Major Surface Protein Research Articles

Increased Virulence of Outer Membrane Porin Mutants of Mycobacterium abscessus.

Chronic pulmonary infections caused by non-tuberculous mycobacteria of the Mycobacterium abscessus complex (MABSC) are emerging as a global health problem and pose a threat to susceptible individuals with structural lung disease such as cystic fibrosis. The molecular mechanisms underlying the pathogenicity and intrinsic resistance of MABSC to antibiotics remain largely unknown. The involvement of Msp-type porins in the virulence and biocide resistance of some rapidly growing non-tuberculous mycobacteria and the finding of deletions and rearrangements in the porin genes of serially collected MABSC isolates from cystic fibrosis patients prompted us to investigate the contribution of these major surface proteins to MABSC infection. Inactivation by allelic replacement of the each of the two Msp-type porin genes of M. abscessus subsp. massiliense CIP108297, mmpA and mmpB, led to a marked increase in the virulence and pathogenicity of both mutants in murine macrophages and infected mice. Neither of the mutants were found to be significantly more resistant to antibiotics. These results suggest that adaptation to the host environment rather than antibiotic pressure is the key driver of the emergence of porin mutants during infection.

Molecular detection and phylogenetic analyses of Anaplasma spp. in Haemaphysalis longicornis from goats in four provinces of China

Anaplasma species, which are distributed worldwide, are gram-negative obligate intracellular tick-borne bacteria that pose a threat to human and animal health. Haemaphysalis longicornis ticks play a vital role as vectors in the transmission of Anaplasma pathogens. However, the Anaplasma species carried by H. longicornis in China are yet to be characterized. In this study, 1074 H. longicornis specimens were collected from goats in four provinces of China from 2018 to 2019 and divided into 371 sample pools. All tick sample pools were examined for the presence of Anaplasma species via nested PCR amplification of 16S ribosomal RNA, major surface protein 4 (msp4), or citric acid synthase (gltA) genes, which were sequenced to determine the molecular and phylogenetic characteristics of the isolates. The overall Anaplasma spp-positive rate of H. longicornis was determined to be 26.68% (99/371). The percentage prevalence of A. phagocytophilum-like1, A. bovis, A. ovis, A. marginale, and A. capra were 1.08% (4/371), 13.21% (49/371), 13.21% (49/371), 1.35% (5/371), and 10.24% (38/371), respectively, and the co-infection rate of two or more types of Anaplasma was 6.47% (24/371). Phylogenetic analyses led to the classification of A. phagocytophilum into an A. phagocytophilum-like1 (Anaplasma sp. Japan) group. Anaplasma bovis sequences obtained in this study were 99.8–100% identical to those of an earlier strain isolated from a Chinese tick (GenBank accession no. KP314251). Anaplasma ovis sequences showed 99.3–99.6% identity to an A. ovis human strain identified from a Cypriot patient (GenBank accession no. FJ460443). Only one msp4 sequence of A. marginale was detected and was grouped with those of other A. marginale isolates, and these A. capra isolates obtained in this present study may be zoonotic. The detection and characterization of four Anaplasma species in H. longicornis in this study have added to the current knowledge of the parasite and provided data on multiple Anaplasma species with veterinary and medical significance from four provinces of China.

Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles.

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

Molecular and recombinant characterization of major surface protein 5 from Anaplasma marginale

Anaplasmosis is a tick-borne disease caused by the intracellular rickettsia Anaplasma marginale, which affects cattle and other ruminants in both tropical and subtropical regions of the world, and also causing tremendous economic losses due to decreasing livestock production. The major surface protein 5 (MSP5) of A. marginale is an immunodominant and highly conserved protein encoding by a single gene. In the present study, the complete full-length of the msp5 coding sequence of A. marginale Thailand strain was cloned and determined at a size of 633 bp. Phylogenetic analysis based on neigh-joining (NJ) method showed that the msp5 sequence Thailand strains were clearly distributed in 3rd clade and conserved when compared with other strains. The results showed 9 haplotypes of the msp5 genes, and the entropy analysis of MSP5 amino acid sequences displayed 92 high entropy peaks with value ranging from 0.198 to 0.845 Additionally, a recombinant MSP5 of A. marginale (rAmMSP5) was over-expressed in the E. coli BL21 Star™ (DE3) host cell, affinity purified, and found in SDS-PAGE at a molecular weight of 26 kDa. The antigenicity of rAmMSP5 (26 kDa) and AmMSP5 (19 kDa) was recognized by rabbit anti-rAmMSP5 antisera and A. marginale-infected cattle sera. Both rAmMSP5 and AmMSP5 were perceived by these sera manifesting that recombinant and native AmMSP5 have conserved epitopes. Immunofluorescence technique using rabbit anti-rAmMSP5 antisera exhibited that the AmMSP5 is distributed on both the membrane and the outside of infected erythrocytes. Therefore, the recombinant MSP5 could be used for the development of immunodiagnostic assays and vaccine purposes for controlling anaplasmosis.

Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

Molecular and serological detection of Anaplasma infection in carrier cattle in north India.

Anaplasma marginale infection in cattle (n=216) in the states of Uttar Pradesh and Uttarakhand, North India was screened by microscopy and nested-polymerase chain reaction (PCR). Two recombinant proteins viz. major surface protein (MSP) 5 and MSP2 of A. marginale were expressed in Escherichia coli and their potential in the detection of antibodies to Anaplasma species in the cattle was evaluated by immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). The MSP5 IgG ELISA results were compared with competitive (c) inhibition ELISA. Microscopy being the least sensitive diagnostic test detected 12.0% of animals positive for A. marginale infection while nested-PCR detected 87.9% of these animals as positive for A. marginale infection. The recombinant MSP5 antigen showed positive reactivity in 170/190 nested-PCR confirmed positive animals (sensitivity 89.5%) with specificity of 77.0%. In comparison, the recombinant MSP2 antigen showed lesser sensitivity and specificity of 79.0% and 69.2%, respectively. The cELISA was more sensitive and specific than IgG-ELISA. However, molecular detection by msp5 nested-PCR was highly sensitive and reliable for detection of carrier cattle for Anaplasma infection. The study indicated that a large cattle population (87.9%) was carrier for A. marginale infection in this region of the country.

Tick-Borne Hemoparasites of Sheep: A Molecular Research in Turkey.

Tick-borne diseases (TBDs) indulge in severe economic losses in the livestock industry by adversely affecting the small ruminant breeding in tropical and subtropical zone countries, including Turkey. Turkey encompasses a wide land area representing diverse climatic conditions. The present study explored the presence and distribution of Babesia ovis, Theileria ovis, Theileria lestoquardi, Anaplasma ovis, Anaplasma phagocytophilum and the co-occurrence status of these pathogens. A total of 299 sheep blood samples were collected from fifteen provinces located in six different geographical regions in Turkey. PCR analyses were executed using species-specific primers based on Babesia ovis BoSSU rRNA, Theileria ovis ToSSU rRNA, Theileria lestoquardi 18S rRNA, Anaplasma ovis Major Surface Protein (AoMSP4), and Anaplasma phagocytophilum 16S rRNA genes. Overall, 219 (73.24%) sheep were found to be infected with at least one of the following protozoan and rickettsial pathogens; B. ovis, A. ovis, T. ovis, and A. phagocytophilum. Theileria lestoquardi was not detected in any blood sample. The global prevalence of B. ovis, A. ovis, T. ovis, and A. phagocytophilum was estimated to be 2.68%, 16.05%, 41.47%, and 57.19%, respectively. Besides this, dual (24.41%), triple (9.03%), and quadruple (0.67%) co-infections were detected in the study. Phylogenetic analysis revealed significant nucleotide sequence identities between the sequences obtained in this study and the sequences registered in the GenBank. This study provides relevant data regarding the predominance of ovine tick-borne protozoan and rickettsial agents in Turkey. A high molecular prevalence of tick-borne pathogens (TBPs) was identified in the study. This situation indicates that TBPs should be screened continuously, and necessary control measures should be taken to prevent diseases caused by tick-borne protozoan and rickettsial agents.

Low dose recombinant full-length circumsporozoite protein-based Plasmodium falciparum vaccine is well-tolerated and highly immunogenic in phase 1 first-in-human clinical testing

Plasmodium falciparum circumsporozoite protein (CSP) is a major sporozoite surface protein and a key target of pre-erythrocytic malaria subunit vaccines. A full-length recombinant CSP (rCSP) based strategy could be advantageous, as this antigen includes a region critical to sporozoite cell attachment and hepatocyte invasion. The adjuvant Glucopyranosyl Lipid A-liposome Quillaja saponaria 21 (GLA-LSQ) functions as a TLR4 agonist, promotes antigen-specific TH1 responses and stimulates cytotoxic T cell production. To date, one study has reported the clinical acceptability of GLA-LSQ. We present interim results of a phase 1 first-in-human dose-escalation clinical trial of full-length rCSP vaccine given with or without GLA-LSQ adjuvant. Participants experienced only mild to moderate related solicited adverse events. The lowest adjuvanted vaccine dose achieved >90-fold rise in geometric mean anti-CSP IgG antibody titer. These favorable safety and immunogenicity results confirm the immunostimulatory capacity of this relatively new adjuvant and support next steps in clinical product development.Trial registration: ClinicalTrials.gov Identifier NCT03589794 (registered 18 July 2018)

Identification of a universal antigen epitope of influenza A virus using peptide microarray

BackgroundHemagglutinin is a major surface protein in influenza A virus (IAV), and HA2 is relative conserved among different IAVs. It will be meaningful to identify broad-spectrum epitopes based on the HA2 protein.ResultsOverlapping peptides of the HA2 protein of the H5N1 IAV A/Mallard/Huadong/S/2005 were synthesized and loaded on modified silica gel film to form a microarray, and antisera against different subtypes of IAVs were used to screen universal epitopes. The selected epitope was further confirmed by western blotting using anti-peptide immune serum and viruses rescued with amino acid substitution. The results showed that 485-FYHKCDNECME-495 of the H5 14th peptide in HA2 had broad-spectrum binding activity with antisera against H1, H3, H4, H5, H6, H7, H8, H9, and H10 subtype IAV. Substitution of amino acids (K or D) in rescued viruses resulted in decreased serum binding, indicating that they were critical residues for serum binding activity. In Immune Epitope Database, some epitopes containing 14–4 peptide were confirmed as MHC-II-restricted CD4 T cell epitope and had effects on releasing IL-2 or IFN.ConclusionThe identified epitope should be a novel universal target for detection and vaccine design and its ability to generate immune protection needs further exploration.

Mucosal Delivery of Recombinant Vesicular Stomatitis Virus Vectors Expressing Envelope Proteins of Respiratory Syncytial Virus Induces Protective Immunity in Cotton Rats.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 103 PFU. In contrast, doses greater than 106 PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection.IMPORTANCE Even after decades of intensive research efforts, a safe and efficacious RSV vaccine remains elusive. Expression of heterologous antigens from rVSV vectors has demonstrated several practical and safety advantages over other virus vector systems and live attenuated vaccines. In this study, we developed safe and efficacious vaccine candidates by expressing the two major immunogenic RSV surface proteins in rVSV vectors and delivering them mucosally in a prime-boost regimen. The main immune parameter responsible for protection was the antibody response. These vaccine candidates induced complete protection of both the upper and lower respiratory tracts.

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi

BackgroundTick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi.MethodsIn a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively.ResultsThe prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected .ConclusionsThe findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil.

Anaplasma marginale is an obligate intracellular Gram-negative bacterium found in ruminants' erythrocytes and is the etiological agent of bovine anaplasmosis. The bacterium's genetic diversity has been characterized based on sequences of major surface proteins (MSPs), such as MSP1α. The aim of the present study was to investigate the genetic diversity of A. marginale in cattle in the state of Maranhão, northeastern Brazil. To this end, 343 blood samples were harvested and subjected to iELISA assays using the recombinant surface protein MSP5. Out of 343 blood samples, 235 (68.5%) were randomly chosen and submitted to DNA extraction, qPCR and conventional PCR targeting the msp1α gene to determine amino acid sequences and classify the genotypes. The iELISA results showed 81.34% seropositivity (279/343), whereas qPCR revealed 224 positive samples (95.32%). Among these qPCR-positive samples, 67.4% (151/224) were also positive in the cPCR. Among the 50 obtained sequences, 21 strains had not been previously reported. Regarding the genotypes, H (26/50) and E (18/50) were identified most often, while genotypes F and C were only identified twice each and B and G once each. In conclusion, high prevalence and genetic diversity for A. marginale were observed in dairy cattle herds in the state of Maranhão.

Correlating the Structure and Activity of Y. pestis Ail in a Bacterial Cell Envelope

Understanding microbe-host interactions at the molecular level is a major goal of fundamental biology and therapeutic drug development. Structural biology strives to capture biomolecular structures in action, but the samples are often highly simplified versions of the complex native environment. Here, we present an Escherichia coli model system that allows us to probe the structure and function of Ail, the major surface protein of the deadly pathogen Yersinia pestis. We show that cell surface expression of Ail produces Y. pestis virulence phenotypes in E. coli, including resistance to human serum, cosedimentation of human vitronectin, and pellicle formation. Moreover, isolated bacterial cell envelopes, encompassing inner and outer membranes, yield high-resolution solid-state NMR spectra that reflect the structure of Ail and reveal Ail sites that are sensitive to the bacterial membrane environment and involved in the interactions with human serum components. The data capture the structure and function of Ail in a bacterial outer membrane and set the stage for probing its interactions with the complex milieu of immune response proteins present in human serum.

Microscopic And Molecular Identification of Anaplasma Ovis in Small Ruminants in Duhok Province in Kurdistan Region of Iraq

In last ten years, there has been a developing enthusiasm for microscopic organisms from the genus Anaplasma, particularly the species A. ovis. It is associated with the pathogenic action of these microscopic organisms in livestock. Anaplasma ovis is a tick-borne obligate intracellular rickettsial bacterium that causes anaplasmosis in domestic and wild small ruminants. The samples of the present study were collected from small ruminants from inside seven distinct regions (Akre, Simele, Zummar, Feshchapoor, Deraboon, Bajed Kandal,Karoda)of Duhok province, 389 (goats 75 and sheep 314) during the period of April and May 2018, blood sample were taken and thin smear was formed, after Giemsa’s staining the slide is observed under microscope. In this study used Giemsa stain for microscopic examination out of 389 animals 250 were found positive for Anaplasma ovis infection with a prevalence rate of 64.26 % and 139 of them were negative with a prevalence rate of 35.73 %. According to the species of animals, the highest prevalence of A. ovis infection in animals by using microscopic examination was 67.83 %, 213 positive sample from total 314 blood samples from sheep and lowest prevalence was 49.33 %, 37 positive sample from total 75 blood samples from goats. PCR analysis of 100 blood samples obtained from total 250 positive blood samples after DNA extraction and measure of concentration and purity we used 2 primers that target major surface protein 4 (MSP4) in A. ovis genomic DNA. The results of PCR test with major surface protein 4 primer was 83 samples positive from total 100 samples, According to the species of animals, the highest prevalence of A. ovis was 83.7 %, 72 positive sample from total 86 blood samples from sheep and lowest prevalence was 78.5 %, 11 positive sample from total 14 blood samples from goats.

PSV-1 A Subcutaneous Ear Implant to Deliver an Anaplasmosis Vaccine to Dairy Steers

Abstract Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to beef production. The objective of the study was to test the ability of a single-dose delivery platform to produce long-lasting immunity against anaplasmosis infections. Twelve Holstein steers were administered a 3-stage, single-dose vaccine against Anaplasma marginale major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously for immune priming, a biodegradable polyanhydride depot for intermediate slow-release of the vaccine for boosting the immune response, and an immune-isolated vaccine platform for extended antigen release deposited subcutaneously in the ear. Six calves were randomly assigned to two vaccine constructs that featured rods and implants containing a combination of two adjuvants (DEAE-Dextran and Quil-A). The remaining 6 calves were randomly assigned to two vaccine constructs that featured rods and implants containing the same adjuvant (DEAE-Dextran or Quil-A). Twenty months post-implantation, calves were challenged with Anaplasma marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Outcome variables (fever, PCV, bacteremia) were analyzed using a linear mixed model (PROC GLMMIX; SAS University Edition v9.04.01, SAS Institute, Cary, NC). Two-tailed chi-squared tests were used to compare need for antibiotic intervention between vaccine constructs. Calves within the combination adjuvant construct group (DEAE-D and Quil-A) exhibited significantly (P = .006) higher PCV than calves within the single adjuvant construct group (DEAE-Dextran or Quil-A). Likewise, calves within the combination adjuvant group required significantly (P = .014) fewer antibiotic interventions compared with calves in the single adjuvant group. Findings of this study suggest that calves exhibited significantly diminished clinical signs of anaplasmosis when vaccine was delivered with a combination of adjuvants as opposed to a single adjuvant in a single-dose delivery platform.

Prevalence of antibodies to Anaplasma in cattle and buffaloes of different organized herds in India.

Bovine anaplasmosis is one of the most important tick borne disease in ruminants causing huge economic loss to the dairy industry. A cross-sectional study was carried out to detect serum antibodies to Anaplasma infection in cattle and buffaloes housed in 14 organized herds located at various climatic zones spreading over 9 different states in India. A total of 911 serum samples, collected from 667 cattle and 244 buffaloes, were subjected to a competitive enzyme linked immune-sorbent assay detecting an epitope of major surface protein 5 (MSP5) of Anaplasma. The overall true prevalence was 48.72% (95% CI 45.13-52.32%). The prevalence rate was higher in cattle (51.58%) than buffaloes (40.89%) and the difference was statistically significant (p < 0.05). Indigenous cattle (59.30%) showed higher seropositivity than crossbreed (57.16%) and exotic cattle breeds (42.28%). Although statistically not significant, female (52.37%) showed higher seropositivity than male (46.43%). Similarly, significant difference in prevalence (p < 0.05) was observed for animals reared in different climatic zones with highest prevalence recorded in arid zone (90.49%) and lowest in semi-arid zone (29.83%). Very wide variation in prevalence (9.95-100%) was recorded between farms. The present study indicates endemicity of Anaplasma in India, similar to other tropical and sub-tropical countries of the world. Endemic instability was recorded in some of the studied farms suggesting possibility of outbreak of new clinical cases resulting in economic loss. Therefore, suitable policies and procedures for prevention and control of Anaplasma infection should be adopted in these farms.

A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing

Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1-infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

A novel lamprey antibody sequence to multimerize and increase the immunogenicity of recombinant viral and bacterial vaccine antigens

Hemagglutinin, the major surface protein of influenza viruses, was recombinantly expressed in eukaryotic cells as a monomer instead of its native trimer, and was only immunogenic when administered with an adjuvant [Pion et al. 2014]. In order to multimerize this antigen to increase its immunogenicity, a cysteine-rich peptide sequence found at the extreme C-terminus of lamprey variable lymphocyte receptor (VLR)-B antibodies was fused to various recombinant hemagglutinin (rHA) proteins from A and B influenza virus strains. The rHA-Lamp fusion (rHA fused to the lamprey sequence) protein was expressed in Leishmania tarentolae and Chinese hamster ovary (CHO) cells and shown to produce several multimeric forms. The multimers produced were very stable and more immunogenic in mice than monomeric rHA. The lamprey VLR-B sequence was also used to multimerize the neuraminidase (NA) of influenza viruses expressed in CHO cells. For some viral strains, the NA was expressed as a tetramer like the native viral NA form. In addition, the lamprey VLR-B sequence was fused with two surface antigens of Shigella flexneri 2a, the invasion plasmid antigen D and a double mutated soluble form of the membrane expression of the invasion plasmid antigen H namely MxiH. The fusion proteins were expressed in Escherichia coli to produce the respective multimer protein forms. The resulting proteins had similar multimeric forms as rHA-Lamp protein and were more immunogenic in mice than the monomer forms. In conclusion, the VLR-B sequence can be used to increase the immunogenicity of recombinant viral and bacterial antigens, thus negating the need for adjuvants.

Genetic diversity of major surface protein 1a of Anaplasma marginale in dairy cattle.

Major surface protein 1 alpha (Msp1α) is a stable genetic marker for identifying Anaplasma marginale. Our aims were to investigate the genetic diversity of A. marginale based on microsatellites and tandem repeats of the msp1α gene and to determine the phylogenetic relationships among six isolates obtained from 63 dairy cows positive for the pathogen from Turkey and from strains worldwide. Msp1α microsatellite analysis revealed infection with three genotypes (B, C, G). Genotype B was found in cows held in an intensive management system, while genotypes C and G were found in grazing cows. Microsatellite sequences produced SD-ATG distances of 19 and 23 nucleotides, with 23 nucleotides predominating, showing high infection potential. Fourteen different tandem repeats of A. marginale were found in the samples, and four msp1α gene repeats designated Tr4, Tr5, Tr6 and Tr7 were newly described. The majority of A. marginale isolates exhibited more than three Msp1a tandem repeats, with a maximum of seven. The Msp1a tandem repeats α-73-73-73-73-73-73 were commonly observed in intensively managed cows. The phylogenetic analysis revealed A. marginale strains from Turkey clustering with those of the Americas, Africa, Asia and Europe, suggesting a history of cattle trade and transport.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus invitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.