Context: Plants and animals are being altered genetically for special purposes now that DNA codes and laboratory mechanisms for manipulating genetics are widely understood and available. Some food plants, for example, have been engineered to produce a bacterial toxin to improve insect resistance. Now transgenic crops are being explored to produce medicines or industrial compounds, such as plastics.
One active subject of research is the potential to use engineered corn or other plants to produce oral vaccines. This approach has several advantages compared to traditional methods, including reduced risks of contamination by animal pathogens, increased stability and shelf life, easier administration (swallowing vs. injection) and a stronger response to pathogens that enter or attack the body through the digestive tract.
Vaccines work by priming the immune system to rapidly recognize and attack foreign proteins found in (or produced by) bacteria, viruses or other microorganisms. Some foreign proteins do not provoke enough of an immune response by themselves to make an effective vaccine. In these situations, other compounds (called adjuvants) are sometimes added to increase the immune response against the targeted proteins. LT-B, a protein extracted from E. coli, is an effective adjuvant in animal studies (Moroveca et al. 2007).
If successful oral vaccines are marketed using an LT-B adjuvant, public exposure to LT-B through the vaccine would be intentional and presumably helpful in spurring the immune response against the target antigen (foreign protein).
However, unintended public exposures are also possible. LT-B produced in genetically engineered food crops could contaminate food supplies through cross-pollination or seed mixing. Farm workers could be exposed by breathing or ingesting grain dust.
Since LT-B is meant to enhance the immune system's response to other proteins administered with it, frequent exposure to low-dose LT-B could cause allergic responses to other food proteins being eaten at the same time as the LT-B. Frequent exposures to low-dose LT-B could also interfere or modify the response to LT-B based vaccines.
Assessing risks of LT-B will be necessary to protect workers and the public. An important first step is to determine the lowest dose of LT-B that produces a detectable immune system effect in laboratory animals. The most sensitive indicator would be the “immune priming” dose – the lowest exposure dose of LT-B for which a later exposure results in an increased immune system response. |
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What did they do? The research team performed two experiments with mice. In the first, they examined immune system response to LT-B across a range of doses, while in the second they confirmed the first study's results and then determined if 10-100 times lower doses also provoked immune system response.
In the first study, four experimental groups of mice were intermittently (days 0, 7 and 21) fed corn pellets containing LT-B at four different doses (0.02, 0.2, 2 or 20 micrograms (µg)) of LT-B per feeding. To determine if these doses had primed the animals' immune systems, the researchers waited until day 49 (28 days after the last previous dose) and gave a 20 µg pellet to all experimental groups and one unexposed control group. The other control group was never given LT-B.
In the second study, the same procedure was followed on different mice, except some groups were fed much lower doses of LT-B: 0.0002, 0.002, 0.02 and 20 µg. The lower doses were 1% and 10% of the dose found to stimulate a response in the first study. The highest dose in this study (20 µg) was a “positive control,” intended to show the experiment was working properly.
In both experiments, blood and fecal samples were collected from each mouse before the first dose and weekly thereafter. At the end of experiment two (days 57-59), mice lungs were rinsed with saline to collect antibodies in the respiratory tract. LT-B antibodies in blood serum (IgA, IgG), fecal matter (IgA) and lung fluid (IgA) were measured using ELISA assays. IgG is the primary type of antibody produced by the immune system. IgA antibody is produced by mucosal surfaces that line the respiratory and gastrointestinal tracts.
What did they find? In the first experiment, serum IgG antibody released after exposure to the final 20 µg "booster" dose of LT-B was found to increase in a dose-dependent manner with the size of earlier intermittent LT-B exposures. Levels peaked on day 55, six days after the final booster dose of 20 µg.
The highest IgA levels in serum were found after earlier exposures to the intermediate level of 0.2 µg, rather than the higher doses of either 2 µg or 20 µg–-a non-monotonic dose-response relationship.
Like serum IgA, fecal IgA in the 0.2 µg group was higher on day 55 than the 2 µg group and nearly as high as the 20 µg group. The lowest dose that caused statistically significant increases in antibody production was 0.02 µg.
In the second experiment, 20 µg again provoked strong antibody production. The lowest dose to provoke an increased response (as compared to the control) was 0.02 µg. No effects were seen at .002 and .0002 µg. Lung fluid showed significantly increased IgA in the 0.02 µg and 20 µg groups as compared with control.
Across both experiments at the 0.02 µg dose, 5 of 8 mice (62.5%) demonstrated immune priming for serum IgA, 3 of 8 (37.5%) for serum IgG and 2 of 8 (25%) for fecal IgA, where immune priming was defined as a four-fold increase in antibody production as compared to controls. That serum IgA was the most sensitive measure of oral LT-B immune system priming is not surprising given that IgA antibodies are produced in mucosal tissues like the gastrointestinal tract. |
