Developing a safe and effective malaria vaccine is critical to reducing the spread and resurgence of this deadly disease, especially in children. In recent years, vaccine technology has seen more development of subunit protein, peptide, and nucleic acid vaccines. This is due to their inherent safety, the ability to tailor their immune response, simple storage requirements, easier production, and lower cost compared to using attenuated and inactivated organism-based approaches. However, these new vaccine technologies generally have low efficacy. Subunit vaccines, due to their weak immunogenicity, often necessitate advanced delivery vectors and/or the use of adjuvants.

A new area of vaccine development involves the design of synthetic micro- and nano-particles and adjuvants that can stimulate immune cells directly through their physical and chemical properties. Further, the unique and complex life cycle of the Plasmodium organism, with multiple stages and varying epitopes/antigens presented by the parasite, is another challenge for malaria vaccine development. Targeting multistage antigens simultaneously is therefore critical for an effective malaria vaccine. Here, we rationally design a layer-by-layer (LbL) antigen delivery platform (we called LbL NP) specifically engineered for malaria vaccines. A biocompatible modified chitosan nanoparticle (trimethyl chitosan, TMC) was synthesized and utilized for LbL loading and release of multiple malaria antigens from pre-erythrocytic and erythrocytic stages. LbL NP served as antigen/protein delivery vehicle and was demonstrated to induce the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice studies as compared to multiple controls.

From immunogenicity studies, it was concluded that two doses of intramuscular injection with a longer interval (4 weeks) than traditional malaria vaccine candidate dosing would be the vaccination potential for LbL NP vaccine candidates. Furthermore, in PfCSP/Py parasite challenge studies, we demonstrated protective efficacy using LbL NP. These LbL NP provided a significant adjuvant effect since they may induce an innate immune response that led to a potent adaptive immunity to mediate the non-specific anti-malarial effect. Most importantly, the delivery of CSP full-length protein stimulated long-lasting protective immune responses even after the booster immunization 4 weeks later in mice.

Malaria kills over 260,000 children under five years old in Africa every year. The first malaria vaccine RTS,S/AS01 (Mosquirix®), an advanced recombinant protein-based vaccine was approved in 2021 for children under 5 years old. However, compared with other vaccinations, RTS, S/AS01 has only modest efficacy in preventing approximately 30% of severe malaria cases after a series of four injections (1). The recombinant protein is a pre-erythrocytic stage circumsporozoite protein (CSP). It targets parasites before they can infect the liver, but this is only relevant for one stage of the parasite’s complex life cycle. One of the primary difficulties in malaria vaccination is the complexity of the multistage life cycle of Plasmodium and the
intricate host-parasite interactions during the course of malaria infection. An optimal malaria vaccine would efficiently target multiple stages of the parasite life cycle (2). Further, vaccines offer another tool that could take pressure off the continued use of combined malaria treatment drugs if one drug becomes resistant (2).

Due to the weak immunogenicity of investigational subunit vaccines, they often require advanced delivery vectors and/or the use of adjuvants (3). The design of novel adjuvant or nanoparticle delivery vectors that can stimulate immune cells and enhance vaccine efficacy has brought hope for future vaccine development. Improving the efficiency of vaccines by a combination of adjuvants and advanced delivery systems based on controlled release technology is also one of the major priorities of the World Health Organization program for vaccine development (4). The goal is to develop a controlled
release system to induce protective immune responses as soon as possible after the first immunization, while also providing prolonged immunity with negated or reduced administration of boosts.

To develop a more effective malaria vaccine with a protective immune response and delivery of multiple life cycle stage antigens, we describe here a trimethyl chitosan-based layer-by layer (LbL) nano-assembly vaccine platform (LbL NP) that enables the LBL delivery and release of multiple malaria
antigens in a controllable manner. We have successfully constructed the LbL NP with efficient loading of different stages of antigens. It encapsulates a Plasmodium falciparum malaria parasite blood stage apical membrane antigen PfAMA-1 or merozoite surface antigen PfMSP-1 inside the core. The preerythrocytic stage antigen PfCSP (full length) is absorbed and stabilized on the shell layer of LbL construct. The size of the LbL NP vaccine candidates can be tuned from 200 nm to 400 nm, which is suitable for intramuscular injection (5). The highly positively charged surface of the trimethyl chitosan nanoparticles is beneficial for loading multiple antigens and confers greater solubility due to the trimethylation on the chitosan surface.

A set of LbL NP was synthesized for encapsulation and loading with pre-erythrocytic and erythrocytic stage antigens at high efficiency of 70%-98%. The release of antigens was controlled between several days to months by tuning the charge and layer composition of the construct. The released antigens were characterized to verify that they maintained their stability and antigenicity. Most importantly, LbL NP served as antigen/protein delivery vehicles and induced the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice, as compared to other adjuvants.

Two doses of intramuscular injection with a longer interval (4 weeks) than other current vaccine candidates between them induced a high titer of the humoral response against PfCSP. Furthermore, 5 of 6 mice were protected against a malaria challenge after receiving a booster of LbL NP delivery of full length of CSP as the vaccine candidate. Finally, general biosafety and dose tolerance studies demonstrated that LbL NP could be applied safely at less than 5 mg/kg with no significant adverse effects. 

We evaluated the tolerability of a trimethylated chitosan nanoparticles administered intramuscularly to male Sprague‐Dawley rats twice over 14 days. In total, sixteen male SpragueDawley rats were assigned to 4 groups (vehicle control or three dose levels of nanoparticle [n=4/group]) as shown in Table-1.

Clinical observations were recorded up to once daily and body weights were assessed prior to dosing and at least twice weekly thereafter. The tissues/organs were also collected and weighed from all animals: heart, liver, kidney, and muscle tissues at the site of administration. Tissues/organs were processed using standard H&E staining. Microscopic evaluations of tissues/organs were conducted by a qualified veterinary pathologist. 

We formulated vaccine candidates by loading one pre-erythrocytic protective antigen PfCSP and two blood stage antigens PfAMA-1 and PfMSP-1 in the LbL NP structure as described in section 2.3. Three formulations were delivered for the animal studies for either two or three doses by intramuscular
injections to compare the vaccine candidate performance (Table 1). They were LbL NP-CSP (TMC-TPP encapsulated PfCSP); LbL NP-AMA-1/CSP-1 (TMC-TPP encapsulated AMA-1 inside of core, and PfCSP in the outside layer); LbL NP-CSP/AMA-1/MSP-1 (TMC-TPP encapsulated MSP-1 with second layer of AMA-1 and the outside layer is CSP). Additionally, another two adjuvants Montanide ISA 720 [Seppic Inc. (9)], a natural metabolizable nonmineral oil and a highly refined emulsifier of mannite monooleate family, and 7DW8-5 (10), a recently identified novel analog of a-galactosylceramide (aGalCer) that enhances the level of malaria-specific protective immune were used to compare and incorporated with these LbL
NP formulations.

In total, 24 female BALB/c mice (n=4 per group) were immunized intramuscularly with each formulation twice or three times at a 3-week interval to determine the vaccine candidate potential dosing. For each animal, an injection containing 10 μg of protein was administered for each dose. Three weeks after the boost, mouse sera were collected for serology analysis of the antibody titers of PfCSP, PfMSP-1, and PfAMA-1 for each formulation and the numbers of IFN-gsecreting T cells in spleens of mice immunized with antigens by intramuscular injection was measured by IFN-g enzyme
linked immunospot (ELISPOT) assay (10).

We prepared nine vaccine candidate samples for nine group of animals with two doses for each group of animals and one group using unloaded LbL NP as a control. These samples included two control adjuvant groups (Montanide ISA 720 VG ST and 7DW8-5). 10 groups (n=6 for each group, nine vaccine candidates and one control) of 8-10 weeks old female BALB/c mice were immunized intramuscularly with each formulation twice with a
4-week interval between two doses. For each animal, an injection containing 10 μg of protein was applied for each dose. Four weeks after the two doses, naïve as well as immunized mice were challenged with 1000 transgenic PfCSP/Py sporozoites intravenously. The infectivity of PfCSP/Py Spz was determined by the presence or absence of parasites (parasitemia) in the blood of the challenged mice. This was done by way of microscopic examination of Giemsa-stained thin blood smears made from one drop of blood extracted from the tail vein of the mice from day 4 to day 12 post-Spz challenge.

It is important that the delivery vehicle used for antigen delivery is highly stable and uniformly dispersed in the human blood system. The limited solubility of chitosan and chitosan-based materials, therefore, hinders their use and application in a wide range of biological environments. The reductive methylation of chitosan for obtaining N, N, N-trimethyl chitosan (TMC) is a good strategy for overcoming such limitations because TMC can be soluble in distilled water, in PBS solution, and in alkaline or acidic aqueous solutions. The solubility of TMC across the range of pH is due to the shifting in charge density originated by methylation of primary amino groups on chitosan. Also, the methylation of chitosan results in a high positive charge on the
surface of TMC which is beneficial for the loading of negatively charged biological molecules. FTIR and NMR analysis of our synthesis indicate that the TMC was successfully prepared and trimethylation was successful.

The ionic gelation method is considered the most suitable method for protein loading on TMC NPs. The presence of crosslinker TPP and surface coating chemistries of PSS or HA have been evaluated to select the optimized composition for development of malaria vaccine candidates. The use of TPP as
a crosslinker was found necessary for successful protein encapsulation within the core of the nanoparticle. PSS as a surface protection coating was found to moderately decrease the loading amount of core and second layer proteins. However, this was determined acceptable since it is used as a protective layer to prevent outer layer proteins from immediate release and degradation. PSS has a very strong affinity to TMC as compared to HA during the formation of NPs. As a result, if we apply PSS as the protective layer, the amount of PSS needs to be limited to less than 0.05 mg per mg of TMC to avoid large precipitations.

Release testing also confirmed that undesired burst release was lower if we decreased the protect layer PSS amount. HA performed similar function as PSS, but it provided more benefits that it reduced precipitation of nanoparticle when we used the same amount of coating as PSS. And it also helped in
prolonged the release of loaded protein compared with using PSS as the protected layer. HA could be the most beneficial for the formation of LbL NP vaccine candidate. Combined, we concluded that the protective layer is necessary and required for achieving long-term release profiles.

Three multiple-stage malaria life cycle antigens were successfully encapsulated and loaded on TMC nanoparticles. These antigens are pre-erythrocytic stage antigens (i) circumsporozoite protein (CSP; the major antigen on the sporozoite surface and its fragments have been included in the most clinically advanced malaria vaccine RTS, S) (12). However, RTS, S does not include the N-terminal region of CSP. Adoptive transfer of a monoclonal antibody specific for the N-terminus of the P. falciparum CSP, strongly inhibits the infection of rodent malaria sporozoites expressing the N-terminus of P. falciparum CSP (13). The erythrocytic stage antigens (ii) apical membrane antigen 1 (AMA1; involved in merozoite invasion of red blood cells and essential to the proliferation and survival of the malarial parasite, and its antibodies have shown to be protective (14), and (iii) merozoite surface protein 1 (MSP1; highly immunogenic in humans and numerous studies suggest it is an effective target for a protective immune response (15). We worked with GenScript for synthesis of the plasmid to produce full size of CSP protein.

As widely known, a wide variety of factors regulate and influence gene expression levels, and GenScript used OptimumGene™ algorithm to consider as many of these factors as possible, producing the single gene that can reach the highest possible level of expression. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. They increased the codon usage bias in E. coli by upgrading the Codon Adaptation Index (CAI) to 0.74. CAI of 1.0 is considered to be perfect in the desired expression organism.

ELISA results demonstrated that both free and entrapped protein after release from NPs possessed similar responses to their antibodies. The binding strength/affinity of released proteins to their respective receptors is an important factor when determining the efficacy of the developed vaccine. Binding, specificity, affinity, kinetics, and active binding concentration were determined from the shape of produced surface plasmon resonance imaging sensorgrams. These observations demonstrated that released PfCSP maintained binding properties to corresponding antibodies 2A10 and 3C1
following loading and release in the LbL NPs.

Many candidate vaccines evaluated to date fail to achieve protection against certain human pathogens, such as malaria, and this is primarily due to their poor cellular immunogenicity (16). As a result, it is important that newly developed adjuvant LbL NP may add value when it is used in a stand-alone manner or in combination with existing adjuvants such as ISA 720 (9) and 7DW8-5 (16). Here, we found in vivo immunogenicity tests using LbL NPs as antigen/protein delivery vehicles demonstrated immunoadjuvant properties. The LbL NP formulation groups showed the greatest PfCSP specific T-cell
responses in mice and also strong titers of humoral responses.

Specific IgG was detected in all mice receiving vaccine formulation with sera dilutions between 4,000 and 20,000 after 2 doses. Finally, we challenged with P. yoelli parasites that express only PfCSP, and therefore, we saw the protective immune response targeted against PfCSP only. When we determined the level of protection by the presence or absence of parasitemia in thin blood smears, we found that 5 of 6 mice were protected against malaria challenge after boost of LbL NP delivery of full length of CSP as the vaccine candidate. Thus, we systematically demonstrated that intramuscular injection of LbL NP leads to a more potent adjuvant effect than commercial ISA720 in the efficacy studies. However, if we establish PfCSP/PfAMA-1/PfMSP-1 triple transgenic parasites and challenge them, the LbL NP expressing the three proteins may exert a better efficacy compared to a single proteinexpressing LbL NP vaccine. Although the protective immunity induced by PfCSP (one antigen) may be weaker, a combined protective immunity induced by all 3 proteins may be more potent due to additive or synergistic effect. Also, it is rare to see protection lasting for more than 4 weeks after a booster dose. To the best of our knowledge, there have been no other malaria vaccines found that can sustain sterile protection for more than 2 weeks.

Also, we observed that LbL NPs are potentially a good adjuvant candidate for vaccine delivery in order to obtain long-lasting protection. The LbL NP found may elicit an innate immune response that was potent in mediating non-specific anti-malarial effects. In the safety studies, we found LbL NP at a
dose of less than 5 mg/ml was also determined biocompatible and safe in male Sprague-Dawley rats. While these studies suggest a protective response using LBL NP as the delivery vector, additional studies are necessary to fully understand the potential of the nanoparticle approach due to the smaller number of mice per group in this study. 

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Authors

Yang Xu 1*, Ziyou Zhou 1, Brad Brooks 1, Tammy Ferguson 1, Judy Obliosca 1, Jing Huang 2,3, Izumi Kaneko 4, Shiroh Iwanaga 5, Masao Yuda 4, Yukiko Tsuji 2, Huitang Zhang 6, Christina C. Luo 6, Xunqing Jiang 6, Xiang-Peng Kong 6, Moriya Tsuji 2,3 and Christopher K. Tison 1

1 Luna Labs USA, Biotech Group, Charlottesville, VA, United States,
2 HIV and Malaria Vaccine Program, Aaron Diamond AIDS Research Center, New York, NY, United States,
3 Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY, United States,
4 Department of Medical Zoology, Mie University Graduate School of Medicine, Mie, Japan,
5 Research Institute for Microbial Diseases, Osaka University, Osaka, Japan,
6 Department of Biochemistry and Molecular Pharmacology, New York University (NYU) Grossman School of Medicine, New York, NY, United States

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Copyright © 2022 Xu, Zhou, Brooks, Ferguson, Obliosca, Huang, Kaneko, Iwanaga, Yuda, Tsuji, Zhang, Luo, Jiang, Kong, Tsuji and Tison.

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Xu Y, Zhou Z, Brooks B, Ferguson T, Obliosca J, Huang J, Kaneko I, Iwanaga S, Yuda M, Tsuji Y, Zhang H, Luo CC, Jiang X, Kong XP, Tsuji M, Tison CK. Layer-by-Layer Delivery of Multiple Antigens Using Trimethyl Chitosan Nanoparticles as a Malaria Vaccine Candidate. Front Immunol. 2022 Aug 17;13:900080. doi: 10.3389/fimmu.2022.900080. PMID: 36059505; PMCID: PMC9428560.