundreds of putative autoantigens were printed on microscopic glass slides [16–19]. We customized the content of these arrays. The first array studied was comprised of many of the known target autoantigens identified for systemic lupus erythematosus [16]. To study MS, all the known myelin proteins in their recombinant forms, their peptide epitopes including post-translationally modified variants were printed on slides [17]. Using a two-stage system after ‘printing’ the slides, fluid taken either from serum or from cerebrospinal fluid (CSF) was first applied to the surface of these slides. A second stage with fluorescent antihuman immunoglobulin was then applied and the slides were scanned for their fluorescence. See Fig. 1 showing a ‘heatmap’ indicating the major antibody responses in the spinal fluid of individuals with relapsing remitting (RR) MS [19].

image

Figure 1. Autoantibody responses in the spinal fluid of patients with relapsing remitting multiple sclerosis. Analysis was performed on cerebrospinal fluid from relapsing remitting multiple sclerosis (RRMS) and other neurologic disease (OND) patients. Statistical Analysis of Microarrays identified significant differences in antibodies in RRMS compared with OND. Samples are arranged with hierarchical clustering, and displayed as a heat map. RRMS patients demonstrated significantly increased autoantibodies against various myelin epitopes including CRYAB protein and peptides (J37, Golli-myelin basic protein isoform J37); PLP, proteolipid protein; MBP, myelin basic protein; HSP, heat shock protein; Abeta, amyloid beta). A false discovery rate threshold of 1.9% and numerator threshold of 2.0 were used. Prediction Analysis of Microarrays yielded a classification model with 21 markers, with cross-validated sensitivity of 10/12 = 83% and specificity of 11/12 = 92% [19]. With permission from Nature Publishing.

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Quantitative measures of antibodies to various myelin components were obtained and validated with alternate methods of measurement [16, 17]. We were able to reveal the major targets of the immune response to myelin in various models of MS, collectively called experimental autoimmune encephalomyelitis [17], as well as the major targets of the adaptive immune response in the spinal fluid of patients with RRMS [18, 19].

Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

We chose to develop a technology based on immunization with DNA encoding a specific antigen that had first been discovered in the early 1990s [2]. In 1990, Wolff et al. [20] showed that naked DNA could transfect cells in vivo. This breakthrough led to the first successful vaccine in animal models when Ulmer et al. [21] injected a naked DNA encoding influenza, and conferred protection against infection. Since then there have been numerous attempts to use DNA vaccines to induce what might be considered conventional Jennerian immunization against a variety of microbial targets. For the past 20 years, investigators have been developing DNA vaccines to induce immunity to microbial infections including HIV [22], rabies [23], ebola [24] and influenza [21]. There are no approved DNA-based vaccines as yet.

In 1996, we published experiments where a DNA vaccine was first used to treat autoimmune disease [25]. The vaccine targeted a key portion of the T-cell receptor that is found on pathogenic T cells that cause paralysis in experimental autoimmune encephalomyelitis (EAE), a collection of autoimmune diseases that bears some important resemblances with MS. A variable region gene of the T-cell receptor, V beta 8.2, is rearranged, and its product is expressed on pathogenic T cells that induce EAE in H-2u mice after immunization with myelin basic protein (MBP). Vaccination of these mice with naked DNA encoding V beta 8.2 protected mice from EAE. Analysis of T cells reacting to the pathogenic portion of the MBP molecule indicated that in the vaccinated mice there was a reduction in the Th1 cytokines such as interferon-γ (IFN-γ). In parallel, there was an elevation in the production of interleukin (IL)-4, a Th2 cytokine associated with suppression of disease. A novel feature of DNA immunization for autoimmune disease, modification of the autoimmune response from Th1 to Th2, indicated that this approach might be relevant for treatment of autoimmune diseases like MS, type 1 diabetes and rheumatoid arthritis, if the particular critical V region genes could be identified [25]. However, given the diversity of T-cell receptor rearrangements it became clear that the approach to targeting autoimmunity via ‘critical’ T-cell receptors was going to be a daunting task. Hence, efforts quickly evolved towards trying to reduce autoimmune responses to pathological autoantigens.

Several groups attempted DNA vaccination with plasmids encoding various myelin antigens in various models of EAE. The Karolinska group showed that EAE was suppressed with a DNA vaccine encoding an immunodominant portion of MBP for the Lewis rat, fused to a dimerized synthetic analogue of the immunoglobulin G (IgG)-binding B domain of staphylococcal protein A [26].

We demonstrated that a DNA vaccine encoding an unmodified myelin epitope, PLP139–151 was shown to suppress EAE, with a reduction in the degree and frequency of paralysis in mice. Proliferative responses and production of the quintessential Th1 cytokine, IFN-γ, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15 and IFN-γ were reduced. The mechanism underlying this reduction in both severity and incidence of paralysis and the reduction in Th1 cytokines involved diminished co-stimulation of T cells. DNA immunization with a plasmid encoding proteolipid protein (PLP) peptide 139–151 altered expression of the co-stimulatory molecules, CD80, and CD86 on antigen presenting cells in the spleen [27].

We also explored whether co-administration of plasmids encoding Th2 cytokines might enhance DNA vaccination with plasmids encoding myelin proteins [28]. Using a combination of local gene delivery and tolerizing DNA vaccination, we demonstrated that co-delivery of the gene encoding IL-4 gene and a DNA plasmid encoding the self-peptide PLP 139–151 protected against development of EAE. Mechanistic studies revealed that IL-4 expressed from the naked DNA is secreted and acts locally on autoreactive T cells via activation of STAT6 to shift their cytokine profile from T helper 1 (Th1) to Th2. This approach with a DNA vaccine plus co-delivery of the gene for IL-4 reversed established EAE and diminished paralysis in a second model of EAE induced with myelin oligodendrocyte glycoprotein (MOG) [28].

Some experiments with DNA vaccination in EAE lead to worsening of disease. CpG motifs in bacterial DNA were shown to activate the immune system via toll-like receptors and to directly activate T and B cells [29]. Raz et al. [30] showed that CpG sequences were critical for induction of DNA vaccination via induction of Th1 responses. Fujinami et al. [31] recognized that oligonucleotide motifs, termed CpG, could induce Th1 cytokines. They showed that bacterially derived DNA plasmids, termed pCMV, could induce IL-6, IL-12 and IFN-γ. They also showed that pCMV injection also enhanced R-EAE with increased IFN-γ and IL-6 responses [31]. In contrast, Lobell et al. [32] at the Karolinska showed that plasmids without any of these so-called CpG motifs were incapable of protecting mice from EAE. Thus, treatment with a DNA vaccine encoding MBP peptide 68–85 and containing three CpG motifs with their characteristic 5′-AACGTT-3′ sequence, suppressed paralysis in EAE. A DNA vaccine without any of these CpG motifs failed to suppress EAE in two different models of EAE, one induced with a peptide to MBP and the other with a peptide to MOG. The issue was therefore raised whether CpG motifs were beneficial or not in inverse vaccination for autoimmune disease.

An oligonucleotide motif termed GpG to suppress Th1 T-cell responses

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

The flagship cytokine of Th1 immunity is IFN-γ [33]. Although administration of IFN-γ is associated with protection from paralysis in EAE [33, 34], administration of this quintessential Th1 cytokine leads to worsening of MS [35]. Segal et al. [36] showed that EAE could be induced with lymphocytes that were first sensitized to PLP 139–151, and were then activated with CpG. Our own work in agreement with that of Lobell et al. [32] showed that at least for EAE, CpG oligonucleotides in bacterial plasmids could by themselves suppress EAE. We demonstrated that injection of plasmid DNA could suppress the prototypic T-cell-mediated autoimmune disease, EAE, by inducing IFN-γ [37]. Given the potential danger of inducing a Th1 response, with high IFN-γ production when we translated this approach to MS and to other diseases where Th1 responses are undesirable, we decided to design an oligonucleotide motif that might actually compete with the CpG motif. We predicted that replacing CpG motifs with a competitor might be the best course of action, given that we wanted to avoid amplifying Th1 immunity in those with autoimmune disease.

We discovered that a GpG oligonucleotide, with a single base switch from CpG to GpG, which effectively inhibited the activation of Th1 T cells was associated with autoimmune disease [38]. A CpG oligonucleotide with the sequence 5′-TGACTGTGAACGTTAGAGATGA-3′ was changed to a GpG oligonucleotide, 5′-TGACTGTGAAGGTTAGAGATGA-3′. The immunomodulatory GpG-ODN suppresses the severity of EAE in mice, a prototypic Th1-mediated animal disease model for MS. The GpG oligonucleotide stoichiometrically competed with CpG for binding to its receptor the toll-like receptor 9, TLR9. The combination of stimulatory CpG oligonucleotides with inhibitory GpG oligonucleotides resulted in a marked reduction in phosphorylation of IkB- at Ser32. Overall, GpG oligonucleotides effectively competed with CpG oligonucleotides to inhibit Th1 responses [38].

Matching the inverse vaccine to targets detected on autoantibody arrays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

We used the two-pronged approach to treat autoimmune disease using large-scale analysis of the specificity of autoantibody responses in EAE to guide selection of the DNA vaccine used to suppress EAE. To survey autoantibody responses in EAE, we used our antigen microarrays containing a spectrum of proteins and peptides derived from the myelin sheath, the target of the autoimmune response in EAE and the 2304-feature myelin proteome arrays contain 232 distinct antigens, including proteins and sets of overlapping peptides representing MBP, PLP, MOG, myelin-associated oligodendrocytic basic protein (MBOP), oligodendrocyte-specific protein (OSP), aB-crystallin, cyclic nucleotide phosphodiesterase (CNPase) and Golli-MBP. We used these customized myelin arrays to profile autoantibody responses in serum derived from mice with EAE. Images of representative arrays are presented in Fig. 2.

image

Figure 2. The diversity of autoantibody responses in acute experimental autoimmune encephalomyelitis (EAE) predicts subsequent disease activity. Hierarchical clustering of antigen features with statistically significant differences in myelin proteome array reactivity between sera derived from groups of normal control mice and from groups of mice upon recovery from acute EAE induced with proteolipid protein 139−151 (day 17), myelin basic protein 85−99 (day 22) or spinal cord homogenate (day 25). Mice were later scored daily for 10 weeks to determine the number of relapses for each mouse (indicated in parentheses). The average relapse rates for mice included in the primary subnodes of the dendrogram, and P-values by Mann–Whitney test for the differences in relapse rate between these nodes, are indicated [17]. With permission from Nature Publishing.

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We applied our myelin proteome arrays to study the evolution of the autoreactive B-cell responses in EAE. Based on results from these arrays showing large-scale spreading of the immune response to multiple myelin antigens in EAE, we devised a DNA vaccine with plasmids encoding four myelin proteins, MBP, PLP, myelin oligodendroglial glycoprotein and myelin-associated glycoprotein. This vaccine referred to as a ‘cocktail’ vaccine was given to mice after the first attack of paralysis. Beginning on day 17 after recovery from the acute paralytic attack in EAE (7–8 days after disease onset), mice were injected intramuscularly on a weekly basis with control therapies or DNA encoding this cocktail of array-determined myelin targets The vaccine with plasmids encoding the four myelin proteins reduced relapses when given after the initial attack of paralysis; see Fig. 3. Autoantibody responses to myelin proteins were reduced on a large scale; see Fig. 3.

image

Figure 3. Tolerizing DNA vaccines reduce autoantibody epitope spreading. At day 7 after onset of and after partial recovery from acute paralytic experimental autoimmune encephalomyelitis (day 17) induced with proteolipid protein (PLP) 139−151, SJL/J mice were treated weekly with phosphate buffered saline vehicle, empty pTARGET vector, pTARGET expressing myelin basic protein, PLP, myelin oligodendrocyte glycoprotein and MAG (cocktail) or pTARGET expressing the cocktail and interleukin-4. After the 10-week treatment, serum was obtained, array analysis carried out and Statistical Analysis of Microarrays was used to identify and analyse the hierarchical cluster to order antigen features [17]. With permission from Nature Publishing

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We next tested to see what would happen when we continued to optimize the DNA vaccination strategy, employing in ongoing EAE an optimized set of antigens, the cocktail of four plasmids encoding four different myelin proteins, a gene for IL-4 and the GpG oligonucleotide [39]. The results of this experiment were illuminating and quite positive. In chronic relapsing EAE, combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG-ODN induced a shift of the autoreactive T-cell response towards a protective Th2 cytokine pattern [39]. Myelin microarrays demonstrated that tolerizing DNA vaccination plus GpG-ODN further decreased antimyelin autoantibody epitope spreading and shifted the autoreactive B-cell response to a protective IgG1 isotype, indicating a shift from a Th1 response to a Th2 response. Moreover, the addition of GpG-ODN to the inverse vaccine effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models [39]. Thus, a method was in hand that reduced both the frequency of relapses when given after the first attack and overall reduced disease progression caused by the recurrent relapses.

Inverse vaccines with increased GpG, high antigen expression and intracellular localization

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

In 2001, we reported that nonobese diabetic (NOD) mice injected intramuscularly with a bacterial plasmid encoding the insulin B chain peptide showed significantly lower disease incidence and delayed onset of disease when compared with controls [40]. Protection was mediated by insulin B (9–23)-specific downregulation of IFN-γ. We have pursued this observation performing experiments with a succession of plasmid to optimize therapeutic efficacy. We gave particular attention to installing GpG constructs in the plasmid backbone, to assessing whether higher levels of antigen expression were beneficial and to assessing whether localizing the site of expression to intracellular locations was of benefit.

The backbone of the plasmid for the inverse vaccine had its CpG motifs eliminated and reconstituted with GpG motifs. Twelve CpG sequences were eliminated from the original plasmid by converting the cytosine to guanosine. This CpG-reduced plasmid, where CpGs were converted to GpGs was named pBHT1 [41]. Using this plasmid, we studied NOD mice at various stages of the disease to compare plasmids encoding distinct islet antigens that have been implicated in the pathogenesis of T1D. The pBHT1 backbone by itself, without an autoantigen insert could not modulate experimental type 1 diabetes mellitus in the NOD mice.

We identified pDNA-encoding proinsulin II as the most potent at reducing type 1 diabetes in both preventive situations before hyperglycaemia was present and in treatment models, where blood glucose levels were between 190 and 250 mg mL–1. Furthermore, we were able to elucidate an optimal frequency of dosing, optimal levels of protein expression and whether intracellular localization of the antigen contributed to efficacy or not [41]. Targeting of the proinsulin II protein product to the cytoplasm by removing the signal sequence from preproinsulin II proved to be the best strategy for delaying diabetes onset. Furthermore, the higher expression vector, created by adding a chimeric intron upstream of the proinsulin II gene sequence provided more robust protection from type 1 diabetes. This chimeric intron is composed of the 5′ donor splice site from the first intron of the human β-globin gene and the branch and 3′ acceptor splice site from the intron of an Ig gene H chain V region. Weekly, biweekly and monthly dosing frequencies were all efficacious [41].

We undertook further studies to analyse the mechanism of action of this inverse vaccine. As reported, administration of a single injection of proinsulin II DNA to hyperglycaemic mice significantly decreased the number of B9-23-specific, IFN-γ producing cells compared with the hyperglycaemic phosphate buffered saline control mice (= 0.02). Furthermore islet-associated antibody (IAA) levels, reflecting humoral immune responses to insulin, were reduced in NOD mice treated with the 10 μg dose of the inverse vaccine [41].

The conclusions from these studies on NOD mice, formed the basis for the design of a multicentre phase I clinical trial testing pDNA encoding human proinsulin II, termed BHT-3021, in individuals with a diagnosis of T1D, which began in October 2006.

Human trials with an inverse vaccine in MS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

An initial clinical trial with an inverse vaccine was attempted in 30 individuals with RRMS and secondary progressive MS at four clinical sites in North America. Although preclinical testing indicated that an inverse vaccine using multiple plasmids encoding multiple myelin proteins would be optimal, after discussions with regulatory authorities in the United States and Europe, it was decided to test a vaccine, termed BHT-3009, encoding just one protein – MBP. BHT-3009 was administered intramuscularly at weeks 1, 3, 5 and 9 after randomization into one of three groups: placebo, BHT-3009 alone or BHT-3009 with the oral statin drug, atorvastatin. Atorvastatin was added in one group because there were indications that it may act as an adjuvant to enhance the efficacy of inverse immunization [42].

The inverse vaccine, BHT-3009, was safe and well tolerated. There were favourable trends on brain magnetic resonance imaging (MRI), with a trend towards a reduction in gadolinium lesion activity and volume after treatment relative to the placebo group. There was no additional benefit from atorvastatin. We observed beneficial antigen-specific immune changes including a marked decrease in proliferation of IFN-γ producing, myelin-reactive CD4+ T cells from peripheral blood [Fig. 4] and a reduction in titres of myelin-specific autoantibodies from CSF as assessed by protein microarrays [42].

image

Figure 4. Example of decreased T-cell response with BHT-3009. An example of one patient whose myelin basic protein (MBP)- and proteolipid protein (PLP)-specific T-cell proliferative response decreased in response to BHT-3009 is shown. Proliferation was measured using a dye dilution method with the vital dye 5,6-carboxyfluorescein diacetate succinimidyl ester. Peripheral blood mononuclear cells were incubated with a variety of antigens and controls, but for simplicity, only the responses to tetanus toxoid (TT), MBP83-99 peptide and a PLP peptide mix are shown. The upper three panels correspond to the baseline response; the middle three, to the week 9 response; and the bottom three, to the week 50 response. Proliferating interferon (IFN)-positive CD4+ T cells are shown in the upper left quadrant of each fluorescent-activated cell sorter plot. Numbers in red indicate the percentage of cells in each quadrant. A dramatic decrease in IFN-positive cells specific for MBP and PLP is demonstrated by week 9 and persists till week 50. Importantly, the response to TT is unchanged with dosing, confirming the antigen-specific nature of BHT-3009 [42]. Permission obtained from Archives of Neurology.

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A phase 2 trial was then performed in patients with RRMS with the inverse vaccine, encoding MBP, termed BHT-3009. As reported in 2008, compared with placebo, in the 267 patient analysis population the median 4-week rate of new enhancing lesions during weeks 28–48 was 50% lower with 0.5 mg of BHT-3009 (= 0.07) and during weeks 8–48 was 61% lower with 0.5 mg of BHT-3009 (= 0.05). The mean volume of enhancing lesions at week 48 was 51% lower on 0.5 mg of BHT-3009 compared with the placebo (= 0.02). No significant improvement in MRI lesion parameters was observed with 1.5 mg of BHT-3009. Dramatic reductions in 23 myelin-specific autoantibodies in the 0.5-mg BHT-3009 arm were observed in CSF, but not with placebo or 1.5 mg of BHT-3009 [43]; see Fig. 5.

image

Figure 5. Treatment with 0.5 mg of BHT-3009 is associated with a reduction in antimyelin antibody titres. Myelin array analysis was performed to quantify antimyelin peptide antibodies in baseline and post-treatment samples in patients treated with 0.5 mg of BHT-3009. Antibodies with pre- to post-treatment changes within each treatment group are represented either as fold change in intensity with increases false coloured red, no change false coloured black, and decreases false coloured green. Grey represents samples with no data available. Significant Analysis of Microarrays was applied to identify statistical differences in antibody reactivity between the pre- and post-treatment samples, and identified 23 myelin peptides (listed to right of heat map) that exhibited overall significant changes (all were decreases) in the post-treated as compared with pretreated cerebrospinal fluid samples in patients treated with 0.5 mg of BHT-3009 (false discovery rate, q < 0.1). There were no statistically significant differences between pre- and post-reactivity in placebo-treated patients. Myelin basic protein peptide sequences are derived from and numbered based on the 18.5-kDa isoform (Genbank accession no.: AAA59562), with the exception of peptides denoted by *s which are numbered based on the 21.5-kDa isoform (Genbank accession no.: AAA59564). MOG, myelin oligodendrocyte glycoprotein; MOBP, myelin-associated oligodendrocytic basic protein; OSP, oligodendrocyte-specific protein. With permission from Annals of Neurology.

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We had the opportunity to study a predefined subset of 80 patients who contributed CSF for protein array analysis at baseline, and CSF at week 44 for repeat protein array analysis. This allowed us to determine whether treatment with BHT-3009 had an effect on the levels of antimyelin autoantibodies. Treatment with the 0.5-mg BHT-3009 dose was associated with a significant decrease in the autoantibody titres to 23 myelin autoantigens, whereas treatment with placebo did not result in a statistically significant net change in any of the antimyelin autoantibodies measured in the CSF. Antibody levels to MBP peptides decreased with 0.5 mg of BHT-3009. We also saw a fall in autoantibodies binding to other components of the myelin sheath in RRMS including aB-crystallin, PLP, MOG, MBOP and OSP. The antibody levels of various components of the myelin sheath decreased in a statistically significant manner as determined by the Statistical Analysis of Microarrays algorithm [43].

We studied whether baseline antibody profile in the CSF predicted response to the study drug. In a prospectively defined protocol we described that ‘in the upper half of the anti-MBP reactive patients (= 13 on placebo, = 11 on 0.5-mg BHT-3009 and = 14 on 1.5-mg BHT-3009), there was a significantly lower number of new Gd-enhancing lesions per patient in MRIs of weeks 28–48 with 0.5 mg of BHT-3009 compared with placebo (mean ± SD, 2.5 ± 4.03 on 0.5-mg BHT-3009 vs. 3.3 ± 4.59 on placebo; = 0.02). In contrast, in this subgroup, there was no significant difference between 1.5-mg BHT-3009 and placebo [43]. Further analysis of baseline antibody profiles has allowed us to identify a serum antibody profile involving MBP where high responders not only had significant reductions in parameters of MRI, but also had reductions in relapse rate in the 0.50-mg group (in preparation). Further trials are planned in both patients with RRMS as well as those patients preidentified by their high responder profiles in a serum assay identifying the detailed immune response to different components of MBP.

Human trials with inverse vaccines in type 1 diabetes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

In June 2009, Gottlieb and colleagues reported results of a trial with an inverse vaccine encoding proinsulin in patients, aged 18–40 with type 1 diabetes. Patients were randomized into placebo or treatment groups and were given 12 weekly injections of various doses of BHT-3021. In all dosage groups at 6 months we reported stability of C-peptide as compared with levels seen in the placebo group. Levels of haemoglobin A1c, were stabilized at all doses compared with the placebo. Full results of the trial at 1 year will be reported in 2010. Based on the results reported in June 2009, a partnership to continue development of this inverse vaccine for type 1 diabetes mellitus was consummated between Bayhill Therapeutics and Genentech, a division of Roche [44].

Further human trials with inverse vaccines

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

In addition to further trials in RRMS and type 1 diabetes mellitus, a plasmid to the α-chain of the acetylcholine receptor was effective in models of experimental autoimmune myasthenia gravis models in mice and rats. This approach will now be taken forward into the clinic in patients with myasthenia gravis. Another neurological condition, neuromyelitis optica, where there is a strong autoantibody response targeting the water channel, aquaporin-4 is being planned. As autoantigens are identified in other diseases inverse vaccines will be developed to treat these conditions in time. Initially diseases where there is clear evidence of a dominant immune response against one protein will have the highest priorities. Over time, as we get more experience with inverse vaccines in man, we shall attempt therapy with multiple plasmids to inhibit responses to several autoantigens on a broad front. One hope is that we will be able to use this approach for reducing specific immune responses instead of current therapies for autoimmune diseases (Fig. 6).

image

Figure 6. CpG motifs in red are reduced and replaced by GpG motifs shown in blue. DNA encoding autoantigen is inserted into the GpG-enriched plasmid. Trials are planned to perform inverse vaccination to acetylcholine receptor in myasthenia gravis, and to aquaporin-4 in neuromyelitis optica.

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Many of the strongest drugs now applied to treat autoimmunity were first approved for cancer therapy or have shown promise for reducing transplant rejection. Such therapies are associated with severe opportunistic infections such as progressive multifocal leukoencephalopathy, which has been reported with Rituxan and Natalizumab [4, 12–14]. There are other complications with these drugs that have been taken from the realms of cancer therapy or therapy to reduce transplant rejection. These complications include increased risk of cancer and other opportunistic infections besides progressive multifocal leukoencephalopathy. One drug, the anti-CD52 approved for chronic lymphocytic leukaemia has shown stellar results in phase 2 trials for RRMS. Yet, 40% of patients taking the drug for RRMS develop thyroiditis, either Hashimoto’s, Graves or idiopathic thrombocytopaenic purpura [6, 7]. Another drug anti-CD3, approved for transplant therapy, induced skin rashes and activation of Epstein–Barr virus in patients undergoing therapy for type 1 diabetes [8–10].

In contrast, inverse vaccination offers the promise of specifically turning the ‘off’ switch’ for unwanted immune responses, while leaving the rest of the immune system intact. These unwanted complications from the current therapies are perhaps inextricably tied to the fact that they were originally developed to intentionally modulate important components of the immune response, some of them being necessary for effective immune surveillance.

It is wise to remember at this early juncture in the development of inverse vaccination, what Jenner said over three centuries ago,

Thus far have I proceeded in an inquiry founded, as it must appear, on the basis of experiment; in which, however, conjecture has been occasionally admitted in order to present to persons well situated for such discussions, objects for a more minute investigation. In the mean time I shall myself continue to prosecute this inquiry, encouraged by the hope of its becoming essentially beneficial to mankind’ [1].

Only further experimentation in man with these inverse vaccines will allow us to see whether this new variation of Jenner’s idea of vaccination will achieve its intended goal to specifically reduce unwanted autoimmune responses, leaving the rest of the immune system intact.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References

The author’s colleagues William Robinson, P.J. Utz and Hideki Garren have been working on this every step of the way since their first meeting at Stanford in the late 1990s. Peggy Ho and Paulo Fontoura made seminal discoveries on the GpG motif. Colleagues at Bayhill Therapeutics particularly Nanette Solvason, Michael Leviten, Robert King and JoAnne Quan have played critical roles in the development of DNA vaccines. None of this would have happened without support from the National Institutes of Health, the Juvenile Diabetes Research Foundation, the National Multiple Sclerosis Society and the loyal investors in Bayhill Therapeutics.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Identification of targets in particular autoimmune diseases for inverse vaccination
  5. Development of inverse vaccines to attenuate specific ongoing adaptive autoimmunity
  6. An oligonucleotide motif termed GpG to suppress Th1 T-cell responses
  7. Matching the inverse vaccine to targets detected on autoantibody arrays
  8. Inverse vaccines with increased GpG, high antigen expression and intracellular localization
  9. Human trials with an inverse vaccine in MS
  10. Human trials with inverse vaccines in type 1 diabetes
  11. Further human trials with inverse vaccines
  12. Acknowledgements
  13. Conflict of interest statement
  14. References