The HIV-1 antisense protein (ASP) induces CD8 T cell responses during chronic infection.

BACKGROUND: CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. RESULTS: A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells was also demonstrated by tetramer staining using cells from an HLA-A*02 HIV-infected patient. CONCLUSION: Our results provide the first description of CD8+ T cell-mediated immune responses to ASP in HIV-1-infected patients, demonstrating that ASP is expressed during infection. Our identification of epitopes within ASP has implications for designing HIV vaccines.

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study.

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.

Expression of HIV-1 antigens in plants as potential subunit vaccines.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. RESULTS: Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. CONCLUSION: Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

Production of tumor necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human immunodeficiency virus.

The production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta by the monocytic cell line THP-1, productively infected with HIV-1, was investigated using specific RIA and Northern blot analysis. HIV-infected cells, like uninfected cells, did not constitutively produce any detectable amounts of protein or mRNA for TNF alpha or IL-1 beta. After stimulation with LPS or a combination of LPS plus IFN-gamma, TNF alpha and IL-1 beta were detected in tissue culture supernatants and cell lysates and transcripts for both cytokines were seen on Northern blots. No significant difference in production of these two cytokines was observed between uninfected and chronically infected cells. Acutely HIV-infected cells, however, showed phenotypic changes compatible with maturation and an increase in TNF alpha and IL-1 beta mRNA production, and released significantly higher levels of TNF alpha and IL-1 beta compared with chronically infected or uninfected cells. Furthermore, LPS stimulation of HIV-infected cells increased virus production. These results suggest that HIV-infected monocytic cells may produce increased amounts of TNF alpha and IL-1 beta in response to stimuli that could be present in vivo.

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

Reactivation of latent human immunodeficiency virus 1 by human herpesvirus 6 infection.

Infection of the ACH-2 line of human leukemic T cells carrying latent Human immunodeficiency virus 1 (HIV-1) with Human herpesvirus 6 (HHV-6) resulted in an increase in reverse transcriptase (RT) activity, a marker of HIV-1 activation, in the culture supernatant. A similar effect was obtained with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The RT activity reached a peak at 24 hrs post infection (p.i.) and then declined, suggesting that the cells underwent lysis. The HIV-1 antigen was co-expressed with an early-late HHV-6 product, but not always with an immediate-early (IE) HHV-6 product, suggesting that one or more IE gene products were involved in the activation of latent HIV-1 in ACH-2 cells.

Influence of aluminum-based adjuvant on the immune response to multiantigenic formulation.

Several adjuvants have been described and tested in humans. However, the aluminum-based adjuvants remain the most widely used component in vaccines today. Emerging data suggest that aluminum phosphate and aluminum hydroxide adjuvants do not promote a strong commitment to the helper T cell type 2 (Th2) pathway when they are coadministered with some Th1 adjuvants. In this regard, subtle differences between both aluminum-based adjuvants have been demonstrated. We have previously shown that subcutaneous immunization, in aluminum phosphate, of a mixture comprising the surface and core antigens of hepatitis B virus (HBV) and the multiepitopic protein CR3 of human immunodeficiency virus type 1 elicits a CR3-specific Th1 immune response. In these experiments, the antigens were adjuvated at the same time. As the final selection of the best adjuvant should be based on experimental evidence, we asked whether aluminum hydroxide allows a better Th1 immune deviation than aluminum phosphate. We also studied several ways to mix the antigens and the impact on CR3-specific interferon (IFN)-gamma secretion. Our findings indicate that aluminum hydroxide allows better Th1 immunodeviation than aluminum phosphate adjuvant for the mixture of HBV antigens and CR3. In addition, CR3-specific IFN-gamma secretion of the various formulations tested was the same irrespective of the order in which the antigens were combined.

New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity.

We have developed an effective and optimally safe microculture method for rapid and convenient assay of the in vitro cytopathic effects of human immunodeficiency virus (HIV-1) on human lymphoblastoid or other suitable host cells. The assay procedure is applicable to the evaluation of drug effects on in vitro infections induced directly in cultured host cells by cell-free HIV-1 or by coculture with H9 cells chronically infected with HIV-1. The assay uses a newly developed tetrazolium reagent that is metabolically reduced by viable cells to yield a soluble, colored formazan product measurable by conventional colorimetric techniques. This simple microassay minimizes the number of plate manipulations typically required with other assay methods and, coupled with computerized data collection and analysis, facilitates large-scale screening of agents for potential antiviral activity. To support and enhance the discovery of new anti-HIV-1 agents, the National Cancer Institute is offering investigators worldwide the opportunity to submit new candidate agents for anti-HIV-1 screening with this method.

Expression and purification of p24, the core protein of HIV, using a baculovirus-insect cell expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been genetically manipulated to yield a recombinant virus capable of expressing p24, the major core protein of HIV-1, in insect cell culture. The expressed product is a p24 protein flanked by short regions of p17 at the amino terminus and p12 at the carboxy terminus. It has been identified and characterized using monoclonal antibodies on Western blots and by amino-terminal sequence analysis. The presence of p24 in the soluble fraction of infected cells following lysis by detergent or sonication, combined with a high level of expression (in excess of 50 mg/l of culture) facilitates the enrichment of large quantities of recombinant HIV antigen in a simple two-step procedure involving ammonium sulphate fractionation and gel filtration. p24 antigen purified in this way is shown to be an efficient diagnostic reagent.

Early diagnosis of HIV infection in infants.

Eighteen infants born to anti-HIV-positive mothers were tested bimonthly for immunoglobulin M (IgM) anti-HIV by Western blot and HIV p24 antigen (Ag) by enzyme-linked immunosorbent assay (ELISA) in order to determine the role of these markers in the early diagnosis of HIV infection. Twelve healthy infants were also studied as a control group. In 11 out of 18 children (61.1%) an IgM response was demonstrable, in 13 out of 18 (72.2%) IgM anti-HIV and/or p24 antigen (Ag) were detected. Two patterns of IgM response were identified: a precocious IgM positivity (group of five children positive at birth) and a later appearance of IgM, always within the third month (six cases). Early p24 antigenemia occurred in one infant. Three out of four children who developed antigenemia after birth were symptomatic within the sixth month. No clinical or immunological abnormalities were found among the three children who were persistently negative for both IgM anti-HIV and p24 Ag. Serial IgM anti-HIV and p24 Ag testing may be helpful in the early identification of HIV-infected patients.

A novel coculture system for evaluating anti-HIV drugs.

OBJECTIVE: To develop a useful system for evaluating novel anti-HIV drugs. DESIGN: The activity of most antiviral compounds in cell-free HIV infection systems has been evaluated. However, the inhibitory effects on both the process of HIV induction and viral dissemination to uninfected cells have not been fully investigated. We have therefore developed a new cocultivation system using chronically HIV-infected monocytes and CD4+ T-lymphocytes in the presence of tumor necrosis factor (TNF). METHODS: We designed a cocultivation system using flow cytometry with U1 cells and Molt-4 cells in the presence of TNF. The antiviral activities of several compounds in the cocultivation system and other assay systems were compared. RESULTS: Only pradimicin A and glycyrrhizin showed strong inhibitory activity in the cocultivation system in the presence of TNF, whereas dextran sulfate, curdlan sulfate and N-acetylcysteine exhibited moderate or weak inhibitory activity in the system. 3'-azido-2',3'-dideoxythymidine and 2',3'-dideoxyadenosine were completely ineffective in the system. CONCLUSION: These results support the suggestion that our cocultivation system includes HIV induction in chronically infected monocytes, and the resulting cell-to-cell infection between HIV-infected monocytes and Molt-4 cells or Molt-4 cells and their HIV-converted counterparts. Our new cocultivation system may constitute a useful tool in the identification of novel anti-HIV compounds.

Gene combination raises broad human immunodeficiency virus-specific cytotoxicity.

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli.

A new expression vector (pBB1) has been constructed for the regulated expression of genes in Escherichia coli. Based on the pUC plasmids, the pBB1 carries lacIts allele of the lac repressor gene. This makes it possible to control expression of cloned genes by shifting the temperature from 30 degrees C to 42 degrees C. Thus the vector combines advantages of the pUC plasmids with convenient regulation by temperature. Expression of a fragment of HIV-1 env gene was achieved with the help of this vector and shown by enzyme-linked immunosorbent assay and Western-blot analysis.

Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors.

Recombinant vaccinia viruses (re-VVs) provide an extremely versatile method for the expression of foreign genes in a wide range of cultured cell types of different lineages and species. In the present report, we examine the utility of re-VV vectors for re-protein production in cultured human primary macrophages obtained through in vitro differentiation of peripheral blood monocytes. Primary macrophages supported early stages of the VV infection cycle, including morphologic cytopathic effect, shut-off of host protein synthesis and activation of early viral protein synthesis; however, late stages of infection were blocked, including synthesis of late viral proteins, replication of viral DNA, and production of infectious progeny virions. Abortive infection was observed with several independent VV strains. Using re-VVs containing Escherichia coli lacZ as a reporter gene, we assayed the activities of different classes of VV promoters. Consistent with the results noted above, human primary macrophages supported reporter gene expression driven by an early or intermediate VV promoter, but not by a late promoter; expression was obtained with synthetic bifunctional promoters containing early and/or intermediate components. Primary macrophages also supported the VV/bacteriophage T7 RNA polymerase hybrid gene expression system. The utility of re-VV vectors for production of proteins of biological interest in human primary macrophages was demonstrated using re-VVs encoding human CD4 and the human immunodeficiency virus type-1 envelope glycoprotein.

Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A.

After incubation of H9 cells infected with human immunodeficiency virus (HIV) with pepstatin A at 10(-4) M for 2, 4, or 11 days, the culture medium contained significantly less HIV core antigen (p24) than controls without pepstatin A and no or only borderline activity of reverse transcriptase was detected. In addition, after pepstatin A treatment no infectious HIV at 2 or 4 days and only minimal amounts at 11 days were detectable in the culture medium.

Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12.

In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.

In vitro assays show a dissociation of reverse transcriptase activity and core antigen (p24) production in two HIV-1 isolates from a patient receiving long-term treatment with zidovudine (ZDV).

Two HIV-1 isolates were obtained from a patient receiving long-term treatment with zidovudine (ZDV). The in vitro sensitivity to ZDV triphosphate of the reverse transcriptase (RT) from both isolates appeared to be unchanged compared to that of the LAV-Bru HIV-1 reference strain. When isolates were grown in CEM cells (a T-lymphoblastoid tumor cell line) and their RT activity and core antigen (p24) production were determined, the level of p24 production compared to RT activity was high; in infected CEM cells treated with ZDV, RT activity was at background level while the p24 production was still significant, thus indicating a dissociation of RT activity and core antigen production.

Phenotypic mixing between human immunodeficiency virus and vesicular stomatitis virus or herpes simplex virus.

Superinfection of H9 cells persistently infected with human immunodeficiency virus (HIV) with thermolabile vesicular stomatitis virus (VSV) or herpes simplex virus (HSV) led to the synthesis of hybrid progeny. These phenotypic mixtures were able to infect HeLa or Chinese hamster ovary cell lines, leading to the production of HIV p24 antigen and infectious HIV. This production was abrogated by prior incubation of the phenotypic mixtures with antiserum against VSV or HSV, as well as by incubation of the mixtures at 39 degrees C for 10 h. These results demonstrate that during coinfection of cells with either a RNA or DNA virus, HIV forms hybrid virions composed of the genetic information of HIV and the envelope glycoproteins of the coinfecting viruses.

CD4-modified synthetic peptides containing phenylalanine inhibit HIV-1 infection in vitro.

Phenylalanine-containing peptides from CD4 were synthesized based on chemical similarity with active CD4(81-92)-benzylated peptides. The synthetic peptide FYIFFVEDQKEEDD blocked the binding of gp120 to CD4 and inhibited 50% human immunodeficiency virus (HIV)-induced syncytia formation at a concentration (IC50) of approximately 40-50 microM and HIV p17 expression with an IC50 of approximately 67 microM. The peptide is not toxic to cells in vitro. Moreover, acute toxicity studies carried out in Swiss mice showed the peptide to be nontoxic at a dose of 2,000 mg/kg. This phenylalanine-substituted CD4 peptide may prove to be useful in the treatment of AIDS.

Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2.

The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described. In a whole-blood, erythrocyte agglutination assay format, recombinant scFv antibodies (expressed in Escherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 peptides, were shown to be able to detect efficiently natural antibodies against HIV in human serum. Performance in trials suggests that these single chain reagents have potential as alternatives to existing Fab-peptide chemical conjugates. We also report the construction of an inducible expression vector, pGC, which can be used both in laboratory experiments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as the Fab peptide conjugate in assays where whole (negative) blood was spiked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives. This deficiency was overcome by inclusion of the natural 1C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagent (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a result of this important modification, performance of the extended scFv reagent (for both HIV-1 and HIV-2) equalled that of the current commercial technology in limited trials.