Nina Wedderburn

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Abstract Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium -infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.

Persistent Epstein—Barr virus infection in the common marmoset (Callithrix jacchus)

Epstein-Barr virus (EBV) infection of the common marmoset causes long-term infection, with production of antibodies to virus-induced antigens, without clinical illness. Attempts to show the presence of EBV DNA in saliva of infected animals by PCR were initially unsuccessful, although slot-blot hybridization analysis demonstrated that viral DNA was present. Further investigations showed that most samples of pilocarpine-induced saliva, and 33% of the samples of whole mouth fluids (WMF) tested, were inhibitory to PCR. Similar results were found using human WMF. A method of assessing samples of marmoset WMF for the presence of EBV, by PCR using an EBV BamHI W probe, and removing inhibition with Chelex 100, is described. A total of 202 samples from 21 EBV infected, and seven non-infected animals was tested. Five seropositive animals shed virus on every occasion, and 15 intermittently. Two marmosets, infected as neonates, showed progressively increasing humoral responses to viral antigens, and shed virus on every occasion tested over 3 years. When mated with uninfected animals, the latter seroconverted 4 and 6 weeks later, respectively, and later shed virus into their WMF. The naturally infected animals were paired with naive marmosets, and were able to pass on infection. These results establish that long-term, permissive EBV infection occurs in the common marmoset, and demonstrate again the similarities in the response to EBV between marmoset and man.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV

Epstein‐Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life‐threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95‐8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton‐top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild‐type strains of EBV in the general population than does the standard B95‐8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95‐8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

Hypothesis: a novel route for immortalization of epithelial cells by Epstein-Barr virus

Transfection of primate tissue explants with a specific sub-fragment (p31) of EBV DNA results in epithelial (but no other) cells proliferating indefinitely (becoming ‘immortalized’) without evidence of a ‘growth crisis’. Molecular evidence supports integration of viral information into the host chromosome, and an early genotypic alteration involving specific amplification of a sub-component (IR1) of p31 DNA, followed by apparent loss of viral DNA from chromosomes, consistent with a ‘hit and run’ mechanism. However, analysis at the individual cell level during long-term culture, by FISH techniques, reveals chromosomal alterations, and viral sequences surviving within double minute (DM) bodies. Changing growth patterns occurring at different stages during propagation (>a year in culture) may be explained by sporadic reintegration of surviving viral DNA into the host chromosome. Notably, throughout culture, telomere lengths in chromosomal DNAs do not alter but rather retain the length observed in the primary cell populations. Introduction of a growth stimulating function of EBV, BARF1, into the immortalized, non-clonable epithelial cells under conditions which permit overexpression, allows clonal populations to be derived. Based on the data, mechanisms of immortalization, in the absence of a proven viral oncogene in p31 DNA, and possible genes involved, are considered.

Direct demonstration of persistent Epstein-Barr virus gene expression in peripheral blood of infected common marmosets and analysis of virus-infected tissues in vivo.

Epstein-Barr virus (EBV) infection in animal model systems has been studied previously in marmosets and tamarins using serology and PCR of saliva. Here we directly demonstrated long-term persistence of EBV in the peripheral blood of marmosets by assaying EBER RNA expression. A new reverse transcription-PCR assay, able to distinguish a naturally occurring strain polymorphism in EBER 2 that may be useful as a strain marker for monitoring persistence and interactions between multiple strains in the same animal or person, has been developed. In situ hybridization and immunohistochemistry have also been used to search for EBV-infected cells in the animals. The carrier state in the common marmoset is similar to that of humans in that it is asymptomatic, long-lived and displays a very low level of circulating virus-infected cells. It differs from the human in lacking the characteristic antibody response to EBNA 1.

In vitro immune responses of spleen cells from friend virus infected mice.

Summary The primary immune response to sheep erythrocytes of spleen cells cultured from mice infected with Friend virus was compared with that of normal spleen cell cultures. Significant depression of the response, as measured by the production of haemolytic plaque-forming cells, was first observed when mice had been infected for 3 days prior to cell culture. This depression became more severe as the infection period was prolonged. After 4 days infection in vivo the peak of the in vitro response was reduced to a mean of 11% of normal values. No depression was observed during the first 48 hr of the development of the response in such cultures. The immune defect was shown to exist only in the non-adherent (lymphocyte-like) fraction of the spleen.

Monoclonal antibodies from Epstein-Barr virus-transformed lymphocytes of common marmosets (Callithrix jacchus) immune to malaria.

Abstract The B lymphocytes of the common marmoset Callithrix jacchus can be immortalised by infection with Epstein-Barr virus (EBV) in vitro (Desranges et al., 1976). C. jacchus is susceptible to infection with the blood stages of several species of malaria parasite including the line designated MVF1 (Mitchell et al., 1988) from which it recovers and shows immunity to reinfection. By exploiting these two phenomena, EBV-transformed, marmoset lymphoblastoid cell lines secreting antibodies to malaria parasite antigens have been generated and cloned. We believe this to be the first time that monoclonal antibodies (MAbs) have been raised from common marmosets. Since numerous and diverse human pathogens can infect this small primate in the laboratory, these methods may prove generally applicable for the generation of MAbs whose specificities derive from immune responses to infection.

Plasmodium vivax malaria in the common marmoset, Callithrix jacchus: adaptation and host response to infection.

Infection with Plasmodium vivax was established in splenectomized Callithrix jacchus marmosets by inoculation of parasitized blood from Aotus trivirgatus carrying the Vietnam Palo-Alto line of P. vivax. Subsequent blood passage through intact marmosets resulted in higher peak parasitaemias (about 1% of red cells infected) and the loss of stainable Schüffners dots in infected cells. Primary infections with the adapted line were patent for 74 days or more, and induced both a substantial antibody response, as determined by indirect fluorescence, and some lymphocytosis, but no marked anaemia. Marmosets which had recovered from their primary infection (or in which it was drug-cured) suffered abbreviated patency with low-grade parasitaemia on re-infection.

Plasmodium brasilianum in the common marmoset Callithrix jacchus

Chronic quartan malarial infection has been established in the common marmoset (Callithrix jacchus). Plasmodium brasilianum from a douroucouli monkey (Aotus trivirgatus) was used to infect splenectomized twin animals, passed to an intact animal, and then to 4 other intact adults, 2 pairs of twins. In 2 of the 4 latter animals there was continuing patency with parasitaemias of less than or equal to 0.5% parasitized erythrocytes for 30 weeks. The other 2 had lower initial levels of parasitaemia; in 1 of these parasitaemias remained low or subpatent. All marmosets developed lymphocytosis. One animal became ill 30 weeks after infection with anaemia, weight loss and mild proteinurea, the other 3 remained well. Histological examination showed minor changes in the kidneys; spleens of infected animals showed marked follicular hyperplasia and phagocytosis of pigment. The livers showed sinusoidal hypercellularity and pigment deposition and in splenectomized animals, a marked lymphoid follicular hyperplasia in the portal tracts.

Chronic malarial infection in Balb/C mice. Effect on the immune response to sheep erythrocytes and histological changes in the liver and spleen.

Chronic malarial infection was established in Balb/c mice by following Plasmodium berghei yoelii with P.b. berghei infection. It was found that the IgG plaque-forming cell response to sheep erythrocytes was depressed for at least six months. A preliminary investigation of the histological changes in the spleen and liver is described. The possibility that chronically infected mice could serve as a model for the tropical splenomegaly syndrome is discussed.