Jeffrey A. Guderian

Identification of Human T Cell Antigens for the Development of Vaccines against Mycobacterium tuberculosis

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-γ recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-γ, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.

Applying TLR synergy in immunotherapy: implications in cutaneous leishmaniasis.

Therapy of intracellular pathogens can be complicated by drug toxicity, drug resistance, and the need for prolonged treatment regimens. One approach that has shown promise is immunotherapy. Leishmaniasis, a vector-borne disease ranked among the six most important tropical infectious diseases by the World Health Organization, has been treated clinically with crude or defined vaccine preparations or cytokines, such as IFN-γ and GM-CSF, in combination with chemotherapy. We have attempted to develop an improved and defined immunotherapeutic using a mouse model of cutaneous leishmaniasis. We hypothesized that immunotherapy may be improved by using TLR synergy to enhance the parasite-specific immune response. We formulated L110f, a well-established Leishmania poly-protein vaccine candidate, in conjunction with either monophosphoryl lipid A, a TLR4 agonist, or CpG, a TLR9 agonist, or a combination of these, and evaluated anti-Leishmania immune responses in absence or presence of active disease. Only mice treated with L110f plus monophosphoryl lipid A-CpG were able to induce a strong effective T cell response during disease and subsequently cured lesions and reduced parasite burden when compared with mice treated with L110f and either single adjuvant. Our data help to define a correlate of protection during active infection and indicate TLR synergy to be a potentially valuable tool in treating intracellular infections.

Design, Development and Evaluation of rK28-Based Point-of-Care Tests for Improving Rapid Diagnosis of Visceral Leishmaniasis

Background Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75–85%, specificity of 70–92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. Methodology/Principal Findings Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83–99.61%; K39: 84.30–98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53–98.95%; K39: 85.54–96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46–99.1 in Sudan and 98.1% [95% CI = 89.93–99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25–93.23%] in Sudan and 88.7% [95% CI = 76.97–95.73%] in Bangladesh). Conclusions/Significance Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.

Treatment of canine visceral leishmaniasis by the vaccine Leish-111f+MPL-SE

Immunotherapy of canine visceral leishmaniasis (CVL) may provide an alternative to both marginally effective chemotherapy and undesired euthanasia of infected dogs and could have a great impact not only on animal welfare, but also on control of human disease. Therefore, we examined the potential immunotherapeutic efficacy of the subunit vaccine Leish-111f+MPL-SE, which has undergone rigorous preclinical testing and been demonstrated safe in human clinical trials. Two separate trials were performed in Salvador, Brazil, to evaluate the vaccine for therapeutic efficacy against CVL caused by natural infection: an Open Trial and a Blinded Trial. In the Open Trial 59 dogs with clinically active CVL were sequentially allocated to four groups: group 1 received Leish-111f+MPL-SE; group 2 was treated with Glucantime; group 3 received a combination of the vaccine and Glucantime; and group 4 was given no treatment. At the 6-month assessment, the 13 non-treated dogs had either died or showed no clinical improvement. In contrast, most dogs in groups 1-3 showed initial improvement (100%, 80%, and 92%, respectively). Upon evaluation for a mean of 36 months after therapy, the following cure rates were observed: 75% for group 1 dogs (exact 95% confidence interval [CI] 43-95%), 64% for group 2 dogs (exact 95% CI 31-89%), and 50% for group 3 dogs (exact 95% CI 19-81%). Therapeutic efficacy of the Leish-111f+MPL-SE vaccine was reconfirmed in a subsequent Blinded Trial. The vaccine was effective for mild cases of CVL and was compromised in dogs with severe disease. Although further studies are required to understand mechanisms of action, the Leish-111f+MPL-SE vaccine is a promising tool to control VL in both dogs and humans.

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional TH1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal TH1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional TH1 immune response characterized by CD4+ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo TH1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

ML0405 and ML2331 Are Antigens of Mycobacterium leprae with Potential for Diagnosis of Leprosy

ABSTRACT Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.

Cloning, Characterization, and Serodiagnostic Evaluation of Leishmania infantum Tandem Repeat Proteins

ABSTRACT Visceral leishmaniasis (VL) is a form of leishmaniasis, which is caused by infection with the protozoan parasite Leishmania, and is often fatal unless it is treated. Rapid and accurate diagnosis of VL is important for effective treatment. Here we report the cloning of previously undescribed tandem repeat (TR) proteins of Leishmania infantum and an evaluation of VL patient antibody responses to the corresponding proteins. By screening an L. infantum expression library with sera from human VL patients or infected hamsters, we identified 43 genes encoding B-cell antigens. Surprisingly, 19 of the 43 genes (44%) were TR proteins, and that percentage was significantly higher than that for genes picked randomly from the database. We then expressed the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and the entire LinJ28.2310 protein. These recombinant proteins were all recognized by Sudanese VL patient sera in an enzyme-linked immunosorbent assay. Recombinant LinJ16.1750 (rLinJ16.1750) showed the best performance among these antigens in terms of both sensitivity and specificity. Serological evaluation revealed that 97% (34 of 35) of Sudanese VL patients had significantly elevated antibody levels to rLinJ16.1750. Furthermore, when eight of the patient sera which had low reactivities to rK39 were tested with the novel recombinant antigens, some of the sera showed stronger antibody responses to these antigens than to rK39. Our results suggest that TR regions from the novel L. infantum proteins identified in this study are immunodominant B-cell epitopes and may represent good candidates for serodiagnosis of VL.

In vitro evaluation of TLR4 agonist activity: formulation effects.

Effective in vitro evaluation of vaccine adjuvants would allow higher throughput screening compared to in vivo studies. However, vaccine adjuvants comprise a wide range of structures and formulations ranging from soluble TLR agonists to complex lipid-based formulations. The effects of formulation parameters on in vitro bioactivity assays and the correlations with in vivo adjuvant activity is not well understood. In the present work, we employ the Limulus amebocyte lysate assay and a human macrophage cellular cytokine production assay to demonstrate the differences in in vitro bioactivity of four distinct formulations of the synthetic TLR4 agonist GLA: an aqueous nanosuspension (GLA-AF), an oil-in-water emulsion (GLA-SE), a liposome (GLA-LS), and an alum-adsorbed formulation (GLA-Alum). Furthermore, we demonstrate the importance of the localization of GLA on in vitro potency. By comparing to previous published reports on the in vivo bioactivity of these GLA-containing formulations, we conclude that the most potent activators of the in vitro systems may not be the most potent in vivo adjuvant formulations. Furthermore, we discuss the formulation considerations which should be taken into account when interpreting data from in vitro adjuvant activity assays.

Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli –expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs.

Upregulated expression of B-cell antigen family tandem repeat proteins by Leishmania amastigotes.

ABSTRACT Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, and they are often targets of B-cell responses. Through systematic analyses of whole proteomes, we recently demonstrated that two trypanosomatid parasites, Leishmania infantum and Trypanosoma cruzi, are rich in antigenic proteins with large TR domains. However, the reason that these proteins are antigenic was unclear. Here, by performing molecular, immunological, and bioinformatic characterizations of Leishmania TR proteins, we found two possible factors affecting the antigenicity of these proteins; one factor is their fundamental composition as TR proteins, and the other is regulation of their expression by parasites. Enzyme-linked immunosorbent assays (ELISAs) using recombinant proteins revealed that the copy number of the repeat affects the affinity of binding between antigens and antibodies, as expected based on thermodynamic binding kinetics. Other than containing TR domains, the TR proteins do not share characteristics, such as sequence similarity or biased cellular location predicted by the presence of a signal sequence(s) and/or a transmembrane domain(s). However, the TR proteome contained a higher percentage of proteins upregulated in amastigotes than the whole proteome, and upregulated expression of a TR protein seemed to affect its antigenicity. These results indicate that Leishmania parasites actively utilize the TR protein family for parasitism in mammalian hosts.