Structural and immunogenicity analysis of reconstructed ancestral and consensus P48/45 for cross-species anti malaria transmission-blocking vaccine.

The development of the anti-malaria vaccine holds a promising future in malaria control. One of the anti-malaria vaccine strategies known as the transmission-blocking vaccine (TBV) is to inhibit the parasite transmission between humans and mosquitoes by targeting the parasite gametocyte. Previously, we found that P48/45 included in the 6-Cysteine protein family shared by Plasmodium sp. We also detected vaccine properties possessed by all human-infecting Plasmodium and could be used as a cross-species anti-malaria vaccine. In this study, we investigated the efficacy of P48/45 through the ancestral and consensus reconstruction approach. P48/45 phylogenetic and time tree analysis was done by RAXML and BEAST2. GRASP server and Ugene software were used to reconstruct ancestral and consensus sequences, respectively. The protein structural prediction was made by using a psipred and Rosetta program. Each protein characteristic of P48/45 was analyzed by assessing hydrophobicity and Post-Translational Modification sites. Meanwhile, the Epitope sequence for B-cell, T-cell, and HLA was determined using an immunoinformatics approach. Lastly, molecular docking simulation was done to determine native binding interactions of P48/45-P230. The result showed a distinct protein characteristic of ancestral and consensus sequences. The immunogenicity analysis revealed the number of epitopes in the ancestral sequence is greater than the consensus sequence. The study also found a conserved epitope located in the binding site and consists of specific Post-Translational Modification sites. Hence, our research provides detailed insight into ancestral and consensus P48/45 efficacy for the cross-species anti-malaria vaccine.

The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum.

This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; ∼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides' 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.

A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Structure and function of a malaria transmission blocking vaccine targeting Pfs230 and Pfs230-Pfs48/45 proteins.

Proteins Pfs230 and Pfs48/45 are Plasmodium falciparum transmission-blocking (TB) vaccine candidates that form a membrane-bound protein complex on gametes. The biological role of Pfs230 or the Pfs230-Pfs48/45 complex remains poorly understood. Here, we present the crystal structure of recombinant Pfs230 domain 1 (Pfs230D1M), a 6-cysteine domain, in complex with the Fab fragment of a TB monoclonal antibody (mAb) 4F12. We observed the arrangement of Pfs230 on the surface of macrogametes differed from that on microgametes, and that Pfs230, with no known membrane anchor, may exist on the membrane surface in the absence of Pfs48/45. 4F12 appears to sterically interfere with Pfs230 function. Combining mAbs against different epitopes of Pfs230D1 or of Pfs230D1 and Pfs48/45, significantly increased TB activity. These studies elucidate a mechanism of action of the Pfs230D1 vaccine, model the functional activity induced by a polyclonal antibody response and support the development of TB vaccines targeting Pfs230D1 and Pfs230D1-Pfs48/45.

A replication-competent late liver stage-attenuated human malaria parasite.

Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage-arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2-. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2- liver stages in human liver-chimeric mice. However, P. falciparum mei2- liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2- is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.

A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine.

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

Whole-blood transcriptomic signatures induced during immunization by chloroquine prophylaxis and Plasmodium falciparum sporozoites.

A highly effective vaccine that confers sterile protection to malaria is urgently needed. Immunization under chemoprophylaxis with sporozoites (CPS) consistently confers high levels of protection in the Controlled Human Malaria infection (CHMI) model. To provide a broad, unbiased assessment of the composition and kinetics of direct ex vivo human immune responses to CPS, we profiled whole-blood transcriptomes by RNA-seq before and during CPS immunization and following CHMI challenge. Differential expression of genes enriched in modules related to T cells, NK cells, protein synthesis, and mitochondrial processes were detected in fully protected individuals four weeks after the first immunization. Non-protected individuals demonstrated transcriptomic changes after the third immunization and the day of treatment, with upregulation of interferon and innate inflammatory genes and downregulation of B-cell signatures. Protected individuals demonstrated more significant interactions between blood transcription modules compared to non-protected individuals several weeks after the second and third immunizations. These data provide insight into the molecular and cellular basis of CPS-induced immune protection from P. falciparum infection.

A VAR2CSA:CSP conjugate capable of inducing dual specificity antibody responses.

BACKGROUND: Vaccine antigens targeting specific P. falciparum parasite stages are under pre-clinical and clinical development. It seems plausible that vaccine with multiple specificities will offer higher protection. With this hypothesis, we exploited the Spy-Tag/SpyCatcher conjugation system to make a, post expression, dual antigen conjugate vaccine, comprising two clinically tested antigen candidates (CSP and VAR2CSA). METHODS: The DBL1x-DBL2x-ID2a region of VAR2CSA was genetically fused with SpyTag at N-terminus. The full-length CSP antigen was genetically fused to C-terminal SpyCatcher peptide. The covalent interaction between SpyTag/SpyCatcher enables the formation of DBL1x-DBL2x-ID2a:CSP conjugate vaccine. Immunogenicity and quality of antibody responses induced by the conjugate vaccine, as well as a control CSP-SpyCatcher vaccine, was tested in BALB/c mice. RESULTS: Serum samples obtained from mice immunized with the conjugate vaccine were able to recognize both untagged DBL1x-DBL2x-ID2a as well as CSP antigen. Moreover, the geometric mean anti-CSP antibody titer was 1.9-fold higher in serum (at day 35 and 55 post-first immunization) from mice immunized with the conjugate vaccine, as compared to mice receiving the control vaccine. CONCLUSION: The data obtained in this study serves as proof-of-concept for the simultaneous induction of antibodies directed against individual antigen components in a dual stage anti-malaria vaccine.

Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230.

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria.

Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development.

BACKGROUND: Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS). Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage) antigens that are targeted by these responses have been identified. METHODOLOGY: Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens. CONCLUSIONS: These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates. TRIAL REGISTRATION: ClinicalTrials.gov NCT00870987 ClinicalTrials.gov NCT00392015.

Evaluation of the adjuvant activity of propranolol, a Beta-adrenergic receptor antagonist, on efficacy of a malaria vaccine model in BALB/c mice.

We have previously shown the adjuvant activity of propranolol (PRP) (a beta-adrenergic receptor antagonist) using a vaccine model for Salmonella typhimurium. In this study PRP was used as an adjuvant in combination with Plasmodium berghei (P. berghei) whole blood stage (PWBS) antigens. BALB/c mice were immunized three times with a 2-week interval, either PWBS vaccine alone or in combination with the adjuvant alum or propranolol. The control group received phosphate buffered saline. Evaluation of the cellular and humoral immunity was performed by measurement of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, lymphocyte proliferation, total IgG and IgG2a in the control and immunized groups. Furthermore, Clinical evaluations were carried out by analyze survival rate and parasitemia of the mice. Our results showed that the mice immunized with propranolol induced higher levels of antibody, IFN-γ and TNF-α as well as stronger lymphocyte proliferative responses compared with other groups. This resulted in improved protective immunity against Plasmodium berghei. Administration of the PRP as an adjuvant in combination with the PWBS Antigen vaccine can shift the immune responses to a T helper1 pattern and enhance the protective immunity.

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components.

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates.

The effect of helper epitopes and cellular localization of an antigen on the outcome of gene gun DNA immunization.

In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine.

This study explored the novel use of iron oxide (IO) nanoparticles (<20 nm) as a vaccine delivery platform without additional adjuvants. A recombinant malaria vaccine antigen, the merozoite surface protein 1 (rMSP1), was conjugated to IO nanoparticles (rMSP1-IO). Immunizations in outbred mice with rMSP1-IO achieved 100% responsiveness with antibody titers comparable to those obtained with rMSP1 formulated with a clinically acceptable adjuvant, Montanide ISA51 (2.7×10 vs. 1.6×10; respectively). Only rMSP1-1O could induce significant levels (80%) of parasite inhibitory antibodies. The rMSP1-IO was highly stable at 4°C and was amenable to lyophilization, maintaining its antigenicity, immunogenicity, and ability to induce inhibitory antibodies. Further testing in nonhuman primates, Aotus monkeys, also elicited 100% immune responsiveness and high levels of parasite inhibitory antibodies (55-100% inhibition). No apparent local or systemic toxicity was associated with IO immunizations. Murine macrophages and dendritic cells efficiently (>90%) internalized IO nanoparticles, but only the latter were significantly activated, with elevated expression/secretion of CD86, cytokines (IL-6, TNF-α, IL1-b, IFN-γ, and IL-12), and chemokines (CXCL1, CXCL2, CCL2, CCL3, CCL4, and CXCL10). Thus, the IO nanoparticles is a novel, safe, and effective vaccine platform, with built-in adjuvancy, that is highly stable and field deployable for cost-effective vaccine delivery.

[Synthetic peptide vaccines].

This review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modelling human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. Examples of synthetic peptide vaccine developments against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease) are given.

Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers.

BACKGROUND: Use of the recombinant proteins NefTat and gp120(W61D) formulated with the AS02A adjuvant system was previously shown to protect against AIDS in a rhesus macaque SHIV animal model system. METHODS: Eighty-four HIV uninfected human participants were vaccinated intramuscularly at 0, 1, and 3 months and evaluated for safety. Immune responses were analyzed for the presence of vaccine-induced antibody and T lymphocyte responses. RESULTS: The vaccines were safe and well tolerated at all doses. Nef-, Tat-, and gp120-specific binding antibodies were induced in all individuals that received the respective antigen, lasting up to 9 months after the final immunization. Antibodies able to neutralize the T-cell laboratory-adapted strain of HIV-1(W61D) were detected in the majority of vacinees, but did not neutralize primary isolates. Envelope-specific antibody-dependent cell cytoxicity was detected in most of the individuals receiving gp120. Robust and persistent HIV-specific lymphoproliferative responses were detected against all subunit proteins in the majority of immunized participants. As expected, HIV-specific CD8 T-cell responses were not detected. CONCLUSIONS: Despite the lack of primary isolate neutralizing antibody induction, the observed high frequency and magnitude of other immune responses warrant further work with this vaccine or vaccine components.

DNA vaccine.

The DNA vaccine has proven to be one of the most promising applications in the field of gene therapy. Due to its unique ability to readily induce humoral as well as cellular immune responses, it attracted great interest when the concept was first confirmed in the early 1990s. After thousands of articles related to the DNA vaccine were published, scientists began to realize that although the DNA vaccine is very effective in small animal models, its effectiveness in recent clinical trails is rather disappointing. Therefore, current effort has been shifted to understanding the different performance of the DNA vaccine in mouse and large animal models and on how to transfer the success of the DNA vaccine in small animals to large animals and humans.

Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research.

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.

Immunization of Saimiri sciureus monkeys with Plasmodium falciparum merozoite surface protein-3 and glutamate-rich protein suggests that protection is related to antibody levels.

The immunogenicity and protective efficacy of various antigen-adjuvant formulations derived either from the merozoite-surface protein-3 (MSP-3) or the glutamate-rich protein (GLURP) of Plasmodium falciparum were evaluated in Saimiri sciureus monkeys. These proteins were selected for immunogenicity studies based primarily on their capacity of inducing an antibody-dependent cellular inhibition effect on parasite growth. Some of the S. sciureus monkeys immunized with MSP-3(212-380)-AS02 or GLURP(27-500)-alum were able to fully or partially control parasitaemia upon an experimental P. falciparum [Falciparum Uganda Palo Alto (FUP-SP) strain] blood-stage infection, and this protection was related to the prechallenge antibody titres induced. The data are indicative that MSP-3 and GLURP can induce protective immunity against an experimental P. falciparum infection using adjuvants that are acceptable for human use and this should trigger further studies with those new antigens.