Tuberculosis TB vaccine to prevent reactivation

Abstract

The present invention discloses a vaccine or immunogenic composition that can be administered to latently infected individuals to prevent reactivation of latent tuberculosis infection caused by species of the tuberculosis complex microorganisms (Mycobacterium tuberculosis, M. bovis, M. africanum), The invention is based on a number of M. tuberculosis derived proteins and protein fragments which are constitutively expressed in different stages of the infection. The invention is directed to the use of these polypeptides, immunologically active fragments thereof and the genes encoding them for immunological compositions such as vaccines.

Claims

1. A method for inhibiting reactivation of latent tuberculosis in an animal or a human being, wherein the latent tuberculosis is caused by Mycobacterium tuberculosis, comprising: selecting a latently infected animal or human being having a latent tuberculosis infection to receive an immunogenic composition for inhibiting reactivation of latent tuberculosis; and administering an immunogenic composition to said animal or said human being, wherein: a) said immunogenic composition comprises an M. tuberculosis antigen polypeptide which is constitutively expressed during infection with M. tuberculosis or a nucleic acid encoding said M. tuberculosis antigen polypeptide, b) said immunogenic composition inhibits reactivation of tuberculosis in latently infected individuals, c) said immunogenic composition comprises an adjuvant comprising IC31 or DDA/TDB, with or without poly I:C, and d) wherein the M. tuberculosis antigen polypeptide, which is constitutively expressed, belongs to the ESX-1 secretion system and comprises: a polypeptide sequence set forth in SEQ ID NO:1.

2. The method according to claim 1, wherein the M. tuberculosis antigen polypeptide is lipidated.

3. The method according to claim 1, wherein said immunogenic composition is administered during late stage infection.

4. The method according to claim 1, wherein the M. tuberculosis antigen polypeptide consists of the polypeptide sequence set forth in SEQ ID NO:1 and is fused to an additional antigen polypeptide, which is expressed by bacteria within the mycobacteria family, so as to form a fusion polypeptide.

Description

FIGURE LEGENDS

(1) FIG. 1: The course of a M. tuberculosis infection runs essentially through 3 phases

(2) FIG. 2: Model for post-exposure vaccination to prevent reactivation

(3) FIG. 3: TB vaccination model.

(4) A schematic overview of the model used at the SSI for the testing of post-exposure vaccines. Mice are infected with virulent M. tb by the aerosol route. From weeks 6 to week 12 post infection mice are treated with antibiotics to establish a state of latent TB. The mice are vaccinated 2 to three times with 3 weeks interval initiated at week 10 post infection with the post-exposure vaccine candidates. The mice are allowed time to reactivate the disease and approximately 20 weeks later the lungs are assessed for bacterial numbers to assess the protective efficacy of the vaccine.

(5) FIG. 4: Post-exposure vaccine induced protection by ESAT6 but not Ag85.

(6) Mice were infected, treated and vaccinated according to the schematic overview in example 1. Mice were killed between week 30-40 post infection and at this time point lungs were assessed for bacterial load (FIG. A, C-E) or as displayed in FIG. 4B where the bacterial load was determined at several time points throughout infection for ESAT6. (A and B) Bacterial load of ESAT6 vaccinated compared to control animals. (C) Bacterial load of Ag85B vaccinated compared to control animals. (D) Bacterial of ESAT-6 pepmix vaccinated (pool of overlapping peptides covering the entire ESAT6 sequence) compared to both Ag85B vaccinated and control animals. (E) Protection against reactivation following postexposure vaccination with Ag85B-ESAT-6 (H1) vaccinated compared to non-vaccinated control mice. All data in FIG. 4A, C-E are displayed as dot plots representing each individual animal with the mean depicted whereas each time point in FIG. 4B is representative of 6 individual animals and displayed as meanstandard error of the mean (SEM) (B). All statistical analyses were performed using either an unpaired t-test (FIGS. A-C and E) or Tukey's multiple comparison test (FIG. D) where p<0.05 was considered significant.

(7) FIG. 5: ESAT-6 postexposure vaccination induce polyfunctional T cells.

(8) Cells from infected lungs from non-vaccinated or ESAT-6 vaccinated animals were stimulated in vitro with ESAT-6 prior to staining with anti-CD4, -CD8, -IFN-, -TNF- and -IL-2. (A and B) Cytokine profiles were determined by first dividing the CD4 T cells into IFN- positive (+) or IFN- negative () cells. Both the IFN-+ and IFN- cells were analyzed with respect to the production of TNF- and IL-2. The pie charts (A and B) are colour coded according to the cytokine production profile and summarizes the fractions of the CD4.sup.+ T cell response (out of the ESAT-6 specific CD4 T cells) that are positive for a given cytokine production profile. (C) Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of ESAT-6 specific CD4.sup.+ T cells in non vaccinated mice (grey bars) or ESAT-6 vaccinated mice (Black bars) expressing any combination of cytokines is given for each immunization group. D. Latently infected mice were vaccinated twice with ESAT-6, and 20 weeks after the last vaccination, lungs were assessed for bacterial number to determine protective efficacy. (**p<0.01, One way ANOVA Tukey's multiple comparisons test).

(9) FIG. 6: Pooled analysis of all post-exposure experiments

(10) For an individual experiment where either ESAT6, Rv3871, Ag85B, Rv3905, Rv3445, Rv0569 or Rv2031c (Figure A), Ag85B-ESAT6 (H1) or Ag85B-ESAT6-Rv2660 (H56) (Figure B) was used for post-exposure vaccination the median of the bacterial load of the adjuvant control group was compared to the bacterial load of each individual mouse in a vaccinated group vaccinated with either one of the antigens mentioned above. In figure A and B each dot corresponds to the level of protection i.e. Log 10 CFU conferred by the vaccination compared to the adjuvant control group and consists of several independent experiments. (A) Log 10 protection for the single antigens ESAT6, Rv3871, Ag85B, Rv3905, Rv3445, Rv0569 or Rv2031c (B) or for the hybrid antigens H1 and H56 compared to ESAT6 alone. A statistical analysis was applied for comparisons of medians between the different groups either using the Kruskall Wallis multiple comparison test. p<0.05 was considered significant.

(11) FIG. 7: Effect of post-exposure vaccination with Rv3871 compared to ESAT6 and control animals.

(12) Mice were infected, treated and vaccinated at week 10, 13 and 18 post infection. At week 36 post infection the mice were terminated and lung lymphocytes from both vaccinated and non-vaccinated saline control mice were restimulated in vitro with Rv3871 (FIG. 7A) or ESAT6 (FIG. 7B). IFN- releases assessed by ELISA and samples were performed in triplicated. Data are depicted as meanSEM. The protective efficacy conferred by the vaccines was determined by enumeration of bacteria in the lung cultured from full lung homogenate (n=16-18). FIG. 7C shows data displayed as a dot plot where each dot represents an individual animal and depicted with the median (line).

EXAMPLES

Example 1: Murine TB Model for Vaccination

(13) The Cornell model has widely been used as a murine model for the study of latent TB. This model has been adapted in our laboratory for the testing of the ability of vaccine candidates to prevent reactivation. Mice are initially aerosolly infected with virulent M. tb and at week 6 post infection antibiotic treatment is initiated to reduce the bacterial load. This is to mimic the latent stage of a human infection which does not occur spontaneously in mice. During this latent stage (a stage with continuous low bacterial numbers) the mice are being immunized twice and the ability to prevent reactivation by the vaccine is determined by culturing the spleen and lungs for live M. tb 20 weeks after the last immunization. The long timespan of the experiments is necessary to allow sufficient time for reactivation of the disease which is a prerequisite for readout of vaccine efficacy (FIG. 3).

Example 2: Postexposure Vaccine Induced Protection by ESAT6 but not Ag85

(14) ESAT-6 and Ag85B have proven to be protective in prophylactic vaccination both as single components and also as the fusion molecule Ag85B-ESAT6 (H1). However, when these antigens were tested in the postexposure model (as described above in example 1) only ESAT6 has a protective effect and control bacteria growth during the reactivation phase (FIG. 4). Furthermore, as seen in FIG. 4B ESAT6 protection against reactivation manifests itself as early as W18 post infection and this protection was maintained throughout the course of the experiment (up until week 40 post infection). This is in contrast to what is observed when Ag85B is used as a post exposure vaccine (FIGS. 4C and D), where there is no significant decrease in bacterial load compared to the control. In addition, we evaluated the H1 fusion protein which is composed of the TB antigens Ag85B and ESAT-6 which has shown promising efficacy in a prophylactic setting. When this molecule was used as a post exposure vaccine in the SSI postexposure model it was able to significantly reduce the bacterial numbers (FIG. 4E).

Example 3: Post Exposure Vaccine Induced Protection by ESAT6 Peptide Mix

(15) As shown in the examples above, the ESAT-6 molecule is very active when given postexposure resulting in a decrease in bacterial load compared to the control group and also compared to Ag85B. Furthermore we have shown that ESAT-6 given as a pool of overlapping peptides instead of a recombinant protein also lead to a better protection against reactivation compared to both the control group and Ag85B demonstrating the strong activity of ESAT6, and ability to function as a post exposure vaccine (FIG. 4D).

(16) Overlapping ESAT-6 Peptides (P1-P13) Used for Protection Experiment:

(17) TABLE-US-00002 P1 (SEQIDNO.19) MTEQQWNFAGIEAAA P2 (SEQIDNO.20) NFAGIEAAASAIQGN P3 (SEQIDNO.21) ASAIQGNVTSIHSLL P4 (SEQIDNO.22) NVTSIHSLLDEGKQS P5 (SEQIDNO.23) SLLDEGKQSLTKLAA P6 (SEQIDNO.24) KQSLTKLAAAWGGSG P7 (SEQIDNO.25) AAWGGSGSEAYQGVQ P8 (SEQIDNO.26) GSEAYQGVQQKWDAT P9 (SEQIDNO.27) QQKWDATATELNNAL P10 (SEQIDNO.28) TATELNNALQNLART P11 (SEQIDNO.29) ALQNLARTISEAGQA P12 (SEQIDNO.30) TISEAGQAMASTEGN P13 (SEQIDNO.31) QAMASTEGNVTGMFA

Example 5: Post Exposure Vaccination with ESAT-6 Induce Polyfunctional T Cells

(18) To examine the effect of a post exposure vaccination with ESAT-6 on the cytokine expression profile of the ESAT-6 specific cells, mice were first aerosolly infected with virulent M. tb and at week 6 post infection antibiotic treatment was initiated to reduce the bacterial load and establish a latent infection. During the latent stage the mice were vaccinated (as shown in FIG. 3) three times with 3 weeks interval and the ability of the ESAT-6 vaccine influence the number of polyfunctional T cells and to prevent reactivation of M. tb was determined 20 weeks after the last vaccination. The results showed that there was a substantial ESAT-6 response in the non-vaccinated group, but the cytokine expression profile was markedly different compared to the ESAT-6 vaccinated group (FIG. 5), in particularly in terms of polyfunctional T cells (IFN-+TNF-+IL-2+ CD4 T cells). Thus, compared to the non vaccinated group, we observed decreased numbers of IFN-/TNF- CD4 T cells, and increased numbers of triple positive polyfunctional CD4 T cells co-expressing IFN-/TNF-/IL-2. The increased presence of polyfunctional T cells correlated with decreased bacterial numbers in the lungs of ESAT-6 vaccinated animals (FIG. 5D).

Example 6: Post-Exposure Vaccination with ESAT6 More Consistently Protects Against Reactivation Compared to Other Antigens Associated with Both Early and Late Stage Infection

(19) To determine which antigens most consistently protect against reactivation we made a pooled analysis of normalized data based on all post-exposure experiments conducted. Data sets from individual experiments was normalized by comparing the bacterial load of each individual mice within a group to the median of the control group i.e. each data point represents the difference (Log 10 CFU control median-Log 10CFU vaccine group) between the control median CFU and the CFU of each individual animal (FIG. 6). In FIG. 6A comparison of the pooled data set for protection for the antigens latency associated antigens Rv0569, Rv2031c and the early antigens Ag85B, ESAT6, Rv3871, Rv3905 and Rv3445 of which the two latter are ESAT6 family proteins show that ESAT6 vaccinated animals are significantly better protected against reactivation compared to other antigens evaluated. Furthermore, protective levels attained following post-exposure vaccination with Rv3871, an ESX-1 protein also seem to be elevated compared to the other antigens (FIG. 6A). To further demonstrate the activity of ESAT6 in particular we compared the protection conferred by ESAT6 to the two fusion constructs H1 (Ag85B-ESAT6) and H56 (Ag85B-ESAT6-Rv2660) both of which contain ESAT6 (FIG. 6B). The analysis show that ESAT6 activity still result in protection against reactivation when included in the two above mentioned fusion constructs.

Example 7: Postexposure Vaccination with Another Member of the ESX-1 Family, Rv3871 Seems to have an Inhibitory Effect on the Reactivation Process

(20) We evaluated other members of the ESX-1 family in parallel with ESAT6 and found that Rv3871 postexposure vaccination led to an induction of Rv3871 specific immune response (FIG. 7B) although not to the extent of the ESAT6 induced immune response (FIG. 7A). Nevertheless both ESAT6 and Rv3871 induced immune response were greater compared to saline control animals. The induction of vaccine specific immune response was associated with a lowered (median) bacterial load in both vaccine groups compared to the saline group. This indicated that Rv3871 may have a similar effect in protection against reactivation compared to ESAT6 demonstrated by the similar levels of bacterial numbers in these two groups compared to the somewhat elevated level in the control group (FIG. 7C).

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