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R Chandrashekar

Publications and source records attributed to R Chandrashekar.

At least 37 records · Page 2Linked to original sources

Onchocerca volvulus: parasitologic and serologic responses in experimentally infected chimpanzees and mangabey monkeys.

Six chimpanzees (Pan troglodytes) and six mangabey monkeys (Cercocebus atys) were inoculated with Onchocerca volvulus third-stage larvae (L3) of West African origin. Two chimpanzees each received 200, 300, or 400 L3, while three mangabeys each received either 50 or 250 L3. All six chimpanzees became microfilaria positive between 11 and 25 months postinoculation (PI), while two of the six mangabeys were skin-snip positive at 24 and 37 months PI, respectively. All chimpanzees developed antibodies to two native antigens of 14 and 22 kDa and to the recombinant antigens OV16, OC3.6, and OC9.3. Marked antibody responses were observed in the mangabey monkeys, and in general, the responses were similar to those observed in the chimpanzees. However, in the mangabeys, these responses did not generally manifest themselves until later in the infection. The results of this study suggest that in chimpanzees, the smallest inoculum used, 200 L3, was sufficient to initiate consistent infections that had parasitologic and immunologic parameters equivalent to animals inoculated with larger numbers of larvae. Similarly, inoculation of mangabey monkeys with small numbers of larvae appeared to be as likely to establish infection and induce immunologic responses as did inoculation of larger numbers of larvae. Microfilaria-positive chimpanzees and mangabey monkeys were examined by three conventional imaging techniques (X ray, ultrasound, and magnetic resonance imaging (MRI)), but no adult worms or nodules could be identified in any animal.(ABSTRACT TRUNCATED AT 250 WORDS)

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Effect of ivermectin prophylaxis on antibody responses to Onchocerca volvulus recombinant antigens in experimentally infected chimpanzees.

Antibody responses to recombinant Onchocerca volvulus antigens were studied in experimentally infected chimpanzees. Sera from 3 groups of 6 animals were tested by ELISA with recombinant antigens OC 3.6 and OC 9.3. Groups I and II were treated with 200 micrograms/kg of ivermectin on the day of infection or on day 28, respectively. Group III were untreated controls. Antibodies to OC 3.6 developed during the prepatent period in all 3 groups. In contrast, antibodies to OC 9.3 were usually first detected around the time of onset of patency. Several animals had early antibody responses to OC 9.3, but these animals subsequently failed to develop microfilarial patency. Only 1 of 6 animals in group I produced a strong antibody response to OC 9.3 while all 12 animals in groups II and III developed antibodies to this antigen. Although there was some inconsistency in antibody responses observed in each treatment group, the results suggest that OC 9.3 may be more useful than OC 3.6 for monitoring the efficacy of prophylactic drugs or vaccines for onchocerciasis while OC 3.6 may be useful for detecting exposure to the parasite and early infection, regardless of the later outcome of the infection.

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Molecular characterization of a Brugia malayi intermediate filament protein which is an excretory-secretory product of adult worms.

Filarial parasites release macromolecules into their environment both in vitro and in vivo. These excretory-secretory products (E-S) have been studied with respect to function, vaccination potential, pathogenicity, and ability to serve as antigen targets for diagnostic tests. We have recently described monoclonal antibody OV-1 which binds to an intermediate filament in E-S and circulating antigens of Onchocerca volvulus. OV-1 also binds to cross-reactive antigens of Brugia malayi. Therefore, OV-1 was used to immunoscreen a B. malayi adult worm cDNA library in an attempt to clone a homologue (BMIF). BMIF is a 1664-bp full-length transcript which codes for 505 amino acids. BMIF has 95% sequence homology at the amino-acid level to OV1CF, an O. volvulus intermediate filament that was also selected with OV-1, and 75% homology to Ascaris intermediate filament A. Southern blot analysis suggests that BMIF is confined to a single location in the genomic DNA of B. malayi. Antibodies raised to BMIF identified native antigens in immunoblots of B. malayi adult worms, infective larvae and adult E-S. In addition, the antibody also bound to a 60-kDa antigen in immunoblots of poly(ethylene glycol)-precipitated immune complexes in sera from B. malayi infected patients. Localization studies showed that the antigen encoded by BMIF is present in the hypodermis, developing embryos and muscle of adult B. malayi. These studies show that BMIF is an E-S product of B. malayi adult worms which is detectable in sera from patients with brugian filariasis.

Amino Acid Sequence↗

Molecular characterization of a parasite antigen in sera from onchocerciasis patients that is immunologically cross-reactive with human keratin.

Onchocerca volvulus is a nematode that causes severe dermatitis and blindness in humans. Prior studies have shown that immune complexes in sera from onchocerciasis patients contain parasite antigens with M(r) of 23,000 and 65,000-70,000. Monoclonal antibody OV-1 binds to these antigens and to corresponding antigens in adult worm extracts and in vitro culture supernatants. OV-1 was used to immunoscreen an O. volvulus adult worm cDNA library. Clone OV1CF contains a 1632-bp open-reading frame that codes for a protein with a predicted M(r) of 63,000. The deduced protein sequence of OV1CF has 78% identity with Ascaris lumbricoides intermediate filament A and 40%-50% identity with several mammalian intermediate filaments. Antibodies raised to OV1CF bind to human keratin, and some sera from onchocerciasis patients contain antibodies to OV1CF and to keratin. Thus, immune complexes from onchocerciasis patients contain a parasite antigen that is immunologically cross-reactive with human intermediate filament proteins.

Adolescent↗

Molecular cloning of Brugia malayi antigens for diagnosis of lymphatic filariasis.

Immunological crossreactivity among nematodes has hampered development of specific serodiagnostic assays for lymphatic filariasis. In the present study, we report the molecular cloning and characterization of two filaria-specific recombinant clones (BmM5 and BmM14) with immunodiagnostic potential. BmM5 is a 505-bp cDNA which codes for a protein of 130 residues that ends with an endoplasmic reticulum targeting sequence. BmM14 is closely related to a recently reported clone (SXP-1), and it has 62% homology (deduced amino acid sequence) with a previously described Onchocerca volvulus clone, lambda RAL-2. Glutathione S-transferase fusion proteins of BmM5 and BmM14 were tested in various ELISA formats. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. ELISA studies showed that approximately 90% of 111 sera from Indian and Egyptian patients with brugian and bancroftian filariasis were reactive with both antigens. Nonendemic sera as well as sera from patients with schistosomiasis or intestinal helminths were uniformly nonreactive. Assays based on BmM5 and BmM14 may be useful for large scale screening as an alternative to microfilaria or filarial antigen detection as a means of obtaining a rough index of filariasis endemicity in previously unstudied areas.

Amino Acid Sequence↗

Vaccination with recombinant filarial paramyosin induces partial immunity to Brugia malayi infection in jirds.

Vaccination with irradiated infective larvae induces partial protective immunity to infection with the filarial nematode Brugia malayi in jirds. Prior studies have shown that such immunization stimulates a much stronger antibody response to recombinant and native filarial paramyosin than that seen after normal infection. To determine whether vaccination with recombinant paramyosin could induce protective immunity to larval challenge, jirds were immunized with either B. malayi paramyosin and maltose binding protein (BM5-MBP) (fusion protein of B. malayi paramyosin and maltose-binding protein), MBP alone, or recombinant Dirofilaria immitis paramyosin. Animals were challenged with 100 infective larvae s.c. 8 wk after the second immunization. Necropsies were performed 16 wk after challenge. Vaccination with BM5-MBP induced significant protective immunity; adult worm recoveries, worm lengths, and blood microfilaria counts were reduced in the BM5-MBP group relative to the MBP control group. The reductions in adult worm recoveries (43%) and female worm lengths (10%) were statistically significant (p < 0.05). Interestingly, protective immunity was not induced by immunization with D. immitis paramyosin. Additional studies are needed to identify mechanisms involved in protective immunity induced by BM5-MBP and to understand the differential activity of the two closely related recombinant paramyosin proteins in this model.

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Onchocerca volvulus: monoclonal antibodies to immune complex-associated parasite antigens.

Improved methods for diagnosis of onchocerciasis are needed. We have recently identified immune complex-associated parasite antigens in sera from onchocerciasis patients. The goal of this study was to produce monoclonal antibodies to these antigens that might be used in antigen detection assays. Two monoclonal antibodies (OV-1 and OV-5) that bind to parasite antigens in immunoblots of PEG-precipitated immune complexes from human onchocerciasis sera and to corresponding antigens in adult worm extracts and excretory-secretory products were produced. The target epitopes of the monoclonals are heat stable, resistant to trypsin, and destroyed by Pronase. The two monoclonals produce similar but not identical patterns of binding to immunoblots of Onchocerca volvulus adult worm antigen with major bands at 43-47, 58-63, and 70 kDa. OV-1 and OV-5 appear to bind to two distinct but closely related epitopes, neither of which is phosphorylcholine. Immunoelectron microscopy showed that the epitopes recognized by these monoclonals are widely distributed in adult female worms, but concentrated in the uterus and intestine. Antigen assays based on these antibodies detected parasite antigen in 9 of 14 sera from onchocerciasis patients, but significant background signal was detected in some nonendemic human sera. Thus, although this study has provided new information on parasite antigens in sera from onchocerciasis patients, additional work will be needed to achieve the goal of producing a sensitive and specific antigen diagnostic test for onchocerciasis.

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Preliminary evaluation of recombinant Onchocerca volvulus antigens for serodiagnosis of onchocerciasis.

Serodiagnostic assays for onchocerciasis based on native antigens are hampered by the scarcity of antigen, and they suffer from poor specificity. The present study was designed to evaluate the diagnostic utility of recently described recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 in enzyme immunoassays. The recombinant proteins were expressed as glutathione S-transferase fusions and were tested in several enzyme immunoassay formats to measure immunoglobulin G (IgG) and IgG4 antibodies with sera from patients with onchocerciasis in Nigeria and with various types of control sera. The best results were obtained by measuring IgG4 antibodies to the fusion proteins. Forty of 42 (95%) serum specimens from patients with onchocerciasis were reactive with OC 3.6; the reactivity with OC 9.3 was 81%. Results obtained with sera from experimentally infected chimpanzees suggest that OC 3.6 might be especially useful for detecting prepatent infections in humans, while OC 9.3 mainly detects mature, patent infections. Sera from individuals in Nigeria and the United States residing in areas nonendemic for onchocerciasis were uniformly nonreactive with these antigens in IgG and IgG4 assays, as were sera from patients with bancroftian filariasis, brugian filariasis, loiasis, ascariasis, schistomiasis, and dracunculiasis. These results suggest that enzyme immunoassays based on the recombinant antigens OC 3.6 and OC 9.3 are useful for the diagnosis of onchocerciasis.

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Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.

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Use of a recombinant antigen (OC 3.6 cDNA) for the serological diagnosis of onchocerciasis in exposed Nigerian children.

We evaluated a recombinant antigen (OC 3.6 cDNA) expressed in pMAL TM vector for the diagnosis of onchocerciasis in children living in an endemic focus of forest-type onchocerciasis in Nigeria. Using the Western blot with the maltose-binding fusion protein as antigen, 91 per cent of mf-positive children and 24 per cent of endemic normal children were positive. Furthermore, age was not a limitation to the assay. The practical limitations of the assay for field diagnosis of onchocerciasis is discussed.

Animals↗

Field evaluation of IgG4 serology for the diagnosis of onchocerciasis in children.

IgG4 serology was evaluated for early diagnosis of onchocerciasis in children living within an endemic focus of forest-type onchocerciasis in Nigeria. IgG4 serology proved to be more sensitive than conventional skin snip examination being able to detect 42% of infected children as opposed to 24% by snip examination. Furthermore, age was not a limitation to the technique within the 8-12 y spectrum. We conclude that IgG4 serology in children is a reliable indicator of community microfilarial rate and ongoing transmission of onchocerciasis.

Age Factors↗

Identification of paramyosin as a potential protective antigen against Brugia malayi infection in jirds.

Vaccination of jirds with irradiated infective larvae of Brugia malayi has been reported to provide partial immunity to larval challenge. In the present study, we found that sera from vaccinated animals recognized larval antigens with apparent molecular weights of 97, 55-60, and 10 kDa that were not recognized by sera from infected animals. A B. malayi cDNA expression library in lambda gt11 was screened to identify clones that were preferentially recognized by sera from immunized animals. One of these clones (BM-5) was chosen for further study. BM-5 contains a 2.1 kb DNA insert and produces a fusion protein with a molecular weight of 185 kDa. Antibody, affinity-purified with the BM-5 fusion protein, binds to a 97 kDa native B. malayi antigen. Immunological studies and partial DNA sequence data confirm that BM-5 encodes paramyosin. Recombinant B. malayi paramyosin is strongly recognized by antibodies in sera from jirds that have been immunized either by injection with irradiated larvae or by chemotherapy-abbreviated infection. Most sera from infected jirds do not contain antibody to paramyosin. Additional studies are needed to determine whether paramyosin is actually protective in this filariasis model.

Amino Acid Sequence↗

Molecular cloning and characterization of recombinant parasite antigens for immunodiagnosis of onchocerciasis.

Immunological cross-reactivity among nematodes has hampered the development of specific serodiagnostic assays for onchocerciasis. In the present study, an Onchocerca volvulus adult worm complementary DNA expression library was differentially screened with human sera from patients infected with O. volvulus and with an omnibus anti-nematode serum pool comprised of sera from patients infected with Brugia malayi, Loa loa, Wuchereria bancrofti, Mansonella perstans, Strongyloides stercoralis, Ancylostoma duodenale, Ascaris lumbricoides, and Dracunculus medinensis. Seven Onchocerca-specific clones were identified and screened with individual onchocerciasis patient sera. Additional studies were performed to characterize the most immunoreactive clones, OC 3.6 and OC 9.3. OC 3.6 produced a 152-kD beta-galactosidase fusion protein that was recognized in dot-immunoblots by 54 of 55 sera from onchocerciasis patients (98%). The OC 3.6 DNA insert is 996 bp long with an open reading frame of 627 bp and a 369-bp untranslated 3' end. OC 3.6 is closely related to a previously reported clone (OV 33-3), but it differs from that clone at both the 5' and 3' ends. OC 9.3 contained a novel 565-bp insert and produced a 138-kD fusion protein that was recognized by 46 of 55 sera from onchocerciasis patients (83%). Additional studies are in progress to develop and evaluate immunodiagnostic tests for onchocerciasis based on measurement of antibodies to these promising recombinant antigens.

Amino Acid Sequence↗

Effect of CGP 20376 on Brugia malayi and parasite antigenemia in jirds.

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.

Animals↗

Effect of ivermectin on filariae of Mastomys natalensis.

The efficacy of ivermectin (Iv) was evaluated against four species of filariae, Litomosoides carinii, Acanthocheilonema viteae, Brugia pahangi and Brugia malayi in Mastomys natalensis. Animals with patent infections, induced with L3 larvae, by intravenous (iv) infusion of the respective microfilariae (Mf) (5 x 10(4) Mf per animal) or by intraperitoneal (ip) route (2 x 10(4) Mf per animal) were used in this study. A single dose of Iv (100 micrograms.kg-1) given subcutaneously (sc) to Mastomys infected with L. carinii or A. viteae resulted in the disappearance of microfilaremia within 2 h of treatment. Iv treatment of sc-infected animals with Brugia spp. had no immediate effect on the circulating Mf 60 days post-treatment. In contrast, such treatment of animals infected with Mf by intravenous infusion completely eliminated the larvae of all four species from the circulation. Iv treatment had no significant effect on the Mf of L. carinii, B. pahangi and B. malayi in animals infected by the ip route. However, the drug had dramatic effect in killing the Mf of A. viteae in the peritoneal cavity. Sera from Iv-treated normal or from L. carinii- or A. viteae-infected Mastomys were effective in clearing the circulating Mf of the species when administered to animals with the respective infections. Similar rapid clearance of Mf was seen when the sera were administered to animals infected iv with these larvae. Furthermore, adult females of L. carinii and A. viteae recovered from Mastomys on different days after Iv treatment released smaller numbers of Mf in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

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