Can antibody conjugated nanomicelles alter the prospect of antibody targeted therapy against schistosomiasis mansoni?

BACKGROUND: CLA (conjugated linoleic acid)-mediated activation of the schistosome tegument-associated sphingomyelinase and consequent disruption of the outer membrane might allow host antibodies to access the apical membrane antigens. Here, we investigated a novel approach to enhance specific antibody delivery to concealed surface membrane antigens of Schistosoma mansoni utilising antibody-conjugated-CLA nanomicelle technology. METHODOLOGY/PRINCIPAL FINDINGS: We invented and characterised an amphiphilic CLA-loaded whey protein co-polymer (CLA-W) as an IV injectable protein nanocarrier. Rabbit anti-Schistosoma mansoni infection (anti-SmI) and anti-Schistosoma mansoni alkaline phosphatase specific IgG antibodies were purified from rabbit sera and conjugated to the surface of CLA-W co-polymer to form antibody-conjugated-CLA-W nanomicelles (Ab-CLA-W). We investigated the schistosomicidal effects of CLA-W and Ab-CLA-W in a mouse model of Schistosoma mansoni against early and late stages of infection. Results showed that conjugation of nanomicelles with antibodies, namely anti-SmI, significantly enhanced the micelles' schistosomicidal and anti-pathology activities at both the schistosomula and adult worm stages of the infection resulting in 64.6%-89.9% reductions in worm number; 72.5-94% and 66.4-85.2% reductions in hepatic eggs and granulomas, respectively. Treatment induced overall improvement in liver histopathology, reducing granuloma size and fibrosis and significantly affecting egg viability. Indirect immunofluorescence confirmed CLA-W-mediated antigen exposure on the worm surface. Electron microscopy revealed extensive ultrastructural damage in worm tegument induced by anti-SmI-CLA-W. CONCLUSION/SIGNIFICANCE: The novel antibody-targeted nano-sized CLA delivery system offers great promise for treatment of Schistosoma mansoni infection and control of its transmission. Our in vivo observations confirm an immune-mediated enhanced effect of the schistosomicidal action of CLA and hints at the prospect of nanotechnology-based immunotherapy, not only for schistosomiasis, but also for other parasitic infections in which chemotherapy has been shown to be immune-dependent. The results propose that the immunodominant reactivity of the anti-SmI serum, Schistosoma mansoni fructose biphosphate aldolase, SmFBPA, merits serious attention as a therapeutic and vaccine candidate.

In vitro interactions between the defense systems of resistant and susceptible Biomphalaria alexandrina and sporocysts of Schistosoma mansoni.

Biomphalaria species that act as an intermediate host for Schistosoma mansoni have different degrees of susceptibility and different internal defense system responses against parasites. Of these species, Biomphalaria alexandrina represents the only intermediate host in Egypt. Given the limited data on the efficacy of the B. alexandrina internal defense system in comparison to that of other species, we sought to better understand its defense against S. mansoni. We performed in vitro hemocyte adherence assay using whole hemolymph and in vitro reaction using the hemocyte-free hemolymph of susceptible and resistant snails against transformed mother sporocysts. The results demonstrated that the interacting factors between the parasite and the hemolymph of the resistant and susceptible snails do not act in a similar manner. Destruction of the parasite was a restricted function of the hemocytes among resistant snails only. This study demonstrates the key role played by snail hemocytes as a first line of defense against the parasite. The incubation of the hemocyte-free hemolymph of both susceptible and resistant snails with the sporocysts did not lead to any changes in the sporocysts shape or integrity. This immunological variance demonstrated between susceptible and resistant snails could be useful to differentiate between susceptible and resistant snails in future field studies. In addition, the results may help further studies to explain the process of attraction, encapsulation and subsequent killing of S. mansoni in its intermediate host.

Genetic variation between Biomphalaria alexandrina snails susceptible and resistant to Schistosoma mansoni infection.

Much effort has been made to control schistosomiasis infection in Egypt. However, enduring effects from such strategies have not yet been achieved. In this study, we sought to determine the genetic variability related to the interaction between Biomphalaria alexandrina snails and Schistosoma mansoni. Using RAPD-PCR with eight (10 mers) random primers, we were able to determine the polymorphic markers that differed between snails susceptible and resistant to Schistosoma mansoni infection using five primers out of the eight. Our results suggest that the RAPD-PCR technique is an efficient means by which to compare genomes and to detect genetic variations between schistosomiasis intermediate hosts. The RAPD technique with the above-noted primers can identify genomic markers that are specifically related to the Biomphalaria alexandrina/Schistosoma mansoni relationship in the absence of specific nucleotide sequence information. This approach could be used in epidemiologic surveys to investigate genetic diversity among Biomphalaria alexandrina snails. The ability to determine resistant markers in Biomphalaria alexandrina snails could potentially lead to further studies that use refractory snails as agents to control the spread of schistosomiasis.

Biomphalaria species in Alexandria water channels.

Of the several species of Biomphalaria snails worldwide that serve as the intermediate host for Schistosoma mansoni, Biomphalaria alexandrina is a species that is indigenous to Egypt. Recently, there has been much debate concerning the presence of Biomphalaria glabrata and the hybrid of the species with Biomphalaria alexandrina. Due to this debate, the absence of a clear explanation for the presence of B. glabrata in Egyptian water channels and the probability that they may be reintroduced, we conducted this field study to identify Biomphalaria species present in Alexandria water channels. Laboratory-adapted susceptible snails to Schistosoma mansoni of the following species were used as a reference; Biomphalaria alexandrina, Biomphalaria glabrata and their hybrid. These snails were used to perpetuate the Schistosoma life cycle at the Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Morphological and molecular studies were conducted on these reference snails as well as on the first generation of Biomphalaria snails from two areas in the Alexandria governorate. The morphological study included both external shell morphology and internal anatomy of the renal ridge. The molecular study used a species-specific PCR technique. The results demonstrated that there was an absence of Biomphalaria glabrata and the hybrid from Alexandria water channels. Moreover, the susceptibility patterns of these reference snails were studied by measuring the different parasitological parameters. It was found that Biomphalaria glabrata and the hybrid were significantly more susceptible than Biomphalaria alexandrina to the Egyptian strain of Schistosoma mansoni. The results demonstrated that if Biomphalaria glabrata was reintroduced and adapted to the local environment in Egypt, it would have important epidemiologic impacts that would have a serious effect on the health of Egyptian people.

Effects of intestinal protozoa infection on gastrointestinal transit and contractility in experimental animals.

Three groups of animals each was orally infected with Cryptosporidia parvum oocysts, Enterocytozoon bieneusi spores or Cyclospora cayetanensis oocysts. At the time peak of colonization of infected animals, each group and its corresponding control were infused orally with radioactive isotope. Gastric emptying of isotope was significantly greater in infected compared to controls in both fasted and fed states, to determine the effect of each parasite on the contractility of longitudinal and circular muscle, isometric tension of jejunal segments was recorded. The development of active tension with stretch and the dose-response curve to muscarinic against were significantly increased in the longitudinal muscle of infected animals compared to controls. The circular smooth muscle did not show an increase in contractility. The results suggest that an altered gastrointestinal transit and smooth muscle contractility may be involved in the intestinal protozoa infections' pathophysiology.

The effect of potassium permanganate and sodium dichloroisocyanurate on metacercariae of Fasciola gigantica.

Batches of encysted metacercariae of F. gigantica, adhered to transparent polyethylene sheets, were treated with KMnO4, while others were treated with NaDCC at specific concentrations and exposure times. Assessment of the effects was carried out by the detached percentage and viable metacercariae and by scanning electron microscope (SEM) ultrastructure changes. In addition, their effects on leaves of green vegetables were reported. The results showed that all metacercariae were detached and were dead by exposure to KMnO4 (96%) and NaDCC (100%) were detached from the polyethylene sheets. SEM showed that the deformities in the metacercariae soaked in NaDCC were more severe than those dipped in KMnO4. However, neither KMnO4 nor NaDCC affected the consistency, color, taste or flavor of the vegetables' leaves. The two disinfectants particularly NaDCC, proved to be safe, effective against the encysted metacercariae with no side-effects.

Further studies on autoclaved cercarial vaccine against schistosomiasis: safety, longevity and stability.

The autoclaved cercarial vaccine (ACV) which is a special type of killed vaccine has been reported to induce experimental high level of homologous protective immunity. This study was to adjust the dose and to assess vaccine safety, longevity and stability as well as the possibility of transplacental transmission of immune response from pregnant mice to their offspring. The results showed that two doses of the lowest most effective concentration of ACV that achieved the high percentage reduction of worm burden is safe as demonstrated by absence of any local or systemic side effects, normal blood picture and normal liver and kidney function tests. ACV is stable when kept either at 4 degrees C for six months or at -35 degrees C for up to 12 months and it offered considerable duration of longevity. Offspring of vaccinated mothers didn't show any signs of protection against challenge infection.

Co-agglutination (Co-A): a rapid test for the diagnosis of experimental trichinellosis.

Co-agglutination test (Co-A test) which has a wide application spectrum in bacterial diseases has been used for the detection of some parasitic infections. This work was to evaluate Co-A test as a new diagnostic tool in detection of T. spiralis antigens in both sera and urine of experimentally infected mice as compared with ELISA, for the diagnosis of trichinellosis. The results obtained by Co-A test were closely correlated with those of ELISA. The most important was that both tests were capable to detect T. spiralis antigen in urine of light and heavily infected mice only at the 30th days post infection. So, Co-A is an accurate, easy and rapid test that can be used on large scales for diagnosis of trichinellosis.

Vaccination against congenital toxoplasmosis.

Different types of Toxoplasma gondii vaccines were evaluated using parasitological and histopathological means to induce immunity in Swiss pregnant mice and their pups against the challenge with virulent RH strain. Immunization was performed before mating by using live cyst vaccines (LCV), LCV-IL-2 combination, irradiated cyst vaccine (ICV) and ICV-IL-2 combination. It was demonstrated that pre-immunization with the current vaccines offered significant protection of both dams and pups. The highest level of protection was noticed in mice which received LCV-IL-2, followed by ICV-IL-2, then LCV and the least protection was elicited in dams immunized with ICV alone. The results threw light on the possibility of applying such vaccines not only in mice but also in other mammalian hosts including human.

Bacterial-parasite interaction between Salmonella and each of Fasciola gigantica and Trichinella spiralis.

The possibility of bacterial-parasite interaction between Salmonella typhimurium and the surface of each of Fasciola gigantica metacercariae and Trichinella spiralis larvae was investigated in vitro. Two studies were carried out. In the first, S. typhimurium were incubated in vitro with the metacercariae of F. gigantica. In the second, S. typhimurium was incubated with larvae of T. spiralis. The interactions of S. typhimurium with each of F. gigantica metacercariae and T. spiralis larvae were studied by scanning electron microscope (SEM). In the first study, numerous bacilli were found adhered to the metacercariae surface. In the second study, no S. typhimurium was observed on the wall of T. spiralis larvae. The results indicated that only F. gigantica metacercariae act as a carrier for S. typhimurium with the possibility of occurrence of mixed infections with both organisms. So, both fascioliasis and salmonellosis must be treated concomitantly.