Malaria remains the deadliest parasitic disease worldwide and is responsible every year for over 600,000 deaths, mainly in sub-Saharan Africa (WHO, 2023). The infection, caused by parasites of the genus Plasmodium, begins with the inoculation of the sporozoite stage into the skin by an infected mosquito (Sidjanski and Vanderberg, 1997). Within the skin, sporozoites become activated and move through the dermis until some find and invade a blood vessel (Amino et al., 2006; Vanderberg and Frevert, 2004). Upon entering circulation, parasites arrest in the liver (Tavares et al., 2017), cross the sinusoidal barrier within 2–3 hr (Tavares et al., 2013), and eventually invade hepatocytes. Intracellular parasites replicate massively before being released back into the blood to infect erythrocytes and cause the disease (Sturm et al., 2006; Tarun et al., 2006). The sporozoite surface is covered by a dense coat of the circumsporozoite protein (CSP), shown to be an immunodominant protective antigen in a rodent malaria model (Zavala et al., 1983). This protein has a highly conserved structural organization across diverse Plasmodium species, comprising conserved N- and C-terminal domains and a highly variable and repetitive central region. Due to its surface localization, abundance, conservation between the same species and immunodominant character, CSP has been targeted by the most effective malaria vaccines. For example, RTS,S/AS01 and R21/Matrix M, the only two malaria vaccines recommended by the World Health Organization (WHO), include a fragment of the central repetitive region and the C-terminal domain of the CSP from Plasmodium falciparum (Pf), the most lethal human-infecting parasite (de Almeida et al., 2021). Extensive research has demonstrated a positive correlation between high levels of anti-CSP antibodies (Abs) induced by the RTS,S/AS01 vaccine and efficacy against malaria (White et al., 2013; White et al., 2014; White et al., 2015). Remarkably, anti-CSP monoclonal Abs (mAbs) protect in vivo against sporozoite infection in various experimental settings, including mice (Imkeller et al., 2018; Kisalu et al., 2018; Murugan et al., 2018; Oyen et al., 2017; Potocnjak et al., 1980; Tan et al., 2018; Wang et al., 2020; Yoshida et al., 1980), monkeys (Cochrane et al., 1982), and humans (Gaudinski et al., 2021; Kayentao et al., 2022; Wu et al., 2022).

It has been observed that Abs that target the CSP central repetitive region exhibit a cytotoxic effect on the sporozoites, accompanied by the shedding of the CSP surface coat (Stewart and Vanderberg, 1991; Yoshida et al., 1980), inhibition of motility, loss of infectivity (Hollingdale et al., 1982; Stewart et al., 1986), and parasite death (Aguirre-Botero et al., 2023; Aliprandini et al., 2018). These effects are independent of downstream host immune effectors such as the complement and immune host cells (Aliprandini et al., 2018). Instead, Ab cytotoxicity increases when parasites are moving in 3D substrates such as the matrix of the cutaneous environment (Aguirre-Botero et al., 2023). Accordingly, neutralization of sporozoites by anti-CSP Abs occurs primarily during their migration through the skin (Aguirre-Botero et al., 2023; Aliprandini et al., 2018) and is dependent on sporozoite motility and host cell-wounding activities (Aliprandini et al., 2018), which are both crucial for parasite progression in the dermis (Amino et al., 2008).

Yet, while the role of the skin in anti-CSP Abs-mediated sporozoite neutralization has been increasingly studied, little is known about the mechanisms by which these effectors contribute, outside of the skin, to neutralize sporozoite infection. Potent Abs or high concentrations of them were shown to efficiently target sporozoites in the blood and liver (BL) following intravenous (i.v.) parasite inoculation which bypasses the skin (Aguirre-Botero et al., 2023; Potocnjak et al., 1980; Wang et al., 2020). Ab cytotoxicity is also thought to be responsible for sporozoite neutralization in the liver (Wang et al., 2020), but some highly cytotoxic Abs were not able to significantly decrease infection following an i.v. sporozoite inoculation (Aguirre-Botero et al., 2023). In this study, we analyzed how 3D11, a cytotoxic mAb against the CSP of Plasmodium berghei (Pb) which has been shown to inhibit parasite motility and kill sporozoites in vitro (Aliprandini et al., 2018), as well as to inhibit motility in the skin (Flores-Garcia et al., 2018), affects sporozoite distribution in the BL, hepatocyte infection, and intracellular development. The current investigation provides a comprehensive view of the kinetics and mode of action of 3D11 on multiple steps of liver infection.

The skin plays a major role in parasite-dependent cytotoxicity induced by anti-CSP Abs (Aguirre-Botero et al., 2023; Aliprandini et al., 2018; Flores-Garcia et al., 2018; Vanderberg and Frevert, 2004). In the skin, mAbs inhibit sporozoite motility (Aguirre-Botero et al., 2023; Aliprandini et al., 2018; Flores-Garcia et al., 2018; Vanderberg and Frevert, 2004) and induce CSP shedding, leading to loss of their infectivity and death (Aguirre-Botero et al., 2023; Aliprandini et al., 2018). However, potent mAbs can also provide protection when parasites are introduced directly into the bloodstream (Aguirre-Botero et al., 2023). Despite having lower protective efficiency in the BL than in the skin, this extra-cutaneous neutralizing activity is a characteristic of the most potent protective Abs (Aguirre-Botero et al., 2023), indicating that the killing of skin escapees is critical for sterilizing immunity. In the hepatic environment, these cytotoxic Abs have been shown to prevent parasite egress from sinusoids, hinder cell traversal, and kill sporozoites (Wang et al., 2020). However, the hepatic cytotoxic neutralizing activities of these different Abs do not always match their protective activity.

By combining intravital imaging and cellular assays, we quantified the P. berghei clearance by 3D11 focused on the different steps of parasite development in the liver. We first measured the effect of 3D11 in the BL and compared it to its effect in the skin by controlling the number of sporozoites for the i.v. and skin challenges. This allowed us to show that 3D11-mediated protection mainly unfolds in the skin, leading to sterile protection upon skin challenge, while there is only a tenfold decrease in parasitemia after i.v. challenge (Figure 1A). Thus, these data support the notion that 3D11 is less effective in the bloodstream (Flores-Garcia et al., 2018), as higher concentrations of Abs are required to protect against i.v. challenge compared to skin challenge (Figure 1). Accordingly, previous studies have reported that high amount of 3D11 transferred to mice is required to provide sterile protection after an i.v. challenge (Charoenvit et al., 1991; Potocnjak et al., 1980). To increase protection in the BL, instead of transferring 100 µg of 3D11 by i.p. injection and waiting 24 hr for the sporozoite inoculation, for the ensuing intravital imaging experiments, we transferred 100 µg of 3D11 by i.v. injection and inoculated parasites in the tail vein 10 min after the Ab transfer. This modification increased the protection in one order of magnitude (Figure 2—figure supplements 1 and Figure 3—figure supplement 1).

When bioluminescent parasites are i.v. injected in passively immunized mice, 3D11 does not inhibit parasite arrest in the liver, suggesting that the repeat region of Pb CSP is not involved in the sporozoite homing to the liver. It is striking that once the parasites reach the liver, the bioluminescent signal in 3D11-treated mice only decreases ~30% at 4 hr post-sporozoite inoculation indicating that most parasites are lately killed by 3D11. A deeper and direct analysis of parasite elimination using intravital confocal microscopy showed that 3D11 mAb promptly inhibits parasite motility in the liver. After arresting in the liver in comparable numbers, ~80% of control PbPf sporozoites were motile, while only 40% of susceptible Pb mCherry sporozoites were moving (Figure 3B). However, the reduction in motility only prevented the hepatic parenchyma invasion of ~50% of parasites. These results suggest that 3D11 inhibits sporozoite motility and limits their exit from the liver sinusoids, as previously reported (Wang et al., 2020) but this twofold difference in parasite translocation alone cannot completely explain the ~100-fold reduction of parasitemia observed 3 days later in 3D11-treated versus control groups (Figure 3—figure supplement 1B). During 3 hr of continuous imaging, it was observed that 3D11-susceptible parasites rarely fragmented and died in vivo. These experiments were conducted in mice expressing GFP in endothelial cells, which made the accurate quantification of control PbPf GFP sporozoites difficult. Nonetheless, susceptible Pb mCherry parasites sporozoites were found in comparable numbers to the non-susceptible GFP sporozoites, indicating a low killing activity of 3D11 in the first 3 hr post-i.v. inoculation, and in concordance with the bioluminescence data. This observation contrasts with the findings on the effects of potent PfCSP human mAb (hmAbs) where 20 and 40% of sporozoites died while recording their in vivo effect in the liver (Wang et al., 2020). This discrepancy in parasite killing may be due to differences in Ab potency. However, this level of killing cannot also completely explain the magnitude of anti-CSP-mediated protection in both studies. To dissect the late-killing effect of the 3D11 mAb, we quantified the in vitro effect of this cytotoxic antibody in the process of invasion and sporozoite development inside hepatoma HepG2 cells. As previously described, 3D11 inhibits sporozoite cell invasion in vitro (Hollingdale et al., 1982). In our experimental setup, 5 µg/ml of 3D11 inhibited 88% of cell invasion (Figure 4B, C), which accounts for most of the 95% infection inhibition observed after 15 and 44 hr (Figure 4C). It is important to note that the cytotoxicity of anti-CSP Abs correlates with sporozoite loss of fitness (Aguirre-Botero et al., 2023), quantified by the inhibition of motility, the loss of infectivity, and the killing of sporozoites (Aguirre-Botero et al., 2023; Aliprandini et al., 2018). The dose–response similarity between the inhibition of invasion and the decrease in viability, both measured in the same assay, suggests that the inhibitory activity of 3D11 on cell invasion assays could be a consequence of the cytotoxic effect of this antibody (Figure 4B). However, this does not rule out other mechanisms of invasion inhibition. Additionally, our data also show a lasting effect of 3D11 on intracellular parasites in vitro after Ab removal. This effect is reflected not only in the two- to threefold decrease in parasite numbers between 2 and 15 hr compared to the control using 2.5 and 5 µg/ml of 3D11 (Figure 4D) but also in the decrease of EEF fitness, revealed by its decreased GFP intensity (Figures 4E and 5D). Notably, a similar effect has been previously reported using sera from mice immunized with PfCSP or mAb against Plasmodium yoelii (Py) CSP. Incubation of Pf or Py sporozoites with the immune sera or mAbs not only affected sporozoite invasion in vitro but continued to affect intracellular forms for several days after invasion (Mazier et al., 1986; Nudelman et al., 1989). Additionally, using anti-Pf CSP sera, it was also observed that late EEFs from sera-treated sporozoites had abnormal morphology (Mazier et al., 1986). Altogether, it was thus concluded that the anti-CSP Abs present in the sera had a long-term effect on the parasites (Mazier et al., 1986; Nudelman et al., 1989). Importantly, in our experimental settings and using the mAb 3D11, we did not observe any malformation of the Pb EEFs but rather a decrease in the EEF GFP fluorescence at 44 hr in the 3D11-treated samples compared to the control. These differences could be due to the distinct invasion and developmental process of Pb and Pf, or to the potency of the Abs present in the serum compared to 3D11. The in vivo bioluminescence data suggest that 3D11 only weakly interferes with the development of late EEFs, as the signal of survivors in 3D11-treated mice between 24, 44, and 72 hr only slightly decreased, and this difference was not significative (Figure 2D, Figure 2—figure supplement 1). Accordingly, in vitro, the decrease on intracellular parasite numbers already occurred at 15 hr (Figure 4C, F) and stayed constant until 44 hr (Figure 4C, D). The GFP fluorescence decrease observed at 44 hr in vitro (Figures 4E and 5C, D), could explain the slight decrease in the bioluminescence signal observed in vivo between 24 and 72 hr (Figure 2—figure supplement 1). Finally, the relative percentual increase of the UIS4-negative population in 3D11-treated parasites versus control parasites indicates that the long-term killing could be a consequence of this phenotype. Further experiments are needed to determine whether the same phenotype is occurring in vivo and if the 3D11 lasting neutralizing activity is associated with the inhibition of UIS4 secretion or with the intracellular arrest of PVM-negative transmigratory parasites.

Although 3D11 cytotoxicity could be the primary contributor to the inhibition of parasite entry into the liver parenchyma in vivo and cell invasion in vitro, it is unclear whether it is the sole factor responsible for these effects. MAbs with high cytotoxicity, such as mAb10, do not significantly protect mice after i.v. sporozoite challenge, despite displaying a similar cytotoxic neutralizing activity profile in the liver than protective mAbs (Aguirre-Botero et al., 2023; Wang et al., 2020). CIS43, a hmAb that targets the junctional epitope of Pf CSP, has been found to interfere with its proteolytic cleavage (Kisalu et al., 2018), which is a crucial process for cell invasion (Coppi et al., 2005; Espinosa et al., 2015). Indeed, it has recently been shown using 13 distinct PfCSP hmAbs, that protection after i.v. challenge strongly correlates with high affinity to the junctional region, which contains all three repeat motifs of Pf CSP (NPDP, NVDP, and NANP) (Aguirre-Botero et al., 2023), reinforcing the hypothesis that anti-CSP repeat Abs could hinder the CSP processing at the putative cleavage site region I that is located adjacent to the junctional region of the central repetitive region of Pf CSP (Coppi et al., 2005). 3D11 Fab was shown to preferentially bind to the NPND motifs located mostly at the junction with the N-terminal domain. Still, it also cross-reacts with all repeat motifs, binding to PbCSP in a spiral-like conformation (Kucharska et al., 2020). Thus, further investigation is required to elucidate the role of CSP processing in inhibiting sporozoite invasion by anti-CSP Ab, for example, using parasites lacking the N-terminal domain of CSP (Coppi et al., 2011). To investigate other potential mechanisms by which Abs are neutralizing sporozoites in the BL, it is necessary to use non-cytotoxic conditions. This can be addressed by using a low-cytotoxic mAb, like CIS43, or parasites lacking sporozoite-secreted pore-forming proteins, such as SPECT2. It has been previously demonstrated that Ab-mediated killing depends on the shedding of CSP, which renders the parasite membrane susceptible to its own cell-wounding proteins (Aliprandini et al., 2018). According to this study, Pb and Py SPECT2KO mutants have a higher resistance to the killing activity of CSP Abs (Aliprandini et al., 2018).

In conclusion, this study shows the kinetics of sporozoite elimination by the cytotoxic 3D11 mAb in the liver and quantifies the inhibitory activity of 3D11 in the key steps of hepatic infection. The measurement of the inhibition of invasion and 3D11 cytotoxicity in vitro suggests a potential association between these two activities, but this result does not exclude the concomitant existence of other neutralizing mechanisms. Remarkably, this study reveals a late-killing effect of cytotoxic 3D11 on sporozoites within the liver parenchyma. This late-killing activity was also observed in vitro and it was associated with the increase of a population lacking the PVM developmental marker UIS4. This study shows that cytotoxic 3D11 mAb exerts its protective action outside the skin by inhibiting several critical steps in the hepatic infection and, surprisingly, taking several hours to significantly decrease the hepatic load of parasites. Understanding the mechanisms of sporozoite neutralization by anti-CSP Abs in the host tissues is crucial for better comprehending parasite biology and developing new tools to prevent sporozoite infection.

All data generated or analyzed during this study are included in the manuscript and supporting file.

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Author details

1. Manuela C Aguirre-Botero

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Contribution

   Conceptualization, Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0019-5122

2. Olga Pacios

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Present address

   Microbiology Department - A Coruña Hospital, MicroTM-INIBIC, A Coruña, Spain

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4476-856X

3. Susanna Celli

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8296-895X

4. Eduardo Aliprandini

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Present address

   Affinity Reagents Team, Biologics Engineering, AstraZeneca, Cambridge, United Kingdom

   Contribution

   Supervision, Investigation

   Competing interests

   Eduardo Aliprandini is affiliated with Astrazeneca. The author has no other competing interests to declare

5. Anisha Gladston

   1. Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France
   2. Department of Life Sciences, Imperial College London, London, United Kingdom

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Jean-Michel Thiberge

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Pauline Formaglio

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Present address

   Sorbonne Université, Inserm U1135, CNRS ERL8255, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), 91 10 Boulevard de l'Hôpital, Paris, France

   Contribution

   Supervision, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9612-3157

8. Rogerio Amino

   Institut Pasteur, Université Paris Cité, Malaria Infection and Immunity, BioSPC, Paris, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review and editing

   For correspondence

   rogerio.amino@pasteur.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8086-2932

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