Humanized HLA-DR4 mice fed with the protozoan pathogen of oysters Perkinsus marinus (Dermo) do not develop noticeable pathology but elicit systemic immunity

Abstract

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.

Figure 1. P. marinus is sensitive to gastric pH and does not shed from the intestine.

Panel A, P. marinus parasites (7×10⁵ to 9×10⁵) were cultured for 10 minutes in media adjusted to pH values ranging from 2.0–6.2 and cell viability was measured by trypan blue exclusion. Data represent mean ± SD of two independent experiments. P values are indicated over the plots. Panel B, DR4.EA⁰ mice (n = 10) were fed with 10⁵ live P. marinus and set in 4 clean cages (2–3 mice per cage). Feces were collected at 24 h and 48 h post-feeding. DNA (10 ng) extracted from fecal samples was amplified with a pair of primers targeting NTS domain located between 5S and SSU rRNA genes from P. marinus (307 bp amplicon). Upper panel shows absence of parasite DNA in feces at 24 h and 48 h post-feeding. Lower panel shows that increasing concentration of fecal DNA (10 ng/µl) did not result in detectable PCR signal. Mixture of fecal DNA and purified parasite DNA (2∶1) resulted in positive PCR signal (lower panel) ruling out that potential inhibitory components in fecal material could have led to false negative results. PC, positive control; NC, negative control; MW, DNA molecular markers; Mix, Mixture of fecal DNA and purified P. marinus genomic DNA.

Figure 2. Oral administration of P. marinus does not induce gastrointestinal or lung pathology in DR4.EA⁰ mice.

DR4.EA⁰ mice were fed by gavage with 10⁵ live P. marinus and euthanized at 24 h, 48 h, or 7 days post-feeding (n = 3 mice per time point) for histological examination. Unfed age-matched mice were used as controls (n = 3). Organs fixed in formaline were processed and stained with H&E. Representative images were acquired using light microscopy under 10× objective (100× total visual magnification).

Figure 3. P. marinus does not alter the frequency of T and B cells in the gut.

Groups of DR4.EA⁰ mice were fed with P. marinus once and examined at days 5 or 14 after the feeding (n = 6 mice per time point) or they were fed twice (at two week interval) and examined at day 6 after the second feeding (n = 6). Controls (n = 7) were unfed mice. Lymphocytes isolated from Peyer’s patches (PP), intraepithelial (IEL), and lamina propria (PPL) were stained with mouse CD3, CD4, CD8, and CD19 Abs and analyzed by FACS. Panel A, frequency of B (CD19⁺) and T (CD3⁺) cells in control and P. marinus-fed mice. Panel B, frequency of CD4⁺, CD8⁺, CD4⁺CD8⁺, and CD4⁻CD8⁻ T cell subsets among gated CD3⁺ T cells. Data represent mean ± SD of mice analyzed individually. There were no significant differences for the frequency of lymphocytes in the gut of control mice as compared to mice fed with P. marinus (p>0.05 determine by unpaired t-test).

Figure 4. Humanized mice elicit IgG and IgM responses following oral feeding with P. marinus.

Panel A, IFA antibody titers in naïve (unfed) mice and mice fed with P. marinus (twice at two-week interval) measured at two weeks post-second feeding. Data represent titers in ten mice analyzed individually. Panel B shows representative IFAs.

Figure 5. Western blot analysis of IgG antibodies to P. marinus.

Panel A, P. marinus proteins extracted by thawing-freezing/ultrasonication as described in material and methods were separated in 4–15% SDS-PAGE gradient gel and silver-stained (lane 1); shown are the MW markers in lanes 2 (MagicMark) and 3 (Odyssey); Panel B show the same P. marinus protein sample after probing with sera from naïve mice (left) and from P. marinus-fed mice (right). The arrows indicate the major P. marinus protein bands recognized by IgG serum antibodies from naïve mice (arrows in panel B left, lane 1) and the most abundant P. marinus protein of approximately 60 kDa recognized by IgG serum antibodies from mice fed with P. marinus (arrow in panel B right, lane 1).

Figure 6. DR4.EA⁰ mice fed with P. marinus elicit cellular immunity.

Panel A, groups DR4.EA⁰ mice were fed with P. marinus twice at two-week interval and euthanized six days later. Controls were unfed mice. Spleen cells were stimulated with ConA (2 days) or P. marinus protein extract (4 days) and the levels of IFNγ in cell culture supernatants were measured by ELISA. Data represent mean ± SD of triplicate samples from three pooled spleens. Panel B, in an independent second experiment mice were fed with P. marinus as in panel A or unfed (control). Splenic cells were stimulated in triplicated samples (2 days) with P. marinus protein extracts or ConA and analyzed by ELISPOT. Control cultures were non-stimulated. Data represent mean spot forming units (sfu)/10⁶ cells ± SD of triplicated samples from control (n = 3) and P. marinus-fed mice (n = 5) analyzed individually. T cells from control mice did not produce IFNγ upon stimulation with P. marinus protein extracts (p = 0.07, paired t-test) while T cells from P. marinus-fed responded to stimulation (p = 0.001, paired t-test). The T cell response to polyclonal stimulation with ConA was significantly higher in spleens of P. marinus-fed mice as compared to control mice (p = 0.0005, unpaired t-test). Panel C&D, Frequencies and numbers of B cells (CD19⁺), T cells (CD3⁺), CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell subsets in spleens of mice fed once with P. marinus and examined at day 5 (1× d5) or day 14 (1× d14) post-feeding, mice fed twice with P. marinus (at two-week interval) and examined at day 6 post-feeding (2× d6) or control (unfed) mice. Panel E, Frequency of regulatory Foxp3⁺ T cells in spleens on P. marinus-fed and control mice. Data in panels C–E represent mean ± SD of six mice fed with P. marinus and seven control (unfed) mice analyzed individually. NS, not significant (p>0.05) determined by unpaired t-test.