Immunological analysis of human immunodeficiency virus type 1 envelope glycoprotein proteolytic cleavage.

Two potential cleavage sites have been identified on precursor gp 160 of human immunodeficiency virus type 1. Using antibodies directed against the C-terminus of gp 120, including the sequence between the two sites, we have shown that nonmutated viral and recombinant gp 160 are cleaved at both sites: the great majority of molecules are cleaved at site 1 (Arg-Glu-Lys-Arg), and gp41 can then associate as an oligomer; a minority of molecules are cleaved at site 2 (Lys-Ala-Lys-Arg-Arg) and the corresponding gp41 appears to present as a monomer. This could reflect two different processing pathways for gp41 biosynthesis, one of which only may result in biologically active molecules according to the literature.     

T cell multideterminant regions in the human immunodeficiency virus envelope: toward overcoming the problem of major histocompatibility complex restriction

Helper T cell determinants should be an important component of an anti-human immunodeficiency virus (HIV) vaccine aimed at either antibody or cytotoxic T cell immunity. However, model protein studies have raised concern about the usefulness of any single determinant, because a given determinant is likely to be seen by only a small subset of major histocompatibility complex (MHC) types within the population. Here, we use 44 peptides, including ones predicted and not predicted on the basis of amphipathicity to be potential T cell sites, to locate T cell antigenic determinants recognized by mice of four MHC haplotypes immunized with the whole gp 160 envelope protein. Although the preselection of peptides necessitates caution in a statistical analysis, alpha-amphipathic peptides predominated among sites eliciting the strongest response. Although we have not tested the entire sequence, we have identified six multideterminant regions, in which overlapping peptides are recognized by mice of either three or all four MHC types. Four of the six regions have sequences relatively conserved among HIV-1 isolates. The existence of such multideterminant regions recognized by multiple MHC haplotypes suggests the possibility that use of peptides longer than a minimal determinant and containing several overlapping determinants may be a possible approach to circumvent the serious problem of MHC restriction in peptide vaccines aimed at eliciting T cell immunity.     

RFLP analysis of the rhesus monkey MHC class II DR subregion.

Restriction fragment length polymorphism (RFLP) analysis was performed on a panel of 39 serologically typed DR homozygous monkeys. DNA was digested with the restriction enzyme TaqI and hybridizations were carried out with a human leukocyte antigen (HLA)-DR beta 3'UT-specific probe. In addition a panel of 18 monkeys was analyzed comprising experimental autoimmune encephalomyelitis (EAE) susceptible and nonsusceptible animals. The number of DRB/TaqI fragments detected for the various DR specificities varied from two to six, suggesting that the number of DRB genes per haplotype is not constant. RFLP typing allows that most serologically defined DR specificities can be subdivided. This knowledge was applied to define the DR specificities of the animals used for EAE experiments.     

Enzyme-linked immunoassay for human immunodeficiency virus type 1 envelope glycoprotein 120.

An enzyme-linked immunosorbent assay (ELISA) that can measure picogram quantities of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) in cell culture medium or body fluids has been developed. Recombinant, soluble CD4 immobilized in microtiter trays was used to capture gp120, which was then detected with polyclonal sheep antibody to gp120 followed by biotinylated rabbit anti-sheep immunoglobulin G and an avidin-alkaline phosphatase indicator system. With a reference recombinant gp120, the assay showed a linear relationship between optical density and concentrations ranging from 60 to 6,000 pg/100-microliters well; precision of the assay varied with the concentrations and ranged from +/- 40% with amounts smaller than 200 pg to +/- 10% with amounts larger than 200 pg. In a group of coded samples containing 60 pg (approximately 10(7) molecules) of reference gp120, the assay correctly identified the samples as containing gp120 99% of the time, with no false-positive results recorded for blank samples. Recombinant gp120 prepared in another cell culture system demonstrated a binding coefficient 13-fold lower than that of reference gp120. Mixing standard amounts of reference gp120 with increasing concentrations of human sera reduced assay sensitivity, although the linear relationship between gp120 concentration and optical density remained. With this assay we were able to detect gp120 in HIV-1 suspensions prepared from cultured lymphoblastoid cells and in the sera of HIV-1-infected patients. This ELISA for gp120 should be useful for studying the biological role of gp120 in HIV infection.     

Rabbit major histocompatibility complex. III. Multiple class II DR beta genes and restriction fragment length polymorphism of the class II alpha and beta genes

Several class II alpha and beta chain genes of the rabbit MHC have been cloned and classified into three distinct subregions, R-DP, R-DQ and R-DR, based on their homology to the corresponding HLA-DP, -DQ and -DR genes. The organization of the rabbit MHC class II genes has now been studied in greater detail by analysing genomic DNA of an inbred III/J strain and several other RLA-D homozygous rabbits, with DNA probes derived from cloned R-DR beta genes. Eight previously cloned R-DR beta genes were shown to be allelic forms of five R-DR beta loci. Genomic blot analyses of DNA from seven rabbits homozygous for different RLA haplotypes revealed that the germline contains a total of approximately seven class II beta genes, one DQ beta, one DP beta and five DR beta. Extensive allelic polymorphism was identified by RFLP analysis using DQ and DR probes; limited RFLP was observed with DP probes. RFLP analyses allowed us to distinguish two haplotypes which had not been previously distinguished by MLR. Such RFLP analyses will be useful for identifying MHC 'compatible' rabbits for various immunobiological studies, including transplantation.     

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.     

Major histocompatibility complex class II (HLA-DRB and -DQB) allele frequencies in Botswana: association with human immunodeficiency virus type 1 infection

Southern Africa is facing an unprecedented public health crisis due to the high prevalence of human immunodeficiency virus type 1 (HIV-1). Vaccine development and testing efforts, mainly based on elicitation of HIV-specific T cells, are under way. To understand the role of human leukocyte antigen (HLA) class II alleles in HIV pathogenesis and to facilitate HLA-based HIV-1 vaccine design, we analyzed the frequencies of HLA class II alleles within the southern African country of Botswana. Common HLA class II alleles were identified within the Botswana population through the molecular genotyping of DRB and DQB1 loci. The DRB1 allele groups DRB1*01, DRB1*02/15, DRB1*03, DRB1*11, and DRB1*13 were encountered at frequencies above 20%. Within the DQB1 locus, DQB1*06 (47.7%) was the most common allele group, followed by DQB1*03 (39.2%) and DQB1*04 (25.8%). We found that DRB1*01 was more common in HIV-negative than in HIV-positive individuals and that those who expressed DRB1*08 had lower median viral loads. We demonstrate that the frequencies of certain HLA class II alleles in this Botswana population differ substantially from those in North American populations, including African-Americans. Common allele groups within Botswana cover large percentages of other African populations and could be targeted in regional vaccine designs.     

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.     

Identification of immunoglobulin recombinant elements in human immunodeficiency virus type 1 envelope gene.

Recently, it has been recognised that the amino acid motif VQL(N/V)ES is shared between the second conserved domain of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and the first framework (FW1) of human IgG heavy chain variable region (subgroup III) (VHIII). We have found that nucleotide sequence corresponding to this motif contains some recombination elements characteristic for the genes of human Ig heavy-chain variable region. The possible role of the Ig recombination elements in HIV-1 envelope gene variability, AIDS pathogenesis and vaccine design is discussed.     

Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.

Transmembrane envelope protein (TM) residues 100, 105, and 128 of human immunodeficiency virus type 1 (HIV-1) strain HXB2 are potential sites for asparagine-linked oligosaccharide additions which are conserved among HIV-1 isolates, and all other lentivirus TM proteins. Site-specific mutants of each of the asparagine residues did not eliminate the ability of the virus to infect and replicate in CD4+ cells, but infectivity was reduced with all of these mutants, and syncytia induction was attenuated with two of these mutants. Studies of envelope expression of the mutant with the most severe defect demonstrated no significant effects on envelope protein synthesis, conformation, processing, multimerization, or release into the culture medium, suggesting that N-linked oligosaccharides are important in the specific fusion activity of TM.     

Cross-reaction of anti-simian immunodeficiency virus envelope protein antibodies with human immunoglobulins

It has been recently established that retroviral envelope proteins (REPs) have structural features similar to those of immunoglobulins (Igs). In this study, we asked whether anti-REP antibodies cross-react with human Igs (hIgs). To this end, murine monoclonal antibodies (mMoAbs) that had been raised against a simian immunodeficiency virus (SIV) envelope protein, SIVMac251gp120, were screened for their ability to react with human monoclonal Igs (HMIgs). We show that two HMIgs, RFSJ2 (a rheumatoid factor) and PAMLN6 (a human anti-hIg V region antibody), but not a number of other HMIgs, could be weakly, but consistently, bound by anti-SIVMac251gp120 mMoAbs KK17 and KK46, as judged by indirect enzyme-linked immunosorbent assay and a liquid-phase inhibition immunoassay. Both mMoAbs are specific to amino acid residues in the V3 loop of the SIVMac251gp120. The RFSJ2 Ig heavy-chain V region (VH) is coded in part by a human VH gene, VH3-30.3 and includes the idiotope 7B4 (NKYY), which was previously shown to be present in the gp120 protein of a number of HIV-2 and SIV strains. However, an entirely different VH gene codes the PAMLN6 VH region, opening the possibility that epitope(s) shared between SIVMac251gp120 and hIgs may not be limited to the 7B4 idiotope.     

Presence of human papillomavirus and Epstein-Barr virus in the cervix of women infected with the human immunodeficiency virus

The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was sought in cervical scrapings from 110 human immunodeficiency virus (HIV)-infected women to evaluate the role of these viruses as risk factors for squamous intraepithelial lesions of the cervix. By using PCR, presence of HPV-DNA and EBV-DNA was found in 60.9% (67/110) and in 10% (11/110) of clinical samples, respectively. Identification of oncogenic group of HPV by hybrid capture (HC II, Murex-Digene) indicated the presence of low-risk HPV in 13 (19.4%) patients, high-risk HPV in 28 (41.8%), and both types of HPV in 26 (38.8%) patients. Squamous intraepithelial lesions were present in 59 cases, being low-grade (n = 52) and high-grade (n = 7) lesions. HPV was detected in 84.7% of patients with lesions, in association with low-grade (43/52) and high-grade lesions (7/7), and in 33% of patients without lesions. EBV-DNA was detected in 8 patients with low-grade lesions and in 3 patients without lesions. Concurrent genital HPV and EBV infection was observed in 9 cases. HPV was associated with detection of squamous intraepithelial lesions [OR = 3.55; 95% CI = (1.96; 6.48)]. No significant association was found between presence of EBV and detection of lesions, both in case of EBV infection alone [OR = 1.4; 95% CI = (0. 93; 2.12)] and in case of HPV/EBV combined infection [OR = 0.87; 95%CI = (0.54; 1.42)]. These data confirm the significant role of HPV as risk factor for squamous intraepithelial lesions and suggest that EBV could not be involved in the pathogenesis of the lesions that arise in the cervix of HIV-positive women.     

Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic 'stores' of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac -1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G-Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.     

Assessment of major histocompatibility complex class I interaction with Epstein-Barr virus and human immunodeficiency virus peptides by elevation of membrane H-2 and HLA in peptide loading-deficient cells.

Earlier findings indicate that peptides can affect the expression of major histocompatibility complex (MHC) class I molecules on the surface of cells with defective peptide loading mechanism. We have used peptide induced increase of class I antigen expression to assess peptide interaction with MHC class I molecules. A panel of 41 overlapping synthetic peptides derived from the human immunodeficiency virus-1 (HIV-1) gag protein and 33 nonoverlapping peptides from Epstein-Barr virus (EBV) proteins EBNA-1, 2, 3, 4, 5, 6, LMP, BZLF2, BILF2, BSLF2, BALF4 and BcLF1 was assessed for the ability to enhance the expression of HLA-A2.1, H-2Db, Kb and Dd on the murine RMA-S and human 721.174/T2 (.174/T2) lines by indirect immunofluorescence. Considering doubling of the fluorescence intensity in the peptide-treated samples as positivity, 6 of 39 HIV and 1 of 32 EBV peptides were found to bind to A2.1, 6 of 39 HIV gag and 7 of 16 EBV peptides to Db, 8 of 39 HIV gag and 5 of 16 EBV peptides to Kb and 2 of 39 HIV gag and 1 of 17 EBV peptides to Dd. The sensitivity of the method is comparable to the in vitro class I assembly assay with conformation-dependent monoclonal antibody and is more discriminating than the solid-phase assay. Due to its simplicity this method can also serve for testing large peptide panels for binding capacity to various class I molecules. Moreover, the method provides information about the relevance of in vitro tests for class I assembly in living cells.     

Cerebrospinal fluid analysis in human immunodeficiency virus infection.

Cerebrospinal fluid (CSF) analytes were evaluated in 59 human immunodeficiency virus (HIV+) individuals to assess neurological involvement. Glucose, total protein, cell counts, p24 antigen, CSF: serum albumin/IgG ratios, and oligoclonal bands were measured. Eighty percent of samples showed abnormalities in one or more analyte. In some patients samples, these abnormalities could mimic those of secondary opportunistic infection when none was present. The presence of oligoclonal banding in CSF (31 percent) and disturbances in CSF: serum albumin/IgG ratio (30 percent) were related to decreases in serum CD4+ lymphocytes. Disturbances in CSF: Serum albumin/IgG ratio were also related to severity of non-neurological HIV disease staging. Cerebrospinal fluid oligoclonal bands were distinct from that found in serum in the same subjects. Since immune complexes between immunoglobulins and enzymes are observed in these same patients, these oligoclonal bands may result in artifactually elevated enzyme results secondary to decreased clearance leading to erroneous clinical decisions. There was no significant relationship between any abnormalities and the presence of neurologic disease as established by a wide variety of other studies. It is important to recognize the limits of CSF interpretation in this patient group.     

Differential major histocompatibility complex class II locus expression on human laryngeal epithelium

The survival of a laryngeal allograft will be dependent on the immunological composition of the donor larynx and, in particular, on the expression of major histocompatibility complex (MHC) class II antigens on professional and non-professional antigen-presenting cells. Laryngeal and tonsillar biopsies from normal individuals aged 18-78 years were processed and prepared for quantitative, multiple-colour immunofluorescence using mouse antihuman monoclonal antibodies to human leucocyte antigen (HLA)-DR, HLA-DQ and CD45. The laryngeal epithelium expressed HLA-DR locus products at variable levels, but expression of HLA-DQ was virtually absent. Tonsillar epithelial cells expressed HLA-DR at the basal layer only, while HLA-DQ was similarly not expressed. In contrast, both HLA-DR and -DQ locus products were present on lamina propria and intraepithelial leucocytes in both laryngeal and tonsillar mucosae, although at varying levels. The finding that laryngeal epithelial cells express MHC class II antigens has implications for the survival of laryngeal allografts and suggests that they may require significant immunomodulation. In addition, antigen presentation by epithelial cells has been hypothesized to contribute to the immunoregulatory function of mucosal tissues, and the finding that HLA-DQ locus products are only expressed at low levels by laryngeal epithelium raises questions about the repertoire of peptides to which the mucosal immune system can respond.     

Effects of human T-lymphotropic virus type II on human immunodeficiency virus type 1 phenotypic evolution

Summary.  Phenotypic change and broader coreceptor usage by HIV-1 have been associated with disease progression. HIV-1 coreceptor usage by primary isolates obtained from HIV-1-infected and HIV-1/HTLV-II-coinfected individuals was determined. HIV-1 was isolated from 15 of 20 HIV-1-infected and 17 of 24 HIV-1/HTLV-II-coinfected individuals. None of the isolates from either the HIV-1-infected or the coinfected group infected CCR5Δ32 PBMCs, suggesting that they all were R5-tropic. Further, both spontaneous and PHA-stimulated production of MIP-1β and RANTES were similar in HIV-1-infected and coinfected individuals. These data indicate that coinfection with HTLV-II has no effect on HIV-1 coreceptor usage or ex vivo β-chemokine production. Received December 23, 2000/Accepted May 16, 2001     

Expression of human immunodeficiency virus type 1 Gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing HIV envelope

Recombinant vesicular stomatitis viruses have been developed as high-level expression vectors which serve as effective vaccine vectors in animals (Roberts et al., 1998, J. Virol. 72, 4704-4711; Roberts et al., 1999, J. Virol. 73, 3723-3732). Here we show that two genes can be expressed simultaneously from a single, live-attenuated VSV recombinant. The genes used encode the Pr55(gag) protein precursor of HIV-1 (1.7-kb gene) and an HIV-1 envelope (Env) protein (2.4 kb gene). Our results show that VSV can accommodate up to a 40% increase in genome size with only a threefold reduction in virus titer. Recombinants expressing the Pr55(gag) protein precursor with or without Env protein produced abundant HIV virus-like particles (VLPs) in addition to bullet-shaped VSV particles. HIV Env protein expressed from a VSV recombinant also expressing Gag was specifically incorporated into the HIV VLPs but not into the VSV particles. In contrast, VSV G protein was found in both VSV particles and in HIV VLPs. Such VSV/HIV recombinants producing HIV VLPs with Env protein could be an effective source of HIV-like particles inducing both cellular and antibody-mediated immunity to HIV-1.