Plasmodium Axenically Developed Exo-erythrocytic Forms Immunization Confer Strong Protection against Infectious Sporozoite Challenge

Abstract

Malaria causes nearly a million deaths every year and nearly 50% of the world population is at risk. Irradiated sporozoite vaccination is a proven and successful strategy but difficult to implement. An attenuated whole parasite vaccine is an achievable goal in spite of difficulties. Here we explore the possibility of using exo-erythrocytic forms (EEF) immunization as an attenuated whole parasite vaccine. As proof of principle we used in vitro derived EEF from Plasmodium yoelii (a mouse malaria parasite) for immunization of mice and that these forms confer strong protection against infective sporozoite challenge. Antibodies generated were species specific and not the strain specific. We also show that antibody response is mounted against few antigens. This shall help in narrowing important antigens of liver stage.

Keywords: Plasmodium; Malaria; Liver stage; Vaccine; Exo-Erythrocytic Forms; Attenuated parasite immunization

Abbreviations

CAS: Chemically Attenuated Sporozoites; CTL: Cytotoxic T Lymphocyte; EEF: Exo-Erythrocytic Forms or liver stage parasite; ITV: Infection Treatment Vaccination; GAS/GAP: Genetically Attenuated Sporozoite/parasite; RAS: Radiation Attenuated Sporozoites; WPI: Whole Parasite Immunization

Introduction

Malaria remains serious public health problem for roughly 50% of the world’s population. Morbidity associated with malaria is ~ 0.63 million each year [1]. There is limited repertoire of available drugs that can cure malaria. The problem is further aggravated due to emergence of drug resistant parasite [2]. An urgent need for the effective vaccine is greater than ever. Vaccine candidates tried till-date are mostly based on single antigens [3] except few combinations [4,5] and targeted at individual stages of the malaria life cycle. Limited success was obtained with the vaccines tested to-date [6-9]. The only proven vaccination strategy that provides sterile protection is through the Radiation Attenuated Sporozoites (RAS) vaccine [10-12]. Recently, alternative to RAS was created in the form of Genetically Attenuated Sporozoites (GAS) and successfully tested in small animals [13-15] as well as in humans with limited success [16]. A vaccine targeting the naïve traveler or newcomer to malaria endemic area would require complete protection and this may be achieved using vaccine against pre-erythrocyte stages [10].

Most successful vaccines (smallpox, measles, polio etc.) were empirically developed using the attenuated or inactivated whole pathogens or material derived directly from the infectious agent [17,18]. Except a few [example Hepatitis B Vaccine] [19], recombinant protein vaccines currently are not very successful [6,7,20]. A successful recombinant protein vaccine for malaria is currently unavailable [21]. Under these circumstances to protect the travelers and infant’s naïve to malaria antigens, a non-replicating pre-erythrocytic whole organism vaccine needed. There are several hurdles in obtaining such a vaccine and they must be addressed [22]. The practical questions that need to be answered are: a) Can one produce enough material for large-scale application, which is cost effective and practically feasible. b) Can one administer such a vaccine by the route that is clinically acceptable. c) Can one produce such a vaccine that meets the regulatory norms [22].

Proof of principle of the live attenuated vaccine first time came in 1967, when Nussenz weig and colleagues reported, that immunizing mice with radiation attenuated Plasmodium berghei (P. berghei) sporozoites protected them against challenge with fully infectious sporozoite [12]. Protection was also demonstrated in humans in 1973 [23]. Recent studies in humans immunized with purified, radiation attenuated sporozoite, introduced by intravenous route further confirmed the previous findings [24]. Irradiated sporozoites are able to invade liver cells and transform into exo-erythocytic forms (EEFs) but fail to develop further [25]. A lot has been done to understand how the irradiated sporozoite vaccine works. Nussenzweig [26] and others [27,28] have shown that optimal dose of irradiation is required to achieve protection. A higher dose of irradiation lead to reduction in the number of early EEFs, a prerequisite for protection, while lower dose of irradiation leads to full growth of EEFs and subsequent development of blood stages leading to disease. Treatment with Primaquine a drug that kills the EEFs also abrogates the protection. Important point that emerged from the aforementioned studies was species specific but not strain specific immunity [10]. In the RAS immunization multiple effect or mechanisms, including antibodies, helper cells, lymphocytes, CD4+, CD8+ cytotoxic T lymphocytes (CTLs) [29-31] are involved. Immunity against EEFs, developing inside the liver hepatocytes, arise from the Interferon gamma (IFN-γ) produced by effecter cells [32,33]. IFN-γ based EEF killing is mediated by nitric oxide (NO) [34,35]. A study with normal sporozoite immunization combined with Chloroquine treatment [Infection treatment vaccination, ITV] provided further evidence of EEF antigens importance in inducing the protective immune response [35].

Liver stages were neglected for long time owing to practical difficulties in obtaining pure EEFs from host cells. Since these EEFs are intracellular, they are not direct targets of humoral responses. This has hindered the studies on their antigenic composition unlike to sporozoite stage proteins. The protection offered by RAS immunization is mainly due to T-cells. Availability of methodology for cell free development of EEFs [36] has opened the doors to study the immune response elicited by the partially developed EEFs produced after whole parasite immunization [WPI].

Materials and Methods

Parasite and axenic EEFs

Four to five day old female Anopheles stephensi mosquitoes were blood fed on anesthetized BALB/c mice infected with wild type P. yoelii yoelii 17XNL strain. After the blood meal, mosquitoes were maintained at 23°C and 83% humidity. Between the days fourteen to seventeen post-feeding, mosquitoes were washed in 70% ethanol for five minute and rinsed twice with sterile medium. Salivary glands were dissected and sporozoite were recovered in sterile DMEM medium containing 2x antibiotic-antimycotic (Invitrogen). Transformation was done essentially as described by Kaiser et al. [36]. Characterization of axenic EEFs was done as described previously [36]. In brief, one million sporozoite per well were put in twenty-four well culture plates along with one ml of medium and incubated for twenty-four hours, at 37°C. Transformed parasites were harvested and washed twice with cold PBS before they were administered to mice. Transformation efficiency for each batch was determined by IFA.

Animals and immunization

Female BALB/c mice aged 6-8 weeks were immunized subcutaneously. Immunizations were done with or without adjuvant. In the case of adjuvant, Freund’s complete adjuvant for priming and Freund’s incomplete adjuvant for boosts were used. We used four doses of EEF immunization and for each dose group five animals were used. The control group received equivalent amount of uninfected salivary glands in PBS or adjuvant. The following schedule for immunizations were used, priming on day one, first boost on day fourteen and second boost on day twenty-one. On day thirty-one livers and sera were collected. For CD4/CD8 cells depletion experiments mice were immunized with 30,000 axenic EEFs on the schedule described above, followed by injection of 0.2 mg of anti- CD4 (monoclonal GK1.5) or anti-CD8 (monoclonal YTS169) on days 26, 27, 28 and 29. In all the experiments mice were challenged with 10,000 sporozoite on day 29.

Sporozoite challenge and quantitation of parasite burden

Seven days after the second boost, animals were intravenously injected with 10, 000 P. yoelii 17XNL infectious sporozoites. Forty-four hours post challenge livers of the mice were collected, RNA extracted, c DNA prepared and real-time RT-PCR performed as described previously [37], to determine liver stage burden. There was a difference from previous protocol that we used SYBR green-I dye in place of fluorescent probe. The liver stage burden was determined by estimating the parasite 18S r RNA copy numbers. Parasite 18S r RNA copy numbers were normalized with murine GAPDH copy numbers in the corresponding reactions. The normalized values for each group were compared to the control group to get percentage inhibition. The inhibition obtained with RAS immunization was considered as 100 percent and those with EEF immunization were compared with respect to the 100 percent of RAS immunization. In this study a 90% inhibition of liver stage burden means 90% with respect to RAS immunization (100%, sterile protection) and not with respect to control immunized group.

Indirect fluorescence antibody Test (IFAT)

EEF transformation efficiencies were checked by IFAT using antibody against parasite HSP70 [38] and Myosin A tail domain interacting protein (MTIP). To check the reactivity of anti-EEF sera against in vitro or in vivo transformed parasite we used mouse anti EEF sera together with a rabbit polyclonal antibody against MTIP that recognize an inner membrane complex associated protein [39]. Secondary antibodies against mouse or rabbit IgG was coupled to Alexa 488 or Alexa 594 and used at 1:250 dilutions in 1% BSA/PBS. Nuclei were stained with DAPI (6 amidino 2 phenyllindole).

Western analysis of EEF lysate

P. yoelii yoelii 17XNL 2×10⁵ transformed (~10% efficiency) or untransformed sporozoite were lyses in lysis buffer [150 mM NaCl, 50 mM Tris pH 8.0, Protease inhibitor cocktail (Roche) 1x, and 1% Triton × 100], heated at 95°C for 10 minute and separated on 10% SDS-PAGE (Bio-Rad). Separated proteins were transferred to a PVDF membrane and blocked in 3% BSA/PBS. Membrane was cut in three parts and one part each incubated with anti-EEF sera, anti- CS (monoclonal 2F6) or anti HSP70 (monoclonal 2E6). Membranes washed three times with PBS/0.05% Tween-20 followed by detection with ECL kit (Amersham Biosciences, USA).

ELISPOT

ELISPOT method used here has previously been described [40]. We followed the sections 6.1, 6.2.1, 6.2.2, 6.2.4, and 6.2.6 as described in the study by Carvalho et al.

Results

Immunization with axenic-EEFs protect from infective sporozoite challenge

Immunization with irradiated sporozoite confers complete protection and the development of EEFs is essential. Availability of in vitro method to culture the early liver stage parasites lead us to examine whether axenic EEFs were equally potent, to RAS immunization, in inducing protective immune responses. Animals were immunized subcutaneously with live axenic EEFs without any adjuvant or as a crude antigen emulsion made in adjuvant (dead). Both immunizations lead to highly protective immune response (Table-1, Figure 1) despite the low antibody titers (IFA titers <500). The protection was dose dependent and live parasite immunization gave slightly better protection compared to dead. At highest dose tested (equivalent to 100,000 axenic EEF) live parasite gave 93 + 9.02% while dead (equal number emulsified) gave 84 + 9.30% reduction in parasite burden when compared to RAS immunization that we consider as 100% (Figure 1). Both immunizations regimens either emulsified in Freunds adjuvant or not, gave comparable protection indicating that EEFs alone in absence of any adjuvant can mount strong immune response.