It is well established that immunization of mice with the C-terminal region of MSP-1 is able to induce protective immunity [6, 8, 9]. What is less clear are the protective mechanisms responsible. There is good evidence that antibody plays an important role in the protection induced [5, 6, 10], but the whether the antibody works by directly neutralizing merozoites and blocking erythrocyte invasion or primarily through Fc-mediated actions needs further investigation.
The ability of three monoclonal antibodies specific for the merozoite surface protein MSP-1 to provide protection against infection with the rodent malaria parasite P. yoelii by passive immunization was investigated. In particular we have examined the effect of combining pairs of antibodies on their protective capacity. These antibodies are of the G3 (B6 and F5) and G2a (D3) isotypes. Both B6 and F5 bind to an epitope within the first EGF-like domain of P. yoelii MSP-119, whereas D3 binds to an epitope in the longer MSP142 fragment, which is formed from the two subdomains of MSP133 and MSP119. The epitopes binding B6 and F5 overlap but are not identical [5, 11], but their proximity to the epitope of D3 is unclear since the epitope for this antibody appears to be a conformational epitope that is formed only in the intact MSP-142. In the first step of this study, the minimum protective dose (MPD) was determined for each antibody as 0.6, 0.9 and 1.2 mg for mAbs D3, B6 and F5, respectively. In the second step of the study, the ability of combinations of two antibodies at their MPD to provide protection by passive immunization was measured. Mixtures of D3 and B6 or D3 and F5 antibodies were less effective than the single antibodies alone, whereas a mixture of F5 and B6 was more effective than either of these two antibodies alone.
The mechanism(s) by which these three antibodies provide protection following passive immunization is unknown. There are several mechanisms that have been suggested to explain the protective effect of antibodies to merozoite surface proteins , and a number of possible mechanisms have been identified for antibodies directed against the C-terminal region of Plasmodium falciparum MSP-1 . These mechanisms may depend on the antibody alone, such as inhibition of proteolytic processing of MSP-1 or merozoite agglutination, or depend on an Fc-mediated component such as opsonisation of merozoites and phagocytosis. In both types of mechanism it is likely that the antibody avidity and concentration are important. In the case of antibody mediated inhibition of invasion through inhibition of MSP-1 processing the fine specificity of antibody binding is crucial. Antibodies that bind to the first EGF-like domain, such as B6 and F5 can inhibit processing but the importance of this mechanism in protection against P. yoelii has not been established. If both B6 and F5 mAbs are providing protection through this mechanism, it is not surprising that the protection provided by passive immunization with a mixture of the two is additive since this is equivalent to increasing the concentration of the protective antibody specificity. In fact, the mice in this group fully resolved their parasitaemia since a blood sample that was transferred from each mouse into naïve recipient mice failed to cause infection. However, it is also possible that an acquired immune response as indicated by the lymphocyte proliferation (Table 1) and resulting from the parasite infection may have also contributed to this clearance.
The failure of mixtures of B6 with D3 or F5 with D3 to provide additive protection is more difficult to explain. mAb D3 does not bind to MSP119 alone and yet is very effective at providing protection on passive immunization; a corresponding antibody specific for P. falciparum MSP-1 has not been described or a mechanism evaluated. The suppressive effect of the mixture on the control of parasitaemia highlights the potential complexity of antibody interactions within a polyclonal response. It has already been established that some antibodies, which do not inhibit MSP-1 processing, can be defined as blocking antibodies in this mechanism because they block the binding of the inhibitory antibody [4, 13, 14]. It is not known whether or not the binding of D3 to MSP-1 competes with and blocks the binding of B6 or F5 mAbs, or vice versa. In either case the consequence may be a reduction in the protective capacity of the mixture of antibodies, as seen in the results presented here.
An alternative explanation may reflect the importance of Fc-mediated mechanisms in the protection mediated by passive immunization with antibodies to MSP-1, which may depend on the isotype of the mAb. It has already been established that Fc-mediated effects are important in immunity dependent on antibodies binding to MSP-1 [15, 16], although some studies have shown that this mechanism is not essential [17, 18]. One possible explanation for the results is that the IgG3 isotype has a greater capacity to fix complement than IgG2a . Although the FcγRI receptor of macrophages has a high affinity for IGg2a, it binds IgG3 with fivefold higher affinity [20, 21]. In competition studies, it has been demonstrated that IgG3 can inhibit IgG2a binding to FcγRI receptor . Earlier work with a different IgG3 specific for MSP-1 also showed that it was effective in passive protection . This interpretation would suggest that IgG3 is the best immunoglobulin sub-class for use in passive immunization to eliminate the parasite from the blood.
Smith and Taylor-Robison  measured the level of different immunoglobulin isotypes induced during infections of mice with virulent and avirulent lines of Plasmodium chabaudi and Plasmodium yoelii. A non-lethal infection was characterized by early upregulation of IgG2a and late upregulation of IgG1, whereas in a lethal infection a slow and reduced IgG2a correlated with rapid fatal outcome before G1 synthesis. Unfortunately these authors did not evaluate the IgG3 isotype.
The proliferation of spleen cells following stimulation with MSP-119 was observed for the group of mice that was protected by passive transfer of B6 and F5 antibodies, and which was similar in magnitude to that induced by the mitogen ConA and was not replicated when the spleen cells came from normal BALB/c mice. Since the passive immunization did not result in sterile immunity it is likely that the infection induced an acquired immune response in these animals. This immune response may have contributed to the observed control of infection, in addition to that resulting from the presence of the B6 and F5 antibodies. In fact, antibodies to MSP-119 were not detected by western blotting in the serum samples from the mice in this group taken after the infection had been cleared, suggesting that all of the passively transferred antibodies had been consumed and other mechanisms might have provided the final clearance of parasites.