Oligomeric structure of virion-associated and soluble forms of the simian immunodeficiency virus envelope protein in the prefusion activated conformation.

The envelope proteins (env) of simian immunodeficiency virus (SIV) and HIV type 1 assemble to form noncovalently associated oligomers in the endoplasmic reticulum. After cleavage in a Golgi compartment, oligomeric env complexes are transported to the surface of infected cells, where incorporation into budding virions can occur. Difficulties in obtaining adequate quantities of virions retaining env, as well as the unstable nature and hydrophobicity of the oligomer, may account for the absence of previous biophysical studies to determine the oligomeric valency of membrane-associated env. The aim of this study was to evaluate the oligomeric state of SIV env before membrane-fusion activation. Virion-associated env, obtained by crosslinking and detergent extraction, and non-crosslinked secreted env ectodomain (recombinant gp140) were purified by lentil-lectin chromatography and gel filtration as single predominant species. Sedimentation equilibrium-derived mass values for both forms of SIV env were close to those predicted for trimeric assemblies. Determination of the mass of individual molecules by scanning transmission electron microscopy confirmed that SIV virion-associated env and gp140 formed largely homogeneous populations of trimers. Furthermore, a triangular or tri-lobed morphology was clearly visualized in a subset of the trimers.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140.

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.

Overview of the vaccinia virus expression system.

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and "appropriate" transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.

Preparation of cell cultures and vaccinia virus stocks.

This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.

Generation of recombinant vaccinia viruses.

This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.

Characterization of recombinant vaccinia viruses and their products.

After a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dot-blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.

Preparation of cell cultures and vaccinia virus stocks.

This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.

Generation of recombinant vaccinia viruses.

This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.

Characterization of recombinant vaccinia viruses and their products.

After a recombinant vaccinia virus is made, its DNA and protein products can be analyzed in several ways. Protocols are provided in this unit for identification of the recombinant virus by PCR (with verification of correct insertion of the DNA by Southern blotting) and by dot-blot hybridization. Also, when antibodies are available, protein expression can be analyzed by immunological methods detailed here such as dot blotting with an antibody, immunoblotting and/or immunoprecipitation. In addition, immunostaining can be used for identification of recombinant plaques as well as for determination of the purity of a recombinant virus stock. All of the protocols in this unit can be used for characterization of modified vaccinia virus Ankara (MVA) recombinant viruses.

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts.

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

Characterization of primary isolate-like variants of simian-human immunodeficiency virus.

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.

Characterization of conformation-dependent anti-gp120 murine monoclonal antibodies produced by immunization with monomeric and oligomeric human immunodeficiency virus type 1 envelope proteins.

Twenty-five conformation-dependent monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric forms of the human immunodeficiency virus type 1 (HIV-1) envelope (env) glycoprotein were used to map exposed, immunogenic regions on oligomeric env. Based on MAb cross-competition, reactivity with diverse env proteins, and reactivity with a panel of gp120 mutants, seven distinct epitope clusters were identified. These include the classic CD4 binding site, V1/V2, and V3. in addition, several novel epitope clusters, including one mapping to the N- and C-termini of gp120, were identified. The locations of the seven epitope clusters on the gp120 core structure are proposed.

Recombinant vaccinia viruses. Design, generation, and isolation.

The technologies of recombinant gene expression have greatly enhanced the structural and functional analyses of genetic elements and proteins. Vaccinia virus, a large double-stranded DNA virus and the prototypic and best characterized member of the poxvirus family, has been an instrumental tool among these technologies and the recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins. Vaccinia virus is also the prototype live viral vaccine and serves as the basis for well established viral vectors which have been successfully evaluated as human and animal vaccines for infectious diseases and as anticancer vaccines in a variety of animal model systems. Vaccinia virus technology has also been instrumental in a number of unique applications, from the discovery of new viral receptors to the synthesis and assembly of other viruses in culture. Here we provide a simple and detailed outline of the processes involved in the generation of a typical recombinant vaccinia virus, along with an up to date review of relevant literature.

Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein.

The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found.

Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.

Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein.

The humoral immune response to human immunodeficiency virus type 1 (HIV-1) is often studied by using monomeric or denatured envelope proteins (Env). However, native HIV-1 Env complexes that maintain quaternary structure elicit immune responses that are qualitatively distinct from those seen with monomeric or denatured Env. To more accurately assess the levels and types of antibodies elicited by HIV-1 infection, we developed an antigen capture enzyme-linked immunosorbent assay using a soluble, oligomeric form of HIV-1IIIB Env (gp140) that contains gp120 and the gp41 ectodomain. The gp140, captured by various monoclonal antibodies (MAbs), retained its native oligomeric structure: it bound CD4 and was recognized by MAbs to conformational epitopes in gp120 and gp41, including oligomer-specific epitopes in gp41. We compared the reactivities of clade B and clade E serum samples to captured Env preparations and found that while both reacted equally well with oligomeric gp140, clade B seras reacted more strongly with monomeric gp120 than did clade E samples. However, these differences were minimized when gp120 was captured by a V3 loop MAb, which may lead to increased exposure of the CD4 binding site. We also measured the ability of serum samples to block binding of MAbs to epitopes in gp120 and gp41. Clade B serum samples consistently blocked binding of oligomer-dependent MAbs to gp41 and, to a slightly lesser extent, MAbs to the CD4 binding site in gp120. Clade E serum samples showed equivalent or greater blocking of oligomer-dependent gp41 antibodies and considerably less blocking of CD4-binding-site MAbs. Finally, we found that < 5% of the antibodies in clade B sera bound to epitopes present only in monomeric gp120, 30% bound to epitopes present in both monomeric gp120 and oligomeric gp140, and 70% bound to epitopes present in oligomeric gp140, which includes gp41. Thus, captured oligomeric Env closely reflects the antigenic characteristics of Env protein on the surface of virions and infected cells, retains highly conserved epitopes that are recognized by antibodies raised against different clades, and makes it possible to detect a much greater fraction of total anti-HIV-1 Env activity in sera than does native monomeric gp120.

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies.

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.