Mutations in both gp120 and gp41 are responsible for the broad neutralization resistance of variant human immunodeficiency virus type 1 MN to antibodies directed at V3 and non-V3 epitopes.

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative alpha-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.

Preparation and characterization of human HIV type 1 neutralizing reference sera.

Reference neutralizing antibody (NA) reagents are needed for laboratories to be able to compare results of neutralization assays that will be used to monitor HIV-1 vaccine recipients. In an effort to establish such reference reagents two asymptomatic, seropositive patients were identified with medium to high amounts of cross-reactive NA activity against a number of HIV-1 strains. Sera obtained from each individual at three or four sequential phlebotomies were pooled, and the two pools were each distributed in > 3000 aliquots into glass ampoules and lyophilized, and the ampoules were flame sealed. An HIV-1 antibody-negative reference serum was prepared in a similar fashion after pooling serum from four individuals. Ampoules were tested for uniformity of fill, sterility, moisture content, residual oxygen, stability, infectivity, and presence of antibody. An international collaborative study was conducted to determine the potency of the samples in six laboratories, each using their own neutralization assays and reagents. The results indicated reasonable consistency between laboratories and that both sera have sufficient titers against a variety of strains for use as reference reagents. These reference sera have been included in the World Health Organization (WHO) AIDS Reagent Project and are available through the three AIDS reagent repositories.

The phorbol ester phorbol myristate acetate inhibits human immunodeficiency virus type 1 envelope-mediated fusion by modulating an accessory component(s) in CD4-expressing cells.

The phorbol ester phorbol myristate acetate (PMA) strongly inhibits human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation; it has been suggested that this inhibitory effect is due to the transient downmodulation of the surface-associated CD4 receptors by PMA (I. H. Chowdhury, Y. Koyanagi, S. Kobayashi, Y. Hamamoto, H. Yoshiyama, T. Yoshida, and N. Yamamoto, Virology 176:126-132, 1990). Surprisingly, PMA treatment of cells expressing truncated (A2.01.CD4.401) and hybrid (A2.01.CD4.CD8) CD4 molecules, which are not downmodulated (P. Bedinger, A. Moriarty, R. C. von Borstel II, N. J. Donovan, K. S. Steimer, and D. R. Littman, Nature [London] 334:162-165, 1988), inhibited their fusion with CD4- (12E1) cells expressing vaccinia virus-encoded HIV-1 envelope glycoprotein (gp120-gp41) and with chronically HIV-1-infected H9 (MN, IIIB, or RF) cells. PMA pretreatment of T (12E1) and non-T (HeLa, U937.3, and Epstein-Barr virus-transformed B) cell lines expressing vaccinia virus-encoded CD4 also blocked fusion with 12E1 cells expressing vaccinia virus-encoded gp120-gp41. Interestingly, pretreatment of the gp120-gp41-expressing 12E1 cells with PMA did not alter their fusion with untreated CD4-expressing cells. Although the inhibitory effect of PMA was rapid and treatment for 1.5 h with 5 ng of PMA per ml was sufficient to reduce fusion by more than 50%, the recovery after treatment was slow and more than 40 h was needed before the cells regained half of their fusion potential. The inhibitory effect of PMA was blocked by staurosporine in a dose-dependent fashion, suggesting that it is mediated by protein kinase C. PMA treatment of A2.01.CD4.401 cells reduced the number of infected cells 6.7-fold, as estimated by a quantitative analysis of the HIV-1 MN infection kinetics, probably by affecting the stage of virus entry into cells. CD26 surface expression was not significantly changed by PMA treatment. We conclude that PMA inhibits the CD4-gp120-gp41-mediated fusion by modulating an accessory component(s), different from CD26, in the target CD4-expressing cells. These findings suggest a novel approach for identification of accessory molecules involved in fusion and may have implications for the development of antiviral agents.

Study of viral replication in HIV-1-infected CEM T-cell subclones which are reduced in their ability to form syncytia.

The derivation of ethyl-methanesulfonate (EMS) mutagenized subclones from the CEM T-cell line has been described. These clones expressed CD4 and bound soluble gp120, however, two of the generated clones were markedly reduced in their ability to form syncytia after infection with either gp160-vaccinia vector or cell-free human immunodeficiency virus type 1 (HIV-1). Here, we asked at what stage(s) viral infection is blocked in these cells. Polymerase chain reaction (PCR) analysis revealed that at 6 and 72 h postinfection with HIV-1, cells of the syncytia-deficient clones expressed markedly reduced amounts of viral-specific DNA compared with cells of the parental line or the syncytia-positive clones. Long-term cultures revealed a marked delay in the appearance of reverse transcriptase (RT) activity in the supernatants of these subclones when compared with the parental line and viral replication did not lead to massive cell death. Syncytia formation in HIV-1-infected cultures of the syncytia-deficient subclones was enhanced by tumor necrosis factor alpha (TNF alpha) when added 24 h postinfection. In contrast, pretreatment with TNF alpha for 48 h followed by washing and infection of the cells with HIV-1 augmented syncytia formation of the syncytia-positive subclones, but not of the syncytia-negative subclones. Thus, the EMS-mutagenized subclones may provide a tool to study host cell factors required for the establishment of a productive HIV-1 infection and responsiveness to TNF alpha.

High prevalence of antibodies to the gp120 V3 region principal neutralizing determinant of HIV-1MN in sera from Africa and the Americas.

Neutralizing antibodies (NA) against HIV-1MN and HIV-1IIIB, and antibodies binding to synthetic peptides (BA) derived from the gp120 envelope V3 region principal neutralizing determinants (PND) of the HIV-1MN, HIV-1IIIB, and HIV-1Z3 virus strains were assayed in HIV-1 antibody-positive sera from the United States, Haiti, Brazil, Zaire, and Zimbabwe. The ability of soluble PND peptide to block neutralization of the corresponding virus by representative sera was also tested. In each country, NA and BA titers were highest against the HIV-1MN strain, and compared with other countries, NA and BA titers against HIV-1MN were higher in sera from the United States and Haiti. When NA titers were compared with BA titers against either HIV-1MN or HIV-1IIIB, no correlation was found for the HIV-1IIIB strain, but there was a significant correlation for HIV-1MN. Addition of the HIV-1MN strain peptide to a neutralization assay for HIV-1MN resulted in a four- to tenfold reduction in NA titers in sera from the United States, Zaire, and Brazil. The results suggest that HIV-1MN and closely related variants are prevalent in many parts of the world, and that antibodies directed against the PND account for most of the neutralizing activity in sera of infected individuals.

PIP: Virologists assessed the extent of neutralizing antibody cross-reactivity to multiple virus strains in sera from 112 HIV-1 infected individuals from the US, Brazil, Haiti, Zaire, and Zimbabwe. They also looked at the association between virus neutralization and the level of antibody binding to synthetic peptides representing the HIV-1 gp120 V3 region principal neutralizing determinant (PND) sequences. The 3 strains observed included HIV-1 MN, HIV-1 Z3, and HIV-1 IIIB. Neutralizing antibodies (NA) and antibodies binding to synthetic peptides (BA) titers ranked highest against the PND sequence HIV-1 MN in all countries (p.01). These titers were higher in sera from the US and Haiti than sera from Brazil and Africa (p.05). A significant correlation existed between the NA and BA titers for HIV-1 MN (p.01), but not for HIV-1 IIIB. When the virologists added HIV-1 MN strain peptide to a neutralization assay for HIV-1 MN, NA titers in sera from the US, Zaire, and Brazil fell 4-10 fold. These findings intimated that HIV-1 MN and closely related variants are commonplace in several locations around the world, and that antibodies directed against HIV--1 gp120 V3 region PND sequences make up most of the neutralizing activity in sera of infected individuals. In conclusion, virologists need to conduct more studies that examine the true extent of strain variation worldwide. These studies could lay the groundwork for the development of an effective HIV-1 vaccine.

Re-evaluation of the involvement of the adhesion molecules ICAM-1/LFA-1 in syncytia formation of HIV-1-infected subclones of a CEM T-cell leukemic line.

The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HIV-1)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common beta subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFN gamma); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNF alpha), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNF alpha-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

International collaborative study to compare assays for antibodies that neutralize human immunodeficiency virus.

A variety of techniques are currently in use to measure antibodies that neutralize human immunodeficiency virus (HIV), and standardization of these assays is needed. Eleven laboratories participated in this comparison study of 14 methods for detection of HIV neutralizing antibodies (NA). A panel of 10 coded sera and a positive and a negative control serum were tested in each assay. The 10 coded samples included aliquots of the same sera that were distributed as positive and negative control sera, an aliquot of the WHO reference human anti-HIV-1 serum, and seven other sera from people with HIV-1 infection. Each laboratory reported features of its test methods and results. The results demonstrated excellent within laboratory and between laboratory consistency. The most important variable appeared to be the virus strain used. Cell line, conditions of neutralization or culture, method of endpoint determination, or use of VSV pseudotypes did not appear to be important variables. The results indicate that standardization of HIV-1 NA assays should be readily achievable.

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection.

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.

Chemically induced CD4 mutants of a human T cell line. Evidence for dissociation between binding of HIV I envelope and susceptibility to HIV I infection and syncytia formation.

This study describes the derivation of a series of mutants from the human leukemic cell line CEM using the frame shift mutagen Ethyl-methanesulfonate followed by negative selection with multiple treatments of OKT4A + C, and sorting into CD4-, CD4-dull, and CD4-intermediate mutants. These mutants express reduced CD4 levels ranging from 0 to 60% of the parental line. The mutants were analyzed by staining with a battery of CD4-specific mAb, by assessing their ability to bind soluble gp120, and by their ability to form syncytia after infection with cell-free HIV I virus and a gp160-vaccinia vector. Two groups of particularly interesting mutants were identified: (1) CD4-dull mutants expressing only 5 to 10% of the wild type surface CD4 density, which nevertheless were infectable by HIV I and produced as many syncytia and reverse transcriptase activity as the parental line after infection with gp160-vaccinia or cell free HIV I. (2) CD4-intermediate mutants (30 to 60% of parental CD4 level), which express CD4-epitopes required for interaction with the HIV I envelope protein, yet are markedly deficient in their ability to form syncytia after gp160-vaccinia or HIV I infection. Two of these mutants did form syncytia after transient reconstitution with a wild type CD4 containing vaccinia vector. Inasmuch as they were found to bind soluble gp120 with the same avidity as other, functionally normal, CD4-intermediate mutants, these human T cell mutants may have a reduced susceptibility to HIV I infection due to the absence of a "fusogenic component" or to a structural alteration in a region of the CD4 molecule not required for binding of the HIV I envelope, but for the subsequent fusion and entry process.

Possible beneficial effects of neutralizing antibodies and antibody-dependent, cell-mediated cytotoxicity in human immunodeficiency virus infection.

We studied the relationship between early human immunodeficiency virus type 1 (HIV-1) specific immune responses and pathogenesis of infection in participants enrolled in the multicenter AIDS cohort study (MACS). Sera collected at 6-month intervals for 2 years (visit 1-5) from 39 persons who seroconverted by enzyme-linked immunosorbent assay (ELISA) 6 months (visit 2) after enrollment were examined for isotype-specific Western blot reactivity, neutralizing antibodies (NA) against two divergent strains of HIV-1 (HIV-1IIIB and HIV-1RF), and for antibodies capable of participating in antibody-dependent, cell-mediated cytotoxicity (ADCC). These results were compared with changes in CD4+ cell number and episodes of lymphadenopathy. Twenty-five subjects had antibodies of at least one isotype reactive to at least one HIV-1 protein by Western blot at visit 1, before they became ELISA positive. NA reactive with HIV-1IIIB were detected before those reactive with HIV-1RF. NA were first observed in 11 sera at visit 2, in 22 sera at visit 3, and in 3 sera at visit 4; sera from three patients remained nonneutralizing through visit 5. In most cases, NA were detected after a decline in CD4+ cell numbers. The data are consistent with the interpretation that NA develop after about 16 to 18 months of declining CD4+ cell numbers, following which the rate of decline in CD4+ cell numbers slows. In contrast, HIV-1 envelope antigen-specific ADCC responses were first observed in 11 subjects at visit 1 when all 39 were NA and ELISA negative, in 12 subjects at visit 2, in 13 subjects at visit 3, and 1 subject at visit 4. Early ADCC responses were associated with high mean % CD4+ cell numbers and absence of lymphadenopathy throughout the 2-year observation period. Not all subjects who developed ADCC developed NA. In some subjects, ADCC and NA were detectable for the first time at the same visit, for others ADCC was detectable prior to NA, and for a few NA was detectable prior to ADCC. These findings suggest that ADCC and neutralization are mediated by different antibody populations, that they may partially inhibit the progress of HIV-1 infection, and that the late appearance of NA may relate to the failure of immunity to effect recovery from this infection.

Human immunodeficiency virus neutralizing antibodies in sera from North Americans and Africans.

Neutralizing antibodies (NAs) against four isolates of human immunodeficiency virus type 1 (HIV-1) were assayed in HIV-1 antibody positive sera from the United States, Haiti, Zimbabwe, and Zaire. Overall, there were NAs detected in 95, 81, 60, and 73% of sera with reciprocal geometric mean titers (GMTs) of 626, 23, 10, and 20, respectively, against HIV-1MN, HIV-1IIIB, HIV-1RF, and HIV-1Z3. Sera from North America had significantly higher NA titers against HIV-1MN. In each country, the highest antibody titers observed were against the MN strain. Otherwise, sera from the U.S. neutralized most strongly HIV-1IIIB, sera from Zaire neutralized most strongly HIV-1Z3, and sera from Zimbabwe had equal titers against all three viruses. The differences between countries were reflected in analyses of NA titers of subgroups classified on the basis of clinical status, indicating that the differences were not likely to be related to differences in clinical status of the patients being tested. Some of this antigenic variation is reflective of known genetic diversity, while some is not. The results suggest that undefined preserved and variable regions containing neutralization epitope(s) exist. These data do not indicate a need to define antigenic subtypes of HIV-1 at present. The existence of conserved neutralization epitope(s) may indicate the potential for broad immunogenicity of appropriately selected vaccine antigens.

Varicella-zoster virus-specific HLA-restricted cytotoxicity of normal immune adult lymphocytes after in vitro stimulation.

Varicella-zoster virus (VZV)-specific cytotoxic T cell responses were studied in 35 presumably immune and two nonimmune adult donors and in two patients with herpes zoster. Peripheral blood lymphocytes (PBLs) were stimulated for five or 12 days with autologous, irradiated, VZV-infected PBLs. Cytotoxicity was measured in a chromium-release assay. Target cells were usually cryopreserved, phytohemagglutinin-stimulated PBLs, either infected with VZV or uninfected. In testing the presumably immune adults, VZV-specific cytotoxicity was observed in 32 (91%) of 35 cases after five days and in 19 (90%) of 21 cases after 12 d of stimulation. Lysis of HLA-matched target cells was significantly greater than that of mismatched, VZV-infected target cells after both intervals. Responses were similar when PBLs from two patients with acute zoster were tested after in vitro stimulation and in one of those two tested without in vitro stimulation.

Detection of human immunodeficiency virus core protein in plasma by enzyme immunoassay. Association of antigenemia with symptomatic disease and T-helper cell depletion.

A sensitive enzyme immunoassay was developed for detecting human immunodeficiency virus (HIV) core antigen. Assay sensitivity was 3.67 pmol/L of purified HIV core protein, and 1 or 100 in-vitro infectious units/mL of HIV in purified virus preparations or cell culture supernatants, respectively. Enzyme immunoassay sensitivity exceeded that of reverse transcriptase assay by 1000-fold. Core antigen was detected in whole plasma from 41% of symptomatic subjects and 13% of asymptomatic subjects seropositive for HIV. After plasma fractionation, antigenemia was found in 60% of symptomatic subjects and in 33% of asymptomatic subjects seropositive for HIV. Fifty-seven percent of samples from which HIV could be isolated in lymphocyte culture had detectable quantities of core antigen in plasma. However, at least 87% of samples with measurable antigen in plasma had HIV isolated from lymphocyte cultures. Antigenemia was associated with reduced T-cell number and symptomatic disease, and may be a useful marker for disease progression.

Five-year follow-up study of recipients of live varicella vaccine using enhanced neutralization and fluorescent antibody membrane antigen assays.

Twenty-six healthy children susceptible to varicella were inoculated with the OKA strain of live attenuated varicella vaccine. All of the recipients showed good antibody responses without adverse clinical reactions. The seropositivity rate 5 years after vaccination was 100% by the enhanced neutralization test and 96% by the fluorescent antibody membrane antigen (FAMA) test. None of the recipients contracted varicella in spite of documented contact exposure. None of the children developed herpes zoster during the 5-year observation period. The results suggest that attenuated varicella vaccine has long-term protective efficacy.

Comparison of the Raji cell line fluorescent antibody to membrane antigen test and the enzyme-linked immunosorbent assay for determination of immunity to varicella-zoster virus.

A prospective study was performed comparing the fluorescent antibody to membrane antigen (FAMA) test and the enzyme-linked immunosorbent assay (ELISA) for identifying susceptibility and seroconversion to varicella-zoster virus (VZV) infection. A total of 75 sera were collected from index cases and from sibling and parent contacts in 10 families. Varicella-zoster virus-infected human diploid embryonic fibroblasts and continuous lymphoblastoid cells (Raji cells) were compared as indicator cells in the FAMA test. Equivalent results were obtained with both types of cell. Results of the FAMA test and the ELISA were identical in two ways. (i) The same 11 individuals were initally defined as susceptible (seronegative), and 9 of them (82%) developed fourfold rises in antibody titers, clinical varicella, or both. (ii) Of 21 immune (seropositive) individuals, 4 developed fourfold antibody rises by FAMA tests, and 3 of these 4 responded by ELISA. Infection was asymptomatic in these individuals. The geometric mean titer by ELISA was significantly higher than by the FAMA test. The results indicated that the ELISA and the FAMA test have similar capacities to define susceptibility to varicella-zoster virus and that subclinical infection with varicella-zoster virus may be common.