Mats A. A. Persson

Identification of a Third Human Polyomavirus

ABSTRACT We have previously reported on a system for large-scale molecular virus screening of clinical samples. As part of an effort to systematically search for unrecognized human pathogens, the technology was applied for virus screening of human respiratory tract samples. This resulted in the identification of a previously unknown polyomavirus provisionally named KI polyomavirus. The virus is phylogenetically related to other primate polyomaviruses in the early region of the genome but has very little homology (<30% amino acid identity) to known polyomaviruses in the late region. The virus was found by PCR in 6 (1%) of 637 nasopharyngeal aspirates and in 1 (0.5%) of 192 fecal samples but was not detected in sets of urine and blood samples. Since polyomaviruses have oncogenic potential and may produce severe disease in immunosuppressed individuals, continued searching for the virus in different medical contexts is important. This finding further illustrates how unbiased screening of respiratory tract samples can be used for the discovery of diverse virus types.

Migration and differentiation of neural precursor cells can be directed by microglia.

Recent reports have supported the existence of neural stem cells in the adult mammalian CNS. Important features of such cells are self-renewal and multipotency, i.e., they can give rise to neurons, astrocytes, and oligodendrocytes and thus in principle replace lost cells in the CNS. Observations in several animal models of CNS diseases have shown that by unknown mechanisms endogenous as well as exogenous precursor cells preferentially migrate to damaged areas. Microglia are immunoreactive cells of nonneural lineage resident in the CNS. After injury to the CNS, microglia are rapidly activated and found concentrated at the sites of injury. In the present article we show, in two different assays, that soluble factors released from mouse microglial cells direct the migration of neural CNS precursor cells. We also provide evidence that microglia have the capacity to influence the differentiation of both adult and embryonic neural precursor cells toward a neuronal phenotype. Given that an invariant feature of pathological processes in CNS is the activation of microglia, these results indicate an important and unique role for microglia in directing the replacement of damaged or lost cells in the CNS.

The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

ABSTRACT Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.

Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC50 values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523–535 region of E2, known to be important for the E2–CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523–535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes.

Recombinant human monoclonal antibodies against different conformational epitopes of the E2 envelope glycoprotein of hepatitis C virus that inhibit its interaction with CD81.

The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81-E2 interaction in vitro; all four were positive at 0.3-0.5 microg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2-CD81 interaction, of which one appeared immunodominant in this donor.

Cerebrospinal fluid interleukin-6 activity in HIV infection and inflammatory and noninflammatory diseases of the nervous system

Interleukin-6 (IL-6) activity was measured in the cerebrospinal fluid (CSF) of patients at different stages of human immunodeficiency (HIV) virus infection and of patients with multiple sclerosis (MS) or other inflammatory (OID) and noninflammatory neurological diseases (OND). In the advanced stages of HIV infection and in OID, IL-6 was detected more frequently (80 and 75% of the cases) and at higher concentrations than in the early stages of HIV infection. MS and OND (44, 48, and 44% of cases). Analysis of CSF and paired sera indicated that IL-6 production can be compartmentalized to either of the fluids. Evidence that altered blood-brain barrier functions can, at least in part, influence the CSF IL-6 levels was found in OID patients. No association was evident between intrathecal immunoglobulin synthesis and CSF IL-6 levels. Interleukin-1 (IL-1) levels were detectable in a minority of the samples from neurological patients; one OID patient had high levels of both CSF IL-1 and IL-6.

Tumor-Specific Bacteriophages Induce Tumor Destruction through Activation of Tumor-Associated Macrophages

We recently reported that administration of tumor-specific bacteriophages initiates infiltration of neutrophilic granulocytes with subsequent regression of established B16 tumors. The aim of the current study was to investigate the mechanism of action of bacteriophage-induced tumor regression and to examine possible stimulatory effects of bacteriophages on macrophages. We observed that the mechanism of phage-induced tumor regression is TLR dependent as no signs of tumor destruction or neutrophil infiltration were observed in tumors in MyD88−/− mice in which TLR signaling is abolished. The microenvironment of bacteriophage-treated tumors was further analyzed by gene profiling through applying a low-density array preferentially designed to detect genes expressed by activated APCs, which demonstrated that the M2-polarized tumor microenvironment switched to a more M1-polarized milieu following phage treatment. Bacteriophage stimulation induced secretion of proinflammatory cytokines in both normal mouse macrophages and tumor-associated macrophages (TAMs) and increased expression of molecules involved in Ag presentation and costimulation. Furthermore, mouse neutrophils selectively migrated toward mediators secreted by bacteriophage-stimulated TAMs. Under these conditions, the neutrophils also exhibited increased cytotoxicity toward B16 mouse melanoma target cells. These results describe a close interplay of the innate immune system in which bacteriophages, located to the tumor microenvironment due to their specificity, stimulate TAMs to secrete factors that promote recruitment of neutrophils and potentiate neutrophil-mediated tumor destruction.

Enzyme-linked immunosorbent assay for subclass distribution of human IgG and IgA antigen-specific antibodies.

In this study we describe an ELISA using monoclonal antibodies to IgG 1, 2, 3, 4, IgA1 and IgA2 for determining the subclass distribution of human-specific antibodies. No cross-reactivity of the subclass-specific reagents under the conditions used was observed. The sensitivity was 0.5 ng/ml for IgG1, 3, 4; 1.5 ng/ml for IgG2 and 50 ng/ml for IgA1 and IgA2. The reproducibility as described by the coefficient of variation calculated on repeated runs was 8-26% if the data were obtained by relating the absorbance values to a positive serum run in the assay, 17-58% when relating the OD figures to those of a standard myeloma plate. The method may be considered semiquantitative with high sensitivity and specificity, easy to handle and with small day-to-day variation. The assay has been applied to a number of antigens of protein and polysaccharide nature.

Tumor specific phage particles promote tumor regression in a mouse melanoma model

Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2Kb tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2Kb tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-γ as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.