Introduction

Malaria is a lethal disease that kills hundreds of thousands of people every year (Zhang et al., 2017). The deadliest cases of malaria are caused by the Plasmodium falciparum parasite, making this organism the subject of intensive research (CDC, 2015). Plasmodium has a very complex life cycle, including phases in both human and Anopheles mosquito hosts. Unfortunately, scientists have yet to develop an effective vaccine against malaria.

One of the major issues in developing a malaria vaccine is the fact that Plasmodium has such a complex life cycle (Hoffman et al., 2015). Proteins may be differentially expressed depending on the parasite’s life cycle stage. This means that a major cell surface antigen, ideal for antibody binding and the initiation of an immune response, might only be expressed in one stage of the parasite’s infection. Research into malaria vaccines often focuses on single parasite stages, such as peptide-based vaccines SPf66 and PEV3a that target the blood stage, or vaccines against malaria’s sexual stage (which are aimed against transmission to mosquitoes) (Hill, 2011).

Currently the best vaccine in circulation is RTS,S/AS01 (Mosquirix) (Zhang et al., 2017). This vaccine consists of the P. falciparum circumsporozoite protein (CSP) fused to hepatitis B and is effective in 50% of child recipients. CSP is a Plasmodium cell-surface protein that consists of an N-terminus region, C-terminus region, and a central repeat region. Antibodies against the repeat region bind with a higher efficacy and result in lower parasitemia rates in infected mice, when compared to antibodies against the N-terminus region. Despite this success, antibodies against the C-terminus have been found to be ineffective (Scally et al., 2018).

To generate a more effective malaria vaccine, it may be necessary to target multiple antigens simultaneously. This is known as a combination vaccine and may be the next step forward in the fight against malaria. If an individual’s immune system can be primed to recognize multiple malaria antigens from multiple infection stages, then the immune response to malaria may be heightened. Due to the success of the PfCSP RTS,S/AS01 vaccine, the development of a combination vaccine might be eased by the inclusion of antibodies against PfCSP.

The Cystine-Rich Protective Antigen (PfCyRPA) is a protein similar in some respects to PfCSP, but it is expressed in the Plasmodium merozoite phase. PfCyRPA is part of a complex that includes Reticulocyte binding-like Homologous protein 5 (PfRH5) and RH5-interacting protein (PfRipr) (Favuzza et al., 2017). PfCyRPA is important for merozoite invasion of human erythrocytes, analogous to how PfCSP functions during human hepatocyte invasion (Coppi et al., 2011). Both PfCyRPA and PfCSP hold potential as antigenic targets for malaria vaccines, thus making the inclusion of both proteins in a combination vaccine a promising research opportunity. This vaccine, if effective, will help reduce the harmful effects of malaria infection within individuals. Additionally, if the number of malaria gametocytes can be reduced in an infected individual, the risk of Plasmodium transmission to a mosquito may be reduced as well.

Hypothesis

This proposal seeks to better understand how antibodies against PfCSP and PfCyRPA might act in combination as a vaccine against malaria. The underlying hypothesis is that antibodies against these cell-surface antigens would interact synergistically to produce a vaccine more effective against malaria than the current RTS,S/AS01 version. Research into this hypothesis will help bridge the current gap in knowledge regarding synergistic combinations of α-PfCSP and α-PfCyRPA antibodies.

Specific Aims

To generate a combination vaccine, one must first identify a set of antigens suitable for combination. This grant proposal has two main aims. The first focuses on the production of antibodies to the merozoite PfCyRPA protein and the efficacy of these antibodies. This aim will test the prediction that PfCyRPA is an immunodominant merozoite cell surface antigen to which antibodies bind with a high efficacy. If this prediction is supported, antibodies against PfCyRPA will significantly reduce parasitemia during P. yoelii infection of mice and reduce the rate of successful human cell invasion by P. falciparum.

The second aim focuses on examining how PfCyRPA antibodies act in combination with PfCSP antibodies and how effective this combination is against malaria infection. This aim will test the prediction that PfCyRPA and PfCSP antibodies act synergistically using isobolograms P. yoelii expressing PfCyRPA and PfCSP infection of mice. If this prediction is supported, the P. yoelii infection of mice will be inhibited with a combination immunization such that the percentage of parasitemia is reduced as compared to immunization with a single antibody type (or control immunization lacking antibodies). Additionally, the isobologram will show that a combination of PfCyRPA and PfCSP antibodies does not improve sporozoite or merozoite invasion of human hepatocytes or erythrocytes (respectively).

Experimental Proposal

Specific Aim 1:

The purpose of this aim is to raise monoclonal antibodies against key cell surface antigen PfCyRPA for the merozoite stage of malaria infection. The experimental design will be similar to that of Zhang et al,  2017, where the researchers used mice to raise antibodies against PfCSP and tested the efficacy of these antibodies. The same anti-PfCSP antibodies from Zhang et al. will be used in both aims of this experimental proposal.

Production and Purification of PfCyRPA

First, the DNA sequence of PfCyRPA will be isolated from merozoites. The sequence will be amplified using PCR and treated with EcoR I and Not I restriction enzyme primers. This will result in in vitro PfCyRPA DNA with EcoR I and Not I restriction enzyme-recognizing DNA sequences on either side of the gene. Next, the DNA will be treated with EcoR I and Not I enzymes, resulting in the cleavage of that sequence. This will be inserted into the pET20b vector (which confers ampicillin resistance) and transformed into E. coli cells. The E. coli cells will be raised on ampicillin-deficient agar, a condition which will inhibit the growth of cells lacking the plasmid. The plasmid will be purified from a bacterial colony and sequenced. 

The success of this experiment will be determined by the plasmid sequencing. If the sequencing shows that the PfCyRPA gene was correctly inserted, then the experiment will be considered successful and will proceed to the next stage. If the sequencing shows that the gene insertion did not occur properly, then the experiment will be repeated. After the proper plasmid sequence has been confirmed, the PfCyRPA protein will be isolated from bacterial cells. This will be done by lysing the bacterial cells and incubating the contents with Nickel-NTA agarose beads. The beads will bind to the PfCyRPA protein. The solution will then be eluted and run on SDS-PAGE to ensure protein purity.

Hybridoma Production and in vitro Antibody Efficacy

To induce the proliferation of PfCyRPA antibody-producing B cells, mice will be immunized with the PfCyRPA protein. After seven weeks of weekly immunizations, B cells from the mice’s lymph node will be isolated. The cells will be fused to a myeloma cell line to generate antibody-producing hybridomas. Hybridomas will be separated via dilution and cloned, and the affinity of the antibodies they produce to PfCyRPA will be tested. Antibody specificity to PfCyRPA will be tested by first coating ELISA wells with pure PfCyRPA protein, then incubating the wells with the hybridoma-produced monoclonal antibodies. Antibodies not bound to PfCyRPA will be washed off. Next, an anti-mouse IgG enzyme-linked secondary antibody will be added to the wells. Secondary antibodies not bound to the primary antibodies will be washed off. A substrate will be added to the wells and will react with the secondary antibody enzyme. This reaction will produce a color change. The more prominent the color change, the higher the antibody specificity to PfCyRPA is. Antibodies will be added at multiple concentrations (including a no-antibody control to establish a baseline level of enzyme activity in the wells).

Antibody Efficacy in vivo

After selecting antibodies that bind to PfCyRPA, P. yoelii parasites (which can infect mice) will be genetically modified to express PfCyRPA during their merozoite infectious phase. PfCyRPA will be inserted at the P. yoelii CyRPA gene locus via double cross-over homologous recombination, in the same manner as that used by Zhang et al. (2016) for the generation of PfCSP-bearing P. yoelii parasites. First, mice will be immunized by each of the PfCyRPA antibodies (or a no-antibody control immunization). Next, live PfCyRPA/Py parasites will be injected into the immunized mice and allowed to infect the mice. Three days after injection, blood will be collected from the mice and stained with a Giemsa stain. The stain will be used to show merozoite invasion of erythrocytes. This will be conducted to assess the efficacy of the PfCyRPA antibodies during parasite infection through a quantitative metric: percentage of parasitemia (the proportion of invaded erythrocytes to total erythrocytes). A high percentage of parasitemia value is indicative of an ineffective vaccine, whereas a lower value is indicative of a more effective vaccine.

Antibody-Dependent Inhibition of Plasmodium Invasion

Both PfCSP and PfCyRPA are involved in P. falciparum invasion of host cells. This likely contributes to the anti-malarial activities of anti-PfCSP and anti-PfCyRPA antibodies; these antibodies may hinder Plasmodium invasion of hepatocytes and erythrocytes. The more effective antibodies will likely reduce the rate of sporozoite and merozoite invasion of human cells. To investigate this in more detail, video microscopy of P. falciparum invasion of human cells will be conducted. This will include analysis of both hepatocyte and erythrocyte invasion. 

The human hepatocyte cell line (HC-04) is susceptible to P. falciparum sporozoite invasion in vitro (Sattabongkot et al., 2006). This cell line will be used as a model for in vitro sporozoite invasion and the effects of anti-PfCSP antibodies on said invasion.

Live malaria sporozoites will be added to cell cultures of HC-04. In addition, the most effective concentration of each anti-PfCSP antibody will be added to the solution (or no antibody as a control). Sporozoites will be observed during hepatocyte invasion. To quantify invasion success, the percentage of successful invasions/ observed invasion attempts will be noted. The lower the % success, the more effective a given antibody is. 

Erythrocytes will be extracted from live human participants to examine the impact of anti-PfCyRPA antibodies on merozoite invasion. The procedure will be similar to those of the HC-04 invasion experiments, where antibodies will be added to the erythrocyte/merozoite mixture (or no antibodies as a control). Percentage of invasion success will be quantified. 

Specific Aim 2:

After a few promising antibodies have been generated using specific aim 1, the antibodies will be tested alongside antibodies that bind to PfCSP (from Zhang et al., 2017). By analyzing combinations of these antibodies, it will be possible to identify the most synergistic combinations which may be applied to a future malaria vaccine. The methods described here are similar to those used by Bustamante et al.(2017). Bustamante et al. used isobolograms to test various combinations of malaria antibodies. Here, isobolograms will be used to ensure that certain combinations of antibodies do not act antagonistically.

α-PfCSP and α-PfCyRPA Antibody Combination Efficacies in vitro

To test the efficacy of a combination of anti-PfCSP and anti-PfCyRPA antibodies, isobolograms will be used. The maximum inhibitory concentration of the antibodies will be determined using a dose-response assay, where various concentrations of each antibody will be tested against P. falciparum sporozoites and merozoites. In this experiment, application of PfCyRPA antibodies to sporozoites is not expected to impact sporozoite invasion of hepatocytes because these antibodies likely do not bind to proteins directly involved in the invasion process. Likewise, PfCSP antibodies are not expected to impact merozoite invasion of erythrocytes. Thus, the main purpose of this experiment is to ensure that combinations of α-PfCSP and α-PfCyRPA antibodies do not act in an antagonistic manner or increase sporozoite or merozoite invasion of human cells. If certain combinations of antibodies are found to act antagonistically, then these combinations will be ruled out for use in future vaccines.

The IC50 will be determined as half of the maximum inhibitory concentration (the lowest antibody concentration at which Plasmodium invasion of human cells is maximally hindered) and will be used as the standard dilution for each antibody. PfCSP and PfCyRPA antibodies will be diluted to ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5, and then each dilution combination will be further diluted using six two-fold dilutions. In this experiment, the 5:0 and 0:5 ratios will serve as controls for each antibody. Each of the five antibody dilutions will be added to a culture containing either human hepatocytes or erythrocytes and either sporozoites or merozoites (respectively) and allowed to incubate for 24 hours. Afterwards, the cultures will be fixed, and the rate of Plasmodium invasion of cells will be examined. 

The isobologram will be used to determine the FIC50, which is calculated by dividing the IC50 of antibody A in combination with antibody B by the IC50 of antibody A alone. A FIC50 value greater than 1 indicates that the antibodies are less effective in combination and are antagonistic. A FIC50 value less than 1 indicates that the antibodies are more effective in combination and are synergistic.

α-PfCSP and α-PfCyRPA Antibody Combination Efficacies in vivo

To further test the efficacy of the antibody combinations, mice will be immunized with antibody combinations daily for a week, and then injected with P. yoelii bearing PfCSP and PfCyRPA in a procedure similar to that used in specific aim 1. This P. yoelii mutant model will be generated by inserting the PfCyRPA gene into PfCSP-bearing P. yoelii parasites (in a manner similar to that described by Zhang et al., 2016). As a control, some mice will receive a vaccination with one, or neither, antibody type. Blood samples will be taken from the mice and stained with Giemsa dye to analyze merozoite invasion of erythrocytes. The lower the percentage of parasitemia, the more effective the vaccine combination is against malaria. If the underlying hypothesis (that certain combinations of PfCSP and PfCyRPA antibodies act synergistically) is supported, then some mice injected with PfCSP and PfCyRPA combination vaccines will experience a lower percentage of parasitemia rates compared to mice injected with either antibody alone or no antibody. This in vivo test of antibody efficacy stands in contrast to the isobologram in vitro test because the antibodies in this test are expected to interact synergistically. The isobologram will used to examine Plasmodium invasion at individual stages, whereas the in vivo experiment here will be used to examine Plasmodium invasion throughout the parasite’s pathogenesis.

Conclusion

The research proposed here will prove important to the development of more effective malaria vaccines. If the overall goal of this research (identification of a synergistic antibody combination) is achieved, novel vaccines may be developed to combat multiple stages of malaria pathogenesis. These vaccines will inhibit Plasmodium invasion of both hepatocytes and erythrocytes and make Plasmodium more visible to the immune system. Despite these potential benefits, the research proposed here does have some limitations. PfCyRPA is part of a larger complex that includes the PfRH5 and PfRipr proteins. It has been suggested that antibodies against both PfCyRPA and PfRH5 may act synergistically; thus, future studies should seek to understand if vaccines against the merozoite stage of Plasmodium might benefit from the inclusion of both PfCyRPA and PfRH5 antibodies (Favuzza et al., 2017). Additionally, this proposal only targets two stages of Plasmodium pathogenesis. If sporozoites manage to evade the immune system and infect hepatocytes, then they are free to produce a large number of merozoites. The same can be said for merozoite infection of erythrocytes. Future studies should investigate the potential for inducing an immune response against infected hepatocytes and erythrocytes to hinder the division of Plasmodium. This might entail targeting of endogenous antigens presented by infected human cells. Research has been conducted to understand the production of endogenous antigens in infected hepatocytes and found that Plasmodium minimizes the presentation of antigens (Montagna et al., 2014). This study also found that antigens localized to the cytoplasm of infected cells can induce a CD8+ T-cell immune response, indicating that endogenous antigens do hold some immunologic potential. Overall, gaining a greater understanding of which malaria proteins to target will be important for developing better malaria vaccines.