Anti-Budding Assay: Development of a high throughput screen to identify neutralizing antibodies that block Chikungunya virus (CHIKV) release

Objective: No licensed CHIKV vaccines or effective therapeutic agents are currently available. However, some CHIKV-specific monoclonal antibodies (mAbs) are highly effective in animal models, both prophylactically and therapeutically. This is thought to be largely mediated via blocking of CHIKV entry into cells. However, we and others have shown that inhibition of viral release/ budding is also a major mechanism for CHIKV control. We aimed to develop a high throughput in vitro screening assay to efficiently identify "antibudding"mAbs. Design and Methodology: An assay for quantification of viral budding from infected cells was optimised by varying cell line, cell density, multiplicity of infection (MOI), incubation periods, NH4Cl concentration and plate type. The assay utilized our novel, fully replication -competent, attenuated CHIKV nano-luciferase (nluc) reporter virus (CHIKV 181/25 E2nluc). The optimised assay was used to screen CHIKV+, Zika virus (ZIKV)+, CHIKV-/ZIKV- sera, and cloned memory B-cells from a CHIKV+ individual. Results: Optimal conditions involved use of rhabdomyosarcoma (RD) cells, bulk-infected at MOI 1 for 2hrs, removal of residual virus, resuspension in media containing 20mM NH4Cl, seeding at 2.5x104cells/well into 96-well plates and luminometry after 18hrs. Inter-plate coefficient of variability CV scores and Z' values remained <15% and >0.5 respectively, indicative of a valid assay. Most CHIKV+ sera displayed potent antibudding activity, two displayed no significant activity, and there was no ZIKV cross-reactivity. Of 800 memory B-cell clones, 13 exhibited significant anti-budding antibody activity. Conclusions: We developed a sensitive, reproducible, Biosafety level (BSL-2) safe, high throughput CHIKV antibudding assay useful for screening both polyclonal sera and monoclonal antibodies.

Los anticuerpos monoclonales en la caracterizacion y vigilancia de los virus de la rabia en America Latina y el Caribe / Monoclonal antibodies for rabies virus characterization and epidemiological surveillance in Latin America and the Caribbean

As one of the activities of the Rabies Reference Laboratories Consortium of the Pan AMerican Health Organization, a technical consultation meeting was held in late 1999 where well-known experts from Europe, North America, and South America analyzed the contributions to rabies epidemiological surveillance in Latin America and the Caribbean made by techniques of antigenic typing based on monoclonal antibodies and by techniques of genetic typing based on gene sequencing (AU)

Detection of dengue infection in patients investigated for leptospirosis in Barbados

The annual incidence of leptospirosis in Barbados is approximately 13 severe cases/100,000. The peak incidence occurs in October to December of each year, coinciding with the months of heaviest rainfall. During the second half of 1995, the epidemic of dengue type 1 infection produced almost 1.000 laboratory-confirmed cases. During the same period, leptospirosis mortality was twice the average, suggesting that some cases of leptospirosis were being misdiagnosed and treated inappropriately. Sera from patients investigated for dengue or leptospirosis were analyzed retrospectively to determine the extent of misdiagnosis. During 1995 and 1996, 31 of 139 and 29 of 93 patients, respectively, were confirmed as having leptospirosis. Sera from the remaining leptospirosis-negative patients were tested for IgM antibodies to dengue virus. During 1995 and 1996, 48 of 108 patients and 21 of 64 patients, respectively, were found to have dengue. In 1997, sera from all patients investigated for leptospirosis were treated prospectively for IgM antibodies to dengue: 38 of 92 leptospirosis-negative patients (41 percent) were dengue IgM-positive, while 2 of 25 leptospirosis cases also had serologic evidence suggesting acute dengue infection. A second large outbreak of dengue caused by serotype 2 occurred in 1997 dengue epidemics in Barbados, dengue cases outnumbered leptospirosis cases investigated in the leptospirosis diagnostic protocol. During, 1997, patients investigated but negative for dengue were also tested for anti-leptospiral IgM: 7.3 percent (19 of 262) were IgM-positive. Substantial misdiagnosis of both dengue and leptospirosis can occur and greater public awareness and clinical suspicion of the similar presentations of these two diseases are necessary. (AU)

Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica

Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (Mabs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-bourne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independently regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus haemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDA tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-bourne encephalitis virus, determination of domain C was more problematic: however, at least four epitopes and biochemical characteristics consistent with C-domain epitopes(AU)

Radioimmunotherapy of interleukin-2R - expressing adult T-cell leukemia with yttrium-90-labeled anti-tac

Adult T-cell leukemia (ATL) is a malignancy of mature lumphocytes caused by the retrovirus human T-cell lymphotropic virus-I. It is an aggressive leukemia with a median survival time of 9 months: no chemotherapy regimen appears successful inducing long-term disease-free survival. The scientific basis of the present study is the ATL cells express high-affinity interleukin-2 receptors identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this differnce, we administered anti-Tac armed with Yttrium-90 (Y) to 18 patients with ATL initially (first 9 patients) in a phase I dose-escalation trial and subsequently (second group of 9 patients) in a phase II trial involving a uniform 10-mCi dose of Y-labeled anti-Tac. Patients undergoing a remission were permitted to receive up to eight additional doses. At the 5-to 15-mCi doses used, 9 of 16 evaluable patients responded to Y anti-Tac with a partial (7 patients) or complete (2 patients) remission. The responses observed represent improved efficacy in terms of length of remission when compared with previous results with unmodified anti-Tac. Clinically meaningful (> grade 3) toxicity was largely limited to the hematopoietic system. In conclusion, radioimmunotherapy with Y anti-Tac directed toward the IL-2R expressed on ATL cells may provide a useful approach for treatment of this aggressive malignancy.(AU)

Trichuriasis: spontaneous and stimulated Th1/Th2 cytokine production by isolated T-cells - abstract

Although trichuriasis is the most prevalent known infection in the CARICOM countries, only a small proportion of infected children acquire intense infection, i.e., Trichuris Dysentry Syndrome (TDS). Hypotheses to be investigated are that there is a specific T-Cell anergy to the parasite in such hosts or that they have general predisposition to the "TH2" T-helper-cell response, which would be in keeping with the TDS local anaphylactic response we have shown by various techniques in our other reports. We investigated 3 groups of children: controls from a surgical private practice unlikely to have ever been significantly infected TDS cases. We used the ELISPOT technique with primary and secondary monoclonal antibodies to show cytokine production by cells separated from peripheral blood or colonic mucosal biopsies, expressing the final result in numbers of positive cells per 100,000 CD3+ cells (T-cells). Interferon-gamma detection was taken as indicative of the TH1 phenotype and Interleukin 4 (IL4) as indicative of TH2. Spontaneous production in short-term culture was compared with that after stimulation ionophore, staphylococcal enterotoxin B (superantigen) or T. trichiura excretory-secretory (ES) antigen. There was great variability in proportions of cytokine positive cells after culture, both spontaneously and after stimulation. There was also variability in the rank of potency of a given stimulant to "the T-cells of different children. ES antigen provoked the greatest IL4 response for one active TDS case, refuting the anergy hypothesis. The active cases had two highest productions (spontaneous) of interferon-gamma and (stimulated) of IL4. No significant difference in Th1/Th2 phenotypic profile emerged between the control and ex-TDS groups. We conclude that it is not innate character in T-helper-cell response to mitogen or antigen that accounts for predisposition to intense trichuriasis (AU)

Cytotoxic activities of recombinant immunotoxins composed of pseudomonas toxin or diphtheria toxin toward lymphocytes from patients with adult T-cell leukemia

We have previously shown that the recombinant single-chain immunotoxin anti-Tac (Fv)-PE40, composed of the variabe domains of the anti-Tac monoclonal antibody in a single-chain form joined to a derivative of pseudomonas exotoxin (PE), is cytotoxic toward malignant cells form adult T-cell leukemia (ATL) patients. Using this assay, we have now compared the activity of anti-Tac(Fv)-PE40 with that of an improved version, anti-Tac (Fv)-PE40KDEL which contains an altered carboxyl terminus, and also with two chimeric toxins made with diphtheria toxin (DT). One of these is a fusion of amino acids 1-388 of DT with anti-Tac(Fv) and is termed DT388-anti-Tac(Fv). The other, DT388-IL2, contains interleukin 2 (IL2) at the carboxyl terminus of the same DT derivative. We incubated these toxin with malignant ATL peripheral blood mononuclear cells (PBMCs) for 1-3 days and then measured [3H]leucine incorporation. We found that anti-Tac(Fv)-PE40KDEL was the most cytotoxic agent and was followed in decreasing order of activity by anti-Tac(Fv)-PE40, DT388-anti-Tac(Fv), and finally DT388-IL2. Trypan blue staining showed that inhibition of protein synthesis correlated with cell death. Time course studies show that the recombinant toxins containing anti-Tac(Fv)-PE40DEL was 30 minutes. Normal PBMCs were resistant to all four toxins. Recombinant immunotoxins made with anti-Tac merit further study as potential reagents in the treatment of ATL.(AU)

Segregation of human T cell lymphotropic virus type I and II infections by antibody reactivity to unique viral epitopes

A recombinant protein of the human T cell lymphotropic virus type I (HTLV-I) gp46 outer membrane envelope, MTA-4 (residues 129-203), reacted by Western blot with sera from HTLV-I-infected individuals from the United States and Jamaica but not with 24 (10 percent) of 242 Japanese sera. A related gp46 recombinant protein, MTA-1 (residues 162-209), reacted with all 58 sera from HTLV-I-infected US and Jamaican individuals and 238 of 242 sera from infected Japanese (combined sensitivity of 99 percent). Neither recombinant showed reactivity to sera from HTLV-II-infected individuals or uninfected controls. The reactivity of recombinant proteins containing the region of HTLV-II gp46 analogous to MTA-1 was also evaluated by Western blot: GH2-K15 (residues 157-205) and GH2-K55 (residues 162-205) reacted with 88 (98 percent) and 89 (99 percent), respectively, of 90 sera from HTLV-II-infected individuals but not with sera from HTLV-I-infected individuals or uninfected controls. These recombinant proteins should permit the development of assays to unambiguously confirm and differentiate HTLV-I and HTLV-II infections. (AU)

Leptospires isolated from toads and frogs on the island of Barbados

Four pathogenic strains of leptospires were isolated from the kidneys of toads (Bufo marinus) and seven from frogs (Eleutherodactylus johnstonei). Isolates from two toads and one frog belonged to serovar bim, the causative agent of most cases of severe leptospirosis on Barbados. The other eight strains belonged to a new serovar within the Australis serogroup. The name bajan is proposed for this new serovar of Leptospira interrogans. (AU)

Retention of leptospiral agglutinins and long-term response to administration of monoclonal antibodies in vervet monkeys (Cercopithecus aethiops) on Barbados

In a study of 21 wild-caught Barbadian vervet monkeys (Cercopithecus aethiops sabaeus), naturally-acquired leptospiral agglutinins were found to persist for over five years. Groups of seropositive as well as seronegative vervets were given a placebo, or full-strength monoclonal antibodies MCA F12C3 (Icterohaemorrhagiae copenhageni), or diluted F12C3 MCAs. They were challenged 24 hours later with a suspension of highly virulent leptospires (copenhageni) administered intraperitoneally. Immunoprotection was evident in animals receiving full strength MCAs as measured by their failure to develop any sunstantial antibody response and by their lower geometric mean titres over a period of 142 weeks (maximum GMT of 113 compared with a maximum of 1280 in the placebo group). Diluted MCAs had little or no protective value. The serological response of the monkeys which were seropositive at capture to challenge with virulent copenhageni antigen was strongly anamnestic both in those given MCAs and those given placebo. None of the naturally or experimentally infected vervets showed clinical signs of leptospiral illness. (AU)

Warthin's tumour in Jamaica: incidence, electron microscopy and immunoenzyme studies

Warthin's tumour has traditionally had a strong male association, and has been said to be rare in Blacks. Current studies describe a newtrend; a rise in females, strongly linked to cigarette smoking. The tumour has eosinophilic epithelial cells packed with distinctive mitochondria, and a lymphoid stroma. Immunological investigations have demonstrated polyclonal B cells and macrophages. Views differ as to whether B or T cells predominate. Between 1958 and 1989, the Jamaica Cancer Registry recorded 491 benign and malignant salivary gland tumours. There were 18 cases of Warthin's tumour (3.7 percent), with a male:female ratio of 5:1. The low proportion of females is similar to the trend for female lung cancer in Kingston & St. Andrew. A case of Warthin's tumour was studied by light and electron microscopy and immunoenzyme methods. The epithelial cells contained numerous mitochondria with stacked cristae, as previously described. Similar morphology occurs in oncocytic tumours; riboflavin-deficient rats and mice; rats given non-lethal doses of hypoglycin; dogs treated with annatto extracts; and hibernating or starving frogs. The mitochondrial changes may be an adaptive response. The immunoenzyme studies utilized newly available monoclonalantibodies: UCHLI, L26, 4KB5, MT1 and LN2. The reaction patterns indicate a distribution of B and T cells in a manner expected in a lymph node. The interaction between mitochondrial changes adaptive metabolic pathways, the immune cells and tobacco raises some interesting questions (AU)

The immunophenotyping of non-hodgkin's lymphoma in formalin-fixed paraffin-embedded tissue - abstract

In this study, we have retrospectively determined the immunophenotype of forty-five cases of non-Hodgkin's lymphoma (HNL) diagnosed in the Department of Pathology at the University Hospital of the West Indies (UHWI) between 1984 and 1986. We used an immunoalkaline phosphatase technique with a panel of monoclonal antibodies reactive against T-cells (UCHL 1; MT 1), B cells (L 26; 4KB5; LN 2) and macrophages (Ber.H2). We found that this panel could accurately identify T-cell lymphomas and B cell lymphomas in formalin-fixed paraffin-embedded tissues. Of forty-five cases, twenty-six were of T-cell lineage and twelve were of B-cell lineage. Sixteen of the T-cell lymphomas were human T-cell leukemia-lymphoma virus type 1 (HTLV-1) positive and showed the additional clinical and pathological features of adult T-cell leukemia/lymphoma (ATL). The HTLV-1 negative cases were neither morphologically nor immunologically different from the HTLV-1 positive patients. None of the B cell lymphomas were HTLV-1 positive. The results lend further support to the finding that NHL in Jamaican patients is predominantly of T cell origin and associated with human T cell lymphotropic virus, type 1 (AU)

High human T-cell lymphotropic virus type I proviral DNA load with polyclonal integration in peripheral blood mononuclear cells of French West Indian, Guianese, and African patients with tropical spastic paraparesis

Human T-cell lymphotropic virus type I (HTLV-I) proviral integration status was examined by Southern blot analysis in peripheral blood mononuclear cell (PBMC) DNA from patients presenting a tropical spastic paraparesis (TSP) and serological evidence of HTLV-I infection. Surface phenotype and morphological aspects of PBMC were also studied. A polyclonal HTLV-I proviral integration was found in the PBMC of the 10 patients studied irrespective of their geographical origin (French West Indies, French Guiana, and Africa), the duration of their clincal illness, or the HTLV-I antibody titer. Furthermore, by dilution experiments and hypothesizing that only one copy of HTLV-I proviral DNA is present in one call, we estimated that this HTLV-I integration is present in 3 percent to 15 percent of their PBMC. All 10 TSP/HTLV-I patients studied had an average of 10 percent of thier lymphocytes abnormal, presening either a misshapen nucleus or an adult T-cell leukemia/lymphoma(ATL)-like feature. Moreover, an elevated CD4/CD8 ratio associated with the presence of activated T cells with a high level of DR expression was observed in most patients. The significant frequency of viral-positive PBMC and the important load of HTLV-I proviral DNA that we observed in TSP/HTLV-I patients might play an important role in the pathogenesis of this recently identified clinico-virological entity. (AU)

Monoclonal antibodies to herapin sulphate proteoglycan: development and application to the study of normal tissue and pathological human kidney biopsies - abstract

In an attempt to quantify the immuno-histochemical properties of the glomerular basement membrane (GBM), monoclonal antibodies (mAbs) were prepared against heparin sulphate Proteoglycan (HSPG). The HSPG was isolated from bovine glomerulae. Enzyme-linked immuno-sorbent assays (ELISA) and immunoblotting demonstrated that the mAbs reacted with HSPG. Indirect immuno-fluorescence showed that the mAbs stained renal basement membranes (BMs) and BMs in other organs of normal bovine and human tissues in patterns typical of HSPG. Immuno-inhibition studies, and immuno-blotting of heparin lyase digested HSPG, indicated that the mAbs recognizes HSPG core protein. Human kidney biopsies revealed interesting patterns of staining for HSPG. For instance, in kidney biopsies from patients with acute post-streptococcal glomerulonephritis, intact linear staining for HSPG was noted despite abnormal pathological processes shown by widened capillary loops. On the other hand, in membranous and in diffuse proliferative lupus glomerulonephritis, loss of HSPG staining was demonstrated at sites of immuno-deposition of IgG or C3; whereas, increased staining for HSPG was seen in areas of newly formed GBM. Extensive loss of HSPG was seen in areas of glomerular sclerosis and necrosis. In biopsies from patients with minimal change glomerulonephritis and mesangio-proliferative lupus glomerulonephritis, a normal linear GBM distribution of HSPG was noted. The study demonstrates that immunological injury to the HSPG component of the GBM of the kidneys plays an aetiological role in the pathogenesis of proteinuria in man (AU)

Identification of an antigenic and genetic variant of dengue-4 virus from the Caribbean

Twenty-one dengue (DEN) viruses isolated from the Caribbean (Dominica and Jamaica) during the 1981-1982 epidemic year were distinct serological and genetic variants of DEN-4 virus. These isolates were clearly identified as DEN-4 viruses using type-specific monoclonal antibodies in indirect immunofluorescence assays. However, they either were not neutralized, or were neutralized poorly using hyperimmune mouse ascitic fluids (HMAF) or rhesus monkey serum directed against the H-241 prototype strain of DEN-4 virus isolated in the Philippines in 1956. HMAF prepared against a representative Caribbean isolate, however, neutralized with similar effectiveness the homologoous virus, the H-241 prototype strain, and virus strains isolated from the Pacific and Southeast Asian areas from 1973 to 1984. The Caribbean isolate exhibited no more than 30 percent and 16 percent oligonucleotide spot homology with the H-241 and Bankok viruses, respectively, by RNA fingerprint analysis, while demonstrating 82 percent and 89 percent homology with the Gilbert and Niue Island isolates, respectively. The isolation of dengue viruses which are serologically and genetically distinct from the prototype virus emphasizes the need for continued dengue virus surveillance. The recognition of unique dengue isolates should allow the selection of reference strains and vaccine candidate strains which will induce antibodies that are equally effective in neutralizing virus from all geographic areas. (AU)

Mapping of antigenic sites on human haemoglobin by means of monoclonal antibodies and haemoglobin variants

Monoclonal antibodies specific for human globin chains have been prepared and the following strategy has been applied in delimiting the antigenic sites involved in antibody binding. The structural sites of the human globin subunit that might be recognised by the monoclonal antibody were deduced from comparisons of the primary structures of mamalian globin chains that did or did not react with the antibody. The involvement of individual residues at these specific sites was subsequently tested by reacting the antibody with abnormal human haemoglobins in which there was either a substitution or a structural site recognised by monoclonal antibody HuHb á 3-2 (an antibody that reacts with the adult haemoglobins from man and macaque monkey, but not with those from baboon and mouse) includes the aspartic acid residue at position 52 of the á-globin subunit.(AU)