A malaria vaccine would have a tremendous impact on vulnerable populations, with the potential to save nearly half a million lives annually and prevent over 200 million cases (1). Yet the development of an efficacious vaccine remains elusive. One of the biggest challenges facing malaria vaccine development is the complex life cycle of the parasite (Figure 1). The sporozoite form of the parasite invades hepatocytes in the liver, undergoes schizogony, and then enters the blood stage. In the blood, some of the parasites differentiate to form gametocytes that can be taken up by mosquitoes during a blood meal, resulting in onward parasite transmission. The challenge to vaccinologists is that the parasite expresses antigens that are largely stage-specific during its lifecycle and no single defining vaccine target or even whole organism vaccine can protect against all stages. Despite this, there are multiple opportunities for vaccines to interrupt the parasite life cycle (2). A vaccine that prevents sporozoite colonization of hepatocytes could protect individuals from Plasmodium infection, while a vaccine targeting the blood stage could curb the clinical manifestation of disease, and a gametocyte-targeting vaccine could block transmission to mosquitoes.

Of the six species of Plasmodium that infect humans, P. falciparum causes the greatest mortality and morbidity worldwide (1). In high transmission settings, millions of young children are at risk of dying from severe falciparum malaria until they acquire immunity to severe disease later in childhood. As such, current vaccine efforts are largely focused on P. falciparum. Only one licensed vaccine exists, RTS,S and this vaccine is currently undergoing pilot roll-out in several African countries. The target of RTS,S is the surface circumsporozoite surface protein (CSP) that is expressed on the surface of P. falciparum sporozoites (3). While this vaccine aims to prevent liver stage infection, the results from earlier vaccine trials suggest good immunity in the first six months but then a significant waning of immunity over time, resulting in poor long-term vaccine efficacy (3). This is likely due in part to the low dose of sporozoites inoculated by mosquitoes that fails to reactivate memory B-cells, combined with antigenic polymorphisms in the T-cell epitopes of the CSP (4). Other vaccines target P. falciparum blood stage antigens and they also face significant challenges, primarily due to antigenic polymorphisms that reduce the efficacy of allele-specific vaccines against natural infections (5).

The limitations of current experimental vaccines may reflect a shortcoming in the traditional approach to antigen discovery (6). Candidates, particularly blood stage antigens, are often identified as targets of neutralizing antibodies in immune sera; but the corollary is that this strategy selects for immunodominant epitopes that are under strong immune selection, and consequently, are highly polymorphic. Incorporating conserved and cryptic epitopes (epitopes not normally exposed to the immune system) into vaccines may overcome these challenges.

Here we consider whether epitopes conserved across species can be exploited in vaccine design. This idea may seem heretical given the absence of sterile immunity following lifelong exposure to a single species, and our understanding that the immune response to malaria is largely considered strain-specific. In fact, cross-species immunity has doubtlessly been selected against due to the co-circulation of multiple Plasmodium species competing for the same human host. Competition between parasites likely resulted in the evolution of different virulence and life cycle strategies as a form of mutual adaptation, and within these species-specific adaptations arose antigenic diversity in virulence genes of that parasite. Nevertheless, the shared evolutionary history among the six species of Plasmodium purports that many proteins will be homologous in origin, with common structures and/or functions. As such, it is likely that there are subdominant or even immunologically cryptic epitopes that remain conserved across multiple species. As a vaccine strategy, this presents an opportunity to direct the immune response against these conserved epitopes and exploit them in a cross-species malaria vaccine.

In this review, we discuss the evidence for immunological cross-reactivity between Plasmodium species and the rationale for considering a cross-species vaccine approach. We define heterologous immunity and cross-reactivity as immunological interactions between two different Plasmodium species and not between two strains of the same species. We first consider the clinical outcomes of natural infection in areas co-endemic for multiple species, deliberate human infection studies, and infections in animal models. Next we describe the parasite-specific immune responses to different species of Plasmodium and the antigens that may mediate cross-species immunity. Lastly, we provide a rationale for mapping conserved epitopes in antigens from different species and developing these epitopes as vaccine candidates.

Interactions between different species of Plasmodium are evident from a number of epidemiological studies of naturally exposed populations [reviewed in (7, 8)]. These are often reported as negative interactions, where co-infection with two species exacerbated disease (7), or provide no evidence of interaction at all - infection with one species had no demonstrable impact on the risk or severity of infection from another species (7). Yet concurrent studies from South Asia and Oceania gave rise to findings in support of cross-species immunity (9–11). In particular, there is compelling data that infection with P. vivax confers a degree of clinical protection against P. falciparum. This was observed in a prospective study in Sri Lanka, where the severity of symptoms from P. falciparum infection was lessened following a P. vivax infection, inferred as “clinical tolerance” to the more virulent species (9). Further support for this phenomenon was garnered from cross-sectional and longitudinal studies in Vanuatu where the incidence of severe malaria (severe anemia and cerebral malaria) was much lower than expected for an area hyperendemic for P. falciparum and P. vivax (10). The authors proposed that cross-species immunity may contribute to clinical protection and impact the infection dynamics of these two species (12). Subsequent data from a large-scale prospective analysis of health-center morbidity in Papua New Guinea provided further evidence that P. vivax infection was associated with clinical protection against P. falciparum disease (11). In all of these studies, P. falciparum never protected against P. vivax infection.

The hypothesis that one species could suppress the pathogenicity of another (7) could also account for other unusual epidemiological observations from co-endemic areas. For example, distinct seasonal patterns characterized the incidence of P. falciparum and P. vivax in Vanuatu (13) and between P. falciparum and P. malariae in Nigeria (14), where each species was dominant at different times of the year and appeared to alter the infection dynamics of the other. Inter-species suppression of infection may also explain the recurrence of latent P. vivax or P. malariae following treatment of P. falciparum infections (15), and the low frequency of mixed infections in populations where multiple species co-exist (16, 17). In fact, this led to the suggestion by Cohen (16) that, “If heterologous immunity can indeed greatly reduce the prevalence of mixed infections, as is claimed, then a malaria vaccine need not be specific to each of the species, strains, or antigenic variants of Plasmodium in order to be effective.”

In none of these studies was there evidence that prior infection with one species reduced the risk of subsequent infection with another species. This is consistent with the lack of sterile immunity to any species of malaria, even against different strains within the same species. Rather, the evidence from these population-based studies suggests that the interactions among species may occasionally be protective and reduce the clinical course of disease. Even this cautious interpretation is subject to challenge by the many confounding factors that plague these types of epidemiological studies. It is very difficult to follow precisely the course of infection in individuals, even in longitudinal studies. This limitation is particularly apparent in light of the high frequency of submicroscopic infections revealed in more recent studies using molecular diagnostics (18). We cannot exclude persistent, submicroscopic infections of one species that could impact interpretation of these data. Alternative explanations to cross-species immunity have also been raised, including non-specific antiparasitic effects (19), ecological competition between parasites for the same mosquito host, and density-dependent mechanisms such as competition for red blood cells and nutrients within the human host (7, 16, 20). Given these limitations, we turn to studies with controlled infections in humans and laboratory animals to assess the validity of cross-species immunity.

One of the earliest studies to deliberately infect human volunteers with P. vivax and P. falciparum was from the 1930s (21). Eight volunteers were infected with either P. vivax or P. falciparum from the bite of an infected mosquito then infected with the heterologous parasite either during the incubation period, the clinical phase, or following a recent infection with the first parasite. In the majority of these cases, the second infection was established, with no evidence of sterile immunity in these volunteers. Yet there was no discussion of whether the severity of symptoms during the second infection was affected by the primary infection.

The subsequent era of experimental human infections from 1940 to 1963 centered on malaria therapy treatments of patients with neurosyphilis. Treatment often resulted in multiple sequential infections with homologous or heterologous species, especially if the first treatment did not meet the therapeutic goals or due to limited availability of mosquitoes and patients infected with a particular species as a source of parasites for treatment. This may confound the interpretation of the results when comparing sequential infections to mono-infections since the reason for treatment failure is not known. Furthermore, homologous protection in control subjects was not always assessed. Despite this inherent variability, the malaria therapy cases yielded a wealth of data on the outcomes of infection with different species.

One of the earliest comprehensive reviews of these cases evaluated the effects of a primary malaria infection with P. falciparum, P. ovale, P. malariae, or P. vivax on patients re-infected with either the same or a different species (22). Upon homologous re-infection, the severity of the subsequent infection was significantly reduced but heterologous re-infections gave variable results. The outcome depended on the combination and order of species for the primary and secondary infections. In fifteen patients with a P. vivax infection followed by a P. falciparum infection, no effect on the second infection was observed when peak asexual parasitemia, gametocytemia and fever episodes were compared to single infections in malaria-naïve individuals. Similarly, there was no effect of a P. malariae infection on a subsequent P. falciparum infection (n = 6). However, previous infection with P. vivax led to lower parasite densities and fewer fever episodes during a subsequent P. ovale infection in 15 patients, compared to naïve individuals. When the order of these infections was reversed and P. ovale was given first, there was no effect on fever or parasitemia during the following P. vivax infection; however, the P. vivax infections were self-limiting, and no drug treatment was required.

There was a similar effect when a P. falciparum infection followed a P. ovale infection (n = 11). In these cases, there was obvious modification of the severity of the P. falciparum infection resulting in a much lower proportion of patients requiring treatment and in those that did, a lower therapeutic dose was sufficient. In fact, no curative doses of drugs were needed if the P. falciparum infection was preceded by a P. ovale infection. When the order of these infections was reversed in eleven patients, there was no effect of prior P. falciparum infection on the subsequent P. ovale infections. Only a small number of patients received a P. vivax infection after a P. malariae infection, but the P. vivax infection in 2 of 3 patients resolved spontaneously.

A later review of different patient files from the same time period suggested some cross-reactivity of P. falciparum with P. malariae, but not with P. vivax or P. ovale (23). The frequency of P. falciparum hyperparasitemia (≥ 10,000/μL) and fever was not affected by prior infection with P. vivax or P. ovale but was reduced when the P. falciparum infection was preceded by a P. malariae infection. This latter observation was countered in another review, concluding there was no evidence that past or current P. malariae infection affected P. falciparum asexual parasitemia; yet interestingly, there was an effect on P. falciparum gametocytemia (24). The authors proposed that the balance between asexual parasitemia and gametocytemia could be altered by the presence of the other species.

Collectively, the data from select human experimental studies bolster the evidence for cross-species interactions observed in the field studies, although this is clearly not a consistent occurrence. These studies further highlight the non-reciprocal nature of parasite interactions that appear to be predicated on the temporal sequence of infection and support a mechanism of partial heterologous immunity that can limit disease severity from the secondary infection.

Animal models of malaria offer an analogous approach to investigate cross-species immunity in a controlled environment. Early studies that investigated interactions between P. gallinaceum and P. lophurae in chickens corroborated the findings of the various human studies that heterologous immunity can be non-reciprocal (25). Chickens infected with either P. gallinaceum sporozoites or blood stage parasites followed by infection with homologous parasites [as sporozoites or infected red blood cells (iRBCs)], or iRBCs of the heterologous species P. lophurae, exhibited marked reductions in both homologous and heterologous parasitemia. But when the order of the inoculations was reversed, weak heterologous immunity was observed and only in chickens that were hyperimmune to P. lophurae (following 4 or 5 infections).

There is ample evidence from mouse models that vaccination with attenuated sporozoites can elicit cross-species protection [reviewed in (26, 27)]. Mice immunized with X-irradiated P. berghei sporozoites were completely protected from heterologous challenge with P. vinckei sporozoites and immunization with irradiated P. chabaudi sporozoites induced sterile protection against infection with P. berghei (28). Furthermore, both irradiated and genetically attenuated P. berghei sporozoites inhibited intrahepatic development of P. yoelii sporozoites based on copies of parasite 18S ribosomal RNA quantified by qRT-PCR (29). Even immunization with P. falciparum sporozoites protected 60% of mice from a P. berghei infection (but not a P. yoelii infection) and passive transfer of IgG from these P. falciparum vaccinated mice protected naïve mice from a P. berghei sporozoite challenge (30). Similarly, chemically attenuated P. berghei sporozoites protected mice from challenge with P. yoelii sporozoites (31). In this case, cross-species protection was short-lived and did not last beyond 10 days post-immunization (31).

Likewise, blood stage murine parasites are capable of inducing cross-species immunity, which was reviewed extensively by Richie (7, 8). For instance, protection - measured as reduction in mortality - was observed when P. berghei-vaccinated mice were challenged with P. yoelii and when P. vinckei-vaccinated mice were challenged with P. chabaudi (32). Similar to Taliaferro and Taliaferro’s observations (25) with P. gallinaceum and P. lophurae in chickens, heterologous immunity in mice was non-reciprocal (32). Prior infection with P. berghei or vaccination with formalin-fixed blood-stage parasites reduced mortality in mice from P. yoelii infection, but no protection was observed when the species order was reversed. Similarly, mice vaccinated with P. vinckei were protected from P. chabaudi but not the inverse. One exception was the reciprocal cross-species protection between the blood stages of P. berghei and P. vinckei. After vaccination or infection with either parasite, 40–50% of mice survived a lethal heterologous challenge with the other species. From these studies and others, the genetic background of the mouse is likely to impact cross-protection. P. chabaudi immunization did not protect against P. yoelii challenge in outbred CD-1 mice (32), whereas partial protection was observed in BALB/c mice, and complete protection in C57BL/6 and CBA mice (33). More recently, this was observed using a different vaccination scheme termed ‘controlled infection immunization’ where mice were immunized with one species while under doxycycline chemoprophylaxis then challenged with the heterologous species (34). C57BL/6 mice immunized with P. chabaudi or P. yoelii promoted survival following heterologous challenge with the reciprocal parasite (34). While in BALB/c mice, protection was non-reciprocal; only P. chabaudi immunization could protect against P. yoelii, mirroring the findings from the older study.

Immunity in the studies described above was defined as protection from mortality, but as McColm and Dalton discuss (32), there is evidence of significant modulation of infection between species. This clinical suppression of disease was apparent as reduced parasitemia over the course of infection and delayed mortality relative to controls. Similarly, cerebral malaria was prevented in mice with a P. berghei ANKA infection if they had a co-infection with P. yoelii (but not with P. vinckei or P. berghei NK25) (35). It is important to note that in many of these studies the effects of non-specific anti-disease factors (such as cytokines or hormones) on secondary infections are impossible to separate from specific immune responses. Non-specific immune factors in sera from mice with a malaria infection inhibited in vitro growth of P. falciparum independent of antibody levels (36). Another factor may be hepcidin, which is upregulated in response to a blood stage malaria infection and inhibits a concurrent liver stage infection irrespective of the strain or Plasmodium species (37).

The evidence supporting cross-species protection from human and animal studies validates efforts to explore heterologous vaccine strategies but also begs an understanding of the underlying immune mechanisms. Rather unexpectedly, insight into the immunological basis of cross-reactivity first emerged from attempts to develop species-specific diagnostic tests. In testing the specificity of a complement fixation assay for malaria diagnosis, Kingsbury detected cross-reactivity between P. vivax and P. falciparum antigens (38). Sera from 6 of 12 individuals with acute P. vivax infection reacted to P. falciparum antigens in a precipitin test, and likewise, 5 of 16 sera from patients infected with P. falciparum reacted to P. vivax antigens. However, a later paper by Mayer and Heidelberger (39) suggested that the specificity of the test was compromised by reactivity of sera with human stromata in the antigen preparations. In a different precipitin test developed by Taliaferro et al. (40, 41), sera from patients in Honduras infected with P. vivax reacted with antigens prepared from a P. falciparum-infected placenta. Surprisingly, heterologous reactions were as strong as the homologous ones. These findings were replicated in two separate studies in Honduras with over 500 sera but not in a later study in Puerto Rico with antigens prepared in the same manner (42). The results from the Puerto Rico study were deemed inconclusive and the inconsistency attributed to the generally poor performance of the precipitin test at that time. Serological cross-reactivity was later observed against P. falciparum and P. vivax crude antigens prepared from short-term culture of parasites isolated from infected patients, but the homologous reactions were more intense than the heterologous reactions (43).

With the advent of techniques to fluorescently label antibodies, their recognition of antigens from distinct malaria species could be directly observed under the microscope with the immunofluorescence assay (IFA). In one of the first records of this method applied to the study of human malaria, fluorescently labeled immunoglobulin from a patient with a long-standing P. vivax infection recognized RBCs infected with the simian malaria species P. cynomolgi (although not P. berghei) (44). This finding was replicated in another study where sera from P. vivax-infected patients (n = 4) recognized thin blood smears made from monkeys infected with P. cynomolgi (45). Homologous parasites were recognized much more strongly than heterologous parasites, but cross-reactivity in this case was reciprocal: serum from 5 volunteers infected with P. cynomolgi recognized two strains of P. vivax (Chesson and Venezuelan strains) by IFA using thin blood smears made from infected patients (45). Similarly, antibodies from a laboratory worker following an accidental P. cynomolgi infection recognized thin smears of P. vivax iRBCs as strongly as those infected with P. cynomolgi (44).

Immunological cross-reactivity between P. vivax and P. falciparum was also demonstrated with sera from naturally infected individuals (46). Sera from 9 out of 29 individuals with a P. vivax infection recognized P. falciparum iRBCs, while sera from 11 out of 21 individuals with a P. falciparum infection recognized P. vivax iRBCs. In this same study, cross-reactivity was also observed in individuals deliberately infected with P. vivax or P. falciparum. Based on the antibody titers against homologous versus heterologous iRBCs, P. vivax sera were more cross-reactive against P. falciparum iRBCs than the converse.

In Guatemala, sera from individuals naturally exposed to P. vivax strongly cross-reacted with asexual P. falciparum antigens by ELISA (20/43 positive), IFA (35/36 positive) and by immunoprecipitation assays (32/32 positive) (47). In order to rule out past P. falciparum infection as the source of these antibodies (despite > 99% prevalence of P. vivax), the sera were also tested against the P. falciparum CSP and heat shock protein (HSP) 70 kD-like-molecule repeat peptides by ELISA. Only 2 out of 36 sera samples recognized the PfCSP repeat peptide and 1 out of 33 sera samples recognized the P. falciparum HSP70 kD-like-molecule repeat peptide (48), suggesting the antibodies were truly cross-reactive. In this study, serological recognition of a HSP70 peptide was used to rule out antibodies specific to P. falciparum infection, but this family of proteins contains other epitopes that are shared across Plasmodium species (49). Given their ubiquitous nature, it is possible that these and other conserved housekeeping antigens underpinned some of the cross-reactivity discussed previously. While these may be viable targets of cross-reactive antibodies, their validity as vaccine candidates would depend on whether they elicit functional antibodies.

IFA was also useful to validate the interactions between the different species of rodent malaria and to develop a model of antigenic similarity among these parasites (50). Hyperimmune sera generated by infecting mice three times with either P. berghei, P. yoelii, P. chabaudi, or P. vinckei revealed that the four species were serologically indistinguishable by IFA. Sera from rabbits immunized with soluble antigens from these parasites gave similar results. These findings form the basis of a proposed model of antigenic conservation between the four murine malaria species whereby certain antigens are shared among all four species, some antigens are shared only between the most similar pairs of parasites and then others are specific to each species (50).

Cellular immunity is also likely to play a role in cross-species immunity and may underpin the protective clinical effects (reduced symptoms and disease severity) - yet there is scant data on the potential contribution of T-cells to this immune mechanism. In rodent models, antibody-independent mechanisms clearly influenced susceptibility to heterologous challenge (51). For example, B-cell deficient mice chronically infected with P. yoelii were resistant to lethal challenge with P. chabaudi (51). Cross-reactive T-cell responses are also vital to the heterologous immunity observed in murine malaria models of attenuated sporozoite vaccination. Immunization with radiation-attenuated P. berghei sporozoites protected 79% of mice challenged with P. yoelii sporozoites and immunization with P. yoelii sporozoites protected 63% of mice challenged with P. berghei sporozoites (52). Heterologous protection was dependent on CD8⁺ T-cells whereas antibodies from immunized mice only recognized homologous, but not heterologous, sporozoites. In another study, 100% of mice immunized with genetically attenuated P. yoelii sporozoites were protected against P. berghei sporozoite challenge (53). The authors suggested that late-liver stage arresting sporozoites elicited a broadly protective CD8⁺ T-cell response.

There are few reports of species-transcending T-cells in humans. The most definitive study showed that T-cells isolated from volunteers immunized with attenuated blood stage P. falciparum parasites proliferated in vitro in response to P. knowlesi iRBCs (54). Whether these T-cells have functional activity to protect against heterologous challenge is not known.

The data presented in this review build the case for heterologous immunity elicited by natural or deliberate infection in animals and humans. The mechanism of immunity likely involves both humoral and cellular immunity, but the antigenic determinants are unknown. To translate the observations spanning the last century into viable heterologous vaccine candidates, the conserved targets of immunity must be identified. Given that partial protection is observed in nature and appears to be a relatively rare event, we expect that multiple vaccine candidates will be needed to target different stages of the parasite’s lifecycle. Cross-species vaccines that aim to prevent infection should target sporozoite antigens to inhibit hepatocyte invasion and development, while vaccines that target blood stage antigens could prevent severe disease. Gametocyte antigens are also attractive targets to prevent the onwards transmission of malaria. Below, we review the discovery and current knowledge of potential cross-species antigens from different parasite stages. We summarized the findings from studies using human malaria parasite antigens in Figure 1.

The slow progress in developing a malaria vaccine underscores the many challenges with a traditional vaccine approach. We need to consider alternative, yet complementary strategies. Thus, exploiting rare immune mechanisms like cross-species immunity are worthy of consideration and with our current tools, this is more amenable than ever before. We don’t expect this approach to yield a vaccine that provides sterile immunity to malaria; but if we could emulate the reduction in disease severity observed with heterologous infections in humans and in animal studies, this vaccine could reduce mortality in the most vulnerable populations and allow natural, strain-transcending immunity to develop.

CM and SY jointly wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.