Antigen presentation in retroviral vector-mediated gene transfer in vivo.

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.

Direct injection of a recombinant retroviral vector induces human immunodeficiency virus-specific immune responses in mice and nonhuman primates.

The cytotoxic T-lymphocyte (CTL) response plays an important role in controlling the severity and duration of viral infections. Immunization by direct in vivo administration of retroviral vector particles represents an efficient means of introducing and expressing genes and, subsequently, the proteins they encode in vivo in mammalian cells. In this manner foreign proteins can be provided to the endogenous, class I major histocompatibility complex antigen presentation pathway leading to CTL activation. A nonreplicating recombinant retroviral vector, encoding the human immunodeficiency virus type 1 (HIV-1) IIIB envelope and rev proteins, has been developed and examined for stimulation of immune responses in mouse, rhesus macaque, and baboon models. Animals were immunized by direct intramuscular injection of the retroviral vector particles. Vector-immunized mice, macaques, and baboons generated long-lived CD8+, major histocompatibility complex-restricted CTL responses that were HIV-1 protein specific. The CTL responses were found to be dependent on the ability of the retroviral vector to transduce cells. The vector also elicited HIV-1 envelope-specific antibody responses in mice and baboons. These studies demonstrate the ability of a retroviral vector encoding HIV-1 proteins to stimulate cellular and humoral immune responses and suggest that retrovector immunization may provide an effective means of inducing or augmenting CTL responses in HIV-1-infected individuals.

Cross-reactive lysis of human targets infected with prototypic and clinical human immunodeficiency virus type 1 (HIV-1) strains by murine anti-HIV-1 IIIB env-specific cytotoxic T lymphocytes.

To evaluate the ability of murine anti-human immunodeficiency virus type 1 (HIV-1) IIIB env cytotoxic T lymphocytes (CTL) to recognize and lyse HIV-1-infected cells, we have constructed a human cell line (Hu/Dd) expressing both the CD4 receptor and the murine H-2Dd major histocompatibility complex (MHC) class I protein. This cell line can be productively infected with HIV-1 and can also function as a target for murine CD8+, class I MHC-restricted CTL directed against the envelope glycoprotein of HIV-1 IIIB. The ability of BALB/c anti-HIV-1 IIIB env CTL to specifically recognize and lyse Hu/Dd target cells infected with divergent HIV-1 strains was tested by using both prototypic and clinical HIV-1 strains. CTL generated by immunization of mice with syngeneic cells expressing either the native or V3 loop-deleted (delta V3) envelope glycoprotein from HIV-1 IIIB were able to recognize and specifically lyse Hu/Dd target cells infected with the HIV-1 prototypic isolates IIIB, MN, WMJ II, SF2, and CC as well as several HIV-1 clinical isolates. These results demonstrate that CTL determinants for HIV-1 env exist outside the hypervariable V3 region, anti-HIV-1 IIIB env CTL appear to recognize common determinants on diverse HIV-1 strains, and classification of HIV-1 strains based on neutralizing antibody reactivities does not appear to correspond to CTL recognition and lysis. The results suggest that the cell-mediated components of the immune system may have a broader recognition of divergent HIV-1 strains than do the humoral components.

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells.

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.

Induction of anti-HIV-1 immune responses by retroviral vectors.

Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.

Retroviral vectors as vaccines and immunotherapeutics.

Retroviral vectors have been tested in mice for their ability to induce immune response to the proteins which their genomes encode. Using HIV encoded proteins as a model, potent cytotoxic T lymphocyte (CTL) and antibody responses are seen to these proteins. This apparent efficacy of induction may be due to the relative simplicity of the vector and the manner in which antigens are presented to the immune system. Safety issues and other proposed methods of CTL induction are discussed. The potential application of retroviral vectors as immune stimuli in man seems quite broad (viral diseases and cancer), and the first attempted applications in man are likely to be therapeutic rather than prophylactic.

Neuroleptic malignant syndrome associated with prochlorperazine.

Neuroleptic malignant syndrome occurred in a patient with AIDS being treated with prochlorperazine. We believe this to be the first report of this association. Recognition and specific treatment were delayed in part because of overlap in signs and symptoms of the underlying infectious process. The true incidence of prochlorperazine-induced NMS is unknown, and this reaction may be underrecognized in patients who often have other significant medical illnesses.

Neisseria mucosus septicemia after near-drowning.

Neisseria mucosus is a normal inhabitant of the upper respiratory tract which may rarely cause serious infection. A 21-year-old woman had N mucosus bacteremia and the adult respiratory distress syndrome (ARDS) after a near-drowning episode. Despite appropriate antibiotic therapy and intensive respiratory support, the patient died. Bacteremia with this organism is rare, and we believe it has not previously been reported in association with near-drowning.

Participation of natural killer cells in the recovery of mice from visceral leishmaniasis.

After infection with the protozoan parasite Leishmania donovani, C57BL/6J bg/bg (beige) mice, which are deficient in natural killer (NK) activity, were unable to control splenic parasite loads relative to phenotypically normal C57BL/6J bg/+ and +/+ mice, particularly beyond 21 days of infection. When beige mice were injected intravenously with 2 or 3 X 10(6) syngeneic, cloned NK cells (NKB61B10 cell line), they displayed splenic parasite burdens which did not differ significantly from those of normal controls. In C57BL/6 +/+ mice rendered NK deficient by split-dose irradiation (four weekly, 200-rad doses of gamma irradiation beginning at 4 weeks of age) splenic and hepatic parasite levels were significantly higher than those in nonirradiated controls at 15 days of infection and beyond. In both sets of experiments, relative degrees of hepato- and splenomegaly were not sufficient to account for differences in parasite burdens among NK-deficient and normal mice. Taken together, the results of these experiments suggest that NK cells may contribute to parasite elimination during the acquired-resistance phase of L. donovani infection in mice.

Bone marrow graft rejection as a function of antibody-directed natural killer cells.

There is conclusive evidence that acute bone marrow transplant rejection in lethally irradiated mice is caused by natural killer (NK) cells. The rejection of marrow allografts is exquisitely specific and is controlled by antigenic determinants encoded in or near the H-2 gene complex. The specificity of in vivo marrow graft rejection contrasts with the in vitro specificity pattern of NK cells in cytotoxicity assays. We therefore examined how NK cells cause H-2-specific marrow graft rejection in vivo. Several experimental approaches are presented that suggest that natural antibody, present in responder strains of mice, specifically directs NK cells in an antibody-dependent cytolytic and/or cytostatic reaction, resulting in marrow graft rejection. The following evidence for this mechanism is documented. The ability to reject a marrow graft can be passively transferred by serum from responder to allogeneic nonresponder mice and the specificity of rejection can be mapped within the H-2 region. Serum-induced marrow graft rejection is abrogated following depletion of immunoglobulin, and the serum of responder mice is able to induce a specific antibody-dependent cytotoxic reaction in vitro.

Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo.

We carried out adoptive transfer studies to determine the role of natural killer (NK) cells in resistance to murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV). We transferred leukocytes from adult mice into suckling mice 1 d before injecting them with virus. Resistance was measured by enhancement of survival and reduction of virus multiplication in the spleens of recipient mice. The phenotype of the cell population capable of mediating resistance to MCMV was that of a nylon wool-nonadherent, asialo GM1+, NK 1.2+, Ly-5+, Thy-1-, Ia-, low density lymphocyte; this is the phenotype of an NK cell. Cloned NK cells, but not cloned T cells, provided resistance to MCMV in suckling mice. Cloned NK cells also provided resistance to MCMV in irradiated adult mice, and antibody to asialo GM1, which depletes NK cell activity in vivo, enhanced the synthesis of MCMV in athymic nude mice. Neither adult leukocytes nor cloned NK cells influenced LCMV synthesis in suckling mice. We conclude that a general property of NK cells may be to provide natural resistance to virus infections, and that NK cells can protect mice from MCMV but not from LCMV.

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.

T cells may express multiple activities: specific allohelp, cytolysis, and delayed-type hypersensitivity are expressed by a cloned T-cell line.

A T-cell line that recognizes private specificities encoded in the H-2 IA subregion in the proliferative response, cytotoxic activity, delayed-type hypersensitivity, and allohelp reaction with B cells has been cloned. The individual clones were assayed for ability to express the activities of the parent line. Results show that the cloned sublines tested are able to perform all the activities of the parent line. It is suggested that T cells may express multiple activities and that the apparent monofunctionality of T cells is due to regulation of these activities.

Culture supernatants of a stimulated T-cell line have helper activity that acts synergistically with interleukin 2 in the response of B cells to antigen.

Culture supernatants of an antigen-stimulated long-term alloreactive T cell line, C.C3.11.75, contain a T-cell-replacing factor (TRF) activity for the B-cell response to antigen. These same supernatants show little activity in the T-cell growth assay or the costimulator assay. TRF activity was assayed by using spleen cells that were rigorously depleted of both T cells and macrophages. In this assay preparations containing interleukin 2 and supernatants from stimulated C.C3.11.75 cells are relatively inactive if added alone but show marked synergy when added together. We conclude that the C.C3.11.75 TRF activity is not due to interleukin 1 or to interleukin 2 but to a third factor provisionally designated as (DL)TRF. This activity may be equivalent to the (late-acting) TRF described by Schimpl and Wecker. Evidence is presented suggesting that the helper activity (DL)TRF is a product of the T-cell line.

Fine specificity mapping of two allospecific T cell lines: recognition of private specificities in the H-2 IA subregion.

Two allospecific T-cell lines, C.C3.11.75 (H-2d anti-H-2k) and B6.C.7.76 (H-2b anti-H-2d) established from mixed lymphocyte cultures, were selected by continuous antigenic stimulation over several years. Both cell lines proliferated to alloantigen, provided allohelp to B cells in the humoral antibody response, and one of them showed cytolytic activity. Using spleen cells from various congenic mouse strains, the specificity of antigen recognition was examined in order to examine whether there is a correlation between T cell function and antigen recognition. Results showed that in all functional assays. This shows that there is no obligatory correlation between T cell function and antigen recognition and may suggest that the T cell receptor repertoire is biased towards recognizing private specificities.