Research ArticleLeishmaniasis

Vaccination with Leishmania Hemoglobin Receptor–Encoding DNA Protects Against Visceral Leishmaniasis

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Science Translational Medicine  11 Sep 2013:
Vol. 5, Issue 202, pp. 202ra121
DOI: 10.1126/scitranslmed.3006406

Abstract

Leishmaniasis is a severe infectious disease. Drugs used for leishmaniasis are very toxic, and no vaccine is available. We found that the hemoglobin receptor (HbR) of Leishmania was conserved across various strains of Leishmania, and anti-HbR antibody could be detected in kala-azar patients’ sera. Our results showed that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamsters. Moreover, HbR-DNA immunization stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor–α (TNF-α) with concomitant down-regulation of disease-promoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induced a protective response by generating multifunctional CD4+ and CD8+ T cells. All HbR-DNA–vaccinated hamsters showed sterile protection and survived during an experimental period of 8 months. These findings demonstrate the potential of HbR as a vaccine candidate against visceral leishmaniasis.

INTRODUCTION

Leishmania, a protozoan pathogen, is the causative agent of various forms of leishmaniasis like cutaneous (CL), mucocutaneous, and visceral leishmaniasis (VL), of which VL is fatal (1). Leishmaniasis is a complex disease that affects about 12 million people worldwide; 500,000 new cases are reported annually (2, 3). Drugs used for chemotherapy of leishmaniasis, such as antimonials, miltefosine, paromomycin, and amphotericin B, are very toxic and expensive, and frequent resistance occurs against these drugs in endemic areas (4). Thus, the major thrust of research in this area is to develop an effective vaccine against leishmaniasis. However, no licensed vaccine is available against VL (5, 6).

VL is associated with an impaired T helper 1 (TH1) response characterized by down-regulation of interleukin-12 (IL-12) and interferon-γ (IFN-γ) along with up-regulation of TH2 cytokines such as IL-4 and IL-10 (7, 8). Thus, one goal of vaccine development is evoking a TH1 response against appropriate parasite antigens (9). Recently, considerable progress has been made and large numbers of Leishmania antigens have been tried as potential vaccine candidates with varied immune response and diverse species-specific protection (5, 6).

Leishmania require heme from exogenous sources for growth because of a lack of a complete heme biosynthetic pathway (10). Heme is a critical prosthetic group required by the parasites for several metabolic pathways; thus, the heme acquisition process in Leishmania could be a potential target (11). Previously, we have shown that Leishmania endocytose hemoglobin (Hb) through a high-affinity receptor (HbR) located on the cell surface (12) and that internalized Hb is targeted to the lysosomal compartment by a Rab5- and Rab7-dependent endocytic pathway (13, 14). This receptor system contributes to the survival of Leishmania by generating heme from intracellular degradation of Hb (14, 15). Subsequently, we have shown that HbR is a hexokinase (16) with an N-terminal (HbR-N) extracellular Hb-binding domain (HbR1–126) and a C-terminal (HbR-C) cytoplasmic domain (HbR270–471). Thus, HbR regulates two major functions in parasite: it acts as HbR on cell surface, and it also regulates glycolysis. Compartmentalization of hexokinase in the glycosome and its large phylogenetic distance from hosts make it a prospective target against trypanosomatids (17). Here, we demonstrate that immunization with bifunctional HbR-encoding DNA (HbR-DNA) induces sterile protection against experimental VL.

RESULTS

HbR is naturally immunogenic in VL patients, and anti-HbR inhibits parasite growth

The presence of antibodies against HbR in confirmed VL patients’ sera was detected by Western blot analysis with purified HbR protein. We found that 27 of 30 VL patient sera had antibody against HbR (Fig. 1A). Moreover, mice immunized with HbR-N–DNA or HbR-FL–DNA construct generated anti-HbR antibody (Fig. 1B), and splenocytes isolated from these mice in response to HbR protein showed enhanced production of IFN-γ, IL-12, and TNF-α (tumor necrosis factor–α) in comparison to controls (Fig. 1C). We also found that addition of antibody against HbR-N to the culture medium induced promastigote aggregation, whereas no effect was seen by the addition of antibody against HbR-C (Fig. 1D). Moreover, addition of guinea pig serum as a source of complement along with anti–HbR-N antibody in the culture medium lysed all parasites (Fig. 1D). Similarly, our results showed that 99.5% of promastigote growth was arrested by the addition of HbR1–126 in the medium in comparison to control or by the addition of HbR270–471 (Fig. 1E). These results indicate that blocking the extracellular domain of HbR by appropriate antibody or peptide is detrimental to parasite growth.

Fig. 1 HbR in Leishmania as a vaccine candidate.

(A) Presence of antibody against purified HbR in kala-azar patient sera was detected by Western blot analysis. Sera obtained from normal healthy individuals and anti-HbR antibodies were used as a control. Results are representative of triplicate measurements. (B) Determination of HbR-specific antibody response in the indicated groups of mice after the second immunization on day 27. Results are expressed as mean (n = 12) unit of absorption ± SD. (C) Detection of cytokines secreted by splenocytes isolated from different groups of mice on day 27 after the second immunization with HbR-DNA. Results are expressed as mean levels of cytokine ± SEM (n = 8). (D) Determination of the effect of the indicated anti-HbR antibody on promastigote aggregation. Upper panel shows phase images of HbR-GFP (green fluorescent protein)–expressing L. donovani promastigotes (lower panel). Preimmune serum was used as a control. Results are representative of triplicate measurements. (E) Promastigotes were incubated in the presence of equimolar amounts of the indicated proteins for 12 hours at 23°C, and cell growth was determined by [3H]thymidine incorporation. Results are expressed as means ± SD of triplicate measurements. CPM, counts per minute; GST, glutathione S-transferase. (F) PCR amplification of HbR from various Leishmania species. Amplification of Ld-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) by specific primers (forward: 5′-GAAGTACACGGTGGAGGCTG-3′; reverse: 5′-CGCTGATCACGACCTTCTTC-3′) was used as a control. Lane M, 1-kb DNA ladder (Promega); lane 1, L. amazonensis; lane 2, L. amazonensis var. 7; lane 3, L. amazonensis var. 5; lane 4, L. infantum; lane 5, L. infantum var. 2; lane 6, L. infantum var. 6; lane 7, L. donovani; lane 8, L. major; lane 9, L. major var. 1; lane 10, L. major var. 4; lane 11, L. major var. 4-2; lane 12, L. turanica; lane 13, L. turanica var. 16; lane 14, L. gerbilli; lane 15, L. tropica; lane 16, L. tropica var. 1; lane 17, L. tropica var. 7; lane 18, L. enrietti. Results of the indicated groups were analyzed by two-tailed paired t test, and levels of significance are indicated by P values.

HbR is conserved across various Leishmania species

To determine whether HbR is present in different species of Leishmania, we tried to amplify HbR from genomic DNA of various Leishmania species by polymerase chain reaction (PCR) using Ld-HbR–specific primers. Notably, we detected HbR from genomic DNA of different strains of L. amazonensis, L. infantum, L. donovani, L. major, L. turanica, L. gerbilli, L. tropica, and L. enrietti by PCR (Fig. 1F) with 97 to 100% sequence identity (fig. S1). Thus, HbR is well conserved in various Leishmania species.

HbR-DNA immunization protects both mice and hamsters against VL

To determine the efficacy of HbR as a DNA vaccine, we cloned full-length HbR (HbR-FL) and HbR-N coding sequence into a mammalian expression vector (pcDNA3.1) driven by the cytomegalovirus promoter and injected the respective construct (endotoxin-free) intramuscularly into BALB/c mice or hamsters. We evaluated HbR-N–DNA and HbR-FL–DNA as potential vaccine candidates in both experimental models because the BALB/c mice model of VL is useful in understanding immune dysfunction, and the golden hamster mimics the clinical features of VL (18). Accordingly, HbR-DNA–immunized animals were challenged with virulent strains of L. donovani (AG83), and parasite loads were determined 21 and 60 days after infection in spleens and livers from different groups of animals. Our results showed that in HbR-N–DNA– and HbR-FL–DNA–immunized mice, splenic parasite burden was reduced by 91 and 86% (Fig. 2A) and hepatic parasite burden was inhibited by 94.97 and 92.36% (Fig. 2C), respectively, 21 days after infection compared with infected and vector-immunized controls. However, more than 99% inhibition of parasite loads in the spleen (Fig. 2B) and liver (Fig. 2D) of HbR-N–DNA– and HbR-FL–DNA–immunized mice was observed 60 days after infection in comparison to infected controls. Of these eight mice, no live parasite was detected in the spleen (Fig. 2E) or liver (Fig. 2F) homogenates of six mice immunized with HbR-FL or HbR-N, whereas two vaccinated mice showed negligible parasite load in comparison to untreated control. Moreover, real-time PCR analysis revealed the presence of very low copies of L. donovani–specific genes in the spleen (Fig. 2E, inset) and liver (Fig. 2F, inset) of HbR-FL– or HbR-N–immunized mice in comparison to untreated control.

Fig. 2 Vaccine-mediated protection of virulent L. donovani infection in mice.

BALB/c mice were challenged with virulent L. donovani after immunization with the indicated DNA construct. Phosphate-buffered saline (PBS)–injected infected animals were used as a control. Animals were sacrificed either 21 days (n = 12) or 60 days (n = 8) after infection, and parasite load in the spleen and liver from the respective animals was determined. Results are expressed as Leishman-Donovan units (LDUs) ± SEM of the respective organ in mice. (A) Splenic parasite burden 21 days after infection. (B) Splenic parasite burden 60 days after infection. (C) Hepatic parasite burden 21 days after infection. (D) Hepatic parasite burden 60 days after infection. (E and F) Detection of live parasite load per milligram of spleen (E) and liver (F) homogenates in the indicated groups of mice (inset shows real-time PCR of the respective organ). Results of the indicated groups were analyzed by two-tailed paired t test, and levels of significance are indicated by P values.

We also observed that HbR-N–DNA– and HbR-FL–DNA–vaccinated hamsters had more than 96% inhibition of splenic parasite load (Fig. 3A) and hepatic burden reductions of 95.19 and 88.35%, respectively, 21 days after infection compared to untreated controls (Fig. 3C). However, more than 99% inhibition of parasite loads in the spleen (Fig. 3B) and liver (Fig. 3D) of HbR-N–DNA– and HbR-FL–DNA–immunized hamsters was observed 60 days after infection in comparison to control. Furthermore, no live parasite was detected from the spleen or liver (60 days after infection) homogenates of hamsters immunized with HbR-FL–DNA or HbR-N–DNA, indicating that HbR-DNA vaccination induced sterile protection (fig. S2A). The complete protection in vaccinated hamsters was supported by the fact that all HbR-N–DNA– and HbR-FL–DNA–vaccinated hamsters were healthy and survived against lethal challenge of Leishmania during an experimental period of 260 days (Fig. 3E). In contrast, all hamsters from control groups died within 200 days. The sizes of the liver and spleen of HbR-FL–DNA and HbR-N–DNA–immunized infected hamsters were found to be comparable with those of control animals, demonstrating the superior efficacy of HbR-DNA as vaccine against VL (fig. S2B).

Fig. 3 Vaccine-mediated protection of virulent L. donovani infection in hamsters.

Hamsters were challenged with virulent L. donovani after immunization with the indicated DNA construct. PBS-injected infected animals were used as a control. Animals were sacrificed either 21 days (n = 10) or 60 days (n = 6) after infection, and parasite load in the spleen and liver from the respective animals was determined. Results are expressed as LDU of the respective organ in hamsters along with median. Data were analyzed by the Mann-Whitney test, and levels of significance are indicated by P values. (A) Splenic parasite burden 21 days after infection. (B) Splenic parasite burden 60 days after infection. (C) Hepatic parasite burden 21 days after infection. (D) Hepatic parasite burden 60 days after infection. (E) Survival kinetics of vaccinated and parasite-challenged hamsters (n = 10) during the experimental period of 260 days after infection. Survival distribution was analyzed by log-rank (Mantel-Cox) test, and levels of significance are indicated by P values.

HbR-DNA vaccination stimulates T cell proliferation

To determine whether HbR vaccination can reverse the impaired T cell response observed in VL, we isolated splenocytes from AG83-challenged HbR-DNA–vaccinated animals 21 days after infection and performed T cell proliferation assay. Splenocytes isolated from both HbR-N–DNA– and HbR-FL–DNA–immunized mice showed enhanced T cell proliferation upon antigenic restimulation compared to infected controls (Fig. 4A). Moreover, induced T cell proliferation in vaccinated animals was complemented by about five- and sixfold more secretion of IL-2 by splenocytes isolated from HbR-FL–DNA– and HbR-N–DNA–immunized mice, respectively, compared to the respective untreated controls (Fig. 4B). Similarly, enhanced proliferation of T cells and higher levels of IL-2 transcripts by real-time PCR were detected in HbR-N–DNA– and HbR-FL–DNA–immunized infected hamsters compared to infected controls (fig. S3, A and B).

Fig. 4 HbR-DNA vaccine–stimulated T cell proliferation.

(A) Proliferation of splenocytes isolated 21 days after infection from the indicated groups of mice (n = 10) was measured in response to HbR (5 μg/ml). Results are expressed as mean unit of absorption ± SEM. (B) Functional significance of splenocyte proliferation in response to the indicated antigens was determined by measuring the secretion of IL-2. Results are expressed as mean levels of cytokine ± SEM. Results of the indicated groups were analyzed by two-tailed paired t test, and levels of significance are indicated by P values. CSA, complete soluble antigen of L. donovani.

HbR-DNA vaccination induces TH1-protective response

To understand the nature of immune response generated by HbR-DNA vaccination, we compared the levels of various cytokines secreted by splenocytes isolated from different groups of mice 21 days after infection by cytometric bead array (CBA). Splenocytes isolated from HbR-FL–DNA–immunized mice in response to HbR protein showed about 2.27-fold more IFN-γ, 2-fold more IL-12, and 2.37-fold more TNF-α, whereas cells from HbR-N–DNA–vaccinated mice produced about 1.88-, 2-, and 2.69-fold more IFN-γ, IL-12, and TNF-α, respectively, in comparison to controls (Fig. 5, A to C). In contrast, vaccination with HbR-FL–DNA and HbR-N–DNA inhibited the production of IL-10 by 2.15- and 5-fold (Fig. 5D) and reduced the production of IL-4 by 2.42- and 2.77-fold, respectively, compared to controls (Fig. 5E). Similarly, hamsters vaccinated with HbR-DNAs showed higher expressions of IFN-γ, IL-12, and TNF-α and down-regulation of IL-10 expression compared to controls by real-time PCR. No substantial changes were observed in IL-4 expression between different groups of hamsters (fig. S4, A to E).

Fig. 5 HbR vaccine–induced TH1-protective immune response in mice.

Levels of different cytokines secreted by splenocytes in the culture supernatant were also measured after 48 hours of incubation as described. (A) IL-12. (B) IFN-γ. (C) TNF-α. (D) IL-10. (E) IL-4. Results are expressed as mean levels of cytokines secreted by splenocytes ± SEM (n = 12). (F) Blood was collected from vaccinated and control groups of mice (n = 12) 21 days after infection, and serum was analyzed for the presence of HbR-specific immunoglobulin G1 (IgG1) and IgG2a antibody. Results are expressed as mean unit of absorption ± SEM. Results of the indicated groups were analyzed by two-tailed paired t test, and levels of significance are indicated by P values.

HbR-DNA vaccination induces the production of IgG2a in infected animals

We also observed three- to fourfold higher levels of anti-HbR IgG2a antibodies in the sera of HbR-FL–DNA– and HbR-N–DNA–vaccinated infected animals compared to infected controls. No major difference was observed in HbR-specific IgG1 levels (Fig. 5F). Similar results were also obtained in immunized hamsters (fig. S5).

HbR-DNA vaccination generates multifunctional CD4+ and CD8+ T cells

We analyzed antigen-specific IFN-γ+, TNF-α+, and IL-2+ CD4+ and CD8+ cells by multiparameter flow cytometry using Boolean gating as illustrated in Fig. 6. Our results showed that HbR-DNA–vaccinated mice generated a significantly higher population of antigen-specific CD4+ (Fig. 7A) and CD8+ (Fig. 7C) T cells with very high numbers of IFN-γ+, TNF-α+, or IL-2+ cytokine compared to infected controls. Subsequent Boolean gating analysis by FlowJo software showed that the frequency of triple-positive (IFN-γ+TNF-α+IL-2+) CD4+ (Fig. 7C) and CD8+ (Fig. 7D) cells was significantly higher in the HbR-DNA–immunized group than in infected untreated controls. However, a comparatively higher population of double-positive (IFN-γ+TNF-α+, IFN-γ+IL-2+, or TNF-α+IL-2+) CD4+ and CD8+ cells was found in both HbR-FL–DNA– and HbR-N–DNA–vaccinated mice compared to that of triple-positive cells in vaccinated mice.

Fig. 6 Identification of multifunctional T cell response.

Lymphocytes were gated on FSC-A versus SSC-A followed by CD3+ and subsequently on CD4+ and CD8+ events. Then, stringent gating for each cytokine-positive (IFN-γ, TNF-α, and IL-2) cells in the CD4+ and CD8+ compartments was done, and Boolean gating analysis was performed to create the full array of possible seven combinations in the CD4+ and CD8+ compartments using FlowJo software.

Fig. 7 HbR-DNA vaccine–induced multifunctional T cell response.

To determine antigen-specific cytokine-positive CD4+ and CD8+ responses, HbR-stimulated isolated splenocytes from different groups of mice (day 21 after infection) were stained for different cytokines and various surface markers. Cells were analyzed by multiparameter flow cytometry using Boolean gating strategy with FlowJo software to determine the frequency of multifunctional T cells. (A) Representative plot showing total frequency of IFN-γ–, IL-2–, or TNF-α–producing CD4+ T cells. (B) Frequency of CD4+ T cells expressing each of the seven possible combinations of IFN-γ, IL-2, and TNF-α. (C) Representative plot showing total frequency of IFN-γ–, IL-2–, or TNF-α–producing CD8+ T cells. (D) Frequency of CD8+ T cells expressing each of the seven possible combinations of IFN-γ, IL-2, and TNF-α. Results are expressed as means ± SEM of three independent experiments. Results of the indicated groups were analyzed by two-tailed paired t test, and levels of significance are indicated by P values.

DISCUSSION

The current standard approach to identify prospective antigens for vaccine development is either to inhibit essential transporters or to block some vital metabolic pathways in the parasite. We have found that not only Leishmania acquires heme from HbR-mediated Hb endocytosis (12, 13, 16), but HbR is also a crucial enzyme of glycolytic pathway in the parasite. Because HbR satisfies both criteria, the goal of the present study was to evaluate the potential of HbR as a vaccine candidate against VL.

Several leishmanial antigens, such as A2, LelF-2, KMP-11, CPB, H2A/H2B, ORFF, protein disulfide isomerase, and triose phosphate isomerase, have been tested in either DNA or protein form as vaccine candidates against VL (1926) and have been shown to induce variable protective response in experimental VL. Among different antigens tested, LACK has shown some promise as a vaccine candidate against CL (27) but failed to protect against L. donovani challenge (28), whereas KMP-11 was found to be effective against VL (21). However, both required exogenous adjuvant like IL-12 to achieve protection against CL (27, 29). Because a single antigen has not been found to be protective, a multiantigenic vaccine composed of KMP-11, SMT, A2, and cysteine protease formulated with monophosphoryl lipid A (MPLA) was tested and found to be protective against VL and CL (30). Because formulation of vaccine composed of mixture of antigens is difficult and expensive, attempts were made to develop fusion polypeptide as a vaccine candidate. Immunization with Leish-111f, a fusion protein composed of an L. major homolog of eukaryotic thiol-specific antioxidant and stress-inducible protein-1 (LmSTI1) along with L. braziliensis elongation and initiation factor (LeIF), has offered better protection against CL (31) but failed to protect against canine VL (32). Vaccination with Leish-111f along with GLA-SE (stable emulsion of monophosphoryl lipid A) as an adjuvant (LEISH-F3 + GLA-SE) was found to be safe and immunogenic (33); however, its protective efficacy against VL needs to be established. Thus, some progress has been made, but no vaccine against VL is available.

Because a DNA vaccine has several advantages against intracellular pathogens (34), we have compared the efficacy of two HbR-DNA constructs: one coding for full-length HbR (HbR-FL–DNA) and the other coding specifically for the N-terminal extracellular domain of the receptor (HbR-N–DNA). Our results showed that vaccination of mice and hamsters with both HbR-DNA constructs inhibits more than 99% splenic and hepatic parasite burden in comparison to infected and vector control animals. The marginal decrease in parasite load observed in vector-immunized hamsters could be due to the presence of unmethylated cytosine-phosphate-guanosine motifs in vector plasmid (34). Our results also demonstrate that vaccination with HbR-DNA conferred complete protection against VL in both BALB/c mice and hamsters. This is supported by the finding that all hamsters immunized with HbR-FL–DNA or HbR-N–DNA have survived and remained healthy against the lethal challenge of virulent L. donovani during the experimental period. The very low parasite-specific signal obtained in HbR-DNA–immunized spleen and liver by real-time PCR could be due to the presence of remnant DNA from the dead parasites in the respective organs. Complete protection against VL in both BALB/c mice and hamsters was not observed with any other antigen.

Impaired T cell response and inhibition of IL-2 production have been associated with VL (35). Our results demonstrated enhanced proliferation of splenocytes isolated from both HbR-N–DNA– and HbR-FL–DNA–immunized infected mice and hamsters in response to HbR restimulation in comparison to infected and vector-immunized animals. Moreover, depressed splenic T cell response could be due to impaired IL-2 production (36). Therefore, we have measured the levels of IL-2 secretion by splenocytes isolated from different groups of mice and hamsters. We found significantly higher production of IL-2 by splenocytes isolated from HbR-DNA–vaccinated animals than from control animals. These results demonstrate that full-length and N-terminal HbR-DNA vaccinations may overcome the impaired T cell proliferation observed in VL and generate an active T cell response for parasite clearance.

For any vaccine to be successful against VL, it should skew the immune response toward the TH1 type (9). Therefore, we have compared the levels of TH1 and TH2 cytokines secreted by splenocytes isolated from vaccinated and control groups of animals. Our results showed that immunization with HbR-FL–DNA and HbR-N–DNA induced significantly higher production of TH1-protective cytokines like IL-12, IFN-γ, and TNF-α in comparison to infected and vector-immunized controls. In contrast, vaccination with HbR-FL–DNA and HbR-N–DNA significantly inhibited the production of disease-promoting TH1-suppressive cytokine IL-10 and the TH2 signature cytokine IL-4. Thus, higher amount of IL-12 and higher IFN-γ/IL-10 ratio in vaccinated animals demonstrate the TH1-biased protective immune response generated by HbR-DNAs. We also found higher amount of IL-12 and higher IFN-γ/IL-10 ratio in HbR-N–DNA than in HbR-FL–DNA vaccination, indicating greater TH1-biased protective immune response generated by HbR-N–DNA.

We also found that immunization of HbR-FL–DNA and HbR-N–DNA generated three- to fourfold higher levels of IgG2a anti-HbR antibodies in experimental animals. These results are consistent with enhanced production of IFN-γ in vaccinated animals because IFN-γ directly regulates IgG2a class switching (37). Higher level of IgG2a might also contribute to pathogen clearance (38) in vaccinated animals.

Cellular immune response involving CD4+ and CD8+ T cells is important for controlling infection by intracellular pathogens (39). Recent studies have shown that vaccine-induced generation of multifunctional CD4+ and CD8+ T cells provides better protection against intracellular infections (40, 41). Similarly, we found that HbR-DNA vaccination generates a significantly higher population of antigen-specific multifunctional (IFN-γ+TNF-α+IL-2+) CD4+ and CD8+ T cells in comparison to infected untreated controls. However, the frequency of double-positive (IFN-γ+TNF-α+, IFN-γ+IL-2+, or TNF-α+IL-2+) and single-positive (IFN-γ+, TNF-α+, or IL-2+) CD4+ and CD8+ T cells is higher than that of triple-positive cells in HbR-DNA–vaccinated mice. Thus, HbR-DNA vaccination provides protection against L. donovani infection by generating multifunctional antigen-specific CD4+ and CD8+ T cell response. IFN-γ mediates pathogen killing by activating the microbicidal properties of macrophages by inducing nitric oxide production (42, 43), and the effect is enhanced by TNF-α (44); thus, enhanced production of IFN-γ+TNF-α+ CD4+ and CD8+ T cells by HbR-DNA vaccination must have provided complete protection. Similarly, higher frequency of antigen-specific IFN-γ+IL-2+ CD4+ T cells produced by HbR-DNA immunization might provide durable protection by developing memory response (45). IL-12 is a strong TH1-inducing adjuvant (46); thus, increased levels of IL-12 in HbR-DNA–vaccinated animals might also contribute to parasite clearance. Additionally, multifunctional CD8+ cells generated in HbR-DNA–vaccinated animals could also significantly contribute to parasite clearance because such cells were shown to be better effector for clearance of intracellular pathogens and vaccine development (47, 48). Therefore, it will be of interest to determine the host-protective role of anti-HbR CD4+ and CD8+ T cells in Leishmania infection.

HbR-DNA vaccination induces all arms of protective immune response to achieve complete clearance of the parasites in VL model systems. It remains unclear whether DNA-based vaccines are safe for human use because of the possible integration of foreign DNA. However, no clear evidence of integration has been found, and large numbers of DNA vaccines are under clinical trials. Moreover, the presence of neutralizing antibodies against pathogens in the patient serum is useful for identification of vaccine candidates (49, 50). Antibodies against Leishmania are detected in VL patients (51), and Leishmania infection usually protects the host against subsequent infection (52). On the basis of our findings that anti-HbR antibody present in VL patients’ sera and antibody raised against HbR-N (HbR1–126) specifically lysed the parasites, it will be worthwhile to determine whether anti-HbR antibody has any role in blocking the transmission of the parasite.

In conclusion, our results demonstrate that HbR-DNA offers major advantages over other vaccine candidates against VL because it is functionally important in the parasite life cycle, conserved across various Leishmania species, and naturally immunogenic in VL patients. We have also found that HbR not only evokes protective TH1 response without any adjuvant but also suppresses disease-promoting cytokines to confer complete protection. Moreover, anti-HbR antibody generated in vaccinated animals might kill extracellular parasites during the infection process by blocking Hb endocytosis (14, 53) or by complement-mediated lysis. Successful protection of VL with vaccination of truncated HbR (HbR-N) indicates the feasibility of developing subunit vaccines of HbR against leishmaniasis. Additionally, HbR is conserved across Leishmania species and hence could also be a potential candidate against different forms of leishmaniasis.

MATERIALS AND METHODS

Study design

HbR function is essential for the survival of Leishmania; therefore, we designed DNA construct containing HbR-FL or HbR-N coding sequence and determined the protective efficacy of these vaccine constructs against experimental VL in mice and hamsters. All animals were used with prior approval of the animal ethics committee of the Indian Institute of Chemical Biology (Kolkata, India). Sample size for mouse studies was either 12 or 8 per group, whereas it was 10 or 6 hamsters per group, depending on whether the 21-day or the 60-day model was used. Animal experiments were conducted following the guidelines of the Institutional Animal Ethics Committee. All animal studies were conducted with appropriate control and in a nonblinded manner.

Reagents and chemicals

RPMI 1640, M199, and fetal calf serum (FCS) (Sigma); penicillin-streptomycin solution (Invitrogen); EndoFree Plasmid Mega Kit (Qiagen); anti-mouse CD4, CD8, CD3, TNF-α, and IL-2 (BD Biosciences); anti-mouse IFN-γ (BioLegend); BD Mouse Th1/Th2 Cytokine Kit II and BD CBA Mouse Inflammation Kit (BD Biosciences); LIVE/DEAD Aqua staining dye (Invitrogen); and BrdU Cell Proliferation Kit (Millipore) were used in the present study.

Animals and parasite

Four- to 6-week-old BALB/c mice (The Jackson Laboratory) and golden hamsters (Mesocricetus auratus), reared in pathogen-free institute animal facilities, were used for experimental purposes. AG83 (MHOM/IN/83/AG83) strain of L. donovani was used for experimental purposes. Parasites were maintained in golden hamsters. Promastigotes obtained after transforming amastigotes from infected spleen were maintained in M199 (Sigma) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FCS at 22°C.

Detection of anti-HbR antibody in VL patients

HbR was purified from Leishmania as described previously (16), and confirmed kala-azar patient sera were used with approval of the human ethics committee from Rajendra Memorial Research Institute of Medical Sciences (Patna, India). Purified HbR protein (1 μg) was subjected to SDS–polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and incubated with patient sera (1:100). The membrane was washed and probed with anti-human IgG + IgM antibody conjugated with horseradish peroxidase (HRP).

Presence of HbR in different species of Leishmania

Genomic DNA isolated from 18 different species and substrains of Leishmania was received as a gift from K. P. Chang (Rosalind Franklin University of Medicine and Science, Chicago, IL). Full-length HbR was amplified from genomic DNA of all 18 species and substrains of Leishmania with forward (5′-GTGGATCCATGGCCACCCGCGTGAAC-3′) and reverse (5′-GTGAATTCCTACTTCTTGTTGACGGCCA-3′) primers designed against the start and stop codons of HbR by PCR as described previously (16). The amplified products were subcloned in pGEM-T Easy vector and sequenced with m13 universal primers.

Effect of anti-HbR antibody on promastigote

HbR1–126 (HbR-N) and HbR270–471 (HbR-C) were previously cloned and expressed as GST fusion proteins, and specific antibodies were raised against HbR-N and HbR-C (13, 16). To determine the effect of HbR fragment–specific antibodies, log-phase Leishmania promastigotes (1 × 107 cells) were washed; resuspended in M199 medium containing anti–HbR-N, anti–HbR-C, or preimmune sera (1:100); and incubated for 10 min at 23°C in six-well tissue culture plates. In addition, cells were also incubated in the presence of anti–HbR-N along with young guinea pig serum (1:100) as a source of complement for 10 min at 23°C. Subsequently, the promastigotes were visualized in an LSM-510 meta confocal microscope with an oil immersion objective.

Blocking of promastigote growth by HbR-N

The effects of different fragments of HbR on the growth of Leishmania promastigotes were determined by [3H]thymidine incorporation (14). Briefly, log-phase promastigotes were washed, resuspended in M199 medium (106 cells/200 μl) containing an equimolar amount of GST–HbR-N or GST–HbR-C peptides (942 pmol/ml) along with [3H]thymidine (0.6 μCi/50 μl per well), and incubated for 12 hours at 23°C. Cells were harvested on Printed-Filtermat with cell harvester, and radioactivity incorporated by the cells was determined.

Immunization, infection, and determination of parasite burden in organs

BALB/c mice and hamsters were divided into five groups: (i) uninfected control, (ii) infected control, (iii) immunized with blank vector (pcDNA3.1) and infected, (iv) immunized with HbR-FL–DNA and infected, and (v) immunized with HbR-N–DNA and infected. Mice and hamsters were immunized by intramuscular injection in the hind thigh with a 28-gauge needle with 100 μg of endotoxin-free plasmid DNA construct dissolved in sterile saline on days 1 and 15. On day 27, immunized mice and hamsters were challenged with intracardial injection of 1 × 107 virulent strain of freshly transformed L. donovani (AG83) promastigotes with a 28-gauge needle. Finally, animals were sacrificed either 21 or 60 days after infection, and liver and spleen from different groups were isolated to determine splenic and hepatic parasite burden. The number of parasites present in the respective organ was determined by microscopic evaluation of Giemsa-stained tissue imprints as described previously (54, 55). The parasite burden in the respective organs was expressed as LDU (19).

Determination of live parasite burden by limited dilution

Parasite burden in the respective organs was also determined by serial dilution as described previously (21). Briefly, a weighed portion of spleen or liver from different groups of experimental mice 60 days after infection was dissected out and mildly homogenized in complete M199 medium and resuspended at a final concentration of 1 mg/ml in the same medium. Serial dilutions (fourfold) of the tissue homogenates were incubated at 22°C for 2 to 3 weeks. The presence of viable and motile promastigotes was examined at a 3-day interval. The reciprocal of the highest dilution that was positive for parasites was considered to be the parasite concentration per milligram of tissue.

Detection of splenic and hepatic parasite burden by real-time PCR

The L. donovani DNA copies in the liver and spleen of 60-day post-infection groups of mice were quantitated with real-time PCR (56). Briefly, genomic DNA was isolated from the respective tissues with QIAamp DNA kit (Qiagen). Leishmania DNA was detected with forward (5′-CCTATTTTACACCAACCCCCAGT-3′) and reverse (5′-GGGTAGGGGCGTTCTGCGAAA-3′) primers of minicircle kDNA, and neurotrophin 3 gene (forward: 5′-ACTTCGCAAACCTATGTCCG-3′; reverse: 5′-CCAATTTTTCTCGACAAGGC-3′) was used as an internal control of mouse genomic DNA. The reaction was developed in a final volume of 25 μl with 12.5 μl of Platinum SYBR Green. All samples were tested in duplicate and run in an ABI 7500 Real-Time PCR Detection System with the following thermal conditions: 50°C for 2 min followed by 2 min at 95°C, then 40 cycles at 95°C for 15 s and 60°C for 30 s. The results were analyzed with the comparative Ct method (2−ΔΔCt). Thus, Leishmania DNA amplification in every sample was normalized to mouse gene amplification with the Ct method and expressed as fold difference.

T cell proliferation assay

Spleens were isolated from different experimental groups of mice and hamsters 21 days after infection, and single-cell suspensions of splenocytes were prepared after Ficoll density gradient centrifugation (21). Cells were resuspended in complete RPMI 1640 and plated in triplicate at a concentration of 1 × 105 cells per well in 96-well plates. Subsequently, cells were allowed to proliferate for 3 days at 37°C in a 5% CO2 incubator in the presence or absence of HbR protein (5 μg/ml). Cell proliferation was determined by BrdU (5-bromo-2′-deoxyuridine) incorporation as described previously (57). Complete soluble antigen from Leishmania was used as a control.

Analysis of cytokines secreted into medium by splenocytes isolated from mice

Single-cell suspension of splenocytes from different groups of mice 21 days after infection was prepared as described previously (21). Subsequently, 2.5 × 105 cells per well in complete RPMI 1640 were plated in 96-well plates and incubated for 48 hours at 37°C in a 5% CO2 incubator in the presence or absence of the indicated proteins (5 μg/ml). After 48 hours, culture supernatants were collected by centrifugation. The levels of different cytokines in the culture supernatant were measured by CBA (multiplexing) as described previously (57). Mouse Th1/Th2 Cytokine Kit II and Mouse Inflammation Kit (BD Biosciences) were used for CBA. All the data were analyzed with FCAP Array in a FACSAria II flow cytometer (BD).

Determination of anti-HbR IgG in HbR-immunized mice

Blood was collected from vaccinated and control groups of mice after the second immunization on day 27 before parasite challenge, and serum was analyzed for the presence of HbR-specific antibody as described previously (21). Briefly, wells were coated with HbR-FL protein (2 μg/ml), and sera (1:100 dilution) obtained from different groups of mice were incubated in HbR-coated wells for 2 hours at room temperature. Wells were washed and incubated with biotin-conjugated rat anti-mouse IgG for 1 hour at room temperature. Binding was detected with avidin-conjugated HRP as described previously.

Measurement of HbR-specific IgG1 and IgG2a

To determine HbR-specific IgG1 and IgG2a antibody, different dilutions of the serum prepared from vaccinated and control groups of mice 21 days after infection were incubated in HbR-coated wells as described previously (21). Subsequently, biotin-conjugated rat anti-mouse IgG1 or rat anti-mouse IgG2a was added and incubated for 1 hour at room temperature. Finally, binding was detected with avidin-conjugated HRP as described.

Multiparameter flow cytometry

To determine HbR-DNA–induced generation of antigen-specific CD4+ and CD8+ cells (40), splenocytes (2 × 106 cells per well) were restimulated in vitro for 24 hours at 37°C in the presence of the HbR protein (5 μg/ml). Brefeldin A (10 μg/ml) was added to the cells during the last 4 hours of incubation. To minimize nonspecific staining, cells were incubated with Fc Block (CD16/CD32) for 30 min at 4°C in PBS containing 2% FCS and 0.1% sodium azide. Subsequently, cells were first stained for the cell surface antigens [CD4-PerCP (peridinin chlorophyll protein), CD3–PE (phycoerythrin)–Cy7, and CD8–APC (allophycocyanin)–Cy7] and LIVE/DEAD Aqua (Invitrogen) to exclude dead cells. Cells were washed thoroughly, permeabilized with BD Cytofix/Cytoperm solution for 15 min at 4°C, and then stained for intracellular cytokines with IFN-γ–PB (Pacific Blue), IL-2–PE, and TNF-α–FITC (fluorescein isothiocyanate) according to the manufacturer’s instructions. Cells were gated on forward and side scatter profiles, and live cells were identified on the basis of exclusion of LIVE/DEAD Aqua dye (Invitrogen). Finally, at least 100,000 lymphocytes per sample were analyzed. Sample acquisition was performed with a BD FACSAria II flow cytometer with FACSDiva software. To determine the multifunctional T cell responses, CD4+ and CD8+ events (gated previously on CD3+ cells) were stringently gated for each cytokine-positive population and subsequently analyzed by Boolean gating with FlowJo software.

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 5.01 and SigmaPlot version 12. Student’s two-tailed paired t test or two-tailed Mann-Whitney test was used to determine differences between control and the respective treated groups with 95% confidence intervals. Log-rank (Mantel-Cox) test was used to determine the significant survival distribution between different groups of hamsters. P values less than 0.05 were considered to be significant for all analyses.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/5/202/202ra121/DC1

Fig. S1. Conservation of HbR in different Leishmania species.

Fig. S2. HbR vaccination in hamster induces complete protection.

Fig. S3. HbR vaccination in hamster induces T cell proliferation.

Fig. S4. HbR vaccination in hamster induces TH1 response.

Fig. S5. HbR vaccination in hamster induces higher level of anti-HbR–specific IgG2 antibody.

REFERENCES AND NOTES

  1. Acknowledgments: We thank A. Qadri (National Institute of Immunology) for critically reviewing the manuscript. Genomic DNA isolated from 18 different species and substrains of Leishmania was received as a gift from K. P. Chang (Rosalind Franklin University of Medicine and Science, Chicago, IL). Funding: Supported by core grants from the National Institute of Immunology by Department of Biotechnology; Council of Scientific and Industrial Research (CSIR) and J. C. Bose Fellowship from the Department of Science and Technology, Government of India (to A.M.); and fellowships from the CSIR, Government of India (to R.G., D.G., R.R., R.V., and G.K.). Author contributions: R.G., D.G., R.R., R.V., and G.K. performed all experiments and S.B. has provided VL patient sera. S.R. supervised and analyzed the results of all animal experiments. A.M. conceived the project, designed experiments, interpreted all results, and wrote the manuscript. Competing interests: A.M., S.R., D.G., R.G., and R.R. have filed a patent application (1449/DEL/2013) on “Hemoglobin receptor as novel vaccine for leishmaniasis.” The remaining authors declare that they have no competing interests.
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