M. Tersmette

ASSOCIATION BETWEEN BIOLOGICAL PROPERTIES OF HUMAN IMMUNODEFICIENCY VIRUS VARIANTS AND RISK FOR AIDS AND AIDS MORTALITY

49 individuals seropositive for human immunodeficiency virus (HIV) antibody were studied longitudinally for the relation between in-vitro properties of their sequential HIV isolates and clinical course before and after the development of AIDS. They were classified into three groups according to the syncytium-inducing capacity, replication rate, and host range of their HIV isolates. The most rapid progression to AIDS (median 15 months) and the lowest survival rate following AIDS diagnosis (median survival 12.5 months) were observed in individuals with high-replicating, syncytium-inducing HIV isolates, followed by individuals with high-replicating, non-syncytium-inducing isolates. In contrast, most individuals with low-replicating, non-syncytium-inducing HIV isolates remained symptom-free during the study period (median follow-up until AIDS diagnosis greater than 42 months), and the few individuals from this group in whom AIDS developed were still alive at the end of the study period (median survival greater than 34 months). In addition, AIDS patients from the three groups differed with respect to their symptoms. Zidovudine treatment in the symptom-free period seemed to delay the onset of AIDS in all risk groups, although stabilisation of CD4+ cell numbers was observed only in individuals with non-syncytium-inducing HIV variants.

Zidovudine sensitivity of human immunodeficiency viruses from high-risk, symptom-free individuals during therapy

Human immunodeficiency type 1 isolates from 18 initially symptom-free men who were treated with zidovudine for 2 years were investigated for drug sensitivity. At the start all the men had persistent core antigenaemia; the acquired immunodeficiency syndrome developed in 6 during the study. The polymerase chain reaction was used to detect mutations at residue 215 of reverse transcriptase, a mutation associated with reduced drug sensitivity. After 2 years 16/18 isolates were mutant. However, after about 6 months of treatment the mutation was detected in only 7 isolates, 4 from individuals who subsequently had AIDS. Limited direct virus sensitivity data correlated with the genetic data. The rate of appearance of the 215 mutation seemed to correlate with CD4 counts and viral virulence.

Conversion Rate towards a Syncytium-Inducing (SI) Phenotype during Different Stages of Human Immunodeficiency Virus Type 1 Infection and Prognostic Value of SI Phenotype for Survival after AIDS Diagnosis

The presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants is predictive for accelerated progression to AIDS. This study showed that a 4-year survival with AIDS also occurred significantly more often for patients who lacked SI variants. However, multivariate Cox analysis excluded the predictive value of SI viruses for rapid death as being independent from low CD4+ T cell counts. Incidence of appearance of SI variants was increased in persons with CD4+ T cell counts <500/microliter but remained constant in the strata of CD4+ T cell counts <500/microliter, excluding the possibility that loss of immune control is the only prerequisite for the development of SI HIV-1 variants.

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.

Biphasic rate of CD4+ cell count decline during progression to AIDS correlates with HIV-1 phenotype

ObjectiveTo determine the kinetics of decline of CD4+ lymphocytes in HIV-1-infected asymptomatic homosexual men. MethodsCD4 + lymphocytes were enumerated in a cohort of 187 HIV-1-infected initially asymptomatic homosexual men seen at 3-month intervals over 5 years. During follow-up, 45 men progressed to AIDS (excluding cases presenting with Kaposis sarcoma). Correlation between rate of CD4+ cell decline and presence of a particular HIV-1 biological phenotype was analysed in 43 participants. ResultsCD4+ cell counts declined slowly and continuously in HIV-1-seropositive men who remained asymptomatic during follow-up. A biphasic CD4+ cell count decline was observed in the group who developed AIDS: the decline was slow and steady (5.6 × 106/1 per month, similar to that observed in the asymptomatic group) until 18 months before AIDS diagnosis, but became three to five times faster thereafter. Rapid CD4+ cell decline was significantly related to syncytium-inducing, fast-replicating HIV-1 isolates; during the period of slow and steady CD4+ cell count decline, non-syncytium-inducing isolates were predominant. ConclusionsAt an average of 18 months preceding AIDS diagnosis, a three to fivefold increase in the rate of loss of CD4+ lymphocytes occurs, and may be related to the appearance of a more virulent HIV-1 phenotype.

Immunological and virological markers in individuals progressing from seroconversion to AIDS

Six men were selected from a large cohort of homosexual men participating in a study on HIV infection that was followed from seroconversion to AIDS. The patients were studied retrospectively for immunological functions of T cells, T-cell subset distribution and biological phenotype of HIV. A severe decrease in anti-CD3 monoclonal antibody (MAb)-induced T-cell proliferation at seroconversion was observed in two out of six men. After this acute phase, CD4+ T-cell numbers were in the normal range in the early asymptomatic period; the proliferative response was subnormal, whereas the capacity to generate cytotoxic T cells (CTL) was normal. From seroconversion on, CD4+CD29+ memory T-cell numbers were decreased to approximately 50% of normal values, which may contribute to loss of T-cell reactivity. In the asymptomatic phase only slow-replicating non-syncytium-inducing HIV variants were observed. The T-cell proliferative response further declined with the depletion of naive CD4+ CD45RA+ T cells and CD4+ T-cell numbers started to decline. This second decrease in T-cell function coincided with the emergence of more rapidly replicating, often (four out of six) syncytium-inducing variants. At diagnosis of AIDS, T-cell proliferation and CD4+ T-cell numbers were extremely low in five out of six patients and CTL function had declined in three out of five individuals tested. Circulating CD8+ cells had gradually shifted to an immature CD38+CD28- phenotype. Our findings support the theory that HIV-induced immune dysfunction allows for the emergence of virulent HIV variants associated with CD4+ cell loss and disease.

Human immunodeficiency virus infection studied in CD4-expressing human-murine T-cell hybrids

Human immunodeficiency virus (HIV) infection was studied by means of CD4-expressing human-murine T-cell hybrids, containing a variable amount of human chromosomes. Fusion of the HPRT- murine cell line BW5147 with human T-cell acute lymphoblastic leukemia or normal human blood cells resulted in a panel of human-murine T-cell hybrids. For this study, we used four hybrids containing all or several human chromosomes, which all expressed the CD4 antigen, as assessed by different anti-CD4 monoclonal antibodies (e.g., OKT4A, Leu-3a, and MT151) and, in addition, a variable number of other human T-cell antigens. For infection, HTLV-IIIB-infected H9 cells, pretreated with mitomycin C, and cell-free concentrated supernatants from these cells were used. In cells of inoculated cultures of the CD4+ T-cell hybrids, no viral antigen could be demonstrated. Culture supernatants of inoculated hybrids, except for an initial rise due to the virus inoculum, never showed reverse transcriptase activity above background. Cocultivation of these cell cultures with H9 cells did not result in detectable virus replication. Cocultivation of CD4-expressing hybrid cells with HIV-infected cells did not result in syncytium formation. Moreover, these hybrids were resistent to infection with vesicular stomatitis virus (VSV)-HIV pseudotypes. These findings imply that expression of the CD4 antigen on the cell surface is not sufficient for productive infection with HIV. The infectivity block observed in these hybrids seems to occur at the level of virus penetration, presumably at the stage of membrane fusion events.

Blood Donors with Indeterminate Anti‐p24gagReactivity in HIV‐1 Western Blot: Absence of Infectivity to Transfused Patients and in Virus Culture

Abstract. During a follow‐up period of 23–40 months, 7 regular blood donors had persistently, and 4 had intermittently indeterminate anti‐p24gagreactivity in human immunodeficiency virus (HIV)‐1 Western Blot. Serological testing and viral cultures revealed that these donors had no signs of infection for HIV‐1, HIV‐2, human T‐cell lymphotropic virus (HTLV)‐4, and HTLV‐1. Extensive interviewing and physical examination of these donors revealed neither risk factors, nor signs of HIV infection in the tested donors. Ten recipients, who were transfused with blood products from 6 of these 11 anti‐p24gag‐positive donors, were traced back. Six months after transfusion, no serological or clinical signs of HIV‐1, HIV‐2, or HTLV‐1 infection were observed in these patients. It is concluded that blood donors with persistent or intermittent anti‐p24gagreactivity in HIV‐1 Western Blot, without development of antibodies to other HIV‐encoded proteins in later blood samples, do not transmit the described retroviruses to transfused patients.

Detection of Early Anti-p24 HIV Responses in EIA- and Immunoblot-Negative Individuals

Abstract. A sensitive and specific radioimmunoprecipitation assay was developed for the detection and analysis of anti‐HIV antibody response in human sera with the use of 125I‐labelled purified HIV proteins with subsequent sodium‐dodecylsulfate gel electrophoresis (125I‐RIPA). The 125I‐RIPA was shown to be as specific but at least 1 log more sensitive with respect to the detection of gp41env and p24gag than the immunoblot analysis as tested in serum samples from several risk groups. Sequential sera were obtained from 9 individuals who seroconverted for HIV antibodies. In 4 individuals, antibody to p24gag was detected in earlier serum samples by the I25I‐RIPA than by EIA or immunoblot; in the other 5 individuals, the detection of p24gag concorded in enzyme‐linked immunosorbent assay (EIA), immunoblot and 125I‐RIPA. Moreover, in one of 78 randomly chosen EIA‐negative sera from individuals at high risk, antibodies to p24gag could be detected by the 125I‐RIPA. This early seroconversion was confirmed 3 months later by means of immunoblotting and EIA. The specificity of the 125I‐RIPA was further demonstrated by analyzing sequential EIA‐negative serum samples from 10 individuals at risk for AIDS, collected during 2 years at 3‐monthly intervals. All 80 serum samples were found to be negative in the 125I‐RIPA and the individuals revealed no signs of HIV infection. The I25I‐RIPA technique may be a valuable confirmatory assay in the serology of HIV infections. The sensitivity of this test provides a reliable measure of effective sensitivity when new‐generation screening tests are evaluated. In addition, this technique appears to be a specific and sensitive assay to detect antibodies to p24gag at very early stages of HIV infection.

Thermal Inactivation of Human Immunodeficiency Virus in Lyophilised Blood Products Evaluated by ID50 Titrations

Abstract. Inactivation of human immunodeficiency virus (HIV) in lyophilised small pool cryoprecipitate, factor VIII concentrate, prothrombin complex and C1 ‐esterase inhibitor concentrate by prolonged heat treatment (72 h, 60° C) was studied. Plasma products, inoculated prior to lyophilisation, had infectious titres ranging from 107 to 1010.5. Residual infectivity (TCID50) was assessed by multiple titrations on H9 cells in a macro system and subsequent detection of virus replication by determining reverse transcriptase activity. Kinetics of inactivation showed a biphasic pattern: during the first 8 h a variable TCID50 reduction up to 104.3 was observed, followed by an additional loss of 101–102.7 during the next 64 h. Heat treatment for 72 h resulted in a mean TCID50 reduction of 105. It is concluded that prolonged heat treatment may lead to the adequate prevention of HIV transmission by lyophilised plasma products.