Structure-based design of a Plasmodium vivax Duffy-binding protein immunogen focuses the antibody response to functional epitopes.

The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development.

RBD design increases the functional antibody titers elicited by SARS-CoV-2 spike vaccination.

Most COVID-19 vaccines contain the SARS-CoV-2 spike protein as an antigen, but they lose efficacy as neutralizing antibody titers wane and escape variants emerge. Modifying the spike antigen to increase neutralizing antibody titers would help counteract this decrease in titer. We previously used a structure-based computational design method to identify nine amino acid changes in the receptor-binding domain (RBD) of spike that stabilize the RBD and increase the neutralizing antibody titers elicited by vaccination. Here, we introduce those enhancing amino acid changes into a full-length spike (FL-S-2P) ectodomain representative of most approved vaccine antigens. These amino acid changes can be incorporated into the FL-S-2P protein without negatively effecting expression or stability. Furthermore, the amino acid changes improved functional antibody titers in both mice and monkeys following vaccination. These amino acid changes could increase the duration of protection conferred by most COVID-19 vaccines.

A Self-Assembling Pfs230D1-Ferritin Nanoparticle Vaccine Has Potent and Durable Malaria Transmission-Reducing Activity.

Malaria is caused by eukaryotic protozoan parasites of the genus Plasmodium. There are 249 million new cases and 608,000 deaths annually, and new interventions are desperately needed. Malaria vaccines can be divided into three categories: liver stage, blood stage, or transmission-blocking vaccines. Transmission-blocking vaccines prevent the transmission of disease by the mosquito vector from one human to another. Pfs230 is one of the leading transmission-blocking vaccine antigens for malaria. Here, we describe the development of a 24-copy self-assembling nanoparticle vaccine comprising domain 1 of Pfs230 genetically fused to H. pylori ferritin. The single-component Pfs230D1-ferritin construct forms a stable and homogenous 24-copy nanoparticle with good production yields. The nanoparticle is highly immunogenic, as two low-dose vaccinations of New Zealand White rabbits elicited a potent and durable antibody response with high transmission-reducing activity when formulated in two distinct adjuvants suitable for translation to human use. This single-component 24-copy Pfs230D1-ferritin nanoparticle vaccine has the potential to improve production pipelines and the cost of manufacturing a potent and durable transmission-blocking vaccine for malaria control.

A targetable receptor-binding site on PfCyRPA to aid in the fight against malaria.

Recently, Day et al. identified a receptor-binding site on the malaria parasite protein PfCyRPA that binds the host sugar Neu5Ac, and they found that disrupting this interaction impedes parasite growth. A map of the receptor-binding site identifies an attractive target for antimalarial vaccines and therapeutics.

Sequence-structure-function characterization of the emerging tetracycline destructase family of antibiotic resistance enzymes.

Tetracycline destructases (TDases) are flavin monooxygenases which can confer resistance to all generations of tetracycline antibiotics. The recent increase in the number and diversity of reported TDase sequences enables a deep investigation of the TDase sequence-structure-function landscape. Here, we evaluate the sequence determinants of TDase function through two complementary approaches: (1) constructing profile hidden Markov models to predict new TDases, and (2) using multiple sequence alignments to identify conserved positions important to protein function. Using the HMM-based approach we screened 50 high-scoring candidate sequences in Escherichia coli, leading to the discovery of 13 new TDases. The X-ray crystal structures of two new enzymes from Legionella species were determined, and the ability of anhydrotetracycline to inhibit their tetracycline-inactivating activity was confirmed. Using the MSA-based approach we identified 31 amino acid positions 100% conserved across all known TDase sequences. The roles of these positions were analyzed by alanine-scanning mutagenesis in two TDases, to study the impact on cell and in vitro activity, structure, and stability. These results expand the diversity of TDase sequences and provide valuable insights into the roles of important residues in TDases, and flavin monooxygenases more broadly.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Naturally acquired antibodies against Plasmodium vivax pre-erythrocytic stage vaccine antigens inhibit sporozoite invasion of human hepatocytes in vitro.

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites.

Duffy antigen is expressed during erythropoiesis in Duffy-negative individuals.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.

Host receptor binding induces conformational changes in a PfEMP1 protein.

In this issue of Structure, Raghavan et al. present the cryo-EM structures of the malaria-associated group A PfEMP1 HB3VAR03 head in both host receptor-free and receptor-bound states. The structures are complemented by biophysical analysis and introduce an innovative model in which host-receptor binding induces conformational changes in a PfEMP1 protein.

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Structure-based design of a strain transcending AMA1-RON2L malaria vaccine.

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control.

A potent and durable malaria transmission-blocking vaccine designed from a single-component 60-copy Pfs230D1 nanoparticle.

Malaria transmission-blocking vaccines (TBVs) reduce disease transmission by breaking the continuous cycle of infection between the human host and the mosquito vector. Domain 1 (D1) of Pfs230 is a leading TBV candidate and comprises the majority of transmission-reducing activity (TRA) elicited by Pfs230. Here we show that the fusion of Pfs230D1 to a 60-copy multimer of the catalytic domain of dihydrolipoyl acetyltransferase protein (E2p) results in a single-component nanoparticle composed of 60 copies of the fusion protein with high stability, homogeneity, and production yields. The nanoparticle presents a potent human transmission-blocking epitope within Pfs230D1, indicating the antigen is correctly oriented on the surface of the nanoparticle. Two vaccinations of New Zealand White rabbits with the Pfs230D1 nanoparticle elicited a potent and durable antibody response with high TRA when formulated in two distinct adjuvants suitable for translation to human use. This single-component nanoparticle vaccine may play a key role in malaria control and has the potential to improve production pipelines and the cost of manufacturing of a potent and durable TBV.

A human antibody epitope map of the malaria vaccine antigen Pfs25.

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01. These structures revealed additional epitopes in Pfs25 capable of reducing transmission and expanded this characterization to malaria-exposed humans. This work informs immunogen design to focus the antibody response to transmission-reducing epitopes of Pfs25, enabling development of more potent transmission-blocking vaccines for malaria.

Designing an effective malaria vaccine targeting Plasmodium vivax Duffy-binding protein.

Malaria caused by the Plasmodium vivax parasite is a major global health burden. Immunity against blood-stage infection reduces parasitemia and disease severity. Duffy-binding protein (DBP) is the primary parasite protein responsible for the invasion of red blood cells and it is a leading subunit vaccine candidate. An effective vaccine, however, is still lacking despite decades of interest in DBP as a vaccine candidate. This review discusses the reasons for targeting DBP, the challenges associated with developing a vaccine, and modern structural vaccinology methods that could be used to create an effective DBP vaccine. Next-generation DBP vaccines have the potential to elicit a broadly protective immune response and provide durable and potent protection from P. vivax malaria.

Structure of anhydrotetracycline-bound Tet(X6) reveals the mechanism for inhibition of type 1 tetracycline destructases.

Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline's inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance.

Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P. falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be utilized by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex-vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not impacted by lack of CD44. However, the rate of P. falciparum invasion was substantially reduced in CD44-null cRBCs relative to isogenic wild-type (WT) control cells, validating CD44 as an important host factor for this parasite. We identified two P. falciparum invasion ligands as binding partners for CD44, Erythrocyte Binding Antigen-175 (EBA-175) and EBA-140, and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors-glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model where P. falciparum exploits CD44 as a co-receptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Immunological characterization of a VIR protein family member (VIR-14) in Plasmodium vivax-infected subjects from different epidemiological regions in Africa and South America.

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). Deepening our knowledge of parasite biology and its molecular components is key to develop new tools for malaria control and elimination. This study aims to investigate and characterize a P. vivax protein (PvVir14) for its role in parasite biology and its interactions with the immune system. We collected sera or plasma from P.vivax-infected subjects in Brazil (n = 121) and Cambodia (n = 55), and from P. falciparum-infected subjects in Mali (n = 28), to assess antibody recognition of PvVir14. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. IgG1 and IgG3 most frequently contributed to anti-PvVir14 responses. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21- CD27-) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. When analyzed at a single-cell level, the B cell receptor gene hIGHV3-23 was only seen in subjects with active P.vivax infection where it comprised 20% of V gene usage. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. Specific B cell subsets, anti-PvVir14 circulating antibodies, and NKT cell levels declined after treatment of P. vivax. This study provides the immunological characterization of PvVir14, a unique P. vivax protein, and possible association with acute host's immune responses, providing new information of specific host-parasite interaction. Trial registration: TrialClinicalTrials.gov Identifier: NCT00663546 & ClinicalTrials.gov NCT02334462.

Structure-Based Design of Bisubstrate Tetracycline Destructase Inhibitors That Block Flavin Redox Cycling.

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

Design of a stabilized RBD enables potently neutralizing SARS-CoV-2 single-component nanoparticle vaccines.

Waning immunity and emerging variants necessitate continued vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Improvements in vaccine safety, tolerability, and ease of manufacturing would benefit these efforts. Here, we develop a potent and easily manufactured nanoparticle vaccine displaying the spike receptor-binding domain (RBD). Computational design to stabilize the RBD, eliminate glycosylation, and focus the immune response to neutralizing epitopes results in an RBD immunogen that resolves issues hindering the efficient nanoparticle display of the native RBD. This non-glycosylated RBD can be genetically fused to diverse single-component nanoparticle platforms, maximizing manufacturing ease and flexibility. All engineered RBD nanoparticles elicit potently neutralizing antibodies in mice that far exceed monomeric RBDs. A 60-copy particle (noNAG-RBD-E2p) also elicits potently neutralizing antibodies in non-human primates. The neutralizing antibody titers elicited by noNAG-RBD-E2p are comparable to a benchmark stabilized spike antigen and reach levels against Omicron BA.5 that suggest that it would provide protection against emerging variants.