Earlier this year, the state of Wisconsin considered adding Lactococcus lactis as the state microbe. Wisconsin is known as America’s Dairyland, and L. lactis is part of the cheese-making process. While its run to become the state microbe was ultimately unsuccessful, I wanted to learn more about the L. lactis bacterium. A quick search through PubMed yielded an intriguing paper by Lei et al., and it had nothing to do with converting milk to cheese.
These scientists put forth an interesting idea: Create an edible vaccine for the avian influenza H5N1 strain to not only inoculate humans but other species that harbor and spread the virus. By minimizing the cross-species spread, the hope is to decrease the likelihood the virus will mutate. The avian H5N1 virus has infected humans and caused deaths so preventing outbreaks is important. With an edible vaccine, we can bypass the need to inject humans, chickens and other species.
Why was this bacterium chosen? L. lactis has a history of use in various potential vaccine delivery systems as I found out from the references cited in the paper and summarized on this web site (a nice page about the bacterium). In this Virology paper (1), the authors chose to clone the H5N1 hemagglutinin (HA) antigen into L. lactis expression vectors designated as L1 (an empty vector without HA), L2 (HA is expressed in the cytoplasm), and L3 (HA + secretion signal ssUSP to create a secreted antigen). Expression of HA and its localization was tested using Western blotting. L1 produced no HA, L2 had HA only in the cell lysate and L3 had HA in both the supernatant and cell lysate, confirming correct expression in the bacteria.
To deliver the antigen orally, 109 CFU of an inducible L. lactis strain containing one of the expression vectors was packaged into 4 × 1mm enteric capsules. These enteric capsules and L1, L2 and L3 in solution were subjected to simulated gastrointestinal tract conditions, and the amount of live bacteria remaining was determined to test how well the L. lactis would survive after oral inoculation. The enteric capsules performed well, maintaining the full inoculum, whereas the bacterial solution retained <4 × 104 CFU.
The enteric capsules and L. lactis solutions were administered orally to mice at 0, 2, 4 and 6 weeks. Ten days after the last dose, the serum titer of IgG was measured using an ELISA. Both the capsule and liquid L. lactis immunizations resulted in HA-specific antibodies with the encapsulated bacteria generating a better response than the liquid inoculations. In fact, the capsule-L3 secretory antigen had the highest antibody titer. For mucosal IgA measurements, fecal material was tested using indirect ELISA and mimicked the results of serum antibody levels.
Neutralizing-antibody titers were measured for each of the inoculated groups of mice. Neutralization titer values above 80 are considered good, and only the encapsulated L2 reached and encapsulated L3 exceeded this threshold. The T-cell response was assessed using an IFN-γ ELISpot assay with the L3 capsule generating the largest cellular immune response.
These tests all demonstrated that there was an immune response to the HA antigen generated in all but the control mice (those treated with L1 and PBS) and that the enteric capsules of L2 and L3 elicited a stronger response than a solution of the same L. lactis expression strain. The big question is would the immunity in mice be strong enough to survive a challenge from H5N1 infection? The mice were infected intranasally with lethal doses of H5N1 two weeks after the last oral L. lactis treatment and monitored for weight loss and mortality over 14 days. All the PBS- and L1-treated mice died, 60% of the capsule-L2 and 80% of the L2-solution mice died but the capsule L3-treated mice lost some weight, but all survived the viral challenge.
So what have I learned from this paper?
- Vaccines can be administered orally and induce an immune response (at least in mice). I can understand how an edible vaccine would be useful for inoculating a variety of species even in the wild. However, if there is a human version, could it be lemon-flavored?
- L. lactis is a really cool bacterium, and I will have to build a shrine to its utility—aside from the large stash of Colby and Parmesan cheese in my refrigerator.
- H5N1 better watch out! More testing needs to be done, but this L. lactis vaccine delivery system proved itself in mice and quite dramatically in the face of a lethal dose.
1. Lei, H., Xu, Y., Chen, J., Wei, X. and Lam, D.M. (2010). Immunoprotection against influenza H5N1 virus by oral administration of enteric-coated recombinant Lactococcus lactis mini-capsules. Virology 407 (2), 319–24. PMID: 20850860