Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.     

Vaccination with a purified blood-stage malaria antigen in mice: correlation of protection with T cell mediated immunity.

A purified 230,000 mol wt protein antigen from the lethal mouse malaria parasite Plasmodium yoelii YM which had previously been shown to be highly effective as a vaccine, was tested for its ability to stimulate specific helper T cells and T cells responsible for delayed hypersensitivity. Strong stimulation was found in both assays, but larger doses were required for delayed hypersensitivity, correlating well with the requirements for protection. It is suggested that T stimulation may be a requirement for effective protection by purified antigens in malaria.     

Vaccination against malaria with live parasites

Despite nearly 80 years of vaccine research and control efforts, malaria remains one of the most prevalent of all infectious diseases. The fact that people living in regions in which malaria is endemic eventually develop immunity to the parasite and the disease suggest that it might be possible to develop vaccines against malaria. Although few vaccination trials were conducted with whole parasites, the only protocol that leads to the induction of sterile immunity in humans relies on immunization with attenuated parasites. This observation has spurred the search for subunit vaccines that aim to reproduce this protection. As yet, none of the current candidate subunit vaccines have achieved complete protection reproducibly. This failure, coupled with the recent advent of the genetically modified Plasmodium parasites, has led to a renewed interest in the use of live parasites for vaccination. This article reviews past studies, summarizes recent developments in this field and discusses the challenges to be overcome before mass immunization with live parasites could be envisaged.     

Granulocyte-macrophage colony-stimulating factor-deficient mice have impaired resistance to blood-stage malaria

The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudi AS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-gamma) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-gamma levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-alpha) levels were significantly increased in KO mice and were significantly higher than TNF-alpha levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-gamma and TNF-alpha production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.     

Immunization of mice against blood-stage Plasmodium yoelii malaria with isoelectrically focused antigens and correlation of immunity with T-cell priming in vivo.

Mice were immunized with lethal Plasmodium yoelii blood-stage malaria antigens that had been fractionated by isoelectric focusing using a variety of Ampholines over the range pH 3 to 10. Fractions were tested for their ability to protect against live challenge and to prime for parasite-specific T-cell help. Both activities exhibited three major peaks in the pH regions 4.5, 6.5, and 8, the pH 4.5 peak being the most consistently protective. There was a significant correlation between protection and T-helper-cell priming, particularly with antigens from the first peak, suggesting that T-cell priming represents an important component of the function of some protective malaria vaccines.     

Protective immunity against Plasmodium yoelii malaria induced by immunization with particulate blood-stage antigens.

The Plasmodium yoelii murine model was used to test several combinations of blood-stage antigens and adjuvants for the ability to induce immunity to blood-stage malaria. Upon fractionation of whole blood-stage antigen into soluble and insoluble components, only the particulate antigens (pAg) induced protective immune responses. Of a number of adjuvants tested, Quil A was the most effective. Immunization with pAg plus Quil A induced solid protection against nonlethal and lethal P. yoelii challenge infection. Analysis of cytokine production revealed mRNA for Th1-type cytokines (interleukin 2 [IL-2] and gamma interferon) as well as Th2-type cytokines (IL-4 and IL-10) in the spleens of both protected and susceptible animals. The data suggested that the protective pAg response was associated with the earlier production of cytokine mRNA with a Th2 phenotype somewhat favored. Immunization of B-cell-deficient JHD mice indicated that the protection against P. yoelii induced by pAg immunization was B cell dependent. Although immunization with pAg plus Quil A increased the levels of antigen-specific antibodies of all four immunoglobulin G (IgG) isotypes, protection correlated most closely with the presence of IgG1 and IgG2b antibodies. Sera from pAg-plus-Quil A-immunized animals recognized only a limited subset of six to eight distinct P. yoelii antigens, primarily associated with the pAg fraction. These results provide the basis for the identification and characterization of potential vaccine antigens, selected solely for their ability to immunize against blood-stage malaria.     

Interleukin-15 enhances innate and adaptive immune responses to blood-stage malaria infection in mice

Compared to C57BL/6 wild-type mice, interleukin-15(-/-) (IL-15(-/-)) mice showed delayed clearance of Plasmodium chabaudi AS infection, lower type 1 cytokine production, impaired dendritic cell and NK cell functions, and lower titers of malaria-specific antibodies. Thus, IL-15 supports early control and timely resolution of blood-stage malaria through promotion of Th1-dependent innate and adaptive immune responses.     

Vaccination against Pseudomonas aeruginosa pneumonia in immunocompromised mice

Immunocompromised patients are highly susceptible to infection with Pseudomonas aeruginosa. Our laboratory previously showed that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa lipopolysaccharide O antigen was effective in clearing bacteria and preventing mortality in wild-type mice after intranasal challenge. We were interested in investigating the efficacy of this vaccine strategy in immunocompromised mice. Mice rendered leukopenic or neutropenic by intraperitoneal treatment with cyclophosphamide (Cy) or RB6-8C5 antibody, respectively, were more susceptible to P. aeruginosa pneumonia than their nontreated counterparts, demonstrating 50% lethal doses several logs lower than that in wild-type mice. This hypersusceptiblity was also associated with bacterial dissemination to the liver and spleen and increased lung permeability in Cy mice. Vaccination of the mice prior to treatment resulted in better survival and lower bacterial loads compared to vector-immunized mice. Although the treatments had no effect on antibody titers, this level of protection was still lower than that seen in untreated vaccinated mice. Administration of antibodies directly to the site of infection at the time of bacterial delivery prolonged survival and lowered bacterial loads in the immunocompromised mice. These results demonstrate the importance of white blood cells while still suggesting a critical role for antibodies in protection against P. aeruginosa infection.     

Cell-mediated immunity in mice vaccinated against malaria.

Mice vaccinated with a formalin-fixed preparation of either Plasmodium berghei or P. yoelli exhibited delayed type hypersensitivity (DTH) to the homologous antigen. This manifested itself in increased delayed thickening of antigen-challenged pinnae of the vaccinated mice as compared to the non-vaccinated controls. DTH was also evident in the vaccinated mice using the homing of radio-labelled bone marrow cells (BMC) to the delayed lesion as a criterion of reactivity. When P. yoelii vaccinated mice were given a live infection P. yoelii, a marked migration of BMC into the spleen occurred, with a peak at 48 hr, and it is suggested that this was a systemic response of DTH. The splenic T-cells of P. yoelii-vaccinated animals transformed in vitro with a soluble extract of the homologous parasite. The potential function of cell-mediated mechanisms in immunity to malarial infections is discussed.     

Vaccination of mice against Mycobacterium leprae infection.

Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae. This protection is long lasting and systemic in that immunization in the left flank results in protection in both the left and right footpads. Immunization with Mycobacterium vaccae was ineffective in protecting mice against M. leprae infection, while Mycobacterium bovis BCG provided partial protection. Mycobacterium habana TMC 5135 (now known as Mycobacterium simiae) was found to be as effective as M. leprae in protecting mice against footpad infection.     

Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect

Dendritic cells (DC) suffer a maturation defect following interaction with erythrocytes infected with malaria parasites and become unable to induce protective malaria liver-stage immunity. Here we show that, by contrast, maturation-arrested DC in vitro are capable of the successful induction of antigen-specific gamma interferon (IFN-gamma) and interleukin 4 (IL-4) T-cell responses, antibody responses, and potent protection against lethal blood-stage malaria challenge in vivo. Similar results were found with DC pulsed with intact parasitized Plasmodium yoelii or Plasmodium chabaudi erythrocytes. Cross-strain protection was also induced. High levels of protection (80 to 100%) against lethal challenge were evident from 10 days after a single immunization and maintained up to 120 days. Interestingly, correlation studies versus blood-stage protection at different time points suggest that the immune effector mechanisms associated with protection could change over time. Antibody-independent, T-cell- and IL-12-associated protection was observed early after immunization, followed by antibody and IL-4-associated, IFN-gamma-independent protection in long-term studies. These results indicate that DC, even when clearly susceptible to parasite-induced maturation defect effects in vitro, can be central to the induction of protection against blood-stage malaria in vivo.     

Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice

DC-based vaccination against Leishmania major induces a parasite-specific Th1 response and long-lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi-mature murine pDC that had been isolated from the spleen, as indicated by up-regulation of the co-stimulatory molecules CD86 and CD80, but did not enhance the level of IFN-alpha secretion by pDC. Vaccination of susceptible mice with L. major lysate-pulsed pDC induced highly effective T cell-mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co-activation of pDC with CpG-containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen-pulsed pDC. These findings demonstrate that antigen-loaded pDC are able to induce T cell-mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC-based vaccination against infections.     

The role of inflammasomes in the immunostimulatory effects of particulate vaccine adjuvants

Adjuvants are essential for enhancing and directing immunity to vaccine antigens. Most adjuvants in clinical use are particulates, but how they drive innate and adaptive immune responses is unclear. A major recent advance was the demonstration that particulate adjuvants promote activation of the NLRP3 inflammasome. The mechanisms underlying this activation have been partly resolved and the role of NLRP3 in particulate adjuvant‐induced adaptive immunity is currently the subject of intense interest.     

Vaccination with recombinant vaccinia viruses protects mice against Mycobacterium tuberculosis infection.

A number of subunit-based vaccine candidates have recently begun to erode the exclusive position of Mycobacterium bovis bacillus Calmette-Guérin (BCG), which gives unpredictable and highly variable protection against tuberculosis. In this paper we investigated the protective capacity of the 19,000 MW and 38,000 MW glyco-lipoproteins of M. tuberculosis expressed by recombinant vaccinia viruses in a mouse Mycobacterium tuberculosis infection model. Both proteins were expressed at high levels by recombinant vaccinia-infected cells. In addition, two inoculations of C57B1/6 mice with either recombinant vaccinia virus significantly reduced the bacterial counts in the lungs of M. tuberculosis H37Rv-infected mice, when compared with the group infected with control virus. This is the first report of protection against tuberculous infection using recombinant vaccinia viruses with results that suggest that secreted glyco-lipoproteins in conjunction with the vaccinia vector represent suitable candidates for further vaccine-related studies.     

Deficiency in tumor necrosis factor alpha activity does not impair early protective Th1 responses against blood-stage malaria

Blood-stage Plasmodium chabaudi AS infection was controlled by 4 weeks in mice with deletion of tumor necrosis factor p55 and p75 receptors (TNFR-knockout [KO]) and control wild-type (WT) mice, although female TNFR-KO mice showed slightly but significantly higher parasitemia immediately following the peak. Serum interleukin 12 (IL-12) p70 and gamma interferon (IFN-gamma) levels were similar but tumor necrosis factor alpha levels were significantly higher in TNFR-KO mice than in WT controls. Splenic IL-12 receptor beta1 and beta2 and IFN-gamma mRNA expression, as well as spleen cell production of IFN-gamma and IL-4, were comparable in both mouse types, but IL-10 production was significantly higher in cells from TNFR-KO mice than in cells from WT mice. Lipopolysaccharide-induced NO secretion by splenic macrophages in vitro was significantly reduced but systemic NO3- levels were similar in infected TNFR-KO and WT mice.     

A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice.

We investigated the kinetics of tissue-specific mRNA expression and systemic production of tumor necrosis factor alpha (TNF-alpha) and the kinetics of splenic expression of mRNAs of gamma interferon (INF-gamma) and interleukin-4 (IL-4), cytokines that may regulate TNF-alpha production, during the early phase of blood-stage infection with Plasmodium chabaudi AS. Northern blot analysis revealed that resistant C57BL/6 mice, which clear the infection by 4 weeks, had higher levels of TNF-alpha mRNA in the spleen and liver early during infection that did susceptible A/J mice, which succumb to the disease 10 days after initiation of infection. Treatment of resistant mice with a polyclonal anti-TNF-alpha antibody confirmed the protective role of TNF-alpha early during the course of infection. Furthermore, resistant C57BL/6 mice also expressed high levels of mRNA of IFN-gamma (a Th1 marker) and low levels of mRNA of IL-4 (a Th2 marker) in the spleen, whereas susceptible A/J mice had low levels of IFN-gamma mRNA but high levels of TNF-alpha mRNA in the liver and had high levels of TNF-alpha protein in serum, as measured by enzyme-linked immunosorbent assay, later during infection just before death occurred. These results demonstrate that a Th1-associated increase in TNF-alpha mRNA expression in the spleen early during infection correlates with resistance to P. chabaudi AS, whereas increased TNF-alpha mRNA levels in the liver and excessive levels of the TNF-alpha protein in serum later during infection correlate with susceptibility. Thus, the role of the TNF-alpha during malaria appears to depend on the timing and site of its expression and the presence of cytokines regulating its production.     

Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice

Parasitology Research - Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but...     

Oral immunization with a recombinant malaria protein induces conformational antibodies and protects mice against lethal malaria

The increasing death toll from malaria, due to the decreasing effectiveness of current prophylactic and therapeutic regimens, has sparked a search for alternative methods of control, such as vaccines. Although several single proteins have shown some promise as subunit vaccines against sexual blood stages in experimental systems, it is clear that multicomponent vaccines are required. Many logistic difficulties make such an approach prohibitively expensive. In an effort to try to overcome some of these issues, we examined the possibility of oral immunization as a route for inducing host protective immunity. We report here that oral feeding of a malaria protein induced serum antibody levels similar to those induced by intraperitoneal immunization with Freund's adjuvant. Further, responses to conformational epitopes were induced. In the rodent challenge system, significant levels of protection to lethal challenge with malaria were induced in mice. The protective efficacy was highly correlated with antibody levels, which depended on the antigen dosage and required cholera toxin subunit B as an oral adjuvant. These findings offer new approaches to the development of a malaria vaccine and provide justification for the investigation of transgenic plants as a means of vaccine delivery.