Background
The control of malaria mainly relies on anti-malarial drugs, rapid diagnosis, and vector control measures (such as insecticide-treated bed nets, or indoor insecticide spraying) and these interventions have reduced the mortality of malaria in recent years. However, due to increased resistance of mosquito vectors to insecticides and of parasites to available anti-malarial drugs, there are still 445,000 malaria-associated deaths worldwide every year, mostly because of
Plasmodium falciparum [
1]. Lessons from eradication of infectious diseases such as smallpox from the world [
2,
3] and polio from the Western Hemisphere [
4] have highlighted that to eliminate malaria, a vaccine would need to be included in the anti-malarial control tools. Such a vaccine would be expected to reduce the parasite reservoirs and interrupt malaria transmission [
5].
In the new era of malaria vaccine development, a transmission-blocking vaccine (TBV) is considered an essential type of vaccine for the elimination of malaria [
6,
7]. The aim of TBVs is to induce antibodies in human hosts against antigens expressed either during the sexual stage or antigens found in mosquito vectors. These antibodies must be able to inhibit parasite development in the
Anopheles mosquito midgut, when they are ingested as a part of the blood meal with mature gametocyte stage parasites [
8]. Although TBVs would not directly prevent infection in human host, they could help in elimination of the disease by preventing the transmission of infections [
6,
7,
9,
10]. On this basis, the Malaria Eradication Research Agenda (malERA) Consultative Group has suggested the concept of “vaccine that interrupts malaria transmission (VIMT)” [
6] in which this new proposed vaccine includes the classical TBVs, pre-erythrocytic and asexual blood-stage antigens that are able to protect immunized subjects from infection and also reducing parasite transmission. This type of malaria vaccines may possibly be an important tool for elimination programmes, as well as protecting against epidemics.
The malaria parasite infects both human and mosquito hosts and thus, an effective anti-malarial subunit vaccine may include antigens expressed in multiple stages of the life cycle. The cell-traversal protein for ookinetes and sporozoites (CelTOS) is a unique 25-kDa protein, which is critical for ookinete traversal of the mosquito midgut and for sporozoite infectivity of liver cells in human host [
11]. Earlier works have demonstrated that irradiated sporozoite-immunized volunteers mount a strong immune response to CelTOS, and the immunization of mice with recombinant CelTOS (rCelTOS) induces both arms of the immune responses, as well as reduction in Plasmodium infection [
12‐
15]. Thus, it is a promising transmission- and infection-blocking malaria subunit vaccine candidate [
16,
17], that could induce broadly multiple immune responses to disrupt parasite infection in both human and mosquito hosts.
An ideal vaccine adjuvant is able to induce proper, potent and distinct types of specific immune responses, as well as to augment the quality, efficiency, and longevity of specific immune responses to a given antigens, with marginal toxicity to the immunized subjects. Among different vaccine adjuvants, toll-like receptor (TLR) agonists have shown promise in the clinical trials [
18,
19]. One of the TLR adjuvants consists of synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (cytosine phosphate guanidine), which act as TLR-9 agonists [
20,
21]. This adjuvant can increase the immunogenicity of recombinant protein antigens and improve/increase the function of professional antigen-presenting cells; as a result, inducing innate immunity allow to enhance antibody responses and to generate cellular (Th1 CD4+ T cell and CTL)-mediated responses [
22]. Clinical trials have indicated that this adjuvant is safe when administered with target antigens in commercial Hepatitis B vaccine [
23,
24], allergy [
25], and cancer [
26,
27]. Another Th1 potent TLR adjuvant is Poly I:C (polyinosinic:polycytidylic acid), a synthetic double-stranded RNA (dsRNA), that mimics viral RNAs and activates TLR-3 located within endosomes [
28,
29]. The administration of this adjuvant activates dendritic cells that quickly produce IL-12 and type I IFN, both play a crucial role in the induction of Th1 responses [
30‐
32]. This adjuvant is one of the most important TLR-3 agonists tested against diseases such as HIV [
33,
34], dengue [
35], malaria [
36], and cancer [
37,
38].
The recently updated Malaria Vaccine Technology Road map to 2030 [
39] has suggested that a highly effective vaccine (as a new strategy) is required to prevent disease transmission. On this basis, the idea that CelTOS is one of the important proteins for the traversal of the
Plasmodium species in both human and female mosquito hosts has encouraged further evaluation of whether specific immune responses against rPfCelTOS formulated in different potent vaccine adjuvants could inhibit the infection in
Anopheles stephensi, as the mosquito host. Therefore, the current study aimed to assess the immune responses to expressed rPfCelTOS protein in
Escherichia coli. It was hypothesized that TLRs-based human-use compatible adjuvants, CpG ODN and Poly I:C with rPfCelTOS antigen (the adjuvanted vaccine groups), would enhance the immunogenicity of the recombinant antigen, as well as the avidity and functional transmission-reducing activity (TRA) of anti-rPfCelTOS antibodies compared to antigen alone (the non-adjuvanted group) in immunized BALB/c mouse. This aim was achieved by head-to-head comparison of the experimental mouse groups using conventional and avidity enzyme-linked immunosorbent assay (ELISA) along with standard membrane feeding assay (SMFA). In addition, the second hypothesis was that whether in comparing to adjuvant alone, co-administration of both adjuvants with rPfCelTOS would enhance better acquired immune responses in mice and also TRA in the
An. stephensi mosquito host. This work is an important step towards the development of a PfCelTOS-based vaccine in humans.
Discussion
Whole parasite based malaria vaccine was the first successful vaccine trial that was achieved by irradiated sporozoites in humans in the 1950s, and the results showed protection against both homologous and heterologous parasite challenge [
45]. However, in the global malaria eradication program, further large-scale production of such vaccine was challenging. Therefore, the vaccine approach was moved to “subunit malaria vaccine” that surely needs highly immunogenic parasite antigens [
46]. The subunit malaria vaccines, RTS, S, which has been demonstrated to induce partial protection against malaria in phase III human clinical trials [
47,
48] is supposed to become the first licensed malaria vaccine in very near future. Despite encouraging results, the level of protection is generally considered inadequate to attain the elimination of malaria [
49]. Therefore, there is a great interest in improving available subunit malaria vaccines or developing new efficacious malaria subunit vaccines with better formulations to improve the immunogenicity of the target antigen that could significantly reduce disease transmission. In this concern, PfCelTOS, an advanced VIMT candidate, has a key role in the development of infections in both mosquito and human hosts [
16]. Therefore, in the present work, a comparative study was performed in the immunogenicity and functional activity of rPfCelTOS delivered in Th1-based adjuvants (CpG and Poly I:C) in BALB/c mouse; the results demonstrated the ability of these vaccine formulations to elicit high-avidity antibodies that inhibit
P. falciparum infection in
An. stephensi.
Although early indications in preclinical animal studies of the PfCelTOS as vaccine candidate antigen are encouraging [
15,
50,
51], the effort on the improvement of the vaccine formulations for efficacy induced by a PfCelTOS-based vaccine in human are under active development [
52]. In this concern, it is possible that the achievement of effective PfCelTOS as a TBV may be affected by the poorly immunogenic subunit antigen. Therefore, including potent adjuvants in antigen-specific formulations might induce a robust immune response of the proper type to prevent developing parasites in both human and mosquito hosts [
53]. The present study has again recommended that
E. coli-expressed PfCelTOS is immunogenic in BALB/c mouse; however, in comparison to rPfCelTOS alone, administration of rPfCelTOS with two distinct TLR-based adjuvants (CpG and/or Poly I:C) showed an increase in responses (level of antibody, avidity, and Th1 cytokines). Also, the pattern of immunofluorescence on
P. falciparum sporozoites (Additional file
1: Figure S2) using sera from vaccine groups 1–5 indicated CelTOS specificity.
In preclinical evaluation of candidate vaccine, it is required to consider the qualitative features of the antibody responses during the interpretation of the results such as the IgG isotypes, avidity, and functional activity. In this work, the anti-rPfCelTOS IgG subclass responses in different vaccine mouse groups were evaluated, and in non-adjuvanted group 1 that received antigen without any adjuvant, almost comparable IgG1 (as the indicator of Th2 and humoral response) and IgG2a as well as IgG2b (as the indicator of Th1 and cellular response) were detected, confirming the induction of both humoral and cellular immune responses in this vaccine group. However, in adjuvanted groups 2–4, the IgG2a subclass corresponded to a Th1 response was predominantly observed. IgG2a and IgG2b isotypes in mouse (IgG1 and IgG3 in human, respectively) are the most cytophilic and effective isotypes aiming at mediating phagocyte activation and complement fixation as in the previous studies against blood-stage antigens; these antibodies showed protection in clinical malaria in the field [
54‐
56]. Moreover, similar to previously published work, it was reported that naturally acquired anti-rPfCelTOS response in human subjects revealed the skewed results towards cytophilic IgG1 and IgG3 [
40], indicating that the presence of such isotypes may possibly explain the mechanism by which sporozoites/ookinete may be impaired.
Interestingly, among adjuvanted vaccine groups (2–4), a higher IgG2a and IgG2b responses to rPfCelTOS were observed in the adjuvanted vaccine group 4 receiving antigen with adjuvants, CpG + Poly I:C in combination. Regarding the Th-polarization responses in mouse immunized with rPfCelTOS and according to the value of IgG2a/IgG1, IgG2b/IgG1, and IgG2a + IgG2b/IgG1 + IgG3 ratios, it has been clear that rPfCelTOS alone (group 1) induces mixed Th1/Th2-responses. However, in the adjuvanted groups (2–4) that received the rPfCelTOS-based candidate vaccines formulated with CpG-ODNs, Poly I:C, and/or a combination of CpG + Poly I:C adjuvants, more shifted responses toward the Th1 immunity (cellular immunity) were detected.
In addition, the quality of pathogen-specific antibodies may play a critical role in protection following vaccination against a given pathogen [
57]. In this regard, avidity that is determined as the antigen-binding ability [
58] would be a good indicator, as previous works reported that the anti-CS antibody avidity with the appropriate isotype had a crucial role in mediating protection against malaria in animal model [
59]. Although Th1 potent adjuvants, such as CpG and Poly I:C, increased the high avidity anti-rPfCelTOS-specific cytophilic antibodies (IgG2a and IgG2b), the combination of these two Th1 potent adjuvants (by means of different mechanisms) with recombinant protein revealed a better impact on the magnitude, high avidity, and polarization of responses toward Th1. Besides, comparing the immune responses induced by rPfCelTOS formulated in human-use compatible adjuvants (CpG and Poly I:C) with the CFA/IFA as the reference adjuvant that is inappropriate for humans use [
60,
61] revealed higher and improved responses, indicating that these TLR-based adjuvants are capable of enhancement and improvement of the immunogenicity of rPfCelTOS protein.
Distinct cytokines such as IFN-
γ, TNF, and IL-10 play an essential role in T-cell polarization. In the present work, the profile of the cytokines induced by the rPfCelTOS protein was investigated in mouse, and similar to the obtained results for the humoral immune response, it was found that the immunized mouse groups stimulated a higher level of CelTOS-specific IFN-γ and TNF. In addition, the level of rPfCelTOS-specific IFN-
γ was reasonably higher in mouse vaccinated with rPfCelTOS adjuvanted with CpG, Poly I:C, and CpG + Poly I:C compared to the group vaccinated with rPfCelTOS protein alone, confirming again the induction of the Th1-type immune responses in these mice groups (Fig.
4a). Enhancement of the levels of rPfCelTOS-specific IFN-γ and TNF response (but marginal IL-4 response) parallel with the serological data may support their anti-parasitic role, as shown earlier [
62]. It has been suggested that IL-10 has a critical role in switching from Th1 to Th2 responses. Thus, the low level of IL-10 production as well as the high ratios of IFN-
γ/IL-10 and TNF/IL-10 in vaccine groups confirmed again switching to Th1, and this response is probably involved in controlling the infection as demonstrated before [
63,
64].
Previous studies have revealed that CelTOS, as a pre-erythrocytic malaria vaccine candidate, is able to stimulate strong antibody and T cell responses, which inhibit the development of blood-stage infection in mice [
13,
14,
16]. CelTOS is also expressed on ookinete in mosquito host and has an essential role in ookinete to oocyst transformation that, in fact, represents one of the most severe bottlenecks for the parasite development in mosquito host [
13,
17]. Therefore, to block infection in mosquito host, anti-CelTOS antibodies should have high avidity (ability to bind), specificity, and functional activity. However, subunit protein vaccines may possibly fail to induce strong and sterile immunity, as observed with other TBV subunit candidate, Pfs25 [
65]. Hence, this limitation suggests the significance of testing subunit antigen with proper, potent and strong adjuvants. Therefore, one of the most important qualitative aspects of the antibody responses to rPfCelTOS antigen evaluated in the present investigation was the assessment of anti-rPfCelTOS antibodies to inhibit oocysts development in female
An. stephensi using SMFA. In the present work, IgG raised against
E. coli-expressed PfCelTOS protein from vaccine groups 1–4 demonstrated functional activity on parasite infectivity using
P. falciparum NF54 parasite in
An. stephensi, thereby again validating it as a potent candidate for the malaria TBV subunit. However, among vaccine groups (1–4), antibodies from the adjuvanted groups (2–4) showed an enhancement in oocyst inhibitory effect. More specifically, among the adjuvanted groups, the strongest inhibitory antibodies were from group 4, indicating that co-adminstration of two distinct TLRs (3 and 9) vaccine adjuvants with rPfCelTOS antigen induces better anti-rPfCelTOS-specific antibodies in avidity and functional effects due to generating multiple signalings. In fact, such polyclonal antibodies could bind strongly to CelTOS protein on ookinetes and to inhibit/block further oocysts development in the basal lamina of mosquito host. However, on the contrary to the obtained results with these polyclonal antibodies in the present study, Espinosa et al. [
17] have reported the inhibitory effect of monoclonal antibodies (MAb3C3 and MAb 4D10), but not mice polyclonal sera, against PfCelTOS through
P. falciparum oocyst formation in Anopheles mosquitoes. Therefore, these polyclonal antibodies without further IgG purification showed ability to inhibit oocysts development in mosquito host, supporting the protective effect of anti-rPfCelTOS responses that has been reported previously [
13‐
15,
17,
66] and provide additional evidence of its key role as a target for VIMT development.
Authors’ contributions
SP (Ph.D. student) contributed in all laboratory experiments and analysis of the data and drafted the manuscript. SZ designed the work, supervised all the experimental works, analysed the data and helped write the manuscript. AAM contributed in protein expression and purification, mature P. falciparum gametocytes production in in vitro, SMFA, and analysis of the data. RA contributed in mice immunization and ELISA. NDD, AAR, JJS, and ZG participated in SMFA. All authors read and approved the final manuscript.