A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens.

Caracterização biológica e molecular de populações clonais de cepas do Trypanososma cruzi: 21SF - biodema tipo II e colombiana - biodema tipo III, isolados de camundongos infectados, tratados com benzonidazol e não curados / Biological and molecular characterization of clonal populations Trypanososma cruzi strains: 21SF - biodeme type II and Colombian - biodeme type III, isolated from infected mice treated with benznidazole and uncured

As cepas e clones do Trypanosoma cruzi apresentam diferentes aspectos de resistência e susceptibilidade aos quimioterápicos. Vários estudos vem sendo desenvolvidos para avaliar a resposta de diversas drogas em diferentes cepas protótipos dos Biodemas tipos I, II e III, de acordo com a caracterização biológica. Resultados vêm demonstrando que cepas protótipos do Biodema Tipo I (cepas Y e Perunana) apresentam uma alta susceptibilidade ao tratamento com Benzonidazol e Nifurtimox; cepas do Biodema Tipo II (protótipo: cepa 21SF) demonstram média susceptibilidade; as cepas do Biodema Tipo III (cepa Colombiana) são altamente resistentes. Considerando que as cepas do T.cruzi são populações multiclonais complexas, que diferem nas suas características genéticas e biológicas, clones de duas cepas do T.cruzi foram avaliadas com o objetivo de investigar se o uso da quimioterapia anti–T.cruzi poderia estar levando à seleção de clones resistentes que poderão ou não diferir nos seus caracteres biológicos e moleculares. No presente trabalho investigamos os caracteres biológicas e moleculares de clones da cepa Colombiana (Biodema Tipo III) e 21SF (Biodema Tipo II) do T. cruzi, isolados de animais tratados e não curados em comparação com clones isolados de animais não tratados, com o intuito de investigar se estes clones resistentes à quimioterapia apresentavam diferenças em suas características que pudessem estar justificando tal resistência. Para isto, 18 clones foram isolados de camundongos infectados da cepa Colombiana e 8 clones foram isolados de camundongos infectados com a cepa 21SF. Os resultados mostraram que as características biológicas dos clones isolados da cepa Colombiana foram mantidas; clones da cepa 21SF mostraram diferentes níveis de parasitemia quando comparados com a cepa parental. As características moleculares foram avaliadas a partir dos fragmentos do k-DNA de cada clone isolado, que foram submetidos à técnica de RFLP, utilizando as enzimas de restrição RSA I, HINF I e ECO RI. A análise dos fragmentos de restrição das cepas parentais e dos respectivos clones demonstrou grande similaridade entres os mesmos. A possibilidade de investigar a estrutura molecular utilizando outras técnicas moleculares, poderá contribuir para demonstrar diferenças na resistência dos clones isolados de animais tratados e não não curados, como visto na discussão

Different strains and clones of Trypanosoma cruzi present differents degrees of susceptibility to treatment with chemotherapic drugs. Several studies have been developed to evaluate the response to different drugs concerning the strains prototypes of the Biodemes Types I, II and III according to the biological characterization. Results have shown that the strains prototypes of the Biodeme Type I (Y and Peruvian strains) disclosed a high susceptibility to treatment with Benznidazole and Nifurtimox; strains of the Biodeme Type II (prototype: the 21SF strain) showed a medium susceptibility; the strains of the Biodeme Type III (Colombian strain) were highly resistant. However a variability was detected according to the phase of infection in which the clones were isolated, and varied from 0% to 23,5% for the clones isolated in an early phase and 0% to 16.0% for those isolated in a late phase of infection. This indicates that the clonal populations could differently respond in different phases of treatment. In the present study we investigated the biological and molecular characters of clones of the 21SF strain (Biodeme Type II ) and of the Colombian strain (Biodeme Type III) isolated from mice treated with Benznidazole, but not cured , in comparison with clones isolated from untreated mice, with the objective of to investigate possible differences in the biological and molecular characteristics of these resistant clones. For that 18 clones were isolated from mice infected with the Colombian strain and 08 clones isolated from those infected with the 21SF strain. Results have shown that the biological characteristics were maintained in the Colombian strain; clones of the 21SF strain showed different levels of parasitemia as compared with the parental strain. This behavior is peculiar to clones isolated from the 21SF as shown in previous studies with clones isolated from untreated mice. The molecular characteristics were evaluated through the restriction fragment length polymorphism (RFLP) of the k DNA for each isolate clone, using restriction enzymes RSA I, HINF I and ECO RI...

Cinética de crecimiento in vitro de dos cepas de Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) en células miocárdicas de roedores con diferente susceptibilidad / In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility. Two Trypanosoma cruzi isolates, TCR-4 from Costa Rica and UES-1 from El Salvador, were studied in vitro to compare their infectivity or resistance and intracellular replication in myocardial cells in three strains of mice and rats: NGP white mice, C3 H mice and Sprague Dowley rats. Myocardial cells were cultured on coverslips at 37 degrees C in a humid 10% CO2 atmosphere and then infected at a ratio of one tripomastigote per cell. Samples were studied after 24, 72, 96 and 120 h of infection to determine parasite infection capacity and intracellular multiplication. Both parasites had the highest infection capacity in C3 H mice, followed by NGP mice cells with a very low infection rate. Lastly, almost no Trypanosoma cruzi multiplication was observed in Sprague Dowley rats, suggesting a strong natural resistance in this animal to both strains of the parasite. The UES-1 isolate presented higher multiplication and greater invasion than the TCR-4 strain, showing greater virulence of UES-1 in heart cells, at least in vitro.

[In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility]. / Cinética de crecimiento in vitro de dos cepas de Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) en células miocárdicas de roedores con diferente susceptibilidad.

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility. Two Trypanosoma cruzi isolates, TCR-4 from Costa Rica and UES-1 from El Salvador, were studied in vitro to compare their infectivity or resistance and intracellular replication in myocardial cells in three strains of mice and rats: NGP white mice, C3 H mice and Sprague Dowley rats. Myocardial cells were cultured on coverslips at 37 degrees C in a humid 10% CO2 atmosphere and then infected at a ratio of one tripomastigote per cell. Samples were studied after 24, 72, 96 and 120 h of infection to determine parasite infection capacity and intracellular multiplication. Both parasites had the highest infection capacity in C3 H mice, followed by NGP mice cells with a very low infection rate. Lastly, almost no Trypanosoma cruzi multiplication was observed in Sprague Dowley rats, suggesting a strong natural resistance in this animal to both strains of the parasite. The UES-1 isolate presented higher multiplication and greater invasion than the TCR-4 strain, showing greater virulence of UES-1 in heart cells, at least in vitro.

Test of synergistic interaction between infection and inbreeding in Daphnia magna.

It has been proposed that parasitic infections increase selection against inbred genotypes. We tested this hypothesis experimentally using pairs of selfed and outcrossed sibling lines of the freshwater crustacean Daphnia magna, which can be maintained clonally. We studied the performance of selfed relative to outcrossed sibling clones during repeated pairwise clonal competition in the presence and absence of two species of microsporidian parasites. In 13 of the 14 pairs, the selfed clones did worse than the outcrossed ones in the control treatment, but the presence of either parasite did not result in an overall increase in this difference. Rather, it decreased the performance of the selfed relative to the outcrossed sibling in some pairs and increased it in others. Moreover, the two parasite species did not have the same effect in a given pair. This indicates that, contrary to the hypothesis that parasites generally lead to a decreased performance of inbred genotypes, their effect may depend on the genetic background of the host as well as on the parasite species, and suggests that inbreeding can lead to reduced or increased resistance to parasites. Our findings also indicate that there is variation for specific resistance to different species of parasites in the meta-population from which the hosts for this study were obtained.

Evidence for strong host clone-parasite species interactions in the Daphnia microparasite system.

Organisms are often confronted with multiple enemy species. Defenses against different parasite species may be traded off against each other. However, if resistance is based on (potentially costly) general defense mechanisms, it may be positively correlated among parasites. In an experimental study, we confronted 19 clones from one Daphnia magna population with two bacterial and three microsporidian parasite species. All parasites were isolated from the same pond as the hosts. Host clones were specific in their susceptibility towards different parasite species, and parasite species were host-clone specific in their infectivity, spore production, and virulence, resulting in highly significant host-parasite interactions. Since the Daphnia's resistance to different parasite species showed no obvious correlation, neither general defense mechanisms nor trade-offs in resistance explain our findings. None of the Daphnia clones were resistant to all parasite species, and the average level of resistance was quite similar among clones. This may reflect a cost of defense, so that the cumulative cost of being resistant to all parasite species might be too high.

T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development.

Excreted-secreted Ags (ESA) of Toxoplasma gondii (Tg) play an important role in the stimulation of the host immune system in both acute and chronic infections. To identify the parasite Ag(s) involved in the maintenance of T cell-mediated long term immunity, 40 ESA-specific T cell clones were derived from three chronically infected healthy subjects. All the clones were CD4+ and recognized both ESA and live tachyzoites in a HLA-DR-restricted manner. Conversely, CD4+ tachyzoite-specific T cell clones from the same subjects proliferated in response to ESA, pointing to shared immunodominant Ags between ESA and Tg tachyzoites. By T cell blot analysis using SDS-PAGE-fractionated parasite extracts, the following patterns of reactivity were detected. Of 25 clones, 6 recognized Tg fractions in the 24- to 28-kDa range and proliferated to purified GRA2, 5 reacted with Tg fractions in the 30- to 33-kDa range; and 4 of them proved to be specific for rSAg1. Although surface Ag (SAg1) is not a member of ESA, small amounts of this protein were present in ESA preparation by Western blot. Of 25 clones, 8 responded to Tg fractions in the 50- to 60-kDa range but not to the 55-kDa recombinant rhoptries-2 parasite Ag, and 6 did not react with any Tg fraction but proliferated in response to either ESA or total parasite extracts. In conclusion, CD4+ T cells specific for either ESA (GRA2) or SAg1 may be involved in the maintenance of long term immunity to Tg in healthy chronically infected individuals.

Cryptic infections in mice with the Trypanosoma cruzi CL-14 clone

A infectividade do clone CL-14 do Trypanosoma cruzi para camundongos foi revista utilizando-se como inoculo metaciclicos de cultura em NNN+LIT, pre-incubados ou nao com complemento de cobaio. Nos animais inoculados nao observamos parasitemia patente, mas a presenca do parasito foi confirmada em 30 por cento deles (9/30) atraves de hemocultivo ou xenodiagnostico, este examinado aos 100 dias. A positividade...

Comparative analysis by polymerase chain reaction amplified minicircles of kinetiplast DNA of a stable strain of Trypanosoma cruzi from Säo Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of Säo Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodene 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reation (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght plymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100 per cent. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone.

Differences between B cell and macrophage transformation by the bovine parasite, Theileria annulata: a clonal approach.

Theileria annulata, a tick-transmitted protozoan parasite, infects and transforms cells of the hemopoietic system, particularly those of the B cell and monocyte/macrophage lineages. Here, the effect of infection/transformation on the resulting phenotype was studied using a clonal approach. Three phenotypes of transformed cell lines could be discerned. The first is characterized by surface expression of IgM, CD21, and the B cell epitopes, B-B2 and B-B8, Ig heavy chain gene rearrangement, and mRNA expression. Such lines were obtained from fresh and cultured PBMC and at increased frequency from purified B cells, but never from fetal bone marrow cells. The second phenotype can be distinguished from the first by the absence of Ig heavy chain expression and reduced surface expression of B cell markers (CD21, B-B2, B-B8). Clones with this phenotype were obtained from transformed fetal bone marrow cells only. The third phenotype showed an absence of all of the above B cell markers, including surface IgM, and a lack of Ig heavy chain gene rearrangement. The latter clones could be maintained for several weeks after elimination of T. annulata by BW720c treatment, and they reacquired a macrophage-like phenotype. This implies that parasite-induced dedifferentiation is restricted to monocyte/macrophage, and that B cell markers are indicative of cell lineage progeny. Demonstration of surface IgM on PBMC-derived B cell clones suggests that infection of B cells with T. annulata may be an epigenetic method to immortalize ruminant B cells of a defined Ag specificity.

Contrasting genomic variability between clones from field isolates and laboratory populations of Schistosoma mansoni

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using ramdomly amplified polymorphic DNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43 per cent of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analysis of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates.

Partial inhibition of Theileria parva-infected T-cell proliferation by antisense IL2R alpha-chain RNA expression.

Antisense RNA expression was used to assess the role of the interleukin 2 receptor (IL2R) alpha-chain in proliferation of T cells transformed by infection with the intracellular parasite Theileria parva. Two vectors were constructed, in which part of the bovine IL2R alpha-chain cDNA was inserted in either a sense or antisense orientation in the plasmid pHS1-CAT, which is expressed by induction of the human metallothionein IIA (hMT-IIA) with cadmium (Cd2+). A T. parva-infected T-cell line, TpM(803), known to express the IL2 and IL2R genes in a constitutive way, was permanently transfected with one of the two constructs, and the effect of antisense IL2R alpha-chain RNA expression upon proliferation of TpM(803) cells was tested. Antisense-transfected TpM(803) cells grew much more slowly than sense-transfected cells even in the absence of added Cd2+, but the addition of Cd2+ to the culture medium resulted in further inhibition. A strong reduction in IL2R alpha-chain mRNA was observed in antisense but not in sense-transfected cells. These observations confirm a role for the IL2R alpha-chain in proliferation of T. parva-infected TpM(803) cells.

Disruption of synchrony between parasite growth and host cell division is a determinant of differentiation to the merozoite in Theileria annulata.

The multinucleated macroschizont stage of the protozoon Theileria annulata is an intracellular parasite of bovine leukocytes. The parasite induces the host cell to proliferate, and divides in synchrony with the immortalised host cell. Differentiation to the next stage occurs within the host cell culminating in the release of merozoites and destruction of the leukocyte. In this study clones of Theileria annulata macroschizont-infected cell lines were isolated by limiting dilution and tested for differentiation to the merozoite stage (merogony). Two cloned cell lines underwent differentiation with enhanced efficiency, while two others were of lower efficiency. Quantification was carried out using monoclonal antibodies, which showed that over 90% of the cells in an enhanced cloned cell line could be induced to differentiate. By carrying out induction at 41 degrees C for limited periods of time followed by culture at 37 degrees C evidence was obtained that differentiation to the merozoite is a two-step process: a preliminary reversible phase, followed by a second irreversible phase of differentiation. Analysis of the nuclear number of the macroschizont and the growth rate of the cloned cell lines showed that the ability to differentiate was associated with an increase in nuclear number (size) of the macroschizont, generated by a disruption in the synchrony between parasite growth and host cell division. We believe that these results reveal a relationship between a reduction in parasite division and differentiation, and that there are similarities between stage differentiation in parasites and cellular differentiation in higher eukaryotes.

Participation of natural killer cells in the recovery of mice from visceral leishmaniasis.

After infection with the protozoan parasite Leishmania donovani, C57BL/6J bg/bg (beige) mice, which are deficient in natural killer (NK) activity, were unable to control splenic parasite loads relative to phenotypically normal C57BL/6J bg/+ and +/+ mice, particularly beyond 21 days of infection. When beige mice were injected intravenously with 2 or 3 X 10(6) syngeneic, cloned NK cells (NKB61B10 cell line), they displayed splenic parasite burdens which did not differ significantly from those of normal controls. In C57BL/6 +/+ mice rendered NK deficient by split-dose irradiation (four weekly, 200-rad doses of gamma irradiation beginning at 4 weeks of age) splenic and hepatic parasite levels were significantly higher than those in nonirradiated controls at 15 days of infection and beyond. In both sets of experiments, relative degrees of hepato- and splenomegaly were not sufficient to account for differences in parasite burdens among NK-deficient and normal mice. Taken together, the results of these experiments suggest that NK cells may contribute to parasite elimination during the acquired-resistance phase of L. donovani infection in mice.

Eimeria tenella: in vitro development in irradiated bovine kidney cells.

The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G2 phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

Growth of Trypanosoma cruzi in a cloned macrophage cell line and in a variant defective in oxygen metabolism.

A continuous cloned murine macrophage-like cell line, clone 16 derived from J774, has been found upon appropriate stimulation to be capable of oxidizing glucose by the hexose monophosphate shunt and producing O2- and H2O2. A variant in oxidative metabolism, clone C3C, was selected from this cell line which under similar conditions is unable to produce significant amounts of O2- and H2O2. When cells of the parental clone 16 were infected with epimastigotes of Trypanosoma cruzi, there was significant killing or growth inhibition of the parasites at 3 to 4 days after infection. In contrast, the parasites grew in the oxidative variant, clone C3C. Trypomastigote forms of T. cruzi were found to be only partially killed in the parental clone 16 but grew abundantly in the oxidative variant. Infection of the parental clone, but not the variant, was sufficient to stimulate oxygen metabolism as demonstrated by the increased reduction of nitro blue tetrazolium. Studies on the killing of T. cruzi epimastigotes in cell-free suspension by xanthine-xanthine oxidase indicated that 90% of the killing was catalase sensitive and due to H2O2, with at most 7 to 8% killing which could be inhibited by scavengers of . OH and singlet oxygen (1O2). In the in vitro experiment with H2O2 produced by glucose and glucose oxidase, the 50% lethal doses of epimastigotes and trypomastigotes were 6.0 and 8.7 nmol of H2O2 per min per ml, respectively, indicating that trypomastigotes were more resistant to killing by H2O2 than epimastigotes were. A reconstitution experiment of trypanocidal activity in clone C3C by ingestion of zymosan particles coupled with glucose oxidase showed that H2O2 was essential for this cytocidal process in the macrophage cell line. These results provide clear evidence for killing of an intracellular parasite by a continuous macrophage-like cell line and suggest the importance of the oxidative cytocidal mechanism in this process.

Cloning of African trypanosomes in mice immunosuppressed by cyclophosphamide treatment.

The use of cyclophosphamide-treated mice for purpose of cloning the African trypanosomes was assessed. C3HeB/FeJ mice were injected with 200 mg/kg cyclophosphamide (CY) 24-72 hours prior to infection with a single trypanosome isolated from Trypanosoma rhodesiense infected blood samples. All CY-treated mice exhibited depressed parasite-exposure, which was a period of time sufficient to grow trypanosomes from a single organism to a fulminating parasitemia. Cloning efficiency was routinely 45% in these animals. Thus, our study demonstrates that CT-treated mice are a convenient and efficient vehicle for cloning African trypanosomes. Techniques which facilitate the selection of single trypanosomes from infected blood are also described in this report.