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Biomedical subjects

R R Redfield

Publications and source records attributed to R R Redfield.

At least 19 recordsLinked to original sources
PubMed1992

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.

Amino Acid Sequence↗
PubMed1992

Fine specificity of the humoral immune response to HIV-1 GP160 in HIV-1 infected individuals from Tanzania.

A total of 160 sera from HIV-1 infected individuals from Tanzania were examined for their fine specificity characteristics relative to 9 synthetic peptides that define HIV-1 gp160 epitopes. Immunorecessive and immunodominant epitopes were identified in both gp120 and gp41 based on serologic reactivity of these HIV-1 infected sera. A significant difference in fine specificity among HIV-1 infected individuals from Tanzania and the United States was observed for an immunodominant gp41 epitope. No significant differences in reactivity among asymptomatic vs. symptomatic HIV-1 infected individuals were detected for the selected HIV-1 gp160 epitopes defined by these peptides. The majority of sera from HIV-1 infected Tanzanians contained antibodies that recognized a peptide corresponding to the V3 region of gp120 from the HIV-1 MN isolate. These data suggest that regional isolates of HIV-1 may exist in Tanzania that differ from HIV-1 isolated in the United States. However, based on serology, HIV-1 isolates exhibiting sequences with HIV-1 MN V3 similarity may also be prevalent in Tanzania. The results of this study may be useful for the design of more effective AIDS diagnostic and therapeutic products for use worldwide.

Amino Acid Sequence↗
PubMed1992

Comparison of antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp160 epitopes in sera from HIV-1-infected individuals from Tanzania and from the United States.

In this study, we compared sera from 159 human immunodeficiency virus type 1 (HIV-1)-infected individuals from Tanzania and 103 infected individuals from the United States for antibodies reactive with 10 HIV-1 gp160 epitopes defined by synthetic peptides. Our data indicate that the anti-gp160 antibody fine specificity differs between infected individuals from these two geographically diverse populations. For example, 50% of the Tanzanian sera contained antibodies reactive with an immunodominant HIV-1 gp41 epitope defined by peptide 600-611, whereas 91% of the sera from the United States were reactive. Differences in serologic reactivity between HIV-1-infected individuals from Tanzania and the United States were also observed with gp160 epitopes defined by peptides 503-528 and 846-860. Included among the peptides examined were four which corresponded to the V3 region of gp120. The majority of sera from either country contained antibodies reactive with peptide RP142, whose V3 sequence is based upon that of HIV-1 isolate MN. Further characterization of serologic reactivity suggested that sera from Tanzania were more likely to neutralize HIV-1 isolate IIIB or MN in vitro than were sera from the United States. These differences in antibody fine specificity between HIV-1-infected individuals from Tanzania and the United States suggest that regional isolates of HIV-1 may exist.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease.

Studies of cultivatable human immunodeficiency virus type 1 (HIV-1) from plasma samples from infected patients have shown a correspondence between increasing viral burden and disease progression, but these measurements are selective and thus nonrepresentative of the in vivo viral load. Quantitation of proviral DNA sequences by the polymerase chain reaction in purified CD4+ T cells has shown a similar relationship but does not provide a measure of viral gene expression. We have studied viral DNA, genomic RNA, and spliced mRNA expression of HIV-1 in infected patients with a quantitative polymerase chain reaction assay. Viral RNA expression is detected in all stages of infection. These data show that the natural history of HIV infection is associated with a shift in the balance of viral expression favoring the production of genomic RNA without a preceding period of true viral latency.

Base Sequence↗
PubMed1992

Examination of sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals for antibodies reactive with peptides corresponding to the principal neutralizing determinant of HIV-1 gp120 and for in vitro neutralizing activity.

Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HIV-1 gp120. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines.

Amino Acid Sequence↗
PubMed1992

HIV neutralization assay using polymerase chain reaction-derived molecular signals.

Characterization of the capacity of human polyclonal antibody to neutralize wild-type patient isolates has important implications for vaccine development. We report the development of a polymerase chain reaction-based neutralization assay that quantitatively measures each infection using HIV proviral formation. These molecular end points identified the absence or quantitative diminution of DNA provirus formation as well as a delay in the kinetics of HIV DNA provirus formation. Using both laboratory strain prototype isolates (HIV-1-MN, HIV-IIIb) and primary wild-type patients' isolates, neutralization end points were reproducibly determined. End points were reached within 72 h, thereby minimizing the impact of subsequent rounds of infection on interpretation of results. Although the neutralization titer of polyclonal sera was usually comparable using standard technology, this assay did find isolate-dependent variation in the relationship between p24 production and HIV proviral DNA formation. Finally, we noted the disparity between the ability of human sera to neutralize prototype and wild-type isolates in primary peripheral blood mononuclear cell targets. We believe this assay provides unique opportunities to characterize the initial events of virus-antibody interaction and will help to elucidate clinically relevant neutralization immunoregulatory mechanisms.

Base Sequence↗
PubMed1992

Predictors of HIV-1 disease progression in early- and late-stage patients: the U.S. Army Natural History Cohort. Military Medical Consortium for Applied Retrovirology.

HIV-infected individuals in both early and late stages of HIV disease were evaluated over 2 years to assess temporal trends and determinants of disease progression. The Walter Reed (WR) staging system was used to categorize patients into an early-stage cohort (WR Stages 1 and 2, N = 1183) and a late-stage cohort (WR Stage 5, N = 260) based on the initial clinical evaluation. Progression was defined as the occurrence of Stage 5 disease or beyond for the early cohort and Stage 6 disease or beyond for the late cohort. The cumulative incidence of progression was 15.7% (137 events) for the early-stage cohort, and 53.7% (85 events) for the late-stage cohort. Baseline CD4+ T lymphocyte (T4) count was the most significant marker of progression: 26% of WR Stage 1 or 2 patients with T4 lymphocytes below 500/mm3 progressed, compared with 12% with T4 lymphocytes at or above 500/mm3. In late-stage individuals, 83% with T4 lymphocytes under 200/mm3 progressed, compared with 27% with T4 lymphocytes at or above 200/mm3. Older age was associated with progression in both early- and late-stage groups. Differences in the rates of disease progression were not significant between blacks and whites or between men and women. Two-year rates of progression among the late-stage patients dropped from 78 to 47% between 1986 and 1988. This contrasted with progression rates in the early-stage cohort, which remained stable: 18% for those entering follow-up in 1986 and 17% for those entering follow-up in 1988. These data indicate a significant slowing of HIV disease progression rates and mortality rates among individuals with late-stage disease that is temporally associated with the increased availability and use of therapies. With control of T4 lymphocyte count, age, and calendar time, neither gender nor race was significantly associated with progression in either early- or late-stage patients.

Adolescent↗
PubMed1991

Anoxia induces human immunodeficiency virus expression in infected T cell lines.

The effects of oxygen deprivation, or anoxia, on human immunodeficiency virus (HIV-1) expression in chronically (ACH.2) and acutely (H9/HIV-1-IIIB) infected cell lines was investigated. Temporary cellular anoxia has previously been shown to activate transcription of endogenous type C leukemia virus sequences, resulting in a significant increase in retroviral RNA within the cell (1). Here we report a 15-fold increase in HIV-1-specific RNA in unstimulated ACH.2 T cells within 24 h of anoxia. This induction of RNA is accompanied by an accumulation of intracellular p24 gag protein as well as an increase in envelope protein. Anoxia induces a further increase in total HIV-1 RNA in ACH.2 cells prestimulated to produce virus by phorbol 12-myristate 13-acetate and in H9 T cells acutely infected with HIV-1-IIIB. The induction of RNA in ACH.2 cells appears to be reversible. Anoxic culture for 24 h followed by a 24-h re-oxygenation period results in a return to "resting state" levels of HIV-1 RNA. These data indicate that oxygen tension within the cellular environment modulates HIV-1 expression, providing a model system in which to study the reversible regulation of HIV-1 RNA and viral gene products within the cell.

Blotting, Northern↗
PubMed1991

A phase I evaluation of the safety and immunogenicity of vaccination with recombinant gp160 in patients with early human immunodeficiency virus infection. Military Medical Consortium for Applied Retroviral Research.

BACKGROUND: Despite multiple antiviral humoral and cellular immune responses, infection with the human immunodeficiency virus (HIV) results in a progressively debilitating disease. We hypothesized that a more effective immune response could be generated by post-infection vaccination with HIV-specific antigens. METHODS: We performed a phase I trial of the safety and immunogenicity of a vaccine prepared from molecularly cloned envelope protein, gp160, in 30 volunteer subjects with HIV infection in Walter Reed stage 1 or 2. The vaccine was administered either on days 0, 30, and 120 or on days 0, 30, 60, 120, 150, and 180. HIV-specific humoral and cellular immune responses were measured; local and systemic reactions to vaccination, including general measures of immune function, were monitored. RESULTS: In 19 of the 30 subjects both humoral and cellular immunity to HIV envelope proteins increased in response to vaccination with gp160. Seroconversion to selected envelope epitopes was observed, as were new T-cell proliferative responses to gp160. Response was associated with the CD4 cell count determined before vaccination (13 of 16 subjects [81 percent] with greater than 600 cells per milliliter responded, as compared with 6 of 14 [43 percent] with less than or equal to 600 cells per milliliter; P = 0.07) and with the number of injections administered (87 percent of subjects randomly assigned to receive six injections responded, as compared with 40 percent of those assigned to three injections; P = 0.02). Local reactions at the site of injection were mild. There were no adverse systemic reactions, including diminution of general in vitro or in vivo cellular immune function. After 10 months of follow-up, the mean CD4 count had not decreased in the 19 subjects who responded, but it had decreased by 7.3 percent in the 11 who did not respond. CONCLUSIONS: This gp160 vaccine is safe and immunogenic in volunteer patients with early HIV infection. Although it is too early to know whether this approach will be clinically useful, further scientific and therapeutic evaluation of HIV-specific vaccine therapy is warranted. Similar vaccines may prove to be effective for other chronic infections.

Adolescent↗
PubMed1991

Trends of HIV seroconversion among young adults in the US Army, 1985 to 1989. US Army Retrovirus Research Group.

Because soldiers in the US Army are recurrently tested for the presence of antibody to the human immunodeficiency virus (HIV), HIV seroconversion rates can be directly measured. From November 1985 through October 1989, 429 HIV seroconversions were detected among 718,780 soldiers who contributed 1,088,447 person-years of follow-up time (HIV seroconversion rate, 0.39 per 1000 person-years). Period-specific seroconversion rates declined significantly from 0.49 per 1000 person-years (November 1985 through October 1987) to 0.33 per 1000 person-years (November 1987 through October 1988) to 0.29 per 1000 person-years (November 1988 through October 1989). The HIV seroconversion risk among active-duty soldiers was significantly associated with race/ethnic group, age, gender, and marital status. Based on these trends, we estimate that approximately 220 soldiers (95% confidence interval, 160 to 297 soldiers) were infected with HIV during 1989 and 1990, with potentially fewer in future years.

Adolescent↗
PubMed1991

Clinical performance of a polymerase chain reaction testing algorithm for diagnosis of HIV-1 infection in peripheral blood mononuclear cells.

The clinical performance of a modified polymerase chain reaction (PCR) testing algorithm was evaluated for confirming the presence of HIV-1 proviral DNA in peripheral blood mononuclear cells. A whole cell lysate, rather than phenol-purified DNA, was used for PCR amplification, under systematically optimized conditions designed and verified within each PCR run to detect as few as 10 copies of proviral DNA. A sequential testing algorithm was designed requiring reactivity in duplicate (with corresponding non-reactivity in negative controls) with at least two sets of primers, before reporting a specimen as HIV-1-positive. In 196 specimens from patients staged according to the Walter Reed staging system, the PCR test sensitivity and the coculture isolation rate (in parentheses) were found to be: 97% (71%), 100% (85%), and 100% (76%) in stage 1, stage 2, and stage 3 specimens, respectively; and 100% (100%) in stage 4, 5, and 6 specimens. Results were uniformly negative for PCR and coculture isolation from 21 blind negative specimens and 105 (negative) donor leukopacks. These data indicate that this PCR testing algorithm is more accurate than tissue culture isolation methods, especially with early stage patients, and results in detection of HIV-1 in virtually 100% of seropositive individuals, with no false positives.

Acquired Immunodeficiency Syndrome↗
PubMed1991

HIV vaccine therapy.

The immunoregulatory mechanisms responsible for effective the in vivo post-infection control of HIV are unknown. In the setting of natural HIV infection, host-directed immune responses directed against gp120 are minimal. This includes poor gp120 epitope specific antibody responses to C1, C2 and C3; lack of T-cell recognition and proliferation to gp120; and minimal envelope cytotoxic T-cell responses. The historical important of anti-envelope responses in the control of other viral pathogens, coupled with the paucity of anti-envelope responses elicited as a consequence of HIV infection, formed the basis for our rationale to pursued a research program which focused on post-infection vaccination utilizing HIV enveloped derived products. Recently we have demonstrated the scientific feasibility of post-infection vaccination with an HIV envelope based vaccine to broaden host-directed HIV-specific immune responses to include seroconversion to gp120 C1, C2 and C3; the induction of T-cell recognition to gp160; and cytotoxic T-cell responses to envelope peptides. The in vivo safety and immunogenicity of the product in adult patients with early HIV infection has been demonstrated. An expanded phase II trial with rgp160 (MGS) is ongoing to examine the potential role of HIV-specific vaccine therapy to alter the natural history of HIV infection. Finally, we are exploring the value of post-infection immunization in defining specific immune responses and their in vivo HIV immunoregulatory relevance.

AIDS Vaccines↗
PubMed1991

Induction of human immunodeficiency virus type 1 expression in chronically infected cells is associated primarily with a shift in RNA splicing patterns.

We have analyzed the kinetics of human immunodeficiency virus type 1 (HIV-1) RNA induction in chronically infected T cells and promonocytes. A substantial amount of spliced mRNAs and assembled virions was found in resting cells. Induction increased the steady-state level of total HIV-1 RNA by 4-fold but increased the level of unspliced transcripts by 25-fold. This increase in unspliced RNA was reflected in the amount of virus seen by electron microscopy. These data suggest a mechanism for the induction of HIV-1 RNA in chronically infected cells involving a shift in splicing greatly favoring the stability of unspliced viral RNA with only a modest increase in total viral RNA. Analysis of the relative abundance of transcript classes is critical to the measurement of HIV-1 viral replication kinetics.

Base Sequence↗
PubMed1991

Safety and immunogenicity of multiple conventional immunizations administered during early HIV infection.

Twenty-one asymptomatic adults who had recently received multiple polysaccharide, live viral, and protein-derived vaccines were identified as being infected with human immunodeficiency virus (HIV). The mean subject age was 24 years (range 18-33); 20 of 21 (95%) were male. The mean T4 count was 523/mm3 with a mean T4/T8 ratio of 0.6. Serologic responses to immunization with meningococcus group C, adenovirus types 4 and 7, tetanus, and diphtheria were evaluated for the HIV seropositive subjects and were compared with the responses of similarly vaccinated age-, sex-, and race-matched HIV-seronegative controls. Significantly fewer (p less than 0.03) HIV subjects responded to meningococcus C (bactericidal antibody) and adenovirus 4 (neutralizing antibody) vaccines than did normals; the HIV-infected subjects who did respond produced functional antibody comparable to that of normals. Booster responses of HIV subjects to tetanus and diphtheria were comparable to those of normals. HIV-infected vaccine nonresponders did not differ from HIV-infected responders in total white blood cell, T4, T4/T8, total serum IgG, or delayed-type hypersensitivity skin test reactivity. All HIV subjects had negative cultures for live vaccine viruses (rubella, measles, adenovirus, and poliovirus). Postimmunization, no clinically apparent adverse reactions to vaccination were detected.

Adult↗
PubMed1991

Immunologic parameters in early-stage HIV-seropositive subjects associated with vaccine responsiveness.

Twenty-one asymptomatic HIV-seropositive subjects and 20 HIV-seronegative controls were assessed for their serologic response to multiple live attenuated viral, protein (toxoid), and polysaccharide vaccine antigens. Extensive in vivo and in vitro immunologic evaluations were performed. Factors predictive of immunogen responsiveness by HIV-seropositive patients were found by correlating vaccine responses with results of T- and B-cell functional assays. Eleven HIV-seropositive patients (HIV nonresponsive-NR) responded to only one of three vaccines used for analysis (meningococcus, group C; adenovirus 4, 7; and diphtheria-tetanus) compared with the normals, of whom 100% responded to two or more of the same immunogens. Ten HIV-seropositive patients (HIV responsive-R) responded equivalently to normals. The HIV NR group had distinctive immunologic abnormalities predictive of their poor immunogen responsiveness. These included defects in the T-cell helper function despite normalization of T cell number and defective T-cell suppression of Epstein-Barr virus (EBV) in vitro (HIV NR, 30% suppression; HIV R, 73% suppression; and normals, 95% suppression of EBV-driven immunoglobulin production in vitro). The B cells of the HIV NR groups were also abnormal in vivo. The HIV NR patients' B cells were larger and had increased native response to B-cell growth factor evidenced by increased thymidine incorporation. (HIV-NR, 10,232 +/- 3,003 cpm; HIV R, 5,432 +/- 1,125; normal, 402 +/- 11. HIV NR/HIV R, p less than 0.05; HIV NR/N, p less than 0.001.)(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1991

Genetic comparison of human immunodeficiency virus (HIV-1) isolates by polymerase chain reaction.

Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3'LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3' end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3' LTR segments of the genome were used to compare sequential HIV-1 isolates form six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1 isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.

Acquired Immunodeficiency Syndrome↗