With respect to the process used to select the "bioinformatically selected panel" of 40 proteins, even in retrospect it is difficult to say whether the approach taken was optimal. Of the 15 literature-selected proteins, 8 would have been identified with the bioinformatic selection algorithm. Among the 2 robust hits from the screen, CyRPA would have been selected using the bioinformatic approach whereas Santigen would not have (**Table S1**). This result does suggest that there may be other invasion-blocking candidate antigens which were not identified as part of the "Merozoite invasion sub-network" by Hu et al. (18). It is difficult to say whether our decision to prioritize the moderately polymorphic proteins at the expense of non-polymorphic proteins was the right one. CyRPA and S-antigens, the two robust hits from this study, both came from the literature-selected panel. CyRPA has one SNP of >10% prevalence, which would mean that had it been in the 'bioinformatically-selected' panel it would have been brought forward whether we selected the 40 most polymorphic or the 40 least prevalent genes. S-antigen is so polymorphic that with the data available at the time it was not possible to actually count SNPs, and even with data available now a full discussion of polymorphism in S-antigen is beyond the scope of this paper. Both of these antigens elicit antibodies capable of neutralizing 3D7 and FVO, which differ markedley in their S-antigen sequences, suggesting that polymorphism is not always predictive of strain-specific neutralization.

While this study has focused on the assay of GIA, as one defined mechanistic correlate that is causative of protection against blood-stage P. falciparum (63) it should be recognized that alternative assays of anti-merozoite antibody efficacy do exist, including phagocytosis assays (64), the assay of antibodydependent respiratory burst (65), and the assay of antibodydependent cellularinhibition (66). Antibody performance in each of these assays has been shown to correlate with protection from clinical malaria in the context of naturally-acquired humoral immunity. However, there are technical challenges with using these assays for antibodies derived from different species as in this study. We selected the assay of GIA because our goal was to benchmark against antibodies to RH5, which are functional in this assay.

In summary, no antibody specificity in this study outperformed anti-RH5 antibodies in the GIA assay. Neither did any polyclonal antibody specificity robustly synergize with or enhance the GIA of anti-RH5 antibodies, although additivity with anti-CyRPA IgG was generally consistent. While we attempted to make this screen as comprehensive as possible, we were not able to express every candidate antigen in HEK293 cells, and new antigens continue to be described that were not included in our original list of proteins. Bustamante et al. have very recently reported that SERA9, RAMA, MSRP5, and EBA181 are targets of neutralizing antibody which potentially synergize with anti-RH5 antibodies (54). The RH5-interacting protein (Ripr) is another protein with a strong rationale for testing as a vaccine antigen, but which has not been well studied due to the difficulty of expressing it in bacterial and mammalian cell systems (26, 67). It now remains a priority to test these proteins in human compatible vaccine-delivery systems to find out if their inclusion in a multi-valent vaccine offers a realistic possibility of improving on RH5 alone.

#### ETHICS STATEMENT

Animal experiments and procedures were performed in accordance with the UK Animals (Scientific Procedures)

#### REFERENCES

Act Project License (PPLs 30/2889 and PA7D20B85) and were approved by the University of Oxford Local Ethical Review Body.

## AUTHOR CONTRIBUTIONS

JI: wrote the paper. JI and SD: conceived the study. JI, DA, MS, KW, AD, GL, JC, DP, and MH: produced and characterized proteins. JI, LB, DP, and SdC: performed animal immunization studies. JI, RB, JM, and DQ: performed GIA assays. SS and HB: performed affinity purification and Biacore analysis on CyRPAspecific antibodies. JI and JW: selected genes of interest.

### FUNDING

This work was supported in-part by funding from the European Union's Horizon 2020 research and innovation programme under grant agreement for OptiMalVax [number 733273].

#### ACKNOWLEDGMENTS

The authors are grateful for the assistance of Julie Furze, Adrian Hill, Jing Jin, and Amelia Lias (Jenner Institute, University of Oxford); Dominic Kwiatkowski (Wellcome Trust Centre for Human Genetics, University of Oxford) for useful discussions; Edmond Remarque (BPRC, Netherlands) for providing the BG98 anti-AMA1 antibodies for assay standardization; Yves Durocher for provision of HEK293E cells (CNRC-NRC, Canada); and Gavin Wright for sharing the CyRPA (Gly KO) via Addgene (plasmid number: 50823).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01254/full#supplementary-material

ordered events of erythrocyte invasion by the blood stage malaria parasite. PLoS ONE. (2012) 7:e46160. doi: 10.1371/journal.pone.0046160

**Conflict of Interest Statement:** JI, DA, AD, KW, SC, MH, and SD are named inventors on patent applications relating to RH5 and/or other malaria vaccines, antibodies and/or immunization regimens.

The remaining 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.

Copyright © 2019 Illingworth, Alanine, Brown, Marshall, Bartlett, Silk, Labbé, Quinkert, Cho, Wendler, Pattinson, Barfod, Douglas, Shea, Wright, de Cassan, Higgins and Draper. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Induction of Plasmodium-Specific Immune Responses Using Liposome-Based Vaccines

Aloysious Ssemaganda1 , Ashwini Kumar Giddam1†, Mehfuz Zaman1 , Mariusz Skwarczynski 2 , Istvan Toth2,3,4, Danielle I. Stanisic1 \* and Michael F. Good1 \*

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

#### Edited by:

1

Australia, 4

Ashraful Haque, QIMR Berghofer Medical Research Institute, Australia

#### Reviewed by:

Peter Timmerman, Pepscan, Netherlands Adrian John Frederick Luty, Institut de Recherche Pour le Développement (IRD), France

#### \*Correspondence:

Danielle I. Stanisic<EMAIL_ADDRESS>Michael F. Good<EMAIL_ADDRESS>
#### †Present Address:

Ashwini Kumar Giddam, Q-Pharm, Royal Brisbane and Women's Hospital, Herston, QLD, Australia

#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 19 September 2018 Accepted: 16 January 2019 Published: 01 February 2019

#### Citation:

Ssemaganda A, Giddam AK, Zaman M, Skwarczynski M, Toth I, Stanisic DI and Good MF (2019) Induction of Plasmodium-Specific Immune Responses Using Liposome-Based Vaccines. Front. Immunol. 10:135. doi: 10.3389/fimmu.2019.00135 In the development of vaccines, the ability to initiate both innate and subsequent adaptive immune responses need to be considered. Live attenuated vaccines achieve this naturally, while inactivated and sub-unit vaccines generally require additional help provided through delivery systems and/or adjuvants. Liposomes present an attractive adjuvant/delivery system for antigens. Here, we review the key aspects of immunity against Plasmodium parasites, liposome design considerations and their current application in the development of a malaria vaccine.

Institute for Glycomics, Griffith University, Southport, QLD, Australia, 2 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia, 3 School of Pharmacy, The University of Queensland, Brisbane, QLD,

#### Keywords: malaria, Plasmodium, immunity, adjuvant, liposomes, vaccine

#### INTRODUCTION

Malaria vaccine development has been a focus of research since the 1940s when inoculation with homologous inactivated sporozoites and/or serum resulted in control of parasitemia amongst immunized domestic fowls (1, 2). Follow-up studies also showed that monkeys and ducks were protected against Plasmodium (P) knowlesi and P. lophurae following vaccination with killed, adjuvanted parasites (3, 4). Additionally, in 1967, it was demonstrated for the first time, that immunization with irradiated sporozoites protected mice against P. berghei (5). Despite these early promising findings, an effective vaccine for malaria still eludes scientists with only one vaccine candidate, RTS, S, receiving a positive scientific opinion from European regulators and it is currently approved for use in pilot implementation trials in 3–5 epidemiologically distinct locations in sub-Saharan Africa (6, 7). The complexity of Plasmodium parasites, antigenic polymorphism and failure to maintain long-lived immune responses calls for continued efforts in the search for novel vaccines which can effectively prevent P. falciparum and P. vivax infections (8).

A major bottleneck in the development of vaccines against infectious diseases is the failure to initiate robust innate immune responses and subsequent potentiation and maintenance of downstream adaptive immune responses. This is achieved naturally with live attenuated vaccines while inactivated and sub-unit vaccines require delivery systems and/or adjuvants for efficient presentation to the immune system and additional stimulation to enhance potency (9). To address this, careful selection of adjuvants and delivery systems needs to be considered early in the vaccine development process. However, only a handful of adjuvants have been licensed or tested for use in human vaccines and these are summarized in **Table 1**.

**80**

TABLE 1 | Clinically tested and licensed vaccine adjuvants (10, 11).

First proposed by Gregoriadis and Allison in 1974 as immunological adjuvants (39), liposomes are a promising vaccine adjuvant/antigen delivery system. Historically well-known as drug carriers, liposomes are self-assembling phospholipid vesicles capable of incorporating and protecting antigens from degradation, as well as facilitating antigen delivery to professional antigen presenting cells (APCs) (9, 40– 43). Liposomes generally act by depot formation resulting in enhanced uptake by APCs and subsequent induction of the desired immune responses. To date, the extensive use of liposomes can be attributed to their safety profile, biocompatibility, biodegradability, versatility, and plasticity and therefore they present an attractive platform for malaria vaccine development.

In a natural malaria infection, the acquisition of clinical immunity is slow, spanning several years of repeated exposure, and it is not sterile (44). An ideal vaccine capable of inducing sterile immunity against the different life-cycle stages of malaria will need to induce a qualitatively and/or quantitatively different immune response to that induced during natural infection immunity (45). Since stage-specific immunity to malaria requires humoral and cell-mediated immune responses, the ideal vaccineinduced responses should preferably be comprised of both forms of responses. However, for rational vaccine development, a clear understanding of the complex nature of immunity to malaria is required and this in turn may help to inform the selection of an appropriate adjuvant/delivery system. This review highlights key aspects of the immune response to malaria, design considerations of liposomes, and their current application in malaria vaccine development.

### IMMUNITY TO MALARIA

Plasmodium parasites, the causative agents of malaria, are obligate intracellular organisms which undergo a complex lifecycle in the vertebrate host broadly divided into: the mosquito stage which occurs in the vector; the pre-erythrocytic stage which occurs in the vertebrate host's liver; and the erythrocytic stage which occurs in the blood of the vertebrate host (46). At all lifecycle stages, the immune responses induced following infection differ significantly and a clear understanding of these responses will inform the vaccine development process.

#### Pre-erythrocytic Stage Immunity to Malaria

The pre-erythrocytic stage of malaria infection is clinically quiescent, probably due to the low number of sporozoites inoculated by the mosquito while taking a blood meal. During this stage, studies in mice have shown that antibodies can control infection through immobilization of sporozoites by inhibiting sporozoite motility and subsequent invasion of hepatocytes (47, 48). Following natural infection, studies demonstrated the existence of pre-erythrocytic antigen-specific antibodies to P. falciparum; however, their role remains unclear (49–51). To date, the best model that has enabled the study of pre-erythrocytic immune response mechanisms has utilized irradiated sporozoites in both humans and animals. Radiation-attenuated sporozoites retain the capacity to infect hepatocytes but cannot develop into an erythrocytic infection. Studies in rodent models involving inoculation of radiation-attenuated sporozoites demonstrated that antibodies were involved in the enhanced clearance of sporozoites, reduction in sporozoite motility and inhibition of hepatocyte invasion (52, 53). In clinical studies, induction of antibodies to the circumsporozoite protein (CSP) following immunization with the P. falciparum sporozoite (PfSPZ) vaccine has been shown to partially correlate with protection (54– 58). Furthermore, following immunization with RTS, S/AS01, high CSP-specific antibody titers are induced and are a surrogate measure of protective efficacy for this vaccine candidate (16, 59–63).

Cell-mediated immunity following inoculation of radiationattenuated sporozoites has also been shown to contribute to vaccine-induced sterilizing immunity to Plasmodium infection in both mice and humans (54–58, 64–72). Studies in rodent models, however, indicate that a network of cellular mechanisms mediates immunity to pre-erythrocytic infection (64–66, 68, 70–72). Initial studies in mice immunized with irradiated P. yoelii 17XNL sporozoites reported that cytotoxic CD8+ T cells mediated protection against a wild-type challenge of infectious sporozoites (64, 65). Further studies indicated that IL-12 from liver antigen presenting cells (APCs) stimulated CD8+ T cells and Natural killer (NK) cells to produce interferon (IFN)-γ. IFN-γ, in turn, induces the infected hepatocytes to produce nitric oxide (NO), which subsequently kills the parasite in the hepatocyte (66, 68, 71). In addition, the balance between IL-2, IL-10, IL-12, and IFN-γ results in an inflammatory response that contributes to pre-erythrocytic immunity (70, 72). In human volunteers, both CD4+ and CD8+ T cell responses against pre-erythrocytic antigens have been observed following immunization with irradiated sporozoites (54–58, 67, 69).

Controlled infection immunization (CII) studies, involving experimental sporozoite inoculation via infectious mosquito bites with concurrent chemoprophylaxis provides another model for investigating pre-erythrocytic immunity to malaria. These studies have shown that a protective polyfunctional T cell response to pre-erythrocytic antigens predominantly characterized by the production of IFN-γ, tumor necrosis factor (TNF), and IL-2 is induced following CII (73–76). Purified IgG against P. falciparum sporozoites, obtained from CII trial participants was also shown to inhibit liver stage infection in a humanized liver-chimeric mouse model (73, 75, 77).

#### Erythrocytic Stage Immunity to Malaria

During the erythrocytic stage, clinical signs, and symptoms of malaria manifest as the parasites invade and replicate in RBCs. Studies in animal models have demonstrated that the innate immune system is involved in the initial recognition of bloodstage parasites, promotion of inflammation, inhibition of parasite growth and potentiation of the adaptive immune response (78, 79). Plasmodium falciparum pathogen-associated molecular patterns (PAMPs) such as glycosylphosphatidylinositol (GPI) anchors (Toll-like receptor 2 [TLR2]), hemozoin (NOD-like receptor containing pyrin domain 3 [NLRP3] inflammasome), CpG-containing DNA motifs bound to hemozoin (TLR9) and AT-rich DNA motifs (unidentified cytosolic receptor) have been shown to trigger an inflammatory cascade by binding pattern recognition receptors (PRRs) on the surface of innate immune cells (79–83). This interaction of PAMPs and PRRs results in the production of pro-inflammatory cytokines (IL-12 [p70], IFN-γ and TNF) by APCs, as well as regulatory cytokines (IL-10 and TGF-β) that have been implicated in immunity and pathogenesis to blood-stage malaria infection (79, 84, 85).

Several innate immune cells such as dendritic cells (DCs), macrophages, mast cells, neutrophils, NK cells, natural killer T (NKT) cells, and γδ T cells have been implicated in this initial immune response (85–88). Plasmodium parasites have been shown to modulate DC maturation and function resulting in the induction of regulatory T cells which in turn modulates CD4+ T cell responses, suppressing protective immune responses while averting immune-mediated pathology (85). IL-12 production by DCs has also been implicated in the production of IFN-γ by NK cells and CD4+ Th1 cells resulting in the control of parasite growth (85). Understanding the balance between protective and immunopathologic responses following DC activation and maturation might have favorable implications in designing vaccines to prevent severe malaria (85). In vitro, NK cells have been shown to be an early source of IFN-γ, promoting the destruction of infected red blood cells by activated macrophages (79, 89). γδ T cells and monocytes on the other hand have been associated with elevated levels of TNF, IL- 10, IP-10, IL-6, macrophage inflammatory protein (MIP)-1β and MIP-1α which is linked with severe disease (90). Furthermore, downregulation of γδ T cell responses following repeated exposures to Plasmodia has also been implicated with better tolerance to clinical malaria (91, 92). Given their specificity for restricted TCR ligands, γδ T cells present an attractive target for a vaccine to protect against severe disease (45, 90).

Antibodies play a role in naturally acquired immunity to erythrocytic stage malaria, as it has been shown that the passive transfer of immunoglobulin from immune donors resulted in the reduction of parasitaemia and clinical disease among semiimmune recipients from East Africa as well as non-immune Thai patients (93–95). Antibodies may function by inhibiting merozoite invasion of RBCs (96), binding to pRBCs and enhancing clearance by the spleen (97, 98), as well as opsonizing pRBCs, resulting in phagocytosis by macrophages (99, 100).

Cell-mediated immunity against the erythrocytic stage is primarily mediated by CD4+ T cells, as demonstrated in both murine and human models. Studies showed that mice depleted of CD4+ T cells developed very high parasitaemia and were unable to control the infection compared to mice depleted of CD8+ T cells, which developed mild parasitaemia that subsequently resolved; this indicated a clear role of CD4+ T cells in erythrocytic stage immunity (101, 102). Additionally, adoptive transfer of CD4+ T cells was shown to confer protection and control parasitaemia in immunodeficient mice (103). Further investigation of the role of CD4+ T cells demonstrated that during the acute phase of infection, there was a significant upregulation of an IFN-γ-specific CD4+ T cell (Th1) response followed by an IL-4-specific CD4+ T cell-mediated (Th2) antibody response during the chronic phase (104). These data indicate that early activation of Th1 cells enables control of the infection via effector mechanisms such as macrophages, followed by a Th2 response which activates B cells to clear the parasite in the later stages of the infection in mice (105, 106).

In human studies, the role of CD4+ T cells was demonstrated when volunteers were infected with low doses of blood-stage P. falciparum followed by drug cure (107). In this study, volunteers appeared to be protected against a homologous challenge infection with immunity associated with an IFN-γ-specific CD4+ T cell response and nitric oxide synthase (NOS) production in the absence of detectable antibodies (107). However, a followup study suggested that residual drug may have contributed to the apparent protection (108). Another study showed that stimulation of T cells obtained from children living in Papua New Guinea resulted in parasite-specific IFN-γ and TNF responses, which were associated with protection against clinical episodes of malaria (109). More recently, studies in African children showed that CD4+ T cells may play an important modulatory role in the development of blood-stage immunity (110, 111). Higher IFN-γ/IL-10+ specific-CD4+ T cell responses were observed amongst children heavily exposed to malaria compared to children with low exposure indicating that CD4+ T cells may play an important immunomodulatory role in the pathogenesis of childhood malaria (110). Additionally, the induction of IL-10 producing CD4+ T cells amongst highly exposed children may interfere with the development of immunity, which may have implications for vaccine development (111).

T follicular helper cells (Tfh) are a subset of CD4+ T cells, capable of providing B cell help as well as activating follicular B-cell responses (112–114). Recent studies in mice showed that Tfh cells play a critical role in controlling P. chabaudi blood-stage infection via activation of IL-21 mediated responses (115). Therefore, since humoral responses are critical to the erythrocytic stages of Plasmodium, an in-depth understanding of the activation and maintenance of Tfh cells during malaria will be critical in designing blood-stage vaccines (115).

Regulatory T cells (Tregs) are another CD4+ T cell subset implicated in the maintenance of immune homeostasis and control of excessive pathogen-driven inflammatory responses (116, 117). Following Plasmodium infection, uncontrolled production of pro-inflammatory cytokines is associated with pathology in both mice and humans (118–121) and antiinflammatory cytokines (TGF-β and IL-10) are known to be critical in the modulation of this inflammatory response (118, 120, 122, 123). The immunomodulatory function of IL-10 and TGF-β is associated with Tregs whose role in rodent malaria remains unclear. Some studies have shown that Tregs are critical in the control of pro-inflammatory responses associated with pathology (124, 125) while other studies have associated upregulation of Tregs with detrimental outcomes (124, 126, 127). These discrepancies may have been dependent on the rodent parasite strains utilized in the study (128). Tregs may inhibit protective immune responses resulting in enhanced parasite growth if induced early in infection but may also limit immune-mediated pathology (45, 128–130). Clinical studies have shown that acute infection with Plasmodium parasites resulted in upregulation of Tregs which positively correlated with augmented parasite load and subsequent disease severity amongst these individuals (131–134). Given this background, a vaccine capable of inducing Tregs with the ability to protect against the immunopathology associated with malaria infection would be desirable if parasite persistence is required for the maintenance of protective immune responses. However, since Tregs are known to inhibit protective immune responses, treatment with antimalaria drugs at the time of vaccination may be necessary to "normalize" the pre-existing immune response and ensure induction of the appropriate vaccinespecific responses (45).

The role of CD8+ T cells in defense against blood-stage parasites remains unclear due to the fact that mature RBCs do not express MHC class I molecules. However, in vitro studies have shown that both P. falciparum and P. vivax are able to invade erythroblasts—immature erythrocytes that possess a nucleus and express MHC class I molecules (135, 136). Additionally, studies in mice demonstrated that blood-stage parasite antigens were cross-presented by CD8-α + DCs, inducing parasite-specific CD8+ T cell responses capable of lysing APCs (137). These findings indicated the possible relevance of CD8+ T cells in erythrocytic immunity to Plasmodium. Indeed, additional studies in mice demonstrated that parasitized erythroblasts activated CD8+ T cells in an antigen-specific manner (138). This contactdependent Fas—Fas Ligand (FasL) interaction of CD8+ T cells with the parasitized erythroblasts results in the exposure of phosphatidylserine (PS) on the erythroblast surface (139). Cells displaying PS on their surface are rapidly phagocytosed by macrophages. Thus, CD8+ T cells in conjunction with macrophages are able to mediate immunity to a blood-stage malaria infection in mice (139). Earlier studies in a P. yoelii infection model demonstrated that professional APCs might cross-present parasite-derived peptides on MHC class I to CD8+ T cells leading to cytotoxicity through the production of IFN-γ, perforin and granzyme B (140). Furthermore, parasite-specific CD8+ T cells have also been shown to clear infected reticulocytes, which express MHC class I molecules, via the secretion of IFN-γ and expression of granzyme B (141).

These data highlighted in the studies above provide an insight into the complex nature of immune responses elicited following infection with malaria parasites. Understanding the balance between protective immunity and immunopathology is critical for the development of an ideal vaccine capable of inducing both humoral and cell-mediated immune responses against different life-cycle stages of the malaria parasite. To achieve this balance, careful selection of antigen delivery systems and adjuvants during the vaccine development process is paramount and the pliability of liposome-based platforms can be utilized for this purpose.

#### DESIGN CONSIDERATIONS OF LIPOSOMAL VACCINE FORMULATIONS

Based on their design, liposome vaccine formulations can be tailored to achieve desired immune responses and adjuvant properties by modification of vesicle physicochemical factors (summarized in **Figure 1**), such as lipid composition, charge, PEGylation, antigen encapsulation, and addition of immunomodulators (9, 40–43).

The choice of phospholipid has been shown to enhance the adjuvanticity of liposomes. Long chain lipids tend to form

rigid ordered bilayer structures while those with shorter tails tend to form fluid and disordered vesicles (9). Immunization of animals with liposomes formulated with long chain lipids such as dimethyldioctadecylammonium bromide (DDAB) and distearyol derivative of L-α-phosphatidyl choline (DSPC) resulted in stronger antigen-specific antibody responses when compared to animals that received liposomes formulated with shorter chain lipids (142). Additionally, the stability of the lipid bilayer can be enhanced further with the addition of cholesterol within the liposome formulation resulting in improved antibody responses compared to formulations without cholesterol (143–145).

Positively charged (cationic) liposomes formulated with saturated acyl chain lipids (with a quaternary ammonium head group) have been shown to promote the binding of antigen at the site of injection stimulating interaction with APCs to elicit a robust Th1 cytokine response (146). In contrast, highly fluid unsaturated acyl chain liposomes that are rapidly cleared from the injection site result in lower activation of APCs as measured by the decreased expression of DC co-stimulatory molecules CD40 and CD86 (146). Additionally, cationic liposomes have been shown to promote retention of higher levels of antigen at the injection site, resulting in a depot-effect allowing continuous attraction of APCs and subsequent induction of robust cellmediated immune responses comprised of IFN-γ, IL-2, TNF, and IL-17 (147). On the other hand, negatively charged (anionic) and neutral liposomes were rapidly cleared from the injection site resulting in lower activation of APCs and lower Th1 as well as Th17 cytokine responses (9, 147, 148).

The inclusion of polyethylene glycol (PEG), known as PEGylation, has been extensively used for the stabilization of liposomes (149). PEGylation has been shown to mask the charge, due to the hydrophilic chains of PEG extending out from the surface of the liposomes subsequently reducing the electrostatic retention of antigen to the surface of these vesicles (150, 151). Additionally, PEGylation influences lipid packaging reducing the number of bilayers and resulting in reduced vesicle size (151). This modification of liposome size and antigen adsorption properties by PEG was shown to reduce depot formation, resulting in reduced IgG2b antibody and Th1 (IFN-γ) cytokine responses as well as an elevated Th2 (IL-5) cytokine response compared to non- PEGylated liposome formulations (150, 151).

The particle size of liposomes has also been shown to impact adjuvanticity and direct the development of the resulting cellmediated immune response (42). Studies have shown that the immune response induced following administration of smallsized liposome vesicles (10–100 nm) was skewed toward Th2 whilst larger vesicles (400–2,500 nm) induced a Th1 response characterized by augmented IFN-γ and IgG2a production (152). The differences in the profiles of the induced immune response of large vs. small vesicles could be due to differences in antigen processing and trafficking to lymph nodes. Large-sized vesicles (560 nm) were shown to be more efficiently phagocytosed and processed by macrophages compared to smaller vesicles (155 nm) (153). Additionally, trafficking of liposome particles to lymph nodes has been shown to be size dependent, with small-sized vesicles (20–200 nm) freely draining to and specifically targeting lymph node-resident cells, while large-sized vesicles (500–2,000 nm) require dendritic cells for trafficking from the injection site to lymph nodes (154). More recently, immunization using a formulation containing large-sized cationic liposomes (∼500 nm) resulted in enhanced splenocyte proliferative responses and reduced IL-10 production compared to small sized liposomes (∼100 nm) (155). Interestingly, smaller unilamellar liposomes (70 nm) were reported to stimulate higher IgG titers than larger unilamellar (400 nm) but not large submicron size multilamellar liposomes in mice (156). The potency of multilamellar liposomes can be explained by more efficient antigen protection against degradation in their multiple lipid bilayers.

Liposomes can be modified to incorporate additional lipophilic immunomodulators within or attached to the lipid bilayer to enhance adjuvanticity. Such immunomodulators are crucial in the activation of the cells of the innate immune system via PRRs which recognize PAMPs on the surface of pathogens, subsequently activating the adaptive immune system. The activation of innate immune cells such as dendritic cells and macrophages requires the use of Toll-like receptor (TLR) and NOD-like receptor (NLR) type PRRs to direct a robust immune response (157). Therefore, the use of synthetic PRR agonists has been predicted to be critical in the formulation of liposome-based vaccine adjuvants (9, 158).

Liposome formulations can be customized by incorporating PRR agonists that mediate activation and maturation of APCs which in turn facilitates the uptake and processing of liposome-associated antigens resulting in potent cell-mediated immune responses (9). The most widely used PRR agonist monophosphoryl lipid A (MPLA), a TLR-4 agonist, has been used in licensed vaccine formulations Fendrix (hepatitis B) (159) and Cervarix (human papillomavirus) (160). A synthetic analog of MPLA, 3′ -O-desacyl-4′ -monophosphoryl lipid A formulated with Quillaja saponaria Molina, fraction 21 (QS-21) saponin is included in the liposome-based GSK Adjuvant System 01 (AS01) and has been tested in human studies for the malaria vaccine RTS, S (Mosquirix) (16), as well as a shingles sub-unit vaccine HZ/su which demonstrated over 90% efficacy amongst elderly persons (15). Similarly, liposomes can be tagged with sugars such as mannose to target them to lectin-like molecules on APCs to facilitate phagocytic uptake thereby promoting MHC class II involvement and, via cross presentation, MHC class I. This targeting of liposomes to different uptake pathways may aid in directing the resulting immune response toward a mixed Th1/Th2 response (9, 161).

The route of administration of particulate antigen delivery systems such as liposomes has been shown to affect the type and magnitude of immune response induced. Interestingly liposomes can be even administered orally; however, they need to be extensively modified to improve their stability in the gastrointestinal tract and their mucosa adhesive properties (162). In a cross-sectional study in mice, the intramuscular, intradermal and intralymphatic routes of administration were associated with intermediate to high induction of IgG2a and IFN-γ cytokine production while the subcutaneous route was associated with low elicitation of IgG2a and IFN-γ cytokine production (163). These data indicate that the route of administration is critical in the generation of the desired immune response and should be considered while interpreting immunological data following immunization with liposomebased vaccine formulations. Together, it is evident that the versatility and plasticity of liposomes facilitates the tailoring of the desired immune responses, as well as enhanced adjuvanticity and this can be exploited for the development of a malaria vaccine.

### UTILITY OF LIPOSOMES IN MALARIA VACCINE DEVELOPMENT

Liposomes are increasingly becoming used in a number of malaria vaccine candidates targeting the different life-cycle stages. The use of liposomes in the development of sporozoitestage malaria vaccines dates back to the mid-1980s where tetrapeptide antigens (asparagine-alanine-asparagine-proline) derived from the repetitive region of the circumsporozoite (CS) protein of P. falciparum sporozoites were conjugated to carrier proteins and were incorporated into liposomes. These studies demonstrated that liposomes containing carrier proteinconjugated peptide induced a potent humoral immune response which was further enhanced when lipid A was incorporated in the liposome formulation (164–169). The aforementioned studies laid the foundation for the development of RTS, S, the only vaccine against malaria that has received approval for use in pilot implementation trials in sub-Saharan Africa (7). The RTS, S vaccine construct is made of the central repeat region (amino acids 210-398) (R) and the C-terminal region containing the T-cell epitopes of CSP (T), fused to hepatitis B surface antigen (HBsAg) (S), co-expressed in Saccharomyces cerevisiae yeast and self-assembled with unfused HBsAg antigen (170, 171). These hybrid virus-like particles (VLPs) were co-formulated with GSK's proprietary liposome-based adjuvant system, AS01, which contains potent immunostimulants, MPLA and QS-21 that was selected over AS02, an oil-in-water emulsion adjuvant following evidence of enhanced antigen-specific antibody and CD4+ T-cell responses as well as improved efficacy in large-scale clinical studies (61, 172–175). The level of and mechanism of immunity induced by RTS, S in endemic settings are topics of much research. Over 4 years of follow up, the level of protection ranged from 18 to 36%, depending on the age of the child and whether the child received 3 or 4 doses of vaccine. Protection was clearly greater in the early months after vaccination, but waned rapidly after that and there was a 'negative efficacy' during the 5th year in some children (176). This is a suboptimal response and as such the mechanism of immunity came under great scrutiny. Evidence suggests that the level of antibody to the CS protein and serum levels of IFN-γ post vaccination both correlate with protection (177, 178). A major concern, has been the rapid diminution of antibody levels over time, in the face of parasite exposure. The problem is not that RTS,S is not immunogenic. The adjuvant system is one of the most potent there is for human use and is used elsewhere with great effect. It has made Shingrix a highly successful vaccine where even in the elderly there is 90% efficacy. Also, the problem is not with boosting per se, as each dose of RTS,S is accompanied by a rapid rise in antibody titer. In our view, the main problem is that even though RTS,S has a powerful adjuvant system, it is still not powerful enough. Titres wane after vaccination and natural infection will not boost. The main reasons for this appear to be: (i) antigenic polymorphism; and (ii) that the dose of sporozoites that individuals are exposed to (and which express the CS protein) are simply too low to boost or maintain antibody levels. T cell epitopes present on the CS protein which are incorporated into the vaccine are polymorphic (179), and this polymorphism does contribute to the low efficacy (180). The T-cell epitopes are known to be non-cross-reactive (181). An additional factor may relate to liposome design per se. Indeed, efforts are currently underway to remodel RTS, S such that each HBsAg particle expresses CSP thereby increasing the concentration of parasite antigens (182). Here, CSP-HBsAg fusion proteins were co-expressed in Pichia pastoris yeast which in the presence of a tightly regulated inducible alcohol oxidase (AOX1) promoter allows production of a higher density of hybrid VLPs (182, 183). This vaccine construct, co-formulated with Matrix-M TM, a saponinbased liposomal adjuvant is now undergoing clinical testing NCT029050191 .

In the development of transmission-blocking vaccines, gel core liposomes encapsulating Pfs25, an antigen expressed on zygotes and ookinetes of P. falciparum and a leading transmission-blocking vaccine (TBV) candidate, have been tested in mouse models (184). Gel core liposomes are a stabilized form of liposomes bearing a core of biocompatible polymer inside the lipid vesicle which serves to prevent the rapid release of antigen content from liposomes (184, 185). Following 2 intramuscular injections with gel core liposomes, Pfs25-specific antibody responses were observed in immunized mice, and these were maintained for up to 8 weeks. Additionally, strong Th1 cytokine (IL-2 and IFN-γ) responses were elicited and these responses were augmented when the gel core liposomes were formulated with CpG oligodeoxynucleotide (CpG-ODN) (184, 185).

A cationic adjuvant liposome formulation (CAF01) consisting of DDAB, synthetic mycobacterial cordfactor as an immunomodulator, and merozoite surface protein 1 (MSP1) antigen derived from P. yoelii genomic DNA, PyMSP1, has been tested in pre-clinical studies as a blood-stage malaria vaccine (186). Compared to the Alum adjuvanted vaccine formulation, immunization with CAF01- Py-MSP-1 resulted in significantly higher antibody and IFN-γ cytokine responses. Furthermore, following challenge, immunization with CAF01- PyMSP1 resulted in significant control of parasite growth (186).

Tyagi et al. (187), utilized a liposome-mediated transdermal immunization approach to deliver P. falciparum merozoite surface protein-1 (PfMSP-1) antigens through intact skin to antigen presenting cells in the skin (187). Similar to observations following immunization with CAF01- PyMSP1(186), durable and stronger parasite-specific humoral responses were observed up to 10 weeks post-immunization. Additionally, robust cell-mediated responses critical in immunity against blood-stage malaria parasites were induced following transdermal administration of elastic liposomes loaded with PfMSP-1 antigens when compared to Alum based vaccine formulations (187). Collectively, these data underscore the substantial superiority of liposome-based formulations over aluminum-based vaccine adjuvant formulations in the induction of parasite-specific immune responses to malaria.

More recently, our group formulated liposomes with mannosylated lipid core peptides (MLCPs) as targeting ligands for the delivery of whole blood-stage parasite antigens to professional antigen presenting cells (188). Immunization with these mannosylated liposome formulations resulted in the induction of significant CD8+ T cell responses; immunized mice demonstrated better control of parasitemia as well as extended survival following challenge, when compared with control mice, availing an alternative delivery system for inactivated whole parasite antigens (188). Together, the studies above indicate that liposomes are being considerably used in malaria vaccine development for targeting all of the different life-cycle stages and their pliability can be further explored to develop a multi-stage vaccine.

#### CONCLUDING REMARKS

The quest for a vaccine against malaria continues despite the partial success with RTS, S/AS01, which showed modest efficacy in phase III clinical trials (16). Given the complex network of immune responses elicited following infection with Plasmodium parasites, an ideal vaccine should aim to induce the appropriate life-cycle stage-specific-antibody and cell-mediated responses capable of protecting against disease and immunopathology. The versatility of liposomes can be exploited to achieve an optimal formulation via the use of charged lipids to promote antigen retention at the injection site (depot-effect) (186), inclusion of targeting ligands to promote uptake by professional APCs (188), to control their stability, release of antigen, enhance antigen protection, and include immunomodulators (40, 43). Additionally, as the poor efficacy of most malaria vaccines evaluated thus far in field trials has been attributed to antigenic polymorphism, the use of whole parasite antigens (188) in liposome formulations needs to be explored further in malaria vaccine development. In summary, the modification of vesicle physicochemical properties may be further exploited to design an optimal liposome formulation with a high level of efficacy required for complete eradication of malaria by 2030 (8).

### AUTHOR CONTRIBUTIONS

AS, DS, and MG drafted and reviewed original manuscript. AG, MZ, MS, and IT critically reviewed original manuscript and provided important intellectual content.

#### ACKNOWLEDGMENTS

We wish to thank Dr. Leanne M. Low for critically reviewing this manuscript. We acknowledge the generous support from Griffith

1A Safety and Efficacy Study of R21 +/- ChAd63/MVA ME-TRAP. https:// ClinicalTrials.gov/show/NCT02905019.

University (Griffith University International Postgraduate Research Scholarship and Griffith University Postgraduate Research Scholarship) awarded to AS. We also acknowledge the

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**Conflict of Interest Statement:** 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.

Copyright © 2019 Ssemaganda, Giddam, Zaman, Skwarczynski, Toth, Stanisic and Good. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria

Kirsty L. Wilson1,2, Dodie Pouniotis 2 , Jennifer Hanley 1 , Sue D. Xiang1 , Charles Ma3 , Ross L. Coppel 3 and Magdalena Plebanski 1,2 \*

1 Department of Immunology and Pathology, Faculty of Medicine, Nursing and Health Sciences, Central Clinical School, Monash University, Melbourne, VIC, Australia, 2 School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, Australia, 3 Department of Microbiology, Monash University, Clayton, VIC, Australia

#### Edited by:

Ashraful Haque, QIMR Berghofer Medical Research

#### Institute, Australia Reviewed by:

Adrian John Frederick Luty, Institut de Recherche pour le Développement (IRD), France Danielle Ilona Stanisic, Griffith University, Australia

\*Correspondence: Magdalena Plebanski<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 08 October 2018 Accepted: 08 February 2019 Published: 15 March 2019

#### Citation:

Wilson KL, Pouniotis D, Hanley J, Xiang SD, Ma C, Coppel RL and Plebanski M (2019) A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria. Front. Immunol. 10:331. doi: 10.3389/fimmu.2019.00331 Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50–80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant.

Keywords: blood-stage, malaria, nanoparticles, adjuvant, immunogenicity, protection

### INTRODUCTION

Malaria remains a global health problem, affecting over 200 million people annually with ∼40,000 deaths (1). It is caused by the plasmodium parasite, of which Plasmodium falciparum (P. falciparum) and Plasmodium vivax (P. vivax) are the most widespread. There is an urgent need for the development of an effective and long-lasting malaria vaccine. The most advanced pre-erythrocytic stage vaccine RTS, S provides <40% protection against disease in African populations (1, 2). Since any break-through parasites progressing to the blood-stage sustain transmission, pre-erythrocytic vaccines alone may be insufficient to support complete protection or eradication. Additionally, the blood-stage of malaria is symptomatic of disease, highlighting the need to eradicate parasites at or before this stage.

One of the major challenges to designing an effective vaccine for malaria is the identification of appropriate antigen targets. Many candidates are polymorphic or poorly immunogenic, thus making them inadequate vaccine antigens. One group of potential blood-stage vaccine antigens are the merozoite surface proteins (MSP) (3). MSP1, MSP2, and MSP3 have been used in human vaccine trials alone or in combination vaccines (4– 6) but demonstrate little protective efficacy (6). Similar results have been seen with other leading blood-stage proteins from the merozoite, such as AMA1 which has shown promising humoral immunogenicity (7, 8) but only modest protective efficacy (9). However, there have been very few trials assessing efficacy in humans, particularly in children for blood-stage vaccines and many more are needed. Vaccine development at this stage mainly focuses on generating a neutralizing antibody response to prevent erythrocyte invasion (similar to that seen with naturally acquired immunity) though inducing strong cellular responses may be just as important to promote protection. Therefore, it is important to identify the antigenic epitopes that are not only highly immunogenic but also protective.

MSP4 and MSP5 are candidates for blood-stage vaccines as they show limited antigenic diversity in both P. falciparum (10– 12) and P. vivax (13, 14). MSP5 is largely conserved across both these strains, and also geographic locations of Plasmodium (15), with polymorphisms detected only in specific gene regions (16). Naturally acquired antibodies have been detected to MSP4 (17, 18) and high antibody levels have been associated with a protective role in subsequent malaria seasons (18), though the precise cause of protection in these cohorts is yet to be fully defined. Similarly, naturally acquired antibodies against MSP4 are associated with reduced clinical malaria cases (19) and low level frequency of cross reactive antibodies have been detected to both P. falciparum and P. vivax (20). Though possibly attributed to anti-MSP antibody production, the exact correlates of protection in these studies are yet to be identified, although it does indicate these antigens are good candidates for inclusion in vaccines. Importantly, functional MSP5 specific T cell responses have also been observed to P. falciparum and P. vivax (21). The low antigen diversity of MSP4 and MSP5 and correlation with protection makes them attractive vaccine candidates compared to the large number of other highly polymorphic antigens. In animal studies, the murine equivalent homolog of MSP4 and MSP5, MSP4/5, has shown protection using different vaccine formulations (22–25), and protection is enhanced when administered in combination with MSP1 (26). Most of these studies examine antibodies in the protective response, with less known about the protective role of the cellular immune response. One study showed that the selected adjuvant AFCo1 (synthetic cochleate structures) enhanced both antibodies and T cell responses against MSP4/5, attributable to the induced Th1-like immune responses (27). Thus, it is important for vaccine design to not only aim to induce antibody responses, but also cell mediated immunity.

One possible reason for the low protective responses seen in recent malaria vaccine trials may be due in part to adjuvant or vaccine delivery platform selection. Optimal antigen and adjuvant combinations are imperative for vaccine efficacy. Alum is currently one of the only licensed adjuvants for widespread human use (28) and the RTS, S vaccine in humans contains the proprietary adjuvant AS01, consisting of liposomes, monophosphoryl lipid A (MPLA, the non toxic derivative of LPS) and the saponin QS-21 (29). Both these adjuvants, as well as most adjuvants in development for human use, are pro-inflammatory (inducing cytokines such as interleukin [IL-6] and/or tumor necrosis factor [TNF]) (30). Recent literature suggests that such pro-inflammatory adjuvants could also promote the subsequent expansion of regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), which may be a natural mechanism aimed to turn off excessive inflammation (31). This property may limit the persistence of immunity, as well as potentially increase the frequency of cells such as TNFR2+ Treg associated with the development of severe malaria (32). It is therefore useful to explore whether non-inflammatory adjuvants and vaccine carriers, which do not induce Treg or MDSC, can also induce malaria specific immunity of sufficient type and magnitude to protect against a malaria challenge.

Viral sized 40–50 nm polystyrene nanoparticles (PSNPs) are non-inflammatory and when used as adjuvanting antigen carriers in experimental vaccines, induce high levels of CD4+ and CD8+ T cells, as well as antibodies (33–37). Though there are many different types of nanoparticles currently being used for malaria vaccines (38, 39) [reviewed in Powles et al. (40)], PSNPs in this size range (and negatively charged) can target antigen presenting cells (APCs) in the local lymph nodes to increase uptake and presentation of the vaccine antigen, leading to long lasting protective immune responses in murine studies (33, 34, 36). These PSNPs have been shown to induce high level CD8+ T cell responses when covalently coupled with malaria liver stage peptide epitopes, however not when simply "mixing" the PSNPs with the peptide (31, 41). Moreover, they have been confirmed to be "inert" in terms of failing to activate inflammatory cytokine or MAPK/ERK mediated inflammatory pathways (37, 42) and do not induce TNFR2+ Treg or MDSC (31). These inert and biocompatible PSNPs have repeatedly shown to be safe and welltolerated in numerous animal models at both low and high doses (33, 36, 37, 43, 44), and can be coupled to different protein and peptide antigens with high efficiency and loading capacities (31, 37, 44). Additionally, recent pre-clinical studies progressing the translation of this approach as a cancer vaccine have seen no toxic or inflammatory effects when injecting similar PSNP doses in mice for up to 4 times at weekly intervals (44). Whilst the exact clearance mechanism of these PSNPs is unknown, it is known that nanoparticles in this viral size range allow their excretion from the body via hepatobiliary elimination and ultimately via feces (45).

Herein we assess both the immunogenicity and protective efficacy of blood-stage malaria vaccines in an animal model of malaria, formulated using PSNP antigen carriers using MSP4/5 as the target antigen and compared to formulations with MSP4/5 adjuvanted with Alum or the experimental adjuvant Complete Freund's Adjuvant (CFA).

#### MATERIALS AND METHODS

#### Mice

Six-to-eight-week-old BALB/c and IFN-γ gene knock out (KO) mice were purchased from the Austin Research Institute or Monash Animal Services. Austin Health and the Alfred Medical Research and Education Precinct (AMREP) Animal Ethics Committees approved the use of all animals and procedures.

#### Recombinant MSP4/5 Production

Recombinant MSP4/5 generated in E. coli, described in Kedzierski et al. (22), was kindly provided by Professor R. Coppel for use in these studies. The protein concentration of recombinant MSP4/5 was determined by Bicinchoninic Assay (BCA, Thermo Fisher) following manufacturer's instructions.

#### Conjugation of MSP4/5 to PSNPs

Conjugation of MSP4/5 to PSNPs was performed as described previously (35, 37). Briefly, 40–50 nm carboxylated polystyrene nanoparticles (PSNPs, Polysciences Inc, USA) at a final 1% solids (∼1.9 × 1014 PSNPs/ml, unless otherwise stated) were pre-activated using a 2-N-morpholino-ethanesulfonicacid buffered (MES; 50 mM final, pH 6.2) solution of 1-ethyl-3- (3-dimethylaminopropryl) carbodiimide hydrochloride (EDC; 4 mg/ml final) (Sigma Aldrich) (with sulfo-NHS for some conjugations) for 15 minutes on a rotating wheel at room temperature (RT). MSP4/5 was added (target final concentration of 0.4 or 1 mg/ml) and further incubated for 2–3 hours (h) at RT. The conjugation reaction was stopped by the addition of glycine (7 mg/ml final) for a further 30 minutes at RT. Conjugation mixture was dialyzed using 100–300 kDa dialysis membrane overnight against phosphate buffered saline (PBS) at 4◦C. Dialysis against 100–300 kDa cutoff membrane permits retention of the PSNPs conjugated to the antigen, and any remaining activating agents and unconjugated protein [MSP4/5 is ∼36kDa (24)] are dialyzed out. This process also allows for buffer exchange into a physiological solution, such as PBS, for immunizations. Conjugated PSNPs were stored at 4◦C and sonicated for 15 minutes before use to create a uniform and homogenous formulation for immunizations.

#### Vaccine Formulations and Immunizations

Mice were immunized intradermally (id) once or twice with the following vaccine formulations: 100 µl of either PSNPs conjugated to MSP4/5 or MSP4/5 with adjuvant, 2–3 weeks apart for immunogenicity studies. Adjuvanted vaccine formulations contained either Alum (Rehydragel HPA, General Chemical) at 0.3 g/ml with MSP4/5 in PBS, or a prime with MSP4/5 emulsified in CFA at a 1:1 v/v ratio and boost with Incomplete Freund's Adjuvant (IFA) at a 1:1 v/v ratio. Ten to fourteen days following the last immunization, mice were humanely sacrificed by CO2 asphyxiation. Conjugated and adjuvanted MSP4/5 protein doses, immunization volume and route were matched per experiment, with specific vaccine formulations provided in each of the figure legends. For challenge experiments, mice were injected with parasitized red blood cells (pRBCs) 3 weeks following the last immunization (see detailed procedure below).

### ELISpot

Splenocytes from immunized animals were isolated from 10 to 14 days following the last immunization and assessed by ELISpot for IFN-γ and IL-4 production. Ninety six well multiscreen plates (MAHA, MAIP or MSIP, Millipore) were coated overnight at 4◦C with 5µg/ml anti-mouse IFN-γ (AN18, Mabtech) or anti-mouse IL-4 (BVD4-1d11, Mabtech or BD Biosciences). All wells were washed 5 times with PBS and blocked with RPMI 1640 (Gibco, Life technologies) containing 10% FBS (further supplemented with 100 units/ml penicillin, 100µg/ml streptomycin, 4 mM Lglutamine, 1 M Hepes, and 0.1 mM 2- mercaptoethanol, to make complete media) for 2 h at 37◦C. Splenocytes were added at a final concentration of 0.5 × 106 or 1 × 106 cells per well and co-incubated with recall antigens (MSP4/5 at 15–25µg/ml final for each recall antigen) in complete media for 12–16 h at 37◦C. Media alone and positive control Concanavalin A (1µg/ml final) wells were also added. Following incubation, plates were washed with PBS (MSIP plates) or PBS containing 0.05% Tween 20 (MAHA and MAIP plates) and 1 µg/ml anti-mouse IFNγ-biotin (R4-6A2, Mabtech) or anti-mouse IL-4-biotin (BVD6- 24G2, Mabtech or BD Biosciences) in 0.5% FBS/PBS was added for 2 h at RT. Plates were washed and 1µg/ml Streptavidin-ALP or extravidin-ALP in 0.5% FBS/PBS was added for a further 1– 2 h at RT. Plates were given a final wash with PBS followed by reverse osmosis water and spots were developed using an AP colorimetric kit (Bio-Rad). Once plates were dry, spots were counted using an AID ELISpot Reader system (AutoImmune Diagnostika, GmbH, Germany).

#### ELISA

Sera from immunized animals was collected at endpoint and assessed for antigen specific antibody production by ELISA. Ninety six well plates (polyvinyl chloride micro-titer plates, Costar, or Nunc Maxisorp plates) were coated with 5µg/ml of MSP4/5 in carbonate/bicarbonate coating buffer overnight at 4 ◦C. Plates were washed with 0.05% Tween 20/PBS and blocked with 2% bovine serum albumin (BSA)/PBS or 5% skim milk/PBS and incubated for 1 h at 37◦C. Plates were washed as above and serial dilutions of sera added and incubated for 2 h at 37◦C (or 4 ◦C overnight). Plates were washed and horseradish peroxidase (HRP) conjugated anti-mouse total Ig or IgG antibodies (1/2000 dilution for total IgG/A/M, Zymed, and sheep anti-mouse IgG, Amersham Biosciences, UK) added for 1 h at 37◦C. Where indicated HRP conjugated subclass antibodies were used as the secondary antibody and incubated for 1 h at 37◦C (HRP-IgG1 at final 1/1,000, HRP-IgG2a at final 1/1,000 and IgG2bbiotin at final 1/250 dilution, BD Biosciences). Biotinylated secondary antibodies were followed with a further incubation of streptavidin-HRP (1/1,000, Amersham biosciences) for 1 h at 37◦C. Plates were given a final wash and antibody detection was developed using ABTS or TMB substrate solution before being stopped with 1 M HCl. Absorbance was read at optical density (OD) 405–450 nm on a plate reader (Fluostar Optima, BMG lab technologies, or Multiscan GO, Thermo Fisher). Antibody end titers were calculated closest to the serum dilution at which the OD was equal to the mean of the naïve sera (averaged across naïve mice for all dilutions tested) plus 3 standard deviations.

#### Malaria Infection Challenge

Plasmodium yoelii parasites were cultured in vivo in naïve mice and pRBCs harvested when mice reached a required level of parasitemia (∼4–5%). pRBCs were harvested from infected mice and experimental mice were infected with 5 × 104 or 5 × 105 pRBCs in PBS by intraperitoneal injection (total volume 100 µl, 50 µl either side of abdomen). Blood parasitemia levels were monitored every 1-2 days beginning from day 3 until ∼21–25 days post-infection. Blood parasitemia levels were calculated by venous blood smears that were Giemsa stained and counted using a light microscope. A representative field at 100X magnification was chosen and the number of pRBCs including all ring, merozoite, and schizont stages calculated compared to the total number of RBCs in the field. Mice were humanely euthanized when blood parasitemia levels reached >70%, according to ethical approval, and parasitemia levels and number of surviving mice (% survival) at each time point calculated.

#### Statistics

Statistical analysis was performed as follows; One or twoway ANOVA with post hoc Tukey analysis was used to assess differences between groups for immunogenicity (ELISpot and ELISA) assays. Kaplan Meyer survival curves with log-rank (Mantel-Cox) test comparisons were used for survival analysis. All statistical analyses were performed using Graphpad Prism software (v.7.02) and statistical significance was set at p < 0.05. Data is expressed as mean ± standard deviation (SD) for each group, with group sizes and additional information indicated in the relevant figure legends.

### RESULTS

#### Highly Immunogenic Vaccine Formulations of PSNPs Conjugated to MSP4/5 (PSNPs-MSP4/5) Induce Broad Immune Responses Including Th1 and Th2 Cells and Multiple Antibody Subclasses

Identifying an immunogenic and highly conserved protein for blood-stage malaria remains a challenge for vaccine development. MSP4 and MSP5 are promising blood-stage vaccine candidates in humans, as they are relatively conserved in different strains of Plasmodium compared to other highly polymorphic antigens. The murine equivalent P. yoelii MSP4/5 has been identified as a protective blood-stage antigen in prior murine studies (23, 25, 46), therefore, we wanted to assess this protein using our nanoparticle delivery system.

To assess the immunogenicity of PSNPs-MSP4/5 we compared this vaccine to one containing MSP4/5 mixed with the most widely used adjuvant, Alum. BALB/c mice were immunized twice intradermally at the base of tail and 14 days following the last immunization T cell (from whole splenocytes) and serum antibody responses were measured by ELISpot and ELISA, respectively. PSNPs-MSP4/5 immunization elicited high levels of antigen specific IFN-γ producing T cells, significantly higher than mice immunized with Alum and naïve controls (**Figure 1A**, p < 0.0001). Whilst Alum did not induce significantly higher IFN-γ producing T cells above control conditions, both the PSNPs-MSP4/5 and Alum + MSP4/5 groups induced similar levels of IL-4 producing T cells compared to naïve mice (**Figure 1B**, p < 0.001, p < 0.01, respectively). Alum is known to induce Th2 biased responses which is reflected in the above results. Furthermore, when looking at total antibody levels induced by vaccination, both Alum and PSNPs induced comparable levels of total Ig (**Figure 1C**) and similar levels of IgG1 antibodies (**Figure 1D**), the latter being classically associated with a Th2 response. Mice immunized with PSNPs-MSP4/5 developed higher IgG2a and IgG2b antibodies compared to either the Alum + MSP4/5 or the naïve groups but these responses were not significant (**Figures 1E, F**). These results indicate PSNPs induce a spectrum of IgG antibody subclasses as well as both IFN-γ-producing Th1 and IL-4-producing Th2 cells.

#### Comparable Immunogenicity of PSNPs-MSP4/5 Nanovaccines Against "Gold Standard" Experimental Vaccine Formulations of MSP4/5 in CFA/IFA

The immune responses generated by PSNPs-MSP4/5 immunizations were comparatively similar for Th2, and better for Th1 type responses, compared to the conventional adjuvant Alum. Therefore, we further tested the robustness of these responses against the strong experimental Th1 inducing adjuvant, CFA, followed by a boost with IFA, and compared against one or two immunizations with PSNPs-MSP4/5. MSP4/5 in CFA/IFA induced functional IFN-γ and IL-4 producing T cells, as well as IgG antibodies, significantly higher than naïve mice (**Figures 2A–C**, p < 0.0001, p < 0.001, p < 0.01, respectively). One immunization with PSNPs-MSP4/5 was comparable to MSP4/5 in CFA/IFA for both IFN-γ and IL-4 producing T cell immune responses (**Figures 2A,B**), and significantly higher than naïve responses (**Figures 2A,B,** p < 0.0001). Two immunizations with PSNPs-MSP4/5 elicited the highest levels of IFN-γ and IL-4 T cell responses, significantly higher than all other groups for IFN-γ producing T cells (**Figure 2A**, p < 0.0001 compared to naïve, p < 0.001 compared to CFA/IFA and p < 0.01 compared to one immunization with PSNPs-MSP4/5) and significantly higher than MSP4/5 in CFA/IFA and naïve groups for IL-4 producing T cell responses (**Figure 2B**, p < 0.0001 compared to naïve and p < 0.01 compared to CFA/ IFA). Antibody titers were also different when comparing one vs. two immunizations with PSNPs-MSP4/5. Two immunizations of PSNPs-MSP4/5 elicited

higher antibody titers compared to one immunization with PSNPs-MSP4/5 (**Figure 2C**, not significant) and comparable antibody titers to MSP4/5 in CFA/IFA (**Figure 2C**). MSP4/5 in CFA/IFA induced a significantly higher antibody end titre compared to only one immunization of PSNPs-MSP4/5 (**Figure 2C**, p < 0.05).

Immunization with PSNPs-MSP4/5 showed strong T cell and antibody responses, especially following a two-immunization regime, therefore, we next determined the effect of PSNP dose on the resulting immune response. De-escalating doses of PSNPs (2.1 × 1014, 3.7 × 1013, and 1.7 × 1013 PSNP/ml) conjugated to matched levels of MSP4/5 were used to assess the potential for dose-sparing of the vaccine preparation, for both immunogenicity and protection studies. As IFN-γ has been identified as the key protective cytokine (47), the effect of different doses of PSNPs (conjugated to the same amount of MSP4/5) were assessed for their ability to affect the resulting IFN-γ T cell response. De-escalating doses of the PSNPs were compared to CFA/IFA adjuvated formulations for IFN-γ T cell responses following two immunizations. As

per group.

expected, MSP4/5 in CFA/IFA produced significantly higher T cell responses compared to controls (**Figure 3**, p < 0.05 compared to MSP4/5 alone and naïve). The three doses of PSNPs-MSP4/5 elicited differing IFN-γ T cell responses. Both the highest and lowest PSNP doses (2.1 × 1014 and 1.7 × 1013 PSNPs/ml-MSP4/5) induced IFN-γ producing T cell responses comparable to CFA/IFA adjuvanted vaccines and both significantly higher than control groups (**Figure 3**, p < 0.0001 2.1 × 1014 PSNPs-MSP4/5 compared to MSP4/5 alone, p < 0.001 compared to naïve, p < 0.001 1.7 × 1013 PSNPs-MSP4/5 compared to MSP4/5 alone, p < 0.01, compared to naïve). Unexpectedly, both the highest and lowest PSNP doses also induced significantly higher IFN-γ T cell responses compared to the 3.7 × 1013 PSNPs-MSP4/5 dose (**Figure 3**, p < 0.01 and p < 0.05, respectively), indicating no clear titratable effect of the PSNPs dose.

### Immunization With PSNPs-MSP4/5 Induces Survival Against Blood-Stage Malaria Infection

Not only is it important to identify vaccine formulations that elicit strong T cell and antibody responses, they also need to translate into a clinical effect of blood-stage protection and/or enhanced survival. To investigate this, mice were immunized twice, 3 weeks apart with de-escalating doses of PSNPs-MSP4/5 and MSP4/5 in CFA/IFA. Two weeks following immunizations, mice were then challenged with P. yoelii parasites and monitored for parasitemia levels and overall survival. Mice reached end point when blood smears showed over 70% parasitemia levels. All groups survived significantly longer than naïve mice (**Figure 4A**, p < 0.001 for MSP4/5 in CFA/IFA and p < 0.01 for all doses PSNPs-MSP4/5, compared to naive mice) as shown on the Kaplan-Meyer survival curve. Despite observing different survival curve patterns, there were no significant differences between MSP4/5 in CFA/IFA and the three doses of PSNPs-MSP4/5 up until 21 days postchallenge, indicating these are comparably protective vaccine formulations. In terms of overall survival, the control naïve mice all reached endpoint parasitemia's (**Figure 4B**). All mice in the MSP3/5 in CFA/IFA group resolved their parasitemia levels 6/6 (**Figure 4C**), in comparison to 5/6 mice in both the 3.7 × 1013 and 1.7 × 1013 PSNPs-MSP4/5 groups (**Figures 4E,F**), and 3/6 mice in the 2.1 × 1014 PSNP-MSP4/5 group resolved their parasitemia levels (**Figure 4D**). This suggests the observed partial protection is not dependent on PSNP dose, regardless of significant differences in immune responses induced.

#### Moderate Blood-Stage Malaria Protection Induced by PSNPs-MSP4/5 Is IFN-γ dependent

In the above studies, high levels of IFN-γ and IL-4 producing T cells were induced by PSNPs-MSP4/5 vaccine formulations, as well as antibody responses including diverse IgG subclasses. Generally, functional antibodies have been associated with protective responses in humans for blood-stage malaria, with few studies characterizing the T cell response (48, 49). IFN-γ has been reported to be protective in prior P. yoelii liver stage murine malaria models (50), and P. chabaudi blood-stage infections (51), therefore, we wanted to assess mechanistically the necessity of IFN-γ production in protecting mice from bloodstage malaria immunized with PSNPs-MSP4/5. Wild type (WT) or IFN-γ KO (IFN-γ KO) BALB/c mice were immunized twice, intradermally, 2 weeks apart, with PSNPs-MSP4/5 and 2 weeks following the last immunization, mice were challenged with P. yoelii parasites and monitored for survival and parasitemia levels. As expected, naïve WT mice reached parasitemias over 70% (**Figures 5A,B**). PSNPs-MSP4/5 immunized WT mice were significantly protected in comparison to naive WT mice (**Figure 5A**, p < 0.01), with 3/6 mice protected up to 25 days postinfection and resolving their parasitemia levels (**Figures 5A,C**). Both the naïve and PSNPs-MSP4/5 experimental groups reached parasitemias above 70% in the IFN-γ KO mice (**Figures 5A,D,E**). There was a significant difference in survival between the PSNPs-MSP4/5 immunized WT mice compared to PSNPs-MSP4/5 immunized IFN-γ KO mice (**Figure 5A**, p < 0.01) suggesting protection may be IFN-γ dependent. There was also a significant difference between the immunized WT mice compared to naive WT mice (**Figure 5A**, p< 0.01), but no significant difference between the immunized and naïve IFN-γ KO mice. Some naive IFN-γ KO mice survived for marginally longer than PSNPs-MSP4/5 immunized mice, though this result was not significant.

Furthermore, antibody responses pre-challenge demonstrated high levels of IgG1 antibodies in PSNPs-MSP4/5 immunized mice in both the WT and IFN-γ KO mice, with both inducing antibody titres significantly higher than WT naïve mice and IFN-γ KO naïve mice (**Figure 5F**, p < 0.0001). PSNPs-MSP4/5 immunized WT mice also induced significantly higher IgG1 antibody levels compared to PSNPs-MSP4/5 immunized IFNγ KO mice (**Figure 5F**, p < 0.05). IgG2a and IgG2b induction was impaired in IFN-γ KO mice but not in WT mice when immunized with PSNPs-MSP4/5. IgG2a antibody levels elicited by PSNPs-MSP4/5 in WT mice were significantly higher than WT naïve mice and both IFN-γ KO naïve and immunized mice (**Figure 5G**, p < 0.001). IgG2b antibodies were also significantly higher in the WT PSNPs-MSP4/5 immunized mice compared to WT naïve and IFN-γ KO naïve mice (**Figure 5H**, p < 0.001) and PSNPs-MSP4/5 immunized IFN-γ KO mice (**Figure 5H**, p < 0.01). These results may be explained when considering IgG1 antibodies are induced by IL-4 and associated with Th2 responses and IgG2 antibodies are associated with Th1 responses a and dependent on IFN-γ.

### DISCUSSION

The present study shows immune responses induced by a biocompatible nanoparticle carrier vaccine approach can induce immunity and moderate protection against blood-stage malaria comparable to the "gold standard" experimental adjuvant CFA. This finding adds to the repertoire of possible adjuvants and vaccine approaches in development, by showing that delivering a classical pro-inflammatory danger signal is not necessary to induce immune responses of sufficient magnitude, or of the type, necessary to protect against blood-stage malaria infection. We note, however, that associations of immune responses with protection do not necessarily imply causality. Nonetheless, the above results support the MSP4 and MSP5 families as candidate antigens for vaccine development when used with an appropriate delivery system.

PSNPs have repeatedly shown, in a range of disease models, to be non-inflammatory and capable of inducing robust immune responses without the addition of T helper epitopes or additional stimuli (31, 37, 42, 44, 52). Side by side comparisons of immunogenicity elicited by PSNPs-MSP4/5 showed them to be capable of producing superior cellular responses to MSP4/5 in Alum, inducing balanced Th1 and Th2 responses, as well as comparable responses to MSP4/5 in CFA/IFA vaccine formulations, the latter representing a gold standard level of immune responses in experimental models. Similar results were obtained with MSP4/5 with an alternative production system in Saccharomyces cerevisiae (unpublished).

Mantel Cox Log Rank Test. † Indicates mouse endpoint. Data shown are from 6 mice per experimental group.

Antigen specific IFN-γ production by T cells was predominantly measured in the present study, though as whole splenocytes were used in the assays there is a possibility that IFN-γ production from other cell types (i.e. NK cells) may also be measured, but unlikely to this protein antigen. The cells that respond in ELISpot assays are predominantly T cells, as shown by depletion assays (53–55). Dose-sparing of the PSNP formulations unexpectedly showed that the middle dose induced significantly lower IFN-γ producing T cell responses, compared to the highest and lowest PSNP doses. Therefore, it will be useful in future studies to explore the dose dependency of other types of immune responses.

PSNPs-MSP4/5 formulations showed substantial improvement over MSP4/5 in Alum formulations in the elicitation of specific antibody subclasses such as IgG2a and IgG2b, but not total Ig or IgG1. Although the antibody titers induced by PSNPs-MSP4/5 vaccines were lower than the "gold standard" MSP4/5 in CFA/IFA, PSNPs-MSP4/5 vaccines

immunized twice intradermally at the base of tail (100 µl/immunization), 14 days apart with PBS alone (Naïve) or PSNPs-MSP4/5 (100µg/ml MSP4/5). Fourteen days following the boost immunization, mice were challenged intraperitoneally with 5 × 104 pRBCs from P. yoelii and survival parasitemia levels were monitored starting at 3 days post-challenge. (A) Kaplan-Meier survival curve and parasitemia levels of mice immunized with (B) WT Naïve -•-, (C) WT PSNPs-MSP4/5 -H-, (D) IFNγ KO Naïve - - and (E) IFNγ KO PSNPs-MSP4/5 -▽-. Pre-challenge (F) IgG1, (G) IgG2a, and (H) IgG2b anti-MSP4/5 antibody levels are also shown as mean antibody titer ± SD per group. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 with Mantel Cox Log Rank Test for survival analysis and one-way ANOVA for antibody titer. † Indicates mouse endpoint. Data shown are from 5−6 mice per experimental group.

induced moderate protective immunity compared to CFA/IFA adjuvanted formulations. Though only CFA/IFA adjuvanted formulations achieved complete protection, it has been noted in this model that is difficult to observe complete protection (22, 24, 46). Surprisingly, there were no significant differences in the protection observed between the different PSNP doses, however there was a slightly lower survival rate in the group immunized with the highest dose of PSNPs. Overall, the partial protection observed with PSNPs in this model shows promise for current and future nanoparticle delivery platforms for blood-stage malaria.

Studying the mechanism of the protective immunity against malaria induced by PSNPs-MSP4/5, using knockout mice, demonstrated protection to be IFN-γ dependent. This is consistent with reports in the literature suggesting IFN-γ is the critical mediator of protection in erythrocytic stage infections (47), and for other strains of blood-stage Plasmodium infection (51), though the exact mechanism of protection has yet to be defined. Our results show a change in IgG subclasses elicited by the vaccine, with sustained IgG1 (Th2 associated) production but significantly decreased anti-MSP4/5 levels of IgG2a and IgG2b (Th1 associated) compared to non-immunized controls. These results indicate that the induction of IgG1 alone is not protective in this model (56), as the IFN-γ KO mice are still able to produce IgG1 but are not protected. Previous studies have shown that high levels of both IgG1 and IgG2a are required to mediate protection in the P. yoelii model (57, 58) however both Hirunpetcharat et al. (59) and Oakley et al. (60) showed that IgG1 alone correlates with protection against P. yoelii challenge (59, 60). In contrast, Adame-Gallegos et al. (61) showed that IgG1 did not mediate protection against P. yoelii (61).

One interesting finding in the knock out experiment was that the IFN-γ KO naïve mice had slightly better survival that the immunized KO mice (though this was not significant). Whilst it is possible that this is due to the potency of the parasites themselves, it may also be due to the differences in vaccine induced responses. As these mice do not have any vaccine induced responses, compared with the PSNPs vaccinated mice, there may be other mechanisms compensating for IFN-γ and contributing to the marginally increased survival. A potential cell type that has been identified as having a key role in clearing early parasitemia's are γδ T cells (62). It is possible that without the expansion of αβ T cells as would be expected in vaccinated animals, γδ T cells may expand instead, as well as there being a potential difference in cytokine profiles, especially from innate cells (63). Though it cannot be proven for the present study, the above hypothesis would be of interest to investigate further in this model in future studies. Indeed, in future studies it would be preferable to explore other immune responses induced by these PSNPs vaccines in the context of blood-stage malaria, including investigating other cytokines (i.e., TNF, IL-5, IL-17) in addition to the hallmark Th1 and Th2 cytokines IFN-γ and IL-4, respectively. Likewise, it would be beneficial to examine immune cell types such as B cells, γδ T cells, Th17, Tfh, Tr1, and T regulatory cells, as well as individual subsets within these populations to explore the different effector functions and cytokine profiles within cell subsets.

Plasmodium falciparum derived MSP4 and MSP5 are closely related to the MSP4/5 P. yoelli antigen used in this murine model of malaria. Their attractiveness as an antigen for target development came from the finding that they, and particularly MSP5, are largely conserved across malaria species, and show little polymorphism when analyzing within any single species of malaria parasites (10, 15). However, enthusiasm declined when it was shown that naturally found antibodies against MSP4 or MSP5 had little neutralizing capacity in in vitro red cell parasite growth inhibitory assays (GIA). Other MSP antigens were also shown to be associated with reduced incidence of malaria (64), and have been more widely studied. Although recent studies have shown association of both MSP4 and MSP5 antigens with decreased cases of malaria, specifically severe malaria (18, 19). The present study reinforces the contention that it is possible to deliver vaccine induced protection against malaria blood-stage, focusing on the induction of robust Th1 immunity, and not necessarily particular antibody functions. Of note is the fact that >60% humans naturally exposed to either acute P. falciparum (63%) or P. vivax (67%) malaria infection show IFN-γ responses to PfMSP5, suggesting it is (1) immunogenic in humans, (2) has the potential to be boosted by natural infection, and (3) is potentially a cross-reactive antigen between the two main stains of malaria found in many malaria endemic regions of the globe, including Asia (20). In agreement with previous studies, the current study suggests it is possible to design vaccines capable of inducing such potentially useful T cell responses. Moreover, we show it is possible to do so using carriers and adjuvants that do not need to deliver classical pro-inflammatory signals, a potentially important consideration when vaccinating infants in malaria endemic areas. Given our positive findings with PSNPs-MSP4/5, this supports the usefulness of further exploring other diverse nanoparticles as they are developed, as carriers for MSP antigens, beyond PSNPs. Conventional adjuvants carry the risk of damaging inflammatory responses, as well as data emerging from the literature suggesting that non-live adjuvanted vaccines may be directly associated with unhelpful non-specific effects (65). Overall, we hope the present study will spur further research into non-inflammatory vaccine carriers and adjuvants, as well as the potential for Th1 associated vaccine induced immunity in blood-stage malaria, which may help us re-examine from an additional perspective the nature of the antigens for inclusion in malaria vaccines.

### AUTHOR CONTRIBUTIONS

MP designed and supervised all experiments and analyzed and interpreted all the data. KW designed and performed some of the experiments and analyzed some of the data. DP designed and performed some of the experiments and analyzed some of the data. JH and CM performed some of the experiments. SX designed and supervised some of the experiments and analyzed some of the data. RC planned some of the experiments. MP, KW, and DP wrote the manuscript and all authors contributed segments and editing to the manuscript preparation.

#### Wilson et al. Blood-Stage Protection by Synthetic Nanoparticles

#### REFERENCES

surface protein 5 with protection against clinical malaria. PLoS ONE. (2014) 9:e101737. doi: 10.1371/journal.pone.0101737

size: formulation of a model vaccine for respiratory syncytial virus. Mol Pharm. (2007) 4:73–84. doi: 10.1021/mp060096p

virus antigens. Blood. (2000) 95:241–8. Available online at: http://www. bloodjournal.org

**Conflict of Interest Statement:** Some of the experiments in Figures 3 and 4 were funded by Panvax Pty Ltd. JH worked for Panvax during this period. MP was a Director of Panvax 2001-2007 and PX Biosolutions Pty Ltd 2007-2016.

The remaining 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.

Copyright © 2019 Wilson, Pouniotis, Hanley, Xiang, Ma, Coppel and Plebanski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Combination of RTS,S and Pfs25-IMX313 Induces a Functional Antibody Response Against Malaria Infection and Transmission in Mice

Florian Brod1 \*, Kazutoyo Miura2 , Iona Taylor 1 , Yuanyuan Li 1 , Arianna Marini 1 , Ahmed M. Salman1 , Alexandra J. Spencer 1 , Carole A. Long2 and Sumi Biswas 1 \*

#### Edited by:

Julius Clemence Hafalla, London School of Hygiene and Tropical Medicine, United Kingdom

#### Reviewed by:

Urszula Krzych, Walter Reed Army Institute of Research, United States Olivier Silvie, Institut National de la Santé et de la Recherche Médicale (INSERM), France

#### \*Correspondence:

Florian Brod<EMAIL_ADDRESS>Sumi Biswas<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology

> Received: 31 August 2018 Accepted: 12 November 2018 Published: 04 December 2018

#### Citation:

Brod F, Miura K, Taylor I, Li Y, Marini A, Salman AM, Spencer AJ, Long CA and Biswas S (2018) Combination of RTS,S and Pfs25-IMX313 Induces a Functional Antibody Response Against Malaria Infection and Transmission in Mice. Front. Immunol. 9:2780. doi: 10.3389/fimmu.2018.02780 1 Jenner Institute, University of Oxford, Oxford, United Kingdom, 2 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, United States

The last two decades saw a dramatic reduction in malaria incidence rates, but this decrease has been stalling recently, indicating control measures are starting to fail. An effective vaccine, particularly one with a marked effect on disease transmission, would undoubtedly be an invaluable tool for efforts to control and eliminate malaria. RTS,S/AS01, the most advanced malaria vaccine to date, targets the parasite before it invades the liver and has the potential to prevent malaria disease as well as transmission by preventing blood stage infection and therefore gametocytogenesis. Unfortunately efficacy in a phase III clinical trial was limited and it is widely believed that a malaria vaccine needed to contain multiple antigens from different life-cycle stages to have a realistic chance of success. A recent study in mice has shown that partially efficacious interventions targeting the pre-erythrocytic and the sexual lifecycle stage synergise in eliminating malaria from a population over multiple generations. Hence, the combination of RTS,S/AS01 with a transmission blocking vaccine (TBV) is highly appealing as a pragmatic and powerful way to increase vaccine efficacy. Here we demonstrate that combining Pfs25-IMX313, one of the TBV candidates currently in clinical development, with RTS,S/AS01 readily induces a functional immune response against both antigens in outbred CD1 mice. Formulation of Pfs25-IMX313 in AS01 significantly increased antibody titres when compared to formulation in Alhydrogel, resulting in improved transmission reducing activity in standard membrane feeding assays (SMFA). Upon co-formulation of Pfs25-IMX313 with RTS,S/AS01, the immunogenicity of both vaccines was maintained, and functional assessment of the induced antibody response by SMFA and inhibition of sporozoite invasion assay (ISI) showed no reduction in biological activity against parasites of both lifecycle stages. Should this findings be translatable to human vaccination this could greatly aid efforts to eliminate and eventually eradicate malaria.

Keywords: malaria, vaccine, transmission blocking, pre-erythrocytic, synergy

## INTRODUCTION

Malaria is caused by parasites of the genus Plasmodium, and it is one of the world's oldest and deadliest diseases. According to estimates in the World Health Organisation (WHO) world malaria report 2017 (1) the global number of malaria deaths reached 445,000 in 2016, and there were a total of 216 million malaria cases, an increase of 5 million cases over 2015. This is the first time since 2010 that case numbers increased with respect to the estimates of the previous year indicating that current control measures, which helped to drastically reduce malaria prevalence over the last decade are starting to fail and novel interventions are necessary if malaria eradication is to be achieved (2).

Vaccines are undoubtedly one of the most successful public heath interventions of all time, and played an essential role in the control or eradication of a number of diseases. An efficacious vaccine would be of immense value to all efforts to contain and eventually eradicate malaria. As outlined in the malaria vaccine technology (MVT) roadmap, as well as the malERA research agenda for malaria eradication (3, 4), an ideal malaria vaccine would have two characteristics; it would protect a vaccinated individual from illness as well as have an impact on malaria transmission (5). Plasmodium parasites have an exquisitely complex lifecycle with obligate developmental steps in the human host as well as in the mosquito vector. Roughly, it can be broken down in three different stages, the preerythrocytic stage, the erythrocytic stage or asexual blood stage and the sexual or sporogonic stage. Vaccines with an impact on malaria transmission are often referred to as VIMTs (vaccines that interrupt malaria transmission), and include classic TBVs that target the sexual life-cycle stage of the parasite in the mosquito vector as well as pre-erythrocytic vaccines (PEVs) that prevent blood stage infection and therefore gametocytogenesis. The impact of vaccines targeting the asexual blood stage, which also is the target of naturally acquired immunity, is likely to be more limited. The most advanced malaria vaccine candidate to date RTS,S/AS01 targets the pre-erythrocytic stage antigen circumsporozoite protein (CSP) of P. falciparum, the causative agent of the most severe form of human malaria, with the aim to interrupt the parasite's lifecycle before the establishment of liver stage infection. It is the only malaria vaccine to have completed Phase III clinical trials (6–9) and to have obtained a positive scientific opinion from the European Medicines Agency (EMA) (10). As RTS,S/AS01 targets a stage in the lifecycle before the emergence of symptomatic and transmissible stages, it has the potential to prevent disease as well as transmission. However, efficacy of RTS,S/AS01 in the recently concluded Phase III clinical trial, stayed below the targets formulated in the MVT roadmap (8, 9).

One way to build on the substantial efforts that led to development of RTS,S/AS01 is to combine it with other malaria vaccines currently in development, and indeed consensus is now that a vaccine containing multiple antigens from different life-cycle stages has the most realistic chance of success (11, 12). A recent study using two monoclonal antibodies targeting CSP and the sexual stage antigen Pfs25 at partially efficacious concentrations showed that pre-erythrocytic and sexual stage interventions synergise over multiple generations as the efficacy of PEVs is sensitively dependent on the sporozoite load of an infectious mosquito and TBVs can reduce the number of oocysts that develop in the mosquito midgut and therefore reduce the number of sporozoites that reach the mosquito's salivary gland (13). Therefore, combination of RTS,S/AS01 with a TBV, targeting the sexual stage of the parasite, is particularly attractive. Furthermore, addition of RTS,S/AS01 to a TBV could overcome one of the perceived drawbacks of TBVs, which is the absence of a direct protective effect on the vaccinee, while addition of a TBV to RTS,S/AS01 could increase the effect RTS,S has on malaria transmission, as well as reduce the emergence of escape mutants.

The most advanced candidate antigen for a transmission blocking malaria vaccine is Pfs25. It has been extensively tested in mouse models where immunization with Pfs25 containing vaccines can induce an antibody response that completely blocks parasite transmission to mosquitoes (14). Pfs25, as well as its P. vivax ortholog Pvs25, has been tested in clinical trials (15, 16) making it, alongside Pfs230, one of only two P. falciparum transmission blocking antigens to be tested in humans. This showed that antibodies against Pfs25 reduce malaria transmission in SMFA (15, 17) and that the transmission reducing activity (TRA) is correlated with the induced antibody titres. Unfortunately repeated immunizations resulted in above 50% TRA in only 9 out of 11 volunteers, and this was reduced to 2 out of 11 6 weeks later (17). Similar observations were made in early clinical trials with vaccines targeting CSP, where limited vaccine efficacy correlated with a moderate antibody response. This triggered the research and evaluation of improved vaccine candidates which eventually led to the development of RTS,S/AS01 (18–20). Consequently a number of strategies have been applied to increase the antibody response induced by vaccines targeting Pfs25 (14). These included conjugating Pfs25 to the exoprotein A of Pseudomonas aeruginosa (EPA), which substantially increase antibody titres against Pfs25 in mice (21), but clinical trials using Pfs25-EPA formulated in Allhydrogel found only limited and short lived serum transmission blocking activity in vacinees (16, 17). The shortcoming of Pfs25-EPA/Allhydrogel in clinical trials suggests that further antigen/adjuvant combinations need to be assessed to improve TRA of vaccines targeting Pfs25. One alternative antigen, currently under clinical development, is generated by multimerisation of Pfs25 by fusion to the nanoparticle platform IMX313 (Pfs25-IMX313). This resulted in increased antibody titres and TRA in mice after immunization, either when encoded in viral vectors or as protein in adjuvant formulations (22). Furthermore, RTS,S, the antigenic component of the RTS,S/AS01 vaccine, is delivered with a highly potent adjuvant (AS01) specifically developed to induce very high antibody responses. In this study we show that formulation of Pfs25-IMX313 with AS01 increases antibody responses against Pfs25 in mice. We also show that RTS,S and Pfs25-IMX313 can be combined without any immunological interference in mice, maintaining the level of antibody immunogenicity and functional activity induced by the individual vaccines. This is the first proof of concept of the compatibility of combining RTS,S with a TBV, which could be of great value, particularly in local malaria eradication campaigns.

### RESULTS

### AS01 Increases Induced Antibody Titres After Immunization With Pfs25-IMX313

Pfs25-IMX313 was purified and expressed as previously described (22), formulated in adjuvant and administered to mice, to assess whether the antibody response induced after vaccination with Pfs25-IMX313 could be improved further by using AS01 instead of Alhydrogel as the adjuvant. Three doses of Pfs25-IMX313 were tested in order to measure if there was a dose sparing effect. Ten CD-1 mice per group were immunized on day 0 and day 28 (**Figure 1A**). Blood samples were collected at day 27 and day 42 and anti-Pfs25 IgG titres assessed by ELISA (**Figures 1B,C**). On day 27 after primary immunization, only vaccine formulations containing 4 µg of Pfs25-IMX313 induced a detectable antibody response in all mice. The next lower dose, 0.4 µg of Pfs25-IMX313 in AS01 induced a detectable immune response in 9 out of 10 mice, but the induced antibody titres were significantly lower than those induced by 4 µg of Pfs25-IMX313 in AS01 (p = 0.0118, Dunn's multiple comparison test). The lowest dose tested, 0.04 µg of Pfs25-IMX313 in AS01, induced a detectable anti-Pfs25 IgG response only in 1 out of 10 mice, and the induced antibody titres were significantly lower than those induced by both formulations containing 4 µg of Pfs25-IMX313 (4 µg of Pfs25-IMX313 in AS01 p < 0.0001, 4 µg of Pfs25-IMX313 in Alhydrogel p = 0.0006, Dunn's multiple comparison test).

After boosting, all vaccine formulations induced a detectable immune response in all mice on day 42, while no detectable immune response was induced in the mice immunized with PBS/AS01. The two highest doses of Pfs25-IMX313 in AS01 induced the highest anti-Pfs25 IgG titres. 4 µg of Pfs25-IMX313 in Alhydrogel induced significantly lower titres than the same vaccine dose in AS01 (p = 0.0014). Titres induced by 0.04 µg of Pfs25-IMX313 in AS01 were significantly lower than those induced by the two higher doses in the same adjuvant (4 µg of Pfs25-IMX313 in AS01 p < 0.0001, 0.4 µg of Pfs25-IMX313 in AS01 p = 0.004, Dunn's multiple comparison test), but not than those induced by the 100-fold higher dose of 4 µg of Pfs25- IMX313 in Alhydrogel. Formulation of Pfs25-IMX313 in AS01 therefore clearly increases the vaccine's immunogenicity, but while there is a dose sparing effect when compared to formulation in Alhydrogel, the highest antibody titres are achieved by immunization with the highest tested dose.

#### Antibodies Induced by Pfs25-IMX313/AS01 Are Functionally Active

Functional activity of the induced antibodies after the second immunization was assessed by SMFA. Live mosquitoes were fed with a mixture of P. falciparum gametocyte-infected blood and purified IgG from each group, and the number of oocysts which developed in the midgut was determined after dissection (**Figure 2**). Functional efficacy was calculated as transmission reducing activity (TRA), the reduction in the number of oocysts compared to the negative control group. In a first feed, total IgG concentrations of 750, 150, and 30µg/ml were tested. All groups apart from the one that received 0.04 µg Pfs25-IMX313/AS01 induced statistically significant TRA when compared to the negative control at the two highest IgG concentrations, but only 4 µg Pfs25-IMX313/AS01 induced statistically significant TRA at 30µg/ml. To further dissect differences between groups at lower IgG concentrations a second feed was performed using 250, 83.3, and 27.8µg/ml IgG. This confirmed results from the first feed with only 4 µg Pfs25-IMX313/AS01 inducing statistically significant TRA at all three IgG concentrations, suggesting the high antibody titres observed in this group correlate with improved TRA.

### Adjuvant and Vaccine Dose do Not Affect Antibody Quality

As a means to assess whether differences in TRA between groups were caused by a qualitative or a quantitative difference in the antibody response, the SMFA results were correlated with the amount of Pfs25 specific IgG in the feeder (**Figure 2C**). Datasets for 4 µg Pfs25-IMX313/Alhydrogel, 4 µg Pfs25- IMX313/AS01 and 0.4 µg Pfs25-IMX313/AS01 were analyzed by linear regression. The 0.04 µg Pfs25-IMX313/AS01 group was excluded from the analyses due to the absence of any statistically significant TRA in this group. To determine the transmission reducing efficacy per anti-Pfs25 AU, anti-Pfs25 AU and vaccine groups were used as explanatory variables in a multiple linear regression analysis. The overall fit to the linear regression model was R 2 = 0.78, and anti-Pfs25 AU (p < 0.001) but not vaccine groups (p = 0.1625) showed a significant effect. TRA in all three groups is therefore dependent on the amount of IgG used in SMFA but not on the dose or adjuvant the vaccine was administered in, suggesting that there is no qualitative difference between the antibody responses induced by the different vaccine regimens. This, as well as the differences in the induced antibody titres measured by ELISA (**Figure 1C**), suggests that the difference in induced TRA is due to an quantitative, not a qualitative difference in the antibody response. To confirm this, a similar linear regression analysis as before was performed using the reciprocal dilution factor of the serum pool as one of the explanatory variables, instead of anti-Pfs25 AU (**Figure 2D**). This allows an indirect analysis of the antibody response's efficacy in the undiluted mouse serum from each group, therefore providing a combined assessment of antibody quantity and quality. The overall fit to the linear regression model was R 2 = 0.68 and the serum dilution factor (p < 0.001) as well as the vaccine group (p = 0.0001) showed significant effects. When three groups were compared, 4 µg Pfs25-IMX313/Alhydrogel and 0.4 µg Pfs25-IMX313/AS01, which had both induced a similar antibody titres also appeared to have induced a similarly efficacious response (p = 0.0924), while both of these vaccine formulations induced a significantly less efficacious antibody response than 4 µg Pfs25-IMX313/AS01 (p < 0.001 and p = 0.0026, respectively), which had induced a significantly higher antibody titer. This analysis therefore confirms the observation from the antibody quality analysis, that the observed difference in SMFA and the superior performance of the 4 µg Pfs25- IMX313/AS01 vaccine formulation is due to a quantitative but not a qualitative difference in the induced antibody response.

### Pfs25-IMX313 can be Combined With RTS,S Without Immunological Interference

Having established that AS01 increases the immunogenicity and efficacy of Pfs25-IMX313 as well as determined an optimal dose, we assessed whether Pfs25-IMX313 could be combined with RTS,S/AS01 as a multi-antigen, multi-stage malaria vaccine. Ten CD1 mice per group were immunized on day 0 and day 28 (**Figure 3A**). The mixed vaccine group was immunized with RTS,S/AS01 and Pfs25-IMX313 combined into a single injection, while the co-administered group received both vaccines separately into the left and the right hind leg, respectively. In the latter group, each injection was formulated with only half a dose of AS01, to keep the total amount of administered adjuvant constant. Blood samples were collected at day 27 and day 42 and anti-Pfs25 and anti-CSP IgG titres were assessed by ELISA (**Figure 3**). All Pfs25- IMX313 containing vaccine formulations induced strong anti-Pfs25 antibody responses and all RTS,S containing vaccine formulations induced strong anti-CSP antibody responses. Mixing or co-administration of the two vaccines did not result in any significant reduction in antibody titres when compared to the groups that had received Pfs25-IMX313/AS01 or RTS,S/AS01 alone, suggesting that there is no immune competition between the two vaccines.

### Combination of Pfs25-IMX313 With RTS,S Does not Reduce the Functional Activity of Anti-Pfs25 IgG

Functional activity of anti-Pfs25 IgG was assessed by SMFA (**Figure 4**). In a first feed, IgGs from all Pfs25-IMX313 containing groups were tested at 750, 250, and 83.3µg/ml, groups that had received RTS,S/AS01 or PBS/AS01 were tested at 750µg/ml only. Significant TRA was observed for all groups that received Pfs25-IMX313 at all the tested concentrations. Immunization with RTS,S/AS01 had no statistically significant effect on oocyst intensity. In a second feed we therefore tested lower IgG concentrations (250, 83.3, and 27.8µg/ml) to assess for differences between groups at these lower concentrations. As

FIGURE 2 | Transmission blocking activity of anti Pfs25-IgG induced by immunization with Pfs25-IMX313 in Alhydrogel or AS01. (A,B) Total IgG, purified from pooled serum of each group (2 weeks post boost) was tested in SMFA in two independent experiments, one using total IgG concentrations of 750, 150 and 30µg/ml (A) and one using total IgG concentrations of 250, 83.3, and 27.7µg/ml (B). The transmission blocking monoclonal anti-Pfs25 antibody 4B7 was used as a positive control. Data

(Continued)

FIGURE 2 | points represent the number of oocysts in individual mosquitoes and the lines show the arithmetic mean. X-axis values are µg/ml total IgG in the assay. The results of the two feeds are summarized in the tables. a IgG concentration (µg/ml) in feeder. bArithmetic mean of oocyst intensity from 20 mosquitoes. cPercent inhibition of mean oocyst intensity and the 95% confidence interval (95% CI). dTwo-sided p-values testing whether % inhibition is significantly different from zero. (C,D) The quality of the anti-Pfs25-IgG used in the SMFA was assessed by linear two regression analyses, correlating the Log10-transformed ratios of mean oocyst counts in control and test samples (log-mean ratio, LMR) with the anti-Pfs25 specific IgG level in the feeder (C) or the reciprocal dilution factor from the original serum pool (D). LMR is plotted on the left y-axis. The right y-axis shows the correspondent %TRA. Values on the x-axis are plotted on a square root scale. The black line shows the shared linear fit for IgG from groups of mice immunized with 4 µg Pfs25-IMX313 in Alhydrogel or with 4 µg and 0.4 µg of Pfs25-IMX313 in AS01. Colored lines show linear fits for individual groups. Filled and unfilled shapes show data points from two independent SMFA experiments. Data points marked with an asterisk were excluded from the analysis as they showed 100% TRA (upper plateau level of dose response).

before, at IgG concentrations of 250 and 83.3µg/ml all groups that had received Pfs25-IMX313 showed significant TRA, while immunization with RTS,S/AS01 had no statistically significant effect when compared to immunization with PBS/AS01. At the lowest tested concentration of 27.8µg/ml, only the Pfs25- IMX313 alone group showed significant TRA. Correlation of antibody titres in the feeder with the SMFA results, however, did not indicate a difference in antibody quality between combination and individually administered vaccine (**Figure 4C**). The overall fit to the linear regression model was R 2 = 0.68, and again anti-Pfs25 AU (p = 0.0012) but not vaccine groups (p = 0.7322) showed a significant effect. Correlation of SMFA results with reciprocal serum dilutions furthermore confirmed the quantification of the antibody response by ELISA (**Figure 3C**) in indicating there was no quantitative difference in the IgG response between the groups (R 2 = 0.69, serum dilution factor: p = 0.001, group: p = 0.1275). The observed difference in SMFA is therefore most likely due to a higher concentration of Pfs25 specific IgG in the purified total IgG from the Pfs25-IMX313 alone group, than in the total IgG purified from the groups that received both vaccines (log10 antibody titer of 1 mg/ml purified IgG: mixed = 3.76, co-administered: 3.75, Pfs25-IMX313 alone: 4.06). This is however not reflective of the amount of Pfs25 specific IgG in serum of immunized mice, which was similar in all three groups (arithmetic mean log10 antibody titer: mixed = 4.83 AU, co-administered =4.81 AU, Pfs25-IMX313 alone = 4.86 AU).

### Combination of RTS,S With Pfs25-IMX313 Does not Reduce the Functional Activity of Induced Anti-CSP Antibodies

Functional activity of anti-CSP antibodies was assessed by ISI assay using P.berghei sporozoites expressing PfCSP under the PbCSP promoter (**Figure 5**). Sporozoites were obtained from the salivary glands of infected A. stephensi mosquitoes and added to Huh7 hepatoma cells in the presence of test and control serum. At 1% serum concentration inhibition of sporozoite invasion is detectable in all groups that received RTS,S. There is no statistically significant difference between groups immunized with RTS,S/AS01 alone or mixed or co-administered with Pfs25- IMX313, but all groups that received RTS,S inhibit sporozoite invasion above the assay's limit of sensitivity and inhibition was significantly higher than in the group that had been immunized with Pfs25-IMX313 (mixed p = 0.0011, co-administered p = 0.0154, RTS,S alone p = 0.0015, Dunn's multiple comparison test). To further characterize the anti-CSP antibody response induced by the different vaccine formulations containing RTS,S the log transformed mean ratio of infected hepatocytes in the vaccinated and PBS control groups was plotted against the square root of the anti-CSP endpoint titer in the hepatocyte growth medium, as had been done to analyse SMFA results. For all vaccine groups there was a significant correlation between increased LMR and higher anti-CSP AU (one-tailed Spearman correlation, mixed r = 0.9273, p = 0.0002, co-administered r = 0.612, p = 0.0334, RTS,S alone r = 0.8601, p = 0.0036. In a multiple linear regression analysis anti-CSP AU and vaccine groups were used as explanatory variables. The overall fit to the linear regression model was R 2 = 0.55, and anti-CSP AU (p < 0.001) but not vaccine groups (p = 0.7913) showed a significant effect on the inhibition of sporozoite invasion, indicating that there is no loss of quality in the antibody response when RTS,S is combined with Pfs25-IMX313 rather than administered alone.

### DISCUSSION

The modest efficacy of recent clinical trials using single antigen subunit vaccines support the development of multi-antigen vaccines targeting multiple stages of malaria. Mixing vaccines in a multi-component formulation is an easy process that does not require developing a new product. This means that it is possible to draw from previous experience in product development and clinical trials. This experience is particularly rich in the case of RTS,S the most advanced malaria vaccine candidate to date. Pfs25-IMX313 is a TBV candidate that we developed specifically to overcome the low antibody titres induced by vaccines targeting Pfs25 in past clinical trials (15, 17). Encoded in viral vectors it has now been tested in a clinical trial (ClinicalTrials.gov Identifier: NCT02532049), and it is currently being produced as a nanoparticle to GMP standards to be tested in clinical trials as a protein in adjuvant formulation, which in mice induced higher antibody titres than viral vectors (22). Here we showed that Pfs25-IMX313 can be formulated in AS01, which results in increased antibody titres when compared to formulation in Alhydrogel as well as increased TRA in SMFA. The functional activity of the induced antibodies per AU is similar for both adjuvants, but the overall stronger antibody response induced by Pfs25-IMX313/AS01 results in a substantially improved efficacy over Pfs25-IMX313/ Alhydrogel.

This study also shows that RTS,S and Pfs25-IMX313 can be combined successfully maintaining both the antibody titres

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FIGURE 3 | (B) and 2 weeks post boost (C). Empty shapes show the antibody units of a 1 mg/ml dilution of purified IgG. (D,E) Anti-NANP6C IgG endpoint titres were measured by ELISA at 4 weeks post prime (D) and 2 weeks post boost (E). Data points show antibody titres in individual mice, lines show the median.

and functional activity of antibodies against CSP and Pfs25 compared to the individual vaccines. The perceived lower activity in SMFA of the combined vaccines is explained by the higher amount of antigen specific IgG in the total IgG preparation of the Pfs25-IMX313 alone group (**Figure 3C**), and not an inferior antibody quality in the groups that received both vaccines (**Figure 4C**). Quantification of anti-Pfs25 serum antibody titres by ELISA (**Figure 3C**) and correlation of TRA with reciprocal serum dilutions (**Figure 4D**) showed that a similar amount of anti-Pfs25 specific IgG had been generated in all three groups. However, in the groups that received RTS,S as well as Pfs25- IMX313 there is an additional strong antibody response against the CSP component in RTS,S. A fixed amount of total IgG from these groups will therefore contain a significant amount of anti-CSP antibodies not present in a total IgG preparation from mice that had been immunized with Pfs25-IMX313 alone, and hence a smaller amount of anti-Pfs25 specific IgG. A trend for increased oocyst intensity after immunization with RTS,S/AS01 (**Figure 4**) is not statistically significant, and most likely due to the high variability of the assay at lower inhibition range, rather than to an enhancing effect of RTS,S/AS01 on parasite transmission. This strongly suggests that there is no negative effect on the TRA induced by immunization with Pfs25-IMX313 when the vaccine is either mixed or co-administered with RTS,S/AS01.

The successful combination of the two vaccines contrasts with previous studies where the combination of different viral vectored malaria vaccines did result in strong immune interference (23, 24) and the combination of RTS,S/AS01 with a viral vector PEV (ChAd63/MVA ME-TRAP) failed to improve vaccine efficacy when compared to the two vaccines administered individually and reduced immunogenicity and efficacy against malaria (25). This success might be attributed to shared characteristics and modes of action of RTS,S/AS01 and Pfs25-IMX313. Both vaccines target antigens that are expressed with high abundance on the surface of the sporozoites and zygotes/ookinetes, respectively, both vaccines are produced in yeast and both vaccines use a particulate platform to array the antigen in order to induce a strong antibody response.

The combination of RTS,S/AS01 and Pfs25-IMX313, as we demonstrated here, is of particular interest, for combining a TBV with a vaccine that protects from clinical disease creates a vaccine which reduces disease as well as malaria transmission, as called for in the MVT roadmap and the malERA research agenda for malaria eradication (malERA 3; 5; malERA 4). Furthermore, a recent study in mice, using monoclonal antibodies against CSP and Pfs25, suggests that partially efficacious PEVs and TBVs could be synergistic within a population (13), as the protective efficacy of PEVs is negatively correlated with the parasite density within an infectious mosquito (26), which can be reduced through the presence of anti-Pfs25 antibodies in the human host (27). Finally, not being a target of naturally acquired immunity against malaria and therefore not under immune pressure, the sequence of Pfs25 is highly conserved (28). The selection of P. falciparum variants less affected by anti-CSP antibodies induced by RTS,S/AS01 can therefore likely be reduced through the presence of anti-Pfs25 which reduce the human-to-mosquito transmission of such escape variants. The advanced stage of RTS,S/AS01 and Pfs25-IMX313 as malaria vaccine candidates harbors the intriguing prospect of being able to test these hypothesis, with their potentially far reaching consequences for malaria control, in human clinical trials.

### MATERIAL AND METHODS

#### Vaccines and Adjuvants

RTS,S and AS01 were provided by GSK. Pfs25-IMX313 was expressed and purified as described previously (22). In brief: Pfs25-IMX313 was expressed in Pichia pastoris under the control of the methanol inducible promoter AOX1. N-terminal fusion to the alpha-mating factor secretion signal sequence allowed secretion into the culture media. Following expression, nanoparticles were purified from the culture supernatant using a 5 ml CaptureSelectTM C-tag affinity matrix column.

#### Immunizations

All animal experiments and procedures were performed according to the UK Animals (Scientific Procedures) Act Project License (PPL 30/2889) and approved by the Oxford University Local Ethical Review Committee. Age-matched female CD1 mice (Envigo, United Kingdom), housed in specific pathogen-free environments, were vaccinated with 50 µl of vaccine formulation in one leg intramuscularly (IM). Protein-in-Alhydrogel was formulated as follows: 85 µg Alhydrogel (Brenntag) per dose was mixed with TBS at room temperature for 15 min, antigen was then added and incubated for a further 60 min at room temperature. AS01E was supplied pre-mixed by GSK to be at 1-fold concentration upon mixing with an appropriate amount of antigen. Total protein per dose for each group is detailed in the tables in **Figures 1**, **3**. Animals were immunized on days 0 and 28, and blood samples were collected on days 27 (4 weeks post prime) and 42 (2 weeks post boost). The samples were allowed to clot at 4◦C overnight before centrifugation at 13,000 × g in a benchtop centrifuge and serum was collected for testing.

#### ELISA

Standardized Pfs25 total IgG ELISAs were carried out as described previously (29). In brief, Nunc-Immuno Maxisorp 96 well plates (Thermo Scientific, UK) were coated with 1µg/ml Pfs25 in carbonate-bicarbonate coating buffer (Sigma Aldrich, UK) overnight at 4◦C. Plates were washed with PBS-Tween and blocked with 5% milk in PBS-Tween. Sera were diluted to reach an OD405 in the linear range of the standard curve at the same time an internal control reaches an OD405 of 1. Samples were added in triplicates. An internal control was added

from pooled serum of each group (2 weeks post boost) was tested in SMFA in two independent experiments, one using total IgG concentrations of 750, 250 and 83.3µg/ml (A) and one using total IgG concentrations of 250, 83.3, and 27.7µg/ml. The transmission blocking monoclonal anti-Pfs25 antibody 4B7

(Continued)

FIGURE 4 | was used as a positive control. Data points represent the number of oocysts in individual mosquitoes and the lines show the arithmetic mean. X-axis values are µg/ml total IgG in the assay. The results of the two feeds are summarized in the tables. a IgG concentration (µg/ml) in feeder. bArithmetic mean of oocyst intensity from 20 mosquitoes. cPercent inhibition of mean oocyst intensity and the 95% confidence interval (95% CI). dTwo-sided p-values testing whether % inhibition is significantly different from zero. (C,D) The quality of the anti-Pfs25-IgG used in the SMFA was assessed by linear two regression analyses, correlating the Log10-transformed ratios of mean oocyst counts in control and test samples (log-mean ratio, LMR) with the anti-Pfs25 specific IgG level in the feeder (C) or the reciprocal dilution factor from the original serum pool (D). LMR is plotted on the left y-axis. The right y-axis shows the correspondent %TRA. Values on the x-axis are plotted on a square root scale. The black line shows the shared linear fit for IgG from all groups. Filled and unfilled shapes show data points from the two independent SMFA experiments. Data points marked with an asterisk were excluded from the analysis as they showed 100% TRA (upper plateau level of dose response).

in six replicates. A standard curve was added in duplicates, starting with a 1:1,000 dilution of reference sera diluted down the plate in 1:2 steps 10 times. Plates were incubated for 2 h at room temperature and then washed as before. Goat anti-mouse whole IgG conjugated to alkaline phosphatase (Sigma Aldrich, UK) was added for 1 h at room temperature. Following a final wash, plates were developed by adding p-nitrophenylphosphate at 1 mg/mL in diethanolamine buffer (Pierce, UK) and optical density (OD) was read at 405 nm. Antibody units (AU) were defined as the dilution of a serum sample at which it had the same OD as the internal control. AUs were extrapolated from the samples OD on the linear range of the standard curve.

NANP-repeat endpoint total IgG ELISAs were carried out as described previously (30). Nunc-Immuno Maxisorp 96 well plates (Thermo Scientific, United Kingdom) were coated with 2µg/ml NANP6C peptide in carbonate-bicarbonate coating buffer (Sigma Aldrich, United Kingdom) overnight at 4◦C. Plates were washed with PBS-Tween and blocked with 10% Casein Block (Thermo Scientific, United Kingdom). Sera were diluted at a starting concentration of 1:1,000, added in duplicate, and serially diluted 3-fold. Plates were incubated for 2 h at room temperature and then washed as before. Goat anti-mouse whole IgG conjugated to alkaline phosphatase (Sigma Aldrich, United Kingdom) was added for 1 h at room temperature. Following a final wash, plates were developed by adding p-nitrophenylphosphate at 1 mg/mL in diethanolamine buffer (Pierce, UK) and optical density (OD) was read at 405 nm. Serum antibody endpoint titres were taken as the x-axis intercept of the dilution curve at an absorbance value of 0.15. A monoclonal antibody against CSP (2A10) was included in each assay as a reference control.

### SMFA

The ability of vaccine-induced antibodies to block the development of P. falciparum NF54 strain oocysts in the mosquito midgut was evaluated by SMFA as described previously (31). Stage V gametocytes from a mature gametocyte culture were mixed with normal human serum and normal red blood cells to make a feeding mixture with 0.15–0.2% stage V gametocytemia. Purified IgG was added to these at the concentrations shown in the figures and then fed to 3–6 day old starved female Anopheles stephensi (SDA 500) via a parafilm R membrane. The mosquitoes were maintained for 8 days and then dissected to count the number of oocysts per midgut in 20 mosquitos. The transmission blocking, Pfs25 specific monoclonal antibody 4B7 was used as a positive control (31). Percent reduction in infection intensity was calculated relative to the respective control IgG tested in the same assay.

### Parasite Production

To produce parasites for the ISI assay, donor mice were intraperitoneally injected with a parasite bloodstock. Parasitaemia was monitored through blood sampling by tail tip amputation and thin film preparation. As soon as the parasitaemia exceeded 5%, the mouse was anesthetized by intra muscular injection of ketamine and placed on top of a pot containing approximately 50 female A. stephensi mosquitoes. Mosquitoes were allowed to feed through the mesh covering the pots for at least 10 min. Seven to ten days after the blood meal mosquitoes were allowed to feed on an uninfected mouse, as this had been shown to increase the number of parasites per mosquito. Twenty-one days after the initial feed salivary glands were dissected from the mosquitoes to obtain sporozoites.

### ISI

Functional activity of anti-sporozoite antibodies was assessed by ISI using transgenic Plasmodium berghei parasites expressing PfCSP under the PbCSP promoter, at the PbCSP locus, replacing the PbCSP gene, as well as GFP to allow detection of infected cells by flow cytometry (Salman, in preparation). This strain of transgenic parasites has been validated for use in ISI (32) and in murine malaria challenge models (33). One day prior to mosquito dissection for isolation of sporozoites, HuH7 cells were seeded in a 96 well plate at a concentration of 300,000 cells/ml in a volume of 100 µl/well, resulting in a concentration of 30,000 cells per well. One day later mosquitoes were dissected and salivary glands collected in ice cold RPMI. Sporozoites were released from salivary glands by tissue disruption using a pestle and mortar, and counted in a hemocytometer. Sera were diluted to double the required concentration in 120 µl complete RPMI. The medium was removed from the HuH7 cells seeded the day before, and 50 µl of diluted serum was added to the wells in duplicates. Sporozoites were then diluted to 300,000 sporozoites per ml in complete RPMI and 50 µl were added to each well, resulting in 15,000 sporozoites per well and a ratio of cells to sporozoites of 2:1. To aid sporozoite invasion of the cells, the plate was centrifuged at 500 × g for 5 min before incubation at 37◦C and 5% CO2 overnight in a tissue culture incubator. After 24 h of incubation, cells were detached from the plate by removing medium from the plate and then adding 100 µl of TrypLE TM Express (Gibco) to the wells and incubation for 5 min

FIGURE 5 | Inhibition of sporozoite invasion by anti CSP-serum induced by immunization with RTS,S alone or in combination with Pfs25-IMX313. (A) Serum from individual mice (2 weeks post boost) was mixed with P. berghei sporozoites from mosquito salivary gland dissections, and the inhibition of sporozoite invasion was assessed. Data points show inhibition of sporozoite invasion by serum from individual mice, lines show the median, the dotted line shows the limit of sensitivity. (B) The quality of anti-CSP serum induced in individual mice was assessed by linear regression analysis, correlating the anti-CSP IgG endpoint titres in the culture medium with the log-transformed ratios of infected hepatocytes in control and test samples (log-mean ratio, LMR). LMR is plotted on the left y-axis. The right y-axis shows the correspondent %ISI. Values on the x-axis are plotted on a square root scale. The black line shows the shared linear fit for IgG from all groups. (C) The induced anti-NANP endpoint titres and the correlating %ISI in individual mice are detailed in the table. aLog10-transformed NANP6C endpoint titres as determined by ELISA. b% Inhibition of sporozoite invasion by 1% serum in culture medium.