Evaluation of crude adult Ascaris suum intestinal tract homogenate in inducing protective IgG production against A. suum larvae in BALB/c mice

Highlights

• Immunization elicited a significant increase in larva-directed IgG response.

• No significant difference in parasite loads in either lung or liver tissues seen.

• Larval counts were very low and not reflective of the actual parasite load in mice.

• Correlation of IgG response with protection against parasite cannot be made.

Abstract

Globally, ascariasis ranks as the second leading intestinal helminth infection. However, progress in developing better control strategies, such as vaccines, remains slow-paced. This study aims to measure antibody production and parasite load in male BALB/c mice immunized with crude Ascaris suum intestinal tract homogenate.

Thirty-two (32) mice were randomized into: (1) unvaccinated, uninfected (UU); (2) unvaccinated, infected (UI); (3) vaccinated, uninfected (VU); and (4) vaccinated, infected (VI) groups. A 100-μL vaccine containing 50 μg of homogenized A. suum intestines and Complete Freund's Adjuvant (1:1) were introduced intraperitoneally. Immunizations were done on days 0, 10, and 20. Oral gavage with 1000 embryonated eggs was done on day 30. Blood was obtained at day 40. To measure serum IgG levels, indirect ELISA was done. Microtiter plates were coated with 100 μg larval homogenate, and HRP-conjugated anti-mouse IgG was used as secondary antibody. Parasite load was measured in lung and liver tissues.

Tukey's HSD of signal to cut-off ratios of absorbance readings obtained in indirect ELISA procedure for the 1:200 serum dilution showed statistically significant difference between the UU and VI (p = 0.026) as well as between UI and VI (p = 0.003) groups. No statistically significant difference in parasite load was observed in the lungs (p = 0.074), liver (p = 0.130), and both lungs and liver (p = 0.101).

Immunization elicited a significant larva-directed IgG production. However, there is no significant difference in parasite loads in either lung or liver tissues across all treatment groups as the larval counts obtained from the study were very low and may not be indicative of the actual parasite load in mice.

Introduction

Ascaris sp. is the most common human helminthic parasite. New estimates from the 2015 Global Burden of Disease Study indicate that approximately 761 million people are chronically infected with A. lumbricoides, resulting in 2700 annual deaths from ascariasis. Ascaris infections have a prevalence of 22% in the Philippines, accounting for 44,388 of disability-adjusted life years (DALYs) and <1% of total deaths (Leonardo et al., 2008).

Ascaris infection is contracted via the fecal-oral route. Eggs in the soil develop into first stage larvae (L1) without hatching. After ingestion by the host, the eggs travel to the duodenum where they hatch, and the larvae enter the second stage (L2). The larvae penetrate the mucosa and travel to the liver via the blood, then they develop into the third stage larvae (L3). The larvae migrate to the heart, then the lungs, a process that usually takes around five to six days. The larvae rupture the small capillaries in the lungs and migrate to the trachea. They are swallowed and enter the intestines once again, but as fourth stage larvae (L4), by nine days post-infection. The larvae are fully sexually mature after the final L4 moulting. In their reproduction, eggs are laid in the intestine and excreted via the feces, starting another cycle (Williams, 2011).

Despite the high global prevalence of soil transmitted helminthiasis, no effective vaccines have been developed against it (Hewitson and Maizels, 2014). The development of vaccines against helminth infections has proven to be a great challenge due to the complexity of the immunologic interactions in helminth infections as well as the lack of health policy and financial support on continuing research regarding the prevention and control of helminthiasis (Bethony et al., 2011).

The complexity of helminth life cycles and the high number of antigenic material produced at each developmental stage allows for several possible targets in vaccine development studies. Each larval stage also occupies a specific tissue niche, allowing a wide range of immunologic environments in which vaccines may be designed to intercept the infection. Vaccines have progressed over the years, from live, radiation-attenuated organisms to biochemical fractions recombinant proteins (Hewitson and Maizels, 2014). In many chronic helminth infections, the body's protective Type 2 immune response tend to be restrained by its own immunosuppressive mechanisms, thereby hindering the elimination of the parasite (Hewitson and Maizels, 2014). This immunoregulatory nature of helminth parasites is a reason why despite repeated exposure with infective stage parasites, some individuals fail to develop protective immunity to infection (Hewitson and Maizels, 2014). This poses challenges to finding the right combination of antigen, adjuvant, and route of administration (Hewitson and Maizels, 2014), contributing to the obstacles in helminth vaccine development.

No study as of writing has utilized the adult worm's gut as antigen for immunization. Most vaccine development studies had used larva or egg antigens, and most of them point to the same frailty: the inability of the vaccine to induce a sustained immune response. These antigens are exposed only transiently in the life cycle of the helminth due to multiple cuticle shedding, and the host is not given enough time to mount a robust and sustained immune response. This study explored the possible protective immune response that can be elicited by immunization of hidden antigens. The objective of the study is to measure IgG production and parasite load in BALB/c mice immunized with Ascaris suum crude intestinal tract antigens.