Beyer and colleagues studied the subsite specificity of *Pf*PM2 at S3 – S3' using the combinato‐ rial chemistry‐based peptide libraries discussed in Section 4.1.1 [125]. *Pf*PM2 prefers accom‐ modating bulky hydrophobic residues (e.g., norleucine, leucine, isoleucine and phenylalanine) in all studied subsites except for the S2', where glutamine is the most favored. The peptide sequence comprising the most favored amino acid at each position, in the order of P3 – P3', is nLInL\*LQI (nL = norleucine). A peptidomimetic inhibitor (KPnLSnLΨLQI) designed using the same approach described above exhibits an inhibition of *Pf*PM2 with the *K*i at nanomolar magnitude and a >15‐fold selectivity for *Pf*PM2 over hcatD. In two earlier studies, the catalytic activity of *Pf*PM2 was assessed in cleaving five sets of chromogenic octapeptides; peptide substrates within a particular set differ in amino acids substituted in one of the P4, P3, P2, P2' and P3' positions [126, 127]. The results showed that peptides with large hydrophobic amino acids (e.g. phenylalanine and leucine) residing in P3, P2 and P3' give rise to the highest *k*cat/*K*m values, consistent with the findings from the combinatorial peptide library study. In addi‐ tion, Siripurkpong and colleagues reported that *Pf*PM2 digests a library of random decameric peptides most efficiently when leucine is placed in the P1' position and that the enzyme has comparable kinetic efficiencies when residues of different properties (e.g., serine, methionine, alanine and glutamine) are placed in the P2' position [122], again consistent with the previous findings. Of note, an N‐terminal extension of peptide substrates to P6 enhances the kinetic efficiency of *Pf*PM2, and yet C‐terminally extended peptides manifest no such effect [124]. The possible presence of a similar effect in other PM homologs is unclear yet.

## *5.1.3. Plasmodium falciparum histo‐aspartic proteinase*

HAP is a PM with the catalytic aspartic acid of the N‐terminus replaced by a histidine. Naturally‐occurring *Pf*HAP, purified as a monomeric mature enzyme of ~37 kDa, cleaves hemoglobin‐derived substrates at an optimal pH 5.7 [48]. *Pf*HAP shows nearly no cleavage of native hemoglobin, but is able to digest acid‐denatured globin and to hydrolyze α33‐34 in hemoglobin‐derived peptide substrates [48]. Nonetheless, *Pf*HAP cleaves α33‐34 20‐fold less efficiently than *Pf*PM1 and *Pf*PM2 [48, 113]. The naturally‐occurring *Pf*HAP can be fully inhibited by isovaleryl‐pepstatin (pepstatin A) at 1 μM and by the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) at 1 mM [48].

Catalytically active *Pf*HAP of the recombinant form was obtained using a similar strategy as the one applied to recombinant *Pf*PM1 [128, 129]. The *in vitro* auto‐matured *Pf*HAP retains 4 pro‐ segment residues [128]. It exhibits an optimal catalytic activity at pH 5.2 and lowers kinetic effi‐ ciencies in cleaving hemoglobin‐derived peptides than its naturally‐occurring counterpart [128]. In addition, though pepstatin A at 1 μM completely inactivate the enzyme, PMSF at 1 mM inhib‐ its enzyme activity by only 25% [128]. The apparent differences in enzymatic features between the naturally‐occurring *Pf*HAP and the *in vitro* auto‐matured may be attributable to improper folding of the recombinant protein [128] and/or the inhibition effects of the pro‐segment [120].

A key question remains elusive is whether *Pf*HAP functions as an aspartic or a serine protein‐ ase. Based on results from computational modeling, some view *Pf*HAP as a serine proteinase with a catalytic triad of H34, S37 and D214 [130], and others consider *Pf*HAP an atypical aspar‐ tic proteinase with D214 performing catalysis and H34 stabilizing the intermediate enzyme species [131]. By conducting alanine mutation of these residues related to catalysis, Parr and colleagues showed that D214A renders *Pf*HAP incapable of auto‐maturation, whereas H34A and S37A do not affect auto‐maturation, but lead to a lower kinetic efficiency in cleaving pep‐ tide substrates [132]. These findings support the role of D214 in enzyme catalysis, indicating that *Pf*HAP is an atypical aspartic proteinase.

#### *5.1.4. Plasmepsin 4 orthologs*

To the author's knowledge, no literatures have thus far reported the characteristics of nat‐ urally‐occurring *Pf*PM4. The recombinantly expressed *Pf*PM4 zymogen lacking the puta‐ tive transmembrane motif conducts auto‐maturation under acidic conditions, resulting in a mature form retaining 12 pro‐segment residues [48]. This mature *Pf*PM4 cleaves hemoglobin‐ derived peptides at an optimal pH 5.4 [48]. *Pf*PM4 digests native hemoglobin less efficiently than *Pf*PM1 and *Pf*PM2 and prefers cleaving acid‐denatured globin [48]. Similar to *Pf*PM1 and *Pf*PM2, but unlike *Pf*HAP, *Pf*PM4 is fully inhibited by pepstatin A at sub‐nanomolar magni‐ tude, but not by inhibitors of other types of proteinases [48, 54].

Recombinant PM4s from the other three human malaria parasites and the rodent malarial para‐ site *P. berghei* were similarly produced and activated [54, 126, 133]. The subsite specificity at S3 – S3' of the five PM4 orthologs (i.e., *Pf*PM4, *Po*PM4, *Pv*PM4, *Pm*PM4 and *Pb*PM4) was investigated using combinatorial peptide libraries [125, 133]. All five PM4s unanimously prefer accommodat‐ ing phenylalanine or tyrosine at S1 and S1', except that *Pb*PM4 accommodates norleucine best at S1'. At S3, bulky hydrophobic amino acids, such as leucine, norleucine and phenylalanine, are preferred by all five enzymes. At S3', the acceptance of amino acids by the four human PM4s is broad with isoleucine accommodated best, whereas *Pb*PM4 accommodates aromatic phenylala‐ nine and tryptophan best. For S2 and S2', all five PM4s seem to tolerate amino acids of different properties. Glutamic acid, serine and isoleucine are the most favored at S2; while for glutamine, isoleucine, glutamic acid and arginine, when accommodated in S2', each leads to a considerable peptide cleavage. The peptide sequence comprising the most favored residue by each subsite, in the order of P3 – P3', is IQF\*YIL for *Pf*PM4, is FEF\*YFI for *Po*PM4, is LEF\*FII for *Pv*PM4, is FEF\*FII for *Pm*PM4, and is FEF\*nLSW for *Pb*PM4. Peptidomimetic inhibitors were designed using the same approach described in Section 4.1.1: KPVEFΨRQT for *Pf*PM4, KPLEFΨFRV for *Po*PM4, KPLEFΨYRV for *Pv*PM4, KPFELΨAWT for *Pm*PM4, and KPYEFΨRQF for *Pb*PM4. These compounds unanimously exhibit a selective inhibition of their respective PM4s over hcatD, and inhibit their respective PM4s with the *K*i values at sub‐nanomolar to nanomolar magnitude, except for the one designed for *Pm*PM4, which inhibits *Pm*PM4 with the *K*i at micro‐ molar magnitude. Such a poor inhibition may be due to the incorporation in the P1' position of an alanine that is poorly recognized by *Pm*PM4, indicating the key role of the P1' amino acid in determining the enzyme‐ligand interaction. In another two studies, the subsite specificity of the four human PM4s was analyzed at S3, S2, S2' and S3' using chromogenic octapeptides [54, 126]. The results showed that (1) hydrophobic amino acids (e.g., phenylalanine and isoleucine) are more favored at P3 than smaller hydrophobic, polar and charged amino acids, (2) hydrophobic amino acids are favored at P2, and (3) amino acids of different properties at P2' and P3' are well tolerated. These findings are consistent with the data obtained from the combinatorial peptide library study.

#### **5.2. Non-food vacuole plasmepsins**

*5.1.3. Plasmodium falciparum histo‐aspartic proteinase*

196 Natural Remedies in the Fight Against Parasites

phenylmethylsulfonyl fluoride (PMSF) at 1 mM [48].

that *Pf*HAP is an atypical aspartic proteinase.

tude, but not by inhibitors of other types of proteinases [48, 54].

*5.1.4. Plasmepsin 4 orthologs*

HAP is a PM with the catalytic aspartic acid of the N‐terminus replaced by a histidine. Naturally‐occurring *Pf*HAP, purified as a monomeric mature enzyme of ~37 kDa, cleaves hemoglobin‐derived substrates at an optimal pH 5.7 [48]. *Pf*HAP shows nearly no cleavage of native hemoglobin, but is able to digest acid‐denatured globin and to hydrolyze α33‐34 in hemoglobin‐derived peptide substrates [48]. Nonetheless, *Pf*HAP cleaves α33‐34 20‐fold less efficiently than *Pf*PM1 and *Pf*PM2 [48, 113]. The naturally‐occurring *Pf*HAP can be fully inhibited by isovaleryl‐pepstatin (pepstatin A) at 1 μM and by the serine proteinase inhibitor

Catalytically active *Pf*HAP of the recombinant form was obtained using a similar strategy as the one applied to recombinant *Pf*PM1 [128, 129]. The *in vitro* auto‐matured *Pf*HAP retains 4 pro‐ segment residues [128]. It exhibits an optimal catalytic activity at pH 5.2 and lowers kinetic effi‐ ciencies in cleaving hemoglobin‐derived peptides than its naturally‐occurring counterpart [128]. In addition, though pepstatin A at 1 μM completely inactivate the enzyme, PMSF at 1 mM inhib‐ its enzyme activity by only 25% [128]. The apparent differences in enzymatic features between the naturally‐occurring *Pf*HAP and the *in vitro* auto‐matured may be attributable to improper folding of the recombinant protein [128] and/or the inhibition effects of the pro‐segment [120]. A key question remains elusive is whether *Pf*HAP functions as an aspartic or a serine protein‐ ase. Based on results from computational modeling, some view *Pf*HAP as a serine proteinase with a catalytic triad of H34, S37 and D214 [130], and others consider *Pf*HAP an atypical aspar‐ tic proteinase with D214 performing catalysis and H34 stabilizing the intermediate enzyme species [131]. By conducting alanine mutation of these residues related to catalysis, Parr and colleagues showed that D214A renders *Pf*HAP incapable of auto‐maturation, whereas H34A and S37A do not affect auto‐maturation, but lead to a lower kinetic efficiency in cleaving pep‐ tide substrates [132]. These findings support the role of D214 in enzyme catalysis, indicating

To the author's knowledge, no literatures have thus far reported the characteristics of nat‐ urally‐occurring *Pf*PM4. The recombinantly expressed *Pf*PM4 zymogen lacking the puta‐ tive transmembrane motif conducts auto‐maturation under acidic conditions, resulting in a mature form retaining 12 pro‐segment residues [48]. This mature *Pf*PM4 cleaves hemoglobin‐ derived peptides at an optimal pH 5.4 [48]. *Pf*PM4 digests native hemoglobin less efficiently than *Pf*PM1 and *Pf*PM2 and prefers cleaving acid‐denatured globin [48]. Similar to *Pf*PM1 and *Pf*PM2, but unlike *Pf*HAP, *Pf*PM4 is fully inhibited by pepstatin A at sub‐nanomolar magni‐

Recombinant PM4s from the other three human malaria parasites and the rodent malarial para‐ site *P. berghei* were similarly produced and activated [54, 126, 133]. The subsite specificity at S3 – S3' of the five PM4 orthologs (i.e., *Pf*PM4, *Po*PM4, *Pv*PM4, *Pm*PM4 and *Pb*PM4) was investigated using combinatorial peptide libraries [125, 133]. All five PM4s unanimously prefer accommodat‐ ing phenylalanine or tyrosine at S1 and S1', except that *Pb*PM4 accommodates norleucine best Thus far, enzymatic characterization of non‐FV PMs has been focused on the PM5 ortho‐ logs. PM5 (*Pf*PM5) immunopurified from cultured *P. falciparum* cleaves PEXEL (RxLxQ/ E/D)‐containing substrates at the C‐terminus of leucine at pH 5–7 [64], resembling the pH of the mammalian ER [134]. The PEXEL‐cleaving activity of *Pf*PM5 is partially inhibited by pepstatin A and HIV‐1 PIs (i.e., lopinavir, nelfinavir, ritonavir and saquinavir) with the IC50 values in the high micromolar range [64, 104]. The presence of P3 R and P1 L is key to *Pf*PM5‐catalyzed PEXEL cleavage in that mutations in these two positions (e.g., P3 R‐ to‐A or K and P1 L‐to‐A or I) unanimously inhibit the cleavage, and abolish the export of PEXEL‐containing proteins to host erythrocytes; amino acids in the prime side positions also influence the efficiency of PEXEL cleavage and subsequent protein export [104, 105, 135]. *Pf*PM5 also digests non‐canonical PEXEL motifs (e.g., RxLxxE) at the C‐terminus of P1 L, which in turn, triggers host‐targeted protein export [105]. Likewise, this *Pf*PM5‐cata‐ lyzed non‐canonical PEXEL cleavage and subsequent protein export are blocked by a P3 R‐to‐A mutation [105]. Of note, though deleting neither the P1' nor the P2' amino acid affects enzyme cleavage, protein export efficiency is reduced by these prime side mutations [105]. Taken together, these findings highlight the essential role of P3 R and P1 L in modulating *Pf*PM5‐mediated PEXEL cleavage and the importance of the prime side peptide sequence in directing host‐targeted protein export.

Two constructs of *Pf*PM5 encoding a truncated zymogen (amino acids 37–521) and a mature enzyme (amino acids 84–521) have been recombinantly expressed in *E. coli* [136, 137]. Following *in vitro* protein refolding, both the zymogen and the mature enzyme exhibit cat‐ alytic activity in cleaving PEXEL‐containing peptides at an optimal pH 6.0–6.5 [136, 137]. Indeed, the pro‐segment of *Pf*PM5 was shown to be non‐essential for guiding the proper fold‐ ing of protein [137, 138]. Subsite specificity analysis of the recombinant mature *Pf*PM5 on a peptide series of RxLxE at P2 and P1' showed that when the polar serine is placed at P1', the hydrophobic isoleucine is more favored at P2 than the charged glutamic acid and lysine; and vice versa, when isoleucine is placed at P2, serine is better accommodated at S1' than glutamic acid and the hydrophobic valine [136]. Recombinant *Pf*PM5, like the parasite‐expressed, can only be partially (<50%) inhibited by pepstatin A, nelfinavir or PMSF at 100 mM; however, its catalytic activity is almost fully blocked by Cu2+ or Hg2+ at the sub‐micromolar level [137]. Furthermore, the zymogen and mature form of PM5 (*Pv*PM5‐Thai) from *P. vivax* Thailand

a This column shows the subcellular locations of catalytically active, mature plasmepsins.

b This column shows the optimal catalytic pH; for *Pf*PM2 and *Pf*PM4, digestion of host cytoskeletal proteins is carried out at near neutral pH.

c Digestion of these natural substrates were performed *in vitro* using *recombinant* plasmepsins.

dThis column shows the best amino acids accommodated at subsites in the order of P3 – P3′; \* represents scissile bond between P1 and P1′; x represents any natural amino acid; nL = norleucine.

**Table 2.** Enzymatic properties of plasmepsins.

isolates were recombinantly expressed; the purified *Pv*PM5‐Thai exhibits similar enzymatic features as the recombinant *Pf*PM5 does [139].

The enzymatic properties of PMs discussed in this section are summarized in **Table 2**.

## **6. Plasmepsin-targeted antimalarial drug development**

Two constructs of *Pf*PM5 encoding a truncated zymogen (amino acids 37–521) and a mature enzyme (amino acids 84–521) have been recombinantly expressed in *E. coli* [136, 137]. Following *in vitro* protein refolding, both the zymogen and the mature enzyme exhibit cat‐ alytic activity in cleaving PEXEL‐containing peptides at an optimal pH 6.0–6.5 [136, 137]. Indeed, the pro‐segment of *Pf*PM5 was shown to be non‐essential for guiding the proper fold‐ ing of protein [137, 138]. Subsite specificity analysis of the recombinant mature *Pf*PM5 on a peptide series of RxLxE at P2 and P1' showed that when the polar serine is placed at P1', the hydrophobic isoleucine is more favored at P2 than the charged glutamic acid and lysine; and vice versa, when isoleucine is placed at P2, serine is better accommodated at S1' than glutamic acid and the hydrophobic valine [136]. Recombinant *Pf*PM5, like the parasite‐expressed, can only be partially (<50%) inhibited by pepstatin A, nelfinavir or PMSF at 100 mM; however, its catalytic activity is almost fully blocked by Cu2+ or Hg2+ at the sub‐micromolar level [137]. Furthermore, the zymogen and mature form of PM5 (*Pv*PM5‐Thai) from *P. vivax* Thailand

> **Enzymatic characteristics pHb Natural**

FV, TV 4.7; ~6.8 Hb; Host

FV, TV 4.5; ~6.6 Hbc

*Pv*PM4 Intra‐erythrocytic phase FV, TV 4.5 Hbc LEF\*FII <1 nM (*K*i

*Po*PM4 n.d. FV, TV 4.5 Hbc FEF\*YFI <1 nM (*K*i

*Pm*PM4 n.d. FV, TV 4.5 Hbc FEF\*FII <1 nM (*K*i

ER/NE 6.0–6.5 PEXEL‐

This column shows the optimal catalytic pH; for *Pf*PM2 and *Pf*PM4, digestion of host cytoskeletal proteins is carried

dThis column shows the best amino acids accommodated at subsites in the order of P3 – P3′; \* represents scissile bond

**substrates**

FV, TV 5.0 Hb FSF\*L(Q/S)F <1 nM (*K*i

cytoskeletal proteins

FV, TV 5.7 Hb n.d. 1 μM (fully

; Host cytoskeletal proteinsc

; Host cytoskeletal proteins

containing parasite proteins **Subsite specificityd**

nLInL\*LQI <1 nM (*K*i

IQF\*YIL <1 nM (*K*i

FEF\*nLSW <1 nM (*K*i

RxL\*x(Q/E/D); RxL\*xxE

**Pepstatin A inhibition**

inhibition)

)

)

)

)

)

)

)

~20–30 μM (IC50)

**PM Expression pattern Subcellular**

merizoites; gametocytes

merizoites; gametocytes; oocysts; sporozoites

merizoites; gametocytes;

merizoites; gametocytes; oocysts; sporozoites

*Pb*PM4 n.d. FV, TV 5.0–5.5 Hbc

This column shows the subcellular locations of catalytically active, mature plasmepsins.

between P1 and P1′; x represents any natural amino acid; nL = norleucine.

Digestion of these natural substrates were performed *in vitro* using *recombinant* plasmepsins.

*Pf*PM1 Intra‐erythrocytic phase;

198 Natural Remedies in the Fight Against Parasites

*Pf*PM2 Intra‐erythrocytic phase;

*Pf*HAP Intra‐erythrocytic phase;

sporozoites

*Pf*PM4 Intra‐erythrocytic phase;

*Pf*PM5 Intra‐erythrocytic phase;

sporozoites

out at near neutral pH.

a

b

c

merizoites; gametocytes;

**Table 2.** Enzymatic properties of plasmepsins.

**locationa**

## **6.1. Evaluation of food vacuole plasmepsins as antimalarial drug targets**

The establishment of the role of FV PMs in hemoglobin processing raised the question whether FV PMs can be targets of novel antimalarial drugs. Peptidomimetic compounds developed in the early stage (e.g., pepstatin A, SC‐50083, Ro40‐4388, and HIV‐1 PIs) bind FV PMs tightly and block growth of cultured parasites [46, 51, 140, 141], suggesting that inhibition of FV PMs is a promising antimalarial strategy. Numerous types of FV PM‐targeted compounds, syn‐ thetic or isolated from natural sources, have been assessed for the past two and a half decades based on criteria involving binding affinity and selectivity, inhibition potency to cultured parasite growth, and cytotoxicity to mammalian cell culture (for reviews, see for example [142, 143]). For example, certain hydroxyethylamine derivatives inhibit *Pf*PM1, *Pf*PM2 and *Pf*PM4 in nanomolar magnitude, exhibit a >30‐fold binding selectivity over hcatD, and dis‐ rupt growth of cultured *P. falciparum* with IC50s in the low micromolar range [144, 145]. In a series of studies, several allophenylnorstatine‐based compounds were found to inhibit all four FV *Pf*PMs in nanomolar magnitude, to block parasite growth with IC50s in the low micro‐ molar range, and to have the TD50s (cytotoxicity) in high micromolar magnitude to rat skel‐ etal myoblasts [146–148]. In addition, clinically used HIV‐1 PIs exhibit antimalarial activity on parasites in both the exo‐erythrocytic and the intra‐erythrocytic phases in the sub‐micromolar to low micromolar range [149–151], inhibit *Pf*PM2 and *Pf*PM4 at low micromolar magnitude, and have a >10‐fold selectivity over hcatD [141]. Interestingly, using affinity binding probes coupled to a FV PM inhibitor library, a hydroxyethyl‐based inhibitor was identified that inhibits all four FV *Pf*PMs and the growth of cultured *P. falciparum* with IC50 at ~1 μM [152].

To assess whether FV PMs are appropriate drug targets, *pfpm*4, 1, 2 and *pfhap* were knocked out individually (i.e., Δ*pfpm4*, Δ*pfpm1*, Δ*pfpm2* and Δ*pfhap*), in combination (e.g., Δ*pfpm4/1* and Δ*pfpm1/2/hap*), or together as a whole (i.e., Δ*pfpm4/1/2/hap*). Genetic ablation of any particu‐ lar gene alters neither the mRNA transcription nor the protein expression of the other three paralogs over the course of the intra‐erythrocytic phase [153]. For hemoglobin metabolism, the Δ*pfpm4* strain, but not the Δ*pfpm1*, Δ*pfpm2* or Δ*pfhap*, shows a reduction in hemozoin accumulation in the FV compared to the parent line [154, 155]. Of note, genetic disruption of PM expression does affect the rate of parasite replication in that the Δ*pfpm4*, Δ*pfpm1*, Δ*pfpm2*, Δ*pfpm4/1* and Δ*pfpm4/1/2/hap* strains all exhibit a reduced growth rate in amino‐acid‐rich media compared to the parent line, and that when cultured in amino‐acid‐limited media, the Δ*pfhap* strain also demonstrates a slower growth rate [153–157]. As for cell and subcellular organelle morphology, though no morphological abnormalities are apparent in the Δ*pfpm1* and Δ*pfhap* strains, a portion of the Δ*pfpm2* shows enlarged mitochondria, and a portion of the Δ*pfpm4* exhibits a notable accumulation of electron‐dense, single‐membrane vesicles in the FV [154, 156]; in addition, ceroid‐like multilamellar bodies, and electron‐dense, single‐membrane vesicles are accumulated in the FV of the *Δpfpm4/1/2/hap* strain [155]. Taken together, genetic ablation of *pfpm*s is not lethal to the parasite in cultured conditions despite apparent metabolic and pathological abnormalities, thus it seems that FV PMs may be dispensable for parasite survival; however, one cannot overlook the potential contribution of *Pb*PM4 to the virulence of the parasite in infected mice (see discussion in Section 4.5). Understanding the pathological role of FV PMs in both cell‐based and animal models may lead to a better assessment of the feasibility of PM‐targeted drug development.

To better understand the relationship between enzyme inhibition and anti‐parasitic activity, the effects of known FV PM inhibitors on the growth of PM‐knockout parasites were inves‐ tigated. When pepstatin A was administered to cultured parasite in the intra‐erythrocytic phase, growth of the Δ*pfpm1*, Δ*pfpm2*, Δ*pfhap* and Δ*pfpm4/1* strains is even slightly less sensi‐ tive to the compound than that of the parent line, and yet growth of the FP‐2‐knockout strain is at least one order of magnitude more sensitive to pepstatin A [156, 157]. These findings indicate that the parasite may turn to other proteinases to maintain normal function when the activities of FV PMs are blocked. The effects of HIV‐1 PIs on *in vitro* PM inhibition and blockage of parasite growth have been well established [141, 158]. However, the Δ*pfpm1/2/hap* and Δ*pfpm4/1/2/hap* strains share a comparable sensitivity to five HIV‐1 PIs (i.e., atazanavir, lopinavir, indinavir, ritonavir and saquinavir) with the parent line [155], indicating that FV PMs may not be the target of these inhibitors in the parasite [141]. Such off‐target effects are rather common among developed PM inhibitors of distinct classes (e.g., *C*2 ‐symmetric 1,2‐ dihydroxyethylenes [159], hydroxylethylamine transition‐state isosteres [145] and amidine‐ containing diphenylureas [160]). The authentic targets of these inhibitors in the parasite have been under investigation [161].

Despite that FV PMs are not critical to parasite survival at the blood stage and that certain FV PM inhibitors exhibit their anti‐parasitic activities with an off‐target effect, it is still early to negate FV PM‐targeted drug design given our limited understanding of their functions and character‐ istics. The continuously identified novel functions of FV PMs plus their broad spatio‐temporal expression pattern over the course of the parasite life cycle are worthy of further investigation.

## **6.2. Developing novel antimalarial drugs targeting non-food vacuole plasmepsins**

PM5 has been considered an ideal target for novel antimalarial drug design based on a series of findings: first, ablation of the gene encoding PM5 is lethal to cultured *P. berghei* [104], so is mutation of a catalytic aspartic acid of PM5 to cultured *P. falciparum* [64]; second, PM5 is evolutionarily conserved among *Plasmodium* spp*.* with no identified gene replication or func‐ tional redundancy [44]; third, PM5 shares a low amino acid sequence identity with human aspartic proteinases (e.g., 26% with mature hcatD, and 18% with mature human β‐secretase 1 (hBACE‐1)); and fourth, the expression profile of PM5 spans the entire life cycle of malaria parasites [44, 65, 66].

Two basic components were incorporated in the initial design of PM5 inhibitors: a PEXEL sequence, which provides a moderate fit of compounds to the active site of the enzyme, and a transition‐state peptidomimetic moiety, which gives rise to a tight interaction with the cata‐ lytic residues of proteinases. WEHI‐916, a statine‐based compound mimicking the non‐prime‐ side RVL motif of the PEXEL, shows a strong inhibition (IC50 = ~20 nM) of *Pf*PM5 and *Pv*PM5, a much weaker inhibition of hcatD (IC50 = 25 μM), and a negligible inhibition of hBACE‐1 (IC50>100 μM) [162, 163]. Administration of WEHI‐916 to cultured *P. falciparum* blocks the PEXEL cleavage in a dose‐dependent manner, and impairs protein export to host erythrocytes [162]. Of particular interest, conditioned knockdown of *pfpm5* enhances WEHI‐916‐medi‐ ated inhibition of PEXEL cleavage and the sensitivity of parasite growth to this compound; whereas overexpression of *Pf*PM5 weakens the anti‐parasitic potency of WEHI‐916 [162]. These findings confirm that PM5 is the target of WEHI‐916 in the parasite. Though, WHEI‐916 has only a moderate potency (EC50 = 2.5 μM to the strain 3D7) in killing cultured *P. falciparum*, which may be attributed to its poor membrane permeability [162, 163]. To enhance the anti‐ parasitic potency of WEHI‐916 while retaining its strong binding to PM5, the highly polar P3 arginine in WEHI‐916 was modified to its isostere L‐canavanine, and the N‐terminal sulfon‐ amide was replaced by a carbamate [164, 165]. The resulting compound WEHI‐842 inhibits *Pf*PM5 and *Pv*PM5 more tightly (IC50 = 0.2–0.4 nM), and blocks the PEXEL cleavage and pro‐ tein export more potently than WEHI‐916 [165]. Importantly, WEHI‐842 kills the chloroquine‐ sensitive 3D7 strain and multiple chloroquine‐resistant *P. falciparum* strains with a potency (EC50 = 0.4 μM) one order of magnitude higher than that of WEHI‐916, and yet it exhibits a low cytotoxicity against human cells (TD50>50 μM) [165]. Taken together, WEHI‐842 repre‐ sents a promising lead for developing PM5‐targeted antimalarial drugs.

Our limited knowledge on PMs 6–10 makes it difficult to assess the necessity and impor‐ tance of developing drugs targeting these enzymes. However, the detection of these PMs in multiple stages of the parasite life cycle suggests that their role in malaria pathogenesis is non‐trivial. For future PM‐targeted drug development, the functions and characteristics of PMs 6‐10 warrant further study.

## **7. Concluding remarks**

[154, 156]; in addition, ceroid‐like multilamellar bodies, and electron‐dense, single‐membrane vesicles are accumulated in the FV of the *Δpfpm4/1/2/hap* strain [155]. Taken together, genetic ablation of *pfpm*s is not lethal to the parasite in cultured conditions despite apparent metabolic and pathological abnormalities, thus it seems that FV PMs may be dispensable for parasite survival; however, one cannot overlook the potential contribution of *Pb*PM4 to the virulence of the parasite in infected mice (see discussion in Section 4.5). Understanding the pathological role of FV PMs in both cell‐based and animal models may lead to a better assessment of the

To better understand the relationship between enzyme inhibition and anti‐parasitic activity, the effects of known FV PM inhibitors on the growth of PM‐knockout parasites were inves‐ tigated. When pepstatin A was administered to cultured parasite in the intra‐erythrocytic phase, growth of the Δ*pfpm1*, Δ*pfpm2*, Δ*pfhap* and Δ*pfpm4/1* strains is even slightly less sensi‐ tive to the compound than that of the parent line, and yet growth of the FP‐2‐knockout strain is at least one order of magnitude more sensitive to pepstatin A [156, 157]. These findings indicate that the parasite may turn to other proteinases to maintain normal function when the activities of FV PMs are blocked. The effects of HIV‐1 PIs on *in vitro* PM inhibition and blockage of parasite growth have been well established [141, 158]. However, the Δ*pfpm1/2/hap* and Δ*pfpm4/1/2/hap* strains share a comparable sensitivity to five HIV‐1 PIs (i.e., atazanavir, lopinavir, indinavir, ritonavir and saquinavir) with the parent line [155], indicating that FV PMs may not be the target of these inhibitors in the parasite [141]. Such off‐target effects are

dihydroxyethylenes [159], hydroxylethylamine transition‐state isosteres [145] and amidine‐ containing diphenylureas [160]). The authentic targets of these inhibitors in the parasite have

Despite that FV PMs are not critical to parasite survival at the blood stage and that certain FV PM inhibitors exhibit their anti‐parasitic activities with an off‐target effect, it is still early to negate FV PM‐targeted drug design given our limited understanding of their functions and character‐ istics. The continuously identified novel functions of FV PMs plus their broad spatio‐temporal expression pattern over the course of the parasite life cycle are worthy of further investigation.

PM5 has been considered an ideal target for novel antimalarial drug design based on a series of findings: first, ablation of the gene encoding PM5 is lethal to cultured *P. berghei* [104], so is mutation of a catalytic aspartic acid of PM5 to cultured *P. falciparum* [64]; second, PM5 is evolutionarily conserved among *Plasmodium* spp*.* with no identified gene replication or func‐ tional redundancy [44]; third, PM5 shares a low amino acid sequence identity with human aspartic proteinases (e.g., 26% with mature hcatD, and 18% with mature human β‐secretase 1 (hBACE‐1)); and fourth, the expression profile of PM5 spans the entire life cycle of malaria

Two basic components were incorporated in the initial design of PM5 inhibitors: a PEXEL sequence, which provides a moderate fit of compounds to the active site of the enzyme, and

**6.2. Developing novel antimalarial drugs targeting non-food vacuole plasmepsins**

‐symmetric 1,2‐

rather common among developed PM inhibitors of distinct classes (e.g., *C*2

feasibility of PM‐targeted drug development.

200 Natural Remedies in the Fight Against Parasites

been under investigation [161].

parasites [44, 65, 66].

Malaria, one of the deadliest infectious diseases in history, still poses a serious socio‐economic problem at present. Malaria control has been effectively undertaken from multiple perspectives, including drug‐based disease prevention and treatment, intervention of malaria transmission by the mosquito vector, and usage of vaccine against malaria parasites. Though, the emer‐ gence and quick spread of drug‐resistant parasite strains urges us to identify new antimalarial drug targets. The subject of this review has focused on the aspartic proteinase PM family, the molecular entities deemed novel and promising targets of next‐generation antimalarial drugs.

Discussed here is our understanding of the PM family members on their biosynthesis, bio‐ logical functions and characteristics for the past two and a half decades. Seven groups of PMs have thus far been identified from genome comparison of a series of *Plasmodium* spp*.* infecting rodents, birds, humans and non‐human primates. These PMs, unique in enzymatic feature and spatio‐temporal expression pattern, play multifaceted roles in the pathogenicity of the malaria parasite. Due to the seemingly dispensable role of FV PMs in parasite growth and survival, the focus of PM‐targeted drug development is shifting towards non‐FV PMs. Selective inhibitors of PM5 have been developed and shown strong inhibition potency to parasite growth.

On the other hand, our knowledge on PMs is still quite limited and much needs to be clari‐ fied and explored in the future studies. For example, what is the biological meaning of the presence of four FV PM paralogs in *P. falciparum*? What do the FV PM inhibitors authenti‐ cally target to exert their anti‐parasitic activity? What are other possible roles of PM5 than host‐targeted protein export? What are the functions of PMs 6‐10, and can these enzymes be antimalarial drug targets? What is the likelihood that PMs are used as immunogens in active immunization and that antibodies directed against PMs are used in passive immunization to protect hosts from malaria parasite infection? Successful PM‐targeted drug development replies on a comprehensive understanding of this enzyme family.

## **List of abbreviations**

parasite. Due to the seemingly dispensable role of FV PMs in parasite growth and survival, the focus of PM‐targeted drug development is shifting towards non‐FV PMs. Selective inhibitors

On the other hand, our knowledge on PMs is still quite limited and much needs to be clari‐ fied and explored in the future studies. For example, what is the biological meaning of the presence of four FV PM paralogs in *P. falciparum*? What do the FV PM inhibitors authenti‐ cally target to exert their anti‐parasitic activity? What are other possible roles of PM5 than host‐targeted protein export? What are the functions of PMs 6‐10, and can these enzymes be antimalarial drug targets? What is the likelihood that PMs are used as immunogens in active immunization and that antibodies directed against PMs are used in passive immunization to protect hosts from malaria parasite infection? Successful PM‐targeted drug development

of PM5 have been developed and shown strong inhibition potency to parasite growth.

replies on a comprehensive understanding of this enzyme family.

ACTs artemisinin‐based combination therapies

ALLN *N*‐acetyl‐Leu‐Leu‐norleucinal

DDT dichloro‐diphenyltrichloroethane

EC50 half maximal effective concentration ECM experimental cerebral malaria

E‐64 L‐3‐carboxy‐2,3‐trans‐epoxypropionyl‐leucylamido(4‐guanidino)butane

AN artemisinin BFA brefeldin A Chr. chromosome CQ chloroquine (k)Da (kilo‐)dalton

*E. coli Escherichia coli*

EM electron microscopy ER endoplasmic reticulum

GFP green fluorescence protein HAP Histo‐Aspartic Proteinase

FP falcipain FV food vacuole

Hb hemoglobin

hBACE‐1 human β‐secretase 1 hcatD human cathepsin D

**List of abbreviations**

202 Natural Remedies in the Fight Against Parasites

## **Author details**

#### Peng Liu

Address all correspondence to<EMAIL_ADDRESS>
Department of Neurology, University of Minnesota Medical School, Minneapolis, USA

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216 Natural Remedies in the Fight Against Parasites

## **Vaccination against** *Trichinella spiralis***: Potential, Limitations and Future Directions**

Jonathan I. Andrade-Becerra, Ericka N. Pompa-Mera, Rosa María Ribas-Aparicio and Lilián Yépez-Mulia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66499

#### **Abstract**

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doi:10.2174/1570180053175061

218 Natural Remedies in the Fight Against Parasites

doi:10.1128/AAC.00882‐09

doi:10.1021/jm500797g

nsmb.3061

doi:10.1371/journal.pbio.1001897

2016;**24**(9):1993‐2010. doi:10.1016/j.bmc.2016.03.027

Trichinellosis is a food-borne parasitic disease caused by round worms of the genus *Trichinella*. The majority of human outbreaks are attributed to consumption of raw or undercooked pork meat contaminated with *T. spiralis* muscle larvae. A blockingtransmission vaccine against trichinellosis will allow preventing swine infection and will contribute to disease control. In this chapter, different vaccine candidates so far developed against *T. spiralis*, including first-, second-, and third-generation vaccines, are discussed. Most vaccine candidates are based on a unique antigen mainly from the muscle larva stage, inducing with some exceptions, partial protection although a mix Th1/Th2 immune response is elicited. Therefore, the need for identification of new antigens from different parasite stages focusing on infective intestinal larvae, adult, and newborn larvae stages as well as the evaluation of their protective capacity in pigs is presented. The design of multi-epitope vaccines and the use of adjuvants or immunomodulatory molecules capable to polarize the immune response to a Th2-type-protective response are discussed as imperative elements of modern vaccines. Plant-based vaccines and probiotics as excellent tools for vaccine development against *T. spiralis* are also presented as an attractive platform for veterinary vaccines.

**Keywords:** *Trichinella spiralis*, DNA vaccine, live carriers, edible vaccines, probiotics

## **1. Introduction**

Trichinellosis is a significant global zoonotic disease produced by the nematode species of the genus *Trichinella*. Trichinellosis is an emerging and reemerging disease in many countries [1]. In the international ranking of food-borne parasites, *T. spiralis* was ranked among the

top 10 [2]. *T. spiralis* is the best characterized member of *Trichinella* genus since it is highly infective for sylvatic and domestic animals as well as for humans. Besides, its life cycle can be maintained in experimental animals, providing information about host-parasite relationships and immunity. Infection with *T. spiralis* initiates when the host ingests raw or undercooked meat contaminated with encysted muscle larvae (ML) (**Figure 1**). The larvae are released from muscle tissue by host digestive enzymes in the stomach. Then, ML migrates to the small intestine where they penetrate the intestinal mucosa and undergo four successive molts, becoming mature adult worms. This intestinal phase is the first stage in the host-parasite interplay. At days 1 and 2 post infection, newborn larvae (NBL) are released by female adult worm and spread via the blood and lymphatic systems to striated muscle, where they invade the myofibers, develop into ML, and induce the transformation of infected cells to the nurse-cell complex.

*T. spiralis* continues to be the causative agent in most outbreaks in humans. The majority of outbreaks are attributed to domestic pork maintained in small farms or non-controlled outdoor backyard pigs, where poor husbandry conditions place pigs at high risk. From 1986 to 2009, there were 65,818 cases and 42 deaths reported from 41 countries, 50% of those occurred in Romania, mainly during 1990–1999 [3]. In China, from 2005 to 2009, 15 outbreaks of human trichinellosis with 1387 cases and four deaths were recorded in three provinces of southwestern China. Twelve of these 15 outbreaks were caused by the eating of raw or undercooked pork meat [4]. The animal health situation varies between different countries being Argentina and some Eastern European countries where most of the cases were reported in pigs in 2015 [5].

*T. spiralis* infection induces a complex host immune response against a diversity of stagespecific antigens. Up to now, it is well known that during the intestinal phase of infection, the immune response involves a Th1/Th2 response with predominance of the Th2 phenotype characterized by the production of high levels of cytokines IL-4, IL-5, IL-9, and IL-10 as well as IgE, IgG1, and the mobilization of eosinophils and mast cells. Furthermore, the long-lasting

**Figure 1.** *Trichinella spiralis* life cycle.

infection of muscles with *Trichinella* reflects successful immunomodulation of the immune response, mainly characterized by a Th2 phenotype [6].

Despite the availability of effective and relatively safe drugs such as albendazole and mebendazole for trichinellosis treatment, chemotherapy has several disadvantages such as treatment failure, parasite drug resistance, poor drug absorption in the intestinal lumen, and low bioavailability. Besides, traditional anthelminthic drugs are active against enteric stages of *T. spiralis*, but currently no anthelminthic drug has proven to be effective against the parasite systemic stages [7, 8]. Furthermore, serious side effects including bone marrow suppression and teratogenic effects are observed [7, 8].

An alternative for trichinellosis control is vaccination of livestock. Indeed, veterinary vaccines have already made enormous impacts not only on animal health, welfare, and production but also on human health [9]. Vaccines have been demonstrated to be efficient, reliable, and sustainable method to control parasitic infections, and have been referred as a green solution [10].

The aim of this chapter is to update the advances so far achieved in the development of a transmission-blocking vaccine against trichinellosis to prevent swine infection. Trichinellosis vaccine would make a practical contribution to disease control, reducing the production of residues in meat and food chain, eliminating the risk for the consumer and in some cases to improve the productivity of the individual animal.

The first part of the review presents an overview of *T. spiralis* antigenic molecules proposed as first- and second-generation vaccines, discussing the need for identifying and characterizing antigenic molecules from NBL and adult worm, mainly recognized by *T. spiralis*-infected swine, administered with adjuvants or delivered by carrier systems. The second part provides a description of third-generation vaccines (DNA vaccines) delivered as naked DNA or by carrier systems. Some experimental data recently obtained by our research group using second- and third-generation vaccines will be presented. Finally, the alternative use of adjuvants, multi-epitope vaccines, plants as a system to express antigenic molecules, and probiotics to protect against parasite infection will be discussed too.

## **2. First- and second-generation vaccines against trichinellosis**

The biggest challenge for vaccine development is the identification of the best *T. spiralis* antigens that elicit host-protective immunity in terms of safety and protection at the both enteral and systemic levels. Different antigenic preparations from different parasite stages using different adjuvants have been tested as vaccine candidates. Most information related to immunity elicited by vaccine candidates have been mainly obtained from rodent models and only few studies have been performed in pigs.

## **2.1. First-generation vaccines**

top 10 [2]. *T. spiralis* is the best characterized member of *Trichinella* genus since it is highly infective for sylvatic and domestic animals as well as for humans. Besides, its life cycle can be maintained in experimental animals, providing information about host-parasite relationships and immunity. Infection with *T. spiralis* initiates when the host ingests raw or undercooked meat contaminated with encysted muscle larvae (ML) (**Figure 1**). The larvae are released from muscle tissue by host digestive enzymes in the stomach. Then, ML migrates to the small intestine where they penetrate the intestinal mucosa and undergo four successive molts, becoming mature adult worms. This intestinal phase is the first stage in the host-parasite interplay. At days 1 and 2 post infection, newborn larvae (NBL) are released by female adult worm and spread via the blood and lymphatic systems to striated muscle, where they invade the myofibers, develop into ML, and induce the transformation of infected cells to the nurse-cell

*T. spiralis* continues to be the causative agent in most outbreaks in humans. The majority of outbreaks are attributed to domestic pork maintained in small farms or non-controlled outdoor backyard pigs, where poor husbandry conditions place pigs at high risk. From 1986 to 2009, there were 65,818 cases and 42 deaths reported from 41 countries, 50% of those occurred in Romania, mainly during 1990–1999 [3]. In China, from 2005 to 2009, 15 outbreaks of human trichinellosis with 1387 cases and four deaths were recorded in three provinces of southwestern China. Twelve of these 15 outbreaks were caused by the eating of raw or undercooked pork meat [4]. The animal health situation varies between different countries being Argentina and some Eastern European countries where most of the cases were reported in pigs in 2015 [5].

*T. spiralis* infection induces a complex host immune response against a diversity of stagespecific antigens. Up to now, it is well known that during the intestinal phase of infection, the immune response involves a Th1/Th2 response with predominance of the Th2 phenotype characterized by the production of high levels of cytokines IL-4, IL-5, IL-9, and IL-10 as well as IgE, IgG1, and the mobilization of eosinophils and mast cells. Furthermore, the long-lasting

complex.

220 Natural Remedies in the Fight Against Parasites

**Figure 1.** *Trichinella spiralis* life cycle.

First-generation vaccines developed against trichinellosis include the use of autoclaved *T. spiralis* larvae and inactivated ML administered with complete Freund's adjuvant (CFA). These types of vaccines induced in immunized mice significant ML burden reduction, as well as degeneration and hyalinization of the nurse-cell structure, accompanied by early pericystic fibrosis [11, 12]. In addition, antigenic preparations from the different stages of *T. spiralis* have been used in protection assays in mice. In this way, adult total extracts and ML total extracts provided protection against adult (89–74%) and ML (80%) stages. Importantly, ML total extracts induced the reduction of female fecundity (74%). The combination of adult and ML total extracts reduced the adult and ML load by 96% and 86%, respectively, and 73% reduction in female fecundity [13]. Protection assays performed in pigs have explored the use of excretory/secretory (E/S) antigens from *T. spiralis* ML and NBL total extracts [14, 15]. E/S products administered with CFA or aluminum hydroxide induced moderate protection mainly directed against the fecundity of female worms [14]. On the other hand, NBL killed by freezing and thawing combined with CFA were highly protective in swine (78%) against *T. spiralis* challenge, compared to 40% protection elicited with E/S products of ML [15]. These assays established that in pigs the immune response is mainly directed against fecundity of female worms and to the NBL.

It is worth mentioning that most of the studies have focused on ML antigens. Indeed, ML antigens are released and presented to the host immune system twice: by ingested ML in the intestine, and again when the new generation of ML becomes resident in muscle cells. Besides, ML antigens play an important role in the invasion of intestinal epithelium and therefore in the establishment of the infection in muscle cells. Even more, *T. spiralis* ML surface and E/S antigens are recognized by a wide range of hosts [16]. The carbohydrate epitope tyvelose confers the immunodominance to surface and E/S ML antigens [17]. Anti-tyvelose antibodies inhibit parasite invasion of an *in vitro* model of epithelial cells [18, 19]; however, tyvelose failed to elicit in mice a protective immune response against the enteral phase of infection [20].

Because of their antigenicity, the protection induced by ML surface and E/S products was extensively evaluated in mice [21–23]. In all these assays, partial protection against *T. spiralis* challenge was obtained as assessed by the reduction of adult and ML burden as well as female worm fecundity (35–58%).

A further step was achieved with surface and E/S stage-specific antigens purified by specific monoclonal antibodies [21, 24]. These antigens administered with CFA protected mice against parasite challenge, as determined by ML load reduction (29.6 and 50%, respectively). Protection induced by purified E/S products (49 and 55 kDa) was similar to that achieved when total E/S products were used [21].

Other E/S products, mainly glycoprotein of Mr 53 kDa (gp53) and 43 kDa (gp43), were widely investigated as first-generation vaccines. The role of these glycoproteins as mediators of intestinal epithelial cell invasion and niche establishment of *T. spiralis* has been suggested [18]. Even more, it was shown that antibodies against these glycoproteins inhibit *in the vitro* invasion of intestinal epithelial by *T. spiralis* [19]. Therefore, gp43 and gp53 are considered good vaccine candidates.

## **2.2. Second-generation vaccines**

The 40- and 30-mer peptides derived from gp43 were synthesized and tested in protection assays [25, 26], giving rise to the development of second-generation vaccines against trichinellosis. The 40- and 30-mer synthetic peptides were administered to mice by intranasal (i.n.) or subcutaneous route with adjuvants such as the subunit B of cholera toxin (CTB) or incomplete Freund's adjuvant (IFA). The 40- and 30-mer synthetic peptides induced a significant reduction of adult worm burden against *T. spiralis* infection in comparison to control (36 and 64%, respectively). The immune response was characterized by the production of IgG1. Although the use of the synthetic peptides represents an innovative strategy for vaccine development, protection induced was not higher than that elicited with crude total extracts.

vaccines induced in immunized mice significant ML burden reduction, as well as degeneration and hyalinization of the nurse-cell structure, accompanied by early pericystic fibrosis [11, 12]. In addition, antigenic preparations from the different stages of *T. spiralis* have been used in protection assays in mice. In this way, adult total extracts and ML total extracts provided protection against adult (89–74%) and ML (80%) stages. Importantly, ML total extracts induced the reduction of female fecundity (74%). The combination of adult and ML total extracts reduced the adult and ML load by 96% and 86%, respectively, and 73% reduction in female fecundity [13]. Protection assays performed in pigs have explored the use of excretory/secretory (E/S) antigens from *T. spiralis* ML and NBL total extracts [14, 15]. E/S products administered with CFA or aluminum hydroxide induced moderate protection mainly directed against the fecundity of female worms [14]. On the other hand, NBL killed by freezing and thawing combined with CFA were highly protective in swine (78%) against *T. spiralis* challenge, compared to 40% protection elicited with E/S products of ML [15]. These assays established that in pigs the immune

response is mainly directed against fecundity of female worms and to the NBL.

worm fecundity (35–58%).

222 Natural Remedies in the Fight Against Parasites

when total E/S products were used [21].

**2.2. Second-generation vaccines**

It is worth mentioning that most of the studies have focused on ML antigens. Indeed, ML antigens are released and presented to the host immune system twice: by ingested ML in the intestine, and again when the new generation of ML becomes resident in muscle cells. Besides, ML antigens play an important role in the invasion of intestinal epithelium and therefore in the establishment of the infection in muscle cells. Even more, *T. spiralis* ML surface and E/S antigens are recognized by a wide range of hosts [16]. The carbohydrate epitope tyvelose confers the immunodominance to surface and E/S ML antigens [17]. Anti-tyvelose antibodies inhibit parasite invasion of an *in vitro* model of epithelial cells [18, 19]; however, tyvelose failed to elicit in mice a protective immune response against the enteral phase of infection [20].

Because of their antigenicity, the protection induced by ML surface and E/S products was extensively evaluated in mice [21–23]. In all these assays, partial protection against *T. spiralis* challenge was obtained as assessed by the reduction of adult and ML burden as well as female

A further step was achieved with surface and E/S stage-specific antigens purified by specific monoclonal antibodies [21, 24]. These antigens administered with CFA protected mice against parasite challenge, as determined by ML load reduction (29.6 and 50%, respectively). Protection induced by purified E/S products (49 and 55 kDa) was similar to that achieved

Other E/S products, mainly glycoprotein of Mr 53 kDa (gp53) and 43 kDa (gp43), were widely investigated as first-generation vaccines. The role of these glycoproteins as mediators of intestinal epithelial cell invasion and niche establishment of *T. spiralis* has been suggested [18]. Even more, it was shown that antibodies against these glycoproteins inhibit *in the vitro* invasion of intestinal epithelial by *T. spiralis* [19]. Therefore, gp43 and gp53 are considered good vaccine candidates.

The 40- and 30-mer peptides derived from gp43 were synthesized and tested in protection assays [25, 26], giving rise to the development of second-generation vaccines against The induction of mucosal immunity plays an important aspect to be considered in the design of a blocking-transmission vaccine in which the use of liposomes, viral particles, and bacterial carriers has been used to deliver the selected antigen [27–31].

In this regard, *Salmonella*-based vaccine systems are considered among the most advanced and promising technologies developed to induce immunological protection against enteric pathogens because of their ability to both colonize the small intestine and invade non-phagocytic epithelial cells, thus allowing access to the underlying lymphoid tissue [32]. Taking advance of the use of *Salmonella* as live bacterial carrier, our group developed a *Salmonella* vaccine candidate expressing the 30-mer peptide derived from gp43 (amino acid residues 210–239, designated as Ag30) from *T. spiralis* ML. The autotransporter ShdA was employed to translocate Ag30 peptide to the surface of *S. enterica* serovar Typhimurium SL3261 [27]. Mice immuzed by i.n. route with the recombinant *Salmonella* pAg30 elicited a protective immune response against *T. spiralis* challenge, with 61.83% reduction of the adult burden and production of antigen-specific IgG1 and IL-5 (**Figure 2**). The use of the autotransporter MisL has also been used to translocate Ag30 to the surface of *S. enterica* serovar Typhimurium SL3261. The immunization of mice with the recombinant vaccine (i.n. route) in combination with an intraperitoneal (i.p.) boost with the recombinant protein induced a higher level of protection (76%) against the enteral phase of *T. spiralis* infection [28]. In addition, our group explore the use of the 40-mer peptide of *T. spiralis* gp43 protein (named Ag40) expressed on the surface of *S. enterica* serovar Typhimurium SL3261 using the autotransporter ShdA (*Salmonella* pAg40). Partial protection against *T. spiralis* infection at the enteral level was induced (47%). The use of *Salmonella* pAg30 together with *Salmonella* pAg40 did not elicit higher protection against *T. spiralis* infection (58%) [33].

To enhance the humoral and cellular antigen-specific immune response against *T. spiralis* infection, multiple copies of the minimum binding domain of complement C3 component (P28) were used as molecular adjuvant. For this, *Salmonella* pAg30 vaccine was engineered to express the Ag30 peptide from *T. spiralis* fused to three copies of P28 adjuvant (Ag30-P283 ) and was either expressed on the bacterial surface or secreted to the milieu [31]. *Salmonella* vaccines were administered to mice by i.n. route. Data showed that *Salmonella* strains secreting Ag30-P283 or Ag30 reduced the adult worm burden by 92.8 and 72%, respectively, following the challenge with *T. spiralis* ML compared to 42% achieved by recombinant *Salmonella* displaying Ag30-P283 on the surface (**Figure 2**). The protection induced by secreted Ag30-P283 was associated with a mixed Th1/Th2 with predominance of Th2 phenotype, characterized by the production of IgG1, intestinal IgA antibodies, and IL-5 secretion.

**Figure 2.** Protection in mice induced by recombinant *Salmonella enterica* serovar Typhimurium strains against the challenge with *Trichinella spiralis* muscle larvae. Attenuated *Salmonella* expresses Ag30 or Ag40 derived from the gp43 of *T. spiralis* muscle larvae displayed on the surface of recombinant *Salmonella* strains. In addition, Ag30 was fused to three copies of the molecular adjuvant P28 (Ag30-P28₃) and it was expressed on the bacterial surface or secreted to the medium through OmpT protease site (SCOT).

Some new surface and E/S proteins from ML (some of them expressed in other stages of the parasite) have been identified and their recombinant proteins evaluated as vaccine candidates. The surface protein 28.9 kDa (Ts14-3-3) [34], the E/S protein of 35.5 kDa (TspSP1.2) [35], 54.7 kDa amino peptidase (TsAP) also expressed by adult worms and NBL, located primarily at the cuticle and internal organs of the parasite [36], the protein of 20 kDa (Ts-ES-1) existing in the E/S products of *T. spiralis* adult and ML [37], and paramyosin (Ts-Pmy) [38] among others have been tested in mice. In all cases, partial protection assessed against the enteral and muscle phase of the infection was elicited (35–55%). Interestingly, a multiple epitope vaccine was developed including a highly antigenic epitope of Ts-Pmy (8F7) and an epitope (M7) from Ts-87 antigen (present in adult worm). Epitopes were conjugated to KLH and mixed to formulate a multi-epitope vaccine. This vaccine induced partial protection (35%) against *T. spiralis* infection in mice [30]. Protection elicited was not higher than that obtained with 8F7 or M7 alone.

Importantly, it was shown that ML cannot invade the intestinal epithelial cells *in vitro* cultured unless they are exposed to the intestinal milieu or bile and activated into the intestinal infective larvae (IIL) [39–41]. The identification of IIL molecules provides attractive information not only to elucidate the mechanism of parasite invasion and immune evasion but also to identify possible molecular targets for vaccine. Following this purpose, several IIL molecules have been identified and their protective capacity evaluated. The Tsp10 polypeptide of *T. spiralis* IIL displayed on the surface of T7 phage was injected i.p. and intradermally at different sites of the abdomen, and elicited in immunized mice a Th2-protective response against parasite challenge, reducing the adult and ML load by 62.8 and 78.6%, respectively [29].

Other proteins with significant higher expression in IIL than in ML such as a putative copper/ zinc superoxide dismutase (SODC), adult-specific DNase II, putative low-density lipoprotein receptor domain class A (LDLRA), and secreting receptor (SR) have been identified [41]. More recently, some important proteins were identified in E/S from IIL, such as the gp53 kDa with serine protease activity, multi-cystatin-like domain and cystatin-like protein, deoxyribonuclease II family protein, among others [42]. The protective evaluation of recombinant cystatin-like protein from *T. spiralis* IIL administered in mice with CFA and boosted with recombinant protein with IFA showed 62 and 64% reduction in the number of ML and adult worm, respectively. Interestingly, it was recognized by pig antiserum as early as 15 days post infection [43].

*T. spiralis* protein Nudix hydrolase (TsNd) is an up-regulated gene in IIL compared to ML. Recombinant TsNd emulsified with CFA displayed in mice a 57.7 and 56.9% reduction in adult worms and in ML burden, respectively, after a challenge infection with *T. spiralis* with high IgG1 levels [44].

Although rodent models have provided important knowledge about the immune response elicited against *T. spiralis* and immunogenic molecules recognized by sera from infected animals, it is important to mention two important aspects of trichinellosis that should be taken into account for the development of a vaccine. First, domestic pork consumption still accounts for many trichinellosis outbreaks, mostly in Eastern Europe and Argentina, where backyard pigs are raised under high-risk-rearing practices, especially the feeding of food waste. Second, a small number of studies have characterized adult antigens that stimulate immunity during an early infection and could be effective in host protection. In this way, recent studies have identified proteins from adult and ML that are recognized by sera of pigs experimentally infected with *T. spiralis* [45, 46]. Some proteins common from adult and ML stage have been identified, among them heat-shock proteins (HSPs), enolase, and 5'-nucleotidase. It was shown that HSP70 and a 38 kDa protein (Ts87) that is present in E/S products and on the adult cuticle induced protective immunity in mice assessed by ML burden reduction (38.4 and 29%, respectively) [46, 47].

Some new surface and E/S proteins from ML (some of them expressed in other stages of the parasite) have been identified and their recombinant proteins evaluated as vaccine candidates. The surface protein 28.9 kDa (Ts14-3-3) [34], the E/S protein of 35.5 kDa (TspSP1.2) [35], 54.7 kDa amino peptidase (TsAP) also expressed by adult worms and NBL, located primarily at the cuticle and internal organs of the parasite [36], the protein of 20 kDa (Ts-ES-1) existing in the E/S products of *T. spiralis* adult and ML [37], and paramyosin (Ts-Pmy) [38] among others have been tested in mice. In all cases, partial protection assessed against the enteral and muscle phase of the infection was elicited (35–55%). Interestingly, a multiple epitope vaccine was developed including a highly antigenic epitope of Ts-Pmy (8F7) and an epitope (M7) from Ts-87 antigen (present in adult worm). Epitopes were conjugated to KLH and mixed to formulate a multi-epitope vaccine. This vaccine induced partial protection (35%) against *T. spiralis* infection in mice [30]. Protection elicited was not higher than that obtained with 8F7

**Figure 2.** Protection in mice induced by recombinant *Salmonella enterica* serovar Typhimurium strains against the challenge with *Trichinella spiralis* muscle larvae. Attenuated *Salmonella* expresses Ag30 or Ag40 derived from the gp43 of *T. spiralis* muscle larvae displayed on the surface of recombinant *Salmonella* strains. In addition, Ag30 was fused to three copies of the molecular adjuvant P28 (Ag30-P28₃) and it was expressed on the bacterial surface or secreted to the medium

Importantly, it was shown that ML cannot invade the intestinal epithelial cells *in vitro* cultured unless they are exposed to the intestinal milieu or bile and activated into the intestinal infective larvae (IIL) [39–41]. The identification of IIL molecules provides attractive information not only to elucidate the mechanism of parasite invasion and immune evasion but also to

or M7 alone.

through OmpT protease site (SCOT).

224 Natural Remedies in the Fight Against Parasites

It is worth noting that another important aspect that has to be considered is the anti-fecundity effects and immunity to the NBL described in *T. spiralis*-infected pigs [48]. Therefore, the identification of immunogenic proteins characteristic of NBL is important for the induction of protection and vaccine development that could be applied in swine. In this regard, an immunodominant serine protease, named NBL1, has been identified in NBL, embryos, and larvae before birth within the gravid females [49]. Importantly, sera from pigs experimentally infected with *Trichinella* and pigs immunized with the recombinant C terminal part of NBL1 allowed the recognition and identification of six immunodominant linear epitopes on the protein [50]. These epitopes could be used for the development of subunit and multiple epitope vaccines.

## **2.3. Third-generation vaccines**

DNA vaccines allow the *in vivo* expression of antigens in their native conformation, persistent expression of the desired antigen, and the induction of both humoral and cellular immunity [51]. Up to date, three DNA vaccines for veterinary use have been licensured (against West Nile equine virus, melanoma in dogs, and hematopoietic necrosis virus in salmon) [52], encouraging the improvement of experimental DNA vaccines against trichinellosis.

Several DNA vaccines using the eukaryotic expression vector pcDNA3.1 have been designed against *T. spiralis* infection and the induced immune response in mice evaluated. The 31 kDa E/S antigen of ML (TspE1) and TsNd, an up-regulated gene in IIL compared to ML, have been cloned in pcDNA3.1 vector [53, 54]. Recombinant pcDNA3.1-TsNd vaccine conferred higher levels of protection against *T. spiralis* infection in comparison to pcDNA3.1-TsE1. Vaccination of mice with pcDNA3.1-TsNd showed 40.44% reduction in worm adults and 53.9% reduction in ML burden with the production of a mixed Th1/Th2 systemic immune response and IgA production at the mucosal level [54]. In this case, the use of pcDNA3.1/TsNd did not increase the protection previously conferred by recombinant TsNd (57.7 and 56.9% reduction in adult and ML burden, respectively) [44].

The eukaryotic expression vector pVAX1 has also been used to express different *T. spiralis* antigens such as macrophage migration inhibitory factor (MIF) of *T. spiralis* (TsMIF), the protein domain of multi-cystatin-type 1 (MCD-1) (TsMCD-1), and the co-expression of TsMIF and TsMCD-1. Vaccination of mice with the recombinant vaccines induced low levels of protection (23.17% reduction of ML load) [55]. Slightly higher protection was achieved when pVAX1-ubiquitin vaccine was used (37.95%) [56]. In addition, the recombinant vaccines pVAX1-Ts87 and pVAX1-TsPmy conferred 9.7 and 46.6% protection against parasite challenge [57, 58]. Higher levels of protection (43.8%) were obtained when animals were co-immunized with pVAX1- Ts87 and recombinant Ts87. In both cases, a Th1/Th2 immune response was induced [57].

To avoid degradation of DNA vaccines by nucleases, the use of live carriers has been investigated. In this way, pVAX1-Ts87 and pcDNA3.1/TsNd were delivered by *S*. typhimurium strains SL7207 and SL1344, respectively [59, 60]. Mice immunized with *Salmonella* pcDNA3.1/ TsNd showed higher levels of protection assessed by adult (73.32%) and ML (49.5%) load reduction [60]. In this case, higher protection at the enteral level was achieved than with the use of the DNA vaccine alone (73.32 vs. 40.44%).

## *2.3.1. DNA vaccine encoding Ag30*

Since DNA vaccines have several advantages over protein vaccines, our research group developed a DNA vaccine encoding Ag30 using the pVAX1 vector (pVAX1-Ag30). The intramuscular administration of 50-μg pVAX1-Ag30 induced 54% reduction of adult burden in mice. The use of *Salmonella* to deliver pVAX1-Ag30 failed to elicit higher protection levels at the intestinal level (22%) (data not published).

the protein [50]. These epitopes could be used for the development of subunit and multiple

DNA vaccines allow the *in vivo* expression of antigens in their native conformation, persistent expression of the desired antigen, and the induction of both humoral and cellular immunity [51]. Up to date, three DNA vaccines for veterinary use have been licensured (against West Nile equine virus, melanoma in dogs, and hematopoietic necrosis virus in salmon) [52], encouraging the improvement of experimental DNA vaccines against

Several DNA vaccines using the eukaryotic expression vector pcDNA3.1 have been designed against *T. spiralis* infection and the induced immune response in mice evaluated. The 31 kDa E/S antigen of ML (TspE1) and TsNd, an up-regulated gene in IIL compared to ML, have been cloned in pcDNA3.1 vector [53, 54]. Recombinant pcDNA3.1-TsNd vaccine conferred higher levels of protection against *T. spiralis* infection in comparison to pcDNA3.1-TsE1. Vaccination of mice with pcDNA3.1-TsNd showed 40.44% reduction in worm adults and 53.9% reduction in ML burden with the production of a mixed Th1/Th2 systemic immune response and IgA production at the mucosal level [54]. In this case, the use of pcDNA3.1/TsNd did not increase the protection previously conferred by recombinant TsNd (57.7 and 56.9% reduction in adult

The eukaryotic expression vector pVAX1 has also been used to express different *T. spiralis* antigens such as macrophage migration inhibitory factor (MIF) of *T. spiralis* (TsMIF), the protein domain of multi-cystatin-type 1 (MCD-1) (TsMCD-1), and the co-expression of TsMIF and TsMCD-1. Vaccination of mice with the recombinant vaccines induced low levels of protection (23.17% reduction of ML load) [55]. Slightly higher protection was achieved when pVAX1-ubiquitin vaccine was used (37.95%) [56]. In addition, the recombinant vaccines pVAX1-Ts87 and pVAX1-TsPmy conferred 9.7 and 46.6% protection against parasite challenge [57, 58]. Higher levels of protection (43.8%) were obtained when animals were co-immunized with pVAX1- Ts87 and recombinant Ts87. In both cases, a Th1/Th2 immune response was induced [57].

To avoid degradation of DNA vaccines by nucleases, the use of live carriers has been investigated. In this way, pVAX1-Ts87 and pcDNA3.1/TsNd were delivered by *S*. typhimurium strains SL7207 and SL1344, respectively [59, 60]. Mice immunized with *Salmonella* pcDNA3.1/ TsNd showed higher levels of protection assessed by adult (73.32%) and ML (49.5%) load reduction [60]. In this case, higher protection at the enteral level was achieved than with the

Since DNA vaccines have several advantages over protein vaccines, our research group developed a DNA vaccine encoding Ag30 using the pVAX1 vector (pVAX1-Ag30). The intramuscular administration of 50-μg pVAX1-Ag30 induced 54% reduction of adult burden in mice.

epitope vaccines.

trichinellosis.

**2.3. Third-generation vaccines**

226 Natural Remedies in the Fight Against Parasites

and ML burden, respectively) [44].

use of the DNA vaccine alone (73.32 vs. 40.44%).

*2.3.1. DNA vaccine encoding Ag30*

The use of liposomes as carriers of plasmid DNA has been used for vaccination purposes in various studies, because they act as adjuvants and protect plasmids from the attack of host enzymes [61]. It was our interest to assess the protection elicited by lipoplexes formed with 3-μg pVAXAg30 and cationic liposomes (LLO-LLE plus cholesterol, L-lysyl-octadecanol, and L-lysyl eicosanol). The intranasal administration of the lipoplexes induced in mice very low levels of protection against *T. spiralis* infection (7 and 9% reduction of adult and ML burden) (data not published).

**Table 1.** Protection in mice induced by some second- and third-generation vaccines administered alone or delivered by carriers.

## *2.3.2. Protection induced by some candidate antigens as second- and third-generation vaccines delivered alone or by live carriers*

A summary of protection elicited in mice by some candidate antigens proposed as secondand third-generation vaccines is presented in **Table 1**. Antigen and delivery systems are critical elements that influence the protection level induced by the candidate vaccines. Some antigens such as Ts87 used as second-generation vaccine or as third-generation vaccine delivered by *Salmonella* elicit similar protection against *T. spiralis* infection. In the case of Ag30, it improves the protection induced against the enteral stage of *T. spiralis* when it is administered as second-generation vaccine delivered by attenuated *Salmonella*, particularly when it is fused to the molecular adjuvant P28 and secreted to the medium. For TsNd, no differences in protection are observed with second- and third-generation vaccines; however, it elicits higher protection against *T. spiralis* adult as third-generation vaccine delivered by *Salmonella*. On the other hand, Tsp10, an IIL antigen displayed on the surface of T7 phage, induced the highest protection against the systemic stage of *T. spiralis*; however, at the enteral level the protection was lower. An important aspect to mention is the administration route of these candidate vaccines that is correlated with the protection elicited against the parasite challenge. The secondgeneration vaccine, *Salmonella* pAg30-p283 (secreted) administered by i.n. route, afforded at the intestinal level the highest protection against *T. spiralis* challenge (92.8%), followed by *Salmonella* pAg30 displayed by MisL (second-generation vaccine) (76%), also administered by i.n. route and *Salmonella* pcDNA3.1-TsNd (third-generation vaccine) (73.2%) administered by oral route. On the other hand, Tsp10, an IIL antigen displayed by T7 phage, provided the highest protection against *T. spiralis* ML (78.6%); it was administered by i.p. and intradermal via at multiple sites of mice abdomen. Therefore, the administration route also plays an important aspect to be considered in the vaccine development.

## **3. Adjuvants**

The induction of mucosal immunity plays an important aspect to be considered in the design of a vaccine against *T. spiralis* infection. Therefore, it is desirable that vaccine formulations contain adjuvants or immunomodulatory molecules capable to polarize the immune response to a Th2-type response. Indeed, adjuvants can influence the balance of the induced antibody and cell-mediated immunity so constitute an imperative element of modern vaccines [62].

An adjuvant with documented *in vitro* and *in vivo* Th2-skewing properties is the cholera toxin subunit B (CTB) [63]. CTB has been used as potent immunological adjuvant in the induction of protective Th2-type response by first- and second-generation vaccines against *T. spiralis*. More recently, the second-generation vaccine, gp53 of *T. spiralis* ML, contained into virus-like particles with the influenza matrix protein 1 (M1) as a core protein was administered to mice together with CTB, inducing protective immunity against the parasite challenge [64].

Aluminum hydroxide adjuvant (alum) has also been administered with first- and secondgeneration vaccines. When administered with E/S products from *T. spiralis* ML, although it has been documented in mice to induce Th2 responses, no production of IL-5 was detected [65].

The use of the adjuvant water in oil emulsions Montanide® ISA70 (Seppic, France) has been recommended for administration with first-generation vaccines, since its administration together with ML total extracts induced high level of protection (84.5%) against *T. spiralis* infection [66].

In order to enhance immunity, cytokines genes such as IL-4 have been included into thirdgeneration vaccines (DNA vaccines) and have demonstrated to evoke a Th2-type response [67]. Interestingly, porcine IL-4 has been successfully evaluated as an immunological adjuvant in a vaccine candidate against porcine reproductive and respiratory syndrome virus (PRRSV) [68]. The cytokine IL-33 plays an important role at the mucosal level, inducing expansion of a multipotent progenitor cell population with differentiation into macrophages, basophils, and mast cell populations that promote the development of Th2 cytokine responses [69]. Further studies are necessary to determine the potential of IL-4 and IL-33 as molecular adjuvants in the induction of mucosal-protective immunity against *T. spiralis*.

## **4. Perspectives and future directions**

*2.3.2. Protection induced by some candidate antigens as second- and third-generation vaccines*

A summary of protection elicited in mice by some candidate antigens proposed as secondand third-generation vaccines is presented in **Table 1**. Antigen and delivery systems are critical elements that influence the protection level induced by the candidate vaccines. Some antigens such as Ts87 used as second-generation vaccine or as third-generation vaccine delivered by *Salmonella* elicit similar protection against *T. spiralis* infection. In the case of Ag30, it improves the protection induced against the enteral stage of *T. spiralis* when it is administered as second-generation vaccine delivered by attenuated *Salmonella*, particularly when it is fused to the molecular adjuvant P28 and secreted to the medium. For TsNd, no differences in protection are observed with second- and third-generation vaccines; however, it elicits higher protection against *T. spiralis* adult as third-generation vaccine delivered by *Salmonella*. On the other hand, Tsp10, an IIL antigen displayed on the surface of T7 phage, induced the highest protection against the systemic stage of *T. spiralis*; however, at the enteral level the protection was lower. An important aspect to mention is the administration route of these candidate vaccines that is correlated with the protection elicited against the parasite challenge. The second-

the intestinal level the highest protection against *T. spiralis* challenge (92.8%), followed by *Salmonella* pAg30 displayed by MisL (second-generation vaccine) (76%), also administered by i.n. route and *Salmonella* pcDNA3.1-TsNd (third-generation vaccine) (73.2%) administered by oral route. On the other hand, Tsp10, an IIL antigen displayed by T7 phage, provided the highest protection against *T. spiralis* ML (78.6%); it was administered by i.p. and intradermal via at multiple sites of mice abdomen. Therefore, the administration route also plays an

The induction of mucosal immunity plays an important aspect to be considered in the design of a vaccine against *T. spiralis* infection. Therefore, it is desirable that vaccine formulations contain adjuvants or immunomodulatory molecules capable to polarize the immune response to a Th2-type response. Indeed, adjuvants can influence the balance of the induced antibody and cell-mediated immunity so constitute an imperative element of

An adjuvant with documented *in vitro* and *in vivo* Th2-skewing properties is the cholera toxin subunit B (CTB) [63]. CTB has been used as potent immunological adjuvant in the induction of protective Th2-type response by first- and second-generation vaccines against *T. spiralis*. More recently, the second-generation vaccine, gp53 of *T. spiralis* ML, contained into virus-like particles with the influenza matrix protein 1 (M1) as a core protein was administered to mice together with CTB, inducing protective immunity against the parasite

Aluminum hydroxide adjuvant (alum) has also been administered with first- and secondgeneration vaccines. When administered with E/S products from *T. spiralis* ML, although it has been documented in mice to induce Th2 responses, no production of IL-5 was detected

(secreted) administered by i.n. route, afforded at

*delivered alone or by live carriers*

228 Natural Remedies in the Fight Against Parasites

generation vaccine, *Salmonella* pAg30-p283

**3. Adjuvants**

modern vaccines [62].

challenge [64].

[65].

important aspect to be considered in the vaccine development.

## **4.1. Multi-epitope or polyvalent vaccines against trichinellosis**

*T. spiralis* has a complex life cycle; the immune response elicited by a vaccine based on a unique antigen may not be strong enough to combat the challenging infection, and therefore multi-epitope vaccines against *T. spiralis* have been proposed. In this regard, the combination of three selected epitopes from Ts-Pmy and Ts87 from *T. spiralis* adult elicited in immunized mice IgG and IgG1 production and higher protection (35%) against the parasite challenge in comparison to that induced by individual epitope peptides [47]. To accomplish higher protective immune responses against *T. spiralis*, it will be necessary to design a vaccine with multiepitopes from different parasite stages focusing on NBL and adult stages.

## **4.2. Probiotics in protection against** *T. spiralis* **infection**

It has been demonstrated that probiotics modulate the intestinal environment preventing enteric infections. The lactic acid bacteria *Lactobacillu*s is considered as probiotic; they are part of the commensal bacteria and contribute to the maintenance of immune homeostasis in the gut [70]. The protective role of *L. casei* against high infection dose of *T. spiralis* has been demonstrated in mice inoculated intraperitoneally with the bacteria as assessed by adult (76.7%) and ML (80.9%) load reduction, production of high IgA and IgG anti-*T. spiralis* antibody levels as well as IL-4 [71]. More recently, the protection conferred by different *Lactobacillus* strains, *L. casei, L. plantarum*, and *L. acidophilus*, against *T. spiralis* infection was analyzed. The highest protection was elicited by *L. plantarum*, against adult (69.02%) and ML (87.92%). Interestingly, the authors demonstrated an amelioration of inflammation and damage in the intestine of *T. spiralis*-infected mice inoculated with *L. plantarum* with respect to non-treated-infected animals [72]. So far, the use of probiotics is considered as a new tool for trichinellosis control.

## **4.3. Plant-based veterinary vaccine**

Plant-based vaccines might be used as edible vaccines for sustainable prophylaxis against various important parasitic diseases, including trichinellosis. Recombinant proteins based in plants can be produced in nuclear-transformed plants, synthesized in the cytoplasm, and can be accumulated in different subcellular organelles, or secreted, once an appropriate transit or signal peptides are used [73, 74]. Plants are considered an attractive platform for veterinary vaccines, due to low-cost production, sterile delivery, and cold storage/transportation at ambient temperature, compared to traditional attenuated vaccines, which present some inconvenience in terms of insufficient mass production, residual toxicity, means of transportation, and safety [75]. Antigens administered by oral route are subject to proteolysis in gastrointestinal tract, reducing their bioavailability, and therefore affecting the quality of immune response. Then, vaccine antigens can be protected by plant cell walls from further degradation in the digestive tract, enabling them to reach the gut-associated lymphoid tissue [73].

Many species of plants, including tobaccos, alfalfa, spinach, potatoes, rice, beans, maize, tomatoes, strawberries, and carrots, can be used in plant biotechnology for the expression and production of foreign proteins, remaining stable without the loss of activity for years at room temperature. Hence, plants could be suitable for direct consumption and useful for the development of animal vaccines [74]. In fact, edible vaccines produced in papaya and corn seed induced protection against porcine-cysticercosis (70–90%) and porcine-transmissible gastroenteritis virus (50%) [76, 77]. Even more, edible vaccines can include adjuvants as it was the case for As16-an antigen protective against the roundworm *Ascaris suum* fused with CTB in transgenic rice seeds, resulting in an antibody response [78].

Plant-based vaccines represent an excellent tool for mass prevention especially at the veterinary field; their use in vaccine development against *T. spiralis* remains to be explored.

## **5. Conclusions**

Different vaccine candidates based on antigens from different stages of *T. spiralis*, used as recombinant proteins or as DNA vaccines, delivered alone or by live carriers have been proposed. Most of them with some exceptions have induced partial protection against the enteral and muscle phase of the infection. In these studies, a mixed Th1/Th2 immune response with predominance of a Th2 response has been elicited. Up to now, the second-generation vaccines, *Salmonella* pAg30-p283 (secreted) and T7-Tsp10, have afforded at the intestinal and systemic level, respectively, the highest protection against *T. spiralis* challenge. Protection elicited by the candidate vaccines is influenced by the candidate antigen, delivery system, and administration route. Importantly, search for more useful vaccine candidates that could elicit high protection against *T. spiralis* infection in pigs is required. These vaccines may include antigens from IIL, NBL, and from pre-adult and adult stages of infection, administered alone or as multi-epitope vaccine. The use of adjuvants or immunomodulatory molecules capable to polarize the immune response to a Th2 type should be taken into account in a way to improve the protection induced by candidate vaccines. On the other hand, plant-based vaccines represent an excellent tool that needs to be explored in vaccine development against *T. spiralis* with application at the veterinary field.

## **Acknowledgment**

This work was supported by grant FIS/IMSS/PROT/G11/961.

## **Abbreviations**

mulated in different subcellular organelles, or secreted, once an appropriate transit or signal peptides are used [73, 74]. Plants are considered an attractive platform for veterinary vaccines, due to low-cost production, sterile delivery, and cold storage/transportation at ambient temperature, compared to traditional attenuated vaccines, which present some inconvenience in terms of insufficient mass production, residual toxicity, means of transportation, and safety [75]. Antigens administered by oral route are subject to proteolysis in gastrointestinal tract, reducing their bioavailability, and therefore affecting the quality of immune response. Then, vaccine antigens can be protected by plant cell walls from further degradation in the digestive tract,

Many species of plants, including tobaccos, alfalfa, spinach, potatoes, rice, beans, maize, tomatoes, strawberries, and carrots, can be used in plant biotechnology for the expression and production of foreign proteins, remaining stable without the loss of activity for years at room temperature. Hence, plants could be suitable for direct consumption and useful for the development of animal vaccines [74]. In fact, edible vaccines produced in papaya and corn seed induced protection against porcine-cysticercosis (70–90%) and porcine-transmissible gastroenteritis virus (50%) [76, 77]. Even more, edible vaccines can include adjuvants as it was the case for As16-an antigen protective against the roundworm *Ascaris suum* fused with CTB

Plant-based vaccines represent an excellent tool for mass prevention especially at the veteri-

Different vaccine candidates based on antigens from different stages of *T. spiralis*, used as recombinant proteins or as DNA vaccines, delivered alone or by live carriers have been proposed. Most of them with some exceptions have induced partial protection against the enteral and muscle phase of the infection. In these studies, a mixed Th1/Th2 immune response with predominance of a Th2 response has been elicited. Up to now, the second-generation vaccines, *Salmonella*

tively, the highest protection against *T. spiralis* challenge. Protection elicited by the candidate vaccines is influenced by the candidate antigen, delivery system, and administration route. Importantly, search for more useful vaccine candidates that could elicit high protection against *T. spiralis* infection in pigs is required. These vaccines may include antigens from IIL, NBL, and from pre-adult and adult stages of infection, administered alone or as multi-epitope vaccine. The use of adjuvants or immunomodulatory molecules capable to polarize the immune response to a Th2 type should be taken into account in a way to improve the protection induced by candidate vaccines. On the other hand, plant-based vaccines represent an excellent tool that needs to be explored in vaccine development against *T. spiralis* with application at the veterinary field.

(secreted) and T7-Tsp10, have afforded at the intestinal and systemic level, respec-

nary field; their use in vaccine development against *T. spiralis* remains to be explored.

enabling them to reach the gut-associated lymphoid tissue [73].

230 Natural Remedies in the Fight Against Parasites

in transgenic rice seeds, resulting in an antibody response [78].

This work was supported by grant FIS/IMSS/PROT/G11/961.

**5. Conclusions**

pAg30-p283

**Acknowledgment**

## **Author details**

Jonathan I. Andrade-Becerra1,2, Ericka N. Pompa-Mera2 , Rosa María Ribas-Aparicio1 and Lilián Yépez-Mulia2 \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Laboratory of Production and Biological Control, Department of Microbiology, National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico

2 Investigation Unit in Infectious and Parasitic Diseases, Pediatric Hospital, Mexican Institute of Social Security, Mexico City, Mexico

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