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Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8 + T cells to target parasites in the liver. Such T cells can be induced by 'Prime-and-trap' vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to "trap" the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10-50 µL) and induced protective levels of CSP-specific CD8 + T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development., (© 2024. The Author(s).)

7DW8-5 is a potent glycolipid adjuvant that improves malaria vaccine efficacy in mice by inducing IFN-γ and increasing protective CD8 + T cell responses. The addition of 7DW8-5 was previously shown to improve the efficacy of a CD8 + T cell-mediated heterologous 'prime-and-trap' malaria vaccine against Plasmodium yoelii sporozoite challenge in inbred female mice. Here, we report significant differential sex-specific responses to 7DW8-5 in inbred and outbred mice. Male mice express significantly less IFN-γ and IL-4 compared to females following intravenous 7DW8-5 administration. Additionally, unlike in female mice, 7DW8-5 did not improve the vaccine efficacy against sporozoite challenge in prime-and-trap vaccinated male mice. Our findings highlight the importance of including both female and male sexes in experimental adjuvant studies.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8 + T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3 + Tbet + CD4 + regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically., (Copyright © 2020 Phares, Kotraiah, Karunarathne, Huang, Browne, Buontempo, Mansour, Noe, Wykes, Pannucci, Tsuji and Gutierrez.)

Adenovirus (Ad) is thought to be one of the most promising platforms for a malaria vaccine targeted against its liver stages, because of its ability to induce a strong T-cell response against a transgene. However, a further improvement of this platform is needed in order to elicit another arm of the immunity, i.e. humoral response, against malaria. In order to augment immunogenicity and protective efficacy of Ad-based malaria vaccine, we inserted B-cell, as well as CD4+ T-cell, epitopes of Plasmodium falciparum circumsporozoite protein (PfCSP) into the capsid protein, Hexon, and the core protein, VII (pVII), of Ad, respectively, in addition to the PfCSP transgene. Insertion of PfCSP-derived B cell epitope to Hexon significantly enhanced the epitope-specific antibody response compared to AdPfCSP, an Ad vaccine expressing only PfCSP transgene. PfCSP-derived CD4+ T-cell epitope insertion into pVII augmented not only PfCSP-specific CD4+ T-cell response but also anti-PfCSP antibody response. Finally, mice immunized with AdPfCSP having both Hexon and pVII modifications were more protected than AdPfCSP or Hexon-modified AdPfCSP against challenge with transgenic rodent malaria parasites expressing the PfCSP. Overall, this study has demonstrated that Hexon and pVII-modified AdPfCSP vaccine is a promising malaria vaccine which induces strong PfCSP-specific humoral, CD4+ T-cell, and CD8+ T-cell responses and protects against infection with transgenic malaria parasites expressing the PfCSP., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination., (Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Malaria is a severe infectious disease with relatively high mortality, thus having been a scourge of humanity. There are a few candidate malaria vaccines that have shown a protective efficacy in humans against malaria. One of the candidate human malaria vaccines, which is based on human malaria sporozoites and called PfSPZ Vaccine, has been shown to protect a significant proportion of vaccine recipients from getting malaria. PfSPZ Vaccine elicits a potent response of hepatic CD8+ T cells that are specific for malaria antigens in non-human primates. To further characterize hepatic CD8+ T cells induced by the sporozoite-based malaria vaccine in a mouse model, we have used a cutting-edge Single-cell Barcode (SCBC) assay, a recently emerged approach/method for investigating the nature of T-cells responses during infection or cancer. Using the SCBC technology, we have identified a population of hepatic CD8+ T cells that are polyfunctional at a single cell level only in a group of vaccinated mice upon malaria challenge. The cytokines/chemokines secreted by these polyfunctional CD8+ T-cell subsets include MIP-1α, RANTES, IFN-γ, and/or IL-17A, which have shown to be associated with protective T-cell responses against certain pathogens. Therefore, a successful induction of such polyfunctional hepatic CD8+ T cells may be a key to the development of effective human malaria vaccine. In addition, the SCBC technology could provide a new level of diagnostic that will allow for a more accurate determination of vaccine efficacy.

A CD1d-binding glycolipid, α-Galactosylceramide (αGalCer), activates invariant NK T cells and acts as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying nearly 100-fold stronger CD1d binding affinity. In the current study, 7DW8-5 was found to exert a more potent adjuvant effect than αGalCer for a vaccine based on radiation-attenuated sporozoites of a rodent malaria parasite, Plasmodium yoelii, also referred to as irradiated P. yoelii sporozoites (IrPySpz). 7DW8-5 had a superb adjuvant effect only when the glycolipid and IrPySpz were conjointly administered i.m. Therefore, we evaluated the effect of distinctly different biodistribution patterns of αGalCer and 7DW8-5 on their respective adjuvant activities. Although both glycolipids induce a similar cytokine response in sera of mice injected i.v., after i.m. injection, αGalCer induces a systemic cytokine response, whereas 7DW8-5 is locally trapped by CD1d expressed by dendritic cells (DCs) in draining lymph nodes (dLNs). Moreover, the i.m. coadministration of 7DW8-5 with IrPySpz results in the recruitment of DCs to dLNs and the activation and maturation of DCs. These events cause the potent adjuvant effect of 7DW8-5, resulting in the enhancement of the CD8(+) T cell response induced by IrPySpz and, ultimately, improved protection against malaria. Our study is the first to show that the colocalization of a CD1d-binding invariant NK T cell-stimulatory glycolipid and a vaccine, like radiation-attenuated sporozoites, in dLN-resident DCs upon i.m. conjoint administration governs the potency of the adjuvant effect of the glycolipid., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.

CD8(+) T cells are critical for the control of various intracellular infections and cancers. To date, however, effective T cell-based vaccines remain elusive, due, in part, to the lack of in vivo models that facilitate the dissection of antigen-specific CD8(+) T-cell responses primed by different antigen-presenting cells (APCs). In this study, we generated four lines of H-2K(d) transgenic (K(d) Tg) mice that differed in their expression of H-2K(d): dendritic cells (DCs) only (CD11c-K(d)), macrophages only (huCD68-K(d)), hepatocytes only (Alb-K(d)), or all nucleated cells (major histocompatibility complex-I-K(d)). Immunization of each of these K(d) Tg mouse strains with a synthetic peptide or a recombinant adenovirus expressing a well-known immunodominant, H-2K(d)-restricted CD8(+) T-cell epitope, SYVPSAEQI, which was derived from the circumsporozoite protein of Plasmodium yoelii, promoted distinct SYVPSAEQI-specific CD8(+) T-cell responses. The route of immunization also greatly influenced the magnitude of the epitope-specific CD8(+) T-cell response. These tissue-specific K(d) Tg mice may be valuable tools for determining the mode of induction of CD8(+) T-cell responses by different APCs in vivo and for characterizing the CD8(+) T-cell responses promoted in response to various microbial infections and/or different types of vaccines., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans., ((c) 2009 Elsevier Inc. All rights reserved.)

NKT cells are known to play a role against certain microbial infections, including malaria and HIV, two major global infectious diseases. NKT cells exhibit either protective or pathogenic role against malaria. They are depleted by HIV infection and have a direct pathogenic role against many opportunistic infections common in end-stage AIDS. This review discusses the various features of the interaction between NKT cells and malaria parasites and HIV, and the potential to harness this interaction for therapeutic and vaccine strategies.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

A major malaria vaccine candidate, the circumsporozoite (CS) protein of Plasmodium, is a pre-erythrocytic stage antigen that is attached to the surface of the sporozoites through a glycosylphosphatidylinositol (GPI) anchor. However, here we show that the motif that signals for glycosylphosphatidylinositol anchor addition interferes with the immunogenicity of this protein and reduces protection in mice upon immunization with a recombinant adenovirus. The presence of the glycosylphosphatidylinositol-anchoring motif sequentially affected total circumsporozoite protein production, cellular distribution, antigen processing and secretion, leading to less effective antigen presentation. Consistently, vaccination with an adenovirus recombinant carrying the anchoring motif-disrupted circumsporozoite gene, resulted in significant increase of the number of interferon-gamma (IFN-gamma) producing T cells and specific IgG2a isotype antibodies, ensuing more effective vaccination. Given that the anchoring motif is highly conserved among different species of Plasmodium, anti-malaria subunit vaccines encoded by recombinant vectors that aim at the induction of strong cellular immunity could maximize immunogenicity by removing anchoring motifs.

alpha-Galactosylceramide (alpha-GalCer) is a glycolipid that stimulates natural killer T cells to produce both T helper (Th) 1 and Th2 cytokines. This property enables alpha-GalCer to ameliorate a wide variety of infectious, neoplastic, and autoimmune diseases; however, its effectiveness against any one disease is limited by the opposing activities of the induced Th1 and Th2 cytokines. Here, we report that a synthetic C-glycoside analogue of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer), acts as natural killer T cell ligand in vivo, and stimulates an enhanced Th1-type response in mice. In two disease models requiring Th1-type responses for control, namely malaria and melanoma metastases, alpha-C-GalCer exhibited a 1,000-fold more potent antimalaria activity and a 100-fold more potent antimetastatic activity than alpha-GalCer. Moreover, alpha-C-GalCer consistently stimulated prolonged production of the Th1 cytokines interferon-gamma and interleukin (IL)-12, and decreased production of the Th2 cytokine IL-4 compared with alpha-GalCer. Finally, alpha-C-GalCer's enhanced therapeutic activity required the presence of IL-12, which was needed to stimulate natural killer cells for optimal interferon-gamma production, but did not affect IL-4. Overall, our results suggest that alpha-C-GalCer may one day be an excellent therapeutic option for diseases resolved by Th1-type responses.

T cells are critical mediators of immunity to the pre-erythrocytic stages of malaria parasites. In this review, we survey the role of the various T cell subsets in combating the pre-erythrocytic stages; in particular, the role of NK T cells. Moreover, we show how studies using malaria models have revealed a unique behavior of NK T cells, namely the bridging of innate and adaptive immunity.

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.

T cells from different subsets play a major role in protective immunity against pre-erythrocytic stages of malaria parasites. Exposure of humans and animals to malaria sporozoites induces (alphabeta CD8(+) and CD4(+) T cells specific for antigens expressed in pre-erythrocytic stages of Plasmodium. These T cells inhibit parasite development in the liver, and immunization with subunit vaccines expressing the respective antigenic moieties confers protection against sporozoite challenge. gammadelta and natural killer T cells can also play a role in protective immunity. Recent studies with mice transgenic for the alphabeta T-cell receptor have revealed the existence of complex mechanisms regulating the induction and development of these responses.

Malaria currently ranks among the most prevalent infections in tropical and sub-tropical areas throughout the world with relatively high morbidity and mortality particularly in young children. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with radiation-attenuated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, vaccines based on radiation-attenuated sporozoites are not feasible for large scale application due to lack of in vitro culture system. Therefore, the development of peptide-based subunit vaccines has been undertaken as an alternative approach. Synthetic peptides containing defined B- and T-cell epitopes of different antigens expressed in sporozoites and/or liver stages have been used as subunit vaccines in experimental animal models. They have been shown to be highly immunogenic and capable of inducing protective immunity mediated by antibodies, as well as CD4+ and CD8+ T-cells.

We describe a highly sensitive real-time PCR to detect and measure the development of the liver-stages of malaria parasites in mice infected with sporozoites ranging in number from 25 to more than 164,000, using the same reaction conditions. Furthermore, this assay detects and measures parasite loads in the livers of mice exposed to the bite of a single malaria-infected Anopheles mosquito. This unique method should greatly facilitate studies aimed at evaluating very precisely the efficacy of anti-malarial experimental drug treatments and vaccination regimens in conditions of infection resembling those found in the field.

The antibody response to the recombinant protein, R32tet32, which contained the repetitive sequence (NANP)n of Plasmodium falciparum CSP was determined in C57BL/6 mice during the course of nonlethal infection with Plasmodium yoelii 17X. Marked suppression of the IgG antibody response to R32tet32 occurred when mice were immunized at peak parasitemia (on day 16). In vitro antibody responses of spleen cells from acutely infected mice to R32tet32 were similarly suppressed. Stimulation of normal spleen cells cultured for 5 days with 100 ng/ml of R32tet32 gave an optimal IgG antibody response, but spleen cells from infected mice obtained at peak parasitemia failed to respond to a broad range of antigen concentrations. Cocultivation studies employing enriched lymphocyte populations from infected and uninfected C57BL/6 mice indicated that both T and B cells from infected mice were defective in their response to R32tet32. The response to the repetitive region was restored by the addition of recombinant mouse interleukin-2 (IL-2) at a dose of 50 U/ml to cultures of spleen cells from infected mice.

We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. We now report that intrahepatic HBV replication is also inhibited in mice infected by the malaria species Plasmodium yoelii 17X NL. P. yoelii infection triggers an intrahepatic inflammatory response characterized by the influx of natural killer cells, macrophages, and T cells. During this process, interferon (IFN)-gamma and IFN-alpha/beta suppress HBV gene expression and replication in the liver. Collectively, the data suggest that malaria infection might influence the course and pathogenesis of HBV infection in coinfected humans.

Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, alpha-galactosylceramide (alpha-GalCer), known to selectively activate Valpha14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of alpha-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasites Plasmodium yoelii and Plasmodium berghei. The antimalaria activity mediated by alpha-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-gamma is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.

Our previous reports indicated that C57BL/6 mice infected with a lethal variant of Plasmodium yoelii 17X (P. yoelii 17XL) produced high levels of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma) while mice infected with the nonlethal variant of the parasite did not produce detectable levels of IL-10. In the present study, the involvement of IL-10 and IFN-gamma in exacerbation or regulation of blood-stage malaria was investigated by using the lethal variant of P. yoelii 17XL and monoclonal antibodies (mAb) against the cytokines. C57BL/6 mice were injected intraperitoneally with a neutralizing anti-IL-10 mAb or anti-IFN-gamma mAb after inoculation with P. yoelii 17XL. Treatment of mice with anti-IL-10 mAb resulted in substantial prolongation of survival and 60% of treated mice survived while 100% of control mice died by day 11. On the contrary, treatment of mice with anti-IFN-gamma mAb exacerbated infection and all mice died after an earlier period than those treated with normal rat Ig. No differences in parasitemias were found between treated and untreated mice. To elucidate the involvement of nitric oxide in the host protection or exacerbation, mice were treated with aminoguanidine, an inhibitor of nitric oxide synthetase, after inoculation of P. yoelii 17XL. Neither mortality nor parasitemia was influenced by the treatment. These results indicate that an IFN-gamma response is associated with protective immunity in mice infected with P. yoelii 17XL, while an IL-10 response is associated with disease exacerbation during the infection.

Recombinant adenovirus, expressing the CS protein of Plasmodium yoelii, AdPyCS, was shown to induce a comparable degree of T cell-mediated protection against malaria as a single dose of irradiated P. yoelii sporozoites, causing inhibition of liver stage development. We now report that differently from sporozoite-induced immunity, interferon (IFN)-gamma does not mediate the protective immunity induced by AdPyCS, since a similar degree of protection was observed in AdPyCS immunized mice lacking IFN-gamma-/- and the IFN-gamma receptor (IFN-gammaR-/-) compared to that in wild-type mice. Depletion of CD8+ T cells from these immunized mice almost completely abolished the AdPyCS-induced immunity, indicating that the immunization with AdPyCS induces CD8+ T cell-mediated protective anti-malaria immunity, which is independent of IFN-gamma.

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.

The immunogenicity of a previously constructed replication-defective recombinant adenovirus expressing the CS protein of Plasmodium yoelii was compared with that of irradiated sporozoites. We found that immunization of BALB/c mice with a single dose of this recombinant adenovirus induced a much greater CS-specific T-cell response compared with immunization with irradiated sporozoites. More importantly, we found that this recombinant adenovirus induces similar or higher levels of protective immunity than those induced by irradiated sporozoites, eliciting an appreciable resistance to malaria infection.

Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major histocompatibility complex-restricted 9-mer epitope of the Plasmodium yoelii circumsporozoite protein or the nucleoprotein of influenza virus induces a large epitope-specific CD8(+) T-cell response. This immunization also elicits a high degree of protection against infection with malaria or influenza A virus.

The immunogenicity of a recombinant replication defective adenovirus expressing a major malaria Ag, the circumsporozoite (CS) protein (AdPyCS), was determined using a rodent malaria model. A single immunizing dose of this construct induced a large number of CS-specific CD8+ and CD4+ T cells in the spleens of these animals, particularly when given by the s.c. or i.m. route. A single dose of AdPyCS also induced high titers of Abs to Plasmodium yoelii sporozoites in mice. No other form of presentation of the CS protein given as a single immunizing dose, i.e., irradiated sporozoites, recombinant vaccinia, or influenza virus, etc., elicits comparably high numbers of CS-specific CD8+ T cells. The high concentration of CS-specific CD8+ T cells in the spleen was relatively short-lived, decreasing to half of its original value by 4 wk and to one-third at 8 wk after AdPyCS inoculation. The decrease in splenic CS-specific CD4+ T cells was even more rapid. Most importantly, a single dose of inoculation of AdPyCS into mice rendered them highly resistant to sporozoite challenge, resulting in a 93% inhibition of liver stage development of the parasites. This protective effect was primarily mediated by CD8+ T cells, as shown by depletion of this T cell population, while depletion of the CD4+ T cell population had only a minor effect on anti-plasmodial activity. Moreover, the inoculation of mice with AdPyCS induces sterile immunity in a significant proportion of mice, preventing the occurrence of parasitemia.

In this study we characterized the CD4+ T cell response directed against two distinct epitopes located in the circumsporozoite (CS) protein of Plasmodium yoelii. The immunization of mice with P. yoelii sporozoites induced CD4+ T cells which were mostly directed against one of these peptides, Py-1, previously reported to contain a CD4+ epitope. The CD4+ T cells directed against this immunodominant epitope were mostly of the Th-1 type. Another newly identified peptide, AS44, induced a specific CD4+ T cell response, which was mainly detectable after immunization with the corresponding peptide. Several CD4+ T cell clones, recognizing this epitope, were generated and their lymphokine expression was characterized, as well as their surface markers and their anti-parasite activity in vivo. It was noteworthy that some of these CD4+ T cell clones, which recognize this cryptic epitope and were of different Th subtypes, were shown to have a strong inhibitory effect on the development of liver stages of malaria parasites.

Sporozoites of P. yoelii nigeriensis are 50-100-times more infective to mice than the strain NK65 of P. berghei. To study the mechanisms involved in this striking difference in the infectivity of these closely related species of malaria parasites, we have developed a quantitative PCR targeted to parasite-specific ribosomal RNA. Using this method, we detect RNA from a single sporozoite, and exo-erythorcytic forms of RNA in the livers of mice injected with 200 sporozoites. We find that 20 h after sporozoite injection, there is no significant difference between the amounts of P. berghei and P. yoelii rRNA in the livers of C57/BL6 mice, indicating that these two parasite species invade hepatocytes with similar efficiency. Between 20 and 40 h, however, P. yoelii RNA increases 11 times, while P. berghei RNA increases only 1.6 times. We conclude that the greater infectivity of P. yoelii sporozoites in these mice reflects, at least in part, their superior development in hepatocytes. These data provide for the first time in vivo evidence supporting the notion that species-specificity of malaria is not determined by mechanisms associated with sporozoite attachment and penetration into the hepatocytes.

Six murine T cell clones expressing gamma delta TCR were generated from malaria immunized, alpha beta T cell-deficient mice. Phenotypic characterization of these clones has revealed that, in contrast to conventional alpha beta T cells, there is a considerable degree of heterogeneity among these gamma delta clones with regard to their surface markers and their lymphokine profile. One clone was found to display significant anti-parasite activity in vivo upon adoptive transfer. We attempted to determine whether the protective clone differs in one or more key characteristics from the non-protective clones. Although no obvious pattern peculiar to the protective gamma delta clone was observed, it appears that more than one parameter may, in combination, define a distinct protective phenotype, and thus explain the functional difference between the protective and non-protective gamma delta clones.

We investigated the induction of T-helper cell subsets during the course of lethal or nonlethal bloodstage Plasmodium yoelii 17X infection in C57BL/6 mice, which are relatively susceptible to these intraerythrocytic parasites. C57BL/6 mice infected with the nonlethal variant (PyNL) showed a moderate level of parasitemia and resolution of primary acute infection by week 4. Mice infected with the lethal variant (PyL) developed fulminating parasitemia and ultimately died. T-helper subset function was assessed during infection by determining the kinetics of in vitro production of the Th1-derived cytokine interferon-gamma (IFN-gamma) and the Th2-derived cytokine interleukin 10 (IL-10) by means of bioassay and enzyme-linked immunosorbent assay (ELISA), respectively. Spleen cells obtained from mice infected with PyL within the 1st week of infection produced high levels of IL-10 and IFN-gamma in response to malaria antigen. IL-10 also appeared in sera from PyL-infected mice at the same time at which the in vitro IL-10 response peaked. In contrast, spleen cells from mice infected with PyNL failed to produce IL-10 during the course of infection. CD4+ T-lymphocytes from mice infected with the lethal variant were a major source of IL-10, although non T-cells were also involved in the production of IL-10 during this malaria infection. In addition, the initial burst of IL-10 in response to malaria antigens was seen concomitantly with the production of IFN-gamma within the 1st week of infection. These results indicate that both Th1 and Th2 subsets of T-helper lymphocytes are activated during infection with the lethal variant of P. yoelii and support the contention of other investigators that a strong Th2 response early in infection is associated with the lethal outcome of malaria.

IFN-gamma receptor deficient (IFN-gamma R-/-) mice, immunized with different developmental stages of malaria parasites, were used to define the mechanisms of protection against the various stages of this infection. IFN-gamma R-/- mice failed to develop protective immunity against Plasmodium yoelii sporozoites or liver stages, upon immunization with a single dose of irradiated sporozoites, whereas in immunized wild-type mice, parasite development was strongly inhibited. Immunized wild-type mice expressed high levels of inducible nitric oxide synthase (iNOS) mRNA in their liver, upon challenge with viable sporozoites, whereas only background levels of iNOS were detected in immunized IFN-gamma R-/- mice. In contrast, after immunization with multiple doses of irradiated sporozoites, both IFN-gamma R-/- and wild-type mice mounted an immune response, which strongly inhibited the development of liver stage parasites. In both types of mice, protection occurred in the absence of appreciable expression of liver iNOS mRNA. As for the course of the erythrocytic phase of infection by nonlethal malaria species, P. yoelii yoelii and P. chabaudi adami, we observed only a moderately prolonged parasitemia in IFN-gamma R-/- mice compared with wild-type mice, indicating that IFN-gamma may only play a modest role in immunity against erythrocytic stages. These results indicate that IFN-gamma is the main mediator of the protective mechanism that develops first upon immunization with sporozoites. However, the nature of the anti-parasite mechanism(s) changes in the course of immunization, so that multiple immunizing doses elicit additional protective mechanisms, which are independent of IFN-gamma and its receptor.

The functional role of gamma delta T cells (expressing the gamma delta heterodimeric T-cell receptor for antigen) in infectious diseases remains largely unknown. We have therefore attempted to define the possible role of these T cells in the immune response against the various developmental stages of malaria parasites. For this purpose, we monitored the immune response and the development of liver and blood stages of Plasmodium yoelii, a rodent malaria parasite, in immunized and nonimmunized alpha beta T-cell-deficient and gamma delta T-cell-deficient mice. Immunization of alpha beta T-cell-deficient mice with irradiated sporozoites induced an immune response that significantly inhibited the development of the parasite's liver stages. This inhibitory immune response was abolished by an antibody-mediated transient in vivo depletion of gamma delta T cells. Two gamma delta T-cell clones were derived from malaria-immunized alpha beta T-cell-deficient mice. The adoptive transfer of one of these gamma delta T-cell clones to normal mice inhibited the development of liver stages, following sporozoite inoculation. These results provide evidence for gamma delta T-cell-mediated protective immunity against parasites, in the absence of alpha beta T cells. As for the blood phase of the infection, both normal mice and gamma delta T-cell-deficient mice cleared the blood stages of the nonlethal strain of P. yoelii, while alpha beta T-cell-deficient mice failed to control the parasitemia.

A CD4+ T cell clone (A1.6) was derived from spleen cells of mice immunized with irradiated sporozoites. This T cell clone recognizes an antigen that is shared by sporozoites and blood forms of Plasmodium berghei and differs from the circumsporozoite protein. Clone A1.6 displays cytotoxic activity, produces IFN-gamma and IL-2 in vitro, and recognizes the plasmodial antigen in the context of the class II I-Ed molecule. Passive transfer of this CD4+ clone into naive mice resulted in a high degree of protection against sporozoite challenge.