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Biomedical subjects

M Robert-Guroff

Publications and source records attributed to M Robert-Guroff.

At least 19 recordsLinked to original sources
PubMed1992

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Type- and group-specific continuous antigenic determinants of HTLV. Use of synthetic peptides for serotyping of HTLV-I and -II infection.

The human T lymphotropic viruses (HTLV-I and -II) are relatively common in subpopulations of certain countries, notably intravenous drug abusers in North America. Infections with these malignancy-associated human retroviruses are hard to discriminate with currently available commercial serological tests. We studied the distribution of antigenicity and the degree of cross-reactivity of epitopes in gag and env of the two viruses. Sequences in the carboxyl terminus of the matrix protein (MA) and the middle of the outer glycoprotein (SU) reacted in a type-specific fashion, while sequences from the capsid protein (CA), the carboxyl terminus of SU, and conserved portions of the transmembrane protein (TM) mainly reacted in a group-specific fashion, correlating with the degree of sequence dissimilarity between the two viruses. The serological discrimination obtained with the peptides was evaluated in a panel of 25 sera where infection with HTLV-I or -II had been typed by competition in a p24 enzyme-linked immunosorbent assay (ELISA) or by the polymerase chain reaction (PCR). After processing peptide results in a computer program, a typing result concordant with earlier results was obtained in 21 of 25 sera. Of the remaining five sera, four were labeled "too weak for typing" and one "HTLV of uncertain type" by the program. They did not react sufficiently strongly or clearly with the peptides to allow classification. A combination of synthetic peptides may become useful for serotyping HTLV infection and become an alternative to Western blots for confirmation of HTLV positivity.

Amino Acid Sequence↗
PubMed1990

The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope.

We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for neutralization resistance of the immune-selected variant is more likely a conformational change altering a neutralization epitope at a distant site.

Amino Acid Sequence↗
PubMed1990

Structure and expression of tat-, rev-, and nef-specific transcripts of human immunodeficiency virus type 1 in infected lymphocytes and macrophages.

Primary RNA transcripts from the human immunodeficiency virus type 1 (HIV-1) are processed into mature mRNA by a complex series of splicing events. Viral structural proteins and reverse transcriptase are translated from unspliced or singly spliced transcripts. Proteins which control virus replication, including tat, rev, and nef, are translated from transcripts which are the product of multiple splicing. We have analyzed the composition and relative abundance of the latter transcripts in long-term infected cell lines and in acutely infected peripheral blood cells by amplification with the polymerase chain reaction (PCR) followed by Southern blot, molecular cloning, and DNA sequence analyses. In H9 cells chronically infected with the HIV-1 strain HTLV-IIIB, the predominant of the three kinds of transcripts is those coding for nef. Transcripts with coding potential for rev constituted an intermediate fraction of those analyzed, while those for tat accounted for only a small minority. A similar pattern was observed with Southern blots of PCR-amplified transcripts from peripheral blood lymphocytes acutely infected with HTLV-IIIB. The same general pattern was also observed with PCR-amplified transcripts from peripheral blood monocyte-macrophages infected with an HIV-1 strain (BA-L) able to grow to high titers in macrophages. In these cells, however, the apparent major form of nef transcript contained only the first and third exons of the multiply spliced transcripts and appeared to be generated by either a single or a triple splicing mechanism. As with lymphocytes, tat-specific mRNAs were by far the least abundant. It thus appears that different cell types infected with different strains of HIV-1 maintain a similar balance of expression in which transcripts for nef vastly predominate over those for tat and that those for rev are intermediate in abundance.

Amino Acid Sequence↗
PubMed1990

Persistent infection of rhesus macaques with a molecular clone of human immunodeficiency virus type 2: evidence of minimal genetic drift and low pathogenetic effects.

In an attempt to generate a suitable animal model to study the infectivity and possible pathogenicity of human immunodeficiency viruses, we intravenously inoculated juvenile rhesus macaques and African green monkeys with a molecularly cloned virus, human immunodeficiency virus type 2 HIV-2sbl/isy, as well as with the uncloned HIV-2nih-z virus. Infection was monitored by virus recovery from the peripheral blood cells and by seroconversion against HIV-2 antigens measured by Western immunoblot, radioimmunoprecipitation, and enzyme-linked immunosorbent assay. We successfully infected two out of two macaques with the molecularly cloned virus and one macaque out of two with the HIV-2nih-z. No evidence of infection was seen in the African green monkeys with either virus. We followed the infected animals for 2 years. The animals remained healthy, although we observed intermittent lymphadenopathy and a transient decrease in the absolute number of circulating CD4+ T lymphocytes in both animals infected with the molecularly cloned virus. Virus isolation from the peripheral blood cells of the infected animals was successful only within the first few months after inoculation. Evidence of persistent infection was provided by the detection of proviral DNA by polymerase chain reaction analysis of the blood cells of the inoculated animals and by the stability of antiviral antibody titers. To evaluate the genetic drift of the proviral DNA, we molecularly cloned viruses which were reisolated 1 and 5 months postinoculation from one of these animals. Comparison of the DNA sequences of the envelope genes of both these isolates indicated that a low degree of variation (0.2%) in the envelope protein had occurred in vivo during the 5-month period. These data suggest that the use of HIV-2sbl/isy in rhesus macaques may represent a good animal model system to study prevention of viral infection. In particular, molecularly cloned virus can be manipulated for functional studies of viral genes in the pathogenesis of acquired immune deficiency syndrome and provides a reproducible source of virus for vaccine studies.

Animals↗
PubMed1989

Mother-to-infant transmission of human immunodeficiency virus type 1: association with prematurity or low anti-gp120.

In a prospective study of pregnant women infected with human immunodeficiency virus type 1 (HIV-1) in Brooklyn, New York, USA, 16 (29%) of 55 evaluable infants were infected with HIV-1. 9 infants had paediatric acquired immunodeficiency syndrome, 6 had less severe clinical manifestations of HIV-1 infection, and 1 was symptom-free but was seropositive for HIV-1 beyond 15 months of age. The 10 infants born at 37 weeks of gestation or earlier were at higher risk of HIV-1 infection than infants born at 38 weeks of gestation or later (60% vs 22%) but the median age at appearance of disease was approximately 5 months in both groups. The HIV-1 transmission rate was not associated with predelivery levels of maternal T cells, anti-p24, or neutralising antibodies but it was higher, among full-term infants, for those with mothers in the lowest third of the distribution of anti-gp120 levels (53%). On immunoblot, transmitting mothers lacked a gp120 band but not other bands. Protection was not associated with antibody to recombinant peptides from the hypervariable region of the major neutralising gp120 epitope, and the anti-gp120 endpoint dilution titre was similar in transmitting and non-transmitting mothers. Mothers of uninfected full-term infants appear to confer immunological protection against HIV-1 infection of their offspring by way of a high-affinity antibody to a gp120 epitope, whose specificity has importance for vaccine development and possibly perinatal immunotherapy.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Seroprevalence of HTLV-I in Portugal and evidence of double retrovirus infection of a healthy donor.

The prevalence of antibodies to HTLV-I in 5,475 Portuguese from 6 regions spanning the country was studied. Overall seroprevalence was 0.55%, indicating that Portugal is not an endemic area for this virus. Seropositives were distributed throughout the country, and no geographic clustering was observed. The seroprevalence of individuals who had lived in former Portuguese colonies in Africa (0.7%) was significantly higher than that of individuals who had not been in Africa (0.36%). An increase in seroprevalence with age was noted, and more females than males were antibody-positive, though not significantly so. Serum from one donor (1711), originating from Guinea-Bissau, was shown by Western blot and radioimmune precipitation to react with various proteins of HTLV-I, HIV-1 and -2, and SIV. Based on the serologic profiles and isolation of bona fide HTLV-I from her lymphocytes (confirmed by immunologic analysis, molecular cloning of the provirus and restriction enzyme analysis and sequencing of the DNA), together with the reactivity of her sera with an HIV-2 isolate obtained from her husband, we conclude that this donor was doubly infected with HTLV-I and HIV-2, rather than being the host to an as yet unidentified retrovirus.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells.

In this report, we demonstrate the sequence-specific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced) transcripts. By contrast, the antisense sequence with unmodified normal phosphodiester linkages as well as phosphorothioate oligomers containing sense, random, homopolymeric sequences, or antisense sequence with N3-methylthymidine residues did not have an inhibitory effect on viral expression. Thus, sequence specificity and nuclease resistance were critical for the anti-viral-gene regulatory effect of the antisense molecules. The altered HIV-1 mRNA profile induced by the antisense phosphorothioate oligomer suggests that the mechanism for the inhibition of viral expression is due to an interference with the regulatory gene, rev, by translation arrest.

Base Sequence↗
PubMed1989

Isolate- and group-specific immune responses to the envelope protein of human immunodeficiency virus induced by a live recombinant vaccinia virus in macaques.

The immune responses produced by four macaques inoculated intradermally with a recombinant vaccinia virus that expresses the envelope gene of human immunodeficiency virus (HIV) were analyzed. Antibody capable of immunoprecipitating the glycosylated envelope protein precursor gp160 was detected in all animals within 3 weeks after the primary intradermal vaccination. The level of antibody was increased following a booster inoculation, and the sera from three of the four animals were then capable of immunoprecipitating gp120. Sera from two of the macaques with anti-gp120 antibody were able to prevent syncytium formation mediated by the parent HIV but not that induced by an unrelated HIV isolate. The fusion-inhibiting antibody was directed toward the highly variable central portion of the gp120 molecule since the effect was abrogated by incubation with PB1. The latter is a recombinant protein produced in Escherichia coli containing amino acids 295-474, a major neutralizing epitope. Sera from the three macaques with anti-gp120 neutralized HIV infectivity in an isolate-specific manner. The serum with highest neutralizing activity was also best at inhibiting syncytium formation. In contrast to the isolate-specific nature of the neutralizing antibody, T lymphocytes from immunized animals proliferated in response to divergent HIV isolates as well as to purified gp120. These studies provide baseline immunologic information for further development of recombinant vaccinia virus vectors.

Animals↗
PubMed1988

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.

Amino Acid Sequence↗
PubMed1986

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope.

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.

Antibodies, Viral↗
PubMed1986

Human monoclonal antibody directed against an envelope glycoprotein of human T-cell leukemia virus type I.

We report the production and characterization of a human monoclonal antibody reactive against the major envelope glycoprotein of human T-cell leukemia virus type I (HTLV-I), a virus linked to the etiology of adult T-cell leukemia. We exposed lymph-node cells derived from a patient with adult T-cell leukemia to the Epstein-Barr virus in vitro and obtained a B-cell clone (designated 0.5 alpha) by a limiting dilution technique. The secreted product of 0.5 alpha is a monoclonal antibody (also designated 0.5 alpha; that is IgG1 and has kappa light chains) that binds to the cell membrane of T-cells infected with HTLV-I and lyses them in the presence of complement. The antibody does not react with HTLV-I-negative T cells. In electroblot assays, the monoclonal antibody detects a 46-kDa glycoprotein in disrupted HTLV-I virions and a 34-kDa product following digestion of the viral protein with endoglycosidase F. These molecules have been reported to represent the HTLV-I env gene products. The antibody does not react with HTLV-II and HTLV-III virions. Glycoproteins of 61 and 68 kDa, which are known to be encoded at least in part by the env gene of HTLV-I, are precipitated by the antibody from endogenously radiolabeled HTLV-I-infected HUT 102-B2 and MT-2 cells, respectively. These results suggest that this human monoclonal antibody reacts with an env-encoded glycoprotein of HTLV-I. By using a competition assay with a biotin-labeled 0.5 alpha antibody, we observed that 15 out of 15 patients with adult T-cell leukemia had antibodies that block binding of the 0.5 alpha antibody to HTLV-I virions. This suggests that the antigen detected by 0.5 alpha antibody is a common epitope recognized in HTLV-I-infected individuals in vivo. This antibody, as well as the general strategy for making human monoclonal antibodies reactive against pathogenic retroviruses, may have diagnostic or therapeutic application.

Antibodies, Monoclonal↗
PubMed1986

Neurologic manifestations of human immunodeficiency virus infection in children.

This report describes the neurologic manifestations of 36 children with human immunodeficiency virus (HIV) infection. In this cohort, in 16 of 21 children with acquired immunodeficiency syndrome (AIDS), three of 12 children with AIDS-related complex, and one of three asymptomatic seropositive children, a progressive encephalopathy developed. Neurologic signs were often detected early but tended to worsen coincident with progression of the immunodeficiency. The presence of progressive encephalopathy correlated with the absence of serum neutralizing antibodies to HIV and with a poor, usually fatal, outcome. The incubation period from initial HIV infection in the perinatal period to the onset of progressive encephalopathy varied from 2 months to 5 years. Intrablood-brain barrier synthesis of HIV-specific antibodies was demonstrated in eight of 14 children with AIDS and AIDS-related complex, indicating active brain infection with HIV. In three cases this was unassociated with progressive neurologic signs. Unique neuropathologic findings in children who died with HIV infection further suggest that the progressive encephalopathy is the result of primary and persistent infection of the brain with this retrovirus. These findings broaden the spectrum of HIV infection in children and have important implications for the development of antiviral therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1979

Characterization of a type-C virus produced by co-cultures of human leukemic bone-marrow and fetal canine thymus cells.

The putative human helper virus SKA-21/A204V, isolated by Nooter et al. in 1977 from human leukemic bone-marrow cells following co-culture with normal fetal canine thymus cells, Cf2th, has been characterized with respect to its major viral core protein, reverse transcriptase, and nucleic acid sequences. The results of these analyses show that this virus is not distinguishable from the woolly monkey type-C virus, SSAV-1, by the techniques employed.

Animals↗
PubMed1977

Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase gamma of human lymphoblasts.

An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.

Antigen-Antibody Reactions↗