Immune Reactivity of a 20-mer Peptide Representing the Zika E Glycan Loop Involves the Antigenic Determinants E-152/156/158

Mosquito-borne Zika virus (ZIKV) causes a severe congenital syndrome and neurological disorders in humans. With the aim to develop a live-attenuated ZIKV strain, we generated a chimeric viral clone ZIKALIVax with African MR766-NIID strain as backbone and the envelope E protein of epidemic Brazilian BeH810915 strain. The MR766-NIID residues E-T152/I156/Y158 were introduced into BeH810915 E protein leading to a nonglycosylated ZIKALIVax. Recently, we reported that the residues E-152/156/158 that are part of ZIKV glycan loop (GL) region might have an impact on the availability of neutralizing antibody epitopes on ZIKV surface. In the present study, we evaluated the antigenic reactivity of a synthetic 20-mer peptide representing the ZIKALIVax GL region. The GL-related peptide was effective for the detection of GL-reactive antibody in mouse anti-ZIKALIVax immune serum. We showed that the residue E-158 influences the antigenic reactivity of GL-related peptide. The ZIKALIVax peptide was effective in generating mouse antibodies with reactivity against a recombinant E domain I that encompasses the GL region. The GL peptide-reactive antibodies revealed that antigenic reactivity of E-domain I may be impacted by both residues E-152 and E-156. In conclusion, we proposed a role for the residues E-152/156/158 as key antigenic determinants of ZIKV glycan loop region.

Structural Characterization of Cuta- and Tusavirus: Insight into Protoparvoviruses Capsid Morphology

Several members of the Protoparvovirus genus, capable of infecting humans, have been recently discovered, including cutavirus (CuV) and tusavirus (TuV). To begin the characterization of these viruses, we have used cryo-electron microscopy and image reconstruction to determine their capsid structures to ~2.9 Å resolution, and glycan array and cell-based assays to identify glycans utilized for cellular entry. Structural comparisons show that the CuV and TuV capsids share common features with other parvoviruses, including an eight-stranded anti-parallel β-barrel, depressions at the icosahedral 2-fold and surrounding the 5-fold axes, and a channel at the 5-fold axes. However, the viruses exhibit significant topological differences in their viral protein surface loops. These result in three separated 3-fold protrusions, similar to the bufaviruses also infecting humans, suggesting a host-driven structure evolution. The surface loops contain residues involved in receptor binding, cellular trafficking, and antigenic reactivity in other parvoviruses. In addition, terminal sialic acid was identified as the glycan potentially utilized by both CuV and TuV for cellular entry, with TuV showing additional recognition of poly-sialic acid and sialylated Lewis X (sLeXLeXLeX) motifs reported to be upregulated in neurotropic and cancer cells, respectively. These structures provide a platform for annotating the cellular interactions of these human pathogens.

Comparative Analysis of the Serological Reactivity of Individuals with Clinical History of Malaria using Two Different ELISA Tests

Early diagnosis of malaria reduces disease, prevents deaths, and contributes to decreased malaria transmission. The use of specific and sensitive antigens in the execution of serological diagnostics may have an impact on the transmission of the disease. However, many individuals cannot be easily diagnosed by serological tests due to low levels of antibodies in the serum. Using two different Enzyme-Linked Immunosorbent Assay (ELISA) tests (a commercial and an in-house ELISA), a total of 365 serum samples from individuals with a clinical history of malaria were analyzed. From the serum samples analyzed, 192 (53%) samples from the commercial ELISA and 219 (60%) samples from the in-house ELISA presented positive serological reactivity to malaria. The concordance of the samples tested (n = 365) between both ELISAs was of 67% (n = 242), and with the negative control was 100% (n = 17). We demonstrated that the in-house ELISA showed high antigenic reactivity to Plasmodium falciparum antigens when compared with the commercial ELISA. The degree of concordance of both ELISAs suggested the possibility of existence of other P. falciparum antigens present in the crude extract of P. falciparum that are important in the serological response during malaria infection.

Involvement of the Areae Compositae of the Heart in Endemic Pemphigus Foliaceus

Background: A new variant of endemic pemphigus foliaceus in El Bagre (El Bagre-EPF), Colombia, South America, shares features with Senear-Usher syndrome and occurs in an endemic fashion. Patients affected by El Bagre-EPF have heterogeneous antigenic reactivity not only to the skin but to other organs, including the heart. Here we test for autoantibodies to the areae compositaeof the heart (structure consisting of typical desmosomal amalgamated fascia adherensmolecules)and evaluate any possible clinical correlation. Methods: A case-control study comparing 45 patients and 45 controls from the endemic area, matched by demographics including age, gender, weight, work activities, and comorbidities, was performed. Direct and indirect immunofluorescence, immunohistochemistry, confocal microscopic studies, and echocardiogram studies were completed. Results: The main clinical abnormally seen in the El Bagre-EPF patients was left ventricular hypertrophy in 15/45 patients, compared with no such findings in the control population (P < 0.1). Seventy percent of El Bagre-EPF patients and none of the controls displayed polyclonal autoreactivity using different immunoglobulins and complement to the areae compositae of the heart using different methods and antibodies (P < 0.1). Conclusions: Patients affected by El Bagre-EPF demonstrated autoantibodies to the areae compositae of the heart. This finding was associated with left ventricular hypertrophic cardiomyopathy.The areae compositaemay play a role incell junction tension and the El Bagre-EPF patients’ autoantibodies possibly disrupting these junctions and thereby contributing to the left ventricular hypertrophy.

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains ofErysipelothrix rhusiopathiae

ABSTRACTErysipelothrix rhusiopathiaecauses swine erysipelas, an infection characterized by acute septicemia or chronic endocarditis and polyarthritis. Among 17E. rhusiopathiaeserovars, determined based on heat-stable peptidoglycan antigens, serovars 1 and 2 are most commonly associated with the disease; however, the molecular basis for the association between these serovars and virulence is unknown. To search for the genetic region defining serovar 1a (Fujisawa) strain antigenicity, we examined the 15-kb chromosomal region encompassing a putative pathway for polysaccharide biosynthesis, which was previously identified in theE. rhusiopathiaeFujisawa strain. Six transposon mutants of Fujisawa strain possessing a mutation in this region lost antigenic reactivity with serovar 1a-specific rabbit serum. Sequence analysis of this region in wild-type strains of serovars 1a, 1b, and 2 and serovar N, which lacks serovar-specific antigens, revealed that gene organization was similar among the strains and that serovar 2 strains showed variation. Serovar N strains displayed the same gene organization as the serovar 1a, 1b, or 2 strain and possessed certain mutations in this region. In two of the analyzed serovar N strains, restoration of the mutations via complementation with sequences derived from serovar 1a and 2 strains recovered antigenic reactivity with 1a- and 2-specific rabbit serum, respectively. Several gene mutations in this region resulted in altered capsule expression and attenuation of virulence in mice. These results indicate a functional connection between the biosynthetic pathways for the capsular polysaccharide and peptidoglycan antigens used for serotyping, which may explain variation in virulence among strains of different serovars.

Major histocompatibility complex (DRB3) geneexpression pattern indicates differences in BrucellaabortusS19 vaccine induced immune responsein Karan Fries and Sahiwal cattle

Brucella abortus S19 strain vaccination is most effectively used as a tool to control the brucellosis in cattle. To understand the genetic basis of differences in immune responsiveness after immunization in cattle of different genotypes, we assessedthe expression of MHC-DRB3 antigen receptor molecule in six each female calves of Karan Fries crossbreds (KF, Bos indicus x Bos taurus) and Sahiwal (Bos indicus) vaccinated with Brucella S19. Serum and peripheral blood mononuclear cells (PBMC) were isolated from blood collected on 0(before vaccination) and 7, 14 and 28 day of vaccination. Antigenic response was assessed for these days in both the groups using Rose Bengal Plate form Test (RBPT). At 0d, the calves of both groups showed no antigen agglutination, confirming the calves free from the infection.The serum of 7d onward started showing the agglutination with more strong response in later stages specifically in KF, indicating increased immune response against Brucella. Therefore, RBPT can be used as earliest screening (7d onward) for Brucella antigenic reactivity in both cattle groups. The expression of DRB3 gene started with slight upregulation after vaccination,in general, however without any significant differences between two different genetic groups upto14d.The significant (p Lass Than 0.01) higher expression (8 times) of DRB3was observed in KF than Sahiwal at 28d. The study indicated that antigenic reactivity and MHC-DRB3 expression elicited by Brucella S19 vaccination was more prominent in KFduring initial days, which may provide an extra advantage to the host for antigen binding, thereby better immune protection at later stage.

Antigenic characterisation of lyssaviruses in South Africa

There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and Mokola virus (0.5%). Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.

Prokaryotic Expression and Identication on the 20kDa Protein Gene of Mycobacterium paratuberculosis

The gene encoding 20kDa protein gene from Mycobacterium paratuberculosis C-2, chromosomal DNA was amplified by using polymerase chain reaction (PCR), the PCR product was approximately 520bp DNA segment. The PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-20 was constructed successfully. The purified 20kDa protein gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression plasmid pET28a-20 was constructed. Plasmid containing pET28a-20 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 23kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic reactivity of M. paratuberculosis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of 20kDa protein gene in their prevention against bovine paratuberculosis.

Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4

ABSTRACT The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core β strands (βB to βI) and helical (αA) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed “valleys” between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes.