George V. Hillyer

Fasciola hepatica: Molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein☆

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.

Fasciola antigens as vaccines against fascioliasis and schistosomiasis

Fascioliasis is an important trematode infection of herbivores worldwide with increasing evidence of prevalence as a disease of humans. Vaccination studies with purified native and recombinant Fasciola antigens suggest that this approach to diminished morbidity and mortality and reduced transmission is a realistic goal. Among the major potential vaccine candidates are fatty acid binding protein (FABP), cysteine (cathepsin) proteases, haemoglobulin, leucine aminopeptidase, and a saposin-like protein. In the case of Fasciola hepatica FABP, cross-reaction and cross-protection against Schistosoma mansoni is an important feature. In addition to protective effects with significant worm burden reductions, some vaccine candidates also have anti-fecundity (smaller flukes), anti-pathology (less liver lesions), and anti-embryonation effects. Optimism is tempered by the fact that fascioliasis in humans is an orphan disease and in need of governmental and foundation support.

Increased Vertical Transmission of Human Immunodeficiency Virus from Hepatitis C Virus-Coinfected Mothers

To determine if hepatitis C virus (HCV) infection affects vertical transmission of human immunodeficiency virus (HIV), 487 HIV-infected pregnant women in the prospective, multicenter, Women and Infants Transmission Study had HCV antibody (anti-HCV by second-generation ELISA) and HCV RNA (by quantitative polymerase chain reaction) measured in peripartum maternal plasma; 161 (33%) were anti-HCV-positive. HIV vertical transmission occurred from 42 HCV-infected mothers (26.1%) versus 53 HCV-uninfected mothers (16.3%; odds radio [OR], 1.82; P = .01). In a logistic regression model that included maternal drug use, a potential confounder, HCV infection was marginally associated with perinatal HIV transmission (OR, 1.64; P = .05), whereas drug use was not. Women who transmitted HIV had higher levels of HCV RNA (median, 721,254 copies/mL) than those who did not (337,561 copies/mL; P = .01). Maternal HCV infection is associated with increased HIV vertical transmission. Further studies are needed to ascertain if HCV directly affects perinatal HIV transmission or is a marker for another factor, such as maternal drug use.

Antibody Profiles by EITB and ELISA of Cattle and Sheep Infected with Fasciola hepatica

In evaluating potential mechanisms of immunity in fascioliasis we compared the time-course analysis of the antibody responses between a resistant (cattle) and a susceptible model (sheep). Sera from sheep and cows experimentally infected with F. hepatica were reacted with both somatic (FhWWE) and excretory-secretory (ES) antigens in order to evaluate the antibody repertoires in the 2 different hosts. Analysis of these sera by ELISA showed a significant increase in antibody levels by 2 wk in most infected cattle using both somatic and ES antigens, whereas with most infected sheep antibodies are not clearly detected until week 4. By EITB, both infected sheep and cows recognize major somatic polypeptides in a molecular weight range of 30-38 kDa by 8 wk. Cattle recognized 3 additional major antigens of 56, 64, and 69 kDa as early as 6 wk. Various polypeptides of 20-25 kDa are prominently detected by most sheep and very faintly, if at all, by the cow sera. The sera from both sheep and cows also identify ES polypeptides of 20-28 kDa. The patterns of polypeptides recognized by sheep infected with S. mansoni and challenged with F. hepatica in EITB are almost identical to those with a simple F. hepatica primary infection. No significant differences were detected in the antibody kinetics in ELISA between these 2 groups. Differences and similarities between these models could eventually help determine which antibodies may be predictive of resistance or susceptibility in fascioliasis.

Fasciola hepatica: vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins.

The current study was designed to compare the immunogenic and immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in rabbits infected with the fluke. Levels of specific anti-nFh12 and anti-rFh15 antibodies were significantly higher in the rabbits vaccinated twice compared with non-vaccinated infection controls. A reduction of 40% in worm burdens was found in rabbits immunized with nFh12 and infected 4 weeks after the second immunization. The recombinant vaccine induced lesser levels of protection than the native one, suggesting that both molecules may have slight differences either in immunogenicity or in their configuration. Further biochemical studies are required to define these differences. The mean length of flukes recovered was always smaller in all vaccinated rabbits. In addition, infected control rabbits had higher gamma glutamil transferase (GGT) levels than immunized rabbits. Lastly, gross anatomic observation always showed fewer liver lesions in all vaccinated rabbits than in controls. This finding clearly supports the possibility of vaccination regimes in fasciolosis.

Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins

The current study was designed to test the immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in sheep subsequently infected with the fluke. Thirty lambs were divided into six groups according to various patterns of immunisation and times of infection and necropsy. The antigens were emulsified in Freunds adjuvant. Levels of specific anti-nFh12 and anti-rFh15 antibodies rose rapidly by 2 weeks after the first immunisation and were always significantly higher in immunised-infected sheep than in control-infected sheep. On completion of the trial there was no difference in fluke burden between groups vaccinated with either of the antigens and non-immunised controls. However, worm size and faecal egg counts were significantly diminished in the sheep vaccinated with either of the antigens, suggesting an anti-fecundity effect. This is the first report of experimental vaccination of sheep against F. hepatica with purified native and recombinant antigens related to fatty acid binding proteins.

Early detection of human immunodeficiency virus on dried blood spot specimens: Sensitivity across serial specimens

OBJECTIVE To evaluate the use of dried blood spot (DBS) specimens and the early diagnostic value of the polymerase chain reaction (PCR) for detection of the human immunodeficiency virus (HIV) in DBS specimens collected at predefined age intervals from a large cohort of U.S. infants at risk of congenital or perinatal HIV infection. DESIGN We assayed available DBS specimens (n = 272) obtained during the first 4 months of life from 144 infants (41 infected, 103 uninfected) born to HIV-infected mothers enrolled in the Women and Infants Transmission Study. The DBS PCR results were compared with infant HIV infection status, PCR on liquid blood, and viral culture results. Analyses also included sensitivity and specificity of assay as related to the age of the infant when the specimen was obtained. RESULTS The DBS specimen PCR results were concordant with results from liquid blood specimens and with results from viral culture. The DBS PCR was highly specific for all age groups. Sensitivity in detecting HIV infection status rapidly increased during the first month of life, from 19% (5/26) by 1 week to 96% (25/26) by 1 month of age. Specimens obtained on the day of birth or the next day were the least likely to have detectable HIV DNA. CONCLUSIONS The PCR assay of DBS specimens is a reliable tool for the early diagnosis of HIV infection and has important advantage over that of liquid blood DNA PCR and viral culture. These advantages include a lower volume of blood required for testing, increased safety, and ease of storage or transport of specimens. Thus DBS PCR is a useful test for clinical and epidemiologic tracking of infants at risk of HIV infection.

Seroreversion in human immunodeficiency virus-exposed but uninfected infants

The goal of this study was to describe seroreversion (SR) in a cohort of human immunodeficiency virus-exposed but uninfected infants. Groups of patients who seroreverted very early or late were examined for salient clinical and immunologic characteristics of the mother or infant. The mean time (± s.d.) to seroreversion by enzyme-linked immunoabsorbent assay (ELISA) was 50.1 ± 14.8 weeks, or 11.6 months (n = 84); the range of times to antibody loss by ELISA was 17.9 to 82.0 weeks. The mean time to seroreversion by Western blot was 68.3 ± 12.6 weeks, or 15.8 months (n = 51), with a range of 44.9 to 94.1 weeks. Initial anti-human immunodeficiency virus titer as measured by cord blood ELISA optical density (OD) was found to relate significantly to mean time to seroreversion. No relationship to time to seroreversion was demonstrated for gestational age, maternal or neonatal serum immunoglobulin concentrations, maternal CD4 cell counts, maternal alchol consumption, infantile diarrhea or failure to thrive. The lengthy time to seroreversion seen here demonstrates the 1994 revised Centers for Disease Control and Prevention definition of human immunodeficiency virus infection (based on seropositivity by both ELISA and confirmatory tests persisting beyond 18 months of age) to be accurate in our population. We recommend Western blot testing be used as confirmation for positive ELISAs only after 18 months of age.

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 infants 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.

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 α-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.