Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals.

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.     

Antifusion activity in sera from persons infected with human immunodeficiency virus type 1.

Cell-to-cell fusion plays an important role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infections. An assay to measure the antifusion activity of serum has been developed by using the fusion event that occurs between H9 cells chronically infected with HIV-1 (H9IIIB) and fusion-susceptible MT-2 cells. The endpoint is determined by measuring neutral red uptake in cells after syncytium formation is allowed to occur in the presence of various serum dilutions. The assessment of antifusion activity in serum by neutral red uptake has been shown to correlate with syncytium reduction as determined by direct counting. The optimal number and ratio of cells in the suspension for efficiency and speed of the assay have been determined. With this assay it was shown that 50% of 36 serum specimens capable of neutralizing cell-free virions failed to inhibit syncytium formation. The assay can thus measure a distinct activity in HIV-1-immune human sera which is a subset of neutralization activity. Because of the potential role of this activity in the rate of disease progression and protective immune responses, the antifusion assay will be an important tool for the investigation of disease pathogenesis and for acquired immunodeficiency syndrome vaccine development. The assay can also be applied to the investigation of the pathogenesis of the fusion event at the cellular level. The ability to use absorbance measurements rather than syncytium counts as the endpoint facilitates direct computer-assisted data analysis, which expedites the performance of the assay.     

Neutralization and infectivity characteristics of envelope glycoproteins from human immunodeficiency virus type 1 infected donors whose sera exhibit broadly cross-reactive neutralizing activity

In this study, we tested the hypothesis that donors with broadly cross-reactive HIV-1 neutralizing (BCN) sera are infected with viruses encoding envelope glycoproteins (Envs) with unusual immunogenic properties. Cloned env genes were from samples of donors previously identified as having BCN antibodies (BCN donors) and from other donors not known to have such antibodies (non-BCN donors). Neutralization properties of viruses pseudotyped with BCN and non-BCN Envs were determined using BCN, non-BCN sera and broadly cross-neutralizing monoclonal antibodies (Mabs). BCN sera neutralized with higher frequency and geometric mean titers than non-BCN sera. Viruses pseudotyped with BCN Envs were mostly resistant to neutralization by anti-gp120 Mabs but tended to be more sensitive to the anti-gp41 Mabs, 2F5 and 4E10 than non-BCN Env-pseudotyped viruses. Sequence analysis of clones obtained from sequential samples of two BCN donors revealed respective 2F5 epitope mutations T662A and K665T. The K665T mutation evolved as the predominant genotype in the respective donor, consistent with an escape mutation event. The A662T mutation reduced sensitivity to 4E10, as well as 2F5 and homologous sera, consistent with neutralization escape mutation and targeting of the 2F5 epitope region by the serum. Our study suggests that viruses infecting these BCN donors encoded Envs that may have been unusually competent for induction of antibodies against the membrane proximal epitope region (MPER) of gp41, and these Envs may be useful vaccine components.     

Human immunodeficiency virus type 1 tropism for human macrophages.

Human immunodeficiency virus (HIV) infects cells of the monocyte/macrophage lineage in addition to lymphocytes, and infection of these cells may be responsible for viral persistence and dissemination, encephalopathy of the acquired immunodeficiency syndrome and other sequelae of HIV infection. We have developed an in vitro model utilizing peripheral-blood monocyte-derived macrophages to study HIV-1 infection of macrophages. HIV-1 isolates vary greatly in their ability to infect and replicate in macrophages, from highly restricted to highly productive infection. Productively infected macrophages undergo syncytium formation but remain viable in culture and support sustained levels of virus production for prolonged periods. Transformed monocytoid and lymphoid cell lines, however, show very different patterns of permissiveness for HIV-1 strains and do not reflect their corresponding primary cell types in studies of host cell tropism. Studies on viral entry show that the CD4 molecule, known to be the HIV receptor on lymphoid cells, is expressed at low levels on the surface of macrophages as well, where it functions as the receptor for viral entry. Therefore, differential host cell tropism does not result from the use of an alternative macrophage-specific receptor instead of CD4.     

Cellular latency of human immunodeficiency virus type 1.

The infection of humans by human immunodeficiency virus type 1 is characterized by a prolonged stage of clinical quiescence. This clinically asymptomatic period may be based, in part, on the development of cell populations within the body that maintain human immunodeficiency virus type 1 in a state of latency. Recent advances in the understanding of the molecular mechanisms involved in various forms of cellular latency of human immunodeficiency virus type 1 have begun to shed light on the variable period of asymptomatic infection. The elucidation of cellular retroviral latency, in vivo, will also be critical to the design of novel therapeutic approaches with which to combat human immunodeficiency virus type 1 infections.     

Current status of therapy for human immunodeficiency virus type 1.

The past year has been refinement in our ability to delay the progression of human immunodeficiency virus type 1 infection through the use of nucleoside analogues, singly or in combination. Progress has also been made in our ability to detect drug-resistant isolates of human immunodeficiency virus type 1 and to begin to put the laboratory observations of resistance into a clinical context.     

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins

To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were "oligomer sensitive," whereas mAbs to V2 and the distal epitope of C5 tended to be "monomer sensitive" (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.     

Variability of human immunodeficiency virus type 1

The variability of human immunodeficiency virus type 1 (HIV-1) is very high. To date, three distinct lineages of HIVs, type 1 group M, type 1 group O and type 2 are described, suggesting at least three different zoonotic infections. HIV-1 group M is responsible for the global epidemic of AIDS. At least ten subtypes of HIV-1 group M, labelled A through J, have been discovered. Viral sequences from both the gag and the env gene, particularly a part of gp 120 referred to as the V3 region have been used to identify subtypes of HIV-1 group M. The nucleotide distance between viruses of different subtypes is on average 30% for the env gene. The various subtypes are geographically distributed throughout the world. Some of the subtypes were identified as recombinant or mosaic viruses. The existence of different subtypes of HIV-1 have major implication for vaccination. They may also influence the diagnosis of HIV infection. To date, it is unclear whether subtypes of HIV-1 differ with respect to transmissibility or pathogenicity.     

Predicting human immunodeficiency virus type 1-positive sera by using two enzyme immunoassay kits in a parallel testing format.

Two algorithms for screening sera for antibody to human immunodeficiency virus type 1 were compared for their efficiency in identifying a true-positive sample in a population with heterogeneous risk factors, using the criteria of specificity and positive predictive value (PPV). In the first algorithm, all sera were screened by using a single enzyme immunoassay (EIA) kit, and a specificity of 98.6% and a PPV of 69.3% was calculated for true-positive sera. The second algorithm employed two different EIA kits in parallel to screen each sample. In the first instance, a specificity and a PPV of 100% was calculated if a positive sample was defined as reactive by both EIA kits; in the second, a specificity of 99.97% and a PPV of 99.4% was obtained if this criterion was extended to include a combination of one reactive and one equivocal result obtained with the two EIA kits.     

Primary human immunodeficiency virus type 1 infection

Primary human immunodeficiency virus type 1 (HIV-1) infection represents the initial stage of disease that immediately follows viral entry into the body. Primary infection is frequently accompanied by an acute retroviral syndrome with associated high levels of plasma HIV-1 RNA and the development of host immune responses. The identification of subjects during this period requires a high index of suspicion and an understanding of how to make the diagnosis, as standard HIV-1 antibody tests can initially be negative. Identifying these people provides a unique opportunity for early counseling to reduce further transmission, facilitates entry into care, and allows for further study of the immunopathogenesis of disease and the potential role of early antiretroviral therapy.     

RNA packaging signal of human immunodeficiency virus type 1.

Cells infected with a recombinant vaccinia virus carrying the gag and pol regions of the human immunodeficiency virus type 1 genome (Vac-gag/pol) released human immunodeficiency virus (HIV)-like particles containing HIV-specific RNA. However, cells infected with another recombinant vaccinia, Vac-gag/pol-dP, derived through the deletion of an 85-base region (nucleotide positions 679-763) of the HIV genome between the primer binding site and the gag initiation codon of Vac-gag/pol, produced HIV-like particles devoid of the HIV-specific RNA. This 85-base deletion was suggested to cause the collapse of a stable stem-loop structure of 46 bases (751-796) around the gag initiation codon. To examine the role of the stem-loop structure in the packaging of RNAs, we constructed a vaccinia vector plasmid that carried this 46-base sequence followed by the Sendai virus nucleocapsid (NP) gene. When both Vac-gag/pol-dP and this plasmid were introduced into cells, HIV-like particles released from the cells contained the NP gene RNA. However, another vaccinia vector plasmid, which carried the 46-base sequence in the midst of the NP gene, could not supply RNA for incorporation into HIV-like particles. Computer analysis of this plasmid sequence suggested that the 46-base sequence cannot form the stem-loop structure. These findings suggest that the stem-loop structure formed by the 46-base sequence is crucial as a packaging signal.     

Envelope glycoproteins are dispensable for insertion of host HLA-DR molecules within nascent human immunodeficiency virus type 1 particles

HLA-DR is a host-derived protein present at the surface of HIV-1. To clarify the mechanism through which this molecule is inserted within viruses, we monitored whether the incorporation process might be influenced by the level of virus-encoded envelope (Env) glycoproteins. Wild-type virions and viruses either lacking or bearing lower levels of Env were produced in different cell types. Results from a virus capture test indicate that HLA-DR is efficiently incorporated and at comparable levels in the tested virus preparations. Therefore, Env does not play an active role in the acquisition of host HLA-DR by emerging HIV-1 particles.     

Comparison of 10 enzyme immunoassays for detection of antibody to human immunodeficiency virus type 2 in West African sera.

The efficacies of nine enzyme-linked immunosorbent assays (EIA) for antibody to human immunodeficiency virus type 1 (HIV-1) and one EIA for antibody to HIV-2 in detecting antibody to HIV-2 were studied. The competitive EIAs for antibody to HIV-1 were less sensitive than the indirect EIAs. The overall prevalence of positive results was between 28 and 51% with the competitive EIAs and between 70 and 93% with the indirect EIAs. Most of the EIAs were less sensitive in detecting antibody to HIV-2 in sera from people with acquired immunodeficiency syndrome-like diseases than in sera from symptomless individuals. The results indicate that there is a high degree of cross-reactivity between HIV-1 and HIV-2 by EIA, indicating that serotype specificity must be determined by Western blot (immunoblot) with both sets of viral antigens. The results are relevant for discussing public health strategies, especially the screening of blood donors; competitive EIAs for antibody to HIV-1 are not sensitive enough to be used in areas where HIV-2 is prevalent (West Africa).     

Evaluation of rapid test for detection of antibody to human immunodeficiency virus type 1 and type 2.

The Genie HIV-1/HIV-2 rapid assay was compared with an enzyme-linked immunosorbent assay and Western blot (immunoblot) by using 540 serum specimens from four different populations. The Genie HIV-1/HIV-2 assay was easy to perform, required no equipment, provided visual results within 10 min, and demonstrated excellent sensitivity and specificity compared with the Western blot.     

Blocking-ELISA for detection of specific antibodies to the glycoproteins of the human immunodeficiency virus type 1 (HIV-1)

A blocking ELISA was developed to confirm the specificity of screening tests for anti-HIV-1 antibodies. A murine monoclonal antibody (McAb) raised against recombinant gp160 was used in combination with a commercial technique (ELA-VIA-1). After determining the optimal experimental conditions, the assay was applied to 92 samples presenting different reactivities by Western blot (WB) analysis. All the sera containing antibodies to gp160/gp120 (53) were positive in our assay. The six patients who sero converted showed a low positivity by ELAVIA-1 (optical density near the cutoff value) reacted by blocking-ELAVIA-1 with an McAb binding inhibition greater than 85%. By contrast, negative samples (29) and specimens that exhibited reactivity only against gag-proteins (10) were not detected (McAb binding inhibition smaller than 15%). This sensitive and specific blocking-ELAVIA-1 represents a convenient alternative to WB as a confirmatory test. The technique is time-saving and inexpensive and can easily be integrated with a screening test for diagnostic or epidemiologic studies on HIV-1 infection.     

Inactivation of human immunodeficiency virus type 1 by the amine oxidase-peroxidase system.

Human immunodeficiency virus type 1 (HIV-1) is rapidly inactivated by exposure to a naturally occurring antimicrobial system consisting of peroxidase, H2O2, and a halide. Among the potential sources of H2O2 is the amine oxidase system in which mono-, di-, and polyamines are oxidatively deaminated with the formation of H2O2. The polyamine spermine is present at exceptionally high concentrations in semen. We report here that spermine, spermidine, and, to a lesser degree, the synthetic polyamine 15-deoxyspergualin are viricidal to HIV-1 when combined with amine oxidase and myeloperoxidase. Antiviral activity required each component of the spermine-amine oxidase-peroxidase system and was inhibited by azide (a peroxidase inhibitor) and by catalase but not by superoxide dismutase. Heat treatment of catalase largely abolished its inhibitory effect. These findings implicate H2O2 formed by the amine oxidase system in the antiviral effect and raise the possibility that the polyamine-amine oxidase-peroxidase system influences the survival of HIV-1 in semen and in the vaginal canal.