Gene Therapy (1999) 6, 1128–1138  1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Augmentation of natural immunity to a pro- inflammatory (TNF-alpha) by targeted DNA vaccine confers long-lasting resistance to experimental autoimmune encephalomyelitis

G Wildbaum and N Karin Department of Immunology, Rappaport Family Institute for Research in the Medical Sciences, Bruce Rappaport Faculty of Medicine, Technion, POB 9697, Haifa 31096, Israel

TNF-␣ is thought to be a key pro-inflammatory cytokine in titer and conferred EAE resistance. These antibodies were -mediated autoimmune diseases, particularly in rheu- found to be neutralizing in vitro and capable of inhibiting matoid arthritis (RA) and multiple sclerosis (MS). Experi- the development of disease when transferred to other EAE mental autoimmune encephalomyelitis (EAE) serves as an rats. Thus, modulation of EAE with TNF-␣ DNA vaccines animal model for MS. The current study observes a notable enhances the regulation of natural immunity to a self pro- TNF-␣-specific antibody titer generated during the course inflammatory cytokine and provides a tool by which the of EAE, apparently not sufficient to prevent the develop- immune system is encouraged to elicit anti-self protective ment of disease. Administration of TNF-␣-naked DNA vac- immunity to restrain its own harmful reactivity when such cine enhanced the production of TNF-␣-specific antibody a response is needed.

Keywords: TNF-␣; DNA vaccination; natural immunity; neutralizing antibodies; experimental autoimmune encephalomyel- itis; CNS

Introduction gamma (IFN-␥) and TNF-␣, and, to a much lesser extent, IL-4 and IL-10; Th2 cells that produce IL-4, Experimental autoimmune encephalomyelitis (EAE), is IL-10 and IL-13 and, to a much lesser extent, IFN-␥ and an autoimmune disease of the central nervous system TNF-␣.12–21 Th1 cells selected in response to various auto- (CNS) which serves as a model for the human disease, antigens transfer T cell-mediated autoimmune diseases, multiple sclerosis (MS), since in both diseases circulating whereas IL-4-secreting Th2 cells, selected in response to leukocytes penetrate the blood–brain barrier and damage these same antigens, either inhibit or exert no profound myelin resulting in impaired nerve conduction and paral- effect on the inflammatory process.16,22–34 High levels of 1,2 ysis. We have previously used molecular biological IFN-␥ and low levels of IL-4 positively select for Th1 techniques to follow leukocyte trafficking to the site of cells, whereas low levels of IFN-␥ together with high lev- inflammation in the CNS of EAE rats, and suggested a els of IL-4 mediate Th2 selection.12–17 In a recent study, model that characterizes this process as a sequential we isolated mRNA encoding -inducing multi-step event in which a primary influx of T cells factor (IGIF, IL-18) from the EAE brain, generated neu- interact with its target antigen to activate the blood–brain tralizing antibodies against its gene product and explored barrier and initiate a secondary influx to commence the the critical role of IGIF in T cell selection and regulation 3 disease. We have also developed various ways to inhibit of disease.35 The protective administration of IGIF-spe- the accumulation of the secondary influx at the target cific antibodies selects low IFN-␥, low TNF-␣, high IL-4- organ and thus block, or even reverse an ongoing dis- producing T cells.35 While the role of IFN-␥ in the mani- 4–7 4 ease. This includes adhesion molecule blockade, T cell festation of EAE is enigmatic, the pivotal role of TNF-␣ 5 receptor antagonism, and soluble peptide therapy using has been well studied. TNF-␣ is produced by activated non-pathogenic tolerizing analogs of the encephalitog- T cells (mostly Th1) and , and its elevated 5–7 enic determinant. Accumulation of the secondary expression at the site of inflammation occurs during the influx at the site of inflammation is dependent on various critical phase of disease,11 at the time when the ‘second- pro-inflammatory and that are ary influx’ of leukocytes is apparent.3 Except for a single produced at the site of inflammation.8–11 + recent study carried out in genetically modified ani- Based on their cytokine profile, CD4 T cells can be mals,36 all investigators agree that TNF-␣ contributes to divided into Th1 cells that produce large amounts of the pro-inflammatory process in EAE, and probably MS.25,37–51 Thus, inhibition of TNF-␣ activity by either neutralizing antibodies or soluble TNF receptor therapy, 25,42,45,47–51 Correspondence: N Karin effectively prevents, or even reverses EAE. Received 4 November 1998; accepted 1 February 1999 Overexpression of TNF-␣ in the CNS aggravated the dis- Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1129 ease,46 whereas genetically impaired expression of this attained its maximal severity (day 13), and marked gene inhibited the development and progression of dis- regression following recovery (Figure 1d). ease.43 Its pivotal role in T cell-mediated autoimmune dis- eases makes TNF-␣ a favorable target for clinical trials Prevention of EAE using TNF-␣ naked DNA vaccines aiming at inhibiting MS and rheumatoid arthritis (RA). Cloned PCR products of rat TNF-␣, obtained as described From a clinical perspective, a major disadvantage in tre- above, were ligated into a pcDNA3 eukaryotic expression ating these chronic diseases with xenogenic neutralizing vector and used as constructs for naked DNA vacci- antibodies, or even chimeric humanized antibodies nation. Rats were subjected to 3 weekly injections of the (reviewed in Ref. 52), lies in their immunogenicity. Fol- above construct. Control rats were either injected with lowing repeated immunization, even these engineered the pcDNA3 vector alone, or with PBS. Two months after antibodies do trigger an apparently allotypic response. the last immunization all rats were immunized with p68– We have recently utilized the novel technology of naked 86/CFA to induce active EAE. All control (PBS- DNA vaccination53–62 to generate self-specific protective immunized) and pcDNA3-vaccinated rats developed immunity against C-C chemokines and inhibit EAE.63 active disease that persisted for 5–6 days (Figure 2, six of Resistance could be transferred by C-C -spe- six in each group with a maximum clinical score of cific neutralizing antibodies.63 The apparent advantage of 3 ± 0.28 in control, 2.83 ± 0.18 in pcDNA3-immunized this method of therapy motivated us to evaluate its thera- rats). In contrast, rats injected with the TNF-␣-naked peutic potential against TNF-␣. DNA vaccine were resistant to EAE (incidence of two of six with a maximum clinical score of 0.33 ± 0.2, P Ͻ 0.003 for treatment with TNF-␣-naked DNA vaccine compared Results with either control or pcDNA3 treatments). Thus, the subsequent in vivo immune response to TNF-␣-naked Levels of mRNA encoding TNF-␣ increase in the DNA vaccine augmented in rats during the development inflamed brain before onset and during disease of EAE prevented the disease. progression RT-PCR analyses of the levels of mRNA encoding Lewis rats immunized with guinea-pig myelin basic pro- TNF-␣ in the brain and spinal cord of naked DNA-vacci- tein (MBP) emulsified in complete Freund’s adjuvant nated EAE-resistant rats (samples were obtained 12 days (CFA) develop the active form of EAE. At the onset of after active EAE induction) revealed a marked decrease disease, the immune T cell response is directed against a (data not shown), which was probably due to the marked major encephalitogenic determinant comprising the resi- reduction in the pro-inflammatory process, which occurs dues 68–86 of the peptide and then to a secondary deter- in the CNS of control EAE rats. minant encompassed by the residues 87–89 of this pep- tide.64–66 We generated long-term CD4+ T cell lines in Generation of natural immunity to TNF-␣ in EAE rats response to each of the above determinants.3,5 We named The development of anti-self immunity to TNF-␣ in EAE these lines according to their target determinant, L68–86 rats was evaluated. Just before active induction of disease and L87–99.3,5 Each line is encephalitogenic, that is the (day 0) and when EAE attained its maximum severity line is capable of transferring EAE to naive recipients. (day 13) blood samples were analyzed for the production Rats injected with L68–86 developed transferred EAE of antibodies against TNF-␣. Rats with developing EAE that persisted for 5–6 days (Figure 1a). Before adoptive displayed a significantly increased TNF-␣-specific anti- transfer of disease (day 0), and at various time-points body titer as compared with rats immunized in hind foot (before the onset of disease (day 3), at the day of onset pads with CFA alone (Figure 3d versus b, log2 Ab titer (day 5), the peak (day 7), following recovery (day 10), of 11 ± 0.85 versus 7 ± 0.66, P Ͻ 0.05). These results are and 10 days after recovery (day 20)) midbrain–brain stem remarkable since both groups exhibited an extensive local samples were obtained from six different rats at each inflammatory process at the site of CFA immunization time-point. From each sample mRNA was isolated and (hind foot pads), with enhanced levels of TNF-␣ mRNA subjected to RT-PCR analysis using specific oligonucleo- (data not shown). Nevertheless, only rats with tide primers which we constructed. Each amplification developing EAE manifested an apparent level of mRNA was calibrated to ␤-actin and verified by Southern blot- encoding TNF-␣ at the CNS that substantially increased ting analysis. This enabled semi-quantitative analysis of at the time when clinical disease attained maximum the levels of mRNA encoding TNF-␣ at the site of severity (Figure 1d). In contrast, rats immunized with inflammation during different stages of the disease. Fig- CFA alone did not exhibit notable levels of mRNA enco- ure 1c shows representative results from each time-point ding TNF-␣ in the brain samples at any time-point (0, 8, of the experiment. mRNA encoding TNF-␣ was apparent 13 and 21 days after CFA administration, data not in EAE brains at the onset of disease (day 5), peaked at shown). Thus, only the elevated levels of TNF-␣ mRNA the time when clinical disease attained its maximal sever- at the CNS enabled the triggering of an anti-self response ity (day 7), and gradually regressed following recovery. against this pro-inflammatory cytokine. This response Markedly reduced but still notable levels of TNF-␣ was, however, not sufficient to prevent the development mRNA in EAE brains could be observed even 10 days of an autoimmune condition (six of six sick rats, Figure after recovery (Figure 1c). 2). Rats with developing active disease (Figure 1b) mani- fested a similar pattern of mRNA levels at the site of Naked DNA encoding TNF-␣ augments TNF-␣-specific inflammation as those with developing transferred dis- protective immunity ease. That is, mRNA encoding TNF-␣ was apparent at The development of anti-self protective immunity in the CNS just before the onset of disease (day 8) with sub- DNA-vaccinated rats was evaluated. When active EAE stantial augmentation at the time when clinical disease attained its maximum severity (day 13), blood samples Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1130

Figure 1 Levels of mRNA encoding TNF-␣ increase in the inflamed brain before the onset and during disease progression. (a) and (c) Rats were injected with 107 L68–86 cells to develop transferred EAE (a). Before adoptive transfer of disease (day 0), and at various time-points (before the onset of disease (day 3), at the day of onset (day 5), the peak (day 7), following recovery (day 10) and 10 days after recovery (day 20)) mid-brain and brain stem samples from six different rats at each time-point were examined. mRNA was isolated from each sample and subjected to RT-PCR analysis using specific oligonucleotide primers constructed to TNF-␣. Each amplification was calibrated to ␤-actin and verified by Southern blotting analysis (e and f). (c) Shows a representative Southern blot analysis of each time point. (b) and (d) Rats were immunized with p68–86/CFA and developed active EAE (b). Before the induction of disease (day 0), and at various time-points (before the onset of disease (day 8), at the peak (day 13) and 5 days after recovery (day 21)) mid-brain and brain stem samples from six different rats at each time-point were obtained and subjected to RT-PCR as described above. (d) Shows a representative Southern blot analysis from each time-point. Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1131 difference could be observed between the production of TNF-␣-specific antibody titer in TNF-␣ DNA-vaccinated rats that were immunized with p68–86/CFA and those

immunized with CFA alone (blood sera: log2 Ab titer of ± ± Ͻ 26 1.88 versus 13 0.6, P 0.001; SCF: log2Ab titer of 15 ± 1.33 versus 8 ± 0.5, P Ͻ 0.005). Thus, administration of naked DNA encoding TNF-␣ augments the generation of anti-self antibodies against a pro-inflammatory cyto- kine that is involved in the induction and progression of the autoimmune condition.

TNF-␣-specific antibodies generated in naked DNA- vaccinated rats are neutralizing antibodies Since DNA vaccination can potentially elicit both cellular and humoral responses against products of a given con- struct, it is difficult to know which of these responses contributed more to the development of EAE resistance in TNF-␣ DNA-vaccinated rats. Twelve to 13 days after active induction of EAE, when production of anti-self antibodies in naked DNA vaccinated rats attained its maximal titer (Figure 4), antibodies were purified (IgG fraction, Protein-G purification) and evaluated for their in vitro competence to neutralize the cytotoxic effect of TNF-␣ (Figure 5). It appears that IgG from TNF-␣ DNA- vaccinated rats abolished the cytotoxic activity of TNF-␣ on U937 cytotoxic T cells (natural red uptake determined in OD at 570 nm of 0.174 ± 0.008 versus 0.075 ± 0.007 and 0.004 ± 0.006 in the presence of 100 ␮g per well IgG from TNF-␣ DNA-vaccinated rats, normal IgG or PBS and 1000 ␣ ␣ pg/ml TNF- , with backgrounds of 0.168, 0.166 and Figure 2 Prevention of EAE using TNF- -naked DNA vaccines. Rats Ͻ were immunized weekly with the cloned PCR products of TNF-␣ ligated 0.165, respectively, P 0.001 for the comparison of the into a pcDNA3 eukaryotic expression vector, or with the pcDNA3 vector first group with each of the control groups). Thus, anti- alone, or with PBS. Two months after the last immunization all rats were bodies produced in naked DNA-vaccinated rats are neu- immunized with p68–86/CFA to induce active EAE and monitored for tralizing antibodies. clinical signs daily by an observer blind to the treatment protocol. EAE was scored as follows: 0, clinically normal; 1, flaccid tail; 2, hind limb TNF-␣-specific neutralizing antibodies generated in paralysis; 3, front and hind limb paralysis; 4, hind limb and total front limb paralysis. Results are shown as mean clinical score of six rats in naked DNA-vaccinated rats are capable of transferring each group ± s.e. EAE resistance The TNF-␣-specific neutralizing antibodies (previous sec- tion above) were then evaluated for their competence to of representative rats from the experiment described in transfer protection against active EAE (Figure 6). Starting the legend to Figure 2 were analyzed for the production 4 days before the onset of active EAE, rats were chal- of antibodies against TNF-␣ (Figure 3d, e and h), and for lenged with the above antibodies (100 ␮g daily, days 6– the kinetics of antibody production (Figure 4). The 13), or with IgG from rats that were vaccinated with notable anti-self antibody titer against TNF-␣ produced either pcDNA3-TNF-␣ construct, or with pcDNA3 alone. in EAE rats without DNA vaccination (Figure 3d versus Repeated administration of antibodies from TNF-␣ DNA- ± ± Ͻ b, log2 Ab titer of 11 0.85 versus 7 0, P 0.05), which vaccinated rats provided substantial protection from dis- was not sufficient to prevent the development of disease, ease progression (mean maximum score of 1 ± 0.2 in rats was profoundly augmented in naked DNA-vaccinated treated with purified antibodies from TNF-␣ DNA-vacci- ± ± ± rats (Figure 3h versus d and e, log2 Ab titer of 28 1.88 nated donors versus 2.66 0.3 and 2.83 0.816 in rats in rats vaccinated with the TNF-␣ construct versus treated with purified antibodies from PBS- or pcDNA3- 11 ± 0.85 in EAE rats vaccinated with pcDNA3 alone and treated rats, P Ͻ 0.001 for each comparison). Anti-sera 8 ± 0.8 in control EAE rats, P Ͻ 0.0001, for the comparison from rats that were previously administered with of h with either d or e). pcDNA3 vaccine alone and then subjected to active At different time-points: 0, 8, 10, 13, 21 and 35 days induction of EAE, and anti-sera from rats that were pre- after active EAE induction, the kinetics of anti-self TNF- viously administered with pcDNA3-TNF-␣ vaccine and ␣ antibody production in blood sera (Figure 4a) and spi- then subjected to active induction of EAE, both mani- nal cord fluid (SCF) (Figure 4b) was determined. TNF-␣- fested a similar MBP-specific antibody titer (data not specific antibody titer profoundly increased within 8 shown). Yet only TNF-␣ DNA-vaccinated rats exhibited ␣ days of MBP p68–86/CFA immunization (blood sera: log2 a profound TNF- -specific antibody titer. Thus, it is Ab titer of 26 ± 1.88 on day 8 versus 7 ± 1 on day 0, P Ͻ plausible that these antibodies reinstated the resistant ± ± 0.001; SCF: log2 Ab titer of 15 1.3 on day 8 versus 6 1.1 state following anti-sera transfer. on day 0, P Ͻ 0.001). The increase was simultaneous with Taken together, our results show that administration the accelerated levels of TNF-␣ mRNA at the site of of naked DNA encoding TNF-␣ augments the generation inflammation (Figure 1). At this time a highly significant of anti-self antibodies, specific to this pro-inflammatory Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1132

Figure 3 Naked DNA encoding TNF-␣ augments TNF-␣-specific protective immunity. Twelve days after the active induction of disease (with p68– 86/CFA) sera of rats from the experiment described in Figure 2, as well as sera from rats that received the same subsequent set of naked DNA vaccinations but were finally challenged with the emulsion of PBS and CFA without p68–86, or from rats that received the same subsequent set of naked DNA vaccinations but were never challenged with p68–86/CA or CFA, were tested for antibody titer against TNF-␣. Calculation of each titer was done by ␣ comparing the OD measured in wells coated with TNF- with those not coated with this cytokine. Results are shown as mean log2 of four different samples ± s.e.

Figure 4 Kinetics of TNF-␣-specific antibody production in sera and SCF of EAE rats following TNF-␣-naked DNA vaccination. Rats were immunized weekly (three repeated immunizations) with the cloned PCR products of TNF-␣ ligated into a pcDNA3 eukaryotic expression vector. Two months after the last immunization, all rats were immunized with either p68–86/CFA to induce active EAE, or with CFA alone. At different time-points, generation

of anti-self antibody titer was determined in blood sera (a) and SCF (b) as described in the legend to Figure 3. Results are shown as mean log2 of four different samples ± s.e. Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1133

Figure 6 TNF-␣-specific antibodies produced by DNA vaccination pro- vide subsequent protection from severe EAE. Six groups of six rats each Figure 5 TNF-␣-specific antibodies produced by DNA vaccination are were immunized with p68–86/CFA to develop active EAE. Starting 4 days neutralizing antibodies. TNF-␣-specific neutralizing antibodies (IgG frac- before the onset of disease, rats were challenged daily (i.v., days 6–13) tion, Protein-G purification, 100 ␮g per well) purified from sera of rats with 100 ␮g of each of TNF-␣-specific neutralizing antibodies (IgG frac- that were previously vaccinated with TNF-␣-naked DNA vaccine and tion, Protein-G purification) purified from sera of rats that were previously were then subjected to active induction of EAE as described in the legend vaccinated with TNF-␣-naked DNA vaccine, and were then subjected to to Figure 2, control IgG purified from normal rat serum (100 ␮g per well) active induction of EAE as described in the legend to Figure 2. Purified or an equal volume of PBS, were determined for their competence to inhibit IgG fraction from rats that were, or were not vaccinated with pcDNA3 neutral red uptake of U937 cells (4 × 104 per well in a total volume of and then subjected to active induction of disease, as well as sera from 100 ␮l). Results are shown as mean OD at 570 nm ± s.d. control rats that were or were not subjected to active induction of EAE, were all used as controls. EAE was monitored daily by an observer blind to the treatment protocol. Results are shown as mean clinical score of six cytokine, that are capable of blocking the development rats in each group ± s.e. of an experimental autoimmune disease of the CNS and thus providing a tool by which the immune system is encouraged to elicit anti-self protective immunity to under control.22,23,30–32,68–72 The current study demon- restrain its own harmful reactivity when such a response strates, for the first time, the appearance of ‘natural’ anti- is needed. self antibodies to a key pro-inflammatory cytokine, TNF- ␣, during the development of a T cell-mediated auto- Discussion immune disease of the CNS. These antibodies developed in rats immunized with p68–86/CFA and not with the In the process of negative selection in the thymus many, CFA alone (Figure 3), even though both groups exhibited but not all, self-reactive T cells are eliminated. Auto- an extensive local inflammatory process at the site of CFA reactive T cells that escape thymic selection can be ident- immunization. They are neutralizing antibodies (Figure ified in both healthy individuals and those suffering from 5) capable of transferring EAE resistance (Figure 6). The self destructive autoimmune diseases.67 In healthy indi- biological significance of the association between the viduals self-tolerance is maintained in part through elevated levels of TNF-␣ mRNA at a privileged auto- mechanisms acting outside the thymus that keep these immune site (CNS) and the enhancement in anti-self autoreactive lymphocytes under control. Anti-inflamma- response against this pro-inflammatory cytokine is tory cytokines such as TGF-␤, IL-10, IL-4 and IL-13 pro- apparent. After all, an ideal immune system would be duced by antigen-specific regulatory T cells and macro- selected in evolution to centralize its destructive com- phages are involved in restraining the activity of petence against invading microbes rather than against the autoreactive T cells, and for keeping the tolerant state self tissues it was designed to protect.73–75 The underlying Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1134 mechanism by which the immune system distinguishes This provides a new perspective for understanding the a gene product transcribed at a privileged autoimmune role of T cell and B cell selection in induction and mainte- site from the same gene product transcribed at a local site nance of tolerance to self. In the process of negative selec- of inflammation is, however, elusive. A partial expla- tion, self-reactive T cells die when they encounter self- nation has been previously suggested by Cyster and his antigen in the thymus.90,91 Similarly, self-specific pre-B colleagues76 who demonstrated that peripheral clonal cells either die or undergo receptor editing in the bone exclusion of self-reactive B cells occurs at germinal cen- marrow.92 It is believed that those cells escaping central ters of lymph nodes that drain tissues lacking immune tolerance are subjected to various mechanisms acting out- surveillance (ie immune privileged areas), where compe- side the thymus or the bone marrow to keep them under tition for follicular niches do not exclude self-reactive control. This type of control has been termed peripheral cells from the recirculating B cell repertoire. The ‘natural’ tolerance. T cell anergy,69 active suppression,31,68–70 T cell production of anti-self antibodies to TNF-␣ in EAE sus- deletion93,94 and generation of anti-idiotypic immunity95 ceptible rats, while it may modulate the degree of dis- have been described as key mechanisms that contribute ease, was however, not sufficient to prevent the develop- to the maintenance of peripheral tolerance. The current ment of an autoimmune condition (six of six sick rats, study suggests, for the first time, that self-specific T and Figure 2). B cells, capable of mounting self-specific immunity An ideal method of treating a disease caused by a mal- against pro-inflammatory mediators, escape central toler- function of the immune system in distinguishing self ance to provide the immune system with a powerful tool from foreign would be to encourage this system to elicit with which it keeps its dangerous anti-self activity under self-protective immunity and thus restrain its own harm- control and thus maintains tolerance to self in the periph- ful reactivity when such a response is needed. This task ery. Moreover, as microbes and self-components are con- has been achieved in the current study using the novel structed from similar ‘building blocks’ and as central technology of naked DNA vaccination. A current use of selection manifests its own limitations, anti-self immun- endogenous cytokine genes in immunization protocols is ity cannot be avoided, but rather has to be restrained by for the increase of the pro-inflammatory (Th1) immune peripheral mechanisms. Moreover, it could well be that response against infectious agents such as tuberculosis, a substantial increase in the competence of the immune HIV and allergens such as mite proteins.60,77–82 This tech- system to effectively to limit its T and B cell repertoire nology has also proved successful in eliciting immunity would result in a constrained ability effectively to con- to self-antigens such as T cell receptor V genes and the front infectious diseases. The case of natural immunity to shift towards a Th2 cell response.62 In another recent TNF-␣, evoked during the course of a T cell-mediated study, transforming beta (TGF-␤) DNA autoimmune disease, demonstrates how the immune sys- vaccines were evaluated for their competence to inhibit tem has evolved to benefit from its own limited com- the development of cell wall-induced arthritis.83 TGF-␤ is petence effectively to select against self reactivity. a well known inhibitory modulator of the immune sys- From a clinical perspective, a major disadvantage in tem. Thus, the administration of TGF-␤ prevented EAE, treating chronic diseases with xenogenic neutralizing whereas TGF-␤-specific neutralizing antibodies aggra- antibodies lies in their immunogenicity. This has mot- vated expression of the disease.84,85 In contrast to its regu- ivated investigators to develop chimeric humanized anti- latory role in EAE, TGF-␤ has been found to be important bodies (reviewed in Ref. 52), and monoclonal antibodies for the manifestation of arthritis. Thus, TGF-␤-specific engineered with human Ig heavy and light chain yeast neutralizing antibodies suppressed, rather than artificial chromosome (YAC).96 However, following enhanced, the development of cell wall-induced repeated immunization, these engineered antibodies do arthritis.86 Along with these data, a TGF-␤-specific naked trigger an apparently allotypic response. The therapeutic DNA vaccine has recently been found to be capable of strategy suggested by our study has an advantage over suppressing this disease.83 TGF-␤ DNA vaccines were the above methods since it resulted in the generation of also found to be beneficial in inhibiting the development immunity to autologous antigen only during the course of experimental colitis87 and SLE.88 Since DNA vaccines of disease at the time when mRNA levels encoding the represent a novel means of expressing antigens in vivo pro-inflammatory cytokine are profoundly elicited at the for the generation of both humoral and cellular immune site of inflammation (Figures 1, 2, 3 and 4). responses55,59,89 it is difficult to know which of these The possible effect of TNF-␣-naked DNA vaccination responses contributed more to the development of EAE on the ability of an individual to confront infectious dis- resistance in TNF-␣ DNA-vaccinated rats. The traditional eases has not yet been evaluated. Nevertheless, the obser- implication of naked DNA vaccines with the aug- vation that CFA immunization alone does not elicit TNF- mentation of Th1 immunity80,81 makes data analysis even ␣-specific immunity in DNA-vaccinated rats (Figure 3) more complicated. Nevertheless, our data showing that implies that elicitation of natural immunity following self-specific neutralizing antibodies to TNF-␣ produced naked DNA vaccination is not induced if TNF-␣ is not by naked DNA vaccination can provide subsequent pro- transcribed at the autoimmune site (Figure 3). This gives tection from severe EAE (Figure 6) strongly implies their TNF-␣-naked DNA vaccination an apparent advantage as pivotal role in the prevention of EAE. The mechanism a candidate for clinical trials. by which TNF-␣-specific naked DNA vaccines augment production of anti-self neutralizing antibodies is not yet Materials and methods fully elucidated. We do not exclude the possibility that naked DNA vaccination elicits the activation of self- Rats reactive T cells that help production of autoreactive anti- Female Lewis rats, approximately 6 weeks old, were pur- bodies to TNF-␣ when this cytokine is profoundly tran- chased from Harlan (Jerusalem, Israel) and maintained scribed at an autoimmune privileged area. under SPF conditions in our animal facility. Augmentation of natural immunity to a pro-inflammatory cytokine G Wildbaum and N Karin 1135 Peptide antigens Rat ␤-actin antisense: 5Ј-CCGGCCAGCCAAGTCCA- Myelin basic protein (MBP) p68–86, Y G S L P Q K S Q GACG-3Ј R S Q D E N P V, was synthesized on a MilliGen 9050 peptide synthesizer (Burlington, MA) by standard 9- The cycle profile was denaturation at 95°C for 60 s, fluorenylmethoxycarbonyl chemistry. Peptides were pur- annealing at 55°C for 60 s, and elongation at 72°C for 60 ified by high performance liquid chromatography. Struc- s. Amplified products were subjected to electrophoresis, ture was confirmed by amino acid analysis and mass transferred on to nylon membranes (MagnaGraph nylon spectroscopy. Only peptides that were greater than 95% transfer membrane, msi, Westborough, MA, USA), fixed pure were used in our study. with ultraviolet light (120 mJ) and hybridized with 106 c.p.m./ml of ␣32P-labeled DNA fragments encoding the Immunizations and active disease induction full-length PCR product of the TNF-␣ and of ␤-actin Rats were immunized subcutaneously in the hind foot (random priming; Amersham, Arlington Heights, IL). pads with 0.1 ml of MBP epitope 68–86 (p68–86) dis- PCR products were used as probes only after each PCR solved in PBS (1.5 mg/ml) and emulsified with an equal product was cloned and its sequence was verified as volume of CFA (incomplete Freund’s adjuvant sup- described below. plemented with 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra in oil (Difco Laboratories, Detroit, MI, USA). Rats were then monitored daily for clinical signs Cloning and sequencing of PCR products by an observer blind to the treatment protocol. EAE was Each of the amplified PCR products described above was scored as follows: 0, clinically normal; 1, flaccid tail; 2, cloned into a pUC57/T vector (T-cloning Kit No. K1212, hind limb paralysis; 3, hind limb and partial front limb MBI Fermentas, Vilnius, Lithuania) and transformed to paralysis; 4, hind limb and total front limb paralysis. E. coli according to the manufacturer’s protocol. Each clone was then sequenced (Sequenase ver 2; USB, Cleve- Induction of transferred EAE land, OH, USA) according to the manufacturer’s proto- EAE was induced by immunizing Lewis rats col. PCR products were selected to be used as constructs (intraperitoneally) with 108 activated spleen cells from for naked DNA vaccination only after cloning and EAE donors obtained as follows: 9 days after induction sequence verification. of active EAE, splenic cells were cultured (12 × 106/ml) at 37°C in humidified air containing 7.5% CO for 2 days 2 DNA vaccination in stimulation medium containing Dulbecco’s modified DNA vaccination was performed according to Waisman Eagle’s medium (Gibco BRL Life Technology) sup- 62 − et al with some modifications as recently described else- plemented with 2-mercaptoethanol (5 × 10 5 m), l-gluta- where.63 Sequenced PCR products were transferred into a mine (2 mm), sodium pyruvate (1 mm), penicillin (100 pcDNA3 vector (Invitrogen, San Diego, CA, USA). Large- ␮g/ml), streptomycin (100 ␮g/ml), 1% syngeneic serum scale preparation of plasmid DNA was conducted using and 20–30 ␮g/ml of the immunizing epitope. Then, cells Mega prep (Qiagen, Chatsworth, CA, USA). Cardiotoxin were separated on a Ficoll gradient (Sigma, St Louis, MO, (Sigma) was injected into the tibialis anterior muscle of USA), resuspended in PBS and injected into naive recipi- 6 to 8-week-old female Lewis rats (10 ␮m per leg). One ents. week following injection rats were injected with 100 ␮g Reverse transcriptase polymerase chain reaction (RT- DNA in PBS. Four to 5 days after the first immunization PCR) analysis one rat from each group was killed and mRNA levels RT-PCR analysis, verified by Southern blotting, was util- encoding the relevant cytokine were verified using RT- ized on brain samples according to the protocol we PCR on tibialis anterior muscle samples. Thereafter, described elsewhere with some modifications.3 Rats were naked DNA vaccines were administered three to five times with intervals of 6–7 days between each injection. killed by CO2 narcosis. Brain samples containing mainly the midbrain and brain stem were obtained after per- fusion of the rat with 160–180 ml of ice-cold phosphate Purification of antibodies and determination of TNF-␣- buffer saline (PBS) injected into the left ventricle follow- specific antibody titer ing an incision in the right atrium. Each sample was Antibodies from rat sera were purified (IgG fraction) homogenized. Total RNA was extracted using the Tri-Zol using a High-Trap Protein G column (Pharmacia, Piscata- procedure (Gibco BRL Life Technologies) according to way, NJ, USA) according to the manufacturer’s protocol. the manufacturer’s protocol. mRNA was then isolated Then, antibody titer to TNF-␣ was determined by a direct ( No. 1741985; Boehringer Mannheim, Mannheim, ELISA assay: ELISA plates (Nunc, Roskilde, Denmark) Germany), and reverse transcribed into first-strand 3 were coated with 50 ng per well recombinant rat TNF- cDNA exactly described in detail elsewhere. First-strand ␣ (Genzyme, Cambridge, MA, USA). Serum from DNA cDNA was then subjected to 35 cycles of PCR amplifi- vaccinated rats was added in serial dilutions from 25 to cation using specific oligonucleotide primers which we 30 ␣ 2 to wells that were, or were not, previously coated with designed based on the published sequence of rat TNF- TNF-␣. Calculation of each titer was done by comparing as follows: the OD measured in wells coated with TNF-␣ with those Rat TNF-␣ sense: 5Ј-ATGAGCACAGAAAGCATGAT-3Ј not coated with this cytokine. Goat anti-rat alkaline phos- Rat TNF-␣ antisense: 5Ј-TCACAG AGCAATGAC phatase-conjugated antibodies (Sigma) were used as a TCCAAA-3Ј. labeled antibody. P-nitrophenyl phosphate (p-NPP) ␤ (Sigma) was used as a soluble alkaline phosphatase sub- All RNA samples were calibrated to -actin: strate. Results of triplicate experiments were calculated ␤ Ј Ј ± Rat -actin sense: 5 -CATCGTGGGCCGCTCTAGGCA-3 as log 2 antibody titer s.e. 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