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.

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques.

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.

DNA priming and recombinant pox virus boosters for an AIDS vaccine.

Eight different protocols for immunization have been compared for the ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Relationship between anti-Tax antibody responses and cocultivatable virus in HTLV-I-infected rabbits.

The presence of anti-Tax antibody responses in human T cell leukemia virus type I (HTLV-I)-infected individuals has been correlated with increased proviral load, increased risk of transmitting infection, and increased risk of developing tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). In this study, a rabbit model of HTLV-I infection was used to determine whether anti-Tax antibody responses could predict the presence of virus with the potential to replicate. Seven of 14 HTLV-I-infected rabbits developed anti-Tax antibody responses. The onset of Tax reactivity was variable, but once detected remained constant throughout the remainder of the 60-week course of the study. All anti-Tax antibody positive rabbits produced virus as measured by p19 expression upon coculture, while p19 was detected in only one of the Tax antibody negative animals. Thus the presence of an anti-Tax antibody response correlates with p19 expression following cocultivation, and may be a useful predictor of virus replication in HTLV-I infected individuals.

Induction in vitro of primary cytotoxic T-lymphocyte responses with DNA encoding herpes simplex virus proteins.

Vaccines which successfully protect against virus infections usually need to induce a broadly reactive immune response which includes the induction of cytotoxic T lymphocytes (CTL). In this study, we have used a convenient in vitro approach to investigate if plasmid DNAs encoding proteins of herpes simplex virus (HSV) are capable of inducing primary CD8+ CTL. Dendritic cells or macrophages were transfected with either plasmid DNA encoding glycoprotein B or DNA encoding the immediate-early protein ICP27. These antigen-presenting cells (APC) were then used to stimulate enriched populations of naive T cells in microcultures for 5 days in vitro. Antigen-specific CD8+ CTL which reacted both with specific protein-expressing targets and with syngeneic targets infected with HSV could be demonstrated. Dendritic cells, as APC, generated the maximal responses, but such cells needed to be transfected with DNA in the presence of a cationic lipid. However, macrophages could act as APC when they were exposed to purified DNA. HSV-primed splenocytes were also shown to generate specific CTL responses when they were stimulated with purified DNA encoding ICP27. The novel approach described in this paper promises to be extremely useful, since defining immunogenicity profiles and identifying epitopes on viral proteins should be easier and more convenient when working with DNA and investigating variables in vitro. This is particularly the case with complex viruses such as HSV, most of whose encoded proteins have yet to be isolated in sufficient quantity or purity to perform in vivo immunological studies.

Cell fusion activity of the simian immunodeficiency virus envelope protein is modulated by the intracytoplasmic domain.

The processing and biological activity of envelope glycoproteins of pathogenic and nonpathogenic simian immunodeficiency viruses (SIVs) was compared using recombinant vaccinia viruses (rVVs). The env glycoprotein of the nonpathogenic SIVmac1A11 virus caused much larger and more numerous syncytia than the glycoprotein of the pathogenic SIVmac239 virus in several CD4+ human cell lines. The env gene of SIVmac239 codes for a full-length transmembrane (TM) protein, while the SIVmac1A11 virus has a TM protein with a markedly truncated cytoplasmic domain. To determine if TM protein truncation alone might affect the biological properties of viral glycoproteins, we constructed a rVV which expresses a SIVmac239 env with a site-specific mutation yielding a truncated TM protein. This truncated env protein induced extensive fusion of rVV-infected HeLa T4 cell monolayers, whereas no fusion was observed for the parental SIVmac239 env recombinant. The truncated glycoprotein also caused larger and more numerous syncytia than the wild-type SIVmac239 glycoprotein in the human cell lines HUT 78 and CEM x 174. The mutation altered env glycoprotein transport, but did not significantly affect cell surface expression levels or the amount of secreted soluble SU protein. In coinfection assays, the full-length SIVmac239 env protein was found to interfere with fusion induced by the truncated envelope protein. The results thus demonstrate that changes in the cytoplasmic domain of the SIVmac envelope protein can markedly affect the ability to induce cell fusion, an activity of the external domains of the TM-SU glycoprotein complex.

Role of the cytoplasmic domains of viral glycoproteins in antibody-induced cell surface mobility.

We have investigated the role of the cytoplasmic domains of the influenza virus hemagglutinin (HA) and the parainfluenza virus type 3 (PI3) fusion (F) glycoproteins as a determinant of their ability to undergo antibody-induced redistribution on plasma membranes. The viral envelope genes were truncated in their cytoplasmic domains by using oligonucleotide-directed mutagenesis and expressed by using recombinant vaccinia viruses. In HeLa cells, the truncated HA (HAt), like the full-length HA, did not cap in response to specific antibody. In CV-1 cells, HAt showed patchy surface immunofluorescence with few caps, whereas full-length HA exhibited capping in many cells in response to bivalent antibody. Quantitation of cap formation indicated a sevenfold decrease in the frequency of capping of HAt in comparison with full-length HA. Similarly, truncated F also exhibited a significant decrease in cap formation in comparison with full-length F. These results indicate that the ability of influenza virus HA and PI3 F to undergo redistribution in response to bivalent antibody has been altered by truncation of the viral glycoproteins and suggest that capping may involve interactions between the cytoplasmic domain of the viral glycoproteins and host cell components.

Host cell-dependent lateral mobility of viral glycoproteins.

The lateral mobility of viral envelope proteins on the plasma membranes of infected cells is an important factor in both virus assembly and pathogenesis. The envelope glycoproteins of measles and human parainfluenza virus are mobile on the surfaces of infected HeLa cells and undergo lateral redistribution in the presence of specific antibody, forming unipolar caps. In contrast, no such redistribution was observed with influenza virus hemagglutinin (HA) or vesicular stomatitis virus (VSV) G glycoproteins on infected HeLa cell surfaces. However, the HA and G glycoproteins were both found to be mobile in the plasma membrane of CV-1 cells, or human or murine peritoneal macrophages. These results indicate that host cell-dependent as well as virus-specific factors are involved in determining viral glycoprotein mobility. No significant differences in the patterns of synthesis of influenza or VSV viral proteins were found in the various cell types examined. The HA and G proteins, when expressed from vaccinia virus recombinants, were each found to be immobile in HeLa cells and mobile in CV-1 cells, thus indicating that the host cell-dependent differences in mobility are an intrinsic property of each viral glycoprotein molecule and not the result of interaction with other viral components. It is suggested that the association of viral glycoproteins with either the cytoskeleton or membrane-associated cellular proteins may be related to the observed differences in lateral mobility.