Protection in treated animals is powered by CD4+ memory T cells that trigger specific cytokine responses after challenge by TB pathogen.
Scientists have developed a prototype tuberculosis vaccine candidate based on an engineered strain of Mycobacterium smegmatis. A team at the Albert Einstein College of Medicine’s Howard Hughes Medical Institute (HHMI) removed the bacterium’s own esx-3 gene region, which is implicated in evasion of bacterial killing by innate immunity, and replaced it with the same region from Mycobacterium tuberculosis.
William R. Jacobs Jr., Ph.D., and colleagues found that the modified M. smegmatis strain remained susceptible to innate immune killing and was highly attenuated in mice, but also stimulated bactericidal immunity against subsequent challenge with virulent M. tuberculosis. They report their studies in Nature Medicine, in a paper titled “A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.”
Mycobacterium tuberculosis (Mtb) uses a number of mechanisms to evade innate and adaptive immune responses, the researchers note. One of the best studied is the mycobacterial type VII secretion system known as esx-1, which has been shown to be involved in a range of effects that promote mycobacterial virulence through secretion of the protein ESAT-6 and other effector proteins. In fact, they continue, the Mtb genome encodes four other potential secretion systems that are paralogs of esx-1. Designated esx-2 through esx-5, the functions of these systems are mostly unknown, but while all mycobacterial species studied to date have at least two of these esx loci, only the esx-3 genes are conserved in all cases, suggesting it does play a crucial function.
Because Mtb isn’t viable if its esx-3 locus is inactivated, the researchers used Mycobacterium smegmatis (Msmeg), which can grow without an intact esx-3 locus, to study this gene region’s influence on the innate and adaptive immune systems of the host. They first analyzed which cytokines were induced in vivo in mice after infection with Msmeg mutants that had deletions of the esx-1 (designated as Δesx-1 strain) or esx-3 loci. The latter mutant was designated IKE (immune killing evasion).
What they found was that whereas parental and Δesx-1 Msmeg organisms induced low or undetectable levels of the interleukin-12 (IL-12) subunit p40 and interferon-γ (IFN-γ), infection with IKE elicited robust secretion of these cytokines. Conversely, IL-6 was strongly induced by parental and Δesx-1 Msmeg but was barely detectable in IKE-infected mice. Interestingly, infecting animals with very high intravenous doses of parental or Δesx-1 Msmeg was invariably fatal (even though Msmeg is generally not considered pathogenic), while animals infected with the IKE mutant survived and cleared their infections without any apparent consequences. Of particular relevance was the finding that the IKE mutant wasn’t even lethal to engineered mice (Rag1−/−) that lack T and B cells, and this suggested that control of infection was effected by an innate immune response.
To investigate this notion further, the team infected Msmeg IKE into a panel of different mouse strains with deficiencies in various innate immune response genes. Of these, only mice without the adaptor protein MyD88 (Myd88−/−) were unable to clear the Msmeg IKE infection, and died.
Histological analyses showed that while animals infected with parenteral Msmeg showed pathology in a range of organs, those infected with IKE showed no notable pathology in the same tissues. With this in mind, the researchers infected the normal, Rag1−/−, and Myd88−/− animals with either parental, Δesx-1, or IKE Msmeg strains. While all the animals showed increasing tissue burdens after infection with the parenteral or Δesx-1 strains, the normal and Rag1−/− mice rapidly cleared IKE from the lungs and kidneys. Only the Myd88−/− animals demonstrated an expansion of IKE bacilli after inoculation. “These findings indicated that a previously unappreciated function of the genes in the esx-3 region is to mediate evasion of a MyD88-dependent innate immune response that normally triggers bactericidal effects and host resistance to Msmeg infection,” the authors write.
To try and investigate the function of Mtb’s own esx-3 locus, they introduced the esx-3 genes from the tuberculosis pathogen into IKE Msmeg using a cosmid, generating a hybrid strain designated IKEPLUS. When IKEPLUS was then used to infect either normal, Rag1−/−, or Myd88−/− mice, it was rapidly cleared from tissues of both immunocompetent and Rag1−/− animals, but was lethal in the MyD88-deficient mice. Measurements of serum cytokine concentrations showed that IKEPLUS induced robust IL-12 p40, p70, and IFN-γ responses similar to those observed with the IKE mutant.
IKE and IKEPLUS display properties, such as high attenuation for virulence in mice, and the induction of a favorable cytokine response, which are theoretically desirable for an antituberculosis vaccine, the researchers state. In the case of IKEPLUS, in particular, the recombinant Msmeg contains a cosmid encoding part of the Mtb genome that has the potential to serve as specific antigens for eliciting adaptive immune reponses that could be recalled by subsequent Mtb infection.
This possibility led the researchers to investigate the effects of Mtb challenge in mice that had been immunized some weeks previously using IKEPLUS. The results were striking. The average time to death after Mtb challenge was 54 days for sham-vaccinated mice and 65 days for BCG-immunized mice, whereas IKEPLUS-immunized mice had a mean survival time of 135 days. In fact, three separate experiments showed that IKEPLUS-immunized animals that survived longest after subsequent Mtb challenge displayed marked reductions in bacterial burden in multiple tissues.
“Notably, the bacterial burdens of all tissues examined in IKEPLUS-immunized mice showed a continuing decline over time in mice that survived >100 days,” the scientists note. And for IKEPLUS-immunized animals that survived for 200 days after Mtb challenge, the tissue burden was more than 1,000-fold lower than that in BCG-vaccinated mice that survived 100 days, and actually became undetectable in the liver. This finding indicates a high level of bactericidal and potentially sterilizing immunity, the authors state.
The unexpectedly potent immune response elicited by intravenously administered IKEPLUS led to a follow-on experiment in which animals were immunized subcutaneously (a more clinically relevant route for human vaccination). First, they immunized mice subcutaneously with BCG or IKEPLUS and eight weeks later challenged them with a high intravenous dose of Mtb. Compared with BCG-immunized mice, those given subcutaneous IKEPLUS had significantly more serum IL-12 p40 upon challenge with Mtb, and survived significantly longer.
The experiment was then repeated, but the mice were challenged a month after IKEPLUS immunization using a low-dose aerosol infection with Mtb to mimic a more physiological setting. In this case mice immunized subcutaneously with IKEPLUS still showed a trend toward longer survival after Mtb challenge compared with BCG-immunized mice, and also demonstrated lower bacterial burden in lung and spleen tissue. Importantly, the reduction in tissue burden in the IKEPLUS-immunized mice was maintained, whereas in BCG-immunized mice tissue burden gradually increased to the level observed in sham-vaccinated animals.
At the level of immune response, the demonstration that IKEPLUS immunization boosts IL-12 p40 and IFN-γ production suggested that enhanced TH1 cell responses were involved in the increased levels of protective immunity after Mtb challenge. Indeed, mice immunized with either IKE or IKEPLUS showed rapid induction of IL-12 p40 and IFN- γ expression within the first two days of high-dose intravenous challenge with Mtb. These data suggested that the protective responses elicited by IKEPLUS immunization were associated with the induction of TH1-type adaptive immunity, which was supported by the finding that Rag1−/− and IL-12 p40-deficient mice showed no significant change in their high susceptibility to Mtb challenge after immunization with IKEPLUS.
Studies in MHC class I- and MHC class II-deficient animals indicated that the protective immunity elicited by IKEPLUS immunization was dependent on MHC class II presentation, “and therefore probably involves responses by CD4+ T cells, the authors remark. To look into this further they tested whether purified T-cell populations from IKEPLUS-immunized mice transferred to naive mice could provide these animals with protection against subsequent Mtb challenge.
Significantly, lung CFU counts in CD4+ T cell-treated animals that were infected with Mtb confirmed that significant protection could be transferred from IKEPLUS-vaccinated donors: in fact, the reduction in CFU among recipients of CD4+ T cells from IKEPLUS-immunized mice was nearly the same as that in mice that had been directly immunized with either intravenous IKEPLUS or subcutaneous BCG. The CD4+ T cell-treated animals also demonstrated increased survival compared with control animals. This led the authors to conclude that the CD4+ T-cell population from IKEPLUS-immunized mice appears to represent a primary repository of memory T cells responsible for activating protective immunity to challenge with Mtb.
“We have demonstrated a major role for the esx-3 locus of Msmeg in modifying the mammalian host immune response, and in so doing we have generated a new and highly effective candidate vaccine for tuberculosis,” the researchers conclude. “To our knowledge, neither BCG nor any of the attenuated Mtb vaccine strains described to date induce such obvious or prolonged bactericidal immunity…Most notably, our observation of apparent sterility in the livers of IKEPLUS-immunized animals surviving past 200 days after Mtb challenge may represent the first documented example of the elimination of this organism from a tissue by immunological mechanisms alone.”
They admit that further improvements will be needed to optimize the efficacy of IKEPLUS vaccination for potential human use, as only 10–20% of IKEPLUS-immunized mice achieved long-term survival after Mtb challenge. Nevertheless, they stress, the high level of bactericidal immunity after even subcutaneous immunization is still encouraging. “Although the magnitude of this immunity was not as great as that observed after intravenous immunization, it was still substantially greater and longer lasting than the protection induced by standard BCG vaccination.”
The team is currently working to both to identify which genes in the Mtb esx-3 cosmid are required for inducing protective immunity, and modify the technology to increase potency of the IKEPLUS vaccine when administered by clinically acceptable routes.
The fact that CD4+ T cells from IKEPLUS-immunized mice transferred into naive mice was enough to provide substantial protection from Mtb strongly suggests that the mechanism of bacterial killing involves a unique CD4+T cell population, they add. “To the best of our knowledge, this is the first example in an Mtb infection model of adoptively transferred immunity from immunized mice to naive mice without previous irradiation for lymphocyte depletion of the recipient mice. This further suggests that the response stimulated by IKEPLUS is qualitatively different from that induced by BCG, or unusually potent.” Nevertheless, the researchers state, while CD4+ T cells seem to be the key memory cells involved in initiating the bactericidal response, it is likely that they do recruit additional effectors that may ultimately be partially or entirely responsible for actually killing the mycobacteria.”