Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Abstract

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by "dormant" M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ(+)/TNF(+)) and IFN-γ(+) CD4(+) T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

FIG 1

Amino acid sequences of Rv1733c synthetic long peptides (SLPs).

FIG 2

(A) Rv1733c p57-84/CpG immunization of HLA-DR3 tg mice. Splenocytes derived from mice immunized with SLP Rv1733c p57-84 in CpG were stimulated in vitro with equimolar amounts of Rv1733c p63-77 (15-mer), p61-80 (20-mer), and p57-84 (28-mer) for 6 h. The percentage of CD4⁺ IFN-γ-producing cells (indicated in each figure) was analyzed by intracellular cytokine staining. (B and C) Rv1733c p57-84/CpG immunization of HLA-DR3 tg mice. Splenocytes derived from mice immunized with Rv1733c p57-84/CpG (B) or with Rv1733c p63-77/CpG (C) were stimulated in vitro with Rv1733c p63-77 (15-mer), p61-80 (20-mer), and p57-84 (28-mer) (0.1-μg/ml or 1.0-μg/ml final concentration). After 6 days, IFN-γ production was analyzed by ELISA. ConA was used as a positive control for in vitro responsiveness, and recombinant protein HPV16 E6 and hsp65 p1-13 (59) were used as negative protein and peptide controls, respectively. (D) Frequency of polyfunctional CD4⁺ T cells. Percentages of IFN-γ-, IL-2-, and/or TNF-producing CD4⁺ T cells in splenocytes of HLA-DR3 mice immunized with Rv1733c p57-84/CpG, analyzed without (left panel) or with (right panel) TNF⁺ cells. Splenocytes were stimulated in vitro with stimuli indicated above each graph. After 6 days, cells were incubated with fresh antigen. After 4 h, brefeldin A was added for overnight (20 h) incubation, after which cells were permeabilized, fixed, stained, and analyzed for intracellular cytokine production. Each symbol represents one mouse. Only CD4⁺ populations of >5 × 10⁴ events were analyzed. No significant cytokine production was detected in naive mice or in CD8⁺ T cells of mice immunized with Rv1733c p57-84/CpG (data not shown). P values were calculated by the Mann-Whitney U test. (E) Frequency of HLA-DR3/p63-77 TM⁺ CD4⁺ T cells. For determination of Rv1733c p63-77-specific CD4⁺ T cells, splenocytes of HLA-DR3 mice immunized with CpG alone (−) or Rv1733c p57-84/CpG were stained for 1 h at RT with HLA-DR3/p63-77 TM⁺ and phycoerythrin-FITC-conjugated anti-CD4. Groups included four mice. All mice were separately analyzed. P values were calculated by the Mann-Whitney U test.

FIG 3

Intracellular IFN-γ production in immunized double transgenic mice. HLA-A2/DR3 double transgenic mice (n = 6) were immunized with the HLA-A2-restricted epitope Ag85B p143-152 (22) and the HLA-DR3-restricted epitope Rv1733c p57-84 (42) in CpG, and IFN-γ production by CD4⁺ T cells (upper panel) and CD8⁺ T cells (lower panel) in splenocytes was analyzed after 6 days of in vitro culture with medium (−) or peptides (5 μg/ml) as indicated on the x axis. Each symbol (^) indicates one mouse. P values were calculated by the Mann-Whitney U test.

FIG 4

In vivo killing induced by M. tuberculosis Ag-derived SLP. HLA-DR3 tg (A) or HLA-A2 (B) mice were left unimmunized (−) or immunized with Rv1733c p57-84 (Rv1733c SLP) (A) in CpG or Ag85B p131-162 (Ag85B SLP) (B) to determine its ability to induce specific CTLs using in vivo cytotoxicity assays. Splenocytes derived from unimmunized mice were split into two equal fractions. Each fraction was differentially labeled with CFSE (target = CFSE^high, 5 μM; control = CFSE^low, 0.02 μM). The target population was pulsed for 2 h with 5 μg/ml Rv1733c p63-77 (A) or 5 μg/ml Ag85B p143-152 (B), and the control population remained unpulsed. The two fractions were mixed in a 1:1 ratio and intravenously injected into the tails of M. tuberculosis antigen-immunized mice. After 2 days, spleens were removed and the ratio of CFSE^low to CFSE^high cells was determined by flow cytometry. Specific killing of pulsed CFSE^high target cells was calculated as follows: [1 − (CFSE^high/CFSE^low)] × 100%. Mice immunized with CpG only did not show any in vivo cytotoxic responses (data not shown). P values were calculated by the Mann-Whitney U test.

FIG 5

Quantification of serum antibodies. Following immunization of HLA-DR3 tg mice with CpG alone (upper panel) or with Rv1733c p57-84/CpG (lower panel), antibody titers (OD at 450 nm [OD₄₅₀]) against Rv1733c p57-84, Rv1733c, or HPV16 E6 were determined by ELISA and are shown with standard errors of the means. As a control, affinity for BSA (0.4% in PBS) alone (■) is shown. Serum dilutions are shown on the x axis. All test groups included six mice. All mice were separately analyzed.

FIG 6

Determination of CFU in M. tuberculosis-infected HLA-DR3 tg mice. CFU were determined for lungs derived from M. tuberculosis-infected HLA-DR3 tg (●) mice s.c. immunized with Rv1733c p57-84 (SLP). Protective efficacies are expressed as bacterial counts. Immunization methods are indicated on the x axis. “pre” indicates final immunization 6 weeks before M. tuberculosis infection, and mice were sacrificed 6 weeks after infection; “post” indicates immunization 2, 4, and 6 weeks after M. tuberculosis infection, and mice were sacrificed 2 weeks after final immunization. BCG/SLP indicates BCG immunization followed after 6 weeks by the first of three immunizations with Rv1733c p57-84 (SLP). Median values are indicated by horizontal bars. CFU are expressed as log₁₀ bacterial counts, each symbol indicating a single mouse. Data shown are representative for three separate experiments.