Biomedical subjects

Paul Goepfert

Publications and source records attributed to Paul Goepfert.

7 recordsLinked to original sources

PubMed2006

Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry.

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

CD3 Complex↗

PubMed2006

Interleukin-2 reconstitutes defective human immunodeficiency virus (HIV), and cytomegalovirus (CMV) specific CD8+ T cell proliferation in HIV infection.

Recent studies indicate that a defective proliferative response of HIV-specific CD8+ T cells is associated with the lack of virologic control in chronic HIV infection in humans. The possible mechanisms that might be responsible for the reduced proliferative potential of HIV-specific CD8+ T cells and conditions conducive to the proliferation of CD8+ T cells were examined in 14 HIV-infected individuals and 7 HIV-uninfected controls using CFSE labeling and flow cytometry techniques, and analyzed data using 2 quantitative measurements: the percentages of proliferating CD8+ T cells (Tp), and the maximum number of cell divisions (Dm) after stimulation. It was found that CD8+ T cells from HIV-infected and -uninfected subjects proliferated equally well after polyclonal stimulation by phylohemagglutinin A (PHA); both groups reached a Tp of 92%-96% and a Dm of 5-8. However, in HIV-infected subjects, proliferation of HIV- and CMV-specific CD8+ T cells was significantly reduced compared to proliferation of CMV- specific CD8+ T cells from HIV-uninfected subjects. These defective proliferative responses of HIV- and CMV-specific CD8+ T cells were restored by the addition of IL-2 at the time of stimulation. These results may have implications for the design of immune modulation strategies in vivo.

Amino Acid Sequence↗

PubMed2005

Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection.

Worldwide HIV-1 vaccine efforts are guided by the principle that HIV-specific T cell responses may provide protection from infection or delay overt disease. However, no clear correlates of T cell-mediated immune protection have been identified. Here, we examine in a HLA-B27(+) HIV seronegative vaccinee persistent HIV-specific vaccine-induced anti-Gag CD4(+) and CD8(+) T cell responses. Although these responses exhibited those characteristics (multifunctionality, appropriate memory phenotype, and targeting of epitopes associated with long-term nonprogression) predicted to correlate with protection from infection, the subject became HIV infected. After HIV infection, the vaccine-induced CD8(+) T cells expanded, but both CD4(+) and CD8(+) T cell responses acquired the functional and phenotypic patterns characteristic of chronic HIV infection. The virus quickly escaped the vaccine-induced T cell response, and the subject progressed more rapidly than expected for someone expressing the HLA-B27 allele. These data suggest that control of HIV by vaccine-elicited HIV-specific T cell responses may be difficult, even when the T cell response has those characteristics predicted to provide optimal protection.

AIDS Vaccines↗

PubMed2005

Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies.

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

AIDS Vaccines↗

PubMed2004

Absence of immunodominant anti-Gag p17 (SL9) responses among Gag CTL-positive, HIV-uninfected vaccine recipients expressing the HLA-A*0201 allele.

According to a number of previous reports, control of HIV replication in humans appears to be linked to the presence of anti-HIV-1 Gag-specific CD8 responses. During the chronic phase of HIV-1 infection, up to 75% of the HIV-infected individuals who express the histocompatibility leukocyte Ag (HLA)-A*0201 recognize the Gag p17 SLYNTVATL (aa residues 77-85) epitope (SL9). However, the role of the anti-SL9 CD8 CTL in controlling HIV-1 infection remains controversial. In this study we determined whether the pattern of SL9 immunodominance in uninfected, HLA-A*0201 HIV vaccine recipients is similar to that seen in chronically HIV-infected subjects. The presence of anti-SL9 responses was determined using a panel of highly sensitive cellular immunoassays, including peptide:MHC tetramer binding, IFN-gamma ELISPOT, and cytokine flow cytometry. Thirteen HLA-A*0201 vaccinees with documented anti-Gag CD8 CTL reactivities were tested, and none had a detectable anti-SL9 response. These findings strongly suggest that the pattern of SL9 epitope immunodominance previously reported among chronically infected, HLA-A*0201-positive patients is not recapitulated in noninfected recipients of Gag-containing canarypox-based candidate vaccines and may be influenced by the relative immunogenicity of these constructs.

AIDS Vaccines↗

PubMed2002

Detection of mucosal antibodies in HIV type 1-infected individuals.

HIV-1-specific mucosal IgA antibodies may correlate with protection in highly exposed but uninfected individuals, but have been detected at highly variable levels in HIV-1-infected individuals. To determine the best assays for detection of IgA antibodies in mucosal samples, rectal washes from 16 HIV-1-infected and 14 uninfected individuals were distributed to six laboratories experienced in detection of mucosal antibodies. Assays for HIV-1-specific IgA and IgG were performed in a blinded fashion by each of the laboratories using modifications of ELISA and chemiluminescence-enhanced Western blotting. Rectal washes contained easily detectable total IgA levels that did not differ between HIV-1-infected and uninfected groups. Irrespective of the assay used, HIV-1-specific IgA antibodies were absent in most samples; only one laboratory identified a higher frequency of positive samples from HIV-1-infected than uninfected individuals. In spite of 10-fold lower levels of total IgG than IgA, all but one laboratory identified HIV-1-specific IgG in most rectal washes of HIV-1-infected individuals. Comparable and readily detectable levels of influenza-specific IgA antibodies were present in nasal, salivary, and rectal secretions from both HIV-1-infected and uninfected individuals. These observations suggest a selective alteration in the production of HIV-1-specific IgA antibodies in HIV-1-infected individuals.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS Vaccine Evaluation Group Protocol 022A.

To test the safety and immunogenicity of a high-titered preparation of ALVAC-HIV vCP205 in both high-risk and low-risk persons and to evaluate variations in dosing schedule, we conducted a multicenter, randomized, double-blind trial of this vector in combination with recombinant subunit gp120 in 150 HIV-1-seronegative volunteers. The high-titered ALVAC vaccine was well tolerated; adverse events were minimal and not influenced by dosing. At day 728, the cumulative probability of a cytotoxic T-lymphocyte (CTL) response was 76% (95% confidence interval [CI]: 64%-89%) among volunteers receiving vaccine, and the net amount attributable to vaccination was 50% (CI: 16%; 74%). The net probability of a repeated positive CTL response by day 728 was 50% (CI: 21%; 64%). There was a significant difference in CTL response at day 182 between volunteers who had received four doses versus three doses of vCP205 (42% vs. 24%, p =.052). The CTL response was similar in high-risk volunteers and vaccinia-naive volunteers compared with vaccinia-immune volunteers. Neutralizing antibody responses were detected in 95% of vaccinees at day 287, with higher geometric mean titers in recipients of sequential versus simultaneous dosing of the two vaccines and in vaccinia-naive volunteers. This high-titered preparation of ALVAC-HIV vCP205 in combination with gp120 was safe and immunogenic in a diverse group of HIV-1-seronegative volunteers.

AIDS Vaccines↗