Dynamic Expedition of Leading Mutations in SARS-CoV-2 Spike Glycoproteins

During the ongoing CoVID-19 epidemic, the continuous genomic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been generating new variants with enhanced transmissibility and immune escape. Being one key target of antibodies, mutations of the spike glycoprotein play a vital role in the trajectory of virus evasion. Here, we present a time-resolved statistical method, dynamic expedition of leading mutations (deLemus), to analyze the evolution dynamics of the spike protein. Together with analysis on single amino-acid polymorphism (SAP), we proposed one L-index to quantify the mutation strength of each amino acid for unravelling mutation pattern of spike glycoprotein. The sites of interest (SOI) with high L-index hold great promise to detect potential signal of emergent variants.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair

Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

Prophylactic Administration of Antibodies Specific for the HPA-1a Epitope Prevents Alloimmunization and Platelet Destruction in a Murine Model of Fetal Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited antigens present on the surface of fetal and neonatal platelets. The human platelet alloantigen HPA-1a (formerly known as the Pl A1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism near the amino terminus of the integrin b3 subunit (known as GPIIIa in the platelet literature) serves as the central target for alloantibody binding, leading to clearance of both fetal and neonatal platelets, thrombocytopenia, and in the most severe cases, spontaneous- or trauma-induced intracranial hemorrhage. Unlike hemolytic disease of the newborn, which occurs in pregnancies subsequent to parturition-induced alloimmunization, an estimated 25% to 50% of FNAIT cases occur without warning during gestation of the first pregnancy. Though long proposed, there are currently no approved therapies for the prevention of FNAIT. We recently described the development of transgenic mice expressing the human HPA-1a allogeneic epitope on a murine GPIIIa backbone. Transfusion of such platelets into wild-type female mice induced the generation of high-titer anti-HPA-1a alloantibodies that can cross the placenta and recapitulate many of the relevant clinical features of FNAIT. To test the hypothesis that rapid elimination of fetal HPA-1a positive platelets from the circulation of a mother who is HPA-1a negative might prevent maternal alloimmunization and the development of FNAIT, we administered either a hyperimmune plasma-derived polyclonal anti-HPA-1a antibody derived from females having previous cases of FNAIT (termed RLYB211), or a novel human monoclonal antibody directed against the HPA-1a epitope (termed RLYB212), to wild-type female mice prior to challenging them with HPA-1a-positive murine platelets. RLYB211 and RLYB212 were each able to effect the rapid removal of HPA-1a-positive platelets from murine circulation and prevent the development of anti-HPA-1a alloantibodies. Importantly, wild-type female mice pretreated with RLYB211 prior to exposure to HPA-1a-positive platelets, and then impregnated by HPA-1a-positive males, gave birth to HPA-1a-positive pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish the potential for prophylactic polyclonal and monoclonal anti-HPA-1a antibody therapy for the prevention of FNAIT in humans. Disclosures Sheridan: Rallybio: Current Employment, Current equity holder in publicly-traded company. Newman: Rallybio: Consultancy, Research Funding.

Genetic Diversity of Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) in Field Isolates from Five Different Areas of the Brazilian Amazon

The Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) has an important role in erythrocyte invasion and has been considered a target for vivax malaria vaccine development. Nonetheless, its genetic diversity remains uncharted in Brazilian malaria-endemic areas. Therefore, we investigated the pvcyrpa genetic polymorphism in 98 field isolates from the Brazilian Amazon and its impact on the antigenicity of predicted B-cell epitopes. Genetic diversity parameters, population genetic analysis, neutrality test and the median-joining network were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. One synonymous and 26 non-synonymous substitutions defined fifty haplotypes. The nucleotide diversity and Tajima’s D values varied across the coding gene. The exon-1 sequence had greater diversity than those of exon-2. Concerning the prediction analysis, seven sequences were predicted as linear B cell epitopes, the majority contained in conformational epitopes. Moreover, important amino acid polymorphism was detected in regions predicted to contain residues participating in B-cell epitopes. Our data suggest that the pvcyrpa gene presents a moderate polymorphism in the studied isolates and such polymorphisms alter amino acid sequences contained in potential B cell epitopes, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide.

Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair

Cooperation between receptors from the NLR superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

A novel strain-specific neutralizing epitope on glycoprotein H of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent MAbs 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the analyzed HCMV strains. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinant derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on expression of the entire ectodomain of gH, whereas 6E3 required residues 1-629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single Met->Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is equally distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 for the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV-strains during pregnancy or after transplantation. Importance HCMV infections are life-threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV-infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH, which only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain-specificity is dependend on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV-strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 for the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.

The strength of selection is consistent across both domains of the MHC class I peptide-binding groove in birds

Background The Major Histocompatibility Complex (MHC) codes for the key vertebrate immune receptors responsible for pathogen recognition. Foreign antigens are recognized via their compatibility to hyper-variable region of the peptide-binding groove (PBR), which consists of two separate protein domains. Specifically, the PBR of the MHC class I receptors, which recognize intra-cellular pathogens, has two α domains encoded by exon 2 (α1) and exon 3 (α2) of the same gene. Most research on avian MHC class I polymorphism has traditionally focused exclusively on exon 3 and comparisons of selection between the two domains have been hampered by the scarcity of molecular data for exon 2. Thus, it is not clear whether the two domains vary in their specificity towards different antigens and whether they are subject to different selective pressure. Results Here, we took advantage of rapidly accumulating genomic resources to test for the differences in selection patterns between both MHC class I domains of the peptide-binding groove in birds. For this purpose, we compiled a dataset of MHC class I exon 2 and 3 sequences for 120 avian species from 46 families. Our phylogenetically-robust approach provided strong evidence for highly consistent levels of selection on the α1 and α2 domains. There were strong correlations in all selection measures (number of positively/negatively selected residues and dN/dS ratios) between both PBR exons. Similar positive associations were found for the level of amino acid polymorphism across the two domains. Conclusions We conclude that the strength of selection and the level of polymorphism are highly consistent between both peptide-binding domains (α1 and α2) of the avian MHC class I.