Sigvard Olofsson

Multiplex real-time PCR for detection of respiratory tract infections

Abstract Background Broad diagnostics of respiratory infection by molecular assays has not yet won acceptance due to technical difficulties and high costs. Objectives To evaluate clinical applicability of multiplex real-time PCR. Study design An assay targeting influenza virus A (IfA) and B (IfB), parainfluenza 1-3 (PIV), human metapneumovirus (MPV), respiratory syncytial virus (RSV), rhinovirus (RV), enterovirus (EV), adenovirus (AdV), human coronaviruses (229E, OC43, NL63), M. pneumoniae and Ch. pneumoniae was developed and run daily on consecutive clinical nasopharyngeal swab samples. Results An etiology was identified in 48% of the 954 samples, with IfA in 25%, RV in 20%, MPV in 10% and M. pneumoniae in 10% of the positive. By a rational procedure costs could be reduced and the customer price set relatively low (€33 per sample). Conclusion Streamlined testing and cost limitation is achievable and probably critical for implementation of a broad molecular diagnostics of respiratory infections.

Glycoconjugate glycans as viral receptors

The carbohydrate parts of cell surface glycoproteins, glycolipids, and proteoglycans constitute receptors for many enveloped as well as non‐enveloped human viruses. The majority of viral receptors of carbohydrate nature are negatively charged, including sulfated glycosaminoglycans (GAGs) or glycans containing sialic acid. Not uncommonly, virus‐carbohydrate interactions are responsible for specific tissue tropism, where the affinity of influenza virus for glycans in the respiratory tract containing (α2‐6)‐linked sialic acid is an important example. Similarly, the number and spacing of sulfates may guide viruses to optimal GAG molecules, although this remains unproven on tissue level. A further understanding of structure and tissue distribution of carbohydrate virus receptors and their viral ligands is essential for elucidating the pathogenesis of such viruses. Also neutral glycans such as histo‐blood group substances may function as virus receptors. Here, natural resistance to a given viral disease may occur in a human subpopulation due to lack of such receptors caused by deletion‐mutants in critical human genes. As regards antiviral applications, the receptor‐destroying enzymes, in contrast to receptor binding proteins, at the surface of, for example, influenza virus have proven to be an excellent target for intervention, which is why sialic acid analogues are now in clinical use both for prophylaxis and treatment.

Prevalence of antibodies to herpes simplex virus in pregnant women in Stockholm in 1969, 1983 and 1989: implications for STD epidemiology.

Prevalence of antibody to herpes simplex virus types 1 and 2 was assessed in consecutive serum samples from a total of 3700 women pregnant in 1969, 1983, or 1989 from the same catchment area in Stockholm. There was little change in seroprevalence of antibody to herpes simplex type 1 in the 3 groups, but age-adjusted herpes simplex virus type 2 antibody prevalence was 19, 33, and 33% respectively. Increase in type 2 seropositivity with age was slight and similar in 1969 and 1989, but steep in 1983, indicating a shift in sexual behaviour. However, rising prevalence in women will be mirrored by rising prevalence in their male partners. The increase from 1969 to 1989 will thus partly be due to higher risk of infection per partner, and cannot be taken as direct evidence of increased rate of partner change during this 20-year period.

Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate

The amino acid residues critical for interaction between herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) and cell surface heparan sulphate (HS) were localized to two separate regions within antigenic site II of this glycoprotein. These amino acids were Arg-143, Arg-145, Arg-147 and Thr-150 in one region and Gly-247 in the other. This conclusion is based on the following observations. (i) Monoclonal antibodies defining gC-1 antigenic site II, and not those reactive with antigenic site I, inhibited HSV-1-induced haemagglutination and virus binding to susceptible cells. (ii) A number of HSV-1 mar mutants, altered at these critical residues, were impaired in attachment to cells. (iii) Synthetic peptides, corresponding to these two regions inhibited virus attachment to cells and infectivity. In addition these peptides were found to agglutinate red blood cells. This agglutination was inhibited by soluble HS, and was prevented by the pretreatment of red blood cells with heparitinase suggesting that cell surface HS was a site of peptide binding. The same was observed with the polycationic substances neomycin and poly-L-lysine. In conclusion, we propose that the regions of gC-1 represented by the HS-binding peptides may form a functional site of a polycationic nature, active in attachment to the polyanionic glycosaminoglycan chain of cell surface HS.

Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response

One of the functions of N-linked glycans of viral glycoproteins is protecting otherwise accessible neutralization epitopes of the viral envelope from neutralizing antibodies. The aim of the present study was to explore the possibility to obtain a more broadly neutralizing immune response by immunizing guinea pigs with gp160 depleted of three N-linked glycans in the CD4-binding domain by site-directed mutagenesis. Mutant and wild type gp160 were formulated into immunostimulating complexes and injected s.c. into guinea pigs. Both preparations induced high serum antibody response to native gp120 and V3 peptides. Both preparations also induced antibodies that bound equally well to the V3 loop or the CD4-binding region, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The sera from animals, immunized with mutated glycoprotein, did not neutralize nonrelated HIV strains better than did sera from animals, immunized with wild type glycoprotein. Instead, a pattern of preferred homologous neutralization was observed, i.e., sera from animals, immunized with mutant gp160, neutralized mutant virus better than wild type virus, and vice versa. These data indicated that elimination of the three N-linked glycans from gp160 resulted in an altered local antigenic conformation but did not uncover hidden neutralization epitopes, broadening the immune response.

Detection and Typing of Herpes Simplex Virus (HSV) in Mucocutaneous Samples by TaqMan PCR Targeting a gB Segment Homologous for HSV Types 1 and 2

ABSTRACT Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled with carboxyfluorescein (FAM) for HSV-1 and JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein) for HSV-2 had a sensitivity similar to that seen in separate reactions. All but one of 217 samples (99.5%) that had been positive by virus culture were positive by TaqMan PCR, with a correct identification of type in all cases. Out of 262 samples negative by virus culture, 48 (18.3%) were positive by TaqMan PCR, with higher Ct values compared with culture positive samples (P < 0.0001). Overall, the Ct values for HSV-1 were lower than for HSV-2 (mean, 25.5 versus 27.9), but to some extent this could be due to weaker fluorescence by JOE. Lower Ct values for HSV-1 were seen also in the 202 genital samples (79 HSV-1, 122 HSV-2, 1 HSV-1 and HSV-2), indicating that HSV-1 replicates as well as HSV-2 in the genital area. HSV-1 constituted 40% of genital infections and was associated with lower mean age (29.2 versus 36.4 years), probably reflecting the fact that recurrent genital HSV-1 infections are rare.

Characterization of a Herpes Simplex Virus Type 2-specified Glycoprotein with Affinity for N-Acetylgalactosamine-specific Lectins and Its Identification as g92K or gG

Extracts from herpes simplex virus type 2 (HSV-2)-infected cells were subjected to affinity chromatography with gel-bound Helix pomatia lectin (HPA). Only one HSV-2-specified glycoprotein was isolated by this procedure and the glycoprotein had an apparent molecular weight of 130 000 (130K). The HPA-binding glycoprotein was genetically mapped, using HSV-1 X HSV-2 intertypic recombinants into the short component of the HSV-2 genome. The mapping position, electrophoretic mobility and the antigenic properties of the HPA-binding protein indicated that it was unrelated to glycoprotein C (gC), which is the HPA-binding glycoprotein in HSV-1-infected cells, and distinct from gE and gD which map in the S component. The glycoprotein was almost quantitatively precipitated by monoclonal antibody AP1, specific for glycoprotein g92K and it also reacted with monoclonal antibody 1206-3, specific for the HSV-2 glycoprotein G previously described. It is concluded that the isolated glycoprotein is identical to g92K and consequently also to the HSV-2-specific glycoprotein G.

Neutralizing Antibody Response During Human Immunodeficiency Virus Type 1 Infection: Type and Group Specificity and Viral Escape

The paradox that group-specific neutralizing antibodies (NA) exist in the majority of human immunodeficiency virus type 1 (HIV-1)-infected patients, whereas the NA response against autologous HIV-1 virus isolates is highly type-specific, motivated us to study the type- and group-specific NA responses generated upon presentation of escape virus, and the viral epitopes involved in the escape. Patients with demonstrable escape virus all developed group-specific NA, which were detectable after a delay and disappeared prior to disease development. The sera tested inhibited the binding of recombinant soluble gp120IIIB to cell-associated CD4, but group-specific virus neutralization required binding of NA to HIV-1 prior to viral attachment to target cells. Consecutive escape virus isolates were tested for sensitivity to neutralization by heterologous sera. Only minor differences were demonstrated, suggesting that the majority of the change in neutralization sensitivity is driven by the selective pressure of type-specific NA. Furthermore, no differences were observed in sensitivity to neutralization by anti-carbohydrate neutralizing monoclonal antibodies or the lectin concanavalin A, indicating a conserved nature of certain carbohydrate neutralization epitopes during escape. Finally the V3 sequence of three sets of consecutive virus isolates were analysed revealing amino acid mutations in V3 sequences of all escape virus isolates. The biological significance of these variations was confirmed further by the demonstration of changes in sensitivity to neutralization by anti-V3 monoclonal antibodies. These results strongly suggest a participation of the NA response against the V3 loop in the immunoselection of escape virus.

LIR-1 expression on lymphocytes, and cytomegalovirus disease in lung-transplant recipients

Human cytomegalovirus infection is a major cause of morbidity after lung transplantation. LIR-1 (leucocyte immunoglobulin-like receptor-1) is an inhibitory cell surface receptor that has high affinity for an MHC class I homologue (UL18) encoded by human cytomegalovirus. We aimed to investigate whether reactivation of human cytomegalovirus affects the expression of LIR-1. We measured LIR-1 expression on peripheral blood lymphocytes from 13 lung-transplant recipients and established human cytomegalovirus load using PCR. Eight patients developed cytomegalovirus disease. The percentage of cells expressing LIR-1 increased in the patients who developed cytomegalovirus disease several weeks before viral DNA was detectable by PCR. Measurement of LIR-1 expression might allow early identification of cytomegalovirus disease in lung-transplant patients.

RAPID SELECTION FOR AN N-LINKED OLIGOSACCHARIDE BY MONOCLONAL ANTIBODIES DIRECTED AGAINST THE V3 LOOP OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

The V3 loop of the human immunodeficiency virus (HIV) surface protein, gp 120, constitutes a principal neutralizing determinant. HIV strains lacking a naturally conserved N-linked oligosaccharide (at position 306) within the V3 loop are highly sensitive to neutralization. We subjected molecular clones of HIV(LAI) lacking this 306N-glycan to in vitro immune selection with MAbs directed against the V3 loop. In all, ten clones were characterized, and all proved resistant to V3-directed neutralization. Sequencing of the V3 loop revealed that six of the clones had become resistant at least partly by reacquisition of the 306N-glycan. Only two of the clones possessed mutations within the binding site of the antibody itself, while the two remaining clones did not display changes within the V3 loop itself. Thus, HIV strains lacking the 306N-glycan primarily develop resistance to V3-directed neutralization through acquisition of the specific oligosaccharide. This demonstrates that protein glycosylation can be a primary modifier of virus antigenicity of possible importance for the interaction of HIV with the host immune response.