A Fasciola hepatica-derived fatty acid binding protein induces protection against schistosomiasis caused by Schistosoma bovis using the adjuvant adaptation (ADAD) vaccination system.

Several efforts have been made to identify anti-schistosomiasis vaccine candidates and new vaccination systems. The fatty acid binding protein (FAPB) has been shown to induce a high level of protection in trematode infection. The adjuvant adaptation (ADAD) vaccination system was used in this study, including recombinant FABP, a natural immunomodulator and saponins. Mice immunised with the ADAD system were able to up-regulate proinflammatory cytokines (IL-1 and IL-6) and induce high IgG2a levels. Moreover, there was a significant reduction in worm burden, egg liver and hepatic lesion in vaccinated mice in two independent experiments involving Schistosoma bovis infected mice. The foregoing data shows that ADAD system using FABP provide a good alternative for triggering an effective immune response against animal schistosomiasis.

Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to a recombinant Fasciola hepatica surface antigen in an endemic area.

The utility of an enzyme-linked immunosorbent assay to determine the sensitization against the trematode Fasciola hepatica in horses from an endemic area (NW Spain) was assessed. Blood samples were collected from 536 horses and tested against a 2.9-kDa recombinant surface protein (FhrAPS) to estimate the presence of IgG antibodies. Data were analysed regarding several intrinsic (age, gender and breed) and extrinsic factors (aptitude and housing). The farm size (number of horses/farm) was also considered. Sixty percent (95% CI 56, 64) of the horses were positive to the FhrAPS-ELISA, with a significantly higher seroprevalence in the mares (67%). Foals reached the lowest percentage of sensitization against the trematode (12%), and a significant positive correlation between the seroprevalence of fasciolosis and the age of the horses was established. When considering all the factors together, the seroprevalence of fasciolosis was initially classified into two groups (nodes) regarding the age of the horses. The node composed of the horses older than 1 year was then divided into two other clusters according to their gender. The mares were finally classified and grouped into two nodes regarding their breed. We concluded that the FhrAPS-ELISA is very useful for the demonstration of specific equine IgG antibodies against F. hepatica. An elevated risk of exposition to this trematode in horses maintained in endemic areas was proven. The possible role of horses as reservoirs for F. hepatica infections is discussed.

Adaptive immune stimulation is required to obtain high protection with fatty acid binding protein vaccine candidate against Fasciola hepatica in Balb/C mice.

Fascioliasis is a parasitic disease that mainly affects cattle and sheep, causing significant economic losses with a great impact in developing countries. Human fascioliasis is becoming more important with the high endemicity in some countries of the world. Previous studies have shown the importance of Fasciola hepatica fatty acid binding proteins (FABP) as protective molecules against fascioliasis in various animal models including mice, rabbits, and sheep. Our studies have shown the protective efficacy of recombinant FABP (rFh15) when the protein is formulated in the adjuvant adaptation system (ADAD), using either natural or synthetic immunomodulators. The ADAD system is most effective when it is used 5 days before each dose of specific vaccine antigen. The results showed survival rates of up to 50% with less severe hepatic lesions and high levels of IgG2a or IFNγ in immunized mice, using the ADAD system, compared to survival rates of 13% with no hepatic lesion reduction and high levels of IgG1 and IL-4 in those mice immunized with the simplified mode (ADADs).

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice.

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.

Reduction in the perinatal HIV transmission: the experience at the Maternal Infant Studies Center and Gamma Projects at the University of Puerto Rico School of Medicine.

The AIDS pandemic had a significant impact in Puerto Rico, especially among the heterosexual populations, in particular women. Women are one of the fastest growing risk groups with HIV/AIDS in the USA and constitute about half of the AIDS cases in the world. During the past 10 years Puerto Rico has ranked among the top 5 jurisdictions in the United States in AIDS cases rates, among men, women and children. In 1987 a universal prenatal HIV screening program was implemented in the University Hospital catchment area consisting of approximately 5,000 deliveries per year. Because of the early identification of pregnant women living with HIV, access to lifesaving clinical research and the implementation of multiple strategies and comprehensive care, the perinatal HIV transmission has been reduced to zero since 1997, with a blip of one case in 2002, and none since then. The availability and access to clinical and behavioral research has been one of the key elements for this success story. The programs involved and responsible for this spectacular outcome, namely the Maternal Infant Studies Center (CEMI-Spanish Acronym) and Gamma Projects at the University of Puerto Rico School of Medicine are described. The cost savings impact of stopping mother-infant perinatal HIV-1 transmission has been calculated to be approximately $34 to $58 million dollars in 10 years. The impact of the effectiveness of these programs in having healthy uninfected infants, prolonging and improving the quality of life of those living with HIV, and providing hope to families affected by this epidemic is incalculable.

Tungiasis: a case report.

This is a case report of a patient who developed a nodule in one foot, which upon biopsy, was diagnosed as tungiasis, a cutaneous infestation caused by a human flea. The natural life cycle, clinical and pathological expressions are discussed.

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice.

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.

A novel Fasciola hepatica saposinlike recombinant protein with immunoprophylactic potential.

A member of the Fasciola hepatica saposinlike/NK-lysin protein family with lytic activity on human peripheral blood mononuclear cells and erythrocytes was recently described. The current study was designed to test the immunoprophylactic potential of this protein termed FhSAP-2 against infection with F. hepatica in rabbits. Two doses of 50 microg of recombinant FhSAP-2 (rFhSAP-2) emulsified in TiterMax were injected subcutaneously on the dorsal surface of 4 rabbits at 2-wk intervals. Four weeks after the second immunization, the rabbits were infected orally with 25 F. hepatica metacercariae. Four non-immunized-infected rabbits were used as controls. An enzyme-linked immunosorbent assay revealed high levels of antibodies to both rFhSAP-2 and F. hepatica excretory-secretory antigens by 2 wk after the first immunization, which were always significantly higher in immunized-infected rabbits than in control-infected rabbits. On the completion of the trial, vaccinated rabbits had 81.2% less flukes than controls. Moreover, F. hepatica egg counts in feces, as well as in bile collected from the gall bladders from vaccinated animals, were lower, 83.8 and 73%, respectively, compared with controls. The vaccinated rabbits also had significantly lower amounts of parasite antigen in stool and bile samples than controls. Last, evaluation of macroscopic liver lesions revealed that the rabbits vaccinated with rFhSAP-2 had milder lesions than the infected-control rabbits. These findings support the hypothesis that this novel rFhSAP-2 protein has immunoprophylactic potential against fascioliasis in rabbits including antifecundity and antipathology effects. This is the first report on experimental vaccination of rabbits against F. hepatica with a purified, defined, recombinant protein related to a member of the saposinlike protein family.

Mother-to-child transmission of HIV-1: strong association with certain maternal HLA-B alleles independent of viral load implicates innate immune mechanisms.

The transmission of HIV-1 from mother to child during pregnancy is unlike other types of HIV-1 transmission because the child shares major histocompatibility complex (MHC) genes with the mother during a time while the mother is induced to tolerate the paternally derived fetal MHC molecules, in part through natural killer (NK) recognition of MHC polymorphisms. The relevance of these immune mechanisms to HIV-1 transmission was assessed by determining the HLA-B alleles of mother and infant. Almost half (48%) of mothers who transmitted with low viral loads had HLA-B*1302, B*3501, B*3503, B*4402, or B*5001 alleles, compared with 8% of nontransmitting mothers (P=0.001). Conversely, 25% of mothers who did not transmit despite high viral loads had B*4901 and B*5301, vs. 5% of transmitting mothers (P=0.003), a pattern of allelic involvement distinct from that influencing HIV-1 infection outcome. The infant's HLA-B alleles did not appear associated with transmission risk. The HLA-B*4901 and B*5301 alleles that were protective in the mother both differed respectively from the otherwise identical susceptibility alleles, B*5001 and B*3501, by 5 amino acids encoding the ligand for the KIR3DL1 NK receptor. These results suggest that the probable molecular basis of the observed association involves definition of the maternal NK recognition repertoire by engagement of NK receptors with polymorphic ligands encoded by maternal HLA-B alleles, and that the placenta is the likely site of the effect that appears to protect against transmission of maternal HIV-1 through interrelating adaptive and innate immune recognition.

Fatty acids bound to Fasciola hepatica 12 kDa fatty acid-binding protein, a candidate vaccine, differ from fatty acids in extracts of adult flukes.

The FA composition of Fasciola hepatica 12 kDA purified native FA-binding protein (nFh12), a candidate vaccine against fascioliasis, is described. The FA chain lengths ranged between 12 and 24 carbons. The principal FA were 16:0, 18:1n-9, 18:0, 20:4n-6, and 20:1n-9. The acids 16:0, 18:1n-9, and 18:0 comprised over half the FA that were bound to the whole FA-binding protein. Small amounts (1.0-2.8%) of iso-anteiso methyl-branched FA also were characterized. Forty-one different FA were identified in extracts of the adult flukes, with the three most abundant FA also being 16:0, 18:1n-9, and 18:0. A similar proportion of saturated vs. unsaturated FA was observed between the whole extract from F. hepatica and the nFh12 protein. However, the n-3/n-6 ratio of PUFA was significantly different, being 1.2 in the whole extract vs. 9.6 in the nFh12 protein complex. The nFh12 protein binds more n-5, n-6, and n-7 PUFA and less n-3 and n-9 PUFA than the whole extract. In addition, cholesterol (56%), sitosterol (36%), and fucosterol (8%) also were bound to the nFh12 protein complex.

Molecular cloning of a member of the Fasciola hepatica saposin-like protein family.

A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic alpha-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2-14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory-secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.

The rapid diagnosis of HIV-1 infection in mothers in Puerto Rico: a crucial testing strategy for maximal reduction of perinatal transmission.

This study was designed to evaluate early post partum rapid HIV testing of infants as surrogates for their mothers. In a screening of 971 infants whose mother's HIV-1 status was not known at delivery, 22 (= 2.26%) were found positive for antibodies by ELISA. Five were new cases and two (40%) were from transmitting mothers. This is in contrast with the UPR Women and Infants Transmission Study (UPR WITS) in which of 186 HIV-1 infected mothers none were transmitters. These were selected among thousands screened for anti-HIV-1 antibodies over a period of almost 5 years studied (September, 1996 through August, 2001). These results clearly indicate that all mothers at delivery should have a rapid test to determine their HIV-1 status to allow in the positive cases rapid intervention strategies to prevent perinatal transmission.

The rapid diagnosis of HIV-1 infection in mothers in Puerto Rico: a crucial testing strategy for maximal reduction of perinatal transmission

This study was designed to evaluate early post partum rapid HIV testing of infants as surrogates for their mothers. In a screening of 971 infants whose mother's HIV-1 status was not known at delivery, 22 (= 2.26) were found positive for antibodies by ELISA. Five were new cases and two (40) were from transmitting mothers. This is in contrast with the UPR Women and Infants Transmission Study (UPR WITS) in which of 186 HIV-1 infected mothers none were transmitters. These were selected among thousands screened for anti-HIV-1 antibodies over a period of almost 5 years studied (September, 1996 through August, 2001). These results clearly indicate that all mothers at delivery should have a rapid test to determine their HIV-1 status to allow in the positive cases rapid intervention strategies to prevent perinatal transmission.

Seroprevalence of Schistosoma mansoni in Puerto Ricans with inflammatory bowel disease

BACKGROUND: The etiology of Inflammatory Bowel Diseases, Crohn's disease (CD) and ulcerative colitis (UC), is unknown. These diseases have a higher incidence in industrialized countries and their pathogenesis involves an over-reaction of the immune system. A genetic factor is believed to predispose to the development of chronic inflammation in response to an unidentified stimulus. Exposure to infections in childhood may modulate future immune responses. Parasitosis, particularly Schistosomiasis, stimulate Th2 immune responses. It has been hypothesized that the absence of these parasitic infections, as seen in economically developed countries, favors a Th1 response that may result in the clinical appearance of Crohn's disease later in life. OBJECTIVE: To determine the prevalence of Schistosoma mansoni antibodies in Puerto Ricans with Inflammatory Bowel Disease and controls. METHODS: Serum from 92 Puerto Ricans with IBD and 106 controls was screened for S. mansoni adult microsomal antigens (MAMA) using the FAST:ELISA assay. Those positive were confirmed with an enzyme-linked immunoelectrotransfer blot test. RESULTS: Seven serum samples (3 UC and 4 controls) were positive for S. mansoni antibodies. There was no significant difference between groups in gender, municipality of origin or seroprevalence of Schistosomiasis. The control group was slightly older than the IBD group. CONCLUSIONS: Our study did not demonstrate an inverse relation between Schistosomiasis and IBD. However, the decreasing prevalence of Schistosomiasis in the general population of Puerto Rico may account for this result.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.