Prime-boost immunization strategies against HIV.
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AIDS Vaccines↗
Biomedical subjects
C M Walker
Publications and source records attributed to C M Walker.
Meniscal regeneration in the long-term after total meniscectomy?
A cohort of 39 patients (28 male, 11 female) that had undergone total meniscectomy as adolescents (mean age 16 years) underwent FISP 3D Magnetic Resonance Imaging at a mean follow up of 30 years. The presence of meniscal tissue was assessed by two independent observers blinded to the operation details. The volume of any meniscal tissue present was calculated. A posterior horn remnant was seen in 57% of medial and 45% of lateral meniscectomy cases. The mean volume of an operated medial meniscal remnant was 0.29 mL compared with a mean volume of 1.15 mL for an intact medial meniscus. The mean volume of an operated lateral meniscal remnant was 0.30 mL compared with 1.07 mL for an intact lateral meniscus. We have shown that the incidence of incomplete excision of the posterior horn is more common after total medial meniscectomy, and that at a mean follow up of 30 years there is no convincing in vivo MRI evidence of long-term meniscal regeneration.
Adolescent↗
Comparative features of hepatitis C virus infection in humans and chimpanzees.
Several features of human HCV infection are recapitulated in the chimpanzee model. Most importantly, the frequency of persistent infection is high in both species, and virus replication occurs despite evidence of cellular and humoral immune responses. A key difference is that necroinflammatory lesions in chronically infected chimpanzees are almost always mild, whereas in humans the disease spectrum is very wide, ranging from mild to severe hepatitis and end-stage cirrhosis requiring transplantation. Understanding the basis for both the similarities and differences in persistent hepatitis C in the two species will probably be important for the development of effective prevention and therapy of HCV infection.
Animals↗
Vaccination with HIV-1 gp120 DNA induces immune responses that are boosted by a recombinant gp120 protein subunit.
Small animals were immunized with plasmid DNA encoding HIV-1 envelope gp120 either intramuscularly by needle injection (mice and guinea pigs) or epidermally with the Accell gene gun (guinea pits). Subsequently, the animals were boosted with a recombinant gp120 protein subunit vaccine in an oil-in-water based adjuvant, MF59. Antibodies and cytotoxic T-lymphocyte (CTL) immune responses to the HIV envelope glycoprotein were observed in animals immunized with gp120 DNA derived from the HIV-1SF2 laboratory strain or from HIV-1 field isolates. Titers of ELISA antibodies and serum neutralizing antibodies against the HIV-1SF2 laboratory isolate were substantially increased in DNA-immunized animals following a single boost with recombinant gp120 protein subunit. This DNA prime/protein subunit boost immunization approach may be important for vaccination against infectious agents such as HIV for which it is difficult to raise strong antiviral humoral responses with DNA vaccination alone.
AIDS Vaccines↗
Virus-specific cytotoxic T-lymphocyte activity elicited by coimmunization with human immunodeficiency virus type 1 genes regulated by the bacteriophage T7 promoter and T7 RNA polymerase protein.
Cytotoxic T lymphocyte (CTL) activity was assessed in mice immunized with DNA plasmids containing the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (pTMIgp120) or p55gag (pTMIgag) gene regulated by the bacteriophage T7 promoter. Immunization with either plasmid resulted in CTL activity against class I major histocompatibility complex-restricted viral epitopes when coadministered with a recombinant vaccinia virus expressing the T7 RNA polymerase protein (T7 RNAP) but not a control vaccinia virus. Recombinant vaccinia-T7 RNAP virus (VTF7-3) could be replaced with a noninfectious source of T7 RNAP. A three-component vaccine consisting of pTMIgag, a recombinant subunit T7 RNAP protein, and a plasmid (pT7T7) encoding T7 RNAP under the control of its own promoter induced gag-specific CTL activity. Intramuscular immunization with the pTMIgag plasmid delivered with either the T7 RNAP protein or pT7T7 plasmid alone also induced HIV-1-specific CTL. Thus, there is adventitious expression of the pT7T7 plasmid in vivo, and enough T7 RNAP is produced to result in production of p24gag protein from the pTMIgag plasmid. The results demonstrate that regulated expression of genes in vivo is possible with this T7-based expression system, and may be useful in vaccine settings where short-term cytoplasmic expression of protein in antigen presenting cells is desired.
AIDS Vaccines↗
Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D.
Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.
Animals↗
Effects of long-acting and short-acting beta-agonists on methacholine dose-response curves in asthmatics.
Regular use of short-acting beta-agonists may decrease control of asthma and increase airway responsiveness to bronchoconstrictor stimuli. The aim of this study was to determine the effects of regular treatment with the long-acting beta-agonist, salmeterol, on the methacholine dose-response curve (DRC) in mild-to-moderate asthmatics. Changes in methacholine airway responsiveness were measured in 14 stable adult asthmatics, randomized in a double-blind, three-way cross-over design to receive salmeterol 50 micrograms, salbutamol 200 micrograms or placebo, each twice daily for 4 days. Two baseline methacholine DRC, were performed, one without premedication and one following a single dose of 200 micrograms salbutamol. Following 4 days of regular treatment, methacholine DRC to plateau were carried out commencing 15 min after the final dose of trial medication. There were no significant differences in mean baseline forced expiratory volume in one second (FEV1) between treatments. Four days treatment with salmeterol and salbutamol shifted the DRC to the right, but salmeterol provided less protection than salbutamol. The point of inflection of the curve from baseline moved 1.9 and 3.2 doubling doses, respectively, compared to placebo (p < or = 0.001), and the provocative concentration of methacholine required to produce a 20% fall in FEV1 (PC20) increased 1.6 and 3.1 doubling doses, respectively (p < or = 0.001). The slope of the DRC was increased slightly by both beta-agonists compared to placebo (log slope 3.11, 3.06 and 2.77 for salmeterol, salbutamol and placebo, respectively). This effect of regular salmeterol on slope was more marked in subjects with lower baseline FEV1. Maximal response plateaus did not differ between the three treatments. These results suggest that regular use either of short- or long-acting beta-agonists could increase the risk of a more precipitous asthma episode associated with "breakthrough" bronchoconstrictor responses, particularly in those with more severe initial airflow obstruction, if subjects are exposed to a sufficiently potent stimulus.
Adrenergic beta-Agonists↗
Role of the flight surgeon in fighter squadron Arctic deployments.
Operational fighter squadrons frequently find themselves deployed to semi-isolated stations in the Arctic. This paper discusses the major issues necessary for flight surgeon consideration. In particular, the areas of pre-deployment planning, preparing for the worst, routine operations, and post deployment actions are discussed. A recent month-long deployment of a 12-ship squadron of F18s with support elements from Bagotvi le, Canada, to Evenes, Norway, is examined. A proposed kit list to support a similar deployment to a semi-isolated station is provided.
Aerospace Medicine↗
Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.
Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.
Animals↗
Patr-A and B, the orthologues of HLA-A and B, present hepatitis C virus epitopes to CD8+ cytotoxic T cells from two chronically infected chimpanzees.
Common chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) show a disease progression similar to that observed for human patients. Although most infected animals develop a chronic hepatitis, virus persistence is associated with an ongoing immune response, for which the beneficial or detrimental effects are uncertain. Lines of virus-specific cytotoxic CD8+ T lymphocytes (CTL) have been previously established from liver biopsies of two common chimpanzees chronically infected with HCV-1. The viral epitopes recognized by six lines of CTL have been defined using synthetic peptides and shown to consist of 8 to 9-residue peptides derived from various viral proteins. Five of the epitopes derive from sequences that vary among strains of HCV. The majority of the corresponding variant epitopes from different HCV strains were either recognized less efficiently or not at all by the CTL, suggesting their response may have limited potential for controlling replication of HCV variants. Complementary DNAs encoding class I alleles of the two common chimpanzees, Patr-A, -B, and -C were cloned, sequenced, and transfected individually into a class I-deficient human cell line. Analysis of peptide presentation by the class I transfectants to CTL identified the Patr class I allotypes that present the six epitopes defined here and an additional epitope defined previously. The assignment of epitopes to class I allotypes based upon analysis of the transfected cells correlates precisely with the segregation of antigen-presenting function within a panel of common chimpanzee cell lines and the expression of class I heavy chains as defined by isoelectric focusing. Five of the HCV-1 epitopes are presented by Patr-B allotypes, two epitopes are presented by a Patr-A allotype, and none is presented by Patr-C allotypes.
Amino Acid Sequence↗
The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species.
A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen-presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species.
Animals↗
HIV-1 p24 Gag-specific cytotoxic T-lymphocyte responses in mice.
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Amino Acid Sequence↗
RANTES, MIP-1 alpha and MIP-1 beta are not involved in the inhibition of HIV-1SF33 replication mediated by CD8+ T-cell clones.
OBJECTIVE: To determine whether CD8+ cells inhibit HIV replication in vitro through the chemokines RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta. DESIGN AND METHODS: CD8+ T-cell clones were screened for their ability to inhibit HIV-1SF33 replication in CD4+ cells using p24 antigen and HIV RNA levels as endpoints. It has been suggested that such inhibition is mediated by three type cc chemokines: RANTES, MIP-1 alpha and MIP-1 beta. To assess whether our T-cell clones inhibited HIV replication through a similar mechanism, the clones' ability to inhibit HIV-1SF33 replication was compared with their secretion of RANTES, MIP-1 alpha and MIP-1 beta. Moreover, we tested the effects of neutralizing antibodies (NAb) against these factors on the anti HIV-1SF33 activity of our clones as well as the direct effect of these recombinant cc-chemokines on HIV-1SF33 replication. RESULTS: The CD8+ T-cell clone; tested differed by their capacity to inhibit HIV-1 replication. We showed no correlation between the ability of these clones to secrete RANTES, MIP-1 alpha and MIP-1 beta and their ability to repress HIV-1SF33 replication. In addition, this inhibitory activity against HIV-1SF33 could not be blocked by NAb directed against these chemokines, nor could these chemokines significantly inhibit HIV-1SF33 replication in acutely infected CD4+ cells in vitro. CONCLUSION: The data indicate that CD8+ cells can inhibit HIV-1SF33 replication in vitro by mechanisms that do not involve either cytotoxicity or RANTES, MIP-1 alpha and MIP-1 beta.
Antibodies↗
Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant.
Hepatitis C virus (HCV) establishes a persistent infection in humans and chimpanzees despite the presence of virus-specific, class I major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes (CTLs) in the liver. The data presented here demonstrate that CTLs directed against a conserved epitope in the HCV nonstructural 3 protein persist in the liver of a chronically infected chimpanzee for at least 2 years after infection. However, these CTLs did not recognize the HCV quasi-species present in the plasma of this animal at week 16 postinfection or at later time points. Escape from the CTL response was facilitated by an aspartic acid to glutamic acid (D-->E) substitution at amino acid position 1449 in all HCV genomes that were sequenced. The results of this study strongly support the concept that CTL responses can select for variant viruses with an enhanced ability to persist in a host and have important implications for the design of vaccines against HCV.
Amino Acid Sequence↗
Hepatitis C virus-specific T lymphocyte responses.
Hepatitis C virus establishes a persistent infection in humans and chimpanzees despite virus-specific cell-mediated immune responses. Available data suggest that these responses do provide some control of ongoing hepatitis C virus replication and are an important factor contributing to chronic hepatitis.
Animals↗
Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus (HCV) infection.
Mechanisms by which HCV evades the cellular immune response in persistently infected humans and chimpanzees are poorly defined, but could involve mutations in epitopes recognized by class I MHC restricted CTLs. To investigate this possibility, we identified an epitope in the NS3 protein of HCV that was recognized by intrahepatic CTLs from a chimpanzee that developed persistent HCV infection after experimental challenge with the virus. Fine mapping studies with truncated synthetic peptides revealed that the epitope was 9 amino acids in length, encompassing residues 1445 to 1454 (GDFDSVIDC) of NS3. This sequence was completely conserved in all full-length NS3 genomes described to date. In view of the fact that the major genotypes of HCV may differ by up to -30% in overall amino acid homology, it appears in contrast that this epitope is highly conserved. The role of CTL escape mutations in HCV persistence was assessed in the virus inoculum used to infect this chimpanzee and in post-inoculation plasma samples. Sequencing of 6-10 M13 clones containing a 232-nucleotide fragment amplified with NS3-specific primers revealed that the epitope in the challenge inoculum and a post-inoculum plasma sample obtained at week 4 were identical to the published sequence of HCV-I. In contrast, all molecular clones sequenced from week 16, 25 and 28 plasma samples contained a single Asp--> Glu (D-->E) amino acid substitution at residue 1449. Significantly, four independently derived CTL clones established from the liver of this chimpanzee at various times up to two years after infection recognized target cells pulsed with a nonameric peptide representing the wild-type HCV-I sequence, but not those pulsed with a peptide containing the D-->E mutation. These data suggest that CTL escape mutations may play a role in viral persistence.
Amino Acid Sequence↗
Induction of HIV-1 envelope (gp120)-specific cytotoxic T lymphocyte responses in mice by recombinant CHO cell-derived gp120 is enhanced by enzymatic removal of N-linked glycans.
Priming of CD8+ cytotoxic T lymphocyte (CTL) responses with recombinant proteins has been facilitated by the development of novel adjuvants that deliver antigens into the class I major histocompatibility complex (MHC) pathway. However, the extent to which secondary structure or glycosylation of these proteins prevents priming of class I MHC-restricted CTL responses is not clear. To address this issue, recombinant HIV-1 gp120 envelope proteins produced in yeast insect, or mammalian cells were compared for the ability to elicit CD8+ CTL activity in mice. Envelope-specific CD8+ T lymphocytes were detected in BALB/c mice immunized with env 2-3, a 55-kDa yeast-derived envelope protein that is not glycosylated and lacks a native conformation. This response was directed against a previously described epitope in the V3 region of gp120, as well as a newly identified epitope located near the carboxy-terminus of the molecule. Similar levels of V3-directed CTL activity were observed in mice immunized with recombinant gp120 produced in insect (Spodoptera fugiperda) cells using a baculovirus expression system (gp120BAC). In contrast, induction of CTL responses was considerably less efficient when mice were immunized with gp120CHO, a native, fully glycosylated envelope protein produced in mammalian CHO cells. Denaturation of gp120CHO prior to immunization was not sufficient to prime CTL responses. However, envelope-specific CD8+ CTL activity was elicited when N-linked glycans were removed by treatment with an endoglycosidase. Possible mechanisms by which N-linked glycans influence delivery or processing of recombinant proteins for class I MHC presentation, and the implications of these findings for the design of subunit vaccines, are discussed.
Amino Acid Sequence↗
Suppression of HIV replication by CD8+ cell clones derived from HIV-infected and uninfected individuals.
CD8+ cell clones have been derived from peripheral blood mononuclear cells of human immunodeficiency virus (HIV)-infected and uninfected individuals. Several of these cloned cells have the ability to suppress HIV replication when cocultured with CD4+ cells acutely infected in the laboratory with HIV or with infected CD4+ cells from infected subjects. Suppression of virus production occurs without killing the target cells. With the CD8+ cell clones studied, this antiviral response correlated with production of a filterable factor that has antiviral activity. These cell clones offer the opportunity for identification of the factor mediating suppression of HIV replication. Moreover, adoptive transfer of cell clones might provide a valuable therapeutic approach for HIV-infected individuals.
CD4-Positive T-Lymphocytes↗