Biomedical subjects

B T Rouse

Publications and source records attributed to B T Rouse.

At least 127 records · Page 7Linked to original sources

PubMed1992

Liposomes as antigen delivery systems in viral immunity.

Since their first description, liposomes have been put to a wide variety of uses. Encapsulation or incorporation of antigens into liposomes markedly enhances the immunogenicity of the antigen. The type of immune response elicited by the liposome is found to depend on their chemical and structural properties. Immunization with viral glycoproteins encapsulated in liposomes has resulted in enhancement of the humoral response seen as a rise in the serum antibody levels which is several fold higher than that elicited by free antigen alone. Furthermore, liposome encapsulation of peptide antigens which are poor immunogens by themselves not only increases the immunogenicity of the peptide but also play an important role in delivery. The adjuvant effect afforded by liposomes can be further enhanced by the concomitant encapsulation of adjuvants like lipid A or muramyl tripeptide-phosphatidylethanolamine. Formation of liposomes with special characteristics such as pH sensitivity has resulted in the use of liposomes to deliver soluble antigen to the cytosol where it can undergo class I pathway of processing and presentation. Therefore liposomes could provide valuable tools to further understand the pathways of antigen processing and the requirements for induction of cell mediated immunity.

Animals↗

PubMed1991

An improved method of loading pH-sensitive liposomes with soluble proteins for class I restricted antigen presentation.

We have recently shown that ovalbumin (OVA) entrapped in pH-sensitive liposomes could sensitize mouse thymoma cells for lysis by MHC class I-restricted cytotoxic T lymphocytes (CTL) (Reddy et al. (1991) J. Immunol. Methods, 141, 157-163). The present studies were designed to optimize the antigen delivery system. A simple freeze-thaw method was developed to load OVA into pH-sensitive liposomes, and the protocol was optimized in terms of the choice of buffer, pH and ionic strength of the medium, lipid composition, lipid and OVA concentrations and the number of freeze-thaw cycles. Under optimized conditions, approximately 25% of OVA could be entrapped in pH-sensitive liposomes at 172 micrograms protein/mg lipid. This compares to only about 5% entrapment (70 micrograms protein/mg lipid) using the previous method. OVA loaded to pH-sensitive liposomes using the improved method led to a sensitive measure of CTL activity. The approach promises to be suitable to measure CTL against less available soluble antigens such as viral proteins.

Animals↗

PubMed1991

pH sensitive liposomes provide an efficient means of sensitizing target cells to class I restricted CTL recognition of a soluble protein.

Exogenous antigens are normally endocytosed and enter the class II pathway of processing and presentation. It had been shown earlier that soluble antigen could be introduced into the class I pathway of processing and presentation by osmotic loading. In this report, we have demonstrated that OVA containing liposomes that destabilize on exposure to low pH, referred to as pH sensitive liposomes, could sensitize target cells to lysis by class I MHC-restricted OVA-specific CTL. However, OVA-containing pH insensitive liposomes, native OVA, or OVA subjected to the same protocol as was used to make the liposomes, failed to sensitize targets to OVA-specific CTL lysis. The pH sensitive liposomal approach was less toxic and more efficient (about 20-fold) in delivering than the osmotic loading approach. The pH liposome approach may prove valuable to study CTL recognition characteristics of less available proteins such as viral proteins.

Animals↗

PubMed1991

MHC II-restricted, CD4+ cytotoxic T lymphocytes specific for herpes simplex virus-1: implications for the development of herpetic stromal keratitis in mice.

Herpetic stromal keratitis (HSK) appears to represent an immunopathological reaction in which CD4+ T cells play a prominent role. However, the exact immunopathological mechanism(s) utilized by CD4+ T cells during HSK remains to be elucidated. In this study, the presence of cytotoxic CD4+ T lymphocytes in the cervical and retropharyngeal lymph nodes of Balb/c mice experiencing HSK was investigated. After in vitro depletion of CD4+ or CD8+ T cells with specific monoclonal antibodies and complement treatment, the cytotoxic functions of the remaining T cell populations were assayed by using target cells expressing either MHC Class I or both Class I and Class II. Our results showed the presence of a distinct cytotoxic T lymphocyte (CTL) population which was CD4+ and demonstrated lytic activity in a Class II-restricted fashion. Furthermore, these cells were able to develop into efficient effector CTL in the absence of CD8+ T lymphocytes as assessed by in vivo depletion experiments. Immunohistochemical methods were also utilized to show the presence of both CD4+ lymphocytes and I-A+ cells in the corneal tissues during HSK. These findings support the notion that direct lysis of infected Class II-bearing corneal cells by CD4+ CTL might be one of the mechanisms leading to stromal immunopathology in herpetic infections.

Animals↗

PubMed1991

Apparent requirement for CD4+ T cells in primary anti-herpes simplex virus cytotoxic T-lymphocyte induction can be overcome by optimal antigen presentation.

Mice depleted in vivo of either CD4+ or CD8+ T cells were used to define the requirement for interaction between the two T subsets for the induction and maturation of a herpes simplex virus (HSV) cytotoxic T-lymphocyte (CTL) response. Whereas C3H mice generated normal CD8+ CTL in the absence of CD4+ T cells, responses were undetectable in BALB/c mice. However, the role of CD4+ T cells appeared to be to supply helper factors for CTL maturation, as the numbers of CTL precursors in CD4-depleted mice were similar to those in nondepleted animals. Moreover, CTL responses were demonstrable if CD4-depleted primed populations were stimulated with antigen or supplied with a source of helper factors. The optimal means of presenting antigen appeared to be via dendritic cells. Our results indicated that CD8+ cells alone were fully capable of differentiating into CTL provided they were appropriately stimulated with antigen. Possibly, the environment necessary for this to occur in vivo is usually lacking, accounting for the fact that in the mouse, it usually is not possible to demonstrate HSV-specific CTL unless cells are cultured or restimulated in vitro.

Animals↗

PubMed1991

Protection against zosteriform spread of herpes simplex virus by monoclonal antibodies.

The in vivo protective role of herpes simplex virus (HSV-1)-specific antibody was analyzed using monoclonal antibodies (MAbs) reactive with discrete antigenic sites on glycoproteins B, C, and D (gB, gC, gD) in the murine zosteriform spread model of HSV-1. All of the anti-gC and anti-gD MAbs, and one of four anti-gB MAbs (B6) were protective. The in vitro abilities of the MAbs to neutralize HSV-1 and mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-1-infected cells were examined as potential mechanistic correlates to in vivo protection. All animals given MAbs at high ADCC unit doses were protected. Some but not all mice given MAbs at high ADCC unit doses were protected. These studies designate specific epitopes recognized by protective antibodies and indicate that protection from the neurologic spread of HSV may be related to neutralization, ADCC, or both. The actual contribution of ADCC and neutralization to in vivo antibody-mediated protection remains unclear.

Animals↗

PubMed1991

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize immediate-early protein ICP27.

The identity of herpes simplex virus type 1 (HSV-1) antigens that serve as targets for cytotoxic T lymphocytes (CTL) and their ability to induce protective immunity remain uncertain. In this article, we report the identification of the immediate-early protein ICP27 as a CTL antigen in H-2d mice but not in H-2k or H-2b mice. Calculation of the frequencies of H-2d-restricted virus-specific CTL demonstrated that approximately one-fourth of the total HSV-1-specific response was directed against ICP27. To define the location of this CTL epitope, four truncated derivatives of the ICP27 gene which place the epitope in a 217-amino-acid region (amino acids 189 to 406) near the central portion of the protein were constructed. Mice immunized with ICP27 were able both to induce HSV-1-specific CTL and to survive a lethal intraperitoneal challenge with virulent HSV-1. However, neither appreciable antibody nor delayed-type hypersensitivity responses were induced in immunized mice, and they were also unable to clear a local epithelial virus challenge. It appears that ICP27, although capable of inducing several aspects of the immune response, is by itself unable to provide complete immunity.

Animals↗

PubMed1990

Role of coexpression of IL-2 and herpes simplex virus proteins in recombinant vaccinia virus vectors on levels of induced immunity.

Vaccinia virus recombinants co-expressing the genes for IL-2 and HSV-1 gC or gD were used to study the potential adjuvant effects of IL-2 on immunity to HSV-1. In addition, constructs were produced which, while expressing IL-2, encoded for reduced levels of HSV-1 gC. Studies with mice revealed that gC and gD could stimulate HSV-1 immunity as measured by neutralizing antibody (NA), lymphoproliferation and viral clearance. IL-2 enhancement of NA and lymphoproliferation was observed only in those groups of mice immunized with the sub-optimal gC construct which co-expressed IL-2. In no instance did the presence of IL-2 augment the ability of mice to clear an epithelial challenge of HSV-1.

Animals↗

PubMed1990

Murine cytotoxic T lymphocytes specific for herpes simplex virus type 1 recognize the immediate early protein ICP4 but not ICP0.

Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2b haplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0. Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproliferation and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.

Animals↗

PubMed1990

Herpes simplex virus type 1-specific immunity induced by peptides corresponding to an antigenic site of glycoprotein B.

Herpes simplex virus (HSV) envelope glycoproteins are the prime targets of adaptive antiviral immunity. Previous investigation identified a protective, neutralizing, glycoprotein B1 (gB-1)-reactive monoclonal antibody (MAb B6) and localized the linear epitope recognized by the MAb to residue 84 of gB-1. Three overlapping peptides (two 20-mers and one 18-mer), together spanning amino acids 63 to 110 of the wild-type sequence of gB-1, were synthesized and analyzed for their ability to stimulate immunity which cross-reacts with HSV-1. All stimulated some level of response. Two peptides, the gB 18-mer and 20.1-mer, were recognized by MAb B6 and HSV-immune antibody but were unable to stimulate virus-neutralizing antibody or serum able to protect against zosteriform spread in vivo. The 20.2-mer peptide, however, which was not recognized by MAb B6 or HSV-generated immune antibody, stimulated the production of neutralizing antibody and serum able to protect against zosteriform spread. Immunization with all of the peptides was able to enhance viral clearance of a low dose of HSV-1 in an ear challenge model and induce antibody reactive in antibody-dependent complement-mediated lysis of HSV-1-infected cells in vitro. These results are the first report of HSV immunity induced by peptides corresponding to gB and indicate that the best immunogen, in terms of stimulating neutralizing antiserum able to protect in vivo against HSV-1, was a peptide not recognized by HSV-immune mechanisms or by the MAb used to localize it.

Amino Acid Sequence↗

PubMed1990

Influence of peptide acylation, liposome incorporation, and synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines.

A peptide corresponding to residues 1 to 23 of glycoprotein D of herpes simplex virus type 1 was chemically synthesized and coupled to a fatty acid carrier by standard Merrifield synthesis procedures. The resulting peptide-palmitic acid conjugate (acylpeptide) exhibited enhanced immunogenicity in mice as compared with that exhibited by the free form of the peptide. Incorporation of the acylpeptide into liposomes further increased the immunogenicity of the peptide, while inclusion of the immunomodulators muramyl tripeptide phosphatidylethanolamine and monophosphoryl lipid A into the same liposome stimulated the strongest response. The humoral immune responses induced by the acylpeptide-liposome construct were greater than those induced by peptide in Freund complete adjuvant, and cellular responses were equal. The acylpeptide-immunomodulator-liposome formulation also induced significant levels of protective immunity, although the immunity was less than that induced by herpes simplex virus infection. Acylated peptides, especially in liposomes, were taken up more effectively by draining lymph nodes, which possibly accounts in part for the enhanced immunogenicity of the peptides. Since the acylpeptide-immunoliposome formulation used was nontoxic, it could represent a useful way to enhance immunogenicity of subunit peptides used for vaccine purpose in humans and animals.

Acylation↗

PubMed1990

Demonstration of MHC class II-restricted cytotoxic T lymphocytes in mice against herpes simplex virus.

In humans CD4+ major histocompatibility complex (MHC) class II-restricted T cells dominate cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV), whereas in the mouse only CD8+ MHC class I-restricted CTL have been reported. In this study, we demonstrate that a minor fraction (around 30%) of the response in draining lymph nodes of acute local HSV infections is attributable to CD4+ CTL mice. Such CTL were identified on the basis of antiserum inhibition studies, negative depletion approaches, as well as their differing antigen processing requirements to CD8+ MHC class I-restricted CTL. A possible role for CD4+ CTL as immunoregulators in local infections is discussed briefly.

Animals↗

PubMed1989

Neurotensin regulation of macrophage colony-stimulating factor-stimulated in vitro myelopoiesis.

Neurotensin, at less than or equal to 10(-9) M, in the presence of an optimal concentration of macrophage CSF (CSF-1), stimulated a dose-dependent enhancement of colony formation by murine marrow-derived mononuclear phagocyte progenitor cells. The additional colonies arose from the cell cycle and Ia Ag-positive subpopulation previously identified as two-signal-dependent progenitors. Two-signal colony formation diminished when the peptide was added at concentrations greater than 10(-9) M. Neurotensin binds specifically to two distinct receptors, a high affinity receptor (KD approximately 10(-9) M) and a lower affinity (KD approximately 10(-7) M) receptor identified as the tuftsin receptor. Rat liver ferritin and an inhibitory tuftsin analog. (ALA1)-tuftsin, which inhibit two-signal colony formation stimulated by tuftsin and tuftsin-like peptides in combination with CSF-1, did not inhibit colony formation stimulated by CSF-1 and 10(-9) M neurotensin. Both inhibitors, however, reversed the loss of two-signal colony growth in the presence of higher neurotensin concentrations. Neurotensin fragment 1-6, unlike ferritin and (ALA1)-tuftsin, inhibited two-signal colony formation stimulated by 10(-9) M neurotensin. However, like ferritin and (ALA1)-tuftsin, fragment 1-6 permitted full expression of two-signal colony formation in the presence of CSF-1 and 10(-7) M neurotensin. The data indicated that occupancy of both receptors at neurotensin concentrations greater than 10(-9) M might be responsible for the diminished progenitor response. The data further support a potential role for neurotensin as an inflammatory mediator. In addition to direct effects on mature phagocytic leukocytes, neurotensin, at least in vitro can influence the production of new mononuclear phagocytes.

Animals↗

PubMed1989

Consequences of exposure to ionizing radiation for effector T cell function in vivo.

The adoptive transfer of actuely primed and memory virus-immune CD8+ T cells causes enhanced meningitis in both cyclophosphamide (Cy) suppressed, and unsuppressed, recipients infected with lymphocytic choriomeningitis virus (LCMV). The severity of meningitis is assessed by counting cells in cerebrospinal fluid (CSF) obtained from the cisterna magna, which allows measurement of significant inflammatory process ranging from 3 to more than 300 times the background number of cells found in mice injected with virus alone. Exposure of the donor immune population to ionizing radiation prior to transfer has shown that activated T cells from mice primed 7 or 8 days previously with virus may still promote a low level of meningitis in unsuppressed recipients following as much as 800 rads, while this effect is lost totally in Cy-suppressed mice at 600 rads. Memory T cells are more susceptible and show no evidence of in vivo effector function in either recipient population subsequent to 400 rads, a dose level which also greatly reduces the efficacy of acutely-primed T cells. The results are interpreted as indicating that heavily irradiated cells that are already fully functional show evidence of primary localization to the CNS and a limited capacity to cause pathology. Secondary localization, and events that require further proliferation of the T cells in vivo, are greatly inhibited by irradiation.

Animals↗

PubMed1989

Recombinant vaccinia virus expressing the herpes simplex virus type 1 glycoprotein C protects mice against herpes simplex virus challenge.

The gene encoding the herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) was isolated and cloned into a vaccinia virus insertion vector, and the resulting vaccinia-gC vector was used to construct a recombinant vaccinia virus that expressed gC (VVgC5). Infection of cells with VVgC5 resulted in cell surface expression of authentic HSV-1 gC. HSV-1 gC-specific neutralizing antibodies were produced in VVgC5-immunized mice, and lymphocytes exhibited an HSV-1-specific proliferation response in vitro following infection. More importantly, VVgC5-immunized mice were resistant to subsequent lethal HSV-1 challenge.

Animals↗

PubMed1989

Evaluation of antiviral immunity using vaccinia virus recombinants expressing cloned genes for herpes simplex virus type 1 glycoproteins.

Immunization of mice with vaccinia virus recombinants expressing the glycoproteins B or D of herpes simplex virus type 1 (HSV-1) induced humoral antibody as well as multiple aspects of HSV-1-specific T lymphocyte-mediated responses. However, vaccinated mice were not completely resistant to HSV-1 challenge and were unable to eliminate an epithelial infection rapidly. Evidence is presented which indicates that immunization with either vaccinia virus recombinant, while inducing the necessary protective populations of CD4+ T lymphocytes, fails to induce the complementing CD8+ cytotoxic T lymphocytes necessary for high levels of protection against a primary HSV-1 infection. These findings are discussed with relevance to the future development of anti-HSV vaccines.

Animals↗

PubMed1989

Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology.

Herpetic stromal keratitis (SK), a frequent cause of visual impairment, is considered to represent an immune-mediated inflammatory response to persistent herpes simplex virus virions or subcomponents within the corneal stroma. The experimental disease in mice involves the essential participation of T lymphocytes, but the role of T-lymphocyte subsets in either mediating or controlling the disease is uncertain. In this report, rat monoclonal antibodies were used to selectively deplete mice in vivo of CD4+ (helper-inducer) and CD8+ (cytotoxic-suppressor) T-cell populations and the effect on herpetic SK was evaluated. As measured by flow cytometry, mice treated with anti-CD4 monoclonal antibody (GK 1.5) were greater than 95% depleted of CD4+ T lymphocytes and mice treated with anti-CD8 monoclonal antibody (2.43) were 90% depleted of CD8+ T lymphocytes. Depleted and nonspecific mouse ascites-treated control mice were infected topically on the corneas with herpes simplex virus type 1, and the induction of various immune parameters during the acute infection was evaluated. CD4+-depleted mice failed to produce either a significant antiviral antibody or delayed-type hypersensitivity response but were capable of producing normal cytotoxic T-lymphocyte responses. In contrast, CD8+-depleted mice produced antiviral antibody and delayed-type hypersensitivity responses comparable with those in control animals, but cytotoxic T-lymphocyte responses were markedly reduced. Clinical observations of the corneas revealed that SK in CD4+-depleted mice was significantly reduced, whereas in CD8+-depleted mice SK developed more rapidly, was more severe, and involved a greater percentage of mice. These observations implicate the CD4+ T-lymphocyte subset as the principal mediators of SK and CD8+ T lymphocytes as possible regulators that control the severity of SK.

Animals↗

PubMed1988

Substance P augmentation of CSF-1-stimulated in vitro myelopoiesis. A two-signal progenitor restricted, tuftsin-like effect.

The inflammatory neuropeptide substance P acted as a costimulant for macrophage CSF-1-induced clonal proliferation of murine marrow-derived two signal-dependent mononuclear phagocyte progenitors. Substance P had no effect on clonal proliferation by progenitors responding solely to CSF-1. Substance P fragment 2-11 had no costimulatory activity; however, SP fragment 1-4 retained the full activity of the parent undecapeptide. Fragment 1-4 (ARG-PRO-LYS-PRO), a peptide containing a PRO residue between two positive charges, is a tuftsin-like (THR-LYS-PRO-ARG) tetrapeptide, and tuftsin exerted an identical costimulatory effect. Substance P, SP:1-4, and tuftsin were optimally effective as costimulants at 10(-7) to 10(-6) M. (ALA1)-tuftsin, an inhibitory analog of tuftsin, was a potent negative regulator of two signal-dependent colony formation. (ALA1)-tuftsin at concentrations less than or equal to 10(-9) M exerted dose-dependent inhibition of the positive effects of optimal concentrations of all of the co-stimulants tested, including bacterial LPS. The inhibitory tetrapeptide was equivalent in activity to ferritin, an established inhibitor of two signal-dependent colony formation. The results indicated that SP may influence myelopoiesis in addition to its other inflammatory and immunopotentiating properties. In addition, a potentially valuable modulator of SP and LPS responses in this system, (ALA1)-tuftsin, was identified.

Amino Acid Sequence↗