Passive immunization and active vaccination against Hendra and Nipah viruses.

Hendra virus and Nipah virus are viral zoonoses first recognized in the mid and late 1990's and are now categorized as the type species of the genus Henipavirus within the family Paramyxoviridae. Their broad species tropism together with their capacity to cause severe and often fatal disease in both humans and animals make Hendra and Nipah "overlap agents" and significant biosecurity threats. The development of effective vaccination strategies to prevent or treat henipavirus infection and disease has been an important area of research. Here, henipavirus active and passive vaccination strategies that have been examined in animal challenge models of Hendra and Nipah virus disease are summarized and discussed.

HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.

A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated "fusin," is a putative G protein-coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target cells. Fusin messenger RNA levels correlated with HIV-1 permissiveness in diverse human cell types. Fusin acted preferentially for T cell line-tropic isolates, in comparison to its activity with macrophagetropic HIV-1 isolates.

CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.

Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic isolates. The chemokines RANTES, MIP-1alpha, and MIP-1beta, which suppress infection by macrophage-tropic isolates, selectively inhibited cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs. CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a fusion cofactor for macrophage-tropic HIV-1 strains.

Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera.

Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3-100.0) and 99.5 (95% CI 98.8-99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3-100.0) and 99.8 (95% CI 99.2-100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the test population.

Human immunodeficiency virus type-1 and chemokines: beyond competition for common cellular receptors.

The chemokines and their receptors have been receiving exceptional attention in recent years following the discoveries that some chemokines could specifically block human immunodeficiency virus type 1 (HIV-1) infection and that certain chemokine receptors were the long-sought coreceptors which, along with CD4, are required for the productive entry of HIV-1 and HIV-2 isolates. Several chemokine receptors or orphan chemokine receptor-like molecules can support the entry of various viral strains, but the clinical significance of the CXCR4 and CCR5 coreceptors appear to overshadow a critical role for any of the other coreceptors and all HIV-1 and HIV-2 strains best employ one or both of these coreceptors. Binding of the HIV-1 envelope glycoprotein gp120 subunit to CD4 and/or an appropriate chemokine receptor triggers conformational changes in the envelope glycoprotein oligomer that allow it to facilitate the fusion of the viral and host cell membranes. During these interactions, gp120 appears to be capable of inducing a variety of signaling events, all of which are still not defined in detail. In addition, the more recently observed dichotomous effects, of both inhibition and enhancement, that chemokines and their receptor signaling events elicit on the HIV-1 entry and replication processes has once again highlighted the intricate and complex balance of factors that govern the pathogenic process. Here, we will review and discuss these new observations summarizing the potential significance these processes may have in HIV-1 infection. Understanding the complexities and significance of the signaling processes that the chemokines and viral products induce may substantially enhance our understanding of HIV-1 pathogenesis, and perhaps facilitate the discovery of new ways for the prevention and treatment of HIV-1 disease.

Chemokine receptors and HIV.

The discovery that chemokine receptors are the human cofactors required along with CD4 for fusion and infection by HIV has opened new directions in AIDS research on mechanisms of viral entry, tropism, and pathogenesis. A possible mechanism of co-receptor function has been demonstrated that involves the formation of a complex on the cell surface between the HIV-1 envelope, CD4, and the coreceptor. Functional studies indicate that this interaction is structurally complex, that it probably involves multiple domains of the coreceptor, and that different virus isolates interact with coreceptors in distinct ways. Other immunodeficiency viruses including simian immunodeficiency virus and feline immunodeficiency virus also utilize chemokine receptors for entry. The identification of genetic polymorphisms helps explain why some people, with alterations in the CCR5 gene that prevent expression, are protected from HIV-1 infection. The discovery of specific HIV-1 fusion coreceptor molecules has not only provided new insights into the mechanisms of viral entry and tropism, but also led to new avenues of investigation on strategies to block HIV infection.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors.

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.

Increased association of glycoprotein 120-CD4 with HIV type 1 coreceptors in the presence of complex-enhanced anti-CD4 monoclonal antibodies.

CD4-specific monoclonal antibodies (CG1, CG7, and CG8), which bind with a 5- to 10-fold higher avidity to preformed CD4-gp120 complexes than to CD4, were previously shown to recognize newly identified conformational epitopes in the D1-CDR3 region of CD4. In the current study, these and other complex-enhanced MAbs were tested in three separate assays of HIV-1 coreceptor (CXCR4/CCR5) recruitment. In these assays, the CD4-specific MAbs CG1, -7, and -8 stabilized the association of coreceptor, gp120, and CD4 in trimolecular complexes. In contrast, the gp120-specific, complex-enhanced MAbs 48d and 17b were inhibitory. These data suggest that conformational changes in the CDR3 region of CD4-D1, induced by gp120 binding, may be involved in coreceptor association and thus play a positive role in the HIV-1 cell fusion process.

Henipaviruses: recent observations on regulation of transcription and the nature of the cell receptor.

Hendra virus (HENV) and Nipah virus (NIPV) are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genetic and biological characteristics that differentiate henipaviruses from other members of the subfamily are summarized. Although they do not display neuraminidase and hemagglutination activities and in that regard resemble viruses in the genus Morbillivirus, several recent observations highlight similarities between henipaviruses and respiroviruses (genus Respirovirus) in structure and replication strategy. First, three-dimensional modeling studies suggest that the external globular head domain of the HENV G protein resembles that of respiroviruses rather than morbilliviruses. Second, the pattern of transcriptional attenuation in HENV-infected cells resembles that observed with Sendai virus, a respirovirus, and differs from that found in cells infected with measles virus, a morbillivirus. Henipaviruses have a broad host range in vitro and in vivo, indicating wide distribution of cellular receptor molecules. The extensive host range has been confirmed in a quantitative in vitro cell-fusion assay using recombinant vaccinia viruses expressing the attachment and fusion proteins of HENV and NIPV. Cell lines of diverse origin and which are permissive in the in vitro cell fusion assay have been identified and the pattern of relative susceptibilities is the same for both HENV and NIPV, implying that both viruses use the same cell receptor. Protease treatment of permissive cells destroys their ability to fuse with cells expressing viral envelope glycoproteins. Virus overlay protein binding assay (VOPBA) and radio-immune precipitation assays confirm that both HENV and NIPV bind to membrane proteins in the 35-50 kD range. Treatment of cell membrane proteins with N-glycosidase eliminates HeV binding activity in VOPBA whereas treatment with neuraminidase has no effect on binding. Thus preliminary evidence suggests that NIPV and HENV bind to the same glycoprotein receptor via a non-sialic acid-dependant mechanism.

Recombinant vaccinia viruses. Design, generation, and isolation.

The technologies of recombinant gene expression have greatly enhanced the structural and functional analyses of genetic elements and proteins. Vaccinia virus, a large double-stranded DNA virus and the prototypic and best characterized member of the poxvirus family, has been an instrumental tool among these technologies and the recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins. Vaccinia virus is also the prototype live viral vaccine and serves as the basis for well established viral vectors which have been successfully evaluated as human and animal vaccines for infectious diseases and as anticancer vaccines in a variety of animal model systems. Vaccinia virus technology has also been instrumental in a number of unique applications, from the discovery of new viral receptors to the synthesis and assembly of other viruses in culture. Here we provide a simple and detailed outline of the processes involved in the generation of a typical recombinant vaccinia virus, along with an up to date review of relevant literature.

Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.

HIV and the 7-transmembrane domain receptors.

The recent discovery of a chemokine receptor, fusin (fusin/CXCR-4), as the long-sought human immunodeficiency virus type 1 (HIV-1) coreceptor opened an entirely new field of aquired immunodeficiency syndrome (AIDS) research on mechanisms of viral entry, tropism and pathogenesis. It was soon followed by the identification of the chemokine receptor CCR-5 as the major macrophage-tropic (M-tropic) HIV-1 coreceptor and the demonstration that other chemokine receptors, CCR-3 and CCR-2b, also may serve as coreceptors, albeit at somewhat lower efficiency. Very recently it was demonstrated that the mechanism of the coreceptor function involves the formation of a complex on the cell surface between the HIV-1 envelope, the primary receptor CD4 and the coreceptor. Thus the prevention of the HIV-1 envelope glycoprotein-mediated fusion by the chemokines RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta, as well as by the recently identified fusin/CXCR-4 ligand, stromal cell-derived factor-1 (SDF-1) could be explained by disruption of that complex. Interestingly, the identification of the HIV-1 coreceptor CCR-5 not only provided new insights into the mechanisms of viral entry and tropism, but also may help in explaining why some people with genetic alterations in CCR-5 are protected from HIV-1 infection.

Substitutions in a homologous region of extracellular loop 2 of CXCR4 and CCR5 alter coreceptor activities for HIV-1 membrane fusion and virus entry.

CXCR4 and CCR5 are the principal coreceptors for human immunodeficiency virus type-1 (HIV-1) infection. Previously, mutagenesis of CXCR4 identified single amino acid changes that either impaired CXCR4's coreceptor activity for CXCR4-dependent (X4) isolate envelope glycoproteins (Env) or expanded its activity, allowing it to serve as a functional coreceptor for CCR5-dependent (R5) isolates. The most potent of these point mutations was an alanine substitution for the aspartic acid residue at position 187 in extracellular loop 2 (ecl-2), and here we show that this mutation also permits a variety of primary R5 isolate Envs, including those of other subtypes (clades), to employ it as a coreceptor. We also examined the corresponding region of CCR5 and demonstrate that the substitution of the serine residue in the homologous ecl-2 position with aspartic acid impairs CCR5 coreceptor activity for isolates across several clades. These results highlight a homologous and critical element in ecl-2, of both the CXCR4 and CCR5 molecules, for their respective coreceptor activities. Charge elimination expands CXCR4 coreceptor activity, while a similar charge introduction can destroy the coreceptor function of CCR5. These findings provide further evidence that there are conserved elements in both CXCR4 and CCR5 involved in coreceptor function.

Consistent and significant inhibition of human immunodeficiency virus type 1 envelope-mediated membrane fusion by beta-chemokines (RANTES) in primary human macrophages.

Infection and entry of CD4(+) cells by human immunodeficiency virus type 1 (HIV-1) requires a coreceptor molecule, which, in concert with CD4, interacts with the viral envelope glycoprotein (Env), leading to membrane fusion. The principal coreceptors are the CCR5 and CXCR4 chemokine receptors. The suppressive effect of beta-chemokines, principally RANTES, on certain HIV-1 isolates was established before the discovery of the CCR5 receptor, and there have since been multiple reports confirming this initial observation. However, the inhibitory effect of beta-chemokines on HIV-1 infection of macrophages has been controversial. The current study focused on this issue in detail, with a reductionist approach, using assays that measure the effect of beta-chemokines solely on Env-mediated fusion. It is shown that under a variety of culture and differentiation conditions, RANTES maintains a significant and consistent inhibitory effect on CCR5-dependent Env-mediated fusion, and the role of these findings is discussed in relation to the role of beta-chemokines in HIV pathogenesis.

CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion.

The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS)

Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation.

The fusogenic activities of enveloped-virus glycoproteins were analyzed by using a quantitative, sensitive, rapid, and highly versatile recombinant vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. One population uniformly expressed vaccinia virus-encoded viral glycoproteins mediating specific binding and fusion activities; the other expressed the corresponding cellular receptor(s). The cytoplasm of one population also contained vaccinia virus-encoded bacteriophage T7 RNA polymerase; the cytoplasm of the other contained a transfected plasmid with the Escherichia coli lacZ gene linked to the T7 promoter. When the two populations were mixed, cell fusion resulted in activation of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase activity was assessed by colorimetric assay of detergent cell lysates or by in situ staining. We applied this approach to study the human immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4 interaction. Beta-Galactosidase was detected within 1 h after cell mixing and accumulated over the next several hours. Cell fusion dependence was demonstrated by the strict requirement for both CD4 and functional Env expression and by the inhibitory effects of known fusion-blocking monoclonal antibodies and pharmacological agents. Quantitative measurements indicated much higher sensitivity compared with analysis of syncytium formation. The assay was used to probe mechanisms of the cell type specificity for Env-CD4-mediated fusion. In agreement with known restrictions, cell fusion occurred only when CD4 was expressed on a human cell type. Membrane vesicle transfer experiments indicated that CD4 initially produced in either human or nonhuman cells was functional when delivered to human cells, suggesting that the fusion deficiency with nonhuman cells was not associated with irreversible defects in CD4. We also demonstrated that the infectivity specificities of different human immunodeficiency virus type 1 isolates for peripheral blood lymphocytes versus continuous CD4+ cell lines were associated with corresponding fusion selectivities of the respective recombinant Env proteins. The assay enabled analysis of the fusogenic activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the paramyxovirus simian virus 5. This system provides a powerful tool to study fusion mechanisms mediated by enveloped-virus glycoproteins, as well as to screen fusion-blocking antibodies and pharmacological agents.

Identification of a specific receptor for plasmin on a group A streptococcus.

Certain group A streptococci demonstrate surface receptors that bind selectively to the key fibrinolytic enzyme, plasmin. These bacteria show no reactivity with the zymogen protein plasminogen or with other serine class proteases, such as trypsin or urokinase. Bacterium-bound plasmin retains its ability to cleave synthetic substrates and its ability to hydrolyze a fibrin clot. The bacterium-bound plasmin is not effectively regulated by its physiological regulator, alpha 2-plasmin inhibitor. This study is the first report of a bacterium-associated receptor for plasmin.

Mapping of the human plasmin domain recognized by the unique plasmin receptor of group A streptococci.

A high-affinity surface receptor for human plasmin has been reported on certain group A streptococci. To map the region of the plasmin molecule that binds to the bacterial receptor, isolated domains of plasmin were tested for their ability to inhibit the binding of intact radiolabeled plasmin to receptor-positive bacteria. Complete inhibition of binding of labeled plasmin to bacteria by isolated heavy chains was achieved, but this inhibition was not as efficient on a molar basis when compared with that of unlabeled plasmin. By contrast, a conformationally altered form of native plasminogen was found to bind to bacteria and was as efficient a competitive inhibitor as intact plasmin was. The results of this study indicate that the selective binding of human plasmin to a group A streptococcus is dependent on structures present in the conformationally altered form of native plasminogen or plasmin that are not found on the native zymogen, the plasminogen with NH2-terminal glutamic acid.