Things we wish we'd known about a lot sooner: TLRs

Most scientists operate under the assumption that basic research is important for human health, even if its applications are not immediately apparent. A paper in the January issue of Nature Medicine demonstrates just how important an understanding of basic immunology can be, and in the process, solves a decades-old vaccine mystery. Delgado et al. take on the issue of why a vaccine against Respiratory Syncytial Virus (RSV) developed forty years ago not only failed to protect infants from the disease, but caused serious illness, and even death in some subjects.

The RSV vaccine consisted of wild-type RSV grown in primate cells, inactivated by formalin fixation, and mixed with alum as an adjuvant. By the time the trial in question started, the researchers had already carried out safety studies in four animal species and adult volunteers. Because RSV is primarily a disease of infants (around half of babies are infected in their first year,) a vaccine is not useful unless it can generate protective immunity in that population. Furthermore, virtually all potential adult volunteers would have already been exposed to the virus many times, obviating the testing or use of the vaccine in that population. The main efficacy study, therefore, took place in infants.

23 babies were given the vaccine, and on the whole, had a measurable increase in their neutralizing antibody titer (6x control) and a big, big increase in their complement-fixing antibody (30x control). The researchers concluded from this that the vaccine was immunogenic, but when the RSV season hit that winter, there was an unexpected result. The vaccinated subjects got far more severely ill when exposed to RSV (80% required hospitalization) and 2 died. Kim et al. did not suggest a mechanism for this result, but in their discussion they refer to something called the "paradoxical vaccine effect," which I've never heard of but apparently happened all the time back then, since they cite failed vaccines against rickettsia, trachoma, mycoplasma, and measles as examples. One has to wonder why they kept trying to make vaccines when the failure rate was so devastating, and whether, given todays safety standards, all of these trials would have ever been allowed.

To investigate the source of RSV's paradoxical vaccine effect, Delgado et al. immunized mice with a range of RSV preparations. One was inactivated by formalin, just like the human vaccine, another other was UV-inactivated, a third vaccine was made of purified protein, and the final preparation contained live replicating virus. When the immunized mice were re-challenged with live virus, only the mice previously exposed to live virus and mice who had received a placebo vaccine avoided enhanced respiratory disease (ERD). The authors went on to show that mice vaccinated with inactivated virus produced antibody with far lower affinity for model epitopes than the mice that received live virus.

Why did affinity maturation fail? First the authors look at draining lymph nodes following immunization and see attenuated upregulation of CD40, CD80, and Cd86 on dendritic cells, decreased CD4 T cell proliferation and cytokine production, and a failure to develop germinal centers. The authors argue that inactivated virus induces these components of immunity poorly because it fails to stimulate TLRs. To support this conclusion, the authors use MyD88 heterozygous mice, and demonstrate that they fail to generate high affinity antibody against live RSV. Whether MyD88 hets actually constitute a good model of reduced TLR activation is not clear, as the authors do no experiments to show that there are actually reduced levels of MyD88 protein or signaling in those mice. Next, Poly(I:C) and LPS are added to the UV-inactivated virus vaccine. Not only does immunization of mice with this preparation result in high affinity neutralizing antibody, but it protects those mice from ERD following re-exposure to RSV.

So if the alum included in the RSV vaccine is not a strong enough adjuvant to induce affinity maturation, why do any vaccines work, since alum is the only adjuvant currently used in humans? Some vaccines, like polio, are live attenuated virus, so they produce their own TLR ligands. The authors argue that the difference between successful and unsuccessful inactivated virus vaccines may be the affinity of the live virus for its cellular receptor. Influenza virus, for example, has low affinity for its receptor, so relatively low affinity antibody can be neutralizing, and indeed, influenza vaccnation doesn't induce affinity maturation. RSV, on the other hand requires a higher affinity antibody for neutralization. Whether or not virus-receptor affinity actually explains vaccine outcome, this paper serves as a nice proof-of-principle for the incorporation of our current knowledge of TLR biology into vaccine development, and addresses a 40-year-old open question in the process.

References:

Delgado MF, Coviello S, Monsalvo AC, Melendi GA, Hernandez JZ, Batalle JP, Diaz L, Trento A, Chang HY, Mitzner W, Ravetch J, Melero JA, Irusta PM, Polack FP. Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease. Nature Medicine 15, 34 - 41 (2008)

Kim, H. W., J. G. Canchola, C. D. Brandt, G. Pyles, R. M. Chanock, K. Jensen, R. H. Parrott. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. American Journal of Epidemiology 89, 422-434 (1969).