H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein

The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated.

Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.

Improved antibody structure prediction by deep learning of side chain conformations

Antibody engineering is becoming increasingly popular in the medical field for the development of diagnostics and immunotherapies. Antibody function relies largely on the recognition and binding of antigenic epitopes via the loops in the complementarity determining regions. Hence, accurate high-resolution modeling of these loops is essential for effective antibody engineering and design. Deep learning methods have previously been shown to effectively predict antibody backbone structures described as a set of inter-residue distances and orientations. However, antigen binding is also dependent on the specific conformations of surface side chains. To address this shortcoming, we created DeepSCAb: a deep learning method that predicts inter-residue geometries as well as side chain dihedrals of the antibody variable fragment. The network requires only sequence as input, rendering our method particularly useful for antibodies without any known backbone conformations. Rotamer predictions use an interpretable self-attention layer, which learns to identify structurally conserved anchor positions across several species. We evaluate the performance of our model for discriminating near-native structures from sets of decoys and find that DeepSCAb outperforms similar methods lacking side chain context. When compared to alternative rotamer repacking methods, which require an input backbone structure, DeepSCAb predicts side chain conformations competitively. Our findings suggest that DeepSCAb improves antibody structure prediction with accurate side chain modeling and is adaptable to applications in docking of antibody-antigen complexes and design of new therapeutic antibody sequences.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74–100% (94.79–100%), 91.9–100% (96.3–100%) and 90.71–100% (93.51–100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5′UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3′UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3′ termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Emergence of equine-like G3 strains as the dominant rotavirus among children under five with diarrhea in Sabah, Malaysia during 2018–2019

Rotavirus infection is a dilemma for developing countries, including Malaysia. Although commercial rotavirus vaccines are available, these are not included in Malaysia’s national immunization program. A scarcity of data about rotavirus genotype distribution could be partially to blame for this policy decision, because there are no data for rotavirus genotype distribution in Malaysia over the past 20 years. From January 2018 to March 2019, we conducted a study to elucidate the rotavirus burden and genotype distribution in the Kota Kinabalu and Kunak districts of the state of Sabah. Stool specimens were collected from children under 5 years of age, and rotavirus antigen in these samples was detected using commercially available kit. Electropherotypes were determined by polyacrylamide gel electrophoresis of genomic RNA. G and P genotypes were determined by RT-PCR using type specific primers. The nucleotide sequence of the amplicons was determined by Sanger sequencing and phylogenetic analysis was performed by neighbor-joining method. Rotavirus was identified in 43 (15.1%) children with watery diarrhea. The male:female ratio (1.9:1) of the rotavirus-infected children clearly showed that it affected predominantly boys, and children 12–23 months of age. The genotypes identified were G3P[8] (74% n = 31), followed by G1P[8] (14% n = 6), G12P[6](7% n = 3), G8P[8](3% n = 1), and GxP[8] (3% n = 1). The predominant rotavirus circulating among the children was the equine-like G3P[8] (59.5% n = 25) with a short electropherotype. Eleven electropherotypes were identified among 34 strains, indicating substantial diversity among the circulating strains. The circulating genotypes were also phylogenetically diverse and related to strains from several different countries. The antigenic epitopes present on VP7 and VP4 of Sabahan G3 and equine-like G3 differed considerably from that of the RotaTeq vaccine strain. Our results also indicate that considerable genetic exchange is occurring in Sabahan strains. Sabah is home to a number of different ethnic groups, some of which culturally are in close contact with animals, which might contribute to the evolution of diverse rotavirus strains. Sabah is also a popular tourist destination, and a large number of tourists from different countries possibly contributes to the diversity of circulating rotavirus genotypes. Considering all these factors which are contributing rotavirus genotype diversity, continuous surveillance of rotavirus strains is of utmost importance to monitor the pre- and post-vaccination efficacy of rotavirus vaccines in Sabah.

Next-Generation Bioinformatics Approaches and Resources for Coronavirus Vaccine Discovery and Development—A Perspective Review

COVID-19 is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To fight this pandemic, which has caused a massive death toll around the globe, researchers are putting efforts into developing an effective vaccine against the pathogen. As genome sequencing projects for several coronavirus strains have been completed, a detailed investigation of the functions of the proteins and their 3D structures has gained increasing attention. These high throughput data are a valuable resource for accelerating the emerging field of immuno-informatics, which is primarily aimed toward the identification of potential antigenic epitopes in viral proteins that can be targeted for the development of a vaccine construct eliciting a high immune response. Bioinformatics platforms and various computational tools and databases are also essential for the identification of promising vaccine targets making the best use of genomic resources, for further experimental validation. The present review focuses on the various stages of the vaccine development process and the vaccines available for COVID-19. Additionally, recent advances in genomic platforms and publicly available bioinformatics resources in coronavirus vaccine discovery together with related immunoinformatics databases and advances in technology are discussed.

Identification of antigenic epitopes on the F and G glycoproteins of bovine respiratory syncytial virus and in vitro assessment of their synthetic peptide vaccine potential

Globally, bovine respiratory disease (BRD) remains the principal reason for mortality of calves over one month of age despite the availability of various vaccines on the UK market. Bovine respiratory syncytial virus (BRSV) was first discovered in the 1970s and is now considered a principal pathogen implicated in the disease complex. Outbreaks occur annually and re-infections are common even in the presence of maternal antibodies. Difficulties have arisen from using both live-attenuated and inactivated vaccines and recent efforts have focused on the development of sub-unit vaccines that are suitable for use in neonatal calves with maternally-derived circulating antibodies. This study was undertaken to identify antigenic epitopes on two of the surface glycoproteins of BRSV, the fusion (F) and attachment (G) proteins, the major surface viral antigens, for inclusion into a novel subunit peptide vaccine. Sequencing and antigenicity prediction of the F and G genes of BRSV revealed 21 areas of potential antigenicity; of which genuine peptide/antisera binding occurred with 4 peptides. Identification of the antigenic components of a vaccine is an important first step in the development of novel BRSV vaccines and this data, therefore, provides the basis for the generation of such vaccines.

Extended live-cell barcoding approach for multiplexed mass cytometry

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. Sample pooling after prior surface staining termed live-cell barcoding is more desirable than intracellular barcoding, where samples are pooled after fixation and permeabilization, since it does not depend on fixation-sensitive antigenic epitopes. In live-cell barcoding, the general approach uses two tags per sample out of a pool of antibodies paired with five palladium (Pd) isotopes in order to preserve appreciable signal-to-noise ratios and achieve higher yields after sample deconvolution. The number of samples that can be pooled in an experiment using live-cell barcoding is limited, due to weak signal intensities associated with Pd isotopes and the relatively low number of available tags. Here, we describe a novel barcoding technique utilizing 10 different tags, seven cadmium (Cd) tags and three Pd tags, with superior signal intensities that do not impinge on lanthanide detection, which enables enhanced pooling of samples with multiple experimental conditions and markedly enhances sample throughput.