R. Stephen Phillips

Suppression of adaptive immunity to heterologous antigens during Plasmodium infection through hemozoin-induced failure of dendritic cell function

Background Dendritic cells (DCs) are central to the initiation and regulation of the adaptive immune response during infection. Modulation of DC function may therefore allow evasion of the immune system by pathogens. Significant depression of the hosts systemic immune response to both concurrent infections and heterologous vaccines has been observed during malaria infection, but the mechanisms underlying this immune hyporesponsiveness are controversial. Results Here, we demonstrate that the blood stages of malaria infection induce a failure of DC function in vitro and in vivo, causing suboptimal activation of T cells involved in heterologous immune responses. This effect on T-cell activation can be transferred to uninfected recipients by DCs isolated from infected mice. Significantly, T cells activated by these DCs subsequently lack effector function, as demonstrated by a failure to migrate to lymphoid-organ follicles, resulting in an absence of B-cell responses to heterologous antigens. Fractionation studies show that hemozoin, rather than infected erythrocyte (red blood cell) membranes, reproduces the effect of intact infected red blood cells on DCs. Furthermore, hemozoin-containing DCs could be identified in T-cell areas of the spleen in vivo. Conclusion Plasmodium infection inhibits the induction of adaptive immunity to heterologous antigens by modulating DC function, providing a potential explanation for epidemiological studies linking endemic malaria with secondary infections and reduced vaccine efficacy.

Malaria Impairs T Cell Clustering and Immune Priming despite Normal Signal 1 from Dendritic Cells

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection.

Leishmania mexicana and Leishmania major: Attenuation of Wild-Type Parasites and Vaccination with the Attenuated Lines

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type parasites.

Protective CD4+ T-cell lines raised against Plasmodium chabaudi show characteristics of either Th1 or Th2 cells

Splenic T‐lymphocyte lines were established in vitro from Plasmodium chabaudi‐infected NIH mice on days 16 and 20 of a primary infection, and from mice after two or three infections. Each line responded specifically to stimulation with a lysed soluble extract of P. chabaudi‐infected erythrocytes (pRBC), and displayed a CD4+ (L3T4+) surface phenotype. Both the day 16 and 20 cell lines, when stimulated in vitro, secreted interleukin‐2 (IL‐2) and γ‐interferon (IFN‐γ), indicative of their belonging to the T helper 1 (Th1) CD4+ subset. In contrast, both lines derived from reinfected mice secreted interleukin‐4 (IL‐4) and provided helper activity in antibody production to P. chabaudi in vitro, and thereby had the characteristics of Th2 cells. All four T‐cell lines provided significant protection to naïve mice infected with P. chabaudi. In immuno‐compromised mice, the day 16 T‐cell line was as protective as in naïve mice whereas the cell line from mice infected twice required the additional transfer of mature naïve splenic B cells to provide protection comparable to that seen in the immunocompetent naïve recipients. The results establish that protective immunity to P. chabaudi may be associated with the induction of CD4+ Tcells of either the Th1 or Th2 subset which confer protection against this malaria parasite by mechanisms independent of, and dependent upon, B‐cell involvement, respectively.

KINETICS OF NITRIC OXIDE PRODUCTION DURING INFECTION AND REINFECTION OF MICE WITH PLASMODIUM CHABAUDI

We have shown previously that at the time of peak primary parasitaemia of P. chabaudi infection in NIH mice, significant levels of nitric oxide are produced, detectable as nitrate in the serum, and that these contribute to the protective immune response to infection. Here, we demonstrate that following reinfection, mice show a markedly diminished ability to produce nitrate. However, if mice are treated with L‐NG‐monomethyl arginine specifically to block nitric oxide metabolism during the primary infection, and are then reinfected, production of nitrate is restored to levels approaching those attained at peak primary parasitaemia. These experiments, together with others we have reported, indicate that whereas nitric oxide appears to play a significant role in control of the primary parasitaemia of P. chabaudi infection, it performs no such function during subsequent patent parasitaemias. Furthermore, they suggest that factors as yet unknown may regulate nitric oxide activity during malaria infection, such that under normal circumstances its production comes under strict control. This is exemplified by the observation that after the burst of nitric oxide activity that coincides with peak primary parasitaemia, there follows a prolonged period of immunological tolerance during which nitrate levels remain low even at secondary challenge infection. This tolerized state is lifted only several months after initial infection, when the nitric oxide activity at reinfection appears to correlate with the size of the parasite challenge and the presence of a patent parasitaemia. The implications of these findings for protective immunity to malaria, malarial immunosuppression, and immunoregulation in general, are discussed.

Gene discovery in Plasmodium chabaudi by genome survey sequencing

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Gentamicin-attenuated Leishmania infantum: cellular immunity production and protection of dogs against experimental canine leishmaniasis

An attenuated line of Leishmania infantum (L. infantum H‐line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H‐line induced significantly higher levels of IFN‐γ and lower levels of IL‐10 compared with those in dogs infected with L. infantum wild type (WT). Anti‐Leishmania‐specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H‐line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H‐line over 24 months post‐inoculation. Dogs which had been immunized with L. infantum H‐line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H‐line was safe and induced a protection which is correlated with cellular immunity in dogs.

Leishmania major H-line attenuated under pressure of gentamicin, induces a Th1 response which protects susceptible BALB/c mice against infection with virulent L. major

An attenuated line of Leishmania major (L. major H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A modification of the previously described method for the generation of attenuated L. major is described, giving rise to attenuated parasites after 8 rather than 12 subpassages. No lesions developed in BALB/c mice infected with L. major H-line, whereas L. major wild-type (WT) induced a Th2 like response with progressive lesions. Analysis of splenocyte IFN-gamma and IL-4 production following stimulation with promastigotes shows that the L. major H-line preferentially induces Th1-like responses and possibly down-regulates Th2 responses in BALB/c mice. L. major H-line parasites remained localized in the skin and draining lymph node, whereas L. major WT parasites disseminated into the visceral organs of BALB/c mice. Mice infected with L. major H-line acquired some resistance against L. major WT. These results show that the attenuated cell line of L. major is not only avirulent but that it may also modulate the host immune response.

Experimental erythrocytic malaria infection induces elevated serum amyloid P production in mice

Experimental blood-stage malaria infection of NIH mice was observed to induce an acute phase response (APR). Infection of mice with either P. chabaudi, P. vinckei (both non-lethal) or P. berghei (lethal infection) resulted in elevated serum amyloid P (SAP) production, the major acute phase protein in mice. Peak production occurred at the peak of the parasitaemia (approximately day 10 post infection). SAP isolated from the serum of P. chabaudi infected mice was shown to inhibit the growth of intra-erythrocytic malaria parasites in vitro. Furthermore, isolated SAP suppressed the proliferative response of splenocytes taken from a naïve mouse to concanavalin A. To assess if SAP had a protective role in vivo during experimental blood-stage infection, IL-6 deficient mice, which have a significantly reduced APR, were infected with P. chabaudi. A significant extension to the primary parasitaemia was observed in IL-6 deficient mice compared to infected wild type mice. These observations demonstrate that blood-stage malaria infection induces a systemic APR and that this may contribute to the immune response to infection in an anti-parasitic or immunomodulatory manner.

Gentamicin-attenuated Leishmania infantum: A clinicopathological study in dogs

The clinicopathological changes following infection with an attenuated line of Leishmania infantum (L. infantum H-line) were evaluated in mixed breed dogs. Two groups of dogs were infected intravenously (i.v.) or intradermally (i.d.) with L. infantum H-line and two control groups were infected i.v. or i.d. with L. infantum wild-type (L. infantum WT). None of the dogs, which were infected i.v. or i.d. with L. infantum H-line, showed any abnormalities during the observation period. In contrast, two out of three dogs, which were infected i.v. with L. infantum WT, developed clinical signs of disease. In addition, no histopathological changes were seen in the liver and spleen of the dogs infected with the attenuated line of parasite, whereas the histopathological changes in the two dogs infected i.v. with L. infantum WT were severe in form and manifested by infiltration of high numbers of inflammatory cells. No promastigotes were found in cultures set up from spleens and livers of dogs infected with L. infantum H-line at 12 months post-infection, whereas promastigotes were seen in the spleen and liver cultures from 2 dogs infected i.v. with L. infantum WT. Serum levels of total IgG anti-Leishmania antibody were raised in all dogs. The antibody level in the serum of dogs infected i.v. with L. infantum WT was higher than that in dogs infected with L. infantum H-line. These results show no clinicopathological abnormalities in the dogs infected with gentamicin-attenuated L. infantum H-line. Moreover, L. infantum H-line induced IgG anti-Leishmania antibody in the dogs.