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Clinical and Vaccine Immunology (CVI) will merge with the American Society for Microbiology (ASM) open-access journal mSphere in January 2018. We commemorate this transition by exploring the history of CVI and that of its predecessor, Clinical and Diagnostic Laboratory Immunology (CDLI), and by acknowledging their contributors. Research on vaccines, clinical immunology, and clinical diagnostic immunology published through mSphere will be available without restrictions to an ever-larger audience, which will expedite progress in the field. ASM remains committed to supporting its members and the research community by facilitating the dissemination of scientific knowledge in these important areas.

With the advent of highly effective antiretroviral therapy (ART), infection with human immunodeficiency virus (HIV) has become a chronic disease rather than a death sentence. Nevertheless, effectively treated individuals have a higher than normal risk for developing noninfectious comorbidities, including cardiovascular and renal disease. Although traditional risk factors of aging as well as treatment toxicity contribute to this risk, many investigators consider chronic HIV-associated inflammation a significant factor in such end-organ disease. Despite effective viral suppression, chronic inflammation persists at levels higher than in uninfected people, yet the stimuli for the inflammation and the mechanism by which inflammation persists and promotes disease pathology remain incompletely understood. This critical gap in scientific understanding complicates and hampers effective decision making about appropriate medical intervention. To better understand the mechanism(s) of chronic immune activation in treated HIV disease, three questions need answers: (1) what is the cause of persistent immune activation during treated HIV infection, (2) what are the best surrogate markers of chronic immune activation in this setting, and (3) what therapeutic intervention(s) could prevent or reverse this process? The NIH sponsored and convened a meeting to discuss the state of knowledge concerning these questions and the best course for developing effective therapeutic strategies. This report summarizes the findings of that NIH meeting.

Highly active antiretroviral treatment has resulted in dramatically increased life expectancy among patients with HIV infection who are now aging while receiving treatment and are at risk of developing chronic diseases associated with advanced age. Similarities between aging and the courses of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome suggest that HIV infection compresses the aging process, perhaps accelerating comorbidities and frailty. In a workshop organized by the Association of Specialty Professors, the Infectious Diseases Society of America, the HIV Medical Association, the National Institute on Aging, and the National Institute on Allergy and Infectious Diseases, researchers in infectious diseases, geriatrics, immunology, and gerontology met to review what is known about HIV infection and aging, to identify research gaps, and to suggest high priority topics for future research. Answers to the questions posed are likely to help prioritize and balance strategies to slow the progression of HIV infection, to address comorbidities and drug toxicity, and to enhance understanding about both HIV infection and aging.

Many aspects of the human immunodeficiency virus (HIV) life cycle are well understood, yet a key paradox remains. The primary targets for HIV are resting CD4 T lymphocytes that are not permissive to HIV replication unless activated by some external and independent event. Numerous mechanisms for this

This study of a subset of women and infants participating in National Institutes of Health Pediatric AIDS Clinical Trials Group protocol 185 evaluated lymphocyte phenotypic markers of immune activation and differentiation to determine their association with the likelihood of human immunodeficiency virus (HIV) transmission from the women to their infants and the potential for early identification and/or prognosis of infection in the infants. Lymphocytes from 215 human immunodeficiency virus type 1 (HIV)-infected women and 192 of their infants were analyzed by flow cytometry with an extended three-color panel of monoclonal antibodies. Women who did not transmit to their infants tended to have higher CD4 + T cells. Most notably, levels of total CD8 + T cells and CD8 + CD38 + cells made significant independent contributions to predicting the risk of mother-to-child transmission. Adjusting for HIV-1 RNA level at entry, a one percentage-point increase in these marker combinations was associated with a nine percent increase in the likelihood of maternal transmission. Total as well as naïve CD4 + T cells were significantly higher in uninfected than infected infants. Total CD8 + cells, as well as CD8 + cells positive for HLA-DR + , CD45 RA + HLA-DR + , and CD28 + HLA-DR + were elevated in infected infants. Detailed immunophenotyping may be helpful in predicting which pregnant HIV-infected women are at increased risk of transmitting HIV to their infants. Increasing differences in lymphocyte subsets between infected and uninfected infants became apparent as early as six weeks of age. Detailed immunophenotyping may be useful in supporting the diagnosis of HIV infection in infants with perinatal HIV exposure.

Background Peripheral blood lymphocyte subsets need to be determined in a large, urban, minority-predominant cohort of healthy children to serve as suitable control subjects for the interpretation of the appearance of these cells in several disease conditions, notably pediatric HIV-1 infection. Objective We sought to determine the distribution of lymphocyte subsets in healthy urban-dwelling infants, children, and adolescents in the United States. Methods Lymphocyte subsets were determined by means of 3-color flow cytometry in a cross-sectional study of 807 HIV-unexposed children from birth through 18 years of age. Results Cell-surface marker analysis demonstrated that age was an extremely important variable in 24 lymphocyte subset distributions measured as percentages or absolute counts—eg, the CD4 (helper) T cell, CD8 (cytotoxic) T cell, CD19 B cell, CD4CD45RACD62L (naive helper) T cell, CD3CD4CD45RO (memory helper) T cell, CD8HLA-DRCD38 (activated cytotoxic) T cell, and CD8CD28 (activation primed cytotoxic) T cell. The testing laboratory proved to be an important variable, indicating the need for using the same laboratory or group of laboratories to assay an individual's blood over time and to assay control and ill or treated populations. Sex and race-ethnicity were much less important. Conclusion The results of this study provide a control population for assessment of the effects of HIV infection on the normal development and distribution of lymphocyte subsets in children of both sexes, all races, and all ethnic backgrounds from birth through 18 years of age in an urban population. This study's findings will also prove invaluable in interpreting the immune changes in children with many other chronic diseases, such as primary immunodeficiency, malignancy, rheumatoid arthritis, and asthma.

The best hope for the prevention of HIV/AIDS, especially in resource-poor countries, is an effective preventive vaccine. Considerable effort is being made to develop a safe and efficacious vaccine that will elicit broad, protective immunity to HIV. Most of the current vaccine development strategies focus on viral subunit immunogens, which tend to be less than optimally immunogenic and thus require methods to enhance their ability to elicit good neutralizing antibody and vigorous cell-mediated responses. Although generalized adjuvants have long been used to enhance the immune response to vaccines, the use of cytokine adjuvants with specific immune modulatory functions allows the manipulation of the host response to both enhance overall immunogenicity and direct the nature of the response toward a Th1 or Th2 pathway. This review describes the various HIV immunogens and cytokine formulations that are being considered and the state of knowledge about in vitro studies, preclinial and clinical trials of these cytokine-adjuvanted HIV vaccines.

We analyzed the role of 4 genes, TCL-1, MTCP-1, TML-1 and ATM, in the early pathogenesis of T cell leukemia, with particular interest in the characteristics of long-standing non-leukemic clonal proliferations in ataxia-telangiectasia (A-T) patients. Five patients were studied: 4 patients had A-T (2 of whom had non-leukemic clonal proliferations [ATCP]), 1 had B cell lymphoma and 1 had T-ALL; a fifth patient with T-PLL did not have A-T. We measured the levels of expression for TCL-1, MTCP-1 and TML-1. TCL-1, not expressed in unstimulated mature T cells, was upregulated in the peripheral blood leukocytes (PBL) of the 2 A-T patients with ATCP. It was also expressed in the malignant cells of the A-T patient with B cell lymphoma and the T-PLL cells of the patient without A-T. In the same cells, MTCP-1 type A was expressed equally in all 5 patients, as well as in the controls; MTCP-1 type B transcripts were not observed. TML-1, also not expressed in unstimulated T cells, was expressed in the PBL of one A-T patient with ATCP and in the leukemic cells of the non-A-T T-PLL patient. These expression patterns were compared to cellular immunophenotypes. The non-leukemic clonal T cell populations had the characteristics of immature T cells. We conclude that TCL-1 and TML-1 play a role in cell proliferation and survival but are not pivotal genes in the progression to malignancy, even when the ATM gene is mutated. Additional genetic alterations must occur to initiate tumorigenesis. © 2001 Wiley-Liss, Inc.

Highly active antiretroviral therapy (HAART) suppresses plasma viremia in most patients with human immunodeficiency virus (HIV) infection. Prospective study of HIV-infected children (n=27) shows that, in 8 of 12 who responded to HAART (>/=0.5 log reduction in plasma HIV RNA), HAART restricted the number of coreceptors used by the predominant HIV isolate (mean number of coreceptors used at baseline was 4, vs. 1 coreceptor used at 6 months after treatment). This decrease was most striking in 6 of 8 children whose HIV coreceptor tropism changed from X4-tropic at baseline to R5-tropic. In 6 of 10 children tested, with plasma HIV RNA levels of

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174–179, 2000. © 2000 Wiley-Liss, Inc.

Immunologic alterations occur during pregnancy, but the effect of pregnancy on HIV infection is controversial. We characterized some of the immunologic alterations with potential to influence HIV disease in 99 infected and 46 uninfected women during pregnancy and up to 6 months post-partum. Immunophenotyping to quantitate the major lymphocyte subsets and determine expression of activation and adhesion molecules on T cells was performed using 3-color staining and laser flow cytometry. Serum neopterin, beta 2-microglobulin, and tumor necrosis factor-alpha (TNF alpha) were quantitated using commercial immunoassays. HIV + pregnant women were compared to uninfected pregnant subjects and to reference ranges established on healthy, HIV-seronegative non-pregnant female controls. Both CD4 and CD8 T cell subsets were increased in HIV-negative pregnant women compared to non-pregnant controls. In HIV-infected pregnant women, CD4 T cells were low and CD8 cells were elevated compared to HIV-negative pregnant and non-pregnant women. Levels of subsets were stable during pregnancy and postpartum in both groups of women. Evidence of peripheral immune activation was found during the later stages of pregnancy. Increases in HLA-DR and CD38 activation antigens on CD8 cells, serum neopterin and beta-2-microglobulin were seen during pregnancy in HIV-negative women. These correlates of immune activation were increased in HIV-infected pregnant women and increased further during pregnancy, paralleling changes seen in uninfected pregnant women. These immunologic alterations may directly or indirectly enhance viral replication, impacting the long-term course of HIV disease.

Human immunodeficiency virus (HIV) type 1 (HIV-1) induces impairment of immune function reflected in reduced lymphocyte proliferative responses. Many other immune changes are induced by HIV-1, but their relationship to lymphocyte functional defects is not known. The present study was designed to correlate functional defects with other HIV disease parameters. Cryopreserved samples from 118 HIV-1-positive subjects and 40 seronegative individuals were examined. The main findings were that impaired proliferative responses to mitogens correlated with (i) decreased cell surface expression of the interleukin-2 receptor (CD25), (ii) increased expression of HLA-DR antigens on CD4 cells, (iii) reduced CD4 and increased CD8 cell numbers, and (iv) increased levels of serum immune complex dissociated p24 antigen. However, impaired function was not associated with increased serum neopterin, beta2-microglobulin, or soluble interleukin-2 receptor or with CD38 antigen expression on lymphocytes. In summary, proliferative functional impairment correlated with some, but not all, immunological changes associated with HIV-1 infection. Most of the phenotypic markers that correlated with altered function are cell surface molecules with significant roles in lymphocyte proliferation and were associated primarily with CD4 cells, compatible with the view that dysregulation of CD4 cells is responsible for impaired function.

We characterized the surface phenotype of B cells from HIV+ children in order to better understand the biology of B cell dysregulation. Twenty-nine HIV-infected, twenty-one exposed, and nineteen age-matched control children were studied for expression of CD5, CD10, CD21, CD23, CD25, CD62L, CD71, and CD69. We conclude that, despite persistent high immunoglobulin levels, total B cells decreased as HIV disease progressed, with selective decreases in CD62L+ and CD23+ B cells. This resulted in an increased proportion of usually minor subpopulations of CD62L- and CD23-negative B cells. We did not find significantly altered B cell expression of other activation/immaturity antigens. This suggests an absolute decrease in a subset of antigen-responsive B cells and a disproportionate increase in a subset of hyperstimulated B cells. These findings provide a biological basis for the paradoxical generalized B cell hyperactivity and specific immune unresponsiveness that are characteristic of HIV infection in children.

The Editors of Clinical and Vaccine Immunology would like to express their thanks and appreciation to the dedicated ad hoc reviewers listed below who have graciously given their time and expertise over the past year. Jacqueline M. Achkar Jeffrey J. Adamovicz Linda B. Adams Diane D. Addie Ben

CD8 T lymphocytes are an important component of the host immune response to human immunodeficiency virus (HIV). To characterize CD8 cell function, we have studied the in vitro phenomenon of CD8 cell-mediated inhibition of HIV replication from autologous, naturally infected CD4 cells. We describe here a reproducible assay of CD8 T cell-mediated inhibition in HIV-infected individuals. The method involves the use of a commercially available cell separation system and anti-CD3 monoclonal antibody to stimulate CD4 cells to produce HIV. Using this technique, we were able to detect HIV production from the CD4 cells of 25 of 27 HIV-infected individuals who had not progressed to AIDS. Further, in vitro CD8 cell-mediated inhibition of HIV production was noted in all of the 25 subjects whose CD4 cells produced viral p24 antigen. This assay may be useful as an in vitro correlate of protective immunity to HIV, with potential application for assessing disease progression, therapeutic efficacy, and immune mechanisms in HIV disease.

A previously observed rise in the plasma viral load postpartum in both treated and untreated HIV-positive women remains unexplained. Virological and immunological markers were evaluated in HIV-negative controls and HIV-positive pregnant women with and without antiretroviral treatment. Plasma HIV RNA, CD4/CD8 T cells, and serum activation markers were sequentially measured during the third trimester, at delivery, and 2 to 8 weeks postpartum in a cohort of HIV-positive pregnant women ( n = 96) enrolled in a maternal-fetal HIV transmission study and a control group of HIV-negative pregnant women ( n = 28). Mean plasma HIV RNA ( P = 0.003) increased from delivery to postpartum, and mean CD4 T cells ( P = 0.002) and serum β2-microglobulin ( P < 0.0001) increased from the third trimester through postpartum among the HIV-positive women. Mean CD8 T cells increased from the third trimester through postpartum in women receiving zidovudine (ZDV) and in those not treated ( P < 0.05) but remained stable in those on highly active antiretroviral therapy (HAART) and the HIV-negative controls. Increases in serum β2-microglobulin were correlated with increases in HIV RNA ( P = 0.01). HIV-positive pregnant women showed postpartum increases in plasma HIV RNA, CD4 T cells, and serum β2-microglobulin regardless of the treatment regimen. The rise in CD4 T cells and β2-microglobulin was also observed in HIV-negative pregnant women, suggesting hormonal changes and/or labor-induced cytokines may contribute to immune activation. Immune activation correlated with increased plasma HIV RNA in postpartum women despite treatment, although HAART appeared to blunt the effect. The observed rise in plasma HIV RNA postpartum, which correlated with markers of immune activation, may have implications for enhanced transmission to infants through early breast-feeding and to sexual partners.

Infection with human T-cell leukemia virus type II (HTLV-II) has been associated with rare chronic T-cell malignancies and has recently been demonstrated in a significant proportion of American intravenous drug abusers (IVDA). Identification of an HTLV-II-infected cohort of IVDA has allowed analysis of the HTLV-II carrier state. We analyzed clinical, hematologic, and immunologic parameters in 21 HTLV-II- infected IVDA, two HTLV-I-infected IVDA, and 20 uninfected control IVDA identified by serologic screening and by analysis of peripheral blood mononuclear cell (PBMC) DNA by polymerase chain reaction (PCR). An elevated absolute lymphocyte count was observed in 4 of 21 HTLV-II- infected IVDA, 1 of 2 HTLV-I-infected IVDA, and 1 of 20 control IVDA. CD8+ T-cell elevation was observed in three of four HTLV-II IVDA with lymphocytosis and one of two HTLV-I-infected IVDA. Activation of CD8+ T cells in HTLV-II-infected IVDA was suggested by an overall increase in CD8+/HLA-DR+ lymphocytes. Cell fractionation and analysis by PCR of HTLV-II-infected carrier blood showed high levels of HTLV-II provirus in unfractionated PBMC and purified T cells and little or no detectable HTLV-II DNA in B cells or monocytes, indicating that T cells were the most likely target of infection in vivo. The frequency of HTLV-II- infected cells was estimated at approximately 1 in 500 cells or less using dilution analysis by PCR of PBMC DNA. Most HTLV-II-infected IVDA are asymptomatic and have no overt hematologic or immunologic abnormalities, although some manifest benign lymphocytosis.

Peripheral blood lymphocyte subsets need to be determined in a large, urban, minority-predominant cohort of healthy children to serve as suitable control subjects for the interpretation of the appearance of these cells in several disease conditions, notably pediatric HIV-1 infection.We sought to determine the distribution of lymphocyte subsets in healthy urban-dwelling infants, children, and adolescents in the United States.Lymphocyte subsets were determined by means of 3-color flow cytometry in a cross-sectional study of 807 HIV-unexposed children from birth through 18 years of age.Cell-surface marker analysis demonstrated that age was an extremely important variable in 24 lymphocyte subset distributions measured as percentages or absolute counts--eg, the CD4 (helper) T cell, CD8 (cytotoxic) T cell, CD19 B cell, CD4CD45RACD62L (naive helper) T cell, CD3CD4CD45RO (memory helper) T cell, CD8HLA-DRCD38 (activated cytotoxic) T cell, and CD8CD28 (activation primed cytotoxic) T cell. The testing laboratory proved to be an important variable, indicating the need for using the same laboratory or group of laboratories to assay an individual's blood over time and to assay control and ill or treated populations. Sex and race-ethnicity were much less important.The results of this study provide a control population for assessment of the effects of HIV infection on the normal development and distribution of lymphocyte subsets in children of both sexes, all races, and all ethnic backgrounds from birth through 18 years of age in an urban population. This study's findings will also prove invaluable in interpreting the immune changes in children with many other chronic diseases, such as primary immunodeficiency, malignancy, rheumatoid arthritis, and asthma.

The use of herbs to treat illness has its roots in an ancient holistic healing tradition that originated in Asia more than 3,000 years ago (10). Largely discounted by 19th and 20th century practitioners of Western medicine, healing practices incorporating herbal remedies, such as traditional Chinese medicine (TCM), Japanese Kampo, and Indian Ayurveda, are rapidly gaining acceptance in the West as we enter the 21st century. Herbal medicines are complex mixtures of minimally processed medicinal plants (e.g., plant parts that are boiled to make a tea). In conjunction with other components of traditional healing philosophies, such as acupuncture or massage, herbal medicines are used to treat a large range of symptoms and ailments, including liver disease, asthma, and other immune problems, menstrual problems, colds, headaches, and various cancers.

We analyzed the role of 4 genes, TCL-1, MTCP-1, TML-1 and ATM, in the early pathogenesis of T cell leukemia, with particular interest in the characteristics of long-standing non-leukemic clonal proliferations in ataxia-telangiectasia (A-T) patients. Five patients were studied: 4 patients had A-T (2 of whom had non-leukemic clonal proliferations [ATCP]), 1 had B cell lymphoma and 1 had T-ALL; a fifth patient with T-PLL did not have A-T. We measured the levels of expression for TCL-1, MTCP-1 and TML-1. TCL-1, not expressed in unstimulated mature T cells, was upregulated in the peripheral blood leukocytes (PBL) of the 2 A-T patients with ATCP. It was also expressed in the malignant cells of the A-T patient with B cell lymphoma and the T-PLL cells of the patient without A-T. In the same cells, MTCP-1 type A was expressed equally in all 5 patients, as well as in the controls; MTCP-1 type B transcripts were not observed. TML-1, also not expressed in unstimulated T cells, was expressed in the PBL of one A-T patient with ATCP and in the leukemic cells of the non-A-T T-PLL patient. These expression patterns were compared to cellular immunophenotypes. The non-leukemic clonal T cell populations had the characteristics of immature T cells. We conclude that TCL-1 and TML-1 play a role in cell proliferation and survival but are not pivotal genes in the progression to malignancy, even when the ATM gene is mutated. Additional genetic alterations must occur to initiate tumorigenesis.

Previous studies have provided conflicting data on the presence of selective pressures in the transmission of a homogeneous maternal viral subpopulation to the infant. Therefore, the purpose of this study was to definitively characterize the human immunodeficiency virus type 1 (HIV-1) quasispecies transmitted in utero and intrapartum. HIV-1 envelope gene diversity from peripheral blood mononuclear cells and plasma was measured during gestation and at delivery in mothers who did and did not transmit HIV perinatally by using a DNA heteroduplex mobility assay. Children were defined as infected in utero or intrapartum based on the timing of the first detection of HIV. Untreated transmitting mothers ( n = 19) had significantly lower HIV-1 quasispecies diversity at delivery than untreated nontransmittting mothers ( n = 18) (median Shannon entropy, 0.711 [0.642 to 0.816] versus 0.853 [0.762 to 0.925], P = 0.005). Eight mothers transmitted a single major env variant to their infants in utero, and one mother transmitted a single major env variant intrapartum. Four mothers transmitted multiple HIV-1 env variants to their infants in utero, and two mothers transmitted multiple env variants intrapartum. The remaining six intrapartum- and two in utero-infected infants had a homogeneous HIV-1 env quasispecies which did not comigrate with their mothers' bands at their first positive time point. In conclusion, in utero transmitters were more likely to transmit single or multiple major maternal viral variants. In contrast, intrapartum transmitters were more likely to transmit minor HIV-1 variants. These data indicate that different selective pressures, depending on the timing of transmission, may be involved in determining the pattern of maternal HIV-1 variant transmission.

Lymphocyte proliferation assays (LPAs) are widely used to assess T-lymphocyte function of patients with human immunodeficiency virus infection and other primary and secondary immunodeficiency disorders. Since these assays require expertise not readily available at all clinical sites, specimens may be shipped to central labs for testing. We conducted a large multicenter study to evaluate the effects of shipping on assay performance and found significant loss of LPA activity. This may lead to erroneous results for individual subjects and introduce bias into multicenter trials.

Objective. HIV-infected children receiving influenza vaccine, pneumococcal vaccine and both vaccines concurrently were studied to examine the effect of immunization on plasma HIV viral load. Methods. Thirteen children received immunizations: pneumococcal vaccine, 5; pneumococcal and influenza vaccines, 7; and influenza vaccine, 1. Most patients (12 of 13) were receiving combination reverse transcriptase inhibitor antiretroviral therapy without protease inhibitors at the time of immunization. Baseline plasma HIV RNA was determined 1 month prior (11 of 13), 2 weeks prior (12 of 13) and on the day of immunization (12 of 13). Plasma HIV RNA was assayed at 2 weeks (11 of 13), 4 weeks (12 of 13) and 3 months (5 of 13) after immunization. T cell activation markers (HLA-DR + , CD38 + and CD45RO + , CD28 + ) were examined for both CD4 + and CD8 + lymphocytes on the day of immunization and 2 weeks after immunization for 11 children. Results. Only one child developed a >0.5-log increase in viral load at any time after immunization. There was no correlation between an increase in viral load and antibody response to pneumococcal vaccine. At least one activation marker increased (>10%) for two children receiving pneumococcal vaccine and two children receiving pneumococcal and influenza vaccines. One of these children experienced an increase in viral load. Conclusion. Immunization with pneumococcal and influenza vaccines, alone or in combination, is rarely associated with a significant increase in HIV plasma RNA in children receiving combination antiretroviral therapy.

The potential role of human immunodeficiency virus type 1 (HIV-1)-specific immune responses in controlling viral replication in vivo has stimulated interest in enhancing virus-specific immunity by vaccinating infected individuals with HIV-1 or its components. These studies were undertaken to define patient populations most likely to respond to vaccination, with the induction of novel HIV-1-specific cellular immune responses, and to compare the safety and immunogenicity of several candidate recombinant HIV-1 envelope vaccines and adjuvants. New lymphoproliferative responses (LPRs) developed in30% of vaccine recipients. LPRs were elicited primarily in study participants with a CD4 cell count350 cells/mm(3) and were usually strain restricted. Responders tended to be more likely than nonresponders to have an undetectable level of HIV-1 RNA at baseline (P=.067). Induction of new cellular immune responses by HIV-1 envelope vaccines is a function of the immunologic stage of disease and baseline plasma HIV-1 RNA level and exhibits considerable vaccine strain specificity.

An external evaluation program for measuring the performance of laboratories testing for cytokines and immune activation markers in biological fluids was developed. Cytokines, chemokines, soluble cytokine receptors, and other soluble markers of immune activation (CSM) were measured in plasma from a healthy human immunodeficiency virus (HIV)-seronegative reference population and from HIV-seropositive individuals as well as in supernatant fluids from in vitro-stimulated human immune cells. The 14 components measured were tumor necrosis factor (TNF) alpha, gamma interferon, interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-10, Rantes, MIP-Ia, MIP-Iβ, soluble TNF receptor II, soluble IL-2 receptor alpha, β 2 -microglobulin, and neopterin. Twelve laboratories associated with the Adult and Pediatric AIDS Clinical Trial Groups participated in the study. The performance features that were evaluated included intralaboratory variability, interlaboratory variability, comparison of reagent sources, and ability to detect CSM in the plasma of normal subjects as well as the changes occurring in disease. The principal findings were as follows: (i) on initial testing, i.e., before participating in the program, laboratories frequently differed markedly in their analytic results; (ii) the quality of testing of a CSM in individual participating laboratories could be assessed; (iii) most commercial kits allowed distinction between normal and abnormal plasma CSM levels and between supernatants of stimulated and unstimulated cells; (iv) different sources of reagents and reference standards frequently provided different absolute values; (v) inexperienced laboratories can benefit from participating in the program; (vi) laboratory performance improved during active participation in the program; and (vii) comparability between analyses conducted at different sites can be ensured by an external proficiency testing program.

RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta are human immunodeficiency virus (HIV) suppressor factors by virtue of their ability to compete with HIV for access to cell surface R5. Their ability to block HIV infection in vitro is unequivocal; however, their role as HIV suppressor factors in vivo is not firmly established. We therefore conducted a study to test the hypothesis that production of these factors in vitro was a correlate of decreased virus burden in vivo. Moreover, we asked whether higher beta chemokine production could be demonstrated with cells from people who are R5D32 heterozygotes, compared with people who are R5 wild-type homozygotes. Our data support the thesis that RANTES, MIP-1alpha, and MIP-1beta production is associated with decreased in vivo virus load. Moreover, enhanced production of these factors may be explained in part by the genetic background of the host.

Acute HIV infection is characterized by the appearance of high concentrations of virus in the peripheral blood. In adults, this high-level viremia spontaneously abates after several weeks. In contrast, after perinatal infection of infants, blood virus levels remain high for many months, during which the concentration of circulating CD4+ lymphocytes remains well above normal values for adults. Here we suggest an explanation for these differences, based on developmental factors including somatic growth and immunological ontogeny. Flow cytometric analysis revealed that at birth the thymus contains elevated levels of mature T lymphocytes, compared to the thymus after 3 months of age. A mathematical model is proposed incorporating immunological and virological data from longitudinally evaluated infants who acquired infection at the time of birth. This model explains the pattern of high-level viremia in infants as resulting from the replication of HIV within the progressively expanding lymphoid cell mass.

CD8 T cell function, lymphocyte surface phenotype, serum markers of immunologic activation, and viral burden were assessed in 75 human immunodeficiency virus (HIV)-infected pregnant women, including 9 who transmitted infection to their infants. Serial studies during and after pregnancy showed no significant differences in levels of cell-surface or serum activation molecules in transmitting compared to nontransmitting mothers, with the exception of a postpartum increase in tumor necrosis factor alpha in transmitting women. The transmitting women had a median plasma viral load of 65,516 RNA copies/mL at delivery versus 5139 in nontransmitting women. During the third trimester, the CD8 cells of 81% of the nontransmitting and 44% of the transmitting mothers suppressed HIV production in vitro by >50%. Women with

One hundred and eighteen HIV-infected homosexual men without AIDS and 40 control seronegative homosexual men were assessed for 23 parameters reflecting immune activation to determine prognostic significance for occurrence of AIDS. Samples cryopreserved in 1987-1989 were analyzed, with AIDS occurrence determined by mid-1992. Cell surface antigens assessed on the major lymphocyte subsets were HLA-DR, CD38, CD71, and CD25. Soluble serum molecules assessed were tumor necrosis factor alpha, soluble TNFalpha receptor II, soluble IL-2 receptor alpha, neopterin, and beta2-microglobulin. Using a proportional hazards model, prognostic markers included decreased CD4 number and percentage; increased sIL-2R, neopterin, and beta2M; increased percentage HLA-DR+ total lymphocytes and CD4+ cells; increased CD38+ total lymphocytes and CD8+ cells; increased CD71+ total lymphocytes and CD4+ cells; and decreased CD25+ total lymphocytes and CD19+ cells. After adjustment for CD4 cell levels, sIL-2R, neopterin, beta2M, and CD25+ CD19 cells remained significant, indicating that additional information about AIDS risk was provided by these markers.

The time of perinatal human immunodeficiency virus type 1 (HIV-1) transmission and the pattern of early plasma viremia as predictors of disease progression were evaluated in infected infants followed from birth. Cox proportional hazards modeling demonstrated that a 1-log higher HIV-1 RNA copy number at birth was associated with a 40% increase in the relative hazard (RH) of developing CDC class A or B symptoms (P = .004), a 60% increase in developing AIDS (P = .01), and an 80% increase in the of risk death (P = .023) over the follow-up period of up to 8 years. the peak HIV-1 RNA copy number for infants during primary viremia was also predictive of progression to AIDS (RH, 9.9; 95% confidence interval [95% CI], 1.8-54.1; P = .008) and death (RH, 6.9; 95% CI, 1.1-43.8; P = .04). The results indicate that high levels of HIV-1 RNA at birth and during primary viremia are associated with early onset of symptoms and rapid disease progression to AIDS and death in perinatally infected children.

Apoptosis continues to be controversial in human immunodeficiency virus (HIV)-induced pathogenesis. To investigate whether apoptosis occurs with HIV exposure with or without subsequent infection, levels of apoptosis were measured in cord blood lymphocytes (CBL) from seven newborns delivered to HIV-infected mothers and seven normal, unexposed newborns. Live cells were costained with antibodies to cell surface markers and the DNA dye 7-amino actinomycin D to immunophenotype apoptotic CBL subsets. Apoptosis was measured in fresh and cultured CBL in the presence and absence of CD3 T-cell receptor stimulation. Compared to the CD4 + CBL from HIV-unexposed newborns, CD4 + CBL from six HIV-exposed, noninfected newborns demonstrated significantly greater apoptosis after overnight culture even in the absence of CD3 stimulation. Compared to HIV-unexposed controls, CD8 + CBL from the six HIV-exposed newborns also demonstrated increased levels of apoptosis after overnight culture without stimulation. The one HIV-infected newborn in this study showed the highest levels of CD4 + and CD8 + apoptotic CBL. The data suggest that levels of apoptosis are increased in infants in association with HIV infection. Furthermore, CD4 + and CD8 + cord blood lymphocytes from HIV-exposed infants behaved differently than T lymphocytes from either normal, unexposed infants or an HIV-infected infant. These results suggest that exposure to HIV or HIV-induced factors increases the levels of apoptosis in CBL.

The impact of social defeat on lymphocyte subpopulations and T helper subsets was investigated in Long Evans rats. CD4 T helper cell subsets with distinct functional properties and different cytokine profiles can be distinguished by using the mAbs OX-22 (anti-CD45RC) and OX-7 (anti-CD90, Thy1.1). Male intruders were exposed for 2, 6, or 48 h to aggressive resident pairs. All intruders were attacked upon introduction and were defeated as indicated by frequent display of full submissive postures. After 2 and 48 h of confrontation, drastic but differential effects on blood leukocyte numbers, CD4 and CD8a cells, and CD4 subsets were evident. However, after 6 h of confrontation most lymphocyte subset numbers corresponded to baseline levels. Focusing on CD4 subsets after 2 h of confrontation, we demonstrated that only the number of the CD45RC-CD90(-) subset declines, whereas neither the number of the CD45RC+CD90(-) subset nor the number of the CD45RC-CD90(+) subset (recent thymic emigrants) was influenced. Con A stimulation of sorted subsets identified the CD45RC-CD90(-) as a poor producer of IFN-gamma. The data clearly demonstrate that social factors might differentially influence not only T cell subsets but also T helper cell subsets with distinct cytokine profiles in a possibly time-dependent manner. Such a stress-induced shift toward a CD45RC+CD90(-)-dominated milieu may have important consequences in interpreting results obtained from mitogenic stimulation of blood lymphocytes and cytokine production profiles measured after such a stimulation. In addition, a shift toward a CD45RC+CD90(-) dominance may modify the type and magnitude of immune response, at least temporarily.

The relationship between viral burden, timing of transmission, and clinical progression was investigated in 110 children at risk for vertical human immunodeficiency virus (HIV) infection using quantitative polymerase chain reaction, coculture, and immune complex-dissociated p24 antigen assay. In a cross-sectional study, the mean HIV DNA copy number in 19 symptomatic children was significantly higher than in 31 infected, asymptomatic children (420 +/- 125 vs. 87 +/- 78; P < .0001). In a second group of 8 vertically infected infants followed prospectively from birth, 4 defined as infected in utero showed a more rapid increase in virus load, an accelerated loss of CD4 cells, and early progression to symptomatic disease (3-12 weeks) compared with 4 children with late in utero or intrapartum transmission (10-31 months). These data suggest that a direct relationship exists between HIV replication, the timing of transmission and onset and progression of HIV disease in vertically infected children.

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.

Two positive selection methods were compared for the ability to capture both the bright and dim subsets of CD8 lymphocytes in mononuclear cell (MC) preparations from ten healthy individuals. The first method utilized anti-CD8-coated magnetic beads; captured cells were then recovered using a polyclonal sheep anti-mouse Fab reagent. At all bead: CD8 cell ratios tested (4:1, 8:1, 16:1), the selected cells were >94% CD8 + , and these CD8 cells were enriched for CD8 bright cells (77–85%) when compared to CD8 cells in the starting MC preparation (68%). The second method utilized anti-CD8-coated culture flasks; captured cells were recovered by physical dislodgement. The recovered cells were >90% CD8 + , and these CD8 cells were modestly enriched for CD8 dim cells (52%) compared to starting CD8 cells (32%). To further enrich for CD8 dim cells, we used these two methods in tandem ( n = 10). MC were first incubated with anti-CD8-coated magnetic beads (4:1 ratio) to obtain a CD8 bright -enriched population (97% of all cells CD8 + , 83% of all cells CD8 bright ). Uncaptured cells were incubated with anti-CD4-coated magnetic beads, and the uncaptured cells from this step were then placed in an anti-CD8-coated flask. The recovered flask-selected cell population was highly enriched for CD8 dim cells (87% of all cells CD8 + , 85% of all cells CD8 dim ). CD8 cells in the CD8 bright population were 94% CD3 + and 6% CD16 + , whereas those in the CD8 dim population were 29% CD3 + and 66% CD16 + . In proliferative studies, CD8 bright cells were preferentially activated by immobilized anti-CD3, whereas CD8 dim cells were preferentially activated by exogenous IL-2. In assays of natural killer activity, CD8 dim cells were markedly more active than CD8 bright cells. This method provides an alternative to cell sorting for obtaining enriched populations of CD8 bright and CD8 dim lymphocytes.

The major histocompatibility complex (MHC) class II deficiency syndrome is a rare immunodeficiency disease associated with defective expression of class II MHC antigens. We have examined the consequences of this defect for the differentiation and functional capabilities of immunoregulatory T-cell subpopulations in an affected patient. Although the number of circulating T cells was normal, there was a striking reduction in the number of CD4+ T cells. Furthermore, purified CD4+ cells from the patient were unable to provide help for antibody secretion. This defect in helper function appeared to be due to the abnormal differentiation of the few CD4+ cells present, virtually all of which expressed the CD4 + HB11 + phenotype characteristic of immature “virgin” T cells. Abnormal development of immunoregulatory CD8+ T cells was also observed. Although increased numbers of CD8+ T cells were present, virtually none had phenotypic properties of suppressor cells (i.e., CD3+/CD8+/9.3-granular lymphocytes that coexpress the Leu-15 or Leu-7 antigens), and purified CD8+ cells from the patient had no suppressor activity. Thus, the absence of class II MHC antigens profoundly disrupts the development of immunoregulatory T cells. We propose that these effects occur by the following mechanisms: (1) the absence of intrathymic class II antigens results in deficient production of CD4+ cells, (2) the CD4+ cells that do emerge from the thymus do not undergo postthymic maturation into CD4+HB11- cells with helper capabilities, and (3) the absence of CD4+HB11- effector cells results in abortive development of suppressor cells involved in feedback suppression.

Lymphocytes were separated from peripheral blood of adult human donors by Ficoll-Hypaque gradient centrifugation and cultured in the presence of nonspecific mitogens (phytohemagglutinin[PHA] and concanavalin A) or specific microbial anatigens (herpes simplex virus [HSV], mumps virus, streptococcal enzymes, and Candida albicans). Exposure of lymphocyte cultures to infectious HSV resulted in almost complete inhibition of blastogenesis ([3/H]thymidine uptake) induced by each of the mitogens and antigens, a finding which suggests that a common mechanism may underlie the inhibitory effect. Several characteristics of the effect of virus on blastogenesis were noted: (1) virus inactivated by heat or ultraviolet irradiation was ineffective; (2) inhibition (is greater than 90%) was greatest in cultures exposed to HSV on or before the addition of PHA; (3) lymphocyte preparations washed free of HSV continued to be refractory to stimulation, an observation indicating that the presence of unabsorbed virions or viral products was not essential; and (4) inhibition was independent of the cell donor's state of humoral immunity to HSV.

Increased permissiveness of macrophages for herpes simplex virus (HSV) replication may be a mechanism for the dissemination and severity of neonatal herpetic infection. We have assessed the replication of HSV in neonatal blood monocytes and placental macrophages using several criteria for viral permissiveness. Assay of production of infectious progeny virus indicated that cord blood monocytes, like adult monocytes, were nonpermissive for HSV (about 1% of cells producing virus). In vitro culture of cord blood monocytes resulted in increased replication of HSV, but no greater extent than virus production in cultured adult cells. HSV infection of fetal placental macrophages was weak but present (4.4% of cells). Assay of production of viral antigens and electron microscopic analysis of structural elements indicated that a larger number of cord blood monocytes and placental macrophages were abortively infected than were productively infected. These results indicate that monocytes and macrophages from human neonates do not show the enhanced permissiveness for HSV demonstrated in newborn mice and suggest that dissemination of herpetic infection in human newborns cannot be explained by increased neonatal monocyte permissiveness for HSV.

SummaryResults from examination of peripheral blood leukocytes from five subjects, three with antibody to HSV and two without, showed that HSV type 1 may replicate in mitogen-stimulated T and B lymphocytes and in continuous lymphoblastoid cell lines derived from either B or T cells. Significant replication was also noted in monocytes from each subject but not in polymorphonuclear leukocyte subpopulations. Replication in monocytes was found to be dependent on certain conditions of culture and viral inoculation. Only adherent cells cultured for 72-hr prior to infection produced significant yields of virus.

Serial measurement ofin vitro immunologic parameters in patients is used to detect change in immune status over time due to disease progression and/or immunodulatory therapy. A statistical method is presented for looking at serial measurements on an individual to detect whether a change in a parameter is outside the bounds of expected within-individual variation. Analysis of variance is used, assuming a normal distribution, to obtain percentiles of the distribution of the absolute difference between consecutive values of immunologic parameters in a healthy population. The assumptions in this analysis are justified from a statistical point of view. We discuss how to use this statistical method to make judgments relevant to clinical immunology, including (1) how to construct a table that can be used to determine quickly if an “interesting” change for some standard immunologic parameters has occurred, (2) whether a linear (additive) or logarithmic (proportional) model for change might be more appropriate for a given parameter, and (3) how to modify the calculations if change is expected in a certain direction or if multiple pre- and/or postevent (clinical change or intervention) measurements are available.

Cytotoxic mechanisms (e.g., natural killer (NK) lysis, antibody-dependent cellular cytotoxicity, and cytotoxic T lymphocyte lysis) play an important role in host defense against various infections and neoplasms. Lymphokine-activated killer (LAK) cytotoxicity, induced in vitro by incubating mononuclear cells with interleukin 2 (IL-2) for 2-5 days, may also represent an important component of the body's cytotoxic repertoire. In 10 patients with congenital cellular immunodeficiencies, including 5 with severe combined immunodeficiency, the mean LAK activity in a 3-hr chromium release assay against Raji target cells was 44 +/- 8.1%, which is equivalent to that observed in normal adults and neonates. In only one case, a patient with reticular dysgenesis, was there absent LAK cell generation. Haploidentical T cell-depleted bone marrow transplantation (BMT) restored LAK activity in this patient. LAK activity was first observed in this patient and two others 3-6 weeks following BMT, prior to other evidence of immunologic engraftment such as lymphocyte proliferation to mitogens, NK activity, or interferon-gamma production. One patient with adenosine deaminase deficiency showed normal levels of LAK activity despite absent NK activity. Three patients with chronic granulomatous disease also had normal LAK activity (57 +/- 14% specific lysis). In 9 patients with acquired immunodeficiency syndrome (AIDS), IL-2 activation resulted in a mean cytotoxic activity of 56 +/- 8.7% toward Raji targets. In addition, 9 patients with pre-AIDS complex also showed normal levels of cytotoxicity (37 +/- 3.3% toward Raji targets), equivalent to that of 8 normal controls, including two healthy homosexual males (mean lysis 38 +/- 3.9%). These results indicate that LAK cells appear early in immunologic ontogeny. Further, the mechanism of lysis is not oxygen dependent since LAK activity was present in the 3 patients with chronic granulomatous disease. The ability to generate LAK in a wide spectrum of immunodeficiencies may indicate that IL-2 could be used in therapy of such disorders.

Testing of cytotoxic function using a panel of natural killer (NK)-sensitive target cells, including a unique herpes simplex virus-infected Raji-cell target, was performed in conjunction with phenotypic cell analysis by dual-color flow cytometry to characterize the NK system. Subjects included in the study were at risk for or infected with the etiologic agent of the acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV). A generalized defect in NK function was temporally correlated with disease manifestations, as evidenced by deficient NK lytic function in patients with AIDS and AIDS-related complex (ARC). Healthy at-risk subjects, including those seropositive for HIV, exhibited robust NK-cell function. Phenotypic analysis revealed that normal proportions of the NK-associated CD16+ (Leu11) Leu7− and CD16+(Leu11)Leu7+ lymphocyte subsets were maintained throughout the clinical progression of HIV infection. However, the proportion and numbers of cells of the CD8+(Leu2)Leu7+ subset were increased in AIDS, ARC, and healthy at-risk subjects, including those seronegative for HIV. These results are consistent with a qualitative defect in the NK system in AIDS, perhaps secondary to CD4-cell depletion and a concomitant lack of essential accessory factors. The elevation in CD8+(Leu2)/Leu7+ cells is not solely the result of HIV infection and may be a general response to viruses and/or other antigenic stimulation.

Strong LAK cell activity induced by interleukin-2 (IL-2) of cord (and adult) mononuclear cells (Pediatr. Res. 19: 271A, 1985) has suggested a possible role for LAK and/or IL-2 therapy in newborns with certain infections. The effect of HSV (type 1) on the LAK effector cell was studied by adding live or irradiated virus (10,000 R) (MOI=10) to 10 cells which have been incubated for 4-6 days with IL-2 (50-100 U/ml). The cells were tested 18-24 hrs later for cytotoxic activity against Cr-labelled K562 and Raji target cells. Live HSV inhibited LAK cytotoxicity of adult cells to K562 by 44% (72 ± 2.4%) to 40 ± 6.2%, n=15) and by 62% against Raji (50 ± 5.6% to 19 ± 4.4%). There was similar inhibition of cord LAK activity. Inhibition was dose dependent and independent of virus replication, since inhibition was observed with irradiated virus. The addition of live virus at the time of the cytotoxic assay or at the beginning of the IL-2 incubation did not affect LAK activity. By contrast, HSV stimulated cytotoxic activity against both targets in the absence of IL-2. The cytotoxicity of adult cells in the presence of live HSV (MOI=10) for 5-7 days increased from 3.3 ± 1.1% in the absence of virus and IL-2 to 19 ± 3.6% against K562 and from 2.2 ± 0.6% to 14 ± 4.4% against Raji targets (n=10). Cord cytotoxicity was stimulated but to a lesser degree. In sum, HSV inhibits LAK effector cell activity independent of virus replication. Further, HSV alone induces natural cytotoxicity which may represent a unique antiviral defense system.