CpG oligodeoxynucleotide enhances immunity against blood-stage malaria infection in mice parenterally immunized with a yeast-expressed 19 kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) formulated in oil-based Montanides.

The 19kDa carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)), an analog of the leading falciparum malaria vaccine candidate, induces protective immunity to challenge infection when formulated with complete/incomplete Freund's adjuvant (CFA/IFA), an adjuvant unsuitable for use in humans. In this study, we investigate Montanide ISA51 and Montanide ISA720 as well as CpG oligodeoxynucleotide (ODN) as adjuvants for induction of immunity to MSP1(19). Mice immunized with MSP1(19) adjuvanted with Montanide ISA51 were protected even though some mice experienced low-grade parasitemia before resolving the infection. Mice immunized with MSP1(19) adjuvanted with Montanide ISA720 showed delayed patent parasitemia with all mice ultimately succumbing to infection. Interestingly, when the synthetic CpG ODN 1826 was included in either Montanide formulation, mice were completely protected with no parasites detected in the blood. MSP1(19)-specific antibodies in MSP1(19)-immunized mice adjuvanted with Montanide ISA51 or Montanide ISA720 showed predominantly IgG1 antibody and low levels of IgG2a. CpG ODN 1826 significantly enhanced both IgG1 and IgG2a antibody responses in Montanide ISA51-adjuvanted mice but significantly enhanced only the IgG2a antibody response in Montanide ISA720-adjuvanted mice. To investigate the relative roles of antibody and CD4(+) T cells in protection, MSP1(19)-immunized mice adjuvanted with Montanide ISA720 and CpG ODN 1826 were depleted of CD4(+) T cells just prior to challenge. Results showed that three of nine immunized/T cell depleted mice died following infection. These results suggest that antibody and CD4(+) T cells are critical for protection following immunization with MSP1(19) adjuvanted with Montanide and CpG ODN and that the formulation of a human malaria vaccine candidate in Montanide ISA720 or ISA51 together with human compatible CpG ODN would be useful for improving efficacy.

Malaria parasite-specific Th1-like T cells simultaneously reduce parasitemia and promote disease.

CD4+ T cells have been implicated in immunity to the blood stages of malaria and cytokines associated with both monocyte and T cell activation have been implicated in disease. To determine whether specific T cells capable of inhibiting parasite growth can also mediate pathology we have transfused populations of Plasmodium berghei-specific T cells into normal and immunodeficient naive mice. We observed that they could inhibit parasite growth but were unable to save the animals which exhibited significantly greater anaemia and weight loss than control infected animals receiving either no T cells or T cells specific for ovalbumin. T cell-dependent tomour necrosis factor (TNF)alpha was a critical component in both parasite killing and disease promotion. Experiments with blocking antibodies demonstrated that all T-cell mediated antiparasitic immunity and all T-cell mediated weight loss was TNF-dependent. Blocking TNF-alpha in mice that received parasite-specific T cells prolonged the survival of the mice. Nitric oxide demonstrated no antiparasite effect, but was involved in the regulation of T-cell mediated weight loss. The data thus show that while parasite-specific CD4+ T cells can significantly limit parasite growth, such an effect need not be beneficial to the host, and that TNF-alpha and nitric oxide are critical effector molecules operating downstream of parasite-specific T cells in both immunity and disease.

Absolute requirement for an active immune response involving B cells and Th cells in immunity to Plasmodium yoelii passively acquired with antibodies to the 19-kDa carboxyl-terminal fragment of merozoite surface protein-1.

Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.

Intranasal immunization with yeast-expressed 19 kD carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (yMSP119) induces protective immunity to blood stage malaria infection in mice.

Variable protection against malaria blood-stage infection has been demonstrated in mice following parenteral immunization with the highly conserved 19 kD carboxylterminal fragment of the merozoite surface protein-1 (MSP119) using CFA/IFA and other adjuvants. Here we show that intranasal immunization of BALB/C mice with yeast expressed Plasmodium yoelii MSP119 plus a mixture of native and recombinant cholera toxin B subunit, could induce serum MSP119-specific antibodies at titres ranging from 20 000 to 2 560 000. The Ig subclass responses were predominantly G1 and G2b. Intranasal immunization led to protection following challenge (peak parasitaemia < 1%) in mice with the highest MSP119-specific titre (>/= 640 000). In two of the three protected mice, a peak parasitaemia of 0.1%-1% was followed by a boost of the antibody response whereas one of the three protected mice did not boost its antibody response after a peak parasitaemia of 0.02%. In unprotected mice, antibody levels rose, then fell, following the detection of parasites in the peripheral blood. CD4+ T cell-depletion abrogated the ability of the mice to boost their antibody response following challenge. These data demonstrate the potential for intranasal immunization with MSP119 to protect against malaria.

Definition of T cell epitopes within the 19 kDa carboxylterminal fragment of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) and their role in immunity to malaria.

MSP1(19) is one of the leading malaria vaccine candidates. However, the mechanism of protection is not clear. To determine whether MSP1(19)-specific effector T cells can control parasitaemia, we analysed the specificity of T cells induced following immunization with recombinant forms of P. yoelii MSP1(19) and asked whether they could protect mice. There was no evidence that effector T cells were capable of protecting since: (1) immunization of mice with yMSP1(19), but not defined epitopes, was able to induce protection; and (2) long term MSP1(19)-specific CD4+ T cell lines were incapable of adoptively transferring protection. In contrast, priming mice with the T cell epitopes resulted in a rapid anamnestic antibody response to MSP1(19) after either challenge with MSP1(19) or parasite. Thus, MSP1(19) contains multiple T cell epitopes but such epitopes are the targets of helper T cells for antibody response but not of identified effector T cells capable of controlling parasitaemia.

Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice after challenge with homologous parasite.

The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4(+) T cells that produce interferon gamma and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasite Plasmodium berghei can reduce but not eliminate parasites in vivo after adoptive transfer. Although cells can persist in vivo for up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected. In vivo activation and division of transferred T cells per se are not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.

Complete protective immunity induced in mice by immunization with the 19-kilodalton carboxyl-terminal fragment of the merozoite surface protein-1 (MSP1[19]) of Plasmodium yoelii expressed in Saccharomyces cerevisiae: correlation of protection with antigen-specific antibody titer, but not with effector CD4+ T cells.

The 19-kDa carboxyl-terminal fragment of the merozoite surface protein-1 (MSP1) is a leading malaria vaccine candidate but is unable to induce immunity in all monkeys or all strains of mice. The mechanism of immunity is unclear, although data show that cell-mediated immunity plays a critical role following immunization with the larger mature MSP1 protein. We optimized a vaccine protocol using the MSP1(19) fragment of Plasmodium yoelii expressed in Saccharomyces cerevisiae, such that following exposure of mice to parasites, they remained undetectable in peripheral blood, whereas control animals all died at very high parasitemia within 10 days. We then depleted the vaccinated mice of >99% of CD4+ T cells by anti-CD4 mAb treatment and could show that infections in most animals remained subpatent following challenge. Furthermore, mice in which the gene for the mu-chain of Ig had been disrupted could not be immunized with MSP1(19). Immunity in normal mice did not depend on the presence of an intact spleen nor production of nitric oxide, persisting unabated when >70% of splenic macrophages were depleted. Thus, while effector CD4+ T cells may contribute to immunity, neither they nor factors associated with a Th1-type cell mediated immune response appeared to play the major role in MSP1(19)-induced protection in normal mice. Furthermore, T cells were not sufficient for immunity in mice lacking B cells. In normal mice, protection correlated with a very high titer of MSP1(19)-specific Abs (>6,400,000), predominantly G1 and G2b, which may function by merozoite neutralization.

The spleen, IgG antibody subsets and immunity to Plasmodium berghei in rats.

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).

Protection of rats against malaria by a transplanted immune spleen.

A number of reports have suggested that the spleen plays a key role in the regulation of immunity to malaria but the role, if any, of other tissues is less clear. Furthermore, numerous functional changes occur in the spleen following malaria infection and it is not known whether the spleen's role relates primarily to its content of malaria-specific lymphocytes or to the altered structure and function that has occurred. To address these issues we have generated splenic chimeras by transplanting spleens between Plasmodium berghei-immune and naive rats. In the absence of a functional spleen, specific immune responses from both isolated splenic and non-splenic cells can partially control infection. However, an immune spleen in a naive rat can solidly protect the animal from malaria and a normal spleen in an otherwise immune rat can provide enhanced protection over the non-splenic state. Thus, in the presence of functional splenic architecture both splenic and non-splenic malaria-specific lymphocytes operate more effectively. However, these studies do demonstrate an important role for non-splenic tissue in immunity at least for P. berghei in the rat. The study could have important implications for induction of protective immune responses by vaccination and suggests that malaria-specific lymphocyte responses induced in the periphery following vaccination could interact with parasites in both spleen-dependent and spleen-independent ways.

Production of mouse immunoglobulin G by a hybrid plant derived from tobacco-mouse cell fusions.

Mouse-tobacco hybrid calli, and complete plants producing mouse gamma-3 heavy and lambda light chains, have been generated by somatic cell fusions of mouse spleen cells and tobacco mesophyll protoplasts. Both gamma 3 and lambda chains were detected in hybrid calli and complete plants by enzyme-linked immunosorbent assay, immunofluorescent staining, and Western blotting. When cellular DNA from hybrid tobacco mesophyll protoplasts was amplified by the polymerase chain reaction (PCR) using two pairs of gamma 3 chain DNA primers and one pair of lambda chain DNA primers, the PCR products contained gamma 3 and lambda chain DNAs, which could be detected by southern blotting and DNA hybridization, using specific synthetic oligonucleotide probes for gamma 3 and lambda respectively. In situ hybridization of hybrid tobacco mesophyll protoplasts with specific recombinant DNA probes of gamma 3 and lambda chains showed the presence of gamma 3 and lambda chain DNAs in the hybrid protoplasts.

Enumeration of interleukin-1 producing monocytes from human peripheral blood mononuclear leukocytes by agar plating technique.

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10(5) monocytes, with mean +/- SEM = 1344 +/- 182 cells/10(5) monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10(5) monocytes/ml, with mean +/- SEM = 42.8 +/- 11.2 U/10(5) monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean +/- SEM = 33.5 +/- 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated (r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-alpha.

Comparison of IgM, IgG and IgA responses to M.leprae specific antigens in leprosy.

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.

Immunologic defects in leprosy patients. II. Interleukin 1, interleukin 2, and interferon production in leprosy patients.

The capabilities of monocytes and lymphocytes in peripheral blood mononuclear leukocytes (PBML) to produce interleukin-1 (IL-1), IL-2, and interferon (IFN), respectively, were evaluated in various types and treatments of leprosy patients. IL-1 production in response to lipopolysaccharide was significantly lower in LL, BL, BB, and BT patients than in normal controls. However, there were no differences in IL-1 levels between TT patients and normal controls. The percentages of nonspecific-esterase-positive cells adhering to the plastic surfaces were not different in LL, BB and TT patients when compared to normal controls. However, they were significantly higher in BT and BL patients than in normal controls. When PBML from leprosy patients were stimulated with concanavalin-A (ConA) for IL-2 production, there were no differences in the IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients compared to normal controls. Similar results were obtained when PBML were stimulated with phytohemagglutinin-P (PHA-P). However, when purified protein derivative (PPD) was used as the stimulating agent, there were significantly lower IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients when compared to normal controls. There were also lower IL-2 levels in untreated BL/LL and BT/TT patients compared to treated BL/LL and BT/TT patients, respectively. PBML were stimulated with PHA-P or ConA for IFN production.(ABSTRACT TRUNCATED AT 250 WORDS)

Agar plating technique for enumeration of IL-2 producing cells in human peripheral blood mononuclear leukocytes.

An agar plating technique for detection and enumeration of IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML) has been developed. This method is based on the principle that PHA-stimulated PBML, as effector cells, secrete interleukin 2 (IL-2) into soft agar containing mouse 3-day Con A blasts as IL-2 dependent responder cells. The IL-2 dependent Con A blasts proliferating around the IL-2 producing cells form colonies or clusters of cells and are easily visualized under a dissecting microscope. The development of IL-2 producing cells was optimum when 1 X 10(6) cells/ml PBML were stimulated with 2 micrograms/ml PHA-P for 4 hours, and when 2.5 X 10(5) cells were co-cultured with 6 X 10(6) Con A blasts in soft agar for 5 days. The average number of IL-2 producing cells in 10 normal healthy controls were 754 +/- 94 (mean +/- S.E.M.) cells/10(6) PBML. The numbers of IL-2 producing cells and the levels of IL-2 produced were highly correlated (r = 0.929). The subpopulation of lymphocytes in the colonies was shown to be mostly murine T-cells, since they were mostly Thy 1.2 positive, CD3 negative and surface immunoglobulin negative. This technique is very simple to perform and provides an accurate and straightforward means to enumerate IL-2 producing cells from human PBML in a variety of human immunologic disorders.

Enumeration of interleukin 2-producing cells from rat spleen.

A method for the enumeration of IL2-producing cells from rat spleen has been developed. Rat spleen cells were stimulated with concanavalin A (Con A), washed, then mixed with IL2-dependent cells (3 day Con A blasts) and plated in soft agar. Clusters of IL2-dependent cells formed around IL2-producing cells, giving colonies which were easy to count under a dissecting microscope. All experimental factors influencing development of colonies of IL2-producing cells surrounded by IL2-dependent cells were evaluated and set up. Optimum number of effector cells was 2.5 x 10(5) cells/culture, while optimum number of IL2-dependent cells was 6 x 10(6) cells/culture. Optimum concentration of Con A for IL2 stimulation was 40 micrograms/ml with an optimal stimulation time of 10 hours. Optimum incubation time for development of IL2-producing cell colonies was 5 days. The number of IL2-producing cells by this technique had a good correlation with the level of IL2 in the cell culture fluid (r = 0.885). When colonies were aspirated from agar and stained by Wright stain, a big purple stained cell at the center was surrounded by small cells in almost all colonies examined. All cells from colonies were fluoresed with anti-mouse Thy 1.2-fluorescein conjugate. However, they were negative with anti-mouse Ig-fluorescein conjugate. The number of IL2-producing cells was 816-2080 cells/10(6) of rat spleen cells with mean +/- S.E.M. = 1404 +/- 154/10(6) cells.