Immunization with select antigens reduces M. leprae infection

Replication and dissemination of M. leprae is limited in PB leprosy patients and most HHC, suggesting that antigens that they recognize are potentially targets of an effective immune response against M. leprae. We previously identified several antigens that fit this criterion.23,24 To investigate if immunization with the antigens ML2028, ML2055 and ML2380 could limit M. leprae infection, mice were immunized with single antigens, or combinations of antigens, formulated with GLA-SE, a Toll-like receptor (TLR) 4 ligand in stable emulsion (SE). Following infection with M. leprae significantly fewer bacteria were recovered from the footpads of immunized mice relative to the numbers recovered from unimmunized control mice (Fig. 1a; p-values <0.05). These data indicate that immunization with the selected antigens elicits responses that protect mice against M. leprae challenge and supports their inclusion within a defined sub-unit vaccine against leprosy.

Fig. 1 Induction of protective anti-M. leprae responses by recombinant antigens formulated in GLA-SE. In a mice were injected subcutaneously with antigens/GLA-SE at biweekly intervals, for a total of three immunizations. One month after the last immunization mice were infected with 1 × 104 M. leprae in each foot, and bacterial burdens determined 12 months later. Results are shown as mean and s.e.m. Mann–Whitney test was used to calculate p-values between each group; n = 7 per group. In b, mice were injected, or not, with BCG vaccine then 1 month later were subsequently injected subcutaneously with LepVax (at biweekly intervals if immunized more than once). Serum and spleens were collected 1 month after the final immunization to determine antigen-specific immune responses Top panel, LEPF1-specific serum IgG1 and IgG2c titers were determined by ELISA. Middle and bottom panels, single-cell suspensions were prepared from each spleen and incubated with 10 μg/ml indicated protein (BCG = BCG lysate; MLCS = M. leprae cell sonicate), then culture supernatants collected 72 h later and IFNγ content determined by ELISA. Responses were corrected by the subtraction of the IFNγ concentration observed in the wells of unimmunized mice. Results are shown as mean and s.e.m; n = 5 per group. Data are representative of two independent experiments. In c, mice were immunized with a mixture of GLA-SE and either recombinant antigens represented in LepVax or with LepVax (either two or three times at biweekly intervals), or with heat-killed M. leprae (HKML) alone (no exogenous adjuvant added). One month after the last immunization mice were infected with 1 × 104 M. leprae in each foot, and bacterial burdens determined 12 months later. Results are shown as mean and s.e.m. Mann–Whitney test was used to calculate p-values between each group; n = 10 per group. *p-value < 0.05 and **p-value < 0.01 versus unimmunized control Full size image

While also increasing the proportion of the population that is likely to respond, combining multiple antigens into a single fusion protein is now commonly used to provide a more consistent production process. We therefore created a single tetravalent 89kD fusion protein, designated LEP-F1, consisting of the ML2028, ML2055 and ML2380 antigens, with the addition of ML2531 to stabilize expression. When mice were immunized with LEP-F1 in conjunction with GLA-SE (LepVax) they raised antibodies against each component indicating that the antigenicity of each component was retained in the fusion (data not shown). Given that the BCG vaccine is routinely used in leprosy-affected regions, we also examined if prior BCG immunization led to any interactions upon LepVax immunization. Mice were either primed, or not, with BCG then immunized with LepVax. Subsequent analyses of the IFNγ recall response to LEP-F1 indicated antigen-specific responses following either immunization scheme (Fig. 1b). Furthermore, mice immunized with LepVax also responded to lysate of BCG and, most importantly, to crude M. leprae antigens (Fig. 1b and data not shown). These data indicate that not only that immunization with Lep Vax raises responses that recognize M. leprae, but that these responses are not adversely affected by prior BCG immunization.

Immunization with LepVax reduces M. leprae burdens

We hypothesized that immunization with LepVax would limit bacterial growth, and therefore evaluated its ability to protect against experimental M. leprae infection. Mice were immunized then infected with M. leprae in the footpad. When assessed 12 months later the bacterial burdens of mice immunized with LepVax were approximately 85% lower than those observed in mice that were injected with the GLA-SE adjuvant formulation alone (Supplementary Figure 1; p-value < 0.05). Immunization with LepVax elicited protection equivalent to the mixture of its individual components, and provided protection when injected two or three times (Fig. 1c; p-values <0.05 versus unimmunized). Statistical analyses between the 2× and 3× LepVax-immunized groups, and the 2 × LepVax and HKML-immunized groups, indicates that they are not significantly different (p-values = 0.07 and 0.14, respectively), but the 3 × LepVax and HKML-immunized groups are (p-value < 0.01). The reason for this vagrancy is unclear. Irrespective of this, our experimental data indicate that the defined subunit LepVax vaccine induces immune responses that significantly limit M. leprae infection.

Immunization with LepVax delays motor nerve function impairment

Many clinicians fear that generation of an inflammatory immune response by vaccination will precipitate leprosy, or leprosy reactions, in infected individuals. Given that the hallmark of leprosy is nerve damage, we evaluated the impact of immunization in nine-banded armadillos. To mimic asymptomatic M. leprae infection, a situation that is likely common in leprosy hyper-endemic regions, armadillos were first infected, then immunized and monitored (Fig. 2). Infected, but untreated armadillos, began to show nerve conduction deficits as early as 4 months after inoculation, and all of the control armadillos had exhibited at least some measurable deficit by 12 months (Fig. 3a). We noticed that in the early stages of disease development conduction deficits could fluctuate and be transient. To account for these fluctuations, we also assessed onset of sustained conduction deficit as defined by exhibiting abnormal readings for three consecutive months. The variable nature of M. leprae infection in these outbred animals became apparent using this parameter, with sustained nerve conduction deficits occurring over a wide range of 6–22 months after infection and 2 of 12 (17%) infected armadillos not actually demonstrating persistent alterations (Fig. 3a). In comparison to previously established compound motor action potential (CMAP) amplitude data in uninfected armadillos (0.9 mV ± 2 SD),13 a lower CMAP was observed among M. leprae-infected animals and this continued to decline over time. Interestingly, BCG immunization of already infected animals led to precipitation of nerve damage. While onset of conduction deficits in BCG vaccinated armadillos occurred at the same time as control untreated animals (Fig. 3a), sustained conduction deficits were more rapidly observed in BCG-vaccinated armadillos than control untreated animals (Fig. 3b). The extent of the dissemination was significant enough that 27% (3 of 11) of the BCG immunized armadillos had to be removed from the study. In stark contrast, LepVax immunization delayed the onset of motor nerve conduction abnormality and reduced the proportion of animals that developed sustained damage (Fig. 3a, b). The CMAP showed a moderate, but significant, improvement in LepVax immunized animals 24 months after infection (Fig. 3c; p-value < 0.05). The improvement generated by LepVax immunization was not be attributable to an adjuvant impact because armadillos treated with the tuberculosis vaccine ID93 + GLA-SE did not exhibit any benefits (Fig. 3a, b). Taken together, these data reveal that LepVax immunization of infected armadillos delayed and alleviated the M. leprae-induced motor nerve damage. Thus, when provides in a post-exposure setting, unlike BCG, LepVax delays and alleviates M. leprae-induced motor nerve damage.

Fig. 2 Experimental set-up to examine post-exposure immunoprophylaxis in M. leprae-infected armadillos. Armadillos underwent intravenous inoculation with M. leprae, then 1 month later were: left untreated (unimmunized); immunized with BCG (BCG) one time; immunized with ID93 + GLA-SE, for a total of three times at monthly intervals or; immunized with LepVax (LEP-F1 + GLA-SE), for a total of three times at monthly intervals. The animals were monitored for compound muscle action potential (CMAP) and motor nerve conduction velocity (NCV) at monthly intervals, underwent three mm skin punches 16 months, and had their posterior tibial nerves (TN) biopsied at 28 months, after M. leprae inoculation. The photograph is the authors own Full size image

Fig. 3 Immunization with LepVax delays M. leprae-induced motor nerve damage. Armadillos were monitored for compound muscle action potential (cMAP) and motor nerve conduction velocity (NCV) at monthly intervals following infection and immunization. In a, the first month at which each animal first showed an abnormal nerve conduction was recorded and in b the time at which three consecutive abnormal readings were obtained for each particular animal is noted. The cumulative percent for each group is shown, and data shown are from one of two similar experiments. In c, CMAP measurements from 24 months after infection are shown. Each point depicts the data from a single armadillo, while the box and vertical bars show the mean and s.e.m, respectively. *p-value < 0.05 versus unimmunized, infected control animals Full size image

Enumeration and physical characterization of tibial nerves

Having demonstrated that LepVax immunization preserved motor nerve function, we then determined its impact on sensory nerves. Electron microscopic examination of peripheral nerves revealed intact and fragmented M. leprae within myelinated axons, Remak Schwann cells and in axoplasm from infected unimmunized and LepVax immunized animals (Fig. 4a). In comparison to the axons from uninfected animals (Fig. 4a, panel i) the axons from infected armadillos exhibited intra axonal edema with loosening of axonal contents (Fig. 4a, panels ii and iii). Although an occasional degenerating Remak bundle and macrophages were identified in infected armadillos, no obvious lymphocytic cell infiltration was observed (Fig. 4a, panels iv, v and vi). Similar to humans, in armadillos the Remak bundles of tibial nerve predominantly carried single axons irrespective of group (Fig. 4b, uninfected: 44%, infected: 43%, immunized: 53%).20 Degenerating Remak Schwann cells were only rarely observed in either uninfected or infected, LepVax-immunized armadillos (<2%). This contrasted with the increase to 5% presence of empty denervated Remak in infected, unimmunized animals. In the uninfected animals, the axon was more compact (481.2 ± 7.4 nm) than those observed in M. leprae infected armadillos (Fig. 4c, p-value < 0.0001). M. leprae infection induced significant dilation of axons, although LepVax immunization significantly reduced this relative to unimmunized armadillos (infected immunized: 686.6 ± 10.17 nm versus infected unimmunized: 718.9 + 9.7 nm; p-value < 0.0001). Residency of M. leprae within the cytoplasm of Schwann cells was also implied by the slight, but proportionate, increase in the ratio of axons to Schwann cells in infected armadillos (unimmunized: 1:2.1 versus immunized: 1:2.3) relative to that observed in uninfected armadillos (1: 1.95). Remak Schwann cell were significantly larger in infected animals in comparison to the Schwann cells observed in uninfected armadillos (Fig. 4d, p-value < 0.0001) and, in contrast to axonal changes, the Schwann cell size in infected animals was not altered by LepVax immunization (Fig. 4d). No remarkable presence of degenerated Schwann cell processes, empty basal lamina tubes or fibroblastic proliferations were observed. Taken together, these data indicate that immunization with LepVax did not precipitate, but rather alleviated, M. leprae infection-induced damage to the tibial nerve.

Fig. 4 Physical characterization and morphometry of tibial nerves in armadillos. Armadillos were infected with M. leprae then had their posterior tibial nerves biopsied 28 months later. In a electron micrographs of a tibial nerve were taken. In (i), both Remak bundles (arrows) and myelinated axon are observed in nerves collected from an uninfected animal, with a Remak axon containing a cluster of mitochondria indicated (Ax). In (ii), a myelinated axon from a M. leprae-infected armadillo (broken arrow) with a M. leprae bacillus (white arrow) bounded by vacuolated axoplasm is shown. In (iii), a microphotograph of a M. leprae-infected tibial nerve section containing Remak bundles (solid black arrows) and a myelinated axon (broken black arrow) can be seen, along with M. leprae in axoplasm and Schwann cell cytoplasm (white arrows) exhibiting edema and degeneration (arrow head). In (iv) a Remak Schwann cell (black arrow) with M. leprae (white arrow) undergoing degeneration is identified. In (v), nerves from a LepVax-immunized armadillo showed an occasional intraneural macrophage (arrow head) adjacent to Remak axons (arrow), and in (vi) a Remak bundle (arrow) without any degenerating changes. Scale bar, 1 µm. in b, axons within Remak bundles in tibial nerves were enumerated. In c, axon diameters were measured. Results are shown as mean and s.e.m, with a minimum of 810 measurements made for each group. In d, Schwann cell diameters were measured. Results are shown as mean and s.e.m, with a minimum of 550 measurements made for each group. ***p-value < 0.0001 Full size image

Characterization and enumeration of cutaneous nerves of distal leg

The cutaneous innervation in M. leprae-infected armadillos followed a pattern comparable to that observed in leprosy patients, with the axons shredding the Schwann cell covering before entering the epidermis (Fig. 5a). In uninfected animals the distal skin exhibited dense epidermal nerve fibers and, regardless of if they had been immunized or not, the epidermal nerve fiber density was not significantly altered in M. leprae-infected animals (Fig. 5b). Remak Schwann cells of the cutaneous nerves in the distal leg were, however, denser in the infected armadillos in comparison to uninfected armadillos, suggesting that proliferation of the Schwann cells was occurring in response to M. leprae infection at cooler distal sites (Fig. 5c,d; p-value = 0.02). Immunization with LepVax slightly reduced the Remak Schwann cells density in the skin relative to unimmunized, infected armadillos, indicating that the vaccine certainly did not exacerbate sensory nerve involvement but rather provided some protective benefit to the cutaneous nerves.