Nasal Immunization with Homogenate and Peptide Antigens Induces Protective Immunity against Trichinella spiralis

Abstract

Mice were successfully immunized against the intestinal nematode Trichinella spiralis by intranasal administration of a 30-mer peptide antigen with cholera toxin B. Immunized mice developed antigen-specific serum immunoglobulin G1, intestinal immunoglobulin A, and a type 2-biased cytokine response. Intranasal immunization therefore generates the Th2-mediated responses required for immunity against intestinal parasites.

Few studies have evaluated mucosal immunization against eukaryote parasites, particularly intestinal nematodes. Oral immunization has generally been unsuccessful without adjuvants (4, 10, 11). Intranasal i.n. vaccine delivery is more effective and requires smaller doses of vaccine (7, 19). Soluble protein antigens alone given i.n. may not induce effective responses (16), but immunity is enhanced by using cholera toxin B subunit (CTB) (6, 17).

The intestinal nematode Trichinella spiralis is a good model for immunological studies (1). Immunity is readily stimulated by systemic immunization with parasite antigen (15). In the present study, nasal administration of T. spiralis antigens was used to see whether an equivalent protection could be induced and to determine whether protection by this route could be obtained with a defined antigen.

Mice were 6- to 8-week-old specific-pathogen-free female NIH strain (Harlan-Olac, Bicester, United Kingdom). Immunity was measured by recovery of adult worms and female worm fecundity (14). Homogenate antigen (CHA) from muscle larvae was prepared as described previously (10). A 30-mer peptide, residues 210 to 239 (RLEMYGSFLAKVMVVNMRIWAVTDNTLQTT) (5) from the T. spiralis 43-kDa antigen was prepared by Fmoc (9-fluorenylmethoxy carbonyl) solid-phase synthesis (3). Initially, CHA was tested for immunogenicity by subcutaneous (s.c.) immunization with complete Freund adjuvant (Sigma, Poole, United Kingdom) before i.n. immunization was attempted. This preliminary experiment showed that the CHA preparation protected mice against a subsequent challenge infection. By day 8, immunized mice harbored a mean of 21 ± 13 worms compared with 125 ± 16 in controls (P < 0.05).

Two sets of experiments with i.n. immunization are described here: one with CHA and one with peptide. In each, one experiment measured protection, and one measured immune responses. For immunization 100 μg of antigen plus 4 μg of CTB (Sigma) in 10 μl was placed directly into the nose.

To assess protection, three groups of 15 mice were used: (i) infection-only controls, (ii) i.n. antigen plus CTB given on days 0, 1, 2, 10, 11, and 12, and (iii) CTB only; all mice were infected with 300 T. spiralis on day 21. Worms were recovered on days 6, 8, and 10 postinfection (p.i.) (five mice/group/day). For the assessment of immune responses, three groups were used: (i) naive controls (10 mice), (ii) antigen plus CTB (25 mice), and (iii) CTB only (25 mice). Five mice/group/day were killed on days 0 and 38 (group 1) and on days 20, 27, 34, and 38 (groups 2 and 3). Five mice from groups 2 and 3 were bled between days 0 and 38.

Intestinal lavage fluid was collected as described previously (11). Antigen-specific immunoglobulin A (IgA; lavage undiluted) and IgG1/IgG2a (plasma diluted, 1:1,000) were measured by enzyme-linked immunosorbent assay (ELISA) (10), with alkaline phosphatase-conjugated goat antimouse IgG1 or IgG2a and biotin-conjugated goat antimouse IgA (Sigma). IgA assays were standardized against mouse IgA (MOPC 315; Sigma) and IgG assays against hyperimmune T. spiralis antisera. Optical density (OD) readings were corrected from standard curves.

Cell culture and cytokine assays were as described previously (10), with 50 μg of CHA or 5 μg of concanavalin A (Sigma)/ml/well. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) ELISAs used paired reagents: (IFN-γ-R4-6A2 and -XMG1.2 and IL-5-TRFK5 and -TRFK4; Pharmingen, San Diego, Calif.). Recombinant IL-5 or IFN-γ standards were included on each plate.

Data are presented as the means ± the standard errors of the mean. Statistical analysis used one-way and two-way analyses of variance and the Spearman rank order correlation test (9). P ≤ 0.05 was considered significant.

Figure 1 shows the number of adult T. spiralis worms from i.n. immunized and control NIH mice. The infection-only controls showed a characteristic pattern of infection, i.e., the numbers of adult worms remained constant between days 6 and 8 p.i., and worms were expelled between day 8 and day 10 p.i. In contrast, after i.n. immunization with CHA+CTB significantly fewer worms were recovered on day 8 p.i. compared to controls (Fig. 1a). Significantly, day 6 worms from immunized mice released significantly fewer larvae (0.68 ± 0.17 larvae/female/h) than infection-only controls (2.14 ± 0.76, P < 0.01). Adjuvant-control worms produced more larvae than those from immunized mice (P > 0.05). Immunized mice produced more CHA-specific IgG1 than adjuvant controls (P < 0.001; Fig. 2), with levels increasing significantly over time (P < 0.01); the levels of IgG2a were low (e.g., <0.2 at day 20). Immunized mice had more antigen-specific intestinal IgA than CTB-only controls (P < 0.001; Fig. 2). Cells from CHA+CTB mice showed no increase in either IFN-γ or IL-5 above control levels until day 34 but then increased with time (P < 0.05; Fig. 4 and Table 1). Levels of IFN-γ were significantly increased relative to CTB and unimmunized controls only when spleen cells (SC) were tested (at day 38, 414 ± 209 pg/ml versus 187 ± 97 and 156 ± 7 pg/ml). SC and mesenteric lymph node cells (MLNC) from CHA+CTB mice released significantly more IL-5 than controls (Fig. 4A and B).

FIG. 1.

Numbers of adult T. spiralis worms (± the standard error [S.E.]) recovered from mice that had been challenged with 300 larvae after i.n. immunization with homogenate antigen plus CTB (CHA+CTB [a]), peptide antigen plus cholera toxin B (peptide+CTB) or CTB only, or infected without immunization. Immunized mice were given 100 μg of CHA or peptide and 4 μg of CTB on days 0, 1, 2, 10, 11, and 12 and challenged 9 days later. CTB only mice received 4 μg of CTB on the same days. ✽, Group mean was significantly lower than those of both the CTB-only and the infection-only groups (P < 0.05).

FIG. 2.

CHA-specific IgG1, IgG2a, and IgA responses in mice immunized with homogenate antigen plus CTB (CHA+CTB) or CTB only. Immunized mice were given 100 μg of CHA and/or 4 μg of CTB on days 0, 1, 2, 10, 11, and 12. Plasma samples and intestinal lavage fluid were collected on days 0, 20, 27, 34, and 38. Anti-CHA antibody was measured by ELISA. OD, OD at 410 nm (IgG) or 450 nm (IgA).

FIG. 4.

Levels of IL-5 (pg/ml ± the standard error [S.E.]) released in vitro from SC (A and C) and MLNC (B and D) of mice immunized i.n. with homogenate (CHA) or peptide antigen. Immunized mice were given 100 μg of CHA and 4 μg of CTB or 100 μg of peptide and 4 μg of CTB on days 0, 1, 2, 10, 11, and 12. Spleens and mesenteric lymph nodes were removed on days 20, 27, 34, and 38, and cell suspensions prepared and stimulated in vitro with 50 mg of CHA (for CHA immunizations)/ml or 5 mg of concanavalin A (for peptide immunizations)/ml. IL-5 was quantified by ELISA. The horizontal dashed line represents the mean IL-5 value of stimulated cells taken from unimmunized mice on days 0 and 38.

TABLE 1.

Levels of the cytokines IFN-γ and IL-5 released in vitro from T. spiralis antigen-stimulated SC and MLNC from mice immunized i.n. with CHA+CTB or CTB only

Cytokine and day	Mean level of cytokine released (U/ml) ± SEMa in:
	SC		MLNC
	CHA+CTB	CTB only	CHA+CTB	CTB only
IFN-γb
34	643*	75 ± 176	110 ± 39	642 ± 3
38	414 ± 209	187 ± 97	133 ± 83	595*
IL-2c
34	276 ± 41	ND	62 ± 24	11 ± 11
38	391 ± 12	5 ± 1	118 ± 12	19 ± 1

^aValues are means ± standard errors, except where an asterisk (*)denotes a single reading. ND, not done.

^bDay 38 naive cells: SC, 156 ± 7; MLNC, 118 ± 26 (values for antigen-stimulated cells from untreated mice).

^cDay 38 naive cells: SC, 52 ± 2; MLNC, 10* (values for antigen-stimulated cells from untreated mice).

Significantly, in preliminary experiments, peptide plus complete Freund adjuvant immunization given s.c. protected mice against subsequent challenge, inducing a strong peptide-specific IgG1 response and a lower IgG2a response (peak ODs at day 34 of 2.28 and 1.18). Data from i.n. immunization are shown in Fig. 1b. Immunized mice showed a significant loss of worms relative to the controls by day 8 (P < 0.01). Day 6 female worm fecundity (0.94 ± 0.06 larvae/female/h) was significantly reduced compared to the controls (1.41 ± 0.18 and 1.42 ± 0.05). Mice immunized with peptide+CTB i.n. produced high levels of peptide-specific IgG1 by day 20, but these levels subsequently declined (Fig. 3). In CTB-treated mice (controls), mean IgG1 levels were ca. OD = 0.8. IgG2a levels in peptide+CTB-immunized mice rose earlier (day 7) and peaked at day 20, but the OD values were lower than IgG1 and not significantly different from controls. Specific IgA (OD = 0.03 to 0.07) was low until day 34 and then rose sharply (0.155 ± 0.04) and was significantly higher the adjuvant controls (0.04 ± 0.005, P < 0.05). Significant IFN-γ responses were seen only in SC from day 20 (347 ± 74 pg/ml), with levels peaking at 1,323 ± 196 pg/ml on day 27 and declining to 19 ± 42 pg/ml on day 38. IL-5 levels rose significantly by day 20 in immunized mice in both SC and MLNC but then declined to control levels by day 38 (Fig. 4C, D). Also, the levels of IL-5 released from MLNC were seemingly greater in peptide-immunized mice compared with CHA-immunized animals.

FIG. 3.

CHA-specific IgG1 (top panel) and IgG2a (bottom panel) responses in mice immunized with peptide antigen plus CTB (peptide+CTB) or CTB only. Immunized mice were given 100 μg of peptide and/or 4 μg of CTB on days 0, 1, 2, 10, 11, and 12 and bled on days 0, 20, 27, 34, and 38. Anti-peptide antibody was measured by ELISA. OD, OD at 410 nm

Cholera toxin (CT) is a potent immunological adjuvant for antigens delivered nasally or orally (11), although nasal delivery seems more effective (8). Experiments in mice with CT as an adjuvant for orally administered antigens have shown that Th2-type responses were induced preferentially in both SC and Peyer's patches, and IgG1, IgE, and sIgA antibodies were stimulated (18, 19, 20). However, Th1 responses have also been reported (2).

Our data show that mice can be protected against an intestinal parasitic infection by i.n. immunization with both crude homogenate antigen and a peptide from the immunodominant 43-kDa antigen. Worm burdens were significantly reduced on day 8 p.i. compared to both infection-only and adjuvant controls, as was female worm fecundity. Immunity to T. spiralis in mice requires Th2-mediated immune responses (1), and the IgG isotype responses seen in immunized mice are consistent with this. Immunization also resulted in significant increases in antigen-specific IgA. In vitro-stimulated MLNC released significant amounts of IL-5 but not IFN-γ, suggesting that mucosal T cells had been appropriately polarized.

Oral immunization by using antigen with CT or CTB has been achieved against the nematodes Trichinella spiralis (4, 10) and Trichuris muris (11). Orally immunized mice showed enhanced mucosal IgA responses but not serum IgG antibodies; nasally immunized mice showed increases in both mucosal IgA and serum IgG1, suggesting induction of a predominantly Th2-mediated response. The cytokine data presented here indicate that i.n. immunization does induce a type 2 response in the mesenteric lymph node.

Successful s.c. immunization with a defined T. spiralis peptide antigen has been reported (12). The protection achieved here with a peptide given i.n. supports the observations of Tsuji et al. (13) with a recombinant 14-kDa antigen from Ascaris suum, although protection in this system must operate systemically and not intestinally.

In summary, we present results here showing for the first time that i.n. immunization is effective in eliciting immunity that operates within the intestine against a parasitic nematode. Encouragingly, immunity by this route of immunization was achieved with a defined peptide as well as a homogenate antigen preparation.