Phenotypic and functional parameters of cellular immunity in a chimpanzee with a naturally acquired simian immunodeficiency virus infection.

The cellular immunologic and virologic status of a chimpanzee, naturally infected with a human immunodeficiency virus type 1 (HIV-1)-like lentivirus (SIVcpz-ant), was compared longitudinally with those of 3 HIV-1-infected and 5 uninfected chimpanzees for a period of 49 months. Evidence of immune deficiency was not observed in the HIV-1-infected chimpanzees, nor could virus be isolated from plasma. Virus could be isolated from plasma of the SIVcpz-ant-infected chimpanzee, but clinical signs of immune deficiency were never observed. Absolute CD4+ cell counts remained relatively stable, but NK cells fluctuated significantly over time and tended to correlate inversely with the virus titer in peripheral blood. Although only CD8+ T cells were directly demonstrated to exert a suppressive effect on viral replication in vitro, the observed fluctuation of NK cells suggests that these cells may also be involved in the interaction with lentivirus infection in this species.

In vivo treatment with a monoclonal chimeric anti-CD4 antibody results in prolonged depletion of circulating CD4+ cells in chimpanzees.

Chimeric M-T412 (cM-T412), an anti-CD4 antibody, was tolerated in chimpanzees at a dosage of 5 mg/kg per day for up to 7 consecutive days, or 5 mg/kg per dose, twice weekly for 4 weeks. All cM-T412-treated chimpanzees showed a prolonged CD4-cell depression. Weak chimpanzee antibody responses to chimeric M-T412 were observed. One of the chimpanzees on the biweekly dosage regimen exhibited a hypersensitivity reaction immediately after receiving its seventh dose. Following supportive treatment, the animal recovered and remained asymptomatic during the non-treatment observation period. The hypersensitivity reaction was not an unexpected response considering the animal received repeated intermittent i.v. administration of a foreign protein. This animal also showed a chimpanzee antibody response to chimeric M-T412 after the seventh dose. Chimeric M-T412 also induced an anti-cM-T412 response in some of the other animals. The level of this response was lower than the anti-mouse responses observed in animals treated with murine anti-CD4. Moreover, the anti-cM-T412 response was mainly directed to idiotypic determinants. The decrease in CD4+ cells observed for all chimeric M-T412-treated chimpanzees is an expected effect of the anti-CD4 antibody. The duration of this CD4+ cell decrease is, however, much longer than observed for other CD4-specific MoAbs described. No selective loss of either memory or naive CD4+ cells was observed after either the single, 7-day or twice-weekly treatments. The CD4+ cell depression was reversible, although individual variation in time to recovery was observed. Therefore, cM-T412 could be a good candidate for clinical use in autoimmune conditions.

Large-scale production of Plasmodium vivax sporozoites.

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2-3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.

Antibodies to the RNase H domain of hepatitis B virus P protein are associated with ongoing viral replication.

Antibodies against the RNase H domain of human hepatitis B virus P protein(s) are frequent markers of acute and chronic virus infection (T. Weimer, K. Weimer, Z.-X. Tu, M.-C. Jung, G. R. Pape, and H. Will, J. Immunol. 143:3750-3756, 1989). In the present study, these antibodies were determined in serial serum samples of experimentally infected chimpanzees and naturally infected human patients with acute and chronic hepatitis B virus infection. Anti-P antibodies were found in the sera of both chimpanzees and humans early in infection shortly after the immunoglobulin M anti-HBc response; they persisted in chronic carriers with ongoing viral replication but declined and disappeared at the time of virus clearance from the sera. These data demonstrate that antibodies to the RNase H domain of the hepatitis B virus P protein are early markers of infection and a signal of ongoing virus replication. Falling titers indicate the decline or end of active virus production and may therefore be a prognostic sign of virus elimination in natural infection and after antiviral therapy.

Human monoclonal antibody Ha6D3, a candidate for treatment of leukaemia? In vitro reactivity of Ha6D3 with leukaemic cells and in vivo applications in a chimpanzee.

The human monoclonal antibody Ha6D3 of the IgM type was used to stain malignant lymphoma cells from peripheral blood in flow cytometry and from cryosections of lymph nodes using the immunoperoxidase technique. It was found to react with peripheral white blood cells of all 12 cases of leukaemia and with lymph node cells of seven out of 11 B cell lymphomas and with the one T cell lymphoma tested so far. For in vivo experiments a batch of 70 mg Ha6D3 was purified and 6 mg Ha6D3 was injected intravenously into a chimpanzee with time intervals of 10 months and 1 month. The side effects observed were shivering, some muscular spasms and variations in the heart frequency. A decrease of lymphocytes of more than 50% was documented by haematogram analysis. The flow cytometry data showed that the Ha6D3 antigen does not modulate. Even after three repeated injections applied in a time interval of several months no immune response to Ha6D3 could be detected in vivo or in vitro. Based on these data we suggest that Ha6D3 may become a candidate for the treatment of certain leukaemias in vivo.

Protective efficacy of a novel hepatitis B vaccine consisting of M (pre-S2 + S) protein particles (a third generation vaccine).

The protective efficacy of a new type of yeast-derived hepatitis B (HB) vaccine (TGP-943, subtype adr), which was formulated from modified M (pre-S2 + S; P31) protein (M-P31c) particles, was investigated in chimpanzees. Animals were injected intramuscularly three times at 4-week intervals with doses of 10 or 40 micrograms (as a protein) of TGP-943. There were no significant differences in the immunogenicity of 10 micrograms compared to that of 40 micrograms of TGP-943 in terms of anti-S antibody response, while the induction and persistence of anti-pre-S2 antibodies seemed dose-related. Chimpanzees, vaccinated with 40 micrograms of TGP-943, produced anti-pre-S2 antibodies 2 weeks after the first injection, which appeared earlier than anti-HBs (S) antibodies. A maximum level of the anti-pre-S2 antibodies was reached 2 weeks after the second injection. Apart from immunization with TGP-943, chimpanzees injected with denatured TGP-943, consisting of 10 micrograms (as a protein) of non-particulate M-P31c antigen, produced anti-pre-S2 antibodies with a non-protecting level of anti-S antibodies (less than 10 mIU ml-1). Five weeks after the third injection, all animals were challenged intravenously with 1000 chimpanzee infectious units of HBV subtype (ayw) and were protected as confirmed by normal serological markers, no signs of infection in the sera and liver biopsies, and no detection of HBV-DNA by PCR method. No side effects from inoculation with TGP-943 or denatured TGP-943 were also encountered in any animals.

Plasmodium falciparum: studies on mature exoerythrocytic forms in the liver of the chimpanzee, Pan troglodytes.

Mature exoerythrocytic forms (EEF) of Plasmodium falciparum from the chimpanzee were examined by light- and transmission electron microscopy from a liver biopsy taken on Day 6 after sporozoite inoculation. Infectivity of the sporozoites obtained from whole mosquitoes which were membrane fed on cultured gametocytes was about 4-6%. In comparison, salivary gland sporozoites added to human hepatocytes in vitro had only a developmental percentage of 0.02 to 0.05% at Day 5. The EEF found in the liver biopsy were not all at the same stage of development. Immature compact parasites were seen simultaneously with stages with fully formed merozoites, indicating a rapid final maturation or asynchrony. At Day 7.5, large numbers of rings were already seen in the peripheral blood, indicating a duration of the liver development of P. falciparum in the chimpanzee of about 5.5-6 days. The process of merogony at the fine structural level was comparable to that described for rodent and other primate parasites in vivo. Compared to the fine structure of EEF in vitro in cultured human hepatocytes, the parasites described here were much more advanced in development. There appeared to be some cell infiltration with collagen deposition around the intracellular parasite; however, no marked degeneration of EEF was observed.

Protective immunisation against hepatitis B with an internal antigen of the virus.

Preparations of hepatitis B virus (HBV) core antigen (HBcAg) synthesised in Escherichia coli have been shown previously to confer partial immunity against infection by the virus [Murray, Bruce, Hinnen, Wingfield, van Eerd, de Reus, and Schellekens: EMBO Journal 3:645-650, 1984]. In a further experiment reported here, immunisation of chimpanzees with a similar preparation of HBcAg that had been treated with sodium dodecyl sulphate in order to expose e antigen epitopes was found to protect one animal completely and another quite substantially upon challenge with the virus. The results are used to support the argument for trials in humans of a vaccine against HBV based upon or containing HBcAg and its e antigen derivative, and in discussion of a more general role for internal antigens in generating immunity against viral infection.

The protection of chimpanzees against hepatitis B viral infection using a recombinant yeast-derived hepatitis B surface antigen.

In order to determine the efficacy of the SmithKline Biologicals recombinant DNA yeast-derived hepatitis B vaccine in inducing protection against hepatitis B infection, two chimpanzees were injected intramuscularly with 20 micrograms according to a 0-, 1-, and 2-month schedule. After the second dose, the vaccinated animals already showed a significant antibody response. One month after the last injection, the animals were challenged with hepatitis B virus. The vaccinated animals were protected while the two unvaccinated controls showed all signs of hepatitis B infection. One year after vaccination, antibodies remained high and were comparable with levels produced by plasma-derived vaccines.

Clinicopathologic study of six cases of meningitis and meningoencephalitis in chimpanzees (Pan troglodytes).

Three fatal cases of purulent meningitis and one fatal case of thromboembolic necrotizing meningoencephalitis occurred in chimpanzees from the Primate Center TNO, The Netherlands. In addition, two apes had clinical signs of meningitis and were successfully treated. The severity of the residual hemiparesis and dysphagia in one of these two apes was such that it was killed for humane reasons. The histopathological diagnosis was chronic active meningoencephalitis. Streptococcus pneumoniae was isolated from five apes and Klebsiella pneumoniae from one. In the majority of cases, the primary site of infection was the upper respiratory tract. After reducing the population density, initiating a vaccination program using a commercially available human polyvalent pneumococcal vaccine, and changing the cleaning procedure of the animal facilities, no other cases of meningitis or meningoencephalitis have occurred in the chimpanzee colony in the ensuing 3.5 years.

Chemical inactivation of hepatitis B virus: the effect of disinfectants on virus-associated DNA polymerase activity, morphology and infectivity.

The inactivation of hepatitis B virus (HBV) using two commercially available disinfectants was analysed. Indirect evidence of virus inactivation was obtained by examining the decrease in HBV-associated DNA polymerase and HBcAg activities after treatment with increasing concentrations of disinfectant. Inactivation was accompanied by the irreversible loss of all morphological forms typically found in hepatitis B-positive sera and in particular 42-nm HBV particles were absent. Physiochemical analysis confirmed that the exposure of HBV to either product resulted in the degradation of virus particle structure. Direct evidence of HBV inactivation was obtained by treatment of virus pellets prior to inoculation into susceptible chimpanzees. No evidence of hepatitis B was found in animals receiving treated HBV thereby confirming the suitability of certain disinfectants for the inactivation of potentially infectious material.

Antiviral and side effects of interferons produced by recombinant DNA techniques as tested in rhesus monkeys.

Human interferon type alpha 2 (HuIFN-alpha 2) produced by Escherichia coli was found to be as active as natural leukocyte interferon in protecting rhesus monkeys against intradermal vaccinia virus infection. HuIFN-beta 1 produced in E. coli had similar but less pronounced activity. HuIFN-alpha 2 induced fever but not leukopenia, while HuIFN-beta 1 had opposite effects. Concurrent treatment with acetosalicylic acid and prednisolone/azothioprine combinations did not interfere with the efficacy of the human interferons.

Calcium ion in cardiac contractility.

Under physiological conditions where the intracellular Ca ion concentration does not exceed 3 X 10(-6) M, the sarcoplasmic reticulum plays a major role in the relaxation process of cardiac muscle; mitochondria do not take up a significant amount of Ca ion during this process. If cardiac muscle undergoes maximum contraction, in which the intracellular Ca ion concentration should reach 10(-4) M, the role of mitochondria in reducing intracellular Ca ion becomes appreciable. The relationship of the tension developed by cardiac glycerinated muscle fibers to the Ca ion concentration resembles the relationship of the amount of bound Ca of cardiac troponin to the Ca ion concentrations, being less steep in its slope compared with those of fast and slow skeletal muscles. This gentle slope seems to reflect the great diversity of affinities for Ca ion of the two Ca-binding sites of cardiac troponin, one being about 100 times that of the other.