Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice

Abstract

Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

Figure 1. A. marginale infection in mice determined by microscopy and qPCR of infected erythrocytes.

Mice erythrocytes free of infection (A) were submitted to A. marginale injection (3×10⁵ rickettsia) and blood smears were visualized after 30 days (B). MSP5 A. marginale copies of infected erythrocytes per µL of blood was quantified by qPCR on days 0, 15 and 30 post-infection (C). The 143-pb MSP5 amplicon is shown in agarose gel electrophoresis (D).

Figure 2. Survival proportions, body weights and infected erythrocytes of mice immunized with synthetic peptides in Anaplasma marginale challenge assay.

FrA and PBS groups mice showed the highest body weight losses from 18th to 22th day after challenge (A), but the survival percentage of these groups where not significantly different among groups (B). Negative controls included non-immunized and unchallenged mice, which have shown no alteration in weight in comparison to the baseline and in mortality (data not shown). Infected erythrocytes for all groups upon challenge with A. marginale are represented as percentage (C) and its infected rate reduction relative to PBS group (D).

Figure 3. Profile of IgG antibody formation during the whole stage of experiment.

Total IgG antibody response of BALB/C mice immunized subcutaneously three times with Am1, Am2, Anaplasma Lysate Antigen (ALA), adjuvant control (FrA) or PBS (infection control) against Am1 (A), Am2 (B) and ALA (C), determined by ELISA. Mice were challenged with 3×10⁵ rickettsia after the 45^th day. Blood samples were collected at 0, 15, 30, 45 and 75 days after immunization. *Statistically significant differences (p<0.001). (D) ALA SDS-PAGE 12% stained with colloidal comassie and western immunobloting of ALA detected by sera of immunized mice with Am1 (lane 1), Am2 (lane 2), ALA (lane 3), Freund adjuvant (lane 4) and PBS (lane 5). Molecular weight (MW, kDa) and the immunodominant antigen are indicated.

Figure 4. Serum IgG isotypes levels and profile.

Specific IgG1 and IgG2a response were analyzed in the serum of mice before challenge (BC) and 30 days after challenge (AC). *Statistically significant differences between IgG1 and IgG2a; ^astatistically significant differences between IgG1 BC and AC; ^bstatistically significant differences between IgG2a, BC and AC (p<0.05).

Figure 5. Effects of immunization on cytokine expression of mice splenocytes.

The Cytokine expression was quantified before challenge (A) and after A. marginale challenge (B) by real-time PCR with focus on mRNA expression of IL-12, IL-18, IFN-γ, TNF-α, IL-10 and TGF-β. Results are presented as the mean ± SE of the fold-change of mRNA in peptides, ALA or FrA immunized mice (n = 3, A; n = 7, B) compared to negative control (PBS-immunized mice). Animals were challenged with 3×10⁵ rickettsia after 45th day. *Statistically significant differences (p<0.05); **p<0.01; ***p<0.0001.