David J. Hockley

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Roles of Matrix, p2, and N-Terminal Myristoylation in Human Immunodeficiency Virus Type 1 Gag Assembly

ABSTRACT Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the >1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.

Comparative Morphology of Gag Protein Structures Produced by Mutants of the gag Gene of Human Immunodeficiency Virus Type 1

Six mutants that differ in the extent of their carboxyterminal sequences and two deletion mutants of the gag gene of HIV-1 have been characterized morphologically following their expression in Spodoptera frugiperda cells using recombinant baculoviruses. Electron microscopy has revealed distinct morphological forms of the Gag protein that can be classified as either (i) particulate, three-dimensional, spherical or tubular shells or (ii) non-particulate, two-dimensional, flat, curved or convoluted sheets. Progressive truncation of the carboxy terminus of Gag was accompanied by changes in the morphology and formation of spherical particles from predominantly C-type assembly and budding at the plasma membrane, through B-type intracytoplasmic assembly, to A-type assembly with budding mainly into cytoplasmic vacuoles. Deletions within the Pr24 CA domain of Gag abolished particle formation but retained association of the protein with the plasma membrane. All of the observed morphologies of the mutant Gag proteins could be accommodated within an icosahedral model for the organization of spherical particles and a basic hexagonal arrangement of assembled Gag protein monomers.

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate. Purified toxoid (not encapsulated) exposed to low pH (2.5) underwent similar changes but re-neutralisation of buffer containing free toxoid, even after one week at pH 2.5 led to some re-folding of protein as determined by fluorescence spectroscopy and gel filtration chromatography. The microencapsulated vaccines were still able to generate an antibody response in mice even after prolonged pre-incubation at 37 degrees C and the apparent absence of detectable toxoid in the vaccine supernate. Electron microscopy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical morphology after incubation in vitro for up to 28 days were able to induce protective antibodies response equal to that of freshly prepared vaccines, which indicates that the toxoid within intact microspheres remained immunogenic. Immunochemical and physico-chemical detection methods, performed on antigen released from PLGA vaccines in vitro, are valuable in providing information on product characteristics but may not be able to predict effectiveness and should be used with in vivo methods to evaluate the stability of such formulations.

OPTIMIZATION OF NB-4 AND HL-60 DIFFERENTIATION FOR USE IN OPSONOPHAGOCYTOSIS ASSAYS

SummaryProduction of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D3 and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.

In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID(50) of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0.05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.

Use of zwitterionic detergent for the preparation of an influenza virus vaccine. 1: Preparation and characterization of disrupted virions.

The zwitterionic, Empigen BB an alkylbetaine based on a C12-C14 alcohol was shown to disrupt influenza A and B viruses in such a way as to retain the biological activity of the surface haemagglutinin (HA) and neuraminidase (NA) antigens. The optimal conditions required to obtain the maximum recovery of HA and NA activity from purified influenza X47 (H3N2) virus concentrate after treatment with Empigen, and the nature and the morphological appearance of the Empigen-treated preparations both before and following a sucrose density gradient purification step, are described. Matrix (M) protein was readily removed during purification, but nucleoprotein (NP) could not be separated from the surface protein activity.

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7.

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg/ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.