Detection of antibodies to a recombinant Cryptosporidium parvum p23 in serum and feces from neonatal calves

Passive transfer of maternal antibodies via colostrum is important to protect newborn ruminants against microbial pathogens. In this study, 10 sets of calf serum, a sample of the colostrum fed to the calf, and serial fecal samples through the first 6 days after birth were collected from arbitrarily selected newborn Holstein heifers. A recombinant Cryptosporidium parvum p23, termed rC7, was used to determine whether anti-C. parvum antibodies can be detected in clinically normal neonates. The results demonstrated that serum, the associated colostrum, and fecal samples contained anti-rC7 antibodies. IgM and IgG1 anti-rC7 tended to be present in highest titers. The presence of specific antibodies to C. parvum was confirmed using Western blots of purified sporozoite membranes probed with serum and colostral whey. Collectively, the results indicated that neonatal calves had antibodies to C. parvum as early as 1 day after birth and suggested that the antibodies were passively transferred.     

Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein

Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.     

Monoclonal anti-idiotypic antibody mimicking the principal neutralization site in HIV-1 GP120 induces HIV-1 neutralizing antibodies in rabbits

A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.     

A novel detection method of Cryptosporidium parvum infection in cattle based on Cryptosporidium parvum virus 1

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry,especially the cattle industry.As there is no specific vaccine or drug against Cryptosporidium,a rapid and accurate method for the detection of C.parvum is of great significance.In this study,colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C.parvum infection in cattle fecal samples.The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold.A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines,respectively.Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution.There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples.The different preservation conditions (room temperature,4℃,and 37℃) and preservation time (7,30,60,and 90 days) were analyzed.The data showed that the strips could be preserved for 90 days at 4℃ and for 60 days at room temperature or 37℃.The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun,China.The results indicated that the rate of a positive test was 5%(6/120).This study provides a rapid and accurate method for detecting C.parvum infection in cattle and humans.     

Pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.     

Single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen P23 Of Cryptosporidium parvum

Cryptosporidium parvum, an apicomplexan parasite transmitted via animal fecal wastes, is the causative agent of cryptosporidiosis. Clones were selected from 2 synthetic na?ve human single-chain variable fragment (scFv) phagemid libraries that bound to the recombinant P23 protein of C. parvum. Panning the Tomlinson I and J phagemid libraries resulted in 6 distinct clones. Two clones had full-length scFv sequences, while the remaining clones were either truncated or missing a section of the heavy chain. Despite these differences, all clones were able to detect both native C. parvum proteins and recombinant P23. None of the selected clones cross-reacted with Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Giardia lamblia (cysts or trophozoites), or with S16, another dominant surface antigen on C. parvum sporozoites. Clones expressed as the scFv-gIIIp fusion construct in soluble form detected C. parvum. Panning from na?ve libraries is a useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.     

Effect of bovine manure on Cryptosporidium parvum oocyst attachment to soil

The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.     

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region

Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.     

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.     

The humoral and cellular immune responses in mice induced by DNA vaccine expressing the sporozoite surface protein of Cryptosporidium parvum

Cryptosporidiosis, a protozoan disease, is caused by Cryptosporidium parvum in animals and humans. To study the humoral and cellular immune responses induced by DNA vaccine expressing the sporozoite surface protein, CP15/60, of Cryptosporidium parvum, the recombinant plasmid containing the CP15/60 gene was injected into tibialis a interior muscle of BALB/c mice. The mice were subsequently given booster doses twice at 3-week intervals. The humoral and cellular immune responses were detected at different times after immunization. The mice were then challenged by inoculation of 1 x 10(6) oocysts of C. parvum. The experimental results have shown that the recombinant plasmid can induce corresponding specific immune responses and thus protect the mice from challenge of the oocysts, suggesting that the recombinant plasmid could be a potential candidate of DNA vaccine.     

An exposed domain in the severe acute respiratory syndrome coronavirus spike protein induces neutralizing antibodies

Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.     

Immunogold labeling of stages of Cryptosporidium parvum recognized by immunoglobulins in hyperimmune bovine colostrum

Ultrathin sections of mouse ileum infected with Cryptosporidium parvum were stained by immunogold techniques. Sections first were stained with polyvalent antibodies in whey from hyperimmune bovine colostrum (HBC), then stained by secondary antibodies in rabbit antibovine IgA, IgM, IgG1, and IgG2, and lastly labeled by goat anti-rabbit gold conjugate. Examination of the immunostained specimens by electron microscopy revealed that each bovine immunoglobulin isotype in the whey recognized antigens in meronts, merozoites, microgametocytes, microgametes, and macrogamonts. Based on these findings it is hypothesized that antigens in all stages of C. parvum provide targets of opportunity for the antiparasitic activity of HBC whey antibodies thereby accounting for its efficacy as an immunotherapeutic agent.     

Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340.

The Epstein-Barr virus membrane antigen gene gp340 was isolated, inserted into several strains of vaccinia virus and expressed under the control of a vaccinia virus promoter. The EBV-derived protein which was produced by the recombinant vaccinia viruses was heavily glycosylated, readily labelled with threonine, could be detected at the surface of infected cells and had a mol. wt. of approximately 340 kd, all of which are properties of the authentic gp340. Polyclonal rabbit antisera against gp340 and an EBV-neutralising anti-gp340 monoclonal antibody both recognised cells infected with the recombinant vaccinia viruses. Moreover, rabbits vaccinated with one of the recombinants produced antibodies that recognised EBV-containing lymphoblastoid cells and neutralised EBV.     

Effect of hyperimmune bovine colostrum raised against Cryptosporidium parvum on infection of guinea pigs by Cryptosporidium wrairi.

Oocysts shedding was markedly reduced in guinea pigs inoculated intraintestinally with Cryptosporidium wrairi sporozoites that had been incubated with hyperimmune bovine colostrum raised to C. parvum when compared with shedding in guinea pigs inoculated with sporozoites incubated in either non-immune bovine colostrum or buffered saline. However oocyst shedding was apparently not reduced in guinea pigs inoculated by gavage with oocysts of C. wrairi and subsequently treated twice daily per os with hyperimmune bovine colostrum. Similarly, oocyst shedding was apparently not reduced by oral treatment with hyperimmune bovine colostrum when treatment was begun simultaneously with inoculation of C. wrairi oocysts.     

Apoptosis in bovine herpesvirus-1 infected bovine peripheral blood mononuclear cells

Apoptosis is a process whereby cells die in a controlled manner in response to various stimuli like cytotoxins, viral antigens and normal physiological signals during differentiation and development. Virus induced immunosuppression has been reported for various viral diseases including Bovine Herpesvirus-1 (BHV-1). In the present study, BHV-1 was found to cause apoptosis in ConA stimulated bovine peripheral blood mononuclear cells (PBMCs). Apoptotic index quantified by fluorescent dyes revealed a significant (P < 0.001) increase in percent apoptotic cells at 2, 24 and 48 hr post infection as compared to their respective non-infected controls. Apoptosis specific internucleosomal laddering in DNA from BHV-1 infected PBMCs was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control non-infected PBMCs.