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S Zolla-Pazner

Publications and source records attributed to S Zolla-Pazner.

At least 73 records · Page 4Linked to original sources
PubMed1997

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.

3T3 Cells↗
PubMed1996

Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies.

Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.

Amino Acid Sequence↗
PubMed1996

Mechanisms contributing to the neutralization of HIV-1.

The phenomenon of virus neutralization is a function of three variables: the antibody (Ab), the virus and the target cell. Variation in any one of these parameters may drastically affect the results of assays for neutralization. In focusing on the virus as a variable in assays for the neutralization of HIV-1, it has been shown that the range of sensitivity of primary HIV-1 isolates is quite large. This may be due to the structure and biology of the virus particle, its density of envelope proteins, its ability to retain or shed these proteins, and its phenotype, determining the type of cells it will infect. The Ab used for neutralization also contributes to the efficiency of neutralization. Thus, the 'match' between the specificity of the Ab and the structure and availability of the epitope on the virus will affect the interaction and contribute to the resultant reduction in virus infectivity. Similarly, the strength of interaction between the virus and the neutralizing Ab, dependent on the affinity of the Ab for the virus epitope, will also be a determining factor. However, other factors contribute to the neutralization sensitivity of primary isolates of HIV-1. One factor that has been almost completely overlooked in the recent literature is the role that the target cell plays in revealing reduced virus infectivity. The facility and mechanism through which different cell types bind a virion and are infected by it will contribute profoundly to the efficiency with which Ab-mediated neutralization can be detected. These factors are discussed below with particular reference to interpreting (and re-interpreting) the current literature on HIV-1 neutralization.

Binding, Competitive↗
PubMed1996

Abnormal serum porphyrin levels in patients with the acquired immunodeficiency syndrome with or without hepatitis C virus infection.

OBJECTIVE: To define the contributions of human immunodeficiency virus (HIV) and hepatitis C virus infections to the development of porphyria cutanea tarda. DESIGN: Analysis of serum porphyrin levels in a cohort of 167 subjects. Serum samples were divided into 4 groups corresponding to the status of HIV and hepatitis C virus infections: positive-positive, positive-negative, negative-positive, and negative-negative. SETTING: Serum samples positive for HIV were obtained from the serum bank of an acquired immunodeficiency syndrome-HIV research center, and HIV-negative samples were obtained from a regional blood center. MAIN OUTCOME MEASURES: Spectrofluorometric measurement of serum porphyrin levels. RESULTS: The median values of porphyrin were 2.31 nmol/L (interquartile range [difference between the 25th and 75th percentiles]: 4.55) in the positive-positive group, 1.99 nmol/L (interquartile range: 1.63) in the positive-negative group, 1.31 nmol/L (interquartile range: 1.58) in the negative-positive group, and 1.14 nmol/L (interquartile range: 0.92) in the negative-negative group. The fluorescence emission spectra of samples with elevated porphyrin levels were identical with that reported for porphyria cutanea tarda. Elevated porphyrin levels were significantly associated with HIV infection (P < .001) and were observed in patients with an elevated level of alanine aminotransferase (P = .03). Infection with hepatitis C virus was also associated with an elevation in porphyrin levels, although the increase was not statistically significant (P = .16). Porphyrin levels in patients positive for HIV were not correlated with serum urea nitrogen or creatinine levels. None of the patients had symptomatic porphyria cutanea tarda. CONCLUSIONS: Factors associated with increased serum porphyrin levels included HIV infection, elevated alanine aminotransferase levels, and, to a lesser extent, hepatitis C virus infection. These findings suggest that patients with the above risk factors are potentially predisposed to the development of symptomatic porphyria cutanea tarda on further exposure to hepatotoxic agents.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1.

This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms.

Antigens, CD↗
PubMed1995

Epitope exposure on functional, oligomeric HIV-1 gp41 molecules.

We have used cells infected with the HIV-1 molecular clone HX10 to study the binding of monoclonal antibodies (mAbs) to different epitopes within the extracellular domain of the HIV-1 transmembrane glycoprotein gp41. Gp41 mAb binding to the infected cells at 4 degrees was variable but weaker than the binding of an anti-gp120/V3 loop mAb and increased substantially for three of the gp41 antibodies at 37 degrees. Treatment of the cells with soluble CD4 (sCD4) at 37 degrees increased gp41 mAb binding to epitopes spanning residues 521-663, implying that these regions had probably been masked by gp120, which following interaction with sCD4 had subsequently dissociated from gp41. By contrast, the binding of a mAb to residues 662-667 which form a neutralization epitope was reduced by sCD4 binding. Another region which has been described as containing a neutralization epitope spans residues 725-750. MAbs to this region bound equally well to noninfected and HIV-infected cells, and binding was not increased in the presence of sCD4. These data strongly imply that this epitope is not exposed on the external surface of the membrane, a finding in accord with the proposed cytoplasmic localization of this region.

Antibodies, Monoclonal↗
PubMed1995

Functional activities of 20 human immunodeficiency virus type 1 (HIV-1)-specific human monoclonal antibodies.

Antibodies that are useful in the treatment of HIV infection should result in virus neutralization or lysis of infected cells but should not enhance infection. In this study, the potential clinical use of 20 HIV-1-specific human monoclonal antibodies (HuMAbs) was determined by measuring their enhancing (C-ADE) activities using HIVLAI as the target virus. Two HuMAbs mediated both C-ADE and ADCC, two exclusively neutralized, and five exclusively mediated ADCC. Ten HuMAbs demonstrated no activity in any of the three assays. Three antibodies that neutralized HIVLAI were tested against HIVSF2; all three also neutralized HIVSF2. Four of five HuMAbs mediating ADCC against HIVLAI that were also tested against HIVSF2 had ADCC activity against HIVSF2. These results demonstrate that many HuMAbs have unique functions, allowing the separation of potentially beneficial and harmful activities. Combinations of HuMAbs with ADCC and neutralizing functions may have therapeutic utility.

Antibodies, Monoclonal↗
PubMed1995

Induction of HIV type 1 neutralizing and env-CD4 blocking antibodies by immunization with genetically engineered HIV type 1-like particles containing unprocessed gp160 glycoproteins.

Genetically engineered, noninfectious HIV-1-like particles containing processed envelope glycoproteins represent potential candidate immunogens for a vaccine against HIV-1. However, since the gp120 glycoprotein is known to be rapidly lost from the surface of infected cells and purified virions as a result of its low-affinity interaction with gp41, shedding of this extracellular subunit could compromise the immunogenic potential of particle-based HIV-1 vaccine candidates. In this study, we demonstrate for the first time the feasibility of producing fully assembled HIV-1-like particles containing only unprocessed gp160 glycoproteins. Monkey kidney Vero cells were transfected with an inducible, human metallothionein-based expression vector containing most of the HIV-1LAI coding sequences that were genetically modified to introduce safety mutations and destroy the major cleavage site of the HIV-1 envelope glycoprotein. A stably-transfected cell line was isolated and shown to secrete HIV-1-like particles containing unprocessed gp160. Immunization with these particles induced HIV-1 cross-neutralizing, syncytium-inhibiting and env-CD4 blocking antibodies. Thus, these novel HIV-1-like particles represent alternative candidate immunogens for the development of a particle-based AIDS vaccine.

AIDS Vaccines↗
PubMed1995

A resting cell assay for improved detection of antibody-mediated neutralization of HIV type 1 primary isolates.

The sensitivity with which antibody-mediated neutralization is detected in vitro is dependent on the virus, the antibody, the target cells, and the culture conditions used in the assay. Using activated and transformed target cells, the ability of various culture-adapted and primary strains of HIV-1 to be neutralized by different polyclonal and monoclonal antibody preparations has been thoroughly studied. However, the vast majority of HIV-1-susceptible CD4+ cells in vivo are not activated or transformed, but are quiescent. Because resting lymphocytes can be infected with HIV-1, we initiated studies to determine (1) if the use of resting lymphocytes as target cells would result in a neutralization assay with increased sensitivity, (2) if the degree of target cell activation had a measurable effect on the sensitivity with which antibody-mediated neutralization could be detected, and (3) whether, using a more sensitive assay, neutralizing antibodies in patients' sera might be detectable that had been below the threshold of detection when using "conventional" assays. The experiments described in the studies below reveal that an inverse relationship exists between the level of target cell activation and the sensitivity with which neutralization can be detected. Moreover, using an assay in which unstimulated peripheral blood mononuclear cells serve as target cells, experiments show that antibody-mediated neutralization of primary and prototype laboratory isolates of HIV-1 can be detected with 10- to 100-fold greater sensitivity than when stimulated peripheral blood mononuclear cells are used as target cells. With this resting cell assay, neutralizing activity can be detected in the sera of HIV-positive subjects that, by previously used "conventional" neutralization assays, was undetectable.

Amino Acid Sequence↗
PubMed1995

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential.

OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies.

AIDS Serodiagnosis↗
PubMed1995

Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry.

The immunologic relatedness of the various human immunodeficiency virus type 1 (HIV-1) clades was determined with 13 human anti-HIV-1 monoclonal antibodies (MAbs) to six immunogenic regions of the HIV-1 structural proteins. The immunoreactivity of the native, oligomeric viral envelope glycoproteins expressed on the surfaces of human peripheral blood mononuclear cells infected in vitro with primary isolates from clades A through E was determined by flow cytometry. Some epitopes in the immunodominant region of gp41 and the C terminus of gp120 appear to be HIV-1 group specific in that they are expressed on the surfaces of cells in cultures infected with the majority of viruses tested from clades A to E. Epitopes within the V3 region appear to be clade restricted. Surprisingly, one MAb to an epitope in the C terminus of gp120 was entirely clade B specific. Staining with anti-V2 and anti-CD4 binding domain (CD4bd) reagents was infrequently detected. Anti-CD4bd MAbs stained only CD4-negative T cells because the CD4bd of gp120 appeared to be complexed with membrane CD4. When present, the epitopes of V2 and the CD4bd appeared to be expressed on cells infected with various clades. Thus, the results suggest that MAbs to gp41, the C terminus, and the V3 loop of gp120 are most useful in serotyping primary isolates of HIV-1, providing group-specific, clade-restricted, and clade-specific reagents. The use of the immunofluorescent method with the reagents described herein distinguishes infection with clade B from that with all other HIV-1 clades. With additional MAbs, this technique will allow a broadly applicable, reproducible, and practical method for serotyping HIV-1.

Amino Acid Sequence↗
PubMed1994

Antibodies to the HIV-1 V3 loop in serum from infected persons contribute a major proportion of immune effector functions including complement activation, antibody binding, and neutralization.

Previous studies have shown that the V3 region of the HIV envelope is both critical to viral functions and immunogenic. However, the relative contribution of anti-V3 antibodies in the sera of infected individuals in mediating immune effector functions directed at whole intact virus and infected cells has not been determined. This study used peptides corresponding to several regions of the HIV envelope as inhibitors of antibody binding and antibody effector functions directed at virions and virus-infected cells in order to assess the relative importance of V3-specific antibodies in sera from infected persons. Approximately 40% of the antibody in serum which could bind to native viral proteins on HIVMN-infected cells was blocked by a peptide corresponding to the central 15 amino acids of the V3 loop. In contrast, little if any blocking of serum antibody binding was observed with peptides corresponding to flanking regions of HIVMN V3 or three regions of gp41. Since antiviral antibody can also activate immune effector functions, we determined whether peptides could block antibody-dependent activation of the complement system by HIV-infected cells or free virus. Surprisingly, the V3 loop peptide blocked 75-95% of complement activation on HIV-infected cells. While the V3 loop peptide also blocked a substantial portion of the neutralizing activity in serum from infected persons for free virus it was again more effective in inhibiting complement-mediated effects on free virus. Accordingly, antibody-dependent, complement-mediated virolysis was inhibited by 61-79%. The results of these experiments indicate that (1) a substantial portion (30-40%) of the antibody in sera from infected persons that is capable of binding to HIV-infected cells and HIV virions is V3-specific, and (2) these V3-specific antibodies are particularly important for complement activation on infected cells and virions. This indicates that the central portion of the V3 loop, while constituting less than 3% of the amino acid sequence of the HIV envelope, apparently provides a major gp160 site for immune effector functions, especially complement activation.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated neutralization of HIV-1.

mAbs specific for the V3 loop of HIV-1 are capable of neutralizing laboratory strains of HIV-1 in vitro. In this report we use surface plasmon resonance and biosensor technology to demonstrate that the ability of V3-specific mAbs to neutralize HIV-1(MN) correlated with the dissociation rate constant of the homologous mAb-gp120 binding interaction. mAbs capable of binding diverse strains of gp120 with similar association rate constants exhibited marked differences in the dissociation rate. The dissociation rate, and not the association rate, was found to be predictive of the neutralization capacity of the mAb. Furthermore, synthetic peptides corresponding to the V3 loop of HIV-1(IIIB, MN) yielded quantitatively similar binding kinetic profiles abrogating the need for purified recombinant gp120 protein and potentially facilitating the screening of more diverse isolates. Biosensor immobilized V3 peptides were found to mimic their conformational structure in solution. This offers advantages to peptides studied by ELISA where some degree of denaturation and masking of epitopes can occur upon adsorption of peptides to plastic surfaces. The impact of amino acid substitutions within epitopes on subsequent mAb binding was dissected by observing alterations in dissociation rates. The technique provides rapid kinetic analyses of V3 Ab binding interactions with diverse V3 sequences, facilitating the efficient screening and selection of those most likely to possess the broadest and most potent HIV-1 neutralizing potentials.

Amino Acid Sequence↗
PubMed1994

Neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-V3 monoclonal antibody, 447-52D.

Human monoclonal antibody 447-52D binds to the V3 determinant of the human immunodeficiency virus type 1 (HIV-1) gp120 external glycoprotein. Its binding requires the expression of the GPxR sequence at the center of the V3 domain. HIV-1 variants that are adapted to replication in T-lymphoid cell lines and express this sequence motif are efficiently neutralized by the antibody (M. K. Gorny, A. J. Conley, S. Karwowska, A. Buchbinder, J.-Y. Xu, E. A. Emini, S. Koenig, and S. Zolla-Pazner, J. Virol. 66:7538-7542, 1992). In the present study, the antiviral activity of 447-52D was further defined with regard to its ability to mediate neutralization of primary HIV-1 clinical isolates. Again, the antibody was found to potently neutralize those isolates that expressed the binding sequence. We confirmed that this determinant is commonly expressed by virus isolates belonging to the subtype (clade) B sequence classification. As such, 447-52D may be useful for prophylactic and immunotherapeutic intervention. In addition, the study demonstrated that neutralization of primary HIV-1 isolates is possible if mediated by an appropriate antibody.

AIDS Vaccines↗
PubMed1994

Human anti-V2 monoclonal antibody that neutralizes primary but not laboratory isolates of human immunodeficiency virus type 1.

A human immunoglobulin G1 lambda monoclonal antibody (MAb), 697-D, was developed that recognizes the V2 region of human immunodeficiency virus type 1 (HIV-1) gp120. Substitutions at amino acid positions 176/177, 179/180, 183/184, and 192 to 194 in the V2 loop of gp120 each completely abolished the binding capacity of 697-D in an enzyme-linked immunosorbent assay format. Competition analysis with three different neutralizing murine anti-V2 MAbs confirmed the specificity of 697-D. The 697-D epitope is primarily conformation dependent, although there was weak reactivity of the MAb with a V2 peptide spanning residues 161 to 180. Treatment of recombinant gp120 HIVIIIB with sodium metaperiodate, which oxidizes carbohydrates, abolished the binding of the MAb, showing the dependence of the epitope on intact carbohydrates. The broad reactivity of 697-D was displayed by its binding to the gp120 molecules from four of four laboratory isolates and five of five primary isolates. The MAb 697-D neutralized three out of four primary isolates but failed to neutralize any of four laboratory strains of HIV-1. 697-D and a human anti-V3 MAb, 447-52-D, displayed similar potency in neutralizing primary isolates, indicating that the V2 region of gp120, like the V3 region and the CD4-binding domain, can induce potent neutralizing antibodies against HIV-1 in humans.

Amino Acid Sequence↗