Immunogenicity and protective potential of a Plasmodium spp. enolase peptide displayed on archaeal gas vesicle nanoparticles

Antibodies against rPfeno were raised in mice using in-house facilities as described earlier [34]. HRP-conjugated mouse and rabbit secondary antibodies were obtained from AbCam. ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)] was obtained from Sigma-Aldrich Co.

GST-tagged-rPfeno was expressed in Escherichia coli and purified as described earlier [17]. Briefly, bacterial culture overexpressing rPfeno were homogenized and clear supernatant was obtained by centrifugation. GST-tagged recombinant protein was purified by passing the supernatant on a glutathione-Sepharose column. The protein was cleaved off its GST tag using thrombin. Purified rPfeno was recovered from the eluate. For raising antisera in mice, a published protocol was used as described earlier [34].

A double-stranded oligonucleotide was synthesized coding for a Pfeno derived 15-amino acid peptide (DGSKNEWGWSKSKLG, containing the Plasmodium ¹⁰⁴EWGWS¹⁰⁸ epitope). The oligonucleaotide sequence was codon-optimized using the codon usage table for predicted genes in the fully sequenced Halobacterium sp. NRC-1 genome [35]. The synthetic DNA fragment was cloned into the Halobacterium sp. pSD104 vector at the AfeI site in gvpC [27], to construct plasmid pSD104::Eno. The gvpC gene region of the constructed plasmid was sequenced to verify the correctness of the insertion sequence.

Plasmid pSD104::Eno was transformed into Halobacterium strain SD109, a strain that lacks the gas vesicle gene cluster. After lysis of cells, Rec-GVNPs displaying the Pfeno epitope were purified from Halobacterium SD109 (pSD104::Eno) by flotation of the GVNP particles using the centrifugally accelerated procedure reported previously [36]. Presence of the GvpC-Pfeno fusion protein was confirmed by Western blotting using Pfeno primary antibody followed by alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, St. Louis, MO, USA).

SDS-PAGE was run using the Laemmli method [37]. Proteins were resolved on a 10 % SDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. For Western blotting, proteins were separated by SDS-PAGE (10 % gels) and transferred to a PVDF membrane using semi-dry western transfer apparatus (Trans-blot SD-cell, Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a constant voltage of 18 volts for 50 min. The membranes were blocked with 5 % skimmed milk in phosphate buffered saline containing 0.05 % Tween-20 for 2 h. The blots were treated with primary antibody. For the detection of Plasmodium yoelii (Pyeno) or Pfeno derived peptide sequence in Rec-GVNPs, anti-rPfeno antibodies were used at 1:1000 dilutions. Anti-rPfeno antibodies were raised in mice as described earlier [34]. Incubation with the primary antibody was followed by washing and incubation with HRP-conjugated secondary antibody. The immunoblots were developed using di-anilino-benzene substrate.

Experiments were performed on male Swiss mice aged 6–8 weeks. Mice were injected with 100 μg of WT-GVNPs or Rec-GVNPs in Freund’s complete adjuvant intra-peritoneally. Subsequent booster doses of 100 μg WT or Rec-GVNPs in Freund’s incomplete adjuvant were administered at 21 days interval. Ten days after the second booster sera from these animals were collected by partial retro-orbital bleed to assay for their reactivity against rPfeno, WT-GVNPs and Rec-GVNPs. All immunized mice and an un-immunized control group of mice were challenged with equal numbers of (approximately 10⁶) P. yoelii 17XL infected RBCs and their survival as well as percent parasitaemia was monitored as described earlier [15].

ELISAs were performed as previously described [34]. Briefly, the antigen in a volume of 100 μl was coated onto a Maxisorp immunoplate and incubated at 37 °C for 2 h. This was followed by washing the plate and blocking the wells with 5 % skimmed milk in PBST (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl containing 0.05 % Tween-20; pH 7.4) for 1 h at room temperature or 4 °C overnight. The blocking solution was discarded and the wells were washed with PBST three times for 5 min each. 100 μl of primary antibody (dilution varied according to experiment) was added to each well and incubated at room temperature for 1 h. The solution was discarded and the wells were washed with PBST three times to remove any unbound antibody. This was followed by the addition of HRP-conjugated anti-mouse secondary antibody at a dilution of 1:1000 and incubated at room temperature for 45 min. The solution was discarded and the wells were washed with PBST thrice. 200 μl of ready-to-use ABTS substrate was added into each well. The colour was allowed to develop for 10–15 min and absorbance was measured as an optical density (OD) at 405 nm on a Tecan Plate Reader.

For the measurements of anti-WT-GVNP (or anti-Rec-GVNP) antibody titres in serum, ELISA plates were coated with 1 µg of the respective antigen and sera were diluted 1:1000. For anti-rPfeno antibody titer determination, plates were coated with 100 ng of rPfeno and serum dilutions used were 1:100.

In order to display the Pfeno antigenic epitope on GVNPs, an oligonucleotide coding for a Pfeno derived peptide containing the desired EWGWS sequence within an 15-amino acid sequence (DGSKNEWGWSKSKLG) was selected. This peptide sequence was BLASTed against the mouse genome (GenBank taxid:10088) to detect if there are homologous sequences present. However, no significant homology in mammalian genome was found. Use of a longer sequence that encompassed the conserved residues on either side of EWGWS ensured that the epitope was displayed in the proper Pfeno context. The oligonucleotide corresponding to the 15aa peptide was inserted at an AfeI site in gvpC in the Halobacterium sp. pSD104 vector, which contains the complete 14-gene gas vesicle gene cluster. The resulting plasmid pSD104::Eno was transformed into Halobacterium strain SD109, deleted for entire the gas vesicle gene cluster. GVNPs were purified from the resulting Halobacterium SD109 (pSD104::Eno) strain and wild-type strain NRC-1 by centrifugally accelerated flotation using a published procedure [27].

Wild-type nanoparticles (WT-GVNPs) differ from recombinant nanoparticles (Rec-GVNPs) in the presence of the Pfeno derived peptide ¹⁰⁴EWGWS¹⁰⁸ in the gvpC gene. For immuno-detection of this epitope in Rec-GVNPs and absence in WT-GVNPs for, samples of purified WT and Rec-GVNPs were analysed on a 10 % SDS-PAGE gel. P. yoelii cell extract (soluble fraction) was included as a positive control for antibody recognition of enolase. The protein profiles of WT and Rec-GVNPs are shown in Fig. 1a. Western blots with anti-rPfeno antibodies (mouse) are shown in Fig. 1b, c. Antibodies recognized enolase (MW ~50 kDa) in the parasite extract. Enolase positive bands at ~20 kDa and around 35–40 kDa were also observed in P. falciparum extracts and were identified by mass spectrometry to be the products of proteolysis of native 50 kDa form [38]. In the nanoparticle lanes, Western blot showed an enolase positive band with an apparent molecular mass of ~71 kDa (marked with *). This was present only in Rec-GVNPs and was absent in WT-GVNPs. The positive reactivity of the ~71 kDa band with anti-rPfeno polyclonal antibodies is due to the presence of the cloned sequence EWGWS in the GvpC-Eno protein found on the GVNPs and confirms the presence of the engineered peptide insert in the Rec-GVNPs.

For comparative analysis of the antibody response, two groups of mice were immunized with WT-GVNPs (n = 5) and Rec-GVNPs (n = 10), respectively. A group of five mice (n = 5) were kept as un-immunized controls. For immunization, 100 µg of GVNPs in complete Freund’s adjuvant were injected. Subsequently, two booster doses (100 µg GVNPs in Freund’s incomplete adjuvant) were administered at an interval of 21 days. Serum samples were collected by partial retro-orbital bleed 10 days after second booster dose.

Antibody responses to the two classes of particles (WT-GVNPs or Rec-GVNPs) were quantified by ELISA using one or the other particle as the antigen. ELISA measurements where the plates were coated with WT-GVNPs (1μg/well), both serum samples gave similar titres of antibodies (Fig. 2a) indicating that both groups of mice produced antibodies against the GVNP component proteins. However, when Rec-GVNPs were used as the antigen, higher antibody titres were observed with serum samples derived from Rec-GVNP-immunized mice (Fig. 2b). This was indicative of antibody response against the Pfeno derived peptide displayed on Rec-GVNPs. However, such a differential antibody response could also arise due to some contaminant in purified Rec-GVNPs. As expected, serum samples from un-immunized control mice did not show any reactivity towards either of the particles when used as antigens (Fig. 2a, b). Lack of differential reactivity towards WT-GVNPs between the two types of serum samples (raised against WT-GVNPs and Rec-GVNPs) suggested that coated WT-GVNPs reacted with the antibodies arising against similar epitopes in both samples. However, the higher reactivity towards Rec-GVNPs in serum samples from Rec-GVNP immunized mice suggested that a fraction of antibodies was directed against the cloned sequence DGSKNEWGWSKSKLG containing EWGWS, the parasite specific insert motif of Pfeno. To determine whether this is indeed the case, the reactivity of sera using rPfeno as an antigen was measured.

To assess the immunogenic potential of Halobacterium gas vesicle nanoparticles displaying the Pfeno derived peptide in the mouse system, sera from WT and Rec-GVNP-immunized mice were subjected to ELISA using rPfeno as an antigen. 100 ng of rPfeno was coated onto each well of the plate and 1:100 diluted sera from immunized and control mice was used as primary antibody. Results of the experiment for serum reactivity of each individual mouse are shown in Fig. 3a. Figure 3b shows the average values for all the animals in a single treatment group. Anti-rPfeno antibody titres were found to be significantly higher in the case of Rec-GVNP-immunized mice as compared to the WT-GVNP group (Fig. 3b). Although, it was expected that displayed Pfeno peptide in the context of gas vesicle nanoparticles (Rec-GVNPs) would elicit a robust antibody response, observations indicate the antibody response was relatively mild which is not unexpected, given the proportionate size of the 5 amino acid sequence compared to the entire GVNP complex.

To evaluate whether the antibodies against the parasite specific insert sequence of Pfeno are protective under in vivo conditions, all three groups of mice, viz. un-immunized (control), WT and Rec-GVNP-immunized mice, were challenged with equal numbers of iRBCs (approximately 10⁶) infected with asynchronous P. yoelii 17XL (lethal strain), which were isolated from infected mice. The parasites were injected 10 days after the second booster dose of nanoparticles. Parasitaemia in these mice was monitored on every second day post parasite injection to assess the effect of immunization on parasite growth. Percent parasitaemia in the control groups of mice (i.e. un-immunized and WT-GVNP-immunized) was much higher as compared to Rec-GVNP-immunized group. Statistical analysis of data using Student’s t-test showed that on days 4 and 6 (post parasite challenge), WT-GVNP-immunized group had significantly higher percent parasitaemia (p = 0.001) as compared to Rec-GVNP-immunized group. The extent of parasite growth retardation in Rec-GVNP immunized mice was approximately 2- to 2.5-fold as compared to the ones immunized with WT-GVNPs (Fig. 4a).

These mice were also monitored for their survival. Survival profiles for all three groups are shown in Fig. 4b. Rec-GVNP-injected mice survived significantly longer compared to WT-GVNP-treated and un-immunized control groups. As stated above, although the antibody titres of anti-EWGWS in Rec-GVNP immunized mice were moderate, observation of significant retardation in parasite growth and prolongation of survival support the view that the Pfeno derived peptide constitutes a protective antigenic epitope against malaria.

Since each Rec-GVNP-injected mouse had a different degree of reactivity with rPfeno on the ELISA based assay (Fig. 3a), it indicated that the antibody titres against rPfeno in each immunized animal was different. If EWGWS was the protective epitope, titres of antibodies against it should have a positive correlation with survival and negative with percent parasitaemia. Anti-rPfeno antibody titres as measured by ELISA were plotted against the number of post parasite challenge days that an animal survived (Fig. 5a). The plot showed a positive correlation (R² = 0.64) (albeit weak) between survival and antibody titres. Antibody titre vs. percent parasitaemia was also plotted. It was expected that anti-EWGWS antibodies would inhibit parasite growth if this sequence constitutes a protective epitope. The plot did show a negative correlation (Fig. 5b), i.e. the increase in antibody titres interfered with the growth and division of the parasite resulting in a negative correlation between percent parasitaemia and anti-rPfeno antibody titres. A negative correlation between percent parasitaemia and number of days a mouse survived post-challenge of the lethal parasite was also observed (Fig. 5c), indicating that the parasite load is a critical factor in the survival of the mice. These results suggested that anti-EWGWS antibodies inhibited the growth of the parasite, resulting in diminished parasitaemia, and that in turn brought about enhanced survival.