The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

BACKGROUND: The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. RESULTS: Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. CONCLUSIONS: We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

Optimizing the design of protein nanoparticles as carriers for vaccine applications.

Successful vaccine development remains a huge challenge for infectious diseases such as malaria, HIV and influenza. As a novel way to present antigenic epitopes to the immune system, we have developed icosahedral self-assembling protein nanoparticles (SAPNs) to serve as a prototypical vaccine platform for infectious diseases. Here we examine some biophysical factors that affect the self-assembly of these nanoparticles, which have as basic building blocks coiled-coil oligomerization domains joined by a short linker region. Relying on in silico computer modeling predictions, we selected five different linker regions from the RCSB protein database that connect oligomerization domains, and then further studied the self-assembly and stability of in vitro produced nanoparticles through biophysical characterization of formed particles. One design in particular, T2i88, revealed excellent self-assembly and homogeneity thus paving the way toward a more optimized nanoparticle for vaccine applications. FROM THE CLINICAL EDITOR: Despite the widespread use of vaccines worldwide, successful development of vaccines against some diseases remains a challenge still. In this article, the authors investigated the physic-chemical and biological properties of icosahedral self-assembling protein nanoparticles (SAPNs), which mimic viral particles, in order to utilize this technology as potential platform for future design of vaccines.

Estimation of recent and long-term malaria transmission in a population by antibody testing to multiple Plasmodium falciparum antigens.

BACKGROUND: Tools that estimate recent and long-term malaria transmission in a population would be highly useful for malaria elimination programs. METHODS: The prevalence of antibodies to 11 Plasmodium falciparum antigens was assessed by cytometric bead assay or enzyme-linked immunosorbent assay in 1000 people in a highland area of Kenya over 14 months, during a period of interrupted malaria transmission. RESULTS: Antibodies differed by antigen in acquisition with age: rapid (>80% antibody positive by age 20 years, 5 antigens), moderate (>40% positive by age 20 years, 3 antigens), or slow (<40% positive by age 20 years, 3 antigens). Antibody seroreversion rates in the 14 months between samples decreased with age rapidly (7 antigens), slowly (3 antigens), or remained high at all ages (schizont extract). Estimated antibody half-lives in individuals >10 years of age were long (40 to >80 years) for 5 antigens, moderate (5-20 years) for 3 antigens, and short (<1 year) for 3 antigens. CONCLUSIONS: Antibodies to P. falciparum antigens in malaria-endemic areas vary by age, antigen, and time since last exposure to P. falciparum. Multiplex P. falciparum antibody testing could provide estimates of long-term and recent malaria transmission and potentially of a population's susceptibility to future clinical malaria.

A field trial to assess a blood-stage malaria vaccine.

BACKGROUND: Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children. METHODS: In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain. RESULTS: The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine. CONCLUSIONS: On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Expression, purification and refolding of a self-assembling protein nanoparticle (SAPN) malaria vaccine.

There are many ways to present antigens to the immune system. We have used a repetitive antigen display technology that relies on the self-assembly of 60 protein chains into a spherical self-assembling protein nanoparticle (SAPN) to develop a vaccine against Plasmodium falciparum malaria. The protein sequence contains selected B- and T-cell epitopes of the circumsporozoite protein of P. falciparum (PfCSP) and, when assembled into a nanoparticle induces strong, long-lived and protective immune responses against the PfCSP. Here we describe the conditions needed for promoting self-assembly of a P. falciparum vaccine nanoparticle, PfCSP-KMY-SAPN, and note pitfalls that may occur when determining conditions for other SAPN vaccines. Attention was paid to selecting processes that were amenable to scale up and cGMP manufacturing.

Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine.

BACKGROUND: A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. METHODS: Inbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. RESULTS: The results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous fashion via transporter associated with antigen processing (TAP) recruitment to the early endosome (EE), and have partially delayed processing through the endoplasmic reticulum, has the potential to induce the long-lived, effector memory CD8+ T-cells as described previously. CONCLUSION: This paper describes the examination of humoral and cellular immune mechanisms induced by PfCSP-SAPN vaccination which result in sterile host protection against a transgenic P. berghei malaria sporozoite expressing the P. falciparum CSP, and which significantly inhibits native P. falciparum sporozoites from invading and developing within cultured human hepatocytes. These results may indicate the type and mode of action of protective antibodies needed to control P. falciparum sporozoites from infecting humans as well as a potential mechanism of induction of protective long-lived effector memory CD8+ T-cells.

Antibodies to Plasmodium falciparum antigens predict a higher risk of malaria but protection from symptoms once parasitemic.

BACKGROUND: Associations between antibody responses to Plasmodium falciparum antigens and protection against symptomatic malaria have been difficult to ascertain, in part because antibodies are potential markers of both exposure to P. falciparum and protection against disease. METHODS: We measured IgG responses to P. falciparum circumsporozoite protein, liver-stage antigen 1, apical-membrane antigen 1 (AMA-1), and merozoite surface proteins (MSP) 1 and 3, in children in Kampala, Uganda, and measured incidence of malaria before and after antibody measurement. RESULTS: Stronger responses to all 5 antigens were associated with an increased risk of clinical malaria (P < .01) because of confounding with prior exposure to P. falciparum. However, with use of another assessment, risk of clinical malaria once parasitemic, stronger responses to AMA-1, MSP-1, and MSP-3 were associated with protection (odds ratios, 0.34, 0.36, and 0.31, respectively, per 10-fold increase; P < .01). Analyses assessing antibodies in combination suggested that any protective effect of antibodies was overestimated by associations between individual responses and protection. CONCLUSIONS: Using the risk of symptomatic malaria once parasitemic as an outcome may improve detection of associations between immune responses and protection from disease. Immunoepidemiology studies designed to detect mechanisms of immune protection should integrate prior exposure into the analysis and evaluate multiple immune responses.

First-in-human assessment of safety and immunogenicity of low and high doses of Plasmodium falciparum malaria protein 013 (FMP013) administered intramuscularly with ALFQ adjuvant in healthy malaria-naïve adults.

The global burden of malaria remains substantial. Circumsporozoite protein (CSP) has been demonstrated to be an effective target antigen, however, improvements that offer more efficacious and more durable protection are still needed. In support of research and development of next-generation malaria vaccines, Walter Reed Army Institute of Research (WRAIR) has developed a CSP-based antigen (FMP013) and a novel adjuvant ALFQ (Army Liposome Formulation containing QS-21). We present a single center, open-label, dose-escalation Phase 1 clinical trial to evaluate the safety and immunogenicity of the FMP013/ALFQ malaria vaccine candidate. In this first-in-human evaluation of both the antigen and adjuvant, we enrolled ten subjects; five received 20 µg FMP013 / 0.5 mL ALFQ (Low dose group), and five received 40 µg FMP013 / 1.0 mL ALFQ (High dose group) on study days 1, 29, and 57. Adverse events and immune responses were assessed during the study period. The clinical safety profile was acceptable and there were no serious adverse events. Both groups exhibited robust humoral and cellular immunological responses, and compared favorably with historical responses reported for RTS,S/AS01. Based on a lower reactogenicity profile, the 20 µg FMP013 / 0.5 mL ALFQ (Low dose) was selected for follow-on efficacy testing by controlled human malaria infection (CHMI) with a separate cohort. Trial Registration:Clinicaltrials.gov Identifier NCT04268420 (Registered February 13, 2020).

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

BACKGROUND: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. METHODS: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. RESULTS: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. CONCLUSIONS: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria.

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

Duration of naturally acquired antibody responses to blood-stage Plasmodium falciparum is age dependent and antigen specific.

Naturally acquired antibody responses provide partial protection from clinical malaria, and blood-stage parasite vaccines under development aim to prime such responses. To investigate the determinants of antibody response longevity, immunoglobulin G (IgG) antibodies to several blood-stage vaccine candidate antigens in the sera of two cohorts of children of up to 6 years of age during the dry seasons of 2003 and 2004 in The Gambia were examined. The first cohort showed that most antibodies were lost within less than 4 months of the first sampling if a persistent infection was not present, so the study of the second-year cohort involved collecting samples from individuals every 2 weeks over a 3-month period. Antibody responses in the second cohort were also influenced by persistent malaria infection, so analysis focused particularly on children in whom parasites were not detected after the first time point. Antibodies to most antigens declined more slowly in children in the oldest age group (>5 years old) and more rapidly in children in the youngest group (<3 years old). However, antibodies to merozoite surface protein 2 were shorter lived than antibodies to other antigens and were not more persistent in older children. The age-specific and antigen-specific differences were not explained by different IgG subclass response profiles, indicating the probable importance of differential longevities of plasma cell populations rather than antibody molecules. It is likely that young children mostly have short-lived plasma cells and thus experience rapid declines in antibody levels but that older children have longer-lasting antibody responses that depend on long-lived plasma cells.

Low prevalence of antibodies to preerythrocytic but not blood-stage Plasmodium falciparum antigens in an area of unstable malaria transmission compared to prevalence in an area of stable malaria transmission.

In areas where levels of transmission of Plasmodium falciparum are high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.

Impact of recombinant adenovirus serotype 35 priming versus boosting of a Plasmodium falciparum protein: characterization of T- and B-cell responses to liver-stage antigen 1.

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.

Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria.

Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Polymorphism patterns in Duffy-binding protein among Thai Plasmodium vivax isolates.

BACKGROUND: The Duffy-binding protein II of Plasmodium vivax (PvDBPII) has been considered as an attractive target for vaccine-mediated immunity despite a possible highly polymorphic nature. Among seven PvDBP domains, domain II has been shown to exhibit a high rate of nonsynonymous polymorphism, which has been suggested to be a potential immune (antibody binding) evasion mechanism. This study aimed to determine the extent of genetic polymorphisms and positive natural selection at domain II of the PvDBP gene among a sampling of Thai P. vivax isolates. METHODS: The PvDBPII gene was PCR amplified and the patterns of polymorphisms were characterized from 30 Thai P. vivax isolates using DNA cloning and sequencing. Phylogenetic analysis of the sequences and positive selection were done using DnaSP ver 4.0 and MEGA ver 4.0 packages. RESULTS: This study demonstrated a high rate of nonsynonymous polymorphism. Using Sal I as the reference strain, a total of 30 point-mutations were observed in the PvDBPII gene among the set of Thai P. vivax isolates, of which 25 nonsynonymous and five synonymous were found. The highest frequency of polymorphism was found in five variant amino acids (residues D384G, R390H, L424I, W437R, I503K) with the variant L424I having the highest frequency. The difference between the rates of nonsynonymous and synonymous mutations estimated by the Nei and Gojobori's method suggested that PvDBPII antigen appears to be under selective pressure. Phylogenetic analysis of PvDBPII Thai P. vivax isolates to others found internationally demonstrated six distinct allele groups. Allele groups 4 and 6 were unique to Thailand. CONCLUSION: Polymorphisms within PvDBPII indicated that Thai vivax malaria parasites are genetically diverse. Phylogenetic analysis of DNA sequences using the Neighbour-Joining method demonstrated that Thai isolates shared distinct alleles with P. vivax isolates from different geographical areas. The study reported here will be valuable for the development of PvDBPII-based malaria vaccine.

Immune response to antigen adsorbed to aluminum hydroxide particles: Effects of co-adsorption of ALF or ALFQ adjuvant to the aluminum-antigen complex.

Aluminum salts have been used as vaccine adjuvants for >50 years, and they are currently present in at least 146 licensed vaccines worldwide. In this study we examined whether adsorption of Army Liposome Formulation (ALF) to an aluminum salt that already has an antigen adsorbed to it might result in improved immune potency of the aluminum-adsorbed antigen. ALF is composed of a family of anionic liposome-based adjuvants, in which the liposomes contain synthetic phospholipids having dimyristoyl fatty acyl groups, cholesterol and monophosphoryl lipid A (MPLA). For certain candidate vaccines, ALF has been added to aluminum hydroxide (AH) gel as a second adjuvant to form ALFA. Here we show that different methods of preparation of ALF changed the physical structures of both ALF and ALFA. Liposomes containing the saponin QS21 (ALFQ) have also been mixed with AH to form ALFQA as an effective combination. In this study, we first adsorbed one of two different antigens to AH, either tetanus toxoid conjugated to 34 copies of a hapten (MorHap), which has been used in a candidate heroin vaccine, or gp140 protein derived from the envelope protein of HIV-1. We then co-adsorbed ALF or ALFQ to the AH to form ALFA or ALFQA. In each case, the immune potency of the antigen adsorbed to AH was greatly increased by co-adsorbing either ALF or ALFQ to the AH. Based on IgG subtype and cytokine analysis by ELISPOT, ALFA induced predominately a Th2-type response and ALFQ and ALFQA each induced more balanced Th1/Th2 responses.

Identification of Immune Signatures of Novel Adjuvant Formulations Using Machine Learning.

Adjuvants have long been critical components of vaccines, but the exact mechanisms of their action and precisely how they alter or enhance vaccine-induced immune responses are often unclear. In this study, we used broad immunoprofiling of antibody, cellular, and cytokine responses, combined with data integration and machine learning to gain insight into the impact of different adjuvant formulations on vaccine-induced immune responses. A Self-Assembling Protein Nanoparticles (SAPN) presenting the malarial circumsporozoite protein (CSP) was used as a model vaccine, adjuvanted with three different liposomal formulations: liposome plus Alum (ALFA), liposome plus QS21 (ALFQ), and both (ALFQA). Using a computational approach to integrate the immunoprofiling data, we identified distinct vaccine-induced immune responses and developed a multivariate model that could predict the adjuvant condition from immune response data alone with 92% accuracy (p = 0.003). The data integration also revealed that commonly used readouts (i.e. serology, frequency of T cells producing IFN-γ, IL2, TNFα) missed important differences between adjuvants. In summary, broad immune-profiling in combination with machine learning methods enabled the reliable and clear definition of immune signatures for different adjuvant formulations, providing a means for quantitatively characterizing the complex roles that adjuvants can play in vaccine-induced immunity. The approach described here provides a powerful tool for identifying potential immune correlates of protection, a prerequisite for the rational pairing of vaccines candidates and adjuvants.

Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine.

To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1ß, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.

Author Correction: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum.

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.