Sequential Vacc-4x and romidepsin during combination antiretroviral therapy (cART): Immune responses to Vacc-4x regions on p24 and changes in HIV reservoirs.

OBJECTIVES: The REDUC clinical study Part B investigated Vacc-4x/rhuGM-CSF therapeutic vaccination prior to HIV latency reversal using romidepsin. The main finding was a statistically significant reduction from baseline in viral reservoir measurements. Here we evaluated HIV-specific functional T-cell responses following Vacc-4x/rhuGM-CSF immunotherapy in relation to virological outcomes on the HIV reservoir. METHODS: This study, conducted in Aarhus, Denmark, enrolled participants (n = 20) with CD4>500 cells/mm3 on cART. Six Vacc-4x (1.2 mg) intradermal immunizations using rhuGM-CSF (60 µg) as adjuvant were followed by 3 weekly intravenous infusions of romidepsin (5 mg/m2). Immune responses were determined by IFN-γ ELISpot, T-cell proliferation to p24 15-mer peptides covering the Vacc-4x region, intracellular cytokine staining (ICS) to the entire HIVGag and viral inhibition. RESULTS: The frequency of participants with CD8+ T-cell proliferation assay positivity was 8/16 (50%) at baseline, 11/15 (73%) post-vaccination, 6/14 (43%) during romidepsin, and 9/15 (60%)post-romidepsin. Participants with CD8+ T-cell proliferation assay positivity post-vaccination showed reductions in total HIV DNA post-vaccination (p = 0.006; q = 0.183), post-latency reversal (p = 0.005; q = 0.183), and CA-RNA reductions post-vaccination (p = 0.015; q = 0.254). Participants (40%) were defined as proliferation 'Responders' having ≥2-fold increase in assay positivity post-baseline. Robust ELISpot baseline responses were found in 87.5% participants. No significant changes were observed in the proportion of polyfunctional CD8+ T-cells to HIVGag by ICS. There was a trend towards increased viral inhibition from baseline to post-vaccination (p = 0.08). CONCLUSIONS: In this 'shock and kill' approach supported by therapeutic vaccination, CD8+ T-cell proliferation represents a valuable means to monitor functional immune responses as part of the path towards functional HIV cure.

Evidence of subtype B-like sequences in the V3 loop region of human immunodeficiency virus type 1 in Kilimanjaro, Tanzania.

The aim of this study was to determine the nucleotide sequence of the third variable (V3) domain of HIV-1 gp120 from strains circulating in the Kilimanjaro region of Tanzania. DNA from this region was amplified from patient peripheral blood lymphocytes using polymerase chain reaction and then subjected to automated DNA sequencing. Subtype A, B, C, and D-like sequences were identified. Subtype B has not previously been described in Tanzania.

Transient Tat activation of the HIVLAV/Lai-1 LTR by primary HIV-1 phenotypic variants in HeLaT4LTRbeta-gal cells.

The rapid/high and slow/low phenotypic variants of primary HIV-1 isolates can be distinguished by their differential co-receptor utilization and their ability to productively infect established cell lines. To reveal possible differences in Tat-mediated transactivation, the potential for primary isolate Tat proteins to transactivate the LTR from the laboratory strain HIVLAV/Lai-1 was examined. Using either cell-mediated or PEG-induced fusion of cells infected with primary HIV-1 isolates and HeLaT4LTRbeta-gal cells, it was clear that the Tat protein encoded by all patient isolates efficiently activated transcription from the HIVLAV/Lai-1 LTR. However, infection of HeLaT4LTRbeta-gal cells by primary HIV-1 isolates was transient, suggesting the development of a postpenetration host control of HIV-1 replication at the level of tat activation, a feature not observed for the laboratory-adapted strain HIVIIIB. Although plasmid vectors based on the HIVLAV/Lai-1 LTR remain useful for the development of susceptible established cell lines for titrating primary HIV-1 isolates, the efficacy of such a system would depend upon the stability/duration of Tat activation.

Intercellular adhesion molecule 3, a candidate human immunodeficiency virus type 1 co-receptor on lymphoid and monocytoid cells.

The CD4 molecule serves as the principal cell surface receptor common to both the human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV). Since binding to CD4 is not sufficient to permit virus entry, HIV 'co-receptors' have been implicated in mediating the fusion of viral and cellular membranes necessary for completing the entry process. In order to identify candidate co-receptor molecules, a panel of monoclonal antibodies (MAbs) directed against adhesion molecules was tested for the ability of the MAbs to inhibit HIV-1-induced cell fusion (syncytium formation) and HIV-1 entry. Certain antibodies directed against CD18, CD11b and CD11c inhibited HIV-1-induced syncytium formation but not entry, in agreement with previous reports. Interestingly, certain antibodies to ICAM-3 (intercellular adhesion molecule 3) (CD50) significantly inhibited HIV-1-specific entry but not syncytium formation using human SupT1 cells. Only one antibody directed against ICAM-3 significantly inhibited HIV-1-induced syncytium formation, entry and infectivity. Our results suggest that certain epitopes of ICAM-3 may be involved in mediating HIV-1-specific entry into lymphoid and monocytoid cells.

Rapid purification of recombinant baculovirus using fluorescence-activated cell sorting.

Expression of foreign proteins in the baculovirus-insect cell expression system has been limited by difficulties in rapid identification and purification of recombinant virus. Although the process of identifying recombinant virus has been greatly facilitated by the introduction of vectors that lead to insect cell co-expression of beta-galactosidase with foreign genes of interest, isolation of pure recombinant virus using plaque purification may still take several weeks to months to accomplish. Using a fluorescent beta-galactosidase substrate, we have established that insect cells harboring recombinant virus can be rapidly isolated using fluorescence-activated cell sorting. Pure recombinant virus can then be readily obtained using this cellular fraction, with a pure viral culture generally obtained within 2-3 weeks of insect cell transfection.

Expression of simian type D retroviral (Mason-Pfizer monkey virus) capsids in insect cells using recombinant baculovirus.

Mason-Pfizer monkey virus (M-PMV) is a primate retrovirus that shows type D morphogenesis in mammalian cells. Immature intracytoplasmic A type particles (ICAPs) preassemble in the infected cell cytoplasm migrate to the plasma membrane and are released by budding. This is in contrast to retroviruses that show type C morphogenesis, where assembly and budding occur concurrently at the plasma membrane. We expressed the M-PMV structural genes (gag-pro-pol) in insect cells using a recombinant baculovirus. The polyprotein precursors assembled predominantly intracellularly, although a small proportion also assembled at the membrane. The protease enzyme was active since mature particles were identified in the culture supernatant. We also expressed the M-PMV mutants, D26N and gag-STOP, which carry a nonfunctional protease or fail to express the protease gene, respectively. These baculovirus recombinants generated a homogeneous population of immature M-PMV capsids having exclusively type D morphogenesis. Sufficient quantities of polyprotein precursors were synthesized to be visualized directly on a Coomassie-stained protein gel, and the capsids were subject to purification. These results provide the first expression of type D retrovirus particles using the baculovirus expression system.

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.

Effect of retroviral proteinase inhibitors on Mason-Pfizer monkey virus maturation and transmembrane glycoprotein cleavage.

Mason-Pfizer monkey virus (M-PMV) is the prototype type D retrovirus which preassembles immature intracytoplasmic type A particles within the infected cell cytoplasm. Intracytoplasmic type A particles are composed of uncleaved polyprotein precursors which upon release are cleaved by the viral proteinase to their constituent mature proteins. This results in a morphological change in the virion described as maturation. We have investigated the role of the viral proteinase in virus maturation and infectivity by inhibiting the function of the enzyme through mutagenesis of the proteinase gene and by using peptide inhibitors originally designed to block human immunodeficiency virus type 1 proteinase activity. Mutation of the active-site aspartic acid, Asp-26, to asparagine abrogated the activity of the M-PMV proteinase but did not affect the assembly of noninfectious, immature virus particles. In mutant virions, the transmembrane glycoprotein (TM) of M-PMV, initially synthesized as a cell-associated gp22, is not cleaved to gp20, as is observed with wild-type virions. This demonstrates that the viral proteinase is responsible for this cleavage event. Hydroxyethylene isostere human immunodeficiency virus type 1 proteinase inhibitors were shown to block M-PMV proteinase cleavage of the TM glycoprotein and Gag-containing precursors in a dose-dependent manner. The TM cleavage event was more sensitive than cleavage of the Gag precursors to inhibition. The infectivity of treated particles was reduced significantly, but experiments showed that inhibition of precursor and TM cleavage may be at least partially reversible. These results demonstrate that the M-PMV aspartyl proteinase is activated in released virions and that the hydroxyethylene isostere proteinase inhibitors used in this study exhibit a broad spectrum of antiretroviral activity.

A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein.

The envelope glycoprotein precursor of retroviruses undergoes proteolytic cleavage in the Golgi complex to yield the mature surface and transmembrane (TM) glycoproteins of the virus. We report here that the TM glycoprotein of Mason-Pfizer monkey virus undergoes a second proteolytic processing event during a late maturation step that can follow virus release and Gag polyprotein cleavage. Cleavage results in the conversion of the cell-associated TM glycoprotein (gp22) to a virus-associated gp20. Processing continues after virus release and yields virions that contain predominantly gp20. A mutation within the active site of the Mason-Pfizer monkey virus aspartyl protease was shown to block both TM glycoprotein cleavage and the processing of the Gag polyprotein precursor. The role of the viral protease in cleavage of the TM glycoprotein localizes the cleavage site to the cytoplasmic domain of this protein. Surprisingly, point mutations within the matrix (MA) coding region of the gag gene can affect the extent to which gp22 is processed to gp20 and in one case [p10(MA)-A79V] results in greater than 90% inhibition of gp22 cleavage. The data provide genetic evidence of a specific interaction between the capsid proteins and the cytoplasmic domain of the TM glycoprotein of a retrovirus. This interaction is required for cytoplasmic domain cleavage to occur and may play a critical role in virus assembly and viral infectivity.

Localization of the receptor gene for type D simian retroviruses on human chromosome 19.

Simian retrovirus (SRV) serotypes 1 to 5 are exogenous type D viruses causing immune suppression in macaque monkeys. These viruses exhibit receptor interference with each other, with two endogenous type D viruses of the langur (PO-1-Lu) and squirrel monkey, and with two type C retroviruses, feline endogenous virus (RD114/CCC) and baboon endogenous virus (BaEV), indicating that each utilizes the same cell surface receptor (M. A. Sommerfelt and R. A. Weiss, Virology 176:58-69, 1990). Vesicular stomatitis virus pseudotype particles bearing envelope glycoproteins of RD114, BaEV, and the seven SRV strains were employed to detect receptors expressed in human-rodent somatic cell hybrids segregating human chromosomes. The only human chromosome common to all the susceptible hybrids was chromosome 19. By using hybrids retaining different fragments of chromosome 19, a provisional subchromosomal localization of the receptor gene was made to 19q13.1-13.2. Antibodies previously reported to be specific to a BaEV receptor (L. Thiry, J. Cogniaux-Leclerc, R. Olislager, S. Sprecher-Goldberger, and P. Burkens, J. Virol. 48:697-708, 1983) did not block BaEV, RD114, or SRV pseudotypes or syncytia. Antibodies to known surface markers determined by genes mapped to chromosome 19 did not block virus-receptor interaction. The identity of the receptor remains to be determined.

Receptor interference groups of 20 retroviruses plating on human cells.

The types of receptors on the surfaces of human and other mammalian cells for 13 C-type and 7 D-type retrovirus strains were determined by interference to the formation of syncytia and the plating of viral pseudotypes. All the D-type simian retroviruses (SRV-1-5, SMRV, PO-1-Lu) share a common receptor which is also utilized by the baboon and cat endogenous C-type viruses (BaEV, RD114). Syncytial cross-interference was also observed in human cells between the gibbon ape leukemia/simian sarcoma associated viruses (GALV/SSAV) and feline leukemia virus subgroup B (FeLV-B). Amphotropic and xenotropic murine leukemia viruses (MLV-A, MLV-X), bovine leukemia virus (BLV), and FeLV-C infect human cells via unique cell surface receptors. Human T-cell leukemia viruses types 1 and 2 (HTLV-1, HTLV-2) share a common receptor with related chimpanzee and simian viruses (STLV). Thus seven distinct receptor groups were delineated on human cells for C-type and D-type retroviruses.

The pH independence of mammalian retrovirus infection.

The pH dependence of early steps in the infection of human and other cells by mammalian retroviruses and retroviral pseudotype particles of vesicular stomatitis virus (VSV) was investigated for 10 strains of retrovirus, including C-type and D-type oncoviruses and human lentiviruses. When cells were treated with weak bases (NH4Cl and amantadine) to raise the pH of endocytic vesicles, only ecotropic murine leukaemia virus (MLV-E) and VSV showed pH-dependent entry. Pretreatment of retrovirus stocks in media below pH 5.0 did not reduce their titres but inactivated VSV to less than 10(-8) of the initial titre. VSV (MLV-E) pseudotype infection in five out of six mouse and rat cell lines was inhibited by NH4Cl, indicating that infection proceeds via receptor-mediated endocytosis. In contrast, NH4Cl treatment has no effect on the infection of XC cells in which MLV-E induces syncytia. It is postulated that the pH-independent entry and cell fusion of XC cells by MLV-E may result from the activity of a cell surface proteinase that cleaves viral gp70 at neutral pH.

Human T cell leukemia viruses use a receptor determined by human chromosome 17.

Human T cell leukemia viruses (HTLV-I and HTLV-II) can infect many cell types in vitro. HTLV-I and HTLV-II use the same cell surface receptor, as shown by interference with syncytium formation and with infection by vesicular stomatitis virus (VSV) pseudotypes bearing the HTLV envelope glycoproteins. Human-mouse somatic cell hybrids were used to determine which human chromosome was required to confer susceptibility to VSV(HTLV) infection. The only human chromosome common to all susceptible cell hybrids was chromosome 17, and the receptor gene was localized to 17cen-qter. Antibodies to surface antigens known to be determined by genes on 17q did not block the HTLV receptor.

Lack of reverse transcriptase activity in serum in sporadic post-transfusional and presumed epidemic or water-borne forms of severe non-A, non-B hepatitis.

Reverse transcriptase (RT) activity was not detected in any serum sample taken from 22 patients with mainly severe non-A, non-B hepatitis (NANBH), using two assays selected to cover the range of known human and animal retroviruses. The study included patients with fulminant and sub-acute hepatic failure, which was was attributed to sporadic, post-transfusional, and presumed epidemic or water-borne epidemiological forms of NANBH. Although we cannot exclude the possibility that some of the agents implicated in NANBH are retroviruses, our negative findings suggest that other agents may be involved at least in the severe forms of NANBH.