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Objective: To investigate whether the specificity of antibody responses to the gp120 V3 domain in HIV-1-infected individuals is related to the variability of this region., Methods: Sera from a cohort of 22 HIV-1-infected Ugandans were tested against peptides derived from each individual's autologous proviral V3 apex sequence. Autologous peptide reactivity was compared with reactivity to peptides derived from two Ugandan consensus sequences and previously isolated US/European and African viruses. Peptides from individuals with heterogeneous V3 apex sequences, representing different HIV-1 variants, were obtained and tested against the corresponding sera., Results: A notable cross-reactivity to different V3 apex peptides was observed. However, in the majority of sera, antibody reactivity to the autologous peptides was found to exceed reactivity to any of the other peptides tested. V3 proviral sequences from the Ugandan cohort studied have been shown to be closely related to the HIV-1MN isolate and thus, their sera gave better reactivity to V3MN and related peptides than to peptides representing other African HIV-1 isolates. In individuals with heterogeneous V3 proviral sequences, we could distinguish divergent antibody responses to the genomic variants differing by single amino acids., Conclusion: Analysis of seroreactivity to peptides might constitute a relevant tool for investigating the variability of the HIV-1 gp120 V3 domain within infected populations and single individuals.

The T-cell-mediated immune response exhibits a crucial function in the control of the intrahepatic proliferation of Echinococcus multilocularis larvae in mice and humans, both being natural intermediate hosts of the parasite. Antigen B (AgB), a metabolized Echinococcus spp. lipoprotein, contributes to the modulation of the T-cell immune response, and distinct sites of the corresponding AgB1, AgB3 and AgB4 genes were shown to be under positive selection pressure. Since several AgB gene variants are present in a single Echinococcus metacestode, we used secondary E. multilocularis infections in BALB/c and in athymic nude mice (devoid of T-cell responses) to analyze the effect of the cellular immune response on the expression and diversity of EmAgB1-EmAgB4 genes. We demonstrated hereby that EmAgB transcripts were less abundant in nude mice during the early phase of infection (at one month post-infection), and that EmAgB2 is simultaneously down-regulated when compared to the other three genes. A negative relationship exists between the level of transcription and diversity of EmAgB genes. Moreover, no excess of non-synonymous substitutions was found among the distinct EmAgB alleles from a single host. Together, these results pointed to the effect of purifying selection, which seemed to eliminate the detrimental AgB variants generated during the development of the metacestode within the peritoneal cavity of its intermediate host.

Background: Although the tprK gene of Treponema pallidum are thought to play a critical role in the pathogenesis of syphilis, the profile of variations in tprK during the development of human syphilis infection have remained unclear. Methods/Principal findings: Through next-generation sequencing, we compared the tprK gene of 14 secondary syphilis patients with that of 14 primary syphilis patients, and the results showed an increased number of variants within the seven V regions of the tprK gene in the secondary syphilis samples. The length of the sequences within each V region also presented a 3-bp changing pattern. Interestingly, the frequencies of predominant sequences within the V regions in the secondary syphilis samples were generally decreased compared with those found in the primary syphilis samples, particularly in the V7 region, where a frequency below 60% was found in up to 57% (8/14) of all secondary samples compared with 7% (1/14) of all primary samples. Moreover, the number of minor variants distributed between frequencies of 10 and 49.9% was increased. The alignment of all amino acid sequences within each V region of the primary and secondary syphilis samples revealed that some amino acid sequences, particularly the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1, were highly stable. Additionally, the amino acid sequences in V6 also exhibited notable intrastrain heterogeneity and were likely to form a strain-specific pattern at the interstrain level. Conclusions: The identification of different profiles of the tprK gene in primary and secondary syphilis patients indicated that the tprK gene of T. pallidum undergoes constant variation to result in the best adaptation to the host. The highly stable peptides found in V1 are likely promising potential vaccine components. The highly heterogenetic regions (e.g., V6) could help to understand the role of tprK in immune evasion. [ABSTRACT FROM AUTHOR]

Introduction: The study presents the results of the genomic surveillance of invasive meningococcal disease (IMD) in the Czech Republic for the period of 2015–2017. Material and methods: The study set includes all available IMD isolates recovered in the Czech Republic and referred to the National Reference Laboratory for Meningococcal Infections in 2015–2017, a total of 89 Neissseria meningitidis isolates—from 2015 (n = 20), 2016 (n = 27), and from 2017 (n = 42). All isolates were studied by whole genome sequencing (WGS). Results: Serogroup B (MenB) was the most common, followed by serogroups C, W, and Y. Altogether 17 clonal complexes were identified, the most common of which was hypervirulent complex cc11, followed by complexes cc32, cc41/44, cc269, and cc865. Over the three study years, hypervirulent cc11 (MenC) showed an upward trend. The WGS method showed two clearly differentiated clusters of N. meningitidis C: P1.5,2:F3-3:ST-11 (cc11). The first cluster is represented by nine isolates, all of which are from 2017. The second cluster consisted of five isolates from 2016 and eight isolates from 2017. Their genetic discordance is illustrated by the changing nadA allele and subsequently by the variance in BAST type. Clonal complex cc269 (MenB) also increased over the time frame. WGS identified the presence of MenB vaccine antigen genes in all B and non-B isolates of N. meningitidis. Altogether 49 different Bexsero antigen sequence types (BAST) were identified and 10 combinations of these have not been previously described in the PubMLST database. Conclusions: The genomic surveillance of IMD in the Czech Republic provides data needed to update immunisation guidelines for this disease. WGS showed a higher discrimination power and provided more accurate data on molecular characteristics and genetic relationships among invasive N. meningitidis isolates. [ABSTRACT FROM AUTHOR]

The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2substitution/site/year (s/s/y) (95% CI 1.362–2.31 x 10−2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS (ω) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM. [ABSTRACT FROM AUTHOR]

Background: In 2015–2016, a cross-sectional carriage survey was performed in Tuscany Region, Italy, during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C clonal complex 11 (MenC:cc11). This study aims to evaluate the genomic profile of meningococcal carriage isolates collected during the survey. Methods: Whole-genome sequencing (WGS) was performed using Illumina MiSeq on 85 cultivated meningococcal carriage isolates received at the Dept. of Infectious Disease, National Institute of Health (Istituto Superiore di Sanità, ISS), as National Reference Laboratory (NRL) for Invasive Meningococcal Disease (IMD). De novo assembled genomes were scanned by the BIGSdb platform to assign: the genotypic profiles, the cgMLST, the vaccine antigen variants and allele types of antimicrobial resistance associated genes, together with denitrification pathway loci. Results: Capsule null and non-groupable meningococci accounted for 52.9% and 10.6%, respectively. Among the remaining carriage isolates, serogroup B was the predominant (71.0%). Serogroup C meningococci were culture negative and unavailable for WGS. Overall, 64 genotypic profiles were identified and, based on cgMLST, isolates clustered according to clonal complexes. Eight isolates (9.4%) harbored at least one gene encoding a 4CMenB vaccine antigen. Mutated penA alleles were found in more than 82%. Finally, complete aniA and norB coding sequences were detected among 71.8% of carriage isolates. Conclusions: Meningococcal carriage isolates collected during the MenC:cc11 outbreak were characterized by an extensive genetic diversity. The lack of outbreak-related isolates among carriage might be attributable to the high transmissibility with low duration of colonization of MenC:cc11 meningococci. [ABSTRACT FROM AUTHOR]

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads–the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process. [ABSTRACT FROM AUTHOR]

The article examines whether the unique camouflage strategy of the fungal genus Pneumocystis is associated with its particular niche within host lungs. Topics discussed include the eight different gene families encoding major surface glycoproteins (Msg) in Pneumocystis species, the structure of the genes belonging to the different Msg families, and the surface structure of Pneumocystis cells.

Background: Despite the progress achieved in scaling-up mass drug administration (MDA) for lymphatic filariasis (LF) in Ghana, communities with persistent LF still exist even after 10 years of community treatment. To understand the reasons for persistence, we conducted a study to assess the status of disease elimination and understand the adherence to interventions including MDA and insecticide treated nets. Methodology and principal findings: We conducted a parasitological and epidemiological cross-sectional study in adults from eight villages still under MDA in the Northern Region savannah and the coastal Western Region of the country. Prevalence of filarial antigen ranged 0 to 32.4% and in five villages the prevalence of night blood microfilaria (mf) was above 1%, ranging from 0 to 5.7%. Median mf density was 67 mf/ml (range: 10–3,560). LF antigen positivity was positively associated with male sex but negatively associated with participating in MDA the previous year. Male sex was also associated with a decreased probability of participating in MDA. A stochastic model (TRANSFIL) was used to assess the expected microfilaria prevalence under different MDA coverage scenarios using historical data on one community in the Western Region. In this example, the model simulations suggested that the slow decline in mf prevalence is what we would expect given high baseline prevalence and a high correlation between MDA adherence from year to year, despite high MDA coverage. Conclusions: There is a need for an integrated quantitative and qualitative research approach to identify the variations in prevalence, associated risk factors and intervention coverage and use levels between and within regions and districts. Such knowledge will help target resources and enhance surveillance to the communities most at risk and to reach the 2020 LF elimination goals in Ghana. [ABSTRACT FROM AUTHOR]

Herd protection, resulting from the interruption of transmission and asymptomatic carriage, is an important element of the effectiveness of vaccines against the meningococcus. Whilst this has been well established for conjugate polysaccharide vaccines directed against the meningococcal capsule, two uncertainties surround the potential herd protection provided by the novel protein-based vaccines that are used in place of serogroup B (MenB) polysaccharide vaccines (i) the strain coverage of such vaccines against carried meningococci, which are highly diverse; and (ii) the generation of a protective immune response in the mucosa. These considerations are essential for realistic estimates of cost-effectiveness of new MenB vaccines. Here the first of these questions is addressed by the whole genome sequence (WGS) analysis of meningococci isolated from healthy military recruits and university students in Greece. The study included a total of 71 MenB isolates obtained from 1420 oropharyngeal single swab samples collected from military recruits and university students on voluntary basis, aged 18–26 years. In addition to WGS analysis to identify genetic lineage and vaccine antigen genes, including the Bexsero Antigen Sequence Type (BAST), the isolates were examined with the serological Meningococcal antigen Typing System (MATS) assay. Comparison of these data demonstrated that the carried meningococcal population was highly diverse with 38% of the carriage isolates showed expression of antigens matching those included in the 4CMenB vaccine. Our data may suggest a limited potential herd immunity to be expected and be driven by an impact on a subset of carriage isolates. [ABSTRACT FROM AUTHOR]

H1N1 is the earliest emerging subtype of influenza A viruses with available genomic sequences, has caused several pandemics and seasonal epidemics, resulting in millions of deaths and enormous economic losses. Timely determination of new antigenic variants is crucial for the vaccine selection and flu prevention. In this study, we chronologically divided the H1N1 strains into several periods in terms of the epidemics and pandemics. Computational models have been constructed to predict antigenic variants based on epidemic and pandemic periods. By sequence analysis, we demonstrated the diverse mutation patterns of HA1 protein on different periods and that an individual model built upon each period can not represent the variations of H1N1 virus. A stacking model was established for the prediction of antigenic variants, combining all the variation patterns across periods, which would help assess a new influenza strain’s antigenicity. Three different feature extraction methods, i.e. residue-based, regional band-based and epitope region-based, were applied on the stacking model to verify its feasibility and robustness. The results showed the capability of determining antigenic variants prediction with accuracy as high as 0.908 which performed better than any of the single models. The prediction performance using the stacking model indicates clear distinctions of mutation patterns and antigenicity between epidemic and pandemic strains. It would also facilitate rapid determination of antigenic variants and influenza surveillance. [ABSTRACT FROM AUTHOR]

Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination. [ABSTRACT FROM AUTHOR]

The article discusses the antigenic variation systems adopted by microbes in the case of Trypanosoma brucei which is the causative agent of African sleeping sickness and has produced variable surface glycoprotein (VSG) for immune avoidance. It states Briggs and colleagues report suggests the role of DNA recombination and repair factors of RNA–DNA hybrids wherein genetic inactivation of the RNaseH1 enzyme promotes VSG antigenic variation. It notes many questions remain unanswered.

The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development. [ABSTRACT FROM AUTHOR]

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise—including whether there is a dedicated and ES-focused mechanism—is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication–transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host. [ABSTRACT FROM AUTHOR]

Background: Due to the high rate of antigenic variation of influenza virus, seasonal characterization of the virus is crucial to assess and monitor the emergence of new pathogenic variants and hence formulate effective control measures. However, no study has yet been conducted in Mozambique to assess genetic, antigenic and antiviral susceptibility profile of influenza virus. Methods: A subset of samples (n = 20) from influenza positive children detected in two hospitals in Maputo city during 2015 season as part of the implementation of influenza surveillance system, were selected. The following assays were performed on these samples: antigenic characterization by hemagglutination inhibition assay, genetic characterization by Sanger sequencing of hemagglutinin (HA) and neuraminidase (NA) and susceptibility to oseltamivir and zanamivir (NA inhibitors) by enzymatic assay. Results: The A(H1N1)pdm09 subtype viruses remained closely related antigenically and genetically to the 2016 vaccine virus A/California/7/2009 and other widely distributed viruses belonging to genetic group 6B. The majority of influenza A(H3N2) viruses studied were antigenically similar to the 2016–2017 vaccine virus, A/Hong Kong/4801/2014, and their HA and NA gene sequences fell into genetic subclade 3C.2a being closely related to viruses circulating in southern Africa. The influenza B viruses were antigenically similar to the 2016 season vaccine virus and HA sequences of all three fell into the B/Yamagata-lineage, clade 3, but contained NA genes of the B/Victoria-lineage. All tested viruses were sensitive to oseltamivir and zanamivir. Conclusion: Overall, all Mozambican influenza A and B viruses were most closely related to Southern African viruses and all were sensitive to oseltamivir and zanamivir. These findings suggest the existence of an ecological niche of influenza viruses within the region and hence highlighting the need for joint epidemiologic and virologic surveillance to monitor the evolution of influenza viruses. [ABSTRACT FROM AUTHOR]

Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision. [ABSTRACT FROM AUTHOR]

Background: Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease. Methodology/Principal findings: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1–91.3%) and identified the most predictive antibody responses. Conclusions/Significance: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level. [ABSTRACT FROM AUTHOR]

Orientia tsutsugamushi (Ots) is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA) of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III) and three spacer conserved domains (SVDI, SVDII/III, SVDIII). The total number (152) of antigenic types (amino acid variants) varied from 14–36 in the six domains of TSA that we studied. Notably, 55% (787/1435) of the predicted CD4 T-cell epitopes (TCEs) from all the six domains had high binding affinities (HBA) to at least one of the prevalent Indian human leukocyte antigen (HLA) alleles. A surprisingly high proportion (61%) of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT) than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients. [ABSTRACT FROM AUTHOR]

An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies in vivo creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth in vitro after conjugation with the maytansinoid DM4. The in vivo effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates. [ABSTRACT FROM AUTHOR]

In the United States, the introduction of the heptavalent pneumococcal conjugate vaccine (PCV) largely eliminated vaccine serotypes (VT); non-vaccine serotypes (NVT) subsequently increased in carriage and disease. Vaccination also disrupts the composition of the pneumococcal pangenome, which includes mobile genetic elements and polymorphic non-capsular antigens important for virulence, transmission, and pneumococcal ecology. Antigenic proteins are of interest for future vaccines; yet, little is known about how the they are affected by PCV use. To investigate the evolutionary impact of vaccination, we assessed recombination, evolution, and pathogen demographic history of 937 pneumococci collected from 1998–2012 among Navajo and White Mountain Apache Native American communities. We analyzed changes in the pneumococcal pangenome, focusing on metabolic loci and 19 polymorphic protein antigens. We found the impact of PCV on the pneumococcal population could be observed in reduced diversity, a smaller pangenome, and changing frequencies of accessory clusters of orthologous groups (COGs). Post-PCV7, diversity rebounded through clonal expansion of NVT lineages and inferred in-migration of two previously unobserved lineages. Accessory COGs frequencies trended toward pre-PCV7 values with increasing time since vaccine introduction. Contemporary frequencies of protein antigen variants are better predicted by pre-PCV7 values (1998–2000) than the preceding period (2006–2008), suggesting balancing selection may have acted in maintaining variant frequencies in this population. Overall, we present the largest genomic analysis of pneumococcal carriage in the United States to date, which includes a snapshot of a true vaccine-naïve community prior to the introduction of PCV7. These data improve our understanding of pneumococcal evolution and emphasize the need to consider pangenome composition when inferring the impact of vaccination and developing future protein-based pneumococcal vaccines. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine. [ABSTRACT FROM AUTHOR]

Subacute sclerosing panencephalitis (SSPE) is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV) that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H) found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3–4 months, SMa79), average (3.5 years, SMa84), and long (18 years, SMa94) disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs) or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4–dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79) were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79) showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a pathogenetic mechanism in SSPE. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi parasite, the causative agent of Chagas disease, infects about six million individuals in more than 20 countries. Monitoring parasite persistence in infected individuals is of utmost importance to develop and evaluate treatments to control the disease. Routine screening for infected human individuals is achieved by serological assays; PCR testing to monitor spontaneous or therapy-induced parasitological cure has limitations due to the low and fluctuating parasitic load in circulating blood. The aim of the present study is to evaluate a newly developed antibody profiling assay as an indirect method to assess parasite persistence based on waning of antibody following spontaneous or therapy-induced clearance of the infection. Methodology/Principal findings: We designed a multiplex serology assay, an array of fifteen optimized T. cruzi antigens, to evaluate antibody diversity in 1654 serum samples from chronic Chagas patients. One specific antibody response (antibody 3, Ab3) showed a strong correlation with T. cruzi parasite persistence as determined by T. cruzi PCR positive results. High and sustained Ab3 signal was strongly associated with PCR positivity in untreated patients, whereas significant and progressive decline in Ab3 signals was observed in BZN-treated patients who cleared parasitemia based on blood PCR results. Conclusion/Significance: Ab3 is a new surrogate biomarker that strongly correlates with parasite persistence in chronic and benznidazole-treated Chagas patients. We hypothesize that Ab3 is induced and maintained by incessant stimulation of the immune system by tissue-based and shed parasites that are not consistently detectable by blood based PCR techniques. Hence, a simple immunoassay measurement of Ab3 could be beneficial for monitoring the infectious status of seropositive patients. [ABSTRACT FROM AUTHOR]

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms. [ABSTRACT FROM AUTHOR]

Influenza virus evolves rapidly due to the accumulated genetic variations on the viral sequence. Unlike in North America and Europe, influenza season in the tropical Southeast Asia spans both the rainy and cool seasons. Thus, influenza epidemiology and viral evolution sometimes differ from other regions, which affect the ever-changing efficacy of the vaccine. To monitor the current circulating influenza viruses in this region, we determined the predominant influenza virus strains circulating in Thailand between January 2016 and June 2017 by screening 7,228 samples from patients with influenza-like illness. During this time, influenza A(H3N2) virus was the predominant influenza virus detected. We then phylogenetically compared the hemagglutinin (HA) gene from a subset of these A(H3N2) strains (n = 62) to the reference sequences and evaluated amino acid changes in the dominant antigenic epitopes on the HA protein structure. The divergence of the circulating A(H3N2) from the A/Hong Kong/4801/2014 vaccine strain formed five genetic groups (designated I to V) within the 3C.2a clade. Our results suggest a marked drift of the current circulating A(H3N2) strains in Thailand, which collectively contributed to the declining predicted vaccine effectiveness (VE) from 74% in 2016 down to 48% in 2017. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). A recombinant vaccine called Bexsero® incorporates four subcapsular antigens (fHbp, NHBA, NadA and PorA) which are used to assign a Bexsero® antigen sequence type (BAST) to each meningococcal strain. The vaccine elicits an immune response against combinations of variants of these antigens which have been grouped into specific BAST profiles that have been shown to have different distributions within geographical locations thus potentially affecting the efficacy of the vaccine. In this study, invasive meningococcal disease isolates from the western seaboard of Australia (Western Australia; WA) were compared to those from the south-eastern seaboard (Victoria; VIC) from 2008 to 2012. Whole-genome sequencing (WGS) of 131 meningococci from VIC and 70 meningococci from WA were analysed for MLST, FetA and BAST profiling. Serogroup B predominated in both jurisdictions and a total of 10 MLST clonal complexes (cc) were shared by both states. Isolates belonging to cc22, cc103 and cc1157 were unique to VIC whilst isolates from cc60 and cc212 were unique to WA. Clonal complex 41/44 represented one-third of the meningococcal population in each state but the predominant ST was locally different: ST-6058 in VIC and ST-146 in WA. Of the 108 BAST profiles identified in this collection, only 9 BASTs were simultaneously observed in both states. A significantly larger proportion of isolates in VIC harboured alleles for the NHBA-2 peptide and fHbp-1, antigenic variants predicted to be covered by the Bexsero® vaccine. The estimate for vaccine coverage in WA (47.1% [95% CI: 41.1–53.1%]) was significantly lower than that in VIC (66.4% [95% CI: 62.3–70.5%]). In conclusion, the antigenic structure of meningococci causing invasive disease in two geographically distinct states of Australia differed significantly during the study period which may affect vaccine effectiveness and highlights the need for representative surveillance when predicting potential impact of meningococcal B vaccines. [ABSTRACT FROM AUTHOR]

Despite very small genomes, mycoplasmas retain large multigene families encoding variable antigens whose exact role in pathogenesis needs to be proven. To understand their in vivo significance, we used Mycoplasma agalactiae as a model exhibiting high-frequency variations of a family of immunodominant Vpma lipoproteins via Xer1-mediated site-specific recombinations. Phase-Locked Mutants (PLMs) expressing single stable Vpma products served as first breakthrough tools in mycoplasmology to study the role of such sophisticated antigenic variation systems. Comparing the general clinical features of sheep infected with a mixture of phase-invariable PLMs (PLMU and PLMY) and the wild type strain, it was earlier concluded that Vpma phase variation is not necessary for infection. Conversely, the current study demonstrates the in vivo indispensability of Vpma switching as inferred from the Vpma phenotypic and genotypic analyses of reisolates obtained during sheep infection and necropsy. PLMY and PLMU stably expressing VpmaY and VpmaU, respectively, for numerous in vitro generations, switched to new Vpma phenotypes inside the sheep. Molecular genetic analysis of selected ‘switchover’ clones confirmed xer1 disruption and revealed complex new rearrangements like chimeras, deletions and duplications in the vpma loci that were previously unknown in type strain PG2. Another novel finding is the differential infection potential of Vpma variants, as local infection sites demonstrated an almost complete dominance of PLMY over PLMU especially during early stages of both conjunctival and intramammary co-challenge infections, indicating a comparatively better in vivo fitness of VpmaY expressors. The data suggest that Vpma antigenic variation is imperative for survival and persistence inside the immunocompetent host, and although Xer1 is necessary for causing Vpma variation in vitro, it is not a virulence factor because alternative Xer1-independent mechanisms operate in vivo, likely under the selection pressure of the host-induced immune response. This singular study highlights exciting new aspects of mycoplasma antigenic variation systems, including the regulation of expression by host factors. [ABSTRACT FROM AUTHOR]

Introduction: Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission. Methods: This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared. Results: The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group. Conclusion: The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains. [ABSTRACT FROM AUTHOR]

The genome of the malaria parasite Plasmodium falciparum contains the surf gene family which encodes large transmembrane proteins of unknown function. While some surf alleles appear to be expressed in sexual stages, others occur in asexual blood stage forms and may be associated to virulence-associated processes and undergo transcriptional switching. We accessed the transcription of surf genes along multiple invasions by real time PCR. Based on the observation of persistent expression of gene surf4.1, we created a parasite line which expresses a conditionally destabilized SURFIN4.1 protein. Upon destabilization of the protein, no interference of parasite growth or morphological changes were detected. However, we observed a strong increase in the transcript quantities of surf4.1 and sometimes of other surf genes in knocked-down parasites. While this effect was reversible when SURFIN4.1 was stabilized again after a few days of destabilization, longer destabilization periods resulted in a transcriptional switch away from surf4.1. When we tested if a longer transcript half-life was responsible for increased transcript detection in SURFIN4.1 knocked-down parasites, no alteration was found compared to control parasite lines. This suggests a specific feedback of the expressed SURFIN protein to its transcript pointing to a novel type of regulation, inedited in Plasmodium. [ABSTRACT FROM AUTHOR]

Background: Improved systematic screening of high-risk groups is a key component of the tuberculosis (TB) elimination strategy endorsed by the World Health Organization (WHO). We used a multiplex microbead immunoassay to measure antibody responses to 28 M. tuberculosis (M.tb) antigens, and assessed whether combinations of antibody responses achieve accuracy thresholds required for a TB screening test. Methods: A random selection of plasma samples obtained from consecutive HIV-negative adults who were admitted to Mulago Hospital in Kampala, Uganda with cough ≥2 weeks’ but <6 months’ duration were analyzed for serological response to 28 M.tb antigens using an in-house multiplex microbead immunoassay. We compared the median difference of the antibody response to each antigen between patients with and without culture-confirmed TB, ranked each antigen according to variable importance (VIM), and assessed the sensitivity and specificity of combinations of antibody responses using an advanced classification algorithm, SuperLearner. Results: Among the 237 patients included in the analysis, 119 (50%) were female, median age was 32 years (IQR 25, 46), and 113 (48%) had TB. Median antibody levels to eight antigens were significantly different between patients with and without TB. A panel including eight of the top ranked antigens had a sensitivity of 90.6% (95% CI 89.4, 93.8) and a specificity of 88.6% (95% CI 78.2, 97.6) (Ag85B, Ag85A, Ag85C, Rv0934-P38, Rv3881, BfrB, Rv3873, and Rv2878c). With sensitivity constrained to be >90%, specificity remained close to 70% with as few as 3 antigens included in the panels. Conclusions: Measuring antibody responses to combinations of antigens could facilitate TB screening and should be further evaluated in populations being targeted for systematic screening. [ABSTRACT FROM AUTHOR]

Four Newcastle disease virus (NDV) strains were isolated from domestic, commercial geese that showed clinical signs that were believed to be the result of NDV infections. The genetic, antigenic, and pathogenic characteristics of the 4 NDVs were compared with those of NDV strains that were isolated from chickens. The complete genomes of 2 of the NDV strains contained 15,186 nucleotides (nt); the other 2 contained 15,192 nt, and exhibited the typical genomic organization of genotype II NDV and molecular characteristics of VIId NDVs. Phylogenetic analysis confirmed that the genotype II and VIId NDVs that were isolated from geese belonged to the same clusters as the corresponding genotypes of the chicken isolates. A serologic assay demonstrated that the antigenic relatedness among the NDVs was associated with their genotypes, rather than their hosts, and that amino acid substitutions in the F and/or HN proteins may contribute to the antigenic differences among these NDV genotypes. Geese infected with genotype VIId NDVs that were isolated from geese and chickens showed similar pathologic characteristics. NDVs that were isolated from geese did not differ in genetic, serologic, and pathogenic characteristics from those isolated from chickens, indicating that these NDVs were derived from chicken NDVs. Given the significance of geese in NDV epidemiology, effective biosecurity measures should be adopted to prevent the interspecies transmission of NDVs. [ABSTRACT FROM AUTHOR]

Objective: To assess the influence of biliary drainage to cholangitis on modified Glasgow Prognostic Score (mGPS) in patients with pancreatic cancer. Methods: mGPS was calculated before and after biliary drainage in 47 consecutive patients with inoperable pancreatic cancer who were receiving chemotherapy. Biliary drainage was indicated for malignant obstructive jaundice that prevented the administration of chemotherapy. To elucidate mGPS values, serum levels of CRP and albumin were measured at the time of diagnosis (before biliary drainage). Overall survival was evaluated and risk factors, which contribute to overall survival, were examined. Results: Biliary drainage was performed in 15 patients. Using values obtained before biliary drainage, there were no significant differences in median survival time between patients with a mGPS of 0 and those with a mGPS of 1 or 2 (10.7 vs. 9.4 months; p = 0.757). However, using values obtained after biliary drainage, median survival time was significantly higher in patients with a mGPS of 0 than in those with a mGPS of 1 or 2 (11.4 vs. 4.7 months; p = 0.002). Multivariate analysis revealed that a mGPS of 1 or 2 (HR: 3.38; 95% CI: 1.35–8.46, p = 0.009), a carbohydrate antigen 19–9 >1000 U/mL (2.52; 1.22–5.23, p = 0.013), a performance status of 2 (7.68; 2.72–21.28, p = 0.001), carcinoembryonic antigen level >10 ng/mL (2.29; 1.13–4.61, p = 0.021) were independently associated with overall survival. Conclusion: mGPS values obtained after biliary drainage appear to be a more reliable indicator of overall survival in patients with inoperable pancreatic cancer. [ABSTRACT FROM AUTHOR]

Background: Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. Methods: In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. Results: We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. Conclusions: Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism. [ABSTRACT FROM AUTHOR]

Background: The fusion (F) protein of RSV is the major vaccine target. This protein undergoes a conformational change from pre-fusion to post-fusion. Both conformations share antigenic sites II and IV. Pre-fusion F has unique antigenic sites p27, ø, α2α3β3β4, and MPE8; whereas, post-fusion F has unique antigenic site I. Our objective was to determine the antigenic variability for RSV/A and RSV/B isolates from contemporary and historical genotypes compared to a historical RSV/A strain. Methods: The F sequences of isolates from GenBank, Houston, and Chile (N = 1,090) were used for this analysis. Sequences were compared pair-wise to a reference sequence, a historical RSV/A Long strain. Variability (calculated as %) was defined as changes at each amino acid (aa) position when compared to the reference sequence. Only aa at antigenic sites with variability ≥5% were reported. Results: A total of 1,090 sequences (822 RSV/A and 268 RSV/B) were analyzed. When compared to the reference F, those domains with the greatest number of non-synonymous changes included the signal peptide, p27, heptad repeat domain 2, antigenic site ø, and the transmembrane domain. RSV/A subgroup had 7 aa changes in the antigenic sites: site I (N = 1), II (N = 1), p27 (N = 4), α2α3β3β4(AM14) (N = 1), ranging in frequency from 7–91%. In comparison, RSV/B had 19 aa changes in antigenic sites: I (N = 3), II (N = 1), p27 (N = 9), ø (N = 4), α2α3β3β4(AM14) (N = 1), and MPE8 (N = 1), ranging in frequency from 79–100%. Discussion: Although antigenic sites of RSV F are generally well conserved, differences are observed when comparing the two subgroups to the reference RSV/A Long strain. Further, these discrepancies are accented in the antigenic sites in pre-fusion F of RSV/B isolates, often occurring with a frequency of 100%. This could be of importance if a monovalent F protein from the historical GA1 genotype of RSV/A is used for vaccine development. [ABSTRACT FROM AUTHOR]

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity. [ABSTRACT FROM AUTHOR]

The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly virulent RHDV field isolate in immunologically naïve laboratory rabbits that were passively immunised 18–24 hours post-challenge with a neutralising monoclonal antibody. After seven passages, two amino acid substitutions in the P2 domain of the capsid protein became fixed within the virus population. Furthermore, a synonymous substitution within the coding sequence of the viral polymerase appeared and was also maintained in all subsequent passages. These findings demonstrate proof-of-concept that RHDV evolution can be experimentally manipulated to select for virus variants with altered phenotypes, in this case partial immune escape. [ABSTRACT FROM AUTHOR]

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. [ABSTRACT FROM AUTHOR]

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma. [ABSTRACT FROM AUTHOR]

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context. [ABSTRACT FROM AUTHOR]

While influenza virus diversity and antigenic drift have been well characterized on a global scale, the factors that influence the virus’ rapid evolution within and between human hosts are less clear. Given the modest effectiveness of seasonal vaccination, vaccine-induced antibody responses could serve as a potent selective pressure for novel influenza variants at the individual or community level. We used next generation sequencing of patient-derived viruses from a randomized, placebo-controlled trial of vaccine efficacy to characterize the diversity of influenza A virus and to define the impact of vaccine-induced immunity on within-host populations. Importantly, this study design allowed us to isolate the impact of vaccination while still studying natural infection. We used pre-season hemagglutination inhibition and neuraminidase inhibition titers to quantify vaccine-induced immunity directly and to assess its impact on intrahost populations. We identified 166 cases of H3N2 influenza over 3 seasons and 5119 person-years. We obtained whole genome sequence data for 119 samples and used a stringent and empirically validated analysis pipeline to identify intrahost single nucleotide variants at ≥1% frequency. Phylogenetic analysis of consensus hemagglutinin and neuraminidase sequences showed no stratification by pre-season HAI and NAI titer, respectively. In our study population, we found that the vast majority of intrahost single nucleotide variants were rare and that very few were found in more than one individual. Most samples had fewer than 15 single nucleotide variants across the entire genome, and the level of diversity did not significantly vary with day of sampling, vaccination status, or pre-season antibody titer. Contrary to what has been suggested in experimental systems, our data indicate that seasonal influenza vaccination has little impact on intrahost diversity in natural infection and that vaccine-induced immunity may be only a minor contributor to antigenic drift at local scales. [ABSTRACT FROM AUTHOR]

Background: Control and elimination of human African trypanosomiasis (HAT) can be accelerated through the use of diagnostic tests that are more accurate and easier to deploy. The goal of this work was to evaluate the immuno-reactivity of antigens and identify candidates to be considered for development of a simple serological test for the detection of Trypanosoma brucei gambiense or T. b. rhodesiense infections, ideally both. Methodology/Principal Findings: The reactivity of 35 antigens was independently evaluated by slot blot and ELISA against sera from both T. b. gambiense and T. b. rhodesiense infected patients and controls. The antigens that were most reactive by both tests to T. b. gambiense sera were the membrane proteins VSG LiTat 1.3, VSG LiTat 1.5 and ISG64. Reactivity to T. b. rhodesiense sera was highest with VSG LiTat 1.3, VSG LiTat 1.5 and SRA, although much lower than with T. b. gambiense samples. The reactivity of all possible combinations of antigens was also calculated. When the slot blot results of 2 antigens were paired, a VSG LiTat 1.3- ISG75 combination performed best on T. b. gambiense sera, while a VSG LiTat 1.3-VSG LiTat 1.5 combination was the most reactive using ELISA. A combination of SRA and either VSG LiTat 1.3 or VSG LiTat 1.5 had the highest reactivity on T. b. rhodesiense sera according to slot blot, while in ELISA, pairing SRA with either GM6 or VSG LiTat 1.3 yielded the best results. Conclusions: This study identified antigens that were highly reactive to T. b. gambiense sera, which could be considered for developing a serological test for gambiense HAT, either individually or in combination. Antigens with potential for inclusion in a test for T. b. rhodesiense HAT were also identified, but because their reactivity was comparatively lower, a search for additional antigens would be required before developing a test for this form of the disease. [ABSTRACT FROM AUTHOR]

Serology-based tests for tuberculosis (TB) diagnosis, though rapid, efficient and easily implemented, have so far shown unsatisfactory levels of sensitivity and specificity, probably due to variations of the antibody response in TB patients. The number and types of seropositive antigens vary from individual to individual. The person-to-person variations of antigen recognition may be linked to genetic polymorphisms of the human leukocyte antigen (HLA) class II alleles. In the present study, we find that there is a significant increase in the frequency of HLA-DRB1*14 (P = 2.5×10−4) among subjects with high antibody response levels compared to those with low antibody levels. HLA-DRB1*15, the most frequent allelic group in the studied active TB population, positively correlates with subjects with low antibody response levels rather than subjects with high antibody response levels (P = 0.005), which indicates the loss of relevant antigens for screening of patients with this allelic group. The potential association between HLA-DRB1 allelic group and individual antigens implies that TB diagnostic yield could be improved by the addition of antigens screened at the proteome scale in infected subjects from the HLA-DRB1*15 allelic group. [ABSTRACT FROM AUTHOR]

Background: Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015. Methods: Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay. Results: A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 102 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106–109 copies/mL, which was markedly higher than the rate observed in the other age groups. Conclusions: The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates. [ABSTRACT FROM AUTHOR]