Immunity to malaria: the antibody response to antigenic variation by Plasmodium knowlesi

Monkey erythrocytes infected with schizonts of Plasmodium knowlesi are agglutinated when mixed with serum taken from chronically infected monkeys (Eaton, 1938). This reaction has proved highly specific and capable of distinguishing antigenic variants of one strain. Chronic infections of P. knowlesi in rhesus monkeys are maintained by a succession of antigenically distinct populations, each population stimulating a specific agglutinin response. Additional agglutinins which are not variant specific are found at low titre in sera taken after an infection has persisted for a month or more. The possible relevance of these observations to protective immunity is discussed.     

Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites.

Rhesus monkeys vaccinated with merozoites in FCA are protected against challenge with several strains and variants of Plasmodium knowlesi. Vaccination induces sterilizing immunity which is species specific. Merozoite-blocking (inhibitory) antibody usually correlates with clinical immunity and protection can be passively transferred with immune sera provided these contain high levels of inhibitory antibody. However, vaccination using adjuvants other than FCA may induce inhibitory antibody without clinical protection. In addition, vaccinated animals may become susceptible to challenge 4-5 weeks after splenectomy, although inhibitory antibody levels are not reduced. These observations indicate that immunity induced by merozoite vaccination involves: (i) merozoite blocking (inhibitory) antibody, (ii) specific antibody or immune complexes acting synergistically with cytotoxic splenic cells stimulated by FCA.     

Properties of protective malarial antibody

Properties of protective malarial antibody have been studied in cultures of P. knowlesi giving average parasite multiplication rates of sixfold in 24 hours. Parasite growth was assessed by incorporation of [³H]-leucine into protein.

Immune serum has little effect upon the growth of intracellular parasites, but prevents reinvasion of red cells and inhibits the succeeding cycle of parasite development. Protective antibody is present in relatively low titre in immune sera even after long immunization and this may explain certain characteristic features of malarial immunity.

Protective antibody in the sera studied is associated with IgG and IgM; its action is not complement dependent, but requires at least two combining sites per molecule. Anti-malarial antibody has several features in common with viral neutralizing antibody.

     

Development of the immune response in the foetal and newborn rhesus monkey: I. Response to bovine serum albumin

1. Foetal rhesus monkeys (at 10–16 weeks gestation) were treated in utero with bovine serum albumin (BSA) and subsequently, as neonates, were tested for immunity or tolerance.

2. Five out of six monkeys treated in utero with 100 mg BSA were tolerant to BSA at challenge.

3. Some foetuses responded to treatment with 1–8 mg BSA in adjuvant with formation of antibody.

4. One monkey treated with a total of 350 mg BSA during the first 3 weeks after birth was tolerant to BSA at challenge; two other neonatal monkeys treated with 100 mg BSA did not show tolerance.

     

Precipitins to candida albicans in chronic mucocutaneous candidiasis studied by crossed immunoelectrophoresis with intermediate gel. Correlation with clinical and immunological findings

The precipitating antibodies in the sera of fifteen patients with chronic mucocutaneous candidiasis were examined by crossed immunoelectrophoresis with intermediate gel. The method permitted identification and quantitation of precipitins against thirty-four of the seventy-eight known antigenic components of Candida albicans.

The sera from every patient contained precipitins and the number of reactivities per serum ranged from two to thirty-nine. All patients had antibodies to antigen 78, a mannan–protein complex. Many sera also possessed antibodies to many other components of the organism, suggesting that some of the yeast cells had been disrupted in the patients' tissues. However, there were no precipitin profiles that characterized patients with specific forms of chronic candidiasis. Instead, in two cases, the antibody profiles appeared to be related to the patients' ability to develop humoral immune responses. Serial studies of patients during remissions and exacerbations showed that there were no consistent changes in antibody activities.

The role of Candida precipitins in chronic candidiasis remains uncertain. Possible functions include prevention of dissemination of the infection from superficial sites, formation of immune complexes in superficial sites and suppression of cell-mediated immunity as suggested by in vitro tests.

     

Eimeria tenella (Gallus domesticus): Immunity to • in Young Fowls

Complete immunity to a challenge dose of 100,000 sporulated oocysts of Eimeria tenella was developed in fowls 14 days after they had received the last of three graded doses of oocysts of this species, whereas uninfected fowls of comparable age were fully susceptible. In fowls similarly immunized, no detectable first-generation schizogony developed from challenge doses of 10 million oocysts administered to each fowl 21 days after the last of the three graded doses had been administered. Precipitating antibodies were demonstrated in some but not all of these immune fowls by the agar-gel diffusion technique. Precipitin bands, developed as the result of infection, showed a reaction of identity with those induced by parenteral injection of schizont antigen and most of the bands appeared to be directed against protein antigens. Cross reactions were observed between E. tenella antiserum and antigens prepared from the species of coccidia, E. tenella from the fowl and the species E. stiedae from the rabbit.

Electrophoretic analysis of serum from immune birds showed an albumin component and four globulin fractions (I-IV); antibody activity was confined to the fraction with the slowest mobility (IV). No significant differences were shown between the electrophoretic analyses at comparable ages of serum from the infected and control groups of fowls between 7 and 63 days of age. The components in both groups altered significantly with time, showing a general rise in protein concentrations. Infected fowls repeatedly showed numerous pyroninophilic cells in the gut mucosa and cells closely resembling globular leucocytes in the deep glands of the caeca.

     

Nonreplicating,Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection by Toxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80 genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion of OMPDC induced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80 Δompdc mutants stimulated a fully protective CD8⁺ T cell-dependent immunity that prevented acute infection by type I and type II strains of T. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdc mutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.     

Review: the cellular basis of the immunity to and immunopathogenesis of tropical theileriosis

The intracellular protozoan parasite Theileria annulata causes a severe and often fatal disease of pure and crossbred cattle in tropical and subtropical countries. Animals that recover from the infection are immune against challenge with homologous parasite strains. In the present review we refer to the role of immunocompetent cells and their products in containing the infection or in facilitating the progress of the disease. Parasite-infected host cells produce cytokines, which, depending on their concentration and timing of production, may enhance the establishment of the infection. Thus, cell lines producing high levels of proinflammatory cytokines cause severe postvaccinal reactions when inoculated into cattle. This may be supported by an aberrant non-specific activation of naive T-cells, leading to the production of high levels of gamma-interferon (IFN-γ). Under these circumstances development of the specific immune response may be inhibited. At this stage, innate immune reactions are operating to contain the infection. Natural killer cells and macrophages may represent the most important part of this immunity. Antibodies and specific T-lymphocytes, CD4⁺ T-cells and cytotoxic T-lymphocytes (CTLs), play the most important role in a challenge infection. In this context, CD4⁺ T-cells produce cytokines required for the clonal expansion of CTLs that kill their target cells in a major histocompatibility complex (MHC) class I-restricted manner. In addition, CD4⁺ T-cells produce macrophage-activating cytokines such as IFN-γ. Such activated macrophages produce mediators such as NO, which destroy the intracellular schizonts. Attempts have been directed toward the identification of parasite antigens involved in the induction of immunity. To date, only a limited number of sporozoite and merozoite antigens have been identified and examined for their immunogenicity, and the protection achieved is partial. An effective vaccine must include schizont proteins, notably, those proteins that are secreted into the host cell cytoplasm because these may have access to the MHC class I and II compartments to be presented to CTLs and CD4⁺ T-cells, respectively. Several schizont proteins have been identified and these are now under investigation. Received: 25 January 1999 / Accepted: 5 February 1999     

Partial isolation of a membrane antigen which induces the formation of antibodies lethal to schistosomes cultured in vitro

Fractions of adult schistosomes (S. mansoni) were tested for their ability to absorb an IgG antibody activity which appears in the sera of rats and Rhesus monkeys infected with the parasite and is lethal to schistosomula cultured in vitro. The same fractions were tested for their immunogenicity in raising lethal antibody when administered to rats in Freund's adjuvant. The results of these experiments indicated that an antigen which both absorbs and induces the formation of lethal antibody is present almost entirely in the water-insoluble portion of adult schistosome homogenates. The antigen appears to be tightly bound to this membrane fraction since the use of strong anionic detergents is necessary in its extraction. Membranes solubilized in sodium dodecyl sulphate were fractionated by gel filtration on Sephadex columns equilibrated with the detergent. The same specific Sephadex fraction was shown to absorb both rat and Rhesus monkey lethal antibodies as well as induce the antibody when administered to rats. Further purification and analysis suggested that the immunogen was one of four proteins with molecular weights estimated between 21,000 and 33,000 Daltons.

Rats vaccinated with partially purified antigen developed levels of lethal antibody similar to those found in animals immune to challenge through previous exposure to the parasite, yet were themselves only marginally resistant to infection. These results indicate that while clearly toxic to schistosomula grown in vitro, lethal antibody operating on its own in vivo is incapable of mediating the rejection of schistosome infections.

     

Immunization against Plasmodium falciparum asexual blood stages using soluble antigens.

Saimiri sciureus monkeys have been successfully immunized against a human malaria parasite, Plasmodium falciparum, using soluble antigens purified from schizont and merozoite extracts. High levels of antibodies reacting with schizont and merozoite specific polypeptides of 140 and 200 kdaltons were detected in the sera of protected monkeys. The five immunized monkeys survived a challenge infection with 5 X 10(7) parasites inducing a fulminant disease in control monkeys.     

The acquisition and loss of antigen-specific cellular immune responsiveness in acute and chronic schistosomiasis in man

To characterize the development and evolution of cellular immune responsiveness in individuals infected with the parasite Schistosoma mansoni, we studied fifteen patients with acute, subacute and chronic schistosomiasis. Lymphocytes from the three acutely infected patients responded vigorously to schistosome antigens in an in vitro blastogenic assay. By contrast, cells from nine chronically infected individuals were essentially unreactive to these same antigens. Patients infected for an intermediate period of time (9 months) generated responses between those of acute and chronic patients.

The diminished responsiveness of chronically infected individuals was specific for schistosome antigens and did not extend to humoral immune responses. Following treatment of the infection with niridazole, these patients temporarily regained responsiveness to schistosome antigens.

From these data we speculate that during the course of this parasitic helminth infection there develops a progressive and specific modulation of antigen recognition and proliferation by lymphocytes to schistosome antigens, and that such diminished immune reactivity may be important in maintaining the unique biological relationship which exists between a host and its parasites.

     

Role of CD8+ cell-produced anti-viral factors in protective immunity in HIV-2-exposed but seronegative macaques resistant to intrarectal SIVsm challenge

The cell‐mediated immune response is likely to be important in controlling HIV/SIV infection. There is evidence that β‐chemokines and other, as yet unknown, anti‐viral factors play a role in host defence against HIV infection. We reported previously that HIV‐2 exposed but seronegative cynomolgus macaques developed SIV‐specific cytotoxic T lymphocytes and were resistant to mucosal SIV challenge. The aim of this study was to examine CD8⁺ cell‐dependent production of β‐chemokines and other anti‐viral factors in these macaques. The animals, selected from among 17 monkeys enrolled in two separate experiments, were either treated with an anti‐viral drug or immunized passively with HIV‐2 antibody‐positive serum. Three of these monkeys were protected against repeated HIV‐2 challenge and were also able to control SIV infection 3 years later. Control samples were obtained from four macaques that became SIV infected and from 39 naïve animals. The three resistant monkeys showed significantly higher production of RANTES and MIP‐1α than the 39 naïve animals. In addition, SIV infection was suppressed by CD8⁺ cell culture supernatants of these monkeys. However, antibodies to chemokines only partially neutralized CD8⁺ cell‐mediated SIV suppression indicating that the anti‐viral activity observed in these monkeys was the result of combined action of several inhibitory factors.     

Protective Effect of Vaccines in Experimental Mycoplasma pneumoniae Disease

The mechanisms of immunity to Mycoplasma pneumoniae were investigated by evaluating different vaccination procedures in an experimental animal model. Hamsters were immunized by intranasal inoculation of broth cultures or by parenteral injections of saline-suspended organisms. All vaccinees received a standardized intranasal challenge which produced pneumonia in 94% of controls. Intranasal immunization with virulent organisms produced a 71% reduction in pneumonia. Subcutaneous and intraperitoneal inoculation of the same organisms yielded 56 and 61% reductions, respectively. Animals similarly immunized with an attenuated strain developed resistance to pneumonia only after intranasal infection. Serum antibody levels did not correlate with protection. Growth-inhibiting activity was demonstrated in bronchial washings of challenged animals, suggesting the development of local antibody in response to infection with M. pneumoniae. Crosschallenge studies were performed in animals vaccinated intranasally with virulent and avirulent variants of the same strain. The avirulent vaccine prevented pneumonia in animals challenged with homologous virulent organisms but not in those receiving an unrelated strain; the virulent vaccine provided protection to both homologous and heterologous challenge. These studies indicate that organism strain variation as well as vaccination technique are important determinants of the immune response to M. pneumoniae.     

Rat monoclonal antibodies which inhibit the in vitro multiplication of plasmodium knowlesi

A total of 28 double cloned monoclonal antibodies specific for Plasmodium knowlesi were raised by fusion of Y3 rat myeloma cells with spleen cells of A0 rats immunized with W1 variant isolated merozoites. Four of these antibodies reacted positively in a solid phase radioimmunoassay against glutaraldehyde-fixed schizonts but gave no detectable reaction on indirect immunofluorescence against methanol-fixed schizonts or merozoites. The remaining 24 antibodies could be divided into 13 distinctive immunofluorescent categories on the basis of their patterns of binding to schizonts and merozoites and reactivity with Plasmodium falciparum. Eight antibodies were studied for their ability to inhibit the in vitro multiplication of W1 P. knowlesi as assessed by parasite incorporation of ³H-amino acids and parasite counts. Partially purified antibody preparations from ascitic fluids were all inhibitory for parasite growth; however, when fully purified antibodies were tested, six of the eight proved to be non-inhibitory. Two of the purified antibodies, both IgG2a isotype, inhibited the in vitro multiplication of P. knowlesi in a dose-dependent manner. Inhibition was not associated with detectable damage to intracellular parasites, suggesting that the inhibitory monoclonal antibodies act by blocking the reinfection of red cells by newly released merozoites. On immunofluorescent analysis both inhibitory antibodies bound to methanol-fixed schizonts, with the intensity increasing for progressively more mature parasites; both reacted diffusely with isolated merozoites, and neither cross-reacted with P. falciparum. Both bound specifically to a single metabolically labelled polypeptide which appears to be a minor parasite component and has an approximate molecular weight of 66,000 when analysed by SDS-PAGE fluorography. The putative protective antigen of P. knowlesi has potential interest as a vaccine against P. knowlesi malaria.     

Lyme Borreliosis in Rhesus Macaques: Effects of Corticosteroids on Spirochetal Load and Isotype Switching of Anti-Borrelia burgdorferi Antibody

Experimental Borrelia burgdorferi infection of rhesus monkeys is an excellent model of Lyme disease and closely parallels the infection in humans. Little is known about the interaction of host immunity with the spirochete in patients with chronic infection. We hypothesized that rapid development of anti-B. burgdorferi antibody in immunocompetent nonhuman primates (NHPs) is the major determinant of the reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by measurement of the spirochetal load by PCR in association with characterization of the anti-B. burgdorferi humoral immune response in immunocompetent NHPs versus that in corticosteroid-treated NHPs. Although anti-B. burgdorferi immunoglobulin G (IgG) antibody was effectively inhibited in dexamethasone (Dex)-treated NHPs, anti-B. burgdorferi IgM antibody levels continued to rise after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This vigorous production of anti-B. burgdorferi IgM antibodies was also studied in vitro by measurement of antibody produced by B. burgdorferi-stimulated peripheral blood mononuclear cells. Despite these high IgM antispirochetal antibodies in Dex-treated NHPs, spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferi IgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.     

Immunological competence in non-human primates: differences observed in four species

Previous studies have shown that the baboon and squirrel monkey are resistant to certain herpesviruses that cause serious infection or induce tumours in the cebus monkey and marmoset. This study was undertaken to evaluate the immunological competence of the four species of nonhuman primates. A variety of clinical immunological techniques were employed to evaluate the bursal, thymicdependent and phagocytic systems of immunity in these species. No primate was found to demonstrate immune competence of the same magnitude observed in normal humans.

The baboon manifested immune responsiveness most closely resembling that of man. These animals displayed no abnormalities in circulating white blood cell populations, ability to form antibody, delayed skin response to mitogens or lymphocyte transformation in vitro. Rebuck skin window (RSW) findings closely resembled those observed in humans. While the squirrel monkey also appeared to be relatively competent, antibody formation, skin test reactivity and RSW responses were slightly less than those observed in the baboon. Cebus monkeys showed several abnormalities which included significant leukopenia, very low serum alpha-1 globulin levels, cutaneous anergy and a poor RSW response. The most marked abnormalities were found in the marmosets that had persistent leucocytosis, absent to poor antibody responses to immunogens, weak responses to phytohaemagglutinin in vivo and in vitro, with a uniquely greater response to pokeweed mitogen in vitro. The poor RSW response was similar to that observed in the squirrel monkey and cebus. While these studies suggest that baboons and squirrel monkeys are relatively intact immunologically, the marmoset and cebus monkey species appear to have significant manifestations of immunological incompetence in association with susceptibility to a variety of infectious and oncogenic agents.

     

Alterations in the Distribution and Proliferative Responses of Rhesus Monkey Peripheral Blood and Spleen Cells During Malaria (Plasmodium knowlesi) Infection

Rhesus monkeys are used frequently as animal models in malaria research, but few studies have evaluated lymphocyte functions in these animals after experimental infections with the primate malarial parasite Plasmodium knowlesi. In this study, the distribution and mitogen responses of mononuclear cells in the peripheral blood and spleens of 16 P. knowlesi-infected rhesus monkeys were followed. All animals included in the study developed acute infections and were bled out with parasitemias of more than 50%. With progression of the infection, alterations in the peripheral blood mononuclear cells were observed, including decreases in the percentage of T cells (measured by E rosette formation) and the total numbers of E and EAC rosette-forming cells per cubic millimeter. In addition, peripheral blood mononuclear cells displayed reduced responses to mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen. Peripheral blood mononuclear cells from infected animals showed similar reductions in mitogen responses when cultured in media containing 15% autologous pre- or postinfection plasma. The mitogen responses of spleen cells did not appear to be affected, but a significant reduction in the proportion of splenic T cells was observed. These lymphocyte changes in P. knowlesi-infected rhesus monkeys are similar to those reported for mice with acute rodent malaria and for humans with chronic Plasmodium falciparum infections.     

An immunological basis for the anaemia of acute Salmonella gallinarum infection of chicken. II. The relationship of the immune response to the development of haemolytic anaemia

The ability of chickens to respond immunologically during acute Salmonella galinarum infection has been examined in relation to the underlying mechanism of the associated haemolytic anaemia.

In spite of the severity and acuteness of the experimental infection, the majority of the chickens showed a marked immune response to lipopolysaccharide (LPS) derived from the challenge organism. Peak antibody titres occurred 5–6 days after infection, coincidentally with in vivo erythrocyte modification and with maximum destruction of erythrocytes. However, those animals which died of the per-acute form of the disease (within 3 days of challenge) showed neither antibody response, nor in vivo erythrocyte modification, and did not develop anaemia.

It was also observed that erythrocytes which were positive to the direct Coombs test persisted in the circulation of surviving chickens for comparatively long periods (on average 4 days).

The evidence is consistent with the hypothesis that an immunologically-mediated mechanism may be responsible for the development of the anaemia.

     

Eimeria tenella (Gallus domesticus): Attempts to Induce a Passive Immunity to • in Young Fowls

Whole serum or γ globulin derived from fowls, either susceptible or immune to coccidiosis (Eimeria tenella), was injected into fully susceptible fowls.

The serum proteins were given by intravenous or intraperitoneal injection and subsequent attempts were made to infect these fowls by giving either a moderate dose of sporulated oocysts per os or a suspension of viable merozoites per rectum.

In the agar-gel double diffusion test, the serum from the donor birds, resistant to E. tenella, formed only weak lines of precipitate when reacting against an antigen prepared from the second schizont stage of the life cycle. However, the immune serum was considered satisfactory because the donor birds on challenge were immune.

Although relatively large amounts of γ globulin were injected into the susceptible fowls (up to 0.88 g. per kg. body weight) and subsequently only mild infections were given, no passively acquired resistance was shown either from the results of the oocyst counts on the faeces or by a macrosopic or microscopic examination of the caeca.

These results are discussed in relation to earlier studies; they show that passively acquired serum antibody at these dose levels did not provide protection.