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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: J Infect Dis. 2010 Apr 1;201(7):1105–1112. doi: 10.1086/651147

Sm-p80 based DNA vaccine protects baboons against Schistosoma mansoni infection to comparable levels achieved by the irradiated cercarial vaccine**

Weidong Zhang 1,#, Gul Ahmad1 #, Workineh Torben 1, Zahid Noor 1, Loc Le 1, Raymond T Damian 2, Roman F Wolf 3, Gary L White 3, Maria Chavez-Suarez 3, Ronald B Podesta 4, Ronald C Kennedy 1,5, Afzal A Siddiqui 1,5
PMCID: PMC2922992  NIHMSID: NIHMS169017  PMID: 20187746

Abstract

No vaccine is available to prevent human schistosomiasis to date. We have targeted a protein of Schistosoma mansoni that plays an important role in the surface membrane renewal process, a mechanism widely believed to be utilized by the parasite as an immune evasion strategy. Sm-p80 antigen is a promising vaccine target because of its documented immunogenicity, protective efficacy and antifecundity effects observed in both experimental murine and nonhuman primate models of this infectious disease. In this study we report that a Sm-p80-based DNA vaccine formulation, in a human use approved vector (VR1020), confers 46% reduction in worm burden in the baboon (Papio anubis) model. Baboons vaccinated with Sm-p80-VR1020 showed 28% decrease in egg production following challenge with the infectious parasite. Sm-p80-VR1020 vaccine elicited robust antigen specific immune responses that included IgG; its subtypes IgG1 and IgG2; IgA and IgM in vaccinated animals. Peripheral blood mononuclear cells (PMBCs) and splenocytes from baboons vaccinated with Sm-p80-VR1020 when stimulated in vitro with recombinant Sm-p80 produced considerably higher levels of Th1 response enhancing cytokines (IL-2, IFN-γ) than Th2 response enhancing cytokines (IL-4, IL-10). PBMCs produced significantly higher number of spot forming units (SFU) for INF-γ than for IL-4 in enzyme-linked immunosorbent spot (ELISPOT) assays. A mixed Th1/Th2 type of humoral and T cell responses were generated following immunization with Sm-p80-VR1020. These findings again highlight the potential of Sm-p80 as a promising vaccine candidate for schistosomiasis.

Schistosomiasis in endemic in 76 different countries and carries an estimated yearly mortality rate of 280,000 [1]. Estimates also indicate that 207 million people are infected and an additional 779 million people are at risk of acquiring this neglected tropical disease [2, 3]. Praziquantel-based morbidity control for schistosomiasis has been useful but there are distinct disadvantages associated with this strategy. These include little impact on the reduction of disease transmission and the inherent danger of development of large scale drug resistance [4–6]. There is now general agreement that durable and sustained reduction in the disease spectrum and transmission can only be obtained through long-term protection via vaccination linked with chemotherapy [5, 7]. An effective anti-schistosome vaccine would contribute greatly to the decrease in morbidity associated with schistosomiasis via protective immune responses leading to reduced worm burdens and decreased egg production [5–10]. To this effect, high protective and antifecundity efficacy of Sm-p80 in both murine [11–13] and nonhuman primate [14, 15] models clearly indicate that this antigen has a great potential as an important vaccine candidate for the reduction of morbidity associated with schistosome infection. Additionally, Sm-p80 was originally identified to be involved in the schistosome immune evasion process of surface membrane biogenesis [16–19], thus Sm-p80 is an important functional protein and represents a unique target to invoke protective immunity against schistosome infection. Using a Sm-p80-based DNA vaccine formulation in VR1020, a vector approved for use in humans, in this pre-clinical vaccine efficacy determination study we report high levels of reduction in worm burden and egg production in baboons that are comparable to levels previously recorded only with the irradiated cercarial vaccine [20].

MATERIALS AND METHODS

Parasites and animals

Schistosoma mansoni-infected snails (Biomphalaria glabrata) were acquired from the Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD. Baboons (Papio anubis), 5.3 to 13.6 years old were obtained from the University of Oklahoma Health Sciences Center baboon breeding colony and housed in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities. Prior to the start of experimentation, baboons were tested for intestinal and blood parasites and for antibodies that were cross-reactive to Sm-p80 and were found negative for both. This study was approved by the Institutional Animal Care and Use Committee.

DNA-vaccine constructs and verification of protein expression in mammalian cells

Full length coding sequence of the large subunit of S. mansoni calpain (Sm-p80) [12, 13, 15, 21] was subcloned into BamHI/BglII sites of VR-1020 (Vical Incorporated, San Diego, CA). The resultant construct was designated as Sm-p80-VR1020. Expression of Sm-p80-VR1020 was determined by transient tranfection in COS-7 [14] and CHO K1 cells [12, 13, 15, 21]. The expressed products in COS-7 and CHO K1 cells were analyzed via polyacrylamide gel electrophoresis and western blotting, as described previously [12, 13]. For DNA vaccination, plasmid DNA was isolated via conventional alkaline lysis method. The plasmid DNA was further purified on Sepharose CL4B columns. The purified DNA was then ethanol precipitated and resuspended in sterile, endotoxin-free saline.

Baboon vaccinations, parasite challenge, worm and egg burden determinations

Six baboons in the experimental group were initially immunized with 500 μg Sm-p80-VR1020 (prepared in PBS). In our previous studies with the baboon model this dosage was found to be optimal in eliciting the protective immunity [14]. At weeks 4, 8, and 12, animals were boosted with 500 μg Sm-p80-VR1020. For the control group, six baboons were immunized with 500 μg of the control plasmid DNA, VR1020 (prepared in PBS) [14, 15]. Baboons in the control group were boosted with 500 μg VR1020 at weeks 4, 8, and 12. In both groups, all plasmid DNA was injected intramuscularly in the quadriceps. At week 16, baboons from both of the groups were challenged with a total of 1000 cercariae of S. mansoni as described earlier [14]. Eight weeks after the challenge, the baboons were euthanized and adult parasites were recovered; and reduction in worm burden was calculated as described previously [14]. Liver and intestine samples were used to determine the number of eggs present in baboons from both groups [14].

Collection of blood and peripheral blood mononuclear cell (PBMC) isolation

Samples of blood from baboons were collected just before the first immunization, at every booster (i.e., 4, 8 and 12 weeks) and 4 weeks after the final immunization, i.e., before challenge (16 weeks). Sera collected from these samplings were used in ELISA assays [14]. Using HISTOPAQUE®-1077 (Sigma-Aldrich, St. Louis, MO), highly enriched populations of PBMCs were isolated from the baboon blood.

Antibody assays

Sera samples from each individual animal were used to determine antibody levels/titers for IgG, IgG subtypes (IgG1, IgG2, IgG3, IgG4), IgM, IgA and IgE antibodies as described previously [14, 15, 22].

PBMC and splenocyte proliferation assays

Splenocytes were isolated from the macerated spleens of individual baboons obtained after the animals were euthanized. PBMCs from the two groups of baboons were isolated, as described above. For the in vitro proliferation assays, the concentration of recombinant protein and incubation period was first optimized. A standard assay was then developed as follows: in a 96-well flat-bottom plate, 5×105 PBMCs or splenocytes in 200μl per well, were stimulated with either 0.5μg ConA or 1.2μg recombinant Sm-p80 or 1.2μg ovalbumin and incubated at 37 °C with 5% CO2. After 48 hours incubation, an aliquot of the supernatant was removed for the estimation of cytokine production and the remainder was used for the MTT assay, as described elsewhere [14].

Estimation of cytokine production by proliferating PBMCs and splenocytes

Using baboon Th1/Th2 ELISA Panel Kit (U-cyTech, The Netherlands), the cytokine production (IL-2, IL-4, IL-10 and IFN-γ) by the proliferating PBMCs and splenocytes were estimated as described previously [14].

ELISPOT

ELISPOT assay was used to estimate IL-4 and IFN-γ secreting cells following in vitro stimulation with recombinant Sm-p80. Briefly, PBMCs from individual baboons were seeded (3×105cells/100μl/well) on the 96 well pre-coated plates (anti-IFN-γ or anti-IL-4, U-cyTech, The Netherlands). The cells were stimulated with either 0.5 μg ConA or 1.2 μg recombinant Sm-p80 or 1.2 μg ovalbumin and incubated at 37°C with 5% CO2 for 48 hours. SFU representing single cells were counted using an ELISPOT Bioreader 5000 (ImmunoBioSystem, The Colony, TX, USA). Antigen-specific SFU per well was calculated by subtracting its individual background value (buffer control well without antigen), by methods described in detail elsewhere [14].

Statistical analyses

Significance between two groups was calculated via independent sample t-test, using the SPSS computer program. Bonferroni adjustments were included for multiple comparisons, to reduce the risk of reaching false conclusions based on chance. P values obtained by these methods were considered significant if they were <0.05.

RESULTS

Protein expression of Sm-p80-VR1020 in COS-7 and CHO K1 cells

Before starting the vaccination with Sm-p80-VR1020, the construct was first characterized and tested for protein expression in COS-7 and CHO K1 cells. Expression of Sm-p80 was observed by western blotting using an anti-Sm-p80 antibody [14]. A distinct 80 kDa band was detected in lysates of COS-7 (Fig 1; Lane A) and CHO K1 cells (Fig 1; Lane B) transiently transfected with Sm-p80-VR1020. No such band was detected in COS-7 or CHO K1 cells transfected with VR1020 alone (data not shown).

Figure 1.

Figure 1

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Protein expression of Sm-p80 in COS-7 and in CHO K1 cells. Western blot of Sm-p80 obtained following transient transfection of the DNA construct, Sm-p80-VR1020, in COS-7 cells (lane A) and CHO K1 cells (lane B). This Sm-p80-VR1020 construct was used in the vaccination experiments.

Reduction in worm burden and in egg production achieved in baboons following vaccination with Sm-p80 in naked DNA immunization protocol

Protective and antifecundity effect of the Sm-p80 DNA construct was examined via four intramuscular injections. Baboons immunized with Sm-p80-VR1020 showed 46.35% reduction in worm burden when compared with baboons that received only the control plasmid, VR1020 (Fig 2A; Table 1). Additionally, in the control group of baboons, the breakup of worm types was found to be as follows: single males (38.31%), single females (17.74%) and paired worms (43.93%). Whereas in the experimental group, the worm count was as follows: single males (43.56%), single females (19.47%) and paired worms (19.47%). No immature worms were recovered in any of the two groups. Distinct antifecundity effect of this vaccine regimen was also observed in this study (Fig. 2B). Baboons vaccinated with Sm-p80-VR1020 showed a reduction in egg production (liver and intestine) by 27.97% (Table 1). These differences in reduction of worm burden (p <0.005) and egg counts (p <0.05) between Sm-p80-VR1020 and control VR1020 groups were found to be statistically significant. Additionally, Sm-p80-VR1020 DNA vaccine was tolerated optimally; baboons from both control and experimental groups did not display any negative behavioral or clinical manifestations. Furthermore, the baboons in both groups did not produce any anti-DNA antibodies (data not shown).

Figure 2.

Figure 2

Figure 2

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Adult worm burden distribution (A) and egg load per gram of liver and intestine of individual baboons (B) for groups of animals immunized with control plasmids, VR1020 (n =6) and with Sm-p80-VR1020 (n =6). Both reduction in worm burden (*P< 0.005) and in egg counts (*P< 0.05) were statistically lower in vaccinated animals.

Table 1.

Anti-worm and antifecundity effects in baboons following immunization with Sm-p80–VR1020

Immunized Group Worm (Mean ± SD)
*P <0.005		 No of eggs/g Liver (mean ± S.D)
**P <0.329		 No of eggs/g Intestine (mean ± S.D)
*P<0.02		 % reduction in worm burden
*P<0.005		 % reduction in egg production (Liver + Intestine)
*P<0.05
VR1020 248.83 ± 76.20 1005.58± 384.69 1265.92± 817.90 - -
Sm-p80-VR1020 133.50±12.03 1299.22± 585.74 336.96 ± 86.48 46.35% 27.97%
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*

Statistically significant;

**

Statistically insignificant

Antibody response to Sm-p80 in immunized baboons

High levels of Sm-p80-specific antibody titers were obtained for the total IgG (Fig 3A) and its subtypes IgG1 (Fig 3BA) and IgG2 (Fig 3CB) in the sera samples obtained from baboons immunized with Sm-p80-VR1020, when compared to control immunized animals. Moderate levels of IgA (Fig 3D) and IgM (Fig 3E) were also observed in the sera of the vaccinated group of animals. IgG3 and IgG4 reactivities were not detected in any of the vaccinated animals. Also, no detectable levels of Sm-p80-specific antibodies (total IgG, IgG subtypes, IgA, IgM) were detected in the group of animals immunized with control plasmid DNA (VR1020). Titers for the total IgG antibodies in the Sm-p80-VR1020 group started to rise at week 4 and reached the highest level at week 12 for all of the six baboons (end point titer = 51,200 for 2 baboons and 25,600 for 4 baboons) (Fig 3A). In the Sm-p80-VR1020 group almost equal levels of antibodies were observed for IgG subtypes, IgG1 and IgG2 (Fig 3B and 3C). The titer for IgG1 in the Sm-p80-VR1020 vaccinated group showed an increase at week 4, reaching peak levels at week 12 in all of the six animals (end point titer = 6400); at week 16, the titers dropped to some degree (Fig 3B). For IgG2 antibody titers gradually started to rise at week 8 and reached a peak at week 16 (end point titer = 6400 in 1 animal and 3200 in 4 animals) (Fig 3C). Titers for IgA antibodies started to rise at week 8 and reached their maximal levels at week 16 (end point titer = 6400 in 4 animals and 3200 in 2 animals) (Fig 3D). Similarly, in the Sm-p80-VR1020 vaccinated group, IgM titers had risen by week 8, peaked at week 12 (end point titer = 3200) and then decreased by week 16 (Fig 3E). No IgE, IgG3 or IgG4 antibodies were detectable in vaccinated animals (data not shown).

Figure 3.

Figure 3

Figure 3

Figure 3

Figure 3

Figure 3

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Titers of anti-Sm-p80 antibodies in immunized baboons. ELISA was performed with sera from each baboon (every four weeks) in their respective groups (VR1020 and Sm-p80-VR1020). Total IgG (A), IgG1 (B), IgG2 (C), IgA (D) and IgM (E) in individual control (JE37, WE62, TR16, JO12 and MA12) and vaccinated (MO34, RO34, LO27, CH40, BA33, and PR57) baboon sera collected every 4 weeks. The values represent the mean of three experiments ± standard error.

T cell proliferative responses and cytokine production in vaccinated baboons

As ascertained by stimulation indices obtained via MTT assays, the PBMCs and splenocytes from the Sm-p80-VR1020 group proliferated 56.4% and 37.8% higher, respectively, compared to the VR1020 controls following stimulation in vitro with the Sm-p80 recombinant protein. However, Sm-p80 driven proliferation of PBMCs and splenocytes was markedly lower when compared to the stimulation induced by ConA. As shown in Table 2, the high degree of proliferation by splenocytes was also correlated by the INF-γ and IL-2 production. Splenocytes from Sm-p80-VR1020 group produced over 8-fold higher levels of IL-2 and 9-fold higher levels of INF-γ compared to the VR1020 immunized controls. IL-4 and IL-10 production by splenocytes was negligible in both groups (Table 2). Similarly high levels of proliferation by PBMCs from the Sm-p80-VR1020 group was also correlated by the INF-γ and IL-2 production; IL-2 was produced at over 82-fold higher level and INF-γ was produced at over 13-fold higher level compared to the VR1020 immunized controls (Table 2). These results were confirmed by ELISPOT analysis of the proliferating PBMCs in response to in vitro stimulation by recombinant Sm-p80. SFU values for individual baboons are shown in Table 3 (IL-4) and Table 4 (INF-γ). In these studies, an average of 22-fold more SFU were detected for INF-γ, compared to IL-4 in baboons vaccinated with Sm-p80-VR1020.

Table 2.

Cytokine production by splenocytes and PBMCs induced by recombinant Sm-p80 after 48 hours of culturing in vitro

Vaccine group IL-4 (pg/mL)* IL-10 (pg/mL)* IL-2 (pg/mL)* IFN-γ (pg/mL)*
Splenocytes
VR1020 52.42±2.42 53.99±5.35 55.33±14.30 72.89±42.93
Sm-p80-VR1020 54.18±7.14 49.03±9.64 447.01±22.96 692. 09±32.73
PBMCs
VR1020 7.91±0.21 0.82±0.61 7.03±0.22 28.02±0.67
Sm-p80-VR1020 9.65±1.56 0.71±0.23 579.77±51.25 384.21±3.64
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*

The values in the table represent mean ± S.D.

Table 3.

ELISPOT detection of IL-4 producing spot forming units from individual baboons induced by recombinant Sm-p80 after 48hrs of culturing in vitro

Baboon Vaccine group IL-4
No stimuli ConA Ovalbumin Sm-p80
JE37 VR1020 3.50±1.50 200.00±33.00 0.50±0.50 0.50±0.50
WE62 VR1020 5.00±0.00 334.00±49.00 1.00 ±0.00 8.50±6.50
TR16 VR1020 6.00±0.00 144.00±0.00 9.50±3.50 9.50±1.50
JO12 VR1020 6.50±1.50 249.00±10.00 4.50±0.50 7.50±1.50
MA95 VR1020 3.00±1.00 66.00±14.00 9.00±1.00 5.00±0.00
MG12 VR1020 8.00±0.00 37.00±7.00 11.00±8.00 6.00±1.00
MO34 Sm-p80-VR1020 9.00±0.00 209.00±25.00 5.00±2.50 11.00±3.00
RO34 Sm-p80-VR1020 4.50±0.50 217.00±02.00 11.50±0.50 7.50±5.50
LO27 Sm-p80-VR1020 7.00±0.00 210.50±45.50 7.50±3.50 3.50±0.50
CH40 Sm-p80-VR1020 6.00±3.00 178.50±11.50 5.50±1.50 6.00±0.00
BA33 Sm-p80-VR1020 4.00±1.00 20.00±02.00 8.00±7.00 4.00±2.00
PR57 Sm-p80-VR1020 6.50±0.50 93.50±02.50 2.00±0.00 5.00±1.00
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The values in the table represent mean ± S.E.

Table 4.

ELISPOT detection of IFN-γ producing spot forming units induced by recombinant Sm-p80 after 48hrs of culturing in vitro

Baboon Vaccine group IFN-γ
No stimuli ConA Ovalbumin Sm-p80
JE37 VR1020 33.25±12.05 183.75±21.80 51.00±8.00 62.80±26.05
WE62 VR1020 7.00±1.78 185.50±80.50 8.00±1.00 18.80±7.77
TR16 VR1020 14.50±8.50 201.00±24.33 8.50±5.50 10.00±2.08
JO12 VR1020 10.50±3.50 210.75±27.00 41.00±0.00 33.50±15.15
MA95 VR1020 13.00±5.60 278.75±16.33 7.50±1.50 35.40±14.44
MG12 VR1020 7.67±2.40 243.00±10.69 5.00±0.00 5.25±0.75
MO34 Sm-p80-VR1020 122.00±34.59 301.00±47.46 221.00±35.00 236.50±35.50
RO34 Sm-p80-VR1020 14.75±6.24 235.75±13.67 49.00±2.00 56.00±6.00
LO27 Sm-p80-VR1020 172.00±0.00 265.25±22.61 148.50±52.50 239.50±4.50
CH40 Sm-p80-VR1020 34.33±15.92 238.50± 0.41 87.50±15.50 114.50±21.50
BA33 Sm-p80-VR1020 10.75±1.49 255.33±50.79 11.00±0.00 22.20±7.72
PR57 Sm-p80-VR1020 35.50±11.50 269.75±43.16 28.50±7.50 125.00±17.00
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The values in the table represent mean ± S.E.

DISCUSSION

Previous data from our laboratory [11–16, 21] and that of others on S. mansoni [23, 24] and on S. japonicum [25, 26] clearly demonstrate that calpain (Sm-p80) is potentially valuable vaccine candidate for the reduction of morbidity associated with schistosomiasis. In our continual efforts to improve the protective and antifecundity efficacy of Sm-p80, in this preclinical study, we have utilized a vaccination regimen that included a vector which is approved for human use (VR1020) and an animal model (baboon) that currently represents the most relevant nonhuman primate model of human clinical manifestations of the disease, schistosomiasis [10]. The baboon can serve as a useful bridge between mouse and human studies [27] and may represent a better predictor of the human situation [10]. Additionally, the DNA vaccine approach has shown promise in a wide array of prophylactic vaccine strategies, for example, against bacteria and viruses [28]; and perhaps to a higher level against several parasitic diseases [29, 30]. Thus in this study a logical and sagacious step was taken to test the most consistent Sm-p80-based DNA vaccine formulation in a baboon model before embarking onto human clinical trials.

Baboons immunized with Sm-p80-VR1020 showed a 46% reduction in worm burden and a 28% reduction in egg production. These results showed higher degree of improvement in protective efficacy compared to vaccination with another Sm-p80-pcDNA3 that exhibited a 38 % reduction in worm burden in baboons [14]. Conversely, the use of Sm-p80-VR1020 vaccine formulation did not change, in a statistically significant fashion, the antifecundity effects obtained previously via the immunization of baboons with Sm-p80-pcDNA3 in which 32 % reduction in egg production was recorded [14]. To test schistosome vaccine formulations, the baboon model has successfully been utilized in the past by many investigators [14, 20, 31–35]. Some examples include immunization of baboons with Sm28GST that resulted in 38% decrease in the number of worms recovered and a 33% reduction in female fecundity [31]. Similarly, immunization of baboons with IrV5 elicited a protection by 28% and visible reduction in the sizes of granulomas [35]. In baboons, radiation-attenuated (RA) cercariae vaccine has been found to be effective to varying degrees, however, multiple exposures are required to elicit high levels of protection [32, 36, 37]. For example, in one study, 50% protection was achieved after three vaccinations with RA vaccine [20] and in another study, 80% protection was achieved after four vaccinations with shorter intervals between the final boost and experimental challenge [35]. Additionally, three exposures with RA vaccine in vervet monkeys resulted in 48% protection, increasing the number to five exposures elicited only 39% protection [20]. Even though the RA vaccine approach has served as a gold standard in elucidating the mechanisms of protection, the inconsistent results with this strategy indicate its limited applicability and its inherent impracticality for use in human populations. Compared to the studies that utilized defined vaccines, Sm-p80-VR1020 vaccine appears to be far superior in its anti-worm and anti-fecundity effects and the regimen used in the present study has attained protection levels that previously can only be achieved by the irradiated cercarial vaccine.

Robust immune responses were generated via the immunization of Sm-p80-VR1020 vaccine formulation that included IgG, IgG1 and IgG2 antibody isotypes, IgA and IgM in vaccinated baboons. Generally, in this study, total IgG and its subtypes (IgG1 and IgG2), responses increased with each subsequent vaccine booster. IgG3, IgG4 and IgE responses were not detectable. Similarly in vaccination studies using Plague antigen LcrV in baboons [38], and in our previous studies with Sm-p80-pcDNA3 [14], IgG3 and IgG4 were found to be under the detection limits. However it is possible that the non-detection of IgG3 and IgG4 (and IgE) could be due to the poor cross-reactivity of human reagents being used for the detection of baboon antibody subtypes since no host-specific secondary antibodies from nonhuman primate origin are available for these subtypes [14]. However, IgM responses to immunization were recorded even though they appeared to be short-lived. Similar tendency for IgM levels was also observed with the RA vaccine in baboons [39]. Interestingly, in this study modest levels of antibody responses were observed for total IgA that was not the case in our previous studies with Sm-p80-pcDNA3 vaccine [14]. Generation of Sm-p80-specific IgA antibodies in this study is interesting since significant association between IgA responses to soluble worm antigen and resistance to reinfection in humans has been reported. [40]. Similar to previous studies [14], in present experiments, IgE was also not detectable in vaccinated animals. As recorded in this study, early emergence with short lives of IgM responses and very low or undetectable levels of IgE were also observed in the “self-curing” S. mansoni-rhesus macaque model [41]. Cumulatively our data on Sm-p80 specific humoral responses indicate that a mixed Th1/Th2 type of priming was achieved following vaccination with Sm-p80-VR1020 vaccine.

Proliferation of PBMC and splenocytes in response to in vitro stimulation with recombinant Sm-p80 resulted in the production of higher levels of Th1 response enhancing cytokines (IL-2, IFN-γ) than the Th2 response enhancing cytokines (IL-4, IL-10). These observations were reinforced by ELISPOT analyses in which several fold higher number of SFUs were detected for INF-γ than for IL-4 in Sm-p80-induced PBMCs. Similar patterns were observed in previous studies using Sm-p80-pcDNA3 vaccine [14]. These results on PBMC and splenocyte proliferation indicate that Sm-p80-VR1020 vaccine formulation is able to elicit a potent Sm-p80-specific T cell response in baboons, as has been observed in the murine [11, 13, 21] and baboon [14] models.

Taken together it appears that both antibodies and INF-γ play an important role in the Sm-p80 mediated protective immunity, however their respective contributions in conferring this protection still need to be elucidated. Furthermore, we have argued that schistosome vaccine candidates should first be exhaustively tested in a nonhuman primate model system before embarking onto human trials [10]. The results of the present study combined with our previous studies in the nonhuman primate model [14] have provided a proof of concept for Sm-p80-based vaccine and we now believe that this vaccine formulation with further optimization is a step closer to be tested for safety and efficacy determination in humans.

Acknowledgments

This work is supported by a grant from the National Institute of Allergy and Infectious Diseases, NIH (R01AI071223) to Afzal A. Siddiqui and by the National Center for Research Resources grant (P40RR012317) to Gary L. White. We thank David W. Carey, for his excellent technical assistance with the baboon studies.

Footnotes

**

Authors do not have any commercial or other associations that might pose a conflict of interest.

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