J. Steven McDougal

The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain

The isolation of clones encoding the human surface protein T4, and the expression of the T4 gene in new cellular environments, have enabled us to examine the role of this protein in the pathogenesis of AIDS. Our studies support a mechanism of AIDS virus infection that initially involves the specific interaction of the AIDS virus with T4 molecules on the cell surface. This association can be demonstrated on T4+ transformed T and B lymphocytes as well as epithelial cells. Furthermore, the presence of T4 on the surface of all human cells examined is sufficient to render these cells susceptible to AIDS virus infection. Our data suggest that the T4-AIDS virus complex is then internalized by receptor-mediated endocytosis. Finally, we find that the T4 gene is expressed in the brain as well as in lymphoid cells, providing an explanation for the dual neurotropic and lymphotropic character of the AIDS virus. In this manner, a T lymphocyte surface protein important in mediating effector cell-target cell interactions has been exploited by a human retrovirus to specifically target the AIDS virus to populations of T4+ cells.

Identification of the residues in human CD4 critical for the binding of HIV.

The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.

Surveillance for HIV-1 incidence using tests for recent infection in resource-constrained countries.

Over the past few years, several assays have been developed for the purpose of estimating HIV-1 incidence from cross-sectional population surveys. The tests detect features of the evolving virological or immunological response to HIV-1 infection that distinguish recent from established infection. Surveillance programmes that collect specimens from population surveys for HIV-1 prevalence can apply some of these tests to the same specimen sets to estimate incidence. We describe these tests and discuss the principle and strategy for implementation of a testing programme for recent infection in surveillance settings. Test-specific prerequisites, such as calibration, validation, and quality assurance, and other test-specific performance characteristics that may influence interpretation, epidemiological considerations that may guide application, and practical operational considerations for implementation in surveillance settings are considered. When properly and judiciously applied, the capacity to estimate incidence from existing programmes that conduct surveillance for prevalent HIV-1 infection will enhance the capacity for more precise and timely analysis of the dynamics of the epidemic and the effectiveness of public health interventions.

ANTIBODY TO LYMPHADENOPATHY-ASSOCIATED VIRUS IN HAEMOPHILIACS WITH AND WITHOUT AIDS

To determine the incidence of antibody to lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III) serum was collected from 25 Georgian patients with hemophilia A 4 patients with hemophilia B 15 hemophiliacs with acquired immunodeficiency syndrome (AIDS) 2 brothers with hemophilia A and lymphadenopathy and their parents the wife and 3 adult children of a hemophiliac with AIDS and 5 controls. Antibody to LAV proteins p25 and p41 was detected in the serum of 18 (72%) of the 25 patients with hemophilia A who used factor VIII in 1980-82 but had no symptoms associated with AIDS. White cell total lymphocyte T lymphocyte T helper and suppressor cell counts and the T helper/T suppressor ratio were not significantly different between seropositive and seronegative hemophilia A patients. The amount of factor VIII concentrate used by seropositive patients however was significantly higher than the amount used by seronegative patients. All patients who received over 150000 units of factor VIII in 1980-82 were seropositive. None of the 4 hemophilia B patients all of whom were being treated with factor IX concentrates had antibodies to LAV/HTLV-III. Of 15 AIDS patients with hemophilia A 5 had antibody to LAV p25 and p41 proteins. All 15 had severe immunosuppression and had received large amounts of factor VIII concentrates. Antibody was not found in the wife and offspring of the hemophilia patient with AIDS or in the parents of the 2 siblings with lymphadenopathy. Patients with hemophilia B in general have not manifested the degree of immune abnormalities seen in hemophilia A. Comparison of these results with data from other studies suggests that the prevalence of LAV antibody in hemophiliacs is greater than that in homosexuals and the same as that found in lymphadenopathy-associated syndrome. These findings support the belief that LAV may be transmitted by some blood products.

INACTIVATION OF HTLV-III/LAV DURING PLASMA FRACTIONATION

The authors present the results of studies on the stability of human T-lymphotropic virus type III (HTLV-III) infectivity to ethanol. In the 1st experiment the residual infectivity of HTLV-III seeded into RPMI-1640 medium or plasma and exposed to ethanol for 10 minutes was measured. In 1 single-donor plasma sample at -23 degrees C infectivity was reduced 10(2)-fold whereas in 3 samples at -5 degrees C infectivity was reduced by 10(2.7) and 10(0.7) in 2 and not reduced in the 3rd sample. In contrast infectivity in medium at 23 degrees C was reduced 10(3)-fold. These results suggest that HTLV-III is more stable to 20% ethanol in plasma than in medium. Some characteristic of the plasma possibly lipid content or protein composition can stabilize the virus to ethanol. Lower temperatures may also stabilize the virus. The 2nd experiment was designed to simulate the precipitation of fractions I+II+III by 20% ethanol at pH 6.9 and -5 degrees C as in a common manufacturing procedure. Samples seeded with HTLV-III were drawn periodically after addition of ethanol to a final concentration of 20% then immediately diluted 1:10 in RPMI-1640 medium and frozen to stop the inactivation. Virus in the control plasma (no ethanol) lost very little infectivity over a 2-hour period while infectivity fell more than 10(3.5) in 5 minutes in an identical sample containing 20% ethanol. Compared with a pH 7 control infectivity was reduced 10(3)-fold in 10 minutes at pH 5.7. These findings indicate that steps in large scale plasma fractionation especially ethanol at -5 degrees C and low pH reduce the risk of infected plasma products.

T-cytotoxic/suppressor cell phenotypes in a group of asymptomatic homosexual men with and without exposure to HTLV-III/LAV

The number of lymphocytes bearing the Leu 2+ or T8+ (suppressor/cytotoxic) phenotype is elevated in asymptomatic homosexual men. By two-color immunofluorescence using paired monoclonal antibodies (alpha-Leu 2 and alpha-Leu 15, alpha-Leu 2 and alpha-Leu 7, alpha-Leu 7 and alpha-Leu 11), we enumerated phenotypic subpopulations that are associated with cytotoxic, suppressor, or natural killer function. Both cytotoxic (Leu 2+15-) and suppressor (bright Leu 2+15+) cell populations are elevated in homosexual men. Homosexual men who have been exposed to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) have higher numbers of Leu 2+15- and Leu 2+7- cells than homosexual men who have not been exposed. Phenotypic subpopulations (dim Leu 2+ Leu 15+ and Leu 7-11+) that are associated with the most potent natural killer activity (against K562 target cells) were not found to be elevated in homosexual men.

CASE FOR CONCLUDING THAT HEAT-TREATED, LICENSED ANTI-HAEMOPHILIC FACTOR IS FREE FROM HTLV-III

Data available from over half a million samples tested in the US suggest that about 0.2% of blood and plasma donors are repeatedly reactive for antibody to lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III) when tested with enzyme-linked immunosorbent assay (ELISA). Thus about 0.2% of the units in plasma pools used to produce antihemophilic factor would contain LAV/HTLV-III with 10(8) particles/ml or 10 (6) ID/ml. Large plasma pools will have a final average virus concentration of 2000 ID/ml because of the 1 in 50 dilution of infectious units with noninfectious units. If viral concentration without loss of infectivity were to occur along with concentration of antihemophilic factor then the factor would contain 2000 ID/ml x 100. Thus any treatment of antihemophilic factor aimed at reducing the risk of transmitting LAV/HTLV-III should be capable of inactivating 2 x 10(5) ID/ml of the virus. (It is noted that all of these assumptions constitute a worst-case scenario especially since the ELISA test is known to have a high proportion of false positives.) Inactivation procedures that reduce LAV/HTLV-III infectivity by 5 logs should therefore provide assurance that the preparation is no longer infectious. Given a minimum temperature of 60 degrees Centigrade and a heating time of 10 hours procedures followed by most factor manufacturers the extrapolated minimum reduction of virus would be 20 logs. There seems to be enough of a safety factor afforded by antihemophilic factor heat treatment to permit the conclusion that infectious LAV/HTLV-III is unlikely to be present in currently licensed heat-treated factor and the use of such products should not result in additional cases of acquired immunodeficiency syndrome (AIDS) in hemophiliacs.

Humans with OKT4-epitope deficiency have a single nucleotide base change in the CD4 gene, resulting in substitution of TRP240 for ARG240☆

The OKT4 epitope of the CD4 cell-surface protein has been shown to be polymorphic in white, black, and Japanese populations. The variable phenotypic expression is due to an alteration of the OKT4 epitope, since those persons lacking reactivity with OKT4 monoclonal antibody (mAb) are reactive with OKT4A-F mAb as well as other mAb specific for CD4. To determine the nature of this polymorphism at the gene level, we sequenced polymerase chain reaction-amplified genomic DNA containing the CD4-V3 and -V4 exons from American black subjects who are OKT4-normal, OKT4-negative heterozygous, or OKT4-negative homozygous. Comparison of the sequences revealed that the two CD4 exons are identical except for a cytosine-to-thymidine transition occurring at nucleotide position 868. This alters the first codon position of mino acid 240 and results in a tryptophan residue replacing an arginine residue. The change was also found in white and Japanese persons who are OKT4-negative.

CD4—gp120 interactions

The three-dimensional structure of the binding domain of the CD4 molecule has been determined and extensive mutational analyses of the respective binding sites on gp120 and CD4 have been completed. The consequences of gp120-CD4 binding with respect to secondary changes in the virion, or the cell, that may be required for infection or that may interfere with cellular function are current active areas of investigation.