Intra-Serotype Polyprotein Variation and its Effect on Antigenicity of Dengue Virus

Dengue virus is a mosquito-borne human pathogen, causing disease that ranges from mild febrile illness to life-threatening hemorrhage fever/ shock syndrome. The altered antigenicity and virulence in the dengue virus, resulting from the accumulation and fixation of the favorable mutations in the genome, is the cause of concern nowadays. The present study focuses on the comparative study of polyproteins of viral strains within each dengue serotype to understand the trend of intra-serotype polyprotein variation and its effect on the antigenicity. Polyprotein sequences of viral strains in each serotype were investigated using multivariate statistical analysis, phylogenetic analysis and multiple sequence alignment methods. Epitope prediction was done by Bepipred-1.0 server and experimental epitope data were extracted from Immune Epitope Database with BLAST search. The study reveals that the polyproteins of viral strains of a serotype have variable amino acid composition that corresponds to the geographical regions of origin. This compositional variation has occurred due to the presence of polymorphic residues at different positions along the polyprotein sequence. The polymorphic residues have also been identified at epitope regions of structural proteins as well as NS1 of viral strains, possessing dissimilar physicochemical properties and occupy surface accessible positions. These positions on epitopes with polymorphic, dissimilar and surface accessible residues might act as putative sites for generation of antigenic variation among viral strains of a serotype of different geographical origin. Thus, these polymorphic residue positions on epitopes might be considered as putative target for development of drug or vaccine, in future.

Global distribution of single amino acid polymorphisms in Plasmodium vivax Duffy-binding-like domain and implications for vaccine development efforts

Plasmodium vivax ( Pv ) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in Pv DBL globally, with an emphasis on India as it remains a major contributor to the global Pv malaria burden. Based on 1358 Pv DBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of Pv DBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the Pv DBL subdomain 2 ( α 1– α 6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of Pv DBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in Pv DBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.

A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease

The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5–mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.

Atomic-Level Dissection of the Polyclonal Immune Response to the Human Platelet Alloantigen, HPA-1a (PlA1)

Alloantibodies to platelet-specific antigens are responsible for two clinically-important bleeding disorders: Post-transfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The HPA-1a/1b (also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP)IIIa is controlled by a Leu33Pro polymorphism, and is responsible for ~80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa isoforms harboring select humanized residues within the PSI and EGF1 domain, and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of murine GPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies, however humanizing distinct amino acids within the linearly distant, but conformationally close, EGF1 domain was required to enable binding of others. Using a series of transgenic mice and recombinant constructs, we reveal previously unsuspected, complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT by providing cells that distinguish specific alloantibody subpopulations, and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents. Disclosures Curtis: Ionis Pharmaceuticals: Consultancy.

High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (PlA1)

Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a–specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti–HPA-1a reagents.

A Polymorphic Residue That Attenuates the Antiviral Potential of Interferon Lambda 4 in Hominid Lineages

As antimicrobial signalling molecules, type III or lambda interferons (IFNλs) are critical for defence against infection by diverse pathogens. Counter-intuitively, expression of one member of the family, IFNλ4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural in-frame nucleotide insertion that abrogates IFNλ4 production improves viral clearance. To further understand how genetic variation between and within species affects IFNλ4 function, we screened a panel of extant coding variants of human IFNλ4 and identified three variants that substantially affect antiviral activity (P70S, L79F and K154E). The most notable variant was K154E, which enhancedin vitroactivity in a range of antiviral and interferon stimulated gene (ISG) assays. This more active E154 variant of IFNλ4 was found only in African Congo rainforest ‘Pygmy’ hunter-gatherers. Remarkably, E154 was highly conserved as the ancestral residue in mammalian IFNλ4s yet K154 is the dominant variant throughout evolution of the hominid genusHomo. Compared to chimpanzee IFNλ4, the human orthologue had reduced activity due to amino acid substitution of glutamic acid with lysine at position 154. Meta-analysis of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFNλ4 is consistent with differences in antiviral gene expressionin vivoduring HCV infection. Mechanistically, our data suggest that human-specific K154 likely affects IFNλ4 activity by reducing secretion and potency. We postulate that evolution of an IFNλ4 with attenuated activity in humans (K154) likely contributes to distinct host-specific responses to and outcomes of infection, such as HCV.

Involvement of the EGF Domain in the Formation of the Human Platelet Alloantigen, HPA-1a

The human platelet alloantigen, HPA-1a (also known as PlA1), is most frequently responsible for several clinically important alloimmune platelet disorders, including neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura, and less frequently, platelet transfusion refractoriness. A single C29523T nucleotide substitution, resulting in a Leu33Pro amino acid polymorphism within PSI domain of the integrin β3 subunit, controls the expression of the HPA-1a/HPA-1b alloantigenic epitopes. Small linear or cyclic synthetic peptides encompassing amino acid 33, however, fail to bind human anti-HPA-1a alloantibodies, leading to the notion that the HPA-1a antigenic determinant is conformationally dependent, and may require noncontiguous regions of the polypeptide chain that are linearly distal to polymorphic residue 33. This concept is supported by the finding that antibodies from some alloimmunized women fail to recognize a Cys435Ala mutant form of β3, and by the observation that the PSI domain of β3 is immediately adjacent in the crystal structure of β3 to the first epidermal growth factor (EGF1 - residues 436-472) domain of the molecule. The purpose of the present investigation, therefore, was to localize the amino acid(s) within EGF1 that contribute to the HPA-1a alloantibody binding interface. Two complementary approaches were employed. In the first, site-directed mutagenesis was used to introduce four amino acid substitutions (T30A, S32P, Q33L, and N39D) into mouse β3 cDNA that have previously been shown to be necessary for reconstituting the human anti-HPA-1a epitope into the murine protein. The resulting mouse β3 integrin subunit containing this "humanized" PSI domain (termed APLDmβ3) was able to bind both the murine HPA-1a-selective monoclonal antibody, SZ21, as well as human maternal anti-HPA-1a antisera. Structural analysis indicates that S469 and Q470 at the C-terminus of EGF1 are immediately adjacent to Leu33 in human β3. Ser469 is conserved in the mouse, however residue 470 is a methionine in the murine protein. Based on these observations, we introduced an additional M470Q amino acid substitution within APLDmβ3 to produce APLDQmβ3. As predicted, binding of mAb SZ21 and human anti-HPA-1a alloantisera toHEK293 cells expressing an aIIb/APLDQmβ3 complex was increased ~30% over that which bound to cells expressing similar levels of aIIb/APLDmβ3. Conversely, substitution of M for Q at position 470 within human β3 resulted in a marked reduction in the ability of SZ21 and human anti-HPA-1a alloantisera to bind. Taken together, ourresults demonstrate that this region of the conformationally close EGF1 domain contributes importantly to the expression of the HPA-1a alloantigenic determinant. These findings may have important implications for diagnostic and therapeutic use of synthetic or recombinant HPA-1a mimetics. Disclosures No relevant conflicts of interest to declare.