**10. DNA vaccines**

deviation. The immunological effects were long lasting immune responses, as they remained

As a sign of increase of T-regulatory cells we noticed an increased GAD65-induced expression of FOXP3 and TGF-β at month 15 in cells from GAD-alum treated patients compared to pla‐ cebo, and the expression of FOXP3 and TGF-β correlated positively in the GAD-alum group but not in the placebo group[77]. Still after 48 months there were clear effects on the immune system suggesting both a Th2 deviation, a decrease of activated T-cells (CD4+CD25+high) but increase of FoxP3-positive regulatory T-cells. Thus, our interpretation is that Diamyd® treat‐

Beside the European phase III trial discussed above [73], a similar trial was started a bit later in USA (US Phase III ClinicalTrials.gov Identifier: NCT00751842 ;Jerry Palmer, PI), with the same design. The recruitment was not so fast as initially only patients >16 years old were accepted, and therefore the recruitment had just finished when the negative results of the European Phase III trial was found. This lead to that the American trial was stopped, before it can give any results. In addition another intervention trial in newly-diagnosed Type 1 diabetic patients aged 3-45 years was performed by TrialNet (TrialNet Intervention ClinicalTrials.gov Identifier: NCT00529399). Patients were randomized in a double-blind controlled study into three arms, one with subcutaneous injections of 20 μg GAD65-alum (Diamyd®) at day 1,30 and 90, a second arm with subcutaneous injections of 20 μg GAD65-alum (Diamyd®) at day 1,30 and placebo at day 90, and a third arm with placebo at all time points The study failed. No effect on C-peptide preservation was found [77]. So far little has been presented from this trial with regard to effects on the immune system. It is difficult to know what the wide age

Because of the positive results in the Swedish Phase II study and the positive results in some prespecified subgroups in the European Phase III trial, new studies are planned. As the Phase III trial failed, GAD-alum will be given as part of combination therapy, which hopefully will give a better effect on the disease process. Thus a new pilot trial is just on its way when GADalum is combined with Vitamin D, which is supposed to positively influence the dendritic cells, contribute to Th2 deviation, but also influence directly beta cell survival and insulin sensitivity. In addition a third drug, anti-inflammatory, will be given to dampen the inflam‐ mation, which might play an important and negative role beside the autoimmune process.

In addition to interventional trials at onset of Type 1 diabetes a pilot trial with the aim to prevent T1D is ongoing in southern Sweden. High risk children have been identified as part of the so called DiPiS (Diabetes Prevention in Skåne) study, in which newborn children in the general population have been screened for auto-antibodies. Children positive for GADA, plus at least one more diabetes-related autoantibody, have been treated with either 20 μg GAD65-alum

ment deviated the immune system towards tolerance against the auto-antigen GAD.

still 48 months after the first injection [75].

502 Type 1 Diabetes

**9.5. Other trials with GAD vaccination**

range, variation in ethnic groups, BMI etc meant for the result.

**9.6. Ongoing or planned GAD-alum studies**

T-cells respond to antigens presented by antigen presenting cells (APCs).DNA-vaccines can be used to present the antigen instead of delivering intact proteins. A protein encoded by a plasmid DNA can either be produced outside the APCs if the pasmidDNA is administered into a muscle, or the plasmidDNA may be taken up by the APCs where the encoded protein is presented [78]. Proteins encoded by DNA vaccines can induce different types of antigenspecific immune responses, and perhaps also some non-specific reactions.

Most common routes of administration are either intramuscular, which is thought to favour Th1 responses, or intradermal, which is thought to favour Th2 response. For treatment of Type 1 diabetes intradermal injection should be most interesting. Another way of skewing the re‐ sponse towards Th2 may be to co-administer plasmids encoding Th2 cytokines.

Promoters from virus, eg Cytomegalovirus, can be used. Certain sequences seem to stimulate Th1 response and should therefore be avoided in treatment of T1D.

So far DNA-vaccines to create tolerance in autoimmune disease have been tried mainly in experimental animals. Plasmid DNA encoding for proinsulin [79] as well as for the insulin B chain [80] have been used for prevention of diabetes in experimental animals. Injection of plasmidDNA encoding for GAD has been shown effective in preventing diabetes in NODmice [81], while similar effect have been seen by combining plasmidDNA encoding for a fusion protein consisting of both GAD, IgG and IL4 [82]. Treatment with a recombinant vaccinia virus expressing GAD ( rVV-GAD65) has also shown to be effective in prevention of autoimmune diabetes in NOD mice by induction of active suppression of effector T-cells [83]. IgG1 anti‐ bodies and IL-4 increased and the IgG2 was unchanged, suggesting a Th2 deviation.Before clinical use there are several problems which need to be solved. Correct dosing is necessary as wrong dose might give increased immune response and a more aggressive disease process In addition it is important to be sure that the DNA is not integrated in the host chromosome. Another problem might be production of antibodies against DNA.
