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Globally, vaccines have emerged as one of the most effective, safe, and cost-effective public health interventions, and are known to save 2–3 million lives, annually. However, despite various commendable efforts, a suitable human malaria vaccine is yet to see the light of the day. The lack of our complete understanding of the molecular mechanisms of pathogenesis and immune protection in malaria appears to be responsible for this state. Earlier, our laboratory has reported that Swiss mice vaccinated with Plasmodium yoelii nigeriensis-total parasite antigens soluble in culture medium and saponin, following a 100% lethal challenge, showed 60% protection. The monoclonal antibodies (MAbs) generated from the splenocytes of these vaccinated/protected mice, following characterization by in vitro merozoite invasion inhibition assay, ex vivo macrophage phagocytosis assay, and in vivo passive transfer of protection test, belonged to 2 distinct groups—a larger group of MAbs inhibited <58% Mz invasion and transferred 30% passive protection, whereas a smaller group of MAbs inhibited 86% Mz invasion and transferred 60% passive protection. Additionally, the MAbs of the smaller group, as compared with the larger one, mediated nearly 2.4-fold enhanced macrophage phagocytosis of infected-erythrocytes, in vitro. These results thus clearly showed a dichotomy among the generated MAbs. An exploration of the phenomenon of dichotomy in protective immunity in malaria by using various hosts and malaria parasite combinations, especially at the level of antibodies, cells, and cytokines, may add new insights to our understanding of the protective immunity, and help in the identification of biomarkers/biosignatures of immune protection and development of future human malaria vaccines.
Malaria, a life-threatening yet treatable and preventable disease, caused by the parasites of the genus Plasmodium, continues to be a major public health problem worldwide. According to WHO Factsheet on the World Malaria Report (2013),1 in the year 2012, an estimated 3.4 billion people were still at risk of malaria, and there were an estimated 207 million malaria cases and 627000 (including 483000 children under the age of 5 y) deaths. And this enormous toll of suffering and death occurred despite the fact that during the period 2000 and 2012, there has been a remarkable progress in world malaria situation resulting in the saving of 3.3 million lives. Based on the reported data in the factsheet, 59 out of 103 countries that had ongoing malaria transmission in the year 2000, are now meeting the Millennium Development Goal target of reversing the incidence of malaria. The emergence of drug-resistant parasites, insecticide-resistant mosquitoes, and the communal and monetary problems have further convoluted the problem of malaria. On the other hand, despite several commendable efforts, a suitable human malaria vaccine is still not available.2 The lack of our complete understanding of the molecular mechanisms of pathogenesis and immune protection in malaria, along with several other problems, continue to be the main reasons behind this state of affairs. Therefore, it is essential to revisit and explore the mechanisms of immune protection in malaria with novel approaches to come-up with some out-of-the-box ideas and potent high-end solutions.
The mechanisms of immune protection in malaria continue to remain elusive, though it is believed to involve both cells and antibodies—nevertheless, evidence is most clear for the latter for blood-stage malaria. In some rodent malarias, and probably in human malarias also, both antibodies and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12 (IL-12), and nitric oxide (NO) are known to protect against the asexual blood stages of malaria.3 Earlier, our laboratory has reported that recombinant human IL-12,4 and recombinant mouse granulocyte-macrophage colony-stimulating factor in combination with methionine-enkephalin5 and its fragment tri-peptide Tyr-Gly-Gly6 can protect against simian and murine malarias, respectively. The immunological paradigm separating CD4+ T helper (Th) cells has helped in the molecular characterization of antimalarial immune response.7 Briefly, Th1 cells produce IL-2, IFN-γ, IL-12, and TNF, and mediate macrophage activation, whereas Th2 cells secrete IL-4, IL-6, and IL-10, and help in antibody production. The nature of CD4+ T cell response in mice and humans is apparently similar, if not identical.8 In humans, antibodies are known to play substantial role in protection from blood-stage of malaria, besides the cell-mediated and innate immune mechanisms.9 Blood-stage malaria, responsible for the majority of morbidity and mortality, following repeated exposures in endemic areas, induces slow-developing infection-induced acquired protective immunity, and over several years, such individuals develop an array of antibodies to several P. falciparum antigens,10 and acquire mechanisms to control the inflammatory responses to the parasites. In mice immunized with recombinant proteins from the C-terminus region of merozoite (Mz) surface protein, protective immunity is primarily mediated by antibodies, and their high serum titers positively correlate with protection.11 Though, as of now, no unequivocal surrogate marker(s) of protection from malaria has been identified, albeit very much warranted, it is now well established that antibodies which inhibit Mz invasion into erythrocytes in vitro, play significant role(s) in protection in vivo,12 and this contention is believed to provide the rational for the development of blood-stage malaria vaccines.13,14 Nevertheless, not much is known about the induction of the protective antibody-mediated immune response and its heterogeneity, the specific identity and physiognomies of the blood-stage antigens, which are responsible for the induction and generation of protective antibodies, both at the molecular and functional levels, and the niceties of immune network that ends-up in protection.
The malaria parasites are awfully complex organisms, and express a large repertoire of antigens on the surface of infected-erythrocytes (IEs),15 which are the targets for naturally acquired protective immunity to malaria.16 These antigens are constantly perceived by the immune system of the host, and are thought to elicit an array of immune response. The application of hybridoma technology that enables the in vitro production of monoclonal antibodies (MAbs) of pre-defined specificity,17 constitutes an effective and handy tool for the dissection, minute examination, and elucidation of the antibody-mediated immune response, and thus allows the application of reductionist approach for the identification of protective malarial antigens.18 A large number of MAbs have been reported against various malaria parasites viz. P. berghei,19 P. chabaudi,20 P. yoelli,21 P. yoelii nigeriensis,22 P. gallinaceum,23 P. knowlesi,24 and P. falciparum.25
In most of these studies—however, enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, and passive transfer of protection test have been used to identify and characterize the positive hybrids and the resultant MAbs. Surprisingly, the characterization of MAbs using both Mz invasion inhibition assay, one of the most accepted in vitro correlate of protective immunity to malaria in vivo, and passive transfer of protection test have apparently not been reported. The invasion of erythrocytes by Mz is an important function for the survival of malaria parasite.26 The gamma globulin from immune adults living for long periods in malaria endemic areas are known to inhibit both Mz invasion in vitro,27 and to transfer passive protection in vivo.27-29 Further, immune serum and purified antibodies from experimentally immunized animals (as evidenced by protection of immunized animals following infective challenge) are also known to inhibit both Mz invasion in vitro30-32 and transfer passive protection in vivo.31,33
To comprehend the mechanisms of protective immunity to malaria, in vivo, various murine immunization/vaccination models have been developed, but apparently all of them are fraught with serious limitations.34,35 The mouse models offer several advantages over the simian models in terms of ease, cost, and ethics. Additionally, mouse immune system is well characterized, allows greater segmentation, and the immunological reagents are easily available. Experience has shown that lethal malaria infections are better suited to study vaccination-induced protective immune mechanisms, whereas non-lethal models are considered best for exploring acquired immunity.36 In our laboratory, a new, novel, and rigorous mouse malaria vaccination model, using P. yoelii nigeriensis total parasite antigens soluble in culture medium (Pyn-SA) and saponin as an adjuvant, has been developed with up to 60% protection against an invariably 100% lethal homologous challenge.31 This host/parasite system was specifically identified and zeroed upon because, unlike various other rodent malaria models, P. yoelii nigeriensis infection in mice, in our hands, invariably resulted in a fulminating infection with consistently 100% mortality within 10 d and, therefore, all the mice that survived the lethal challenge can be expected to have developed the vaccination-induced robust protective immunity only; the slow-developing low-grade infection-induced immunity, observed in other non-lethal rodent malarias cannot be expected to play any role in this model because P. yoelii nigeriensis infection in mice is fast-progressing that would not allow sufficient host-parasite interaction to develop infection-induced protective immunity. Nevertheless, we contend that this novel model needs to be improved and refined by using purified and characterized highly protective antigen(s). Further, in an extended separate similar study,37 these researchers generated and characterized MAbs from the splenocytes of these vaccinated/protected mice. Their one of the most important and striking observation, apparently reported for the first time, has been that the Mz invasion inhibitory activity, in vitro, of purified MAbs generated from the splenocytes of each of the 6 protected mice showed a distinct functional dichotomy—a smaller group of only 2–3 MAbs inhibited >86% invasion in vitro, and transferred 60% passive protection, whereas the other relatively larger group of 6–9 MAbs inhibited <58% Mz invasion and transferred 30% passive protection. Surprisingly, MAbs from each of the 4 mice, which, following challenge, resisted the infection for a while but ultimately developed uncontrollable high parasitemia, also inhibited <58% Mz invasion but transferred only 10% passive protection. Additionally, the smaller group of MAbs consistently augmented nearly 2.4-fold phagocytosis of IEs by mouse elicited peritoneal macrophages, in vitro (unpublished observations). Thus these findings revealed the occurrence of dichotomy in protective malarial MAbs.
The cell-mediated immunity (CMI) is also believed to play a significant role in protection from malaria. There are several cells and molecules such as CD8+ T cells, CD4+ T cells, dendritic cells (DCs), and several cytokines and other mediators which comprise CMI. Based on their functional capabilities, the mouse Th lymphocytes have been divided into 2 well-defined subsets of effector cells and cytokine milieu they produce.38 The Th1 cytokines such as IFN-γ and TNF, are usually associated with inflammatory responses and induce CMI mechanisms. However, on the other hand, IL-4 and IL-5 that help B cells to proliferate and differentiate are Th2 cytokines, and induce humoral-type of immune responses.38 The CD4+ T cells are important for the protective immunity against asxeual stage of malaria parasites; however, the underlying mechanisms through which protective immunity is generated and expressed remain elusive.39 It is now well documented by adoptive transfer experiments that both Th1 and Th2 subsets of CD4+ T cells can impart protection to mice against an infective P. chabaudi chabaudi challenge.39 Thus, from these reports it can be contended that a potential functional dichotomy exists in CD4+ T cells. Our views thus appear to be in consonance with this contention that there is a functional/mechanistic dichotomy in CD4+ T cells that is reflected in mechanisms of immune protection induced by them: nitric oxide appears to be the effector molecule for protection induced by Th1 cells, whereas enhanced specific IgG production appears to be the mechanism of protection by Th2 cells.39 Nonetheless, the recent literature is supportive of the view that stretches the paradigm of Th1 and Th2 to Th17 cells, a new effector T cell, which elaborates a novel family of cytokines, the IL-17 cytokines.40
Kinhikar and Singh,37 in their studies on the phenomenon of dichotomy in protective antibodies in rodent malaria have used saponin as the adjuvant for the vaccination of mice. Nevertheless, it is now increasingly believed that adjuvants exert tremendous effects on both the quality and the quantity of the immune response generated. It is, therefore, expedient to expolre if adjuvants have anything to do with the phenomenon of antibody and/or cytokine dichotomy. The adjuvants may exert their vaccine-associated effect(s) by recruiting professional antigen presenting cells (APCs) to the vaccination site, by increasing the delivery of antigens to APCs, or by activating APCs to produce cytokines, which, in turn, may function as the activating signals to T cells.41 Alum (an aluminum salt) is considered as the most extensively used adjuvant in human vaccines, over a long period of time. The antigens of the pathogens are adsorbed onto the surface of alum molecules, which results in a suspension/emulsion that is administered, intramuscularly. Although it is being extensively used in human vaccines for a long time, so far there is no idea as to how it acts as an adjuvant. However, despite this general contention, apparently, there is a convergence of opinion that aluminum adjuvants exert their actions via sustaining a slow-release antigen(s) repository to the immune system.42 Further, aluminum salts and MF59 may also exert their adjuvant actions by initiating inflammation and permeation of DCs to the vaccination site(s) and by expanding the uptake of related antigens by DCs.43 QS21 and other saponins are thought to exert their adjuvant actions by inducing the DCs to produce IL-12.44 The dependence of the generation of the protective immune response on the quality of antigen and its route of administration has been very well demonstrated in a rodent model of Plasmodium yoelii malaria.45 It is thus apparent that the adjuvants may have the potential to influence both humoral and CMI during vaccination, and various adjuvants exert their effects on the protective immunity in diverse ways, and thus may play potentially important role(s) in the generation of dichotomous immune response.
Herein, apparently for the first time, we have attempted to identify and add an entirely new dimension to our understanding of the mechanisms of immune protection in malaria. More pointedly and specifically, we report that MAbs generated from the splenocytes of Pyn-SA vaccinated/protected mice showed a clear-cut dichotomy, as determined by the well-established functional tests/assays of protective malarial immunity: Mz invasion inhibition, in vitro, macrophage phagocytosis, ex vivo, and passive transfer of immunity, in vivo. Further, from these observations a potential possibility emerges that in these vaccinated/protected mice, in all probability, some other mechanisms of immune protection (both humoral and cellular), and especially those mediated by cytokines, may also show dichotomy. Therefore, further studies along these lines are very much warranted. The knowledge so generated may add significantly to our understanding of the protective immune mechanisms in malaria, which can be expected to go a long way in the identification of potent biomarkers/biosignatures of immune protection in malaria and in the development of new and more effective malaria vaccines for human use.
No potential conflicts of interest were disclosed.
We are grateful to Prof. K.K. Bhutani, Officiating Director, National Institute of Pharmaceutical Education and Research (NIPER), for his help and encouragement. B.P. is grateful to NIPER for financial assistance. This is NIPER communication No. 494.