Multiple Consecutive Infections Might Explain the Lack of Protection by BCG

<div><p>Although contacts between tuberculosis patients may result in multiple consecutive infections (MCI), no experimental animal models consider this fact when used in basic studies. Moreover, the current TB vaccine (BCG) has demonstrated a limited protection in humans. In this study we evaluate the effect of tuberculosis MCI by way of a simple mathematical analysis using data from the low dose aerosol murine experimental model. The results show that a higher number of, or shorter intervals between, multiple consecutive infections reduce the protective effect of BCG. This is due to both the increase in bacillary load at the stationary level of the infection, and the protective immune response induced by the infection itself. This factor must therefore be taken into account when designing new prophylactic strategies as candidate vaccines for the replacement of BCG.</p></div

Influence of the interval between multiple consecutive infections (MCI) on the stationary level.

<p>Picture A shows the evolution of the total bacillary load in the lung (sum) considering 10 consecutive infections only. The data have been adjusted to a polynomic formula: (R² = 0.9988). In Picture B, which show differences between naïve and vaccinated mice, the formula is: (R² = 0,9774).</p

Example of the analysis considering five MCI with intervals of 24

<p>Evolution of the infection at each site is followed over time using a spreadsheet (all data can be found as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094736#pone.0094736.s001" target="_blank">Table S1</a>). We consider the formula where <i>No</i> is the initial dose (100 CFUs), and <i>t</i> is the time in days. We highlight in bold italics the total bacillary load in the lung (sum) at the time when the immune response induced the stationary level in the model of single infection (1,195,374 and 135,952 CFUs for naïve and vaccinated mice, respectively). At this time the stationary level is reached at each infection site where there is a minimal bacillary concentration (135,952 CFUs, in bold). This is why, in contrast to what happens in a single infection model, the first infection stops before reaching 1,195,374 CFUs (i.e. on day 18 at 501,022 CFUs). The stationary level is therefore reached at all sites except site 5, which has not reached the minimal bacillary concentration.</p><p>In the case of BCG-vaccinated mice, the stationary level at each site could be reached earlier as the sum on day 13 is 143,228. This exceeds the level of 135,952 bacilli, and thus the immune response is ready. However, there is insufficient bacillary load locally, which is why the stationary level is not immediately reached at each infection site. This illustrates how important it is for the local bacillary load to have the benefit of the immune response.</p

Evolution of the bacillary load in the model of single or multiple consecutive infections (MCI).

<p>Progression of the bacillary concentration in both naïve (A to C) and BCG-vaccinated mice (D to F) after single infection (A and B) or MCI with 10 (x10) or 40 (x40) infections. The red line represents the sum of the bacillary load at all individual infection sites.</p

Protection after a short-term vaccination in terms of bacillary load obtained in tissues.

<p>The figure shows the reduction of the bacillary load in the lungs and spleen of animals according to their experimental group. The experimental groups are shown as follows: black (unvaccinated animals, control group), white (BCG vaccinated) and striped (RUTI® vaccinated). Significant differences (p<0.05) obtained after the statistical analysis (One-Way Anova) between groups are marked with an asterisk.</p

Experimental design of the experiments.

<p>The figure shows in 4 panels the experimental design of the 4 experiments run. As indicated in the figure, red arrows mean RUTI® vaccination (dotted if boosting) and the blue arrow means BCG vaccination. The purple X represents endpoint (mice sacrifice), the yellow vertical arrow the aerosol challenge and the blue horizontal arrow following-up to evaluate survival.</p

Results of the long-term vaccination experiment.

<p>The experimental groups are shown as follows: black (unvaccinated animals, control group), white (BCG vaccinated), striped (RUTI® vaccinated), chequered (BCG boosted with RUTI®) and with diamonds (RUTI® boosted with RUTI®). Significant differences (p<0.05) between groups are marked with an asterisk.</p

Survival experiment in guinea pigs after short-term vaccination.

<p>Data show the evolution of the protection given by the vaccination. Only the BCG group demonstrated a significant difference when compared with the control under the Kaplan-Meier Survival Analysis.</p

Vaccination soon after infection.

<p>The figure shows the protection induced by vaccinating after challenge. The experimental groups are shown as follows: black (unvaccinated animals, control group), white (BCG day 4), horizontal striped (RUTI® day 4), diagonal striped (RUTI® day 11) and chequered (RUTI® day 4 and 11). Significant differences (p<0.05) respect to the control group are marked with *.</p

<i>M. tuberculosis</i> increases mAIM serum levels in an <i>in</i><i>vivo</i> infection model.

<p>C57BL/6 mice were infected with <i>M. tuberculosis</i> H37Rv through aerosol inoculation. Mice were treated with INH/RIF for 8 weeks (w6 to w14) at which point antibiotic was withdrawn, and infection was allowed to reactivate. mAIM serum levels and bacillary load in the lung and spleen were measured at several time points post-infection (24 h - 21 weeks). A) Representative image of mAIM levels analyzed by Western blot of serum samples. B) Graphs showing spleen and lung bacterial loads at the indicated times (upper graph) and mAIM protein intensity (lower graph) data. Box plots show median values and 5-95 percentile values, from 1µl serum (n=3 to n=5). Fold induction levels were calculated using as reference the serum mAIM from a pool of 5 C57BL/6 uninfected healthy animals, set as 1. *p≤0.05; **p≤0.01; ***p≤0.001 two-way ANOVA.</p