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Effective antiretroviral treatment of HIV-1, defined as continuously undetectable virus in blood, has substantial effects on the infectiousness and spread of HIV.This paper outlines the assessment of the Swedish Reference Group for Antiviral Therapy (RAV) and Public Health Agency of Sweden regarding contagiousness of HIV-infected persons on antiretroviral therapy (ART).

Objective Interferon gamma release assays like Quantiferon Gold In-Tube (QFT) are used to identify individuals infected with Mycobacterium tuberculosis. A dichotomous cut-off (0.35 IU/ml) defines a positive QFT without considering test variability. Our objective was to evaluate the introduction of a borderline range under routine conditions. Methods Results of routine QFT samples from Sweden (2009-2014) were collected. A borderline range (0.20-0.99 IU/ml) was introduced in 2010 recommending a follow-up sample. The association between borderline results and incident active TB within 3 to 24 months was investigated through linkage with the national TB-register. Results Using the recommended QFT cut-off, 75.1 % tests were negative, 21.4% positive and 3.5% indeterminate. In total, 9% (3656/40773) were within the borderline range. In follow-up samples, individuals with initial results between 0.20-0.34 IU/ml and 0.35-0.99 IU/ml displayed negative results below the borderline range (amp;lt;0.20 IU/ml) in 66.1% (230/348) and 42.5% (285/671) respectively, and none developed incident TB. Among 6712 individuals with a positive initial test amp;gt;0.99 IU/ml, 65 (0.97%) developed incident TB within 3-24 months. Conclusions We recommend retesting of subjects with QFT results in the range 0.20-0.99 IU/ml to enhance reliability and validity of the test. Half of the subjects in the borderline range will be negative at a levelamp;lt;0.20 IU/ml when retested and have a very low risk of developing incident active TB. Funding Agencies|Swedish Research council [201602043]; Swedish Heart and Lung Foundation (Oscar II Jubilee foundation) [20150236, 20150237]

Objectives During the last decades, a changing epidemiological pattern of genital herpes simplex virus (HSV) infection has emerged. Primary infection is now caused as often by HSV-1 as by HSV-2. Once established, HSV can be reactivated leading to recurrent mucocutaneous lesions as well as meningitis. Why some otherwise immune-competent individuals experience severe and frequent recurrences is not known, and the immunological mechanism underlying recurrent symptomatic HSV infection is not fully understood. In this study, we investigate and characterise the immune response of patients with first episode of HSV genital infection and its relation to the frequency of symptomatic recurrences. Methods In this cohort study, clinical and immunological data were collected from 29 patients who were followed 1 year after presenting with a first episode of genital or meningeal HSV infection. They were classified by PCR and serology as those with primary HSV-1, primary HSV-2 and non-primary HSV-2 infection. Results HSV-specific interleukin(Il)-4 and Il-10 responses at first visit were higher in primary infected HSV-2 infected patients experiencing lower numbers of recurrences during subsequent year. Conclusions The median number of recurrences following primary HSV-2 genital infection may partly be predicted by the strength of an early HSV-specific IL-4 and IL-10 response.

There is a large and growing worldwide need for reliable tests to diagnose active and latent tuberculosis (TB). Improved methodology for identifying individuals with true latent TB (LTBI), particularly those with a recent infection, would pave the way for targeted prophylactic treatment. The traditionally used tuberculin skin test (TST) is unspecific and impractical. Interferon gamma release assays (IGRA) are more specific than the TST but, like that test, cannot discriminate either between recent and remote TB infection, or between these and a mere immunological memory of previous TB infection. The Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) combines long-term antigen stimulation of whole blood and flow-cytometric analysis with quantification of the expanded T-lymphoblasts and can also be employed for measurement of cytokine responses.

Neither the number of HIV-1 proviruses within individual infected cells in HIV-1–infected patients nor their genetic relatedness within individual infected cells and between cells and plasma virus are well defined. To address these issues we developed a technique to quantify and genetically characterize HIV-1 DNA from single infected cells in vivo. Analysis of peripheral blood CD4 + T cells from nine patients revealed that the majority of infected cells contain only one copy of HIV-1 DNA, implying a limited potential for recombination in virus produced by these cells. The genetic similarity between HIV populations in CD4 + T cells and plasma implies ongoing exchange between these compartments both early and late after infection.

The clinical picture of herpes simplex virus type 2 (HSV-2) infection includes genital blisters and less frequently meningitis, and some individuals suffer from recurrent episodes of these manifestations. We hypothesized that adaptive and/or innate immune functional deficiencies may be a major contributing factor in susceptibility to recurrent HSV-2 meningitis. Ten patients with recurrent HSV-2 meningitis were studied during clinical remission. For comparison, 10 patients with recurrent genital HSV infections as well as 21 HSV-seropositive and 19 HSV-seronegative healthy blood donors were included. HSV-specific T cell blasting and cytokine secretion were evaluated in whole blood cultures. HSV-2-induced NK cell gamma interferon production, dendritic cell Toll-like receptor (TLR) expression, and TLR agonist-induced alpha interferon secretion were analyzed. Patients with recurrent HSV-2 meningitis had elevated T cell blasting and Th1 and Th2 cytokine production in response to HSV antigens compared to those of patients with recurrent genital infections. A somewhat increased NK cell response, increased dendritic cell expression of TLR3 and -9, and increased TLR-induced alpha interferon responses were also noted. Contrary to our expectation, recurrent HSV-2 meningitis patients have increased HSV-specific adaptive and innate immune responses, raising the possibility of immune-mediated pathology in the development of recurrent HSV2 meningitis.

BACKGROUND: The purpose of this study was to estimate the cost-effectiveness of using individual-donor nucleic acid testing (ID-NAT) in addition to serologic tests compared with the sole use of serologic tests for the identification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) among blood donors in Sweden. STUDY DESIGN AND METHODS: The two strategies analyzed were serologic tests and ID-NAT plus serologic tests. A health-economic model was used to estimate the lifetime costs and effects. The effects were measured as infections avoided and quality-adjusted life-years (QALYs) gained. A societal perspective was used. RESULTS: The largest number of viral transmissions occurred with serologic testing only. However, the risks for viral transmissions were very low with both strategies. The total cost was mainly influenced by the cost of the test carried out. The cost of using ID-NAT plus serologic tests compared to serologic tests alone was estimated at Swedish Krona (SEK) 101 million (USD 12.7 million) per avoided viral transmission. The cost per QALY gained was SEK 22 million (USD 2.7 million). CONCLUSION: Using ID-NAT for testing against HBV, HCV, and HIV among blood donors leads to cost-effectiveness ratios that are far beyond what is usually considered cost-effective. The main reason for this is that with current methods, the risks for virus transmission are very low in Sweden.

We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag , env , rev , and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag , env , and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [ 3 H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost ( n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed ( n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [ 3 H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4 + T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8 + T-cell responses. There was strong correlation between the proliferative responses measured by the [ 3 H]thymidine uptake assay and CD4 + T-cell FASCIA-WB ( r = 0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4 + T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8 + T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [ 3 H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [ 3 H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

With new treatments of inflammatory diseases targeting key inflammatory pathways follows an increased risk for infections. The aim of the present study was to identify an immunological readout where consecutive immunizations induce reproducible immune responses. Such a method could be used as a tool to assess drug-induced immunomodulation in individual patients by comparing responses to the immunizations before and after introduction of a specific treatment. Importantly, the vaccine is merely used as a model antigen and protective immunity is not the primary aim of the method. Eleven volunteers were immunized with influenza vaccine three times, four weeks apart. In order to find the optimal readout for the method, immune responses to the immunizations were measured as circulating antigen-specific B-cells, serum antibody titers and avidity, T-cell proliferation and cytokine secretion. The first exposure to the influenza vaccine induced a stronger B- and T-cell responses than the consecutive immunizations. The second and third immunizations induced comparable but lower B-cell responses as measured by ELISPOT. In summary, we have measured immune responsiveness by using repeated immunizations with influenza virus vaccine as the model antigen. The induction of comparable B-cell responses after the second and third serial immunizations provides a possibility to investigate effects on immune responsiveness by immunomodulatory drugs. The method also allows humoral memory and immune competence per se to be studied on a cellular level in different patient groups.

In 2014 the Public Health Agency of Sweden and the Swedish Reference Group for Antiviral Therapy (RAV) conducted a review and analysis of the state of knowledge on the duration of follow-up after exposure to human immunodeficiency virus (HIV). Up until then a follow-up of 12 weeks after exposure had been recommended, but improved tests and new information on early diagnosis motivated a re-evaluation of the national recommendations by experts representing infectious diseases and microbiology, county medical officers, the RAV, the Public Health Agency, and other national authorities. Based on the current state of knowledge the Public Health Agency of Sweden and the RAV recommend, starting in April 2015, a follow-up period of 6 weeks after possible HIV-1 exposure, if HIV testing is performed using laboratory-based combination tests detecting both HIV antibody and antigen. If point-of-care rapid HIV tests are used, a follow-up period of 8 weeks is recommended, because currently available rapid tests have insufficient sensitivity for detection of HIV-1 antigen. A follow-up period of 12 weeks is recommended after a possible exposure for HIV-2, since presently used assays do not include HIV-2 antigens and only limited information is available on the development of HIV antibodies during early HIV-2 infection. If pre- or post-exposure prophylaxis is administered, the follow-up period is recommended to begin after completion of prophylaxis. Even if infection cannot be reliably excluded before the end of the recommended follow-up period, HIV testing should be performed at first contact for persons who seek such testing.

Received 11 February 2005; Revision 19 July 2005; Accepted 2 August 2005 Background: Assessment of CD8 1 T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. Methods: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and –6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE hi PI 2 )i n the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The 51 Cr-release assay and IFN-g ELISpot were performed by standard procedures. Results: A comparison with the Cr-release showed that the two assays were strongly correlated (r 5 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r 5 0.66; P < 0.01). Conclusion: This new assay provides both specific and sensitive results when employed for the detection of HIVspecific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels. q 2005 International Society for Analytical Cytology Key terms: CTL; HIV; cytotoxicity; flow cytometry

The assessment of cytokine production is an important component of studies of cell-mediated immune responses (CMI) to immunological challenges. In this study, we present a method to enhance the detection of cytokine-producing cells by allowing antigen-specific cells to expand in long-term culture. We investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on whole blood as well as peripheral blood mononuclear cells (PBMCs), cultured in the absence or presence of mitogens, superantigens or specific antigens, for intracellular cytokine production (IFNgamma, TNFalpha, IL-2, IL-4, IL-10 and IL-13) by CD4+ and CD8+ T lymphocytes using four-colour flow cytometry. Whole blood was diluted 1/1, 1/2, 1/5 and 1/10, and cultured for 6, 24, 48, 72 and 120 h in the presence of antibodies against the co-stimulatory molecules CD28 and CD49d, and, during the last 4 h of culture, in the presence of brefeldin A. Optimum conditions for detection of a high number of IFNgamma-positive cells were observed after 72 h of culture in blood diluted 1/10. Median frequencies of IFNgamma+ cells obtained after activation by PMA-ionomycin, PHA or SEA-B were 29.3%, 20.0% and 6.8% for CD4+ cells, and 67.8%, 20.6% and 6.8% for CD8+ cells. In blood samples diluted 1/5 or 1/10, and cultured in the presence of cytomegalovirus (CMV) or varicella-zoster virus (VZV), mean peak levels of 2.8% and 1.4% IFNgamma+CD4+ cells were recorded at 120 h. The levels of cells producing cytokines other than IFNgamma were generally much lower and, in the case of IL-4 and IL-13, difficult to distinguish from background levels recorded in cultures with medium only. Kinetic studies of cytokine production by PBMCs showed a pattern similar to that of whole blood with peak levels of IFNgamma-producing cells recorded at 72 h. The increased levels of IFNgamma production after culture for 72 h were due to an expansion of the numbers of cytokine-producing cells responsive to a specific stimulus. Antigen-specific cells are usually present only at low levels in peripheral blood and may not be detected following simple activation for a few hours. To reach a level of detection in such cases, culture of diluted blood for several days is recommended.

A new assay for the detection of specific cell-mediated immune (CMI) responses is described. Whole blood, diluted 1/10 in medium, was cultured in the presence or the absence of specific antigens. Results were assessed by flow cytometric analysis with or without immunophenotyping to detect proliferating lymphoblasts among cultured cells. Interferon-γ, IL-10, and IL-5 in culture supernatants are measured by ELISAs. The assay was evaluated using samples from 37 VZV-antibody-positive children with a history of chickenpox and samples from 15 seronegative children without a history of chickenpox; it displayed a sensitivity of 95% and a specificity of 100% for the detection of varicella-zoster virus (VZV)-specific CMI. The intraassay and interassay variations of the new test were lower than with the conventional assay for CMI, detecting thymidine incorporation in peripheral blood mononuclear cells (PBMCs). Cytokines were detected in only 70% of cultures from VZV-antibody-positive subjects. The cytokine response was restricted to IFN-γ in most cases. The Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA) is a precise and accurate yet simple and convenient test that can be readily employed for the examination of single samples as well as for large-scale studies.

Resting CD4(+) T lymphocytes are an important reservoir for human immunodeficiency virus type 1 (HIV-1) in treated patients with undetectable viremia. The knowledge of viral persistence in these cells is limited, however, for patients without treatment or patients for whom treatment is failing; therefore, this reservoir in such patients was characterized. Virus variants were characterized in 3 subjects who were followed-up from primary HIV-1 infection and 5 treatment-experienced subjects. No founder viral sequences and only a minority of the earlier identified drug-induced mutations were found in the resting T lymphocytes. Instead, the viral sequences were closely related to those detected simultaneously in plasma, except in 2 treatment-experienced subjects. Thus, a turnover and replenishment of this virus reservoir in peripheral blood is likely to occur in most persons with detectable viremia. However, infrequently the virus variants in plasma and resting T cells seem to be derived from independent sources.

A latent pool of HIV-1 is established early in memory CD4+ T lymphocytes and persists during antiretroviral therapy. Also, viral replication may continue in subjects despite undetectable viremia. However, it remains unclear whether this residual replication results in any significant sequence evolution. We were therefore interested in studying the viral evolution and HIV-1 DNA dynamics in subjects with primary infection receiving or not receiving early potent antiretroviral therapy. In 16 subjects, HIV-1 DNA load was monitored from 1 to 23 days, up to 1253 days, after onset of symptoms. Extensive sequential cloning and sequence analysis of the V3 region was performed in four subjects. In the treated subjects a continuous decline in the proviral load was found, corresponding to a half-life of about 6 months. As expected in newly infected individuals the founder virus populations showed high intrasubject sequence similarity. Also, a limited increase in the viral divergence was detected during the first 6 months in three treated subjects. Thereafter, no significant sequence changes were found despite analysis of a large number of clones. Our data thus suggest that early and successful therapy in compliant subjects with primary HIV-1 infection results in a highly restricted viral evolution and a decline in the proviral load close to the decay rate of human memory T lymphocytes.

Objective: To determine the sensitivity of 33 currently available and seven earlier tests for the detection of HIV or HIV antibody in primary HIV-1 infection, to estimate the duration of the 'window period' and the influence of early initiated antiretroviral treatment (ART). Design: A prospective cohort study of 38 patients with primary HIV-1 infection. ART was initiated at a median time of 13 (range 0-23) days after the onset of symptoms in 10 patients. Main outcome measures: The time from infection to onset of symptoms and from onset of symptoms to the appearance of HIV antibody as measured by 36 different tests, and the start and duration of viraemia, as detected by four different tests. Results: The illness appeared 13-15 days after infection in 12 of 15 determinable cases, and seroconversion was detected within 1-2 weeks after the onset of illness by 27 of 30 currently available tests for HIV antibody, in contrast to the 2-7 weeks or more needed by the old tests. HIV RNA appeared during the week preceding the onset of illness and was detected in all subsequent samples, except when ART had been initiated, which also induced a delay of the antibody response. Conclusion: Many tests for HIV or HIV antibody can now be employed for an early confirmation of primary HIV infection (PHI). Currently available screening tests proved much more sensitive than older tests, and seroconversion was usually detected within one month after infection. Consequently, in Sweden we now recommend only 3 months of follow-up after most cases of HIV exposure.

Objectives: To study the natural course of viremia during primary HIV infection (PHI). Method: Eight patients were followed from a median of 5 days from the onset of PHI illness. Plasma HIV-1 RNA levels were measured frequently and the results were fitted to mathematical models. HIV-1 RNA levels were also monitored in nine patients given two reverse transcriptase inhibitors and a protease inhibitor after a median of 7 days from the onset of PHI illness. Results: HIV-1 RNA appeared in the blood during the week preceding onset of PHI illness and increased rapidly during the first viremic phase, reaching a peak at a mean of 7 days after onset of illness. This was followed by a phase of rapidly decreasing levels of HIV-1 RNA to an average of 21 days after onset. Viral density continued to decline thereafter but at a 5- to 50-fold lower rate; a steady-state level was reached at a median of 2 months after onset of PHI. Peak viral density levels correlated significantly with levels measured between days 50 and 600. Initiation of antiretroviral treatment during PHI resulted in rapidly declining levels to below 50 copies/ml. Conclusions: This study demonstrates the kinetic phases of viremia during PHI and indicates two new contributions to the natural history of HIV-1 infection: PHI peak levels correlate with steady-state levels and HIV-1 RNA declines biphasically; an initial rapid decay is usually followed by a slow decay, which is similar to the initial changes seen with antiviral treatment.

A flow cytometric assay for the combined measurement of cell-mediated cytotoxicity and conjugate formation has been developed. Cytolysis is detected by propidium iodide uptake. Target cells, effector cells and conjugates between targets and effectors are separated by post-culture immunophenotyping and their scatter profiles. Pre-assay staining of cells is thus not required. Each cluster of cells can be further examined at the single-cell level by simultaneously performed additional immunophenotyping. Two applications were established: the assessment of NK cell activity against K562 cells and the evaluation of LAK cell cytotoxicity against both K562 and Daudi cells. A comparison with the standard 51Cr release assay for the detection of NK cytotoxicity showed that the two assays were strongly correlated, but the sensitivity of the flow cytometric assay was significantly higher.

Different patterns of temporal evolution in human immunodeficiency virus type 1 V3 and p17 regions are described for eight patients studied during the first years following primary infection. In samples from three patients, a rapid replacement of the major sequence occurred but the original sequence reappeared later simultaneously with clinical deterioration and increased plasma viral load.

Eighty-five subjects with symptomatic primary (P) human immunodeficiency virus (HIV) type 1 infection were analyzed in a retrospective cohort study to investigate the long-term clinical benefit of antiretroviral treatment during PHIV infection. Zidovudine treatment was initiated (PHIV treatment group) in 21 persons a median of 9 days after onset of PHIV symptoms and continued for a median of 55 days (range, 21‐99). Sixty-four subjects did not receive early antiretroviral treatment (PHIV nontreatment group). After follow-up for 3‐10 years, 33 subjects had developed AIDS and 22 subjects had died of AIDS. The median times for progression to AIDS and death were 6.4 and 9.1 years, respectively. Progression rates did not differ between the PHIV treatment and nontreatment groups. Zidovudine treatment initiated during PHIV infection did not improve long-term outcome after symptomatic PHIV infection. Primary (P) human immunodeficiency virus (HIV) type 1 infection is associated with extremely high levels of plasma HIV RNA, and immediate initiation of antiretroviral treatment is generally recommended [1]. Treatment of PHIV infection using combinations of antiretroviral drugs has, however, been evaluated only during short-term follow-up in a limited number of cases [2‐4]. Zidovudine therapy initiated during PHIV infection has been reported in three studies.

Objective To study viral heterogeneity at a very early phase of primary HIV-1 infection. Design Samples were drawn very early during primary HIV-1 infection. A virus population-based approach was used to study the viral heterogeneity in the C2-V3 and p17 regions. Methods Plasma samples (n = 33) were obtained before or shortly after onset of acute symptoms in 15 patients. In all subjects, the first sample was drawn within 10 days after onset of symptoms. Peripheral blood mononuclear cells (PBMC) were available in two patients. The number of polymorphic sites in the C2-V3 (15 patients) and p17 regions (eight patients) were determined by direct sequencing. Results The sequence heterogeneity was restricted in most patients, although only two out of 15 patients had a completely homogeneous C2-V3 sequence. However, pronounced individual differences were seen. Rapid sequence changes occurred during the first month in two patients. In one patient, the major DNA species at day 12 later became the major species in plasma. Conclusions The viral population is seldom completely homogeneous during primary HIV-1 infection, although the heterogeneity is restricted in most, but not all, patients. These individual differences do not seem to be due to sex or viral subtype. Rapid changes of the virus population may occur during primary HIV-1 infection. The DNA species detected in PBMC do not only represent earlier viral quasispecies but are also a potential source of future viral RNA species.

The purpose of this review is to outline the clinical signs of the symptomatic HIV-1 seroconversion, with the emphasis on skin and mucous membranes. Our own experience is discussed in relation to other reports in the literature. Major findings are fever and pharyngitis with several patients also showing nonoozing, superficial ulcers or rather epithelial defects of the oral, genital, or anal mucosa. Uniform skin eruptions usually consist of circumscribed, erythematous, dull to bright red, macular or maculopapular lesions that were oval or rounded in shape ranging from a few millimeters to 1 cm in diameter on the upper thorax, particularly within the collar region and also on the face, forehead, and scalp. Skin lesions are sparse or absent at the periphery of the extremities. In the most typical cases, the exanthema appears following 48–72 h of fever, with a peak during one day, persisting for 5–8 days and a gradual clearing concurrently with the general recovery of the patient. Skin biopsy findings do not ...

Viremia during human immunodeficiency virus type-1 (HIV-1) infection results in progressive impairment of several components of the immune system. Here a unique model of repeated treatment interruptions (TIs) was used with the aim to reveal the effect of controlled short-term viremia on innate stimuli responsiveness and circulating dendritic cells (DCs). Sequential peripheral blood samples from HIV-1-infected patients on combination antiretroviral therapy, subjected to repeated TI cycles as part of a therapeutic DNA vaccination study, were analyzed. In vitro responsiveness of peripheral blood mononuclear cells to toll-like receptor (TLR) stimuli was analyzed by cytokine secretion, and frequencies of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were monitored by flow cytometry. These parameters were found not to be significantly different between the vaccinated and placebo groups. Instead, independent of vaccination altered in vitro TLR responsiveness was observed in parallel with TI cycles. TLR7/8-triggered secretion of IL-12 and IFN-α, as well as TLR9-triggered secretion of IL-12, was hyperactivated. In contrast, expression of IFN-α after TLR9 stimulation decreased during the initial cycle of TI. Reduced frequencies of pDCs and mDCs, compared with baseline, were noted before and during the second TI, respectively. Furthermore, spontaneous ex vivo release of IL-12 from PBMC was noted during cycles of TI. In conclusion, these results suggest that consequences of short-term TI include dysregulated TLR responses and fluctuations in the frequencies of circulating DCs. Knowledge of these immunological factors may influence the continuation of stringent treatment schedules during HIV infections.

Objective: To retrospectively compare the accuracy of combinations of two enzyme-linked immunosorbent assays (ELISAs) with a Western blot based strategy for identification of human immunodeficiency virus type 1 (HIV-1) seropositivity. Materials and methods: 48,977 sera, sent to the National Bacteriological Laboratory, Stockholm, Sweden, for HIV antibody determinations between October 1988 and June 1993, were investigated. All samples were tested in parallel with two different ELISAs, either Abbott Recombinant HIV-1 EIA and Wellcozyme Recombinant Anti-HIV-1 EIA, or Enzygnost Anti-HIV-1/2 and Wellcozyme Recombinant Anti-HIV-1 EIA, or Enzygnost Anti-HIV-1/2 and Wellcozyme Anti-HIV-1+2 EIA. 1565 sera repeatedly reactive by one or both ELISAs were investigated by Western blot (WB). Furthermore, a total of 2820 referred sera, screen reactive at primary laboratories but negative on our combinations of two ELISAs were analysed by WB. Results: Out of 1244 truly HIV antibody positive samples 1203 were WB positive and 41 (3.4%) were WB indeterminate. A sensitivity of 100% was obtained by all three combinations of two ELISAs on examination of these 1244 sera including repeated testing of 5 samples with initially discrepant results. Among 2820 sera from HIV-negative individuals 649 (23%) sera were WB indeterminate. The combination of Enzygnost (indirect test with synthetic peptides) and Wellcozyme (sandwich test with recombinant and synthetic peptides) Anti-HIV 1+2 EIAs was 100% specific when used for analysis of 9111 sera. One of 30,323 HIV-1 antibody negative sera tested was initially reactive on both Enzygnost Anti-HIV 1+2 and Wellcozyme Recombinant Anti-HIV-1 EIA (competitive assay) but was found to be negative by repeated testing, resulting in a specificity of 100% for that combination of ELISAs. Abbott Recombinant Anti-HIV-1 EIA (indirect assay) combined with Wellcozyme Recombinant Anti-HIV-1 EIA was initially falsely reactive with 12 of 8272 sera of which 6 were repeatedly reactive. Conclusions: This large-scale evaluation demonstrates that combinations of two ELISAs based on different test principles and antigens increase the accuracy of the HIV antibody determination and could be used as an alternative or complement to WB.

Background Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Non-human primate models aid to design novel vaccine strategies. We analyzed therefore the dynamics of cellular immune responses induced by a bacille Calmette-Guerin (BCG) / recombinant BCG (rBCG, perfringolysin plus Ag85A/B, TB10.4) prime, subsequent adenoviral (Ad35 expressing Ag85A/B and TB10.4) boosts followed by a challenge with M. tuberculosis (Mtb, Erdman strain) in peripheral blood mononuclear cells from rhesus macaques. T-cell compartment mobilization was defined by CD45RA/CCR7/CD27/CD28 expression on CD4+, CD8αα+ and CD8αβ+ T-cells and functional analysis (STAT-5 phosphorylation) was carried out using multicolor flow cytometry. CD4+ as well as CD8+ T-cells showed a marked decrease in IL-7 receptor alpha-chain (IL-7Rα) expression in vaccinated animals (n=12/12) after the first adenoviral boost, but not in control animals (who received saline). BCG- and rBCG- vaccinated animals showed a dramatic decrease of precursor (CD45RA+CCR7+) and subsequent increase of activated central memory (CD45RA-CCR7+) T-cells in the CD4+, CD8αα+ and CD8αβ+ T-cell compartments 2 weeks after Mtb challenge. In contrast, this pattern was observed in control animals only 7 weeks after Mtb challenge. Expression of IL-7Rα was markedly reduced in CD4+ T-cell subsets two weeks after Mtb challenge in BCG-vaccinated animals, yet not in rBCG-vaccinated animals or control animals. These data show that vaccination profoundly shapes the dynamics of immune memory T-cells associated with Mtb infection.

Background There is a need for reliable markers to diagnose active and latent tuberculosis (TB). The interferon gamma release assays (IGRAs) are compared to the tuberculin skin test (TST) more specific, but cannot discriminate between recent or remote TB infection. Here the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA), which quantifies expanded T-lymphoblasts by flow-cytometric analysis after long-term antigen stimulation of whole blood, is combined with cytokine/chemokine analysis in the supernatant by multiplex technology for diagnosis of Mycobacterium tuberculosis (Mtb) infection. Methods and Findings Consecutive patients with suspected TB (n = 85), with microbiologically verified active pulmonary TB (n = 33), extra pulmonary TB (n = 21), clinical TB (n = 11), presumed latent TB infection (LTBI) (n = 23), patients negative for TB (n = 8) and 21 healthy controls were studied. Blood samples were analyzed with FASCIA and multiplex technology to determine and correlate proliferative responses and the value of 14 cytokines for diagnosis of Mtb infection: IFN- γ, IL-2, TNF-α, IP-10, IL-12, IL-6, IL-4, IL-5, IL-13, IL-17, MIP-1β, GM-CSF, IFN-α2 and IL-10. Cytokine levels for IFN-γ, IP-10, MIP-1β, IL-2, TNF-α, IL-6, IL-10, IL-13 and GM-CSF were significantly higher after stimulation with the Mtb specific antigens ESAT-6 and CFP-10 in patients with active TB compared to healthy controls (p

Immunotherapy in patients with HIV-1 infection aims to restore and broaden immunological competence, reduce viral load and thereby permit longer periods without combined antiretroviral treatment (cART). Twelve HIV-1-infected patients on cART were immunized on the skin with DNA plasmids containing genes of several HIV-1 subtypes with or without the addition of hydroxyurea (HU), or with placebo. The mean net gain of HIV-specific CD8+ T cell responses were higher and broader in the HIV DNA vaccine groups compared to non-vaccinated individuals (p

A better understanding of similarities and differences in the composition of the cellular immune system in non-human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8alpha, CD8beta, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin-7 receptor alpha-chain (IL-7Ralpha) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti-CD3, -CD4, -CD25, -FoxP3, and -IL-7Ralpha monoclonal antibodies. Responsiveness to IL-7 was gauged in a signal transducer and activation of transcription 5 (STAT-5) phosphorylation assay. Human and NHP PBMCs showed a similar T-cell composition pattern with some remarkable differences. Similarities: human and NHP CD4(+) and CD8(+) cells showed a similar STAT-5 phosphorylation pattern in response to IL-7. Multicolour flow cytometric analysis identified a CD4(+) CD8alphaalpha(+) CD8alphabeta(+) T-cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4(+) T-cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5.16% in CD3(+) T cells) of CD8alphaalpha(+) T cells when compared with human donors (1.22% in CD3(+) T cells). NHP CD8alphaalpha(+) T cells produced tumour necrosis factor-alpha / interferon-gamma (TNF-alpha/IFN-gamma) or TNF-alpha, whereas human CD8alphaalpha(+) T cells produced simultaneously TNF-alpha/IFN-gamma and IL-2. A minor percentage of human CD8(+) T cells expressed CD25(bright) and FoxP3 (0.01%). In contrast, 0.07% of NHP CD8(+) T cells exhibited the CD25(bright) FoxP3(+) phenotype. PBMCs from NHPs showed less IL-7Ralpha-positive events in all T-cell subsets including CD4(+) Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long-lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

We investigated HIV-1 vaccine-induced lymphoproliferative responses in healthy volunteers immunized intradermally or intramuscularly (with or without adjuvant granulocyte-macrophage colony-stimulating factor [GM-CSF] protein) with DNA expressing HIV-1 gag, env, rev, and rt at months 0, 1, and 3 using a Biojector and boosted at 9 months with modified vaccinia virus Ankara (MVA) expressing heterologous HIV-1 gag, env, and pol (HIV-MVA). Lymphoproliferative responses to aldrithiol-2 (AT-2)-inactivated-HIV-1 antigen were tested by a [(3)H]thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA-WB) 2 weeks after the HIV-MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed (n = 14). Thirty-five of 38 (92%) vaccinees were reactive by the [(3)H]thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4(+) T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8(+) T-cell responses. There was strong correlation between the proliferative responses measured by the [(3)H]thymidine uptake assay and CD4(+) T-cell FASCIA-WB (r = 0.68; P0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4(+) T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8(+) T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the [(3)H]thymidine uptake assay. The overall HIV-specific T-cell proliferative response in the vaccinees employing any of the assays was 100% (38/38). A standardized FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [(3)H]thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings.

Methods Healthy volunteers were immunized intradermally or intramuscularly (with or without adjuvant GM-CSF protein) with DNA expressing HIV-1 gag, env, rev and rt at months 0, 1 and 3 using a Biojector and were boosted at nine months with an MVA expressing heterologous inserts of HIV-1 gag, env and pol genes. Lymphoproliferative responses to AT-2 inactivated HIV-1 antigen were tested by a 3H-thymidine uptake assay and a flow-cytometric assay of specific cell-mediated immune-response in activated whole blood (FASCIA-WB) two weeks after HIV-1 MVA boost (n = 38). A FASCIA using peripheral blood mononuclear cells (FASCIA-PBMC) was also employed during the later part of the study (n = 14).

Serum samples of 120 patients in different stages of chronic human immunodeficiency virus type 1 (HIV-1) infection, 11 patients with primary HIV-1 infection (PHI), and 49 HIV-1 seronegative homosexual men were analyzed for tumor necrosis factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha), and HIV-1 p24 antigen. Increased levels of IFN-alpha and TNF-alpha were found in some, but not all, cases with PHI. During progressing disease IFN-alpha occurred in serum with increasing frequency and concentration. Raised levels of TNF-alpha were found in all stages of chronic infection, but were less common in patients with AIDS than were raised levels of IFN-alpha. The levels of the two substances were not correlated. There was a correlation between IFN-alpha, but not TNF-alpha, and the occurrence of HIV-1 p24 antigen in serum. These results suggest that IFN-alpha and TNF-alpha are induced by different agents during HIV-1 infection. The findings would be consistent with the hypothesis that IFN-alpha and TNF-alpha are counteracting forces that have important down- and upregulatory effects, respectively, on HIV-1 replication in vivo.

In January 2006, an after-school carer in Stockholm was diagnosed with open pulmonary tuberculosis (TB) after having been symptomatic for 3 months. The aim of this paper is to illustrate the difficulties encountered in estimating recent transmission of TB among children in an immigrant school population. A tuberculin skin test was performed on 261 pupils aged 6-15 y and an additional interferon-gamma release assay was performed on 20 children. In total, 76% of the children were born in Sweden; however, 95% of the parents originated from countries with TB incidence >25/100,000. Three active TB cases were identified, 1 of whom was culture-positive with the same strain as the index case. Latent tuberculous infection (LTBI) was diagnosed in 35 children. However, the increased risk of earlier infection in this population makes it difficult to evaluate when transmission occurred. The magnitude of recent transmission from the index case will thus be uncertain and indications to treat less clear.

BACKGROUND: BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). METHODS AND FINDINGS: Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (

Homosexual men with symptomatic primary HIV-1 infection displayed a pronounced lymphopaenia with significantly depressed numbers of CD3+, CD4+ and CD8+ cells and B cells during the first week of illness. Subsequently, the CD8+ cell counts rose in parallel with numbers of CD3+ cells, atypical lymphocytes and activated (CD38+ and HLA-Dr+) cells to attain maximal levels about a month following onset of illness. In contrast CD4+ and B cell numbers remained low for an extended period of time. Early signs of a host response included a transient appearance of interferon-alpha in the blood and raised levels of neopterin and beta 2-microglobulin (beta 2-M). Neither CD4+/CD8+ cell ratio nor beta 2-M resumed completely normal values during a follow-up period of 2 years. These findings shed some light on pathogenetic events during early HIV-1 infection and suggest that the infection, following the acute symptomatic stage, usually enters a stage of chronic active rather than latent infection.

The kinetics of appearance and specificity of HIV-1 neutralizing antibodies was studied in four individuals. HIV-1 was isolated during symptomatic primary HIV-1 infection and repeatedly thereafter, and tested against autologous sera collected in parallel. Our patients developed isolate-specific low-titer neutralizing antibodies within 2-4 weeks, and the titers to the first isolates increased with time. We documented the emergence of virus variants with reduced sensitivity to neutralization by autologous, but not heterologous, sera in three patients. These virus variants were not, however, resistant to neutralization per se, since they were readily neutralized by the positive control serum. Our patients did not develop antibodies capable of neutralizing the new virus variants during the observation period. This suggests either a failure of the immune system to respond to the new virus variants or a mechanism by which the virus evades detection by the immune system. The emergence of neutralization-resistant virus variants was not directly correlated with disease progression since two patients have remained asymptomatic after the emergence of such virus variants. It is, however, likely that the emergence of virus variants which the patient fails to neutralize in the long run contributes to disease progression.

In preparation for monitoring of vaccine-induced responses, we determined HIV-specific cell-mediated immune responses in 17 treatment naive HIV-1 infected individuals with > 400 CD4+ T cells/ml for at least 5 years including 9 patients with low viral load (VL, 5000 copies/ml). HIV-1 specific IFN-γ-production and cytolytic activity were higher in subjects with low VL. The differences between the two groups were statistically significant for CD4+ Tcell responses to Gag and Nef peptides, tested by a longterm (48 h) ICS assay and of border-line significance for the Gag-specific cytolytic responses measured by a flowcytometry assay and a chromium release assay. We also found a significant inverse correlation between VL and IFN-γ-production by CD8+ T-cells in response to Gag as measured by ICS. The ELISpot IFN-γ response was not significantly different in patients with high and low VL. During a median follow-up period of 2.4 years, 6 of 8 subjects with high VL and 1 of 9 with low VL showed decreasing CD4+ T-cell counts, and ARV treatment was more frequently initiated in the former patient group (5 of 8 versus 1 of 9). The CD4 and CD8 T cell immune responses found to be associated with low VL and stable CD4 counts may be of importance for protection. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Cytokine profile assessment is important to characterize immune responses to pathogens. To identify optimal time points for determination of cytokine profiles, we diluted whole blood 1:10, to enable daily cytokine measurements during one week. Cultures for 10 blood donors were set up in the presence of phytohemagglutinin (PHA), cytomegalovirus (CMV) or Candida. Supernatant levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, interferon-gamma (IFN-gamma), granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha), were determined by multiplex technique, and intracellular cytokine staining (ICS) was employed to detect IFN-gamma, IL-2, IL-4 and IL-13 in CD3+ cells. The multiplex analysis detected representative cytokine profiles for the majority of the cytokines on day 7 by identifying peak levels or good correlation with peak levels, with the exception of IL-2 and TNF-alpha in PHA and CMV cultures and IL-10 in PHA cultures. For these cytokines an extracellular measurement on day 2-3 would be appropriate. The intracellular cytokines showed distinct kinetics for IFN-gamma and IL-2, while IL-4 and IL-13 were not detected at all with ICS. In conclusion, the combination of whole blood cultures with multiplex analysis is a simple and powerful tool that can be used to identify detailed cytokine profiles of specific cell-mediated immune responses.

The level of CD4(+) T-lymphocytes represents a useful marker with which to monitor the progression of HIV infection. Sex and geographical differences in the reference values of lymphocyte subsets have been reported. We have compared two flow cytometric methods (MultiSET and SimulSET) for the quantification of lymphocyte subsets using whole blood from 92 HIV seropositive and 241 seronegative adults, and determined the reference values of lymphocyte subsets in HIV seronegative Tanzanian subjects. In seronegative Tanzanian subjects, the percentages of CD3(+) and CD4(+) T-lymphocytes and the CD4(+):CD8(+) T-lymphocyte ratios were lower while the percentage of natural killer cells was higher compared to the levels of the corresponding parameters reported for Europeans. Seronegative Tanzanian females had significantly higher levels of CD3(+) and CD4(+) T-lymphocytes and CD4(+):CD8(+) T-lymphocyte ratios compared to seronegative males. The correlation coefficients of CD3(+), CD4(+) and CD8(+) T lymphocyte counts and percentages obtained by the two flow cytometric methods were high. The median values of the number of CD4(+) T-lymphocytes obtained by the two methods were not significantly different. In conclusion, determination of the reference values of lymphocyte subsets in HIV seronegative Tanzanian adults showed significant sex differences and differences in percentage values compared to those reported in certain other geographical areas. There was acceptable agreement in the levels of CD4(+) T-lymphocyte values obtained by the two flow cytometric methods.

A broad antibody panel was used for immunophenotyping of human immunodeficiency virus type 1 (HIV-1)-infected patients who were long-term nonprogressors (LTNP). The LTNP were compared with patients in the early phase of infection and patients who had progressed to advanced immunodeficiency. Changes in CD8 + subset distribution were observed mainly at acquisition of HIV-1 infection, whereas CD4 + subset changes appeared during progression of HIV-1 infection. The decreasing levels of CD4 + cells were characterized by an increasing frequency of cells expressing the activation markers HLA-Dr and CD45RO but not the CD28 surface antigen. The LTNP exhibited significant changes compared to HIV-negative patients in almost all markers. Compared to patients in the early phase of infection, the only difference was a relatively lower frequency of CD4 + cells expressing CD26 among the LTNP. The results show that HIV-1-infected persons who have no signs of immunodeficiency despite many years of infection have an immunophenotypic pattern that is substantially different from that of noninfected persons. Despite the long duration of infection, the LTNP exhibit a pattern similar to that of newly infected persons, with the exception of lower expression of CD26 on CD4 + cells.

Background: Comparisons of progression in HIV-1 infection between injecting drug users (IDU) and homosexual men have been inconclusive due to short follow-up periods, often with less well-defined starting points and endpoints. In addition, comparisons of survival after infection have been to some extent obscured by higher non-AIDS mortality in IDU. Methods: In a retrospective cohort study, homo-/bisexual men and IDU were followed, with dates of seroconversion defined within ± 1 year by a previously negative HIV antibody test. Endpoints were CD4 cell count below 200 x 10 6 /l, AIDS, and death from AIDS. Results: Sixty-three homo-/bisexual men and 125 IDU fulfilled the entry criteria, with no significant differences in age at or date for seroconversion. Mean follow-up times were 6.7 and 7.0 years, respectively. The homo-/bisexual group had a significantly accelerated progression rate to all three endpoints: time to CD4 cell count below 200 x 10 6 /l (P= 0.002), to AIDS (P = 0.0003), and to death from AIDS (P< 0.0001). Adjusting for age and sex only made marginal alterations. Ten years after infection, 54% of homosexual men had developed an AIDS-defining condition and 51% had died from AIDS, whereas the corresponding percentages in the IDU group were 26 and 15%, respectively. There was, however, no difference in overall mortality due to an almost constant, non HIV-related, yearly mortality of some 4% in IDU. Conclusions: In our cohort there was a highly significant difference in disease progression and death from AIDS between homo-/bisexual men and IDU. This difference was proposed to be due to the transmission route determining the initial immune response and suggested that this route may have played a more important role than virus variability on the subsequent prognosis.

SUMMARY The proliferative responses of distinct cell subsets from healthy, bacille Calmette–Guérin (BCG)-vaccinated blood donors were assessed after in vitro stimulation with live or UV-killed Mycobacterium tuberculosis and Myco. avium or with soluble extracts obtained from either mycobacterial species. Proliferation of cell subsets was evaluated by flow cytometric determination of 5-bromo-2′-deoxy-uridine incorporation into DNA and simultaneous identification of surface phenotypic markers. In the presence of monocytes, the response to whole (live or killed) bacteria was characterized by a predominant proliferation of CD4+αβ+ T cells and, to a lesser extent, of CD8+αβ+ T cells. Proliferation of CD8+αβ+ T cells was primarily elicited by live rather than killed bacilli (P

Objective: To investigate the prognostic significance of symptomatic primary HIV-1 infection. Design: Prospective study of homosexual men seroconverting to HIV in 1985 and 1986. Patients were followed up at least three times yearly with clinical examinations and T cell subset determinations for an average of 7.2 years. Setting: Research project centred on attenders for treatment and screening for HIV at the Karolinska Institute, Stockholm. Subjects—19 patients presenting with a glandularfever-like illness associated with seroconversion to HIV and 29 asymptomatic seroconverters. Main outcome measures: Progression to Centers for Disease Control and Prevention stage IV disease, CD4 cell count below 200 × 106/l, AIDS, and death from AIDS. Results: Symptomatic seroconverters were significantly more likely to develop Centers for Disease Control and Prevention stage IV disease (95% (upsilon) 66%), CD4 cell counts below 200 × 106/l (84% (upsilon) 55%), and AIDS (58% (upsilon) 28%) and die of AIDS (53% (upsilon) 7%). Conclusion: A glandular-fever-like illness associated with seroconversion to HIV-1 predicts accelerated progression to AIDS and other HIV related diseases.

The biological phenotype of HIV-1 isolates from 21 subjects with primary HIV-1 infection was determined in primary lymphocytes and monocyte-derived macrophages as well as in three T-cell lines (MT-2, HUT78, and C8166). Seven isolates (33%) replicated on at least one cell line and were classified as rapid/high (n = 6) or intermediate (n = 1), while 14 isolates (67%) did not replicate in cell lines and were classified as slow/low. All isolates replicated in primary monocyte-derived macrophage cultures. Studies on 10 pairs consisting of index case and seroconverting sexual partner showed that the biological phenotype of HIV-1 was unchanged in nine transmissions, including 5 pairs where a rapid/high virus was transmitted. Most individuals (5 of 6) infected by a rapid/high virus retained this phenotype after seroconversion. These individuals also had significantly lower CD4 lymphocyte counts 1-3 years after infection. Thus, in contrast to what has been suggested by others, we found that HIV-1 variants with rapid/high phenotype were usually not suppressed, neither during sexual transmission nor after seroconversion in the new host. These findings have important implications for the understanding of HIV-1 transmission and pathogenesis as well as for vaccine design.